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th Annual Minisymposium on Reproductive Biology

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					28th Annual Minisymposium
           on
   Reproductive Biology


         October 15, 2007
         Hilton Garden Inn
        1818 Maple Avenue
         Evanston, IL 60201




         Sponsored by:




     The offices of the President and
      Vice President for Research
                     About the Center for Reproductive Science
The Center for Reproductive Science (CRS) includes faculty whose research is relevant, in a broad sense, to
reproductive biology and human medicine. The Center does not have a discrete physical location, but rather includes
faculty from across the University who share common interests in research and training in the reproductive sciences.

CRS has three missions: 1. To enhance and coordinate research in the reproductive sciences at Northwestern, 2. To
promote the application of this research toward human welfare, and 3. To promote training in reproductive sciences
of future research, teaching and clinical investigators.

CRS includes 44 faculty from 13 departments in the Weinberg College of Arts and Sciences and the McCormick
School of Engineering on the Evanston campus and Children’s Memorial Research Center and the Feinberg School
of Medicine on the Chicago campus. Research areas of current faculty interest include: circadian rhythms; clinical
fertility and infertility; gonadal development and function; growth and development; neural control of sexual
function; neuroendocrinology; ovarian function; prostate biology; reproduction and society; and testis function.

CRS administers several multidisciplinary NIH grants in the reproductive sciences, including the T32 Training
Program in Reproductive Biology (Jon Levine, P.I.), the P01 Program Project Hormonal Signals that Regulate
Ovarian Differentiation (Kelly Mayo, P.I.) and the U54 Cooperative Center Structure Function Relationships in
Reproductive Biology (Teresa Woodruff, P.I.). For further information about the center, please visit the CRS
website at www.northwestern.edu/research/crs/ The CRS Newsletter entitled “Reproduction Matters” is a biannual
publication available on the CRS website.

                                   Center-Sponsored Awards
CRS “Go to Meeting” Award. This is a travel award for undergraduates working in the laboratories of CRS
Members to accompany other lab members to professional meetings such as the annual meeting of the Endocrine
Society, Society for the Study of Reproduction, Society for Neuroscience, etc.

Marcia L. Storch Scholarship for Undergraduate Women Dr. Storch practiced gynecology in New York
City for a number of years and died at her home in Maine. She was always interested in the education of young
women, and stipulated that donations in her name be made to the Center for Reproductive Science for the purpose of
introducing undergraduate women to research. This scholarship is awarded to undergraduate women working in
some aspect of ovarian research and provides a sum for supplies in the designated laboratory.

Go to www.northwestern.edu/research/crs/ for details on how to apply for the CRS “Go to Meeting” and the
“Marcia L. Storch Scholarship for Undergraduate Women” awards. The deadline to apply for both awards is June 1,
2008.

Constance Campbell Memorial Research Award. The awards given at the Annual Minisymposium on
Reproductive Biology are named after Constance Campbell. Connie, a Ph.D. in psychology from University of
                   Illinois-Chicago, was hired as an assistant professor in the department of Biological Sciences at
                   Northwestern in 1974. Her research was on the relationship of sexual and other behaviors to
                   environmental cues. She was promoted to associate professor in the department of
                   Neurobiology and Physiology and administered the undergraduate honors program before her
                   untimely death in 1981. Connie cared deeply about student research. After her death, her
                   family, friends and colleagues raised money which is now used to fund the Constance
                   Campbell Memorial Research Awards for the Minisymposium on Reproductive Biology




Cover Photograph: E 12.5 xy gonad stained with antibodies against laminin to outline testis cord
structure. Laminin also outlines the mesonephic duct and tubules (bottom of frame).Picture courtesy of
Dr. Blanche Capel
                                 Table of Contents

                                                                        Page #
About the Center/Center Sponsored Awards……………….…………………………... Inside Cover
Neena B. Schwartz Lectureship in Reproductive Science….…….…….……...…………………… .2
      Lectureship Recipient, Blanche Capel, Ph.D.…….….………..……………….………..……...3
Northwestern Alumni Speakers……………………....……………………………………...………4
Program for Minisymposium…………..……...………………….………………………………… 5
Abstracts:
         Oral Session…………………………………………………………………………......…12
         Poster Session….…………..…………………………………………………………..…..28
List of Presenters………………………...…………………………………………………………54




                              Minisymposium Overview

           8:00 - 8:25 AM            Registration/Continental Breakfast
           8:25 – 8:30 AM            Welcome/Announcements
           8:30 -8:45 AM             Northwestern Alumni Speaker – Dr. Jose Santiago
           8:45 - 10:15 AM           Oral Session I
           10:15 - 10:30 AM          Coffee Break
           10:30-10:45 AM            Northwestern Alumni Speaker – Dr. Sarah Leupen
           10:45-12:00 PM            Oral Session II
           12:00 -1:00 PM            Lunch
           1:00 - 2:15 PM            Poster Session
           2:15 - 2:30 PM            Northwestern Alumni Speaker – Dr. Jackie Jeruss
           2:30 - 3:45 PM            Oral Session III
           3:45 - 4:30 PM            Wine and Cheese Reception
           4:30 - 4:40 PM            Intro to Keynote Address
           4:40- 5:40 PM             Keynote Address – Dr. Blanche Capel
           5:40 - 5:50 PM            Awards Presentations
           5:50 PM                   Closing Comments/Adjourn
        Neena B. Schwartz Lectureship in Reproductive Science


This year is the 28th anniversary of the Minisymposium on Reproductive Biology. The idea for
the Minisymposium, a day-long event focused on providing trainees with an early opportunity to
present their research, came primarily from Dr. Neena B. Schwartz, William Deering Professor
Emerita of Biological Sciences and founder of the Center for Reproductive Science.

Neena is a native of Baltimore who has spent most of her
research and teaching career in the greater Chicago area.
After obtaining her undergraduate degree from Goucher
College, Neena completed her M.S. and Ph.D. degrees in
physiology at Northwestern University. Her first faculty
position was at the University Of Illinois College Of
Medicine, where she rose to the rank of Professor before
moving to Northwestern University in 1974 as Chair of
the Department of Biological Sciences. Neena founded
the Program in Reproductive Research, now the Center
for Reproductive Science, in 1980, and developed the program into a premier training site for
students and young investigators interested in reproductive endocrinology.

Neena has had a distinguished and productive research career and has made many seminal
contributions toward understanding the hypothalamic-pituitary-gonadal axis and its control. Her
early studies on the rat estrous cycle established many of the basic tenets of cyclical changes in
gonadotropin secretion upon which our current views of the HPG axis are based. Much of
Neena’s research has focused on the issue of differential regulation of pituitary FSH and LH
secretion. It was this interest that led her and the late Cornelia Channing to describe a
nonsteroidal feedback factor from the ovary involved in controlling the secondary FSH surge,
and Neena’s laboratory went on to make many important contributions to the study of ovarian
inhibin.

To honor Dr. Schwartz on the 25th anniversary of the Minisymposium, the Center for
Reproductive Science named the keynote address the Neena B. Schwartz Lectureship in
Reproductive Science. The past lectureships were given to Dr. William F. Crowley Jr., Professor
of Medicine at Harvard Medical School and Massachusetts General Hospital, Dr. J. David Puett,
Regents Professor and Head of the Department of Biochemistry & Molecular Biology at the
University of Georgia, and Dr. JoAnne S. Richards, Professor of Molecular Biology and Cellular
Biology at Baylor College of Medicine. This year, Dr. Blanche Capel, Professor of Cellular
Biology at Duke University will give the keynote address and receive the lectureship award.




                                                2
              28th Minisymposium on Reproductive Biology
          Neena B. Schwartz Lectureship in Reproductive Science




                                             Dr. Blanche Capel
My laboratory has been very interested in the biology of sex determination, and the basic questions it raises about
how patterning decisions are made during organ development.

    Unlike most organs whose fate is pre-determined, the gonad arises as a bipotential primordium that can chose to
follow one of two developmental pathways. ‘Primary sex determination’ refers to the decision within the gonad to
develop as a testis or ovary, and involves initial cell fate decisions coordinated with a dramatic reorganization of the
tissue into testis or ovary morphology. Recent evidence suggests that a balance between Fgf and Wnt signaling
pathways explains the underlying property of ‘bipotentiality’ in cells of the gonad. We have proposed that Sry, the
Y-linked sex-determining gene in mammals, functions by tipping the balance between these signals toward testis
development. Downstream of this event, the pathway is stabilized by feed-back and feed-forward loops and by the
architectural reorganization of gonadal cells – both somatic and germ cells -- into testis or ovarian structure.

We are interested in whether this model underlies sex determination in other species where the molelcular switch
between male and female development is not Sry. To address this question, we are currently investigating whether
antagonistic signals between Fgfs and Wnts are conserved in other vertebrates such as red eared slider turtles where
sex determination is regulated by the temperature of egg incubation. In addition, we are very interested in how
extracellular signals are co-ordinated with intracellular pathways that regulate the fate decision of somatic cells that
differentiate either as Sertoli cells or follicle cells. As Sertoli or follicle cells regulate whether the gonad
differentiates as a testis or ovary, this cell fate decision controls sex determination.

The general viability of mice harboring mutant gonad phenotypes and the unique bipotentiality of the gonad
primordium make the gonad a powerful tool for elucidating the underlying conserved mechanisms at play in the
development of all organ systems. A second principle focus of the lab has been on how cell signaling leads to the
morphological reorganization of the cells in the gonad into testis or ovarian structure. Using live imaging, we are
exploring the architectural patterning of the testis focusing on the role of the vasculature and the de novo
organization of testis cord structures.

 A key outcome of testis organogenesis is the establishment of the germline stem cell niche inside testis cords. Our
efforts are centered on understanding how germ cells normally come under the regulatory control of Sertoli cells
and transition into pro-spermatogonia. To address this question, we are investigating Ter mutants, a classic mouse
mutant in which germ cells do not successfully navigate this transition, but instead undergo a transformation event,
and give rise to teratocarcinomas. Our efforts are focused on how loss of DND1 (the RNA-binding protein
responsible for the Ter phenotype) disrupts the normal regulation between Sertoli and germ cells.




                             For further information about our research, please visit:
                              http://www.cellbio.duke.edu/Faculty/Capel/index.html
                                                           3
                        Northwestern Alumni Guests


                                                Jose Santiago, Ph.D.
                                         Scientific Affairs Area Manager
                                                     Diagnostics Division
                                           Abbott Laboratories, Chicago
      Northwestern University Graduate Student, 1998-2004 ,Woodruff Lab




                   Sarah Leupen, Ph.D.
                   Assistant Professor
                   Department of Zoology
                   Ohio Wesleyan University

                   Northwestern University Graduate Student, 1989-1994, Levine Lab




                                      Jackie Jeruss, M.D., Ph.D
                                                  Assistant Professor
                                    Department of Medicine-Surgery
                Feinberg School of Medicine, Northwestern University

Northwestern University Postdoctoral Fellow, 2001-2003, Woodruff Lab




                                           4
                          PROGRAM FOR
                   28th ANNUAL MINISYMPOSIUM
                               ON
                      REPRODUCTIVE BIOLOGY
                                    October 15, 2007
                             Northshore and Lakeshore Rooms
                                    Hilton Garden Inn


8:00-8:30 AM            Registration                        Atrium
                        Continental Breakfast               Northshore Room
                        Run-through talks                   Northshore Room
                        Set up Posters                      Lakeshore Room & Atrium

8:25 AM                 Opening Remarks – Kristin Meldi, Department of Biochemistry,
                        Molecular Biology and Cell Biology, Northwestern University, Evanston,
                        IL

8:30 AM                 Northwestern Alumni Speaker – Jose Santiago, PhD, Scientific Affairs
                        Area Manager, Abbott Laboratories

ORAL SESSION I: Early Development and Germ Cell Differentiation
Session Moderators: Daniel Trombly, Cell and Molecular Biology, Northwestern University,
           Chicago, IL and Alison Kim, Obstetrics and Gynecology, Northwestern University,
           Evanston, IL

           Abstract #    Presenting authors shown in bold

8:45 AM        1        PRAP/17β Hydroxysteroid Dehydrogenase Type 7 Gene Plays a Crucial
                        Role in Embryonic Development and Fetal Survival, Aurora Shehu1,
                        Jifang Mao1, Gil Gibori1, Julia Halperin1, Jamie Le1, Hiroaki Kiyokawa2,
                        and Geula Gibori1, 1Department of Physiology and Biophysics, College of
                        Medicine, University of Illinois at Chicago; 2Department of Molecular
                        Pharmacology and Biological Chemistry and Robert H. Lurie
                        Comprehensive Cancer Center, Northwestern University, Chicago, IL

9:00 AM        2        Beta-Catenin is Essential for Proper Development of Fetal Ovaries,
                        Chia-Feng Lui1, Nathan Bingham2, Keith Parker2, Humphrey H-C
                        Yao1, 1Department of Veterinary Biosciences, University of Illinois at
                        Urbana-Champaign, IL; 2Department of Internal Medicine, University of
                        Texas Southwestern Medical Center, Dallas, TX

9:15 AM        3        Characterization of A Mouse Mutation Disrupting Murine Homolog of
                        Highly Conserved Germ Cell Regulator—Pumilio, Yanmei Chen and
                        Eugene Yujun Xu, Division of Reproductive Biology Research,
                        Department of OB/GYN, and Center of Genetic Medicine, Northwestern
                        University Feinberg School of Medicine, Chicago, IL

                                                5
9:30 AM          4       Sox3 is Important for the Initiation and Progression of Early
                         Spermatogenesis, M.M. Laronda, M.L. Bernhardt, J.L.Jameson,
                         Division of Endocrinology, Department of Medicine, Feinberg School of
                         Medicine, Northwestern University, Chicago, IL

9:45 AM          5       Transdifferentiation of Spermatogonial Stem Cells into Prostatic
                         Epithelium, Liz Simon, Natalia Kostereva, Rex A. Hess, Marie Claude
                         Hofmann ad Paul S. Cooke, Department of Veterinary Biosciences,
                         University of Illinois at Urbana-Champaign, Urbana, IL

10:00 AM         6       The highly conserved reproductive regulator—murine Boule, like its fly
                         counterpart, is also required for meiotic progression, Chirag Shah,
                         Villian Naeem, Yanmei Chen, Yin Wang, Mike VanGompel, and
                         Eugene Yujun Xu, Division of Reproductive Biology Research,
                         Department of OBGYN and Center for Genetic Medicine, Northwestern
                         University Feinberg School of Medicine

10:15-10:30 AM          Coffee Break

10:30 AM                 Northwestern Alumni Speaker - Dr. Sarah Leupen, Assistant Professor,
                         Department of Zoology, Ohio Wesleyan University.



ORAL SESSION II: Estrogen Receptor Expression and Signaling
        Session Moderators: Candace Tingen, Obstetrics and Gynecology Northwestern
         University, Evanston, IL and Anne Marie Wissman, Neurobiology & Physiology,
         Northwestern University, Evanston, IL

           Abstract #

10:45 AM         7       Activin Regulates Estrogen Receptor Gene Expression in the Mouse
                         Ovary, Jingjing L. Kipp1,3, Signe M. Kilen1,3, Teresa K. Woodruff1,2,3,
                         and Kelly E. Mayo1,3, 1Department of Biochemistry, Molecular Biology
                         and Cell Biology; 2Department of Obstetrics and Gynecology; 3Center
                         for Reproductive Science, Northwestern University, Evanston, IL 60208

11:00 AM         8       Overexpression of Estrogen Receptor Alpha Increases Sensitivity to
                         Methoxychlor in the Mouse Ovary, T. Paulose, J.A. Flaws, Department of
                         Veterinary Biosciences, University of Illinois, Urbana, IL

11:15 AM         9       The CCAAT Enhancer Binding Protein Beta (C/EBP-β) is a Critical
                         Mediator of Estrogen Induced Cell Proliferation in the Uterine Epithelium
                         of Mice, Ramathal C.R.1, Bagchi I.C.2 and Bagchi M.K.3,1, 1Department
                         of Cell and Development Biology, 2Department of Veterinary Bioscience,
                         3
                          Department of Molecular and Integrative Physiology, University of
                         Illinois, Urbana-Champaign, IL

                                                6
11:30 AM        10     Involvment of p21 Activated Kinase in the Estrogen (E2) Regulation of
                       GnRH/LH Secretion, Z. Zhao, and J.E. Levine, Department of
                       Neurobiology and Physiology, Northwestern University, Evanston, IL
                       60208

11:45 AM        11     ERE-Independent ERα Signaling does not Rescue Sexual Behavior but
                       Restores Normal Testosterone Secretion in Male ΕΡαΚΟ Mice, Melissa
                       Chamberlin McDivitt1, Christine Glidewell-Kenney2, Jeffrey Weiss2,
                       Pierre Chambon3, J. Larry Jameson2, Jon E. Levine1

12:00-1:00 PM          Lunch Break

1:00-2:15 PM           POSTER SESSION

 Abstract #

     P1    Regulation of Lactate Dehydrogenase C (Ldhc) Gene Expression, Huanghui Tang,
           Aisha Kung, John McCarrey*, Erwin Goldberg, Department of Biochemistry,
           Molecular Biology and Cell Biology and Cell Biology, Northwestern University,
           Evanston, IL 60201. *Health Science Center at San Antonio, University of Texas, TX
           78229

     P2    Disription of the Sperm-Specific Ldhc Gene is Detrimental to Sperm Function and Male
           Fertility, Chongwen Duan and Erwin Goldberg, Northwestern University, Evanston, IL

     P3    Notch 1 Promotes Type A Spermatogonia Proliferation and Differentiation by Down-
           Regulating p53-mediated Cell Cycle Arrest, 1Natalia Kostereva, 1Kay Carnes and
           1,2
              Marie-Claude Hofmann. 1Department of Veterinary Biosciences and 2Institute for
           Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61802

     P4    Transcription Regulation of Cyp26b1 in a Mouse Sertoli Cell Line, Miranda L.
           Bernhardt, J. Larry Jameson, Division of Endocrinology, Division of Medicine,
           Feinberg School of Medicine, Northwestern University, Chicago, IL

     P5    Silver Nanoparticles Disrupt GDNF Signaling in Male Germ-line Stem Cells,
           1,2
               Benjamin Lucus, 3Laura Braydich-Stolle, 1,2Timothy Lee, 3John Schlager, 3Saber
           Hussain and 1,2Marie-Claude Hofmann, 1Department of Veterinary Biosciences and
           2
             Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 2001
           South Lincoln Avenue, Urbana, IL, 3Applied Biotechnology Branch, Human
           Effectiveness Directorate, Air Force Research Laboratory/HEPB, Wright-Patterson
           Air Force Base, OH 45433

P6         An Alginate Based Culture System Provides an Appropriate Environment for In Vitro
           Ovarian Follicle Development, Elizabeth Parrish1, Teresa Woodruff2, Lonnie D. Shea1,
           1
            Department of Chemical and Biological Engineering, 2Department of Obstetrics and
           Gynecology, Northwestern University, Evanston, IL



                                              7
P7    Long-term Culture of Monkey and Human Preantral Follicle in Three-Dimensional
      Alginate Scaffold, Min Xu1,2, Mary B. Zelinski3,4, Laxmi A. Kondapalli1,2, Richard
      L. Stouffer5, Lonnie D. Shea3,4, Teresa K. Woodruff1,2,4, 1Department of Obstetrics
      and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL
      60611; 2Center for Reproductive Research, Northwestern University, Evanston, IL
      60208; 3Department of Chemical and Biological Engineering, Northwestern
      University, Evanston, IL 60208; 4The Robert H. Lurie Comprehensive Cancer of
      Northwestern University Chicago, IL 60611; 5Division of Reproductive Biology,
      Oregon National Primate Research Center, Oregon Health & Science University,
      Beaverton, Oregon

P8    Aquaporin Expression and Antral Cavity Development are regulated by Matrix
      Stiffness in an In Vitro Ovanian Follicle Culture System, Erin R. West1, Monica A.
      Gomberg2, Teresa K. Woodruff2,3,4, Lonnie D. Shea1,3,4, 1Department of Chemical
      and Biological Engineering, 2Department of Obstetrics and Gynecology, The
      Feinberg School of Medicine, 3Center for Reproductive Research, Northwestern
      University, Evanston, IL, 4The Robert H. Lurie Comprehensive Cancer Center of
      Northwestern University

P9    Activan of Smoothened, a Hedgehog Signaling Transducer, Induces Ectopic
      Appearance of Fetal Leydig cells in Fetal Ovaries, Ivraym Barsoum1, Nathan
      Bingham2, Keith Parker2 and Humphrey H-C Yao3, 1Department of Cell &
      Developmental Biology, University of Illinois, Urbana, IL; 2Department of Internal
      Medicine, University of Texas Southwestern Medical Center, Dallas, TX;
      3
        Department of Veterinary Biosciences, University of Illinois, Urbana, IL

P10   Activan Signaling Influences the Stromal Cell Population in Neonatal Mouse Ovaries,
      Daniel Trombly, Teresa Woodruff, and Kelly E. Mayo, Department of Biochemistry,
      Molecular Biology & Cell Biology and Center for Reproductive Science, Northwestern
      University, Evanston, IL 60208

P11   Activin/inhibin are Involved in the FSH-Regulation Early Folliculogenesis in Mouse,
      Lei Lei, Monica Antenos, Jie Zhu, Teresa K. Woodruff, Department of Obstetrics and
      Gynecology, Northwestern University Feinberg School of Medicine, Evanston, IL
      60208

P12   Abnormal Development in Female Gonads of Fused Toes Mutant Mice, B.Kim, J.S.
      Jorgensen, Department of Veterinary Biosciences, University of Illinois, Urbana, IL

P13   Conditional Knockout of Connexin 43 in Mouse Uterus Uncovers an Essential Role
      of Stromal Cell Gap Junctions in Uterine Angiogenesis during Pregnancy, Mary
      Laws1, Francesco DeMayo3, John Lydon3, Milan K. Bagchi2, and Indrani C. Bagchi1,
      1
        Department of Veterinary Biosciences, 2Department of Molecular and Integrative
      Physiology, University of Illinois at Urbana-Champaign, 3Baylor College of
      Medicine, Houston, TX




                                         8
P14     The Adaptor Protein MyD88 is Essential for E.coli induced Preterm Labor, Yana
        Filipovich, Shi-Jiang Lu, Shizuo Akira and Emmet Hirsch, Departments of Obstetrics
        and Gynecology, Evanston Northwestern Healthcare and Feinberg School of
        Medicine, Northwestern University

  P15   P450 Aromatase-Derived Intra-Uterine Estrogen is Critical for Decidualization,
        Amrita Das1, Srinivasa R. Mantena1, Athilakshmi Kannan1, Milan K. Bagchi2, and
        Indrani C. Bagchi1, 1Department of Veterinary Biosciences, 2Department of
        Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign,
        Urbana, IL

  P16   Hyperphagia is Introduced by the Steroid Milieu of Pregnancy: The Role of Estrogen
        Induced Nuclear Progesterone Receptor, C. Park, Z. Zhao, T.H. Horton, J.E. Levine,
        Northwestern University

  P17   Neonatal Estrogen Exposure Results in Hyperplastic Obesity in Adult Mice, M.A.
        Cimafranca, D. Meling, C.M. Beals, P.S. Cooke, Department of Veterinary
        Biosciences, University of Illinois, Urbana-Champaign, Urbana, IL

  P18   Circadian and Diurnal Activity Rhythm Comparisons in OB/OB and DB/DB Mice,
        D.M. Arble, M.R. Augustine, A.D. Laposky, & F.W. Turek, Sleep & Circadian
        Biology, Northwestern University, Evanston, IL

  P19   Estrogen Experts Rapid Effects on Thermoregulation in Ovariectomized Mice, J.L.
        White, W.C. Levine, J.E. Levine, and T.H. Horton, Department of Neurobiology and
        Physiology and The Center for Reproductive Science, Northwestern University,
        Evanston, IL

  P20   Ovarian Steroidogenic Regulation is Dependent upon ERE-Mediated ERα Signaling,
        D.A. Smith, C. Glidewell-Kenney, J.L. Jameson, Division of Endocrinology,
        Department of Medicine, Feinberg School of Medicine, Northwestern University,
        Chicago, IL

  P21   Follicular Atresia during the Pre-Pubertal Period in Mice is not Due to Classical
        Apoptosis, Candace M. Tingen1 and Dr. Teresa K. Woodruff1,2,3, 1Department of
        Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, IL
        60208, 2Department of Neurobiology and Physiology, Northwestern University,
        Evanston, IL 60208, 3Department of Obstetrics and Gynecology, Feinberg School of
        Medicine, Northwestern University, Chicago, IL 60611, 4Center for Reproductive
        Science, Northwestern University, Evanston, IL 60208

  P22   Methoxychlor Alters the Expression of Selected Apoptotic Factors in the Ovary,
        Weck RL, Gupta RK, Flaws JA, University of Illinois Urbana-Champaign, Urbana,
        Illinois




                                           9
   P23    Cell Cycle Regulators in Mouse Ovarian Antral Follicles are altered by Methoxychlor
          Exposure, Gupta RK, Meachum SH, Yao HH, and Flaws JA, Department of
          Veterninary Biosciences, School of Veterinary Medicine, University of Illinois at
          Urbana-Champaign

   P24    Age-Related Changes in Follicle Growth in Aryl Hydrocarbon Receptor-Deficient Mice,
          I. Hernandez-Ochoa, R.K. Gupta, B.N. Karman, J.A. Flaws, Department of Veterinary
          Biosciences, University of Illinois at Urbana-Champaign, Urbana IL

   P25    Epithelial Lineage of Ovarian Carcinoma in the Laying Hen, Gallus Domesticus,
          Kristine Ansenberger, University of Illinois at Chicago

   P26    From A to Zinc: The Inorganic Physiology of Mammalian Oocytes, Alison M. Kim1,
          Thomas V. O’Halloran2,3, Teresa K. Woodruff1,3,4, 1Department of Obstetrics and
          Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL
          60611; 2Department of Chemistry, Northwestern University, Evanston, IL 60208;
          3
            Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern
          University, Evanston, IL 60208; 4Center for Reproductive Science, Northwestern
          University, Evanston, IL 60208




2:15PM       Northwestern Alumni Speaker- Dr. Jackie Jeruss , Feinberg School of Medicine

ORAL SESSION III: Cell Signaling in Ovarian and Uterine Function
         Session Moderators: Micheal VanGompel, OB/GYN, Northwestern University,
         Chicago, IL and Monica Laronda, Medicine - Endocrinology, Northwestern
         University, Chicago, IL

           Abstract #

2:30 PM        12       Nerve Growth Factor Inducible B (NGFI-B) Represses Inhibin Alpha
                        Expression Independently of DNA-binding in Mouse Grandulosa Cells,
                        Kristin M. Meldi, Anna D. Burkart, PhD., and Kelly E. Mayo, PhD.,
                        Department of Biochemistry, Molecular Biology and Cell Biology, Center
                        for Reproductive Science, Northwestern University, Evanston, IL

2:45 PM        13       Expression of Hypoxia-Inducible Factors in the Ovary: A Novel Role
                        during Ovulation in Mice, Jaeyeon Kim1, Indrani C. Bagchi2, Milan K.
                        Bagchi1, 1Deapartment of Molecular & Intergrative Physiology,
                        2
                          Department of Veterinary Biosciences, University of Illinois at Urbana-
                        Champaign, Urbana, IL

3:00 PM        14       Manipulation of Actin Dynamics Impacts Human Uterine Fibroblast
                        Decidualization, I. Ihnatovych, M. Livak, Z. Strakova, Department of
                        Obstetrics and Gynecology, University of Illinois at Chicago, Chicago,
                        IL 60612
                                               10
3:15 PM           15     The Role of Toll-like Receptor 2 (TLR-2) Activation in Bacterially
                         Induced Preterm Labor in Mice, Vladimir Ilievski, MD1 and Emmet
                         Hirsh, MD1,2, 1Obstetrics and Gynecology, Evanston Northwestern
                         Healthcare, Evanston, IL United States, 60201 and 2Obstetrics and
                         Gynecology, Feinberg School of Medicine, Northwestern University,
                         Chicago, IL, United States, 60611

3:30 PM           16     Downregulation of Dickkopf-1 by Three-Dimensional Culture Leads to
                         Membrane Type-1 Matrix Metalloproteinase Upregulation, Maria V.
                         Barbolina1, Lonnie D. Shea1, M. Sharon Stack2, 1Department of
                         Chemical and Biological Engineering, Northwestern University,
                         Chicago, IL, 2Department of Pathology and Anatomical Sciences,
                         University of Missouri, Columbia, MO

3:45-4:30 PM             Wine and Cheese Reception

4:30-4:40 PM             Introduction of Keynote Speaker – Anne Marie Wissman, Neurobiology
                         & Physiology, Northwestern University, Chicago, IL

                         Presentation of the Neena B. Schwartz Lectureship in Reproductive
                         Science – Dr. Teresa Woodruff, Associate Director, Center for
                         Reproductive Science, Northwestern University, Evanston, IL

4:40- 5:30 PM            Keynote Address – The Battle of the Sexes: Antagonistic Signals Regulate
                         Sex Determination, Blanche Capel, PhD, Professor of Cellular Biology,
                         Duke University.

5:30-5:45 PM             Presentation of Constance Campbell Research Awards -
                         Dr. Teresa Woodruff, Associate Director, Center for Reproductive Science

5:45 PM                  Closing Comments – Kristin Meldi, Biochemistry, Molecular Biology
                         and Cell Biology, Northwestern University, Evanston, IL



                Please take down posters immediately following the Minisymposium




                                                11
                                                                                         Abstract # 1

PRAP/17β-Hydroxysteroid Dehydrogenase Type 7 Gene Plays a Crucial Role in
Embryonic Development and Fetal Survival
Aurora Shehu1, Jifang Mao1, Gil Gibori1, Julia Halperin1, Jamie Le1, Hiroaki Kiyokawa2 and
Geula Gibori1. 1Department of Physiology and Biophysics, College of Medicine, University of
Illinois at Chicago; 2Department of Molecular Pharmacology and Biological Chemistry and
Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL.

Our laboratory has previously cloned and purified a novel 17β-HSD enzyme, (PRAP/17β-HSD7)
which is highly expressed in the corpus luteum of the ovary and converts estrone to estradiol. To
examine the functional significance of PRAP/17β-HSD7in pregnancy, we generated a knockout
mouse model with a deletion of the PRAP/17β-HSD7gene. We anticipated a mouse with severe
fertility defect due to its inability to regulate estrogen levels during pregnancy in the absence of
this enzyme. The heterozygous mutant mice (PRAP/17β-HSD7+/-) appeared normal in their
development and gross anatomy. They were born with the expected Mendelian frequency from
F1 parents and grew to adulthood. They were fertile as indicated by normal estrus cycle, by their
ability to become pregnant and by the size of the litter. In addition, no difference was observed
at both mRNA and protein levels of PRAP/17β-HSD7between the postpartum ovaries of
heterozygous and their wild type littermates. To our surprise, the breeding of heterozygous mice
yielded no viable PRAP/17β-HSD7null mice. However, we found PRAP/17β-HSD7null embryo
in utero at day 8.5 and 9.5 of gestation. These embryos were dramatically smaller in size and
displayed severe disorganization of blood vessels as compared to heterozygous and wild type
embryos. The null embryos were found in utero until day 11.5 with severe resorption. The
cause of this early lethality is unknown, as there is no known function of fetal steroids at
embryonic day 8.5. However this gene is expressed in the placenta where estradiol may play an
important role in vascularization and in the early development of the fetus. Recent reports have
shown that PRAP/17β-HSD7also participates in cholesterol biosynthesis. Since cholesterol is
known to be essential for normal fetal development, PRAP/17β-HSD7 may also play a role early
in development through this pathway. This is the first discovery identifying a role for PRAP/17β-
HSD7gene in early mouse development and raises important questions regarding the role and
significance of this gene’s function.

Supported by NIH grants HD11119 and HD12356 (GG) and APS (JH).




                                                12
                                                                                          Abstract #2

Beta-Catenin Is Essential for Proper Development of Fetal Ovaries Chia-Feng Liu1, Nathan
Bingham2, Keith Parker2, Humphrey H-C Yao1, 1Department of Veterinary Biosciences,
University of Illinois at Urbana-Champaign, IL; 2Department of Internal Medicine, University of
Texas Southwestern Medical Center, Dallas, TX

The development of mammalian ovary is considered as a default process, arising only in the
absence of the testis-determining gene Sry. However, ovary-to-testis sex reversal without the
existence of the Sry gene in human and animal cases counters the default concept of the ovarian
development. To explain these unusual phenomena, an alternative hypothesis was proposed that
a “Z” gene is present in the fetal ovary to suppress testis development and promoting ovarian
development. Several ovarian specific genes have been considered as candidates for the Z gene
and among them, Wnt4 and R-spondin1 are two most possible ones. Wnt4 is one of the Wnt
(wingless related protein) family proteins, which are important for many aspects of embryo
development. At early embryonic stage, Wnt4 is expressed in the fetal gonad of both sexes but
becomes ovary-specific at the time of sex determination. In Wnt4 null mice, ectopic testis
vasculature and production of androgens occurred in fetal ovaries, leading to masculinization of
the XX embryo. Mutations in human WNT4 gene cause virilization phenotypes similar to those
in Wnt4 knockout mice. In addition to Wnt4, R-spondin 1, a member of the orphan ligands, was
recently identified to be essential for ovary development in humans. Patients with mutations in
R-spondin1 gene appear ovary-to-testis sex reversal phenotypes. The appearance of testis
characteristics in the mutation of Wnt4 or R-spondin1 in fetal ovaries suggests that these two
genes own some properties of the Z gene. Interestingly, both Wnt4 and R-spondin1 have been
shown to induce similar intracellular signaling through beta-catenin, indicating a possible
interaction between these two molecules. Based on these findings, we hypothesized that beta-
catenin is essential for the fetal ovary development. To test this hypothesis, we applied a loss-of-
function model to mutate beta-catenin in fetal gonads and then examined the development of
fetal ovaries. Taking advantage of the SF-1/cre (Steroidogenic factor1-cre) mouse line, which
expressed Cre recombinase in somatic cells of fetal gonads around embryonic day 11.5(E11.5),
we avoided the early embryonic death of the universal beta-catenin knockout. In female beta-
catenin conditional knockout newborns, partial female to male sex-reversal phenotypes appeared
with the formation of colemic vessel and an epididymis-like structure, indicating ectopic
production of androgens. Female germ cells were also lost in the absence of beta-catenin. These
phenotypes are identical with those in the Wnt4 knockout female but differed from the R-
spondin1 null phenotypes in humans, which resulted in complete sex reversal. Our results
suggested that Wnt4 might signal through beta-catenin to suppress the masculinization of the
fetal ovaries. In addition, Wnt4 and R-spondin1 probably operate through different signaling
pathways.


Supported by NIH-HD46861 and the March of Dimes Birth Defects Foundation




                                                13
                                                                                       Abstract #3

Characterization of a Mouse Mutation Disrupting Murine Homolog of Highly Conserved
Germ Cell Regulator--Pumilio
Yanmei Chen and Eugene Yujun Xu, Division of Reproductive Biology Research, Department
of OBGYN, and Center for Genetic Medicine, Northwestern University Feinberg School of
Medicine, Chicago, IL.

Pumilio is a member of the highly conserved Puf-family of RNA-binding proteins that act as
translational repressors. Studies in invertebrates and lower vertebrates revealed that Pumilio is
required for multiple stages of germ cell development including stem cell regulation, primordial
germ cell differentiation, meiotic maturation and sperm-egg switch. Two Pumilio genes, Pum1
and Pum2, have been identified in mammal. To determine the functional requirement of murine
homologs of highly conserved RNA binding protein-Pumilio, we used mouse genetic analyses,
blastocyst PCR genotyping method and in vitro culturing system to characterize the phenotype of
a Pumilio-1 (Pum1) gene trap mutation. We found that mice homozygous for the Pum1 mutation
are not present among pups from heterozygotes crosses. Analysis of different stages of embryos
reveal that homozygotes are absent in all stages as early as blastocysts. The lack of Pum1
homozygotes suggests that mammalian Pum1 might play an important role in very early stages
of embryonic development or the formation of zygotes. This role is not dispensable and can’t be
compensated by the other member of Pumilio family--Pum2 protein.

This research is supported by NIH grant U01 HD045871.




                                               14
                                                                                       Abstract #4

Sox3 is Important for the Initiation and Progression of Early Spermatogenesis, M. M.
Laronda, M. L. Bernhardt, J. L. Jameson, Division of Endocrinology, Department of Medicine,
Feinberg School of Medicine, Northwestern University, Chicago, IL

Spermatogenesis is a tightly regulated process in which germ cells progress through various
germ cell stages starting with spermatogonial stem cells (SSCs) and undifferentiated
spermatogonia, and ending with mature spermatozoa. Spermatogenesis has been described as a
process with three phases: proliferation, meiosis and differentiation. In a normal mouse testis,
neonatal germ cells, called gonocytes, migrate to the basement membrane of the testis cord and
are supported by the Sertoli cells shortly after birth. It has been suggested that only a few
gonocytes that line the testis cords maintain their stem cell properties, while others succumb to
spermatogonial differentiation. By post-natal day 10 (P10), the first meiotic phase is initiated
and by P20 the first post-meiotic germ cells are visible. Spermatogenesis continues as the animal
matures and specific germ cell associations are synchronized within tubule cross-sections and are
visible by 8 weeks of age. Our lab has previously published a mouse model with impaired
spermatogenesis. The seminiferous tubules of the Sox3 C57BL/6 knockout model contain very
few germ cells by P20. However, tubules appear to improve, displaying more germ cell
associations, as the animals age. These data imply a delayed initiation of spermatogenesis. Our
lab and others have demonstrated Sox3 expression within a subset of undifferentiated
spermatogonia. RT-PCR analysis comparing P5 through P10 wildtype and Sox3-knockout
animals demonstrates a decrease in genes essential for SSC initiation, maintenance and for
progression through the phases of spermatogenesis. These data indicate the importance of Sox3
in early spermatogenesis. Because of the specific localization of Sox3 expression within the
undifferentiated population and the impact of Sox3 ablation on the testis, ongoing study of this
mouse model will provide a better understanding of the early spermatogenic process and its
impact on the adult testis.

Funding is provided by NIH Grant U01HD043425




                                               15
                                                                                        Abstract #5

Transdifferentiation of Spermatogonial Stem Cells into Prostatic Epithelium,
Liz Simon, Natalia Kostereva, Rex A. Hess, Marie Claude Hofmann and Paul S. Cooke.
Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL;

         Embryonic stem (ES) cells have great medical potential, but their use is constrained by
ethical, moral and legal limitations. This potential problem could be alleviated if stem cells of
other organ systems could be differentiated into tissues that could be used for therapy. In the
testis, spermatogonial stem cells (SSCs) generate differentiated germ cells that ultimately give
rise to sperm. Some recent literature suggests that extended culture of SSCs leads to the
formation of a subpopulation of cells with an ES-like morphology. These cells can be selected
and propagated, and can then differentiate into tissues with some characteristics of
cardiomyocytes, for example, although these cells appear to differ from cardiomyocytes in vivo.
If SSCs can be shown to be capable of differentiating into many or all somatic tissues, this would
pave the way for the use of these SSCs therapeutically, and would avoid complications of using
ES cells. The fetal urogenital sinus (UGS) gives rise to prostate in males, and vagina in females.
UGS consists of urogenital sinus mesenchyme (UGM) surrounding urogenital sinus epithelium
(UGE). During male development, androgen stimulates UGM to induce prostatic development
(formation of prostatic buds) in UGE, which forms prostatic epithelium. UGM is an instructive
inducer, and it can stimulate prostatic differentiation not only in UGE, but in bladder epithelia
(also derived from UGS) as well. Our hypothesis was that SSCs could directly transdifferentiate
into prostatic epithelium when SSCs were removed from their niche and recombined with UGM
and grown as tissue recombinants in vivo. Gonocytes (from 16.5-day male fetuses) or SSCs from
6-day-old neonatal male mice were isolated and recombined with UGM that had been
enzymatically dissociated from UGS of 16.5-day fetuses. Grafts were grown under the renal
capsule of male hosts for a month, then examined by histology and immunohistochemistry.
Gonocytes and SSCs recombined with UGM and grown in male hosts in vivo formed
histologically normal prostate, indicating that SSCs could transdifferentiate into prostatic
epithelium (n=13). Epithelium in these grafts expressed androgen receptor (AR) and NKX3.1, a
transcription factor that is a unique marker for prostatic differentiation; neither AR or NKX3.1
are normally found in SSCs. In addition, SSCs and all other germ cells normally express GCNA,
widely used as a germ cell marker. Epithelial cells in recovered grafts do not express GCNA.
This result, in concert with the AR and NKX3.1 data, indicates that the SSCs have totally lost
their germ cell characteristics and show the phenotypic and molecular markers characteristic of
prostatic differentiation. This study is the first to demonstrate that SSC can be directly
transdifferentiated into other somatic tissues; both gonocytes and SSCs are capable of this
transdifferentiation. These germline stem cells are clearly pluripotent, but their ultimate
developmental potential remains to be established. If SSCs can differentiate into many or all cell
types from all 3 germ layers, it would open up new vistas for using SSCs in regenerative
medicine and treating diseases and injuries like diabetes, Parkinson’s, spinal injuries, etc.


This work was supported by the Billie Field Endowment.




                                               16
                                                                                         Abstract #6

The Highly Conserved Reproductive Regulator—Murine Boule, like its Fly Counterpart, is
also Required for Meiotic Progression, Chirag Shah, Villian Naeem, Yanmei Chen, Yin Wang,
Mike VanGompel, and Eugene Yujun Xu, Division of Reproductive Biology Research,
Department of OBGYN and Center for Genetic Medicine, Northwestern University Feinberg
School of Medicine, Lurie 7-250, 303 E Superior Street, Chicago, IL-60611 USA

Conservation of developmental regulators across large evolutionary distance often reveal key
regulators of developmental processes such as Hox genes for axis formation and Pax 6 for eye
development. One exception to such developmental conservation is reproductive tissues where
reproductive genes and traits often evolve fast and rapid divergence is the common theme of
reproductive evolution. Given the strong selective pressure on reproductive tissues, it remains
unknown if any reproductive-specific regulator could maintain its function across large
evolutionary distance. Here we report a case of functional conservation for reproductive
regulator from insects to mammals. We show that Boule gene is an ancient reproductive
regulators present from primitive multicellular organism to fly and to human, suggesting Boule is
one of the earliest reproductive gene associated with primitive testis in multi-cellular organisms.
To determine if Boule protein remains functionally conserved across metazoan world, we
compared the Boule gene from two distant lineages—fly and mouse. Mouse Boule is similar to
fly Boule in sequence and expression pattern. Mouse Boule mutation is male sterile with no
detectable defect on somatic tissues like its fly counterpart and its spermatogenic development in
the testis is arrested similarly at meiotic entry stage. Hence BOULE represents an unusually
conserved reproductive-specific regulator required for sperm development from fly to mammals.

Supported by NIH grant U01 HD045871.




                                                17
                                                                                          Abstract #7

Activin Regulates Estrogen Receptor Gene Expression in the Mouse Ovary, Jingjing L.
Kipp1,3, Signe M. Kilen1,3, Teresa K. Woodruff1,2,3, and Kelly E. Mayo1,3 ,1Department of
Biochemistry, Molecular Biology and Cell Biology; 2Department of Obstetrics and Gynecology;
3
  Center for Reproductive Science, Northwestern University, Evanston, IL 60208


Activin, a member of the TGF-β superfamily, is an important modulator of FSH synthesis and
secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle
development. In order to identify activin regulated genes in the mouse ovary, a microarray study
was performed using RNAs collected from mouse granulosa cells maintained in primary culture
and treated with PBS, activin A, or activin A plus follistatin (an activin antagonist). Illumina
Sentrix mouse-6 bead chips containing 48,000 probes were used, and 14,641 of those probes
gave positive signals in the granulosa cells. We identified 75 genes that were significantly
stimulated (range 2-18 fold) and 52 genes that were significantly inhibited (range 2-11 fold) by
activin. The effect of activin on these genes was reversed by follistatin. Among the genes
stimulated by activin, one of particular interest was the estrogen receptor β (ERβ), as we
previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback
relationship between these two follicle-regulating hormones. The purpose of this study was to
investigate fully activin A regulation of ER expression. Real time RT-PCR assays on cultured
granulosa cells showed that both ERα and ERβ mRNAs were induced by activin A at 4, 12 and
24 hrs in a dose-responsive manner. Western blots confirmed an increase in their protein levels.
Consistent with increased ERα and ERβ expression, activin A stimulated estradiol-induced
estrogen response element (ERE) promoter activity. Activin A stimulation of ER expression was
a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was
abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ERα
promoter activity. To investigate the effect of activin in vivo and thus its biological significance,
we examined ER expression in inhibin transgenic mice that have decreased activin expression
and discovered that these mice had decreased ERα and ERβ expression in the ovary. We also
found that ER mRNA levels were decreased in MIS-Smad 2 dominant negative mice that have
impaired activin signaling through Smad 2 and siRNAs targeting Smad 2 or Smad 3 suppressed
ERα promoter activation, suggesting that Smad 2 and Smad 3 are involved in regulating ER
levels. Therefore, this study reveals an important role for activin in inducing the expression of
ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.



Supported by Program Project Grant HD91291.




                                                 18
                                                                                          Abstract #8

Overexpression of Estrogen Receptor Alpha Increases Sensitivity to Methoxychlor in the
Mouse Ovary, T. Paulose, J.A. Flaws, Department of Veterinary Biosciences, University of
Illinois, Urbana, IL

Xenobiotic chemicals such as the organochlorine pesticide methoxychlor (MXC) may disrupt the
normal functions of estrogen by either obstructing its normal course of action or interfering in
the physiological response to the hormone. MXC is also known to cause atresia of antral
follicles in mice. To examine whether MXC causes atresia through an estrogen receptor alpha
(ESR1) pathway, we generated a transgenic mouse model in which ESR1 is overexpressed in
several tissues including the ovaries. These mice were generated using the tetracycline
responsive system in which a transgene coding the ESR1 sequence was placed under the
regulatory control of a tetracycline-operator promoter (tet-op). In this model, transcriptional
activation of the ESR1 transgene is only achieved in the presence of a tetracycline responsive
transactivator (tTA) protein. In the presence of doxycycline (DOX), this protein undergoes a
conformational change and is rendered inactive causing ESR1 overexpression to be turned off.
To validate this mouse model, ovaries collected from control mice lacking the ESR1 transgene
and ESR1 overexpressor (ESR1-OE) mice were subjected to real-time polymerase chain reaction
(qPCR) for mRNA expression of ESR1. The results show that ESR1 is significantly increased in
ESR1-OE ovaries compared to controls (control = 0.32 ± 0.07 genomic equivalents (ge); ESR1-
OE = 0.93 ± 0.20 ge; n = 3; p ≤ 0.05). When doxycycline (200mg/kg) was administered via food
to the mice for 3 days, ESR1-OE ovaries and control ovaries expressed similar levels of ESR1,
indicating that the overexpression was successfully turned off (control = 0.19 ± 0.01 ge; ESR1-
OE = 0.21 ± 0.03 ge; n = 3; p = 0.47). To determine whether ESR1 overexpression affects the
ability of mice to respond to MXC, controls and ESR1-OE mice were treated with either vehicle
or MXC (32 and 64 mg/kg/day) for 20 days. The ovaries of these mice were then collected and
subjected to histological evaluation to analyze the percentage of atretic follicles and the thecal
cell thickness of antral follicles. The results indicate that the percentage of atretic follicles was
significantly increased in ESR1-OE and control mice treated with MXC compared to those
treated with vehicle. Interestingly, ESR1-OE treated with MXC had a significantly higher
percentage of atretic follicles than controls treated with MXC (controls treated with vehicle =
9.18%, MXC 32 mg/kg/day = 19.53%, MXC 64 mg/kg/day = 21.58%; ESR1-OE treated with
vehicle = 9.56%, MXC 32 mg/kg/day = 19.5%; MXC 64 mg/kg/day = 37.23%; n = 3; p ≤ 0.05).
Thecal cell thickness was found to be significantly less in ESR1-OE mice treated with MXC than
controls (controls treated with vehicle = 6.13 μm, MXC 64 mg/kg/day = 5.97 μm; ESR-OE
treated with vehicle = 5.95 μm MXC 64 mg/kg/day = 5.1 μm; n = 3; p ≤ 0.05). Collectively,
these results indicate that we have developed a new transgenic animal model in which ESR1
overexpression in the ovaries can be regulated by doxycycline treatment. Further, these results
indicate that ESR1 overexpression increases the sensitivity of the ovaries to MXC.

NIH R21 ES13061 and RO1ES012893




                                                 19
                                                                                        Abstract #9

The CCAAT Enhancer Binding Protein Beta (C/EBP-β) is a Critical Mediator of Estrogen-
induced Cell Proliferation in the Uterine Epithelium of Mice, Ramathal, C. R.1, Bagchi I. C.2,
& Bagchi M. K.3 1 , 1Department of Cell and Developmental Biology, 2 Department of
Veterinary Biosciences, 3 Department of Molecular and Integrative Physiology, University of
Illinois, Urbana-Champaign, IL.

The preparation of the uterus for embryo implantation requires estrogen (E)-induced
proliferation of the uterine epithelial lining during the reproductive cycle. However, the
molecular pathways via which E regulates this cell proliferation event remain unclear. Our recent
studies revealed that CCAAT-enhancer binding protein beta (C/EBPβ) is an E-regulated
transcription factor in the uterine epithelium. Most importantly, ablation of C/EBPβ gene in the
mouse resulted in a complete loss in female fertility and significantly impaired proliferation in
response to E in the uterine epithelium. To determine whether C/EBPβ mediates the proliferative
effects of E at a specific stage of the cell cycle, we analyzed several markers of cell cycle
progression in wild type and C/EBPβ-null uterine epithelial cells. By the criteria of BrdU
incorporation and immunostaining for the PCNA marker, our studies strongly indicated that in
the epithelia of mutant mice the cell cycle progression is primarily arrested at the G1-S phase
transition. Furthermore, the mutant epithelium also displayed a complete blockade in the G2-M
phase transition in response to E as indicated by immunostaining for the mitosis marker
phospho-histone H3. To further analyze the molecular basis of the cell cycle arrest, we
investigated the expression levels of several known regulators of the G1-S transition in wild type
versus C/EBPβ-null uterine epithelium. The levels of mRNAs corresponding to cyclin D1, cdk2,
retinoblastoma and several other G1-S phase regulators remained unaltered in the C/EBPβ-null
epithelium, whereas that of cyclin E mRNA was significantly reduced in the mutant tissue.
Additionally, the level of the mRNA encoding p27, a negative regulator of G1-S progression,
was enhanced and this factor displayed an increased nuclear retention in the C/EBPβ-null uterine
epithelium. Finally, we observed a marked reduction in the expression of the mRNA encoding
transcription factor E2F3 in the C/EBPβ–deficient epithelium while the mRNA levels of two
other major E2F family members, E2F1 and E2F2, remained unchanged. Collectively, our
studies indicated that C/EBPβ critically controls the G1-S transition during uterine epithelial
proliferation by regulating the expression of at least three key G1-S regulatory molecules: cyclin
E, p27, and E2F3. These findings provide novel insights into the molecular pathways via which
E influences uterine epithelial proliferation during the reproductive cycle

Supported by NIH grants to ICB and MKB




                                               20
                                                                                     Abstract #10

Involvement of p21 Activated Kinase in the Estrogen (E2) Regulation of GnRH/LH
Secretion. Z. Zhao, and J. E. Levine, Department of Neurobiology and Physiology,
Northwestern University, Evanston, IL 60208

   The PAKs are serine/threonine kinases that appear to play essential roles in dendrite
morphogenesis, synaptogenesis, and neuroplasticity.          These proteins are activated by
RhoGTPases, and have been shown to mediate rearrangements of the actin cytoskeleton that are
essential for directed dendritic spine outgrowth and synapse formation. In a recent DNA
microarray experiment, we identified PAK3 as a putative E2 responsive gene in the preoptic area
of the rat. Follow-up real-time PCR experiments confirmed that mRNAs encoding PAK1 and
PAK3, the two PAK isoforms expressed predominantly in the brain, are significantly elevated in
the POA of ovariectomized (OVX) rats at 12h after estradiol benzoate (EB) treatment. To assess
the ability of E2 to activate PAK1 and PAK3, we first determined by PCR and western blotting
that their mRNAs and corresponding proteins are expressed in the immortalized GnRH-
producing cell line, the GT1-7 cells. Incubation of cells with a low physiological concentration
of E2 (10 nM) stimulated phosphorylation of PAK within 5 minutes. This effect was maximal at
20 min and maintained for at least 1 hour. Furthermore, neurite outgrowth from GT1-7 cells was
significantly enhanced by 38% ( 81±35 μm of E2 treated, and 59 ± 24 μm of vehicle), and the
proportion of neurite bearing GT1-7 cells was significantly higher (66% ± 17%) at 24 hours
following addition of E2 to cultures compared to 44% ± 22% of vehicle. PCR revealed
expression of both estrogen receptor α (ERα) and ERβ transcripts in GT1-7 cells, and both ERα
and ERβ agonists mimicked the effects of E2 on PAK activation. Vehicle treatment was without
effect on PAK phosphorylation or neurite outgrowth. In in vivo tests, we determined that that
treatment of OVX rats with EB caused a rapid induction of phosphorylated PAK1 (pPAK1)-
Immunoreactive (Ir) neurons in the AVPV within as early as 30 min and 1 h; the acute activation
returned to the baseline level before a second activation at 12 h. In addition, pharmacological
PAK inhibition in female rats was sufficient to cause a delay in the initiation and peak of E-
induced surges of LH. Our findings demonstrate that E2 1) stimulates the expression and
phosphorylation of PAKs in preoptic tissues, 2) rapidly activates PAKs in immortalized GnRH
producing GT1-7 cells, and 3) induces neurite outgrowth in GT1-7 cells. While it remains to be
determined if E2 -induced neurite outgrowth is mediated in part via PAK activation, our
observations nevertheless demonstrate the existence of a potentially important non-genomic
signaling pathway through which E2 may regulate GnRH neurons.


Supported by NIH HD20677, NIH U54HD 041859 and NIH P50HD 44405




                                              21
                                                                                       Abstract #11



ERE-independent ERα Signaling does not Rescue Sexual Behavior but Restores Normal
Testosterone Secretion in Male ERαKO Mice, Melissa Chamberlin McDevitt1, Christine
Glidewell-Kenney2, Jeffrey Weiss2, Pierre Chambon3, J. Larry Jameson2, Jon E. Levine1,
1
  Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois
60208, USA, 2Department of Endocrinology, Metabolism and Molecular Medicine,
Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611 USA, 3Institut de
Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France,
Illkirch Cedex, France.


Estrogen receptor alpha (ERα) mediates estradiol (E2) actions in the male gonads and brain and
is critical for normal male reproductive function. In the classical pathway, ERα binds to estrogen
response elements (EREs) to regulate gene transcription. ERα can also regulate gene
transcription independently of EREs via protein-protein interactions with transcription factors,
and additionally signal via rapid, nongenomic pathways originating at the cell membrane. This
study assessed the degree to which ERE-independent ERα signaling can rescue the disrupted
masculine sexual behaviors and elevated serum testosterone (T) levels that have been shown to
result from ERα gene deletion. We utilized male ERα null mice that possess a ER knock-in
mutation (E207A/G208A; “AA”), in which the mutant ERα is incapable of binding to DNA and
can only signal through ERE-independent pathways (ERα-/AA mice). We found that sexual
behavior, including mounting, is virtually absent in ERα-/- and ERα-/AA males, suggesting that
ERE-independent signaling is insufficient to maintain any degree of normal sexual behavior in
the absence of ERE binding. By contrast, ERE-independent signaling in the ERα-/AA mouse is
sufficient to restore serum T levels to values observed in wild-type males. These data indicate
that binding of ERs to EREs mediates most if not all of E2’s effects on male sexual behavior,
while ERE-independent ERα signaling may mediate E2’s inhibitory effects on T production.

Supported by P01 HD21921, R01 HD20677, and T32 HD07068.




                                                22
                                                                                      Abstract #12



Nerve Growth Factor Inducible B (NGFI-B) Represses Inhibin Alpha Expression
Independently of DNA-binding in Mouse Granulosa Cells, Kristen M. Meldi, Anna D.
Burkart, PhD., and Kelly E. Mayo, PhD., Department of Biochemistry, Molecular Biology and
Cell Biology, Center for Reproductive Science, Northwestern University, Evanston, IL

The preovulatory luteinizing hormone (LH) surge regulates gene expression in the ovary to
permit ovulation. While many genes essential to ovulation are activated by the LH surge, the LH
surge also signals the down-regulation of other genes that must be repressed. The inhibin alpha
subunit gene is a gene that, while induced by follicle stimulating hormone (FSH) during
follicular maturation, is dramatically down-regulated in response to the LH surge. This down-
regulation of inhibin is necessary for the secondary FSH surge to occur on the morning of estrus
and the subsequent recruitment of a new cohort of follicles for the next estrus cycle. Therefore,
investigating the mechanism by which the LH surge down-regulates inhibin alpha expression in
preovulatory granulosa cells is important in understanding ovulation and can be applied to other
genes that undergo similar regulation during the periovulatory period.
We have identified nerve growth factor inducible B (NGFI-B) as a repressor of inhibin alpha
expression in granulosa cells. NGFI-B is a NR4A family orphan nuclear receptor that has been
shown by others to be rapidly induced by the LH surge in granulosa cells of the preovulatory
follicle. Through cell-based transfection assays, we have shown the NGFI-B can repress inhibin
alpha expression in a mouse granulosa (GRMO2) cell line. While this repression is exerted
through a 20-base pair region of the inhibin alpha promoter, gel mobility shift assays could not
detect binding of NGFI-B to this promoter region. Furthermore, a mutant form of NGFI-B
containing a glycine to glutamic acid change that abolishes DNA-binding activity was still
capable of repressing inhibin alpha expression. These results indicate that repression may be
independent of DNA-binding by NGFI-B. Further mutational analysis of the 20-base pair region
of the inhibin alpha promoter identified a GATA factor binding site as essential for NGFI-B’s
repression. We demonstrated that GATA-4 and GATA-6 are expressed in GRMO2 cells and can
activate inhibin alpha expression in these cells. GATA-4 can bind to both of the two GATA
binding sites in the proximal promoter and our experiments suggest that binding to these two
sites is independent. We are now examining the mechanism by which NGFI-B utilizes the
GATA factors to repress inhibin alpha expression in granulosa cells. Coexpression of NGFI-B
and GATA-4 in HeLa cells had no effect on GATA-4 binding to the inhibin alpha promoter in
vitro, as demonstrated in gel mobility shift assays. Using GST-pulldown assays and
coimmunoprecipitations, we are currently determining whether NGFI-B directly interacts with
GATA-4 and GATA-6 as a means to repress inhibin alpha expression through protein-protein
interactions. This will represent a novel mechanism by which a nuclear receptor represses gene
expression in the ovary independently of DNA-binding.

This work is funded by the NIH Specialized Cooperative Centers Program in Reproductive
Research (U54 HD41857) to KEM.




                                               23
                                                                                  Abstract #13

Expression of Hypoxia-Inducible Factors in the Ovary: A Novel Role during Ovulation in
Mice, Jaeyeon Kim1, Indrani C. Bagchi2, Milan K. Bagchi1,1Department of Molecular &
Integrative Physiology, 2Department of Veterinary Biosciences, University of Illinois at Urbana-
Champaign, Urbana, IL

During ovulation in mice, mature ovarian follicles rupture to release fertilizable oocytes. The
steroid hormone progesterone (P), acting via its nuclear receptor, is a major regulator of the
process of follicular rupture. Consequently, the female progesterone receptor (PR)-null mouse
exhibits an anovulatory phenotype in which the follicles fail to rupture and retain “trapped”
oocytes. To identify the P-regulated pathways that control the process of follicular rupture, we
analyzed global changes in gene expression profiles in the ovaries of wild type (WT) and PR-
null mice treated sequentially with pregnant mare serum gonadotropin (PMSG) and human
chorionic gonadotropin (hCG) in a superovulation protocol. This analysis uncovered 216 genes
whose expression was reduced at least 2-fold in the PR-null ovaries compared to the WT ovaries
at a time immediately preceding ovulation. Interestingly, we identified three hypoxia-inducible
factors (HIFs), the α isoforms HIF-1α and HIF-2α, and the β isoform HIF-1β as PR-regulated
genes in the ovary. These transcription factors form αβ heterodimers, which regulate the
transcription of specific cellular genes, thereby mediating adaptive response of the tissue to a
drop in oxygen level. We observed that shortly before follicular rupture, the expression of
mRNAs encoding HIF-1α, HIF-2α and HIF-1β was induced in a PR-dependent manner,
specifically in the granulosa cells of the preovulatory follicles. Western blotting experiments
confirmed an elevation in the level of the HIF-1α protein at the time of ovulation. Most
importantly, an inhibition of the HIF transcriptional activity by echinomycin, a small-molecule
inhibitor that suppresses the binding of a HIF αβ heterodimer to target genes, blocked the
rupture of the preovulatory follicles, thereby drastically reducing the number of ovulated oocytes
in mice. Consistent with this observation, echinomycin also inhibited the expression of several
genes that are known to be critical for ovulation, such as endothelin-2, peroxisome proliferator-
activated receptor γ, a disintegrin and metalloproteinase with thrombospondin-like motifs-1
(ADAMTS-1). Furthermore, echinomycin reduced the expression of vascular endothelial growth
factor A, a factor critical for vascular network formation during ovulation, reinforcing the
concept that HIF signaling is important for follicular rupture. Collectively, our study unveiled a
novel ovarian role for HIFs during the ovulatory period in mice.


Supported by NIH grants




                                               24
                                                                                        Abstract #14

Manipulation of Actin Dynamics Impacts Human Uterine Fibroblast Decidualization,
I. Ihnatovych, M. Livak, Z. Strakova, Department of Obstetrics and Gynecology, University of
Illinois at Chicago, Chicago, IL, 60612

The cytoskeleton organization and function mostly depend on actin-myosin interactions, which
are regulated by phosphorylation of myosin light chain (MLC) and actin dynamics. We have
recently demonstrated that changes in MLC phosphorylation precede differentiation of human
uterine fibroblasts (HuF) into decidual cells (decidualization) and that upregulation of MLC
phosphorylation prevents decidualization. Actin dynamics are governed by actin-binding
proteins, one of which is cofilin. The activity of cofilin is regulated through its phosphorylation
by kinases LIMK1 and LIMK2. Here we show that decidualization induced by a stimulus of
embryonic origin, interleukin-1β (IL-1β) in presence of steroid hormones (H), is accompanied by
an upregulation of LIMK1, a downregulation of LIMK2 and translocation of inactivated
(phosphorylated) cofilin from the nucleus into the cytosol. On the other hand, cAMP (with H)-
induced decidualization is characterized by a significant decrease in phosphorylated cofilin, a
notable translocation of active cofilin into the nucleus and unchanged amounts of LIMK1 and
LIMK2. As cofilin binds to both monomeric (G-) and filamentous (F-) actin, its association with
G- and F-actin in HuF during decidualization was investigated. Immunofluorescence
demonstrated that cofilin mainly co-localized with G-actin in the nucleus as well as in the
cytoplasm, although some spots of co-localization between cofilin and F-actin were detected in
the cytoplasm. A strong association of cofilin with monomeric actin was further confirmed by
Western blot analysis of the G-actin and F-actin fractions. Actin dynamics were manipulated
with jasplakinolide, an actin stabilizing drug causing de novo polymerization of F-actin, and
latrunculin B, an actin depolymerizing drug. HuF cells were pretreated for 1 h with
jasplakinolide (100 nM) or latrunculin B (1 µM) and exposed to decidualization stimuli for 6 d.
Both drugs significantly delayed decidualization induced by IL-1β (and H) as estimated by the
measurement of protein markers of decidualization: insulin-like growth factor binding protein-1
and prolactin. On the other hand, only jasplakinolide inhibited cAMP-induced decidualization,
whereas latrunculin B did not affect it. Analysis of G-/F-actin ratio revealed no change during
decidualization in HuF cells. However, the pretreatment with jasplakinolide caused a significant
decrease in the G-/F-actin ratio, further confirming the importance of actin dynamics for
decidualization. Thus, we conclude that changes in actin dynamics (particularly the stabilization
of F-actin) have a significant negative impact on decidualization.



Supported by NIH Grant HD-44713 to ZS




                                                25
                                                                                            Abstract #15



The Role of Toll-like Receptor 2 (TLR-2) Activation in Bacterially Induced Preterm Labor in Mice

Vladimir Ilievski, MD1 and Emmet Hirsch, MD1, 2. 1Obstetrics and Gynecology, Evanston Northwestern
Healthcare, Evanston, IL, United States, 60201 and 2Obstetrics and Gynecology, Feinberg School of
Medicine, Northwestern University, Chicago, IL, United States, 60611.

OBJECTIVE: Toll-like receptors (TLRs) are membrane-bound proteins that recognize structural
components of bacterial and viral pathogens and initiate host defense responses. TLR-2 is implicated in
the recognition of Gram positive bacteria such as group B -hemolytic streptococcus (GBBS) via their
cellular constituents, including peptidoglycan (PGN). The objective of this project was to investigate in
vivo     the    role    of   TLR-2     in    bacterially-induced  and    inflammation-induced      labor.

METHODS: Inbred TLR2-deficient mice (Tlr2tm1Kir) and control wild type mice (C57BL/6J) on day 14.5
of pregnancy were given intrauterine injections of PGN, killed GBBS or killed E. coli (a Gram negative
bacterium). Preterm delivery (the finding of at least one fetus in the cage or lower vagina within 48 hours
of                           surgery)                               was                           recorded.

     RESULTS:

Number (%) of pregnant dams delivering <48 h after treatment
Treatment (per mouse) E. coli (108 organisms)         GBBS (108 organisms)            PGN (0.25 mg)
Wild-type              4/4 (100%)                     6/10 (60%)                      10/15 (67%)
TLR-2 knockout         7/8 (88%)                      1/13 (8%)                       3/13 (23%)
P value                1.00                           0.019                           0.03



CONCLUSION: E. coli, GBBS and PGN can all induce preterm birth in wild-type mice. Preterm delivery
induced by GBBS and PGN, but not E. coli, is dependent on TLR-2.


Supported by RO1HD41689




                                                     26
                                                                                    Abstract #16

Downregulation of Dickkopf-1 by Three-Dimensional Culture Leads to Membrane Type-1
Matrix Metalloproteinase Upregulation. Maria V. Barbolina 1, Lonnie D. Shea 1, M. Sharon
Stack 2, 1 Department of Chemical and Biological Engineering, Northwestern University,
Chicago, IL, 2 Department of Pathology and Anatomical Sciences, University of Missouri,
Columbia, MO.

Epithelial ovarian carcinoma (EOC) is the leading cause of death from gynecologic malignancy.
Due to difficulties in diagnosing ovarian cancer at early stages, most patients present with
disseminated intraperitoneal metastases. Thus, it is important to understand mechanisms of
invasion and metastasis and develop strategies to prevent tumor cells from spreading. EOC cells
shed from the primary ovarian tumor invade the submesothelial matrix of peritoneal organs to
anchor secondary lesions. As the submesothelial matrix is comprised primarily of interstitial
collagens, the goal of this study was to determine whether contact between metastasizing EOC
cells and three-dimensional collagen I (3DCI) induces changes in gene expression that promote
metastasis. To mimic early events in metastasis, when cells invade into the collagen-rich
submesothelial matrix prior to establishing secondary lesions, cells were cultured atop 3DCI
gels. Our results identified an inhibitor of Wnt signaling, dickkopf-1 (DKK1), as a unique
product downregulated by the 3DCI culture conditions both on RNA and protein levels, as seen
by cDNA microarray, real time RT-PCR, and Western blot analysis. Analysis of human normal
and malignant ovarian epithelial tissues with real time RT-PCR revealed that most of ovarian
cancer tissues in late stages of malignant transformation (III-IV) did not express DKK1 in
contrast to those on early stages of the disease (I-II), suggesting that downregulation of DKK1
may facilitate tumor progression. Consistent with these data, downregulation of DKK1
expression using siRNA led to enhanced collagen invasion, while addition of exogenous DKK1
protein inhibited invasiveness. Loss of DKK1 expression also resulted in enhanced expression of
membrane type-1 matrix metalloproteinase (MT1-MMP), a potent ovarian tumor-associated
collagenase. These data support DKK1 as an invasion suppressor in ovarian cancer metastasis,
functioning via negative regulation of MT1-MMP expression.

Supported by grants from Ovarian Cancer Research Foundation and Illinois Department
of Public Health.




                                              27
                                                                                       Abstract # P1




Regulation of Lactate Dehydrogenase C (Ldhc) Gene Expression, Huanghui Tang, Aisha
Kung, John McCarrey*, Erwin Goldberg, Department of Biochemistry, Molecular Biology and
Cell Biology, Northwestern University, Evanston, IL 60201. *Health Science Center at San
Antonio, University of Texas, TX 78229.

Spermatogenesis is characterized by genes that express exclusively in testis. But the mechanism
governing tissue specificity is largely unknown. To address this question we study the ldhc gene
which is expressed in germ cells from pachytene spermatocytes to spermatozoa and encodes for
a specific lactate dehydrogenase (LDH-C4). Previous work indicated that the 100-bp core
promoter of ldhc is sufficient to drive tissue-specific expression in transgenic mice. Based on
our in vitro experimental data we designed 2 transgenes to confirm whether the palindrome
(PAL) sequence and a GC box in the core promoter region are regulate ldhc expression in vivo.
Our results indicated that both mutated constructs were able to drive efficient testis-specific
expression, suggesting that these two regulatory elements are not essential for tissue-specific
expression. To discover additional cis elements we prepared mutated ldhc promoter-luciferase
reporter constructs for injection into semniferous tubules and in vivo electroporation. Our results
indicated that the GC box mutation and CRE sites caused a 75% and 72% decrease in promoter
activity respectively, while a double mutation of GC box and CRE sites completely abolished
promoter activity. The PAL mutation had no effect on promoter activity. We conclude that there
is functional redundancy in Sp1 and CREB/CREM in regulating ldhc expression. We propose
that a repressor binding to these sites may silence the gene in somatic cells.




Supported by NIH grant HD05863-35




                                                28
                                                                                       Abstract # P2


Disruption of the Sperm-Specific Ldhc Gene is Detrimental to Sperm Function and Male
Fertility, Chongwen Duan and Erwin Goldberg, Northwestern University, Evanston, IL

        The lactate dehydrogenase isozymes are key catalysts in the glycolytic pathway of energy
metabolism and it is well known that the distribution of the LDH isozymes vary in accordance
with the metabolic requirements of different tissues. There are three subunit types (A, B and C)
and exquisite tissue specificity is exemplified by the Ldhc gene. It is abundantly expressed in
male germ cells during spermatogenesis and encodes the only LDH isozyme for which activity
can be detected in spermatozoa. Spermatozoa rely almost exclusively on aerobic glycolysis to
produce the ATP necessary for capacitation, motility and fertilization.
        Why Ldhc gene expression has been conserved in mammalian testes where Ldha and
Ldhb genes are also transcribed remains an enigma. We have chosen targeted disruption of the
Ldhc gene as the method to address the question of why testes and sperm need this unique form
of LDH. Preparation of a targeting construct proved challenging since the intronic sequences
are composed of 43% repetitive elements. The final construct contained Neo sequences bounded
by loxP elements for conditional expression with TK for selection.             Redundancy in this
metabolic pathway would predict a wild-type phenotype in the mutant animal. Reliance on
LDHC for glycolysis in germinal epithelial cells would predict impaired spermatogenesis most
likely during pachytene of the first meiotic division or in early spermatids which prefer lactate
over glucose as energy substrate. We hypothesized that disruption of ATP production via this
metabolic pathway would impair motility and fertilizing capacity of spermatozoa similar to that
observed with GAPDHS null mice. However, here we demonstrate that targeted disruption of
Ldhc affects only male fertility. LDHC has been studied as an immunocontraceptive in females.
The present results not only satisfy proof of principle but also suggest that this protein may be a
useful target in developing a male contraceptive.




This research was supported by NIH HD05863-35




                                                29
                                                                                      Abstract # P3


Notch1 Promotes type A Spermatogonia Proliferation and Differentiation by Down-
regulating p53-mediated Cell Cycle Arrest. 1Natalia Kostereva, 1Kay Carnes and 1, 2Marie-
Claude Hofmann. 1Department of Veterinary Biosciences and 2Institute for Genomic Biology,
University of Illinois at Urbana-Champaign, Urbana, IL 61802

The Notch signaling pathway is involved in a variety of cell fate decisions during development
and after birth. Notch receptors are activated upon ligand binding from neighboring cells through
juxtacrine interactions, and regulate stem cell fate by adjusting the cellular response to other
stimuli instead of direct specification. Therefore, depending on the tissue and environmental
cues, Notch signaling can promote either stem cell self-renewal or differentiation. Upon ligand
binding, the intracellular domain of Notch (NICD) is cleaved and migrates into the nucleus
where it functions as a transcription factor. In the mouse testis, Jagged1/2 is a Sertoli cell
membrane protein that binds to Notch receptors at the surface of germ cells. Different Notch
family receptors are sequentially activated during spermatogenesis, but their exact role is not
known (1). Therefore, the current study was performed to determine the role of Notch1 in this
process. Immunohistochemistry revealed that the cleaved form of Notch1 (NICD) is present in
the cytoplasm of gonocytes at birth, migrates around day 3 into the nuclei, and is retained in the
nuclei of mitotic germ cells as spermatogenesis progresses. In vitro cultures of spermatogonial
stem cells (SSCs) in presence of Jagged1 indicated that Notch1 activation promoted stem cell
proliferation and differentiation into Aaligned spermatogonia, as observed by an increase in the
formation of chains of cells expressing Tex14 and c-Kit, and evaluated by RT-PCR. Because the
presence of NICD seemed to exclude the simultaneous expression of the cell cycle inhibitor p53,
we asked whether Notch1 might allow spermatogonia to proliferate/differentiate by down-
regulating or inactivating p53. Using multiple cellular and molecular approaches, we found that
down-regulation of Notch1 inhibited proliferation/differentiation and induced apoptosis in
spermatogonial stem cells (SSCs) and a spermatogonial stem cell line, C18-4. Conversely, the
activation of Notch1 in SSCs with Jagged1 or valproic acid induced differentiation and down-
regulation of p53 phosphorylation at Ser6 and Ser15, as well as up-regulation of Bcl-xL through
Akt phosphorylation at Ser473. Based on these findings, we conclude that activated Notch1
allows spermatogonial proliferation and differentiation by up-regulating Bcl-xL and preventing
p53-mediated cell cycle arrest.

Supported by NIH HD044543




                                               30
                                                                                       Abstract # P4

Transcriptional Regulation of Cyp26b1 in a Mouse Sertoli Cell Line, Miranda L. Bernhardt,
J. Larry Jameson. Division of Endocrinology, Department of Medicine, Feinberg School of
Medicine, Northwestern University, Chicago, IL.


Retinoic acid (RA) is a key regulator of germ cell fate in the developing gonad. Previous work
has identified the RA-metabolizing cytochrome P450 enzyme Cyp26b1 as a sexually dimorphic
gene required for testis development in the mouse. Cyp26b1 is expressed specifically in the
developing male gonad beginning around embryonic day 12.5 (E12.5), a time when the
bipotential gonad begins to undergo testis-specific differentiation. Degradation of RA by
Cyp26b1 in the developing testis prevents germ cells from entering meiosis and committing to
oogenesis. Because expression of Sox9 (Sry-box containing gene 9), which increases in pre-
Sertoli cells at E11.5, has been shown to regulate several genes involved in differentiation of the
male gonad, we examined whether Sox9 could induce Cyp26b1 expression in both the MSC1
and TM4 mouse Sertoli cell lines. A luciferase reporter construct containing approximately 1.2kb
of the region upstream of mouse Cyp26b1 was stimulated by increasing amounts of co-
transfected Sox9. Using 5’ deletion analysis, we found that a construct containing a 325bp
region of the promoter retained this response to Sox9. Further analysis will be necessary to
identify the Sox9 responsive element(s) within this region. In addition to Sox9, Sf1
(Steroidogenic factor 1) and Dax1 (Dosage-sensitive sex reversal-adrenal hypoplasia congenita
critical region on the X chromosome, gene 1) modulate Cyp26b1 expression. Sf1 induces
reporter activity, while a dose-dependent repression is observed with increasing Dax1
expression. In addition to its well characterized interaction with Dax1, Sf1 has also previously
been shown to interact with Sox9. These data support a role for the sex-determining genes Sox9,
Sf1, and Dax1 in the male-specific transcriptional regulation of Cyp26b1.




                                                31
                                                                                       Abstract #P5

Silver Nanoparticles Disrupt GDNF Signaling in Male Germ-line Stem Cells.
1,2
    Benjamin Lucas, 3Laura Braydich-Stolle, 1,2Timothy Lee, 3John Schlager, 3Saber Hussain and
1,2
    Marie-Claude Hofmann. 1Department of Veterinary Biosciences and 2Institute for Genomic
Biology, University of Illinois at Urbana Champaign, 2001 South Lincoln Avenue, Urbana, IL,
3
  Applied Biotechnology Branch, Human Effectiveness Directorate, Air Force Research
Laboratory/HEPB, Wright-Patterson Air Force Base, OH 45433

Nanosized silver has found anti-microbial uses in paint, bandages, and coatings on surfaces, yet
no one has addressed the potential health risk of these materials, which must be considered since
they have the ability to bind proteins with high affinity. Due to their nanoscale size, inhaled or
ingested nanoparticles readily pass through cellular membranes and accumulate in many organs
such as the brain, liver and testis (1). We have previously shown that the spermatogonial stem
cell line C18-4 is a useful model for studying reproductive nanotoxicity (2). Proliferation of
these cells is mediated by the growth factor glial cell line derived neurotrophic factor (GDNF),
which triggers a Src/PI3 kinase-mediated signaling pathway through the Src family kinase Fyn
(3). Based on these studies, we wanted to evaluate the effects of silver (Ag) nanoparticle size and
surface chemistry on GDNF signaling in C18-4 cells. We used hydrocarbon (HC) and
polysaccharide coated (PS) Ag nanoparticles with 15, 25, and 80 nm diameter. Initial MTS
assays demonstrated that both type of nanoparticles are toxic and disrupt GDNF-mediated stem
cell proliferation in a size-dependent manner. The decrease in cell numbers was not due to
production of reactive oxygen species at any concentration used. Our data indicated that HC-Ag
nanoparticles induced apoptosis, while PS-Ag nanoparticles impaired cell proliferation through a
different process. Using western blotting and a kinase activity assay, we established that PS-Ag
nanoparticles significantly decreased intracellular Fyn kinase phosphorylation and activity. In
conclusion, Ag-nanoparticles impair the proliferation of germ-line stem cells in a dose-dependent
manner, and different surface coatings induce the decrease of cell numbers through different
mechanisms.


Supported by NIH HD044543




                                                32
                                                                                     Abstract # P6

An Alginate Based Culture System Provides An Appropriate Environment For In Vitro
Ovarian Follicle Development, Elizabeth Parrish1, Teresa Woodruff2, Lonnie D. Shea1,
1
  Department of Chemical and Biological Engineering, 2Department of Obstetrics and
Gynecology, Northwestern University, Evanston, IL.

Ovarian follicle development is a complex process that requires bidirectional communication
between a centrally located oocyte and its surrounding somatic cells. Paracrine, autocrine, and
endocrine signaling play vital roles in this developmental process, and numerous signaling
pathways have been identified as key regulators of follicle maturation. We investigate the
influence of an alginate based in vitro culture system on signaling pathway activation during
ovarian follicle development. Primary, two-layered secondary, and multi-layered secondary
follicles were isolated from mouse ovaries for gene expression analysis. Through the use of
Illumina Beadarray technology, gene expression prolifes of in vivo growing follicles at these
three different stages of development were examined and a subset of differentially expressed
genes were identified. Of most interest, insulin-like growth factor 1 and transforming growth
factor beta receptor II were shown to be up-regulated and the glycoprotein and sperm receptor
zona pellucida 3 was shown to be down regulated during folliculogenesis. Biological
interpretation of the microarray data, facilitated by Ingenuity Pathway Analysis, showed that the
beta-estradiol, Akt, and tumor necrosis factor pathways might also be tied to proper
development. Transcript abundance of genes well known to be involved in the developmental
process as well as novel genes uncovered from the microarray analysis was confirmed by real
time PCR. Comparative gene and protein expression analysis between in vivo growing follicles
and in vitro cultured follicles of the newly identified and well-known developmental pathways
allows for determining whether or not the alginate based culture system induces inappropriate
changes in signaling pathway activation. Understanding the regulation of various signaling
pathways on the genetic and protein scales during different stages of follicle maturation and
under varying culture conditions can direct the manipulation of the culture environment towards
a system that more closely mimics the in vivo ovarian environment and allows for enhanced
follicle growth, viability, and maturation.

 Supported by the NSF-GRF and a NIH U54 grant.




                                               33
                                                                                     Abstract # P7


Long-term Culture of Monkey and Human Preantral Follicles in Three-dimensional
Alginate Scaffold, Min Xu1,2, Mary B. Zelinski5, Erin West3,4, Laxmi A. Kondapalli 1,2, Richard
L. Stouffer5, Lonnie D. Shea3,4, Teresa K. Woodruff1,2,4. 1Department of Obstetrics and
Gynecology, Feinberg School of Medicine, Northwestern University, Chicago IL 60611; 2Center
for Reproductive Research, Northwestern University, Evanston, IL 60208; 3Department of
Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208; 4The
Robert H. Lurie Comprehensive Cancer Center of Northwestern University Chicago, IL 60611;
5
  Division of Reproductive Biology, Oregon National Primate Research Center, Oregon Health &
Science University, Beaverton, Oregon

   The human ovarian cortex contains hundreds of thousands of immature follicles. The ability to
mature these follicles in vitro could be of great important for both infertility treatment and
fertility preservation. In this study, we investigate primate (rhesus monkey and human) follicles
growth in a novel three-dimensional alginate scaffold.
   Monkey preantral follicles (0.2mm-0.5mm) were mechanically isolated from ovaries in either
early follicle phase (EFP, days1-4) or late luteal phase (LLP) of the menstrual cycle. Follicles
were encapsulated in 0.5% alginate, and then were randomly divided into two culture conditions.
The basal culture media contains αMEM with 100 mIU/ml rhFSH, 3 mg/ml BSA, 1 mg/ml
bovine fetuin, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium. rhLH (10mIU/ml)
were supplied (Group A) or not (Group B). Alginate beads containing a single follicle were
plated one follicle per well in 48-well plates in 250 μl of culture media. Human preantral
follicles (0.1mm-0.3mm) were isolated from ovarian cortex strips (OCS) after digested with a
mixture of collagenase I and deoxyribonuclease (DNase I) for 1.5-2 hours at 37ºC. All of
follicles were encapsulated in 0.5% alginate, and cultured within the basal culture media same as
above without rhLH supplement. Individual alginate bead containing a single follicle was plated
in 96-well plates in 100 μl of culture media. Half of media were changed every other day, and
follicles were in vitro grown up to 4 weeks.
   Fifty-eight monkey preantral follicles were obtained from EFP monkeys, and 38 follicles were
obtained from LLP monkeys. Twelve human preantral follicles were obtained from two 0.5cm x
0.5cm size human ovarian cortex strips. All of follicles can be grown in vitro in presence of FSH
up to four weeks. Average follicle size increased 142% in monkey, and 264% in human.
Follicles were steroidogenically active in vitro, with androgen, estrogen and progestrone levels
rising through the culture. Follicles collected from the EFP (55.5%) resulted in better survival
rate than those collected from LLP (21.5%) when they were cultured absence of rhLH.


Supported by grant NIH U54-HD41857




                                               34
                                                                                         Abstract # P8

Aquaporin Expression and Antral Cavity Development are Regulated by Matrix Stiffness
in an In Vitro Ovarian Follicle Culture System, Erin R. West1, Monica A. Gomberg2, Teresa
K. Woodruff2,3,4, Lonnie D. Shea1,3,4, 1Department of Chemical and Biological Engineering,
2
  Department of Obstetrics and Gynecology, The Feinberg School of Medicine, 3Center for
Reproductive Research, Northwestern University, Evanston, IL, 4The Robert H. Lurie
Comprehensive Cancer Center of Northwestern University

In vitro follicle culture systems allow for the development of cryopreserved ovarian tissue, and
therefore may provide reproductive options to women facing premature infertility due to cancer
therapies. Three-dimensional culture systems maintain the morphology of the follicle while
allowing for follicle expansion and the exchange of soluble factors with the surrounding culture
medium. Previous studies have demonstrated that follicle growth in a three-dimensional culture
system is regulated by the matrix stiffness. Follicle growth, oocyte quality, embryonic
development, steroid biosynthesis, and development of the fluid-filled antral cavity were all
regulated by stiffness. The antral cavity is a defining characteristic of mature follicles, yet the
mechanisms regulating its formation are not understood. We hypothesized that aquaporins,
transmembrane proteins that facilitate water transport across cell membranes, play a role in
development of the antral cavity, and may be regulated by matrix stiffness. Multilayered
secondary follicles (150-180 μm diameter) were isolated from mice at 16 days of age and
encapsulated in 0.5% (permissive) and 1.5% (non-permissive) alginate matrices. Follicles were
cultured for 2-8 days, at which time surviving follicles were removed from the alginate for RNA
isolation. In addition, RNA was isolated from freshly dissected follicles of sizes 150-180 μm
(preantral) as well as 300-400 μm (antral) from both untreated and pregnant mare serum
gonadotropin- (PMSG-) stimulated mice.

Growth and antrum formation were regulated by the matrix stiffness in these culture conditions,
and aquaporin 7 and 8 expression varied with follicle stage and matrix stiffness. Follicles
cultured in 0.5% alginate increased in diameter 83.0±5.5% by day 8, while those cultured in
1.5% alginate increased in diameter 39.4±2.6%. By day 8 of culture, 46.4% of follicles cultured
in 0.5% alginate and only 4.2% of follicles cultured in 1.5% alginate had formed antral cavities.
Aquaporins 7, 8, and 9 were examined by RT-PCR, and aquaporins 7 and 8 were found to be
present at detectable levels in both freshly isolated and cultured follicles, indicating that
aquaporin 9 is not involved in follicle development. In cultured follicles, the highest level of
aquaporin 7 and 8 expression was observed in follicles collected at day 6 of culture from 1.5%
alginate. These results may indicate that aquaporins are upregulated in non-permissive matrices
to compensate for the increased resistance the follicle encounters in trying to grow in the
relatively stiff matrix. For freshly isolated follicles, lowest expression of both aquaporins 7 and 8
was observed in the PMSG-stimulated antral follicles. This result demonstrates that these
aquaporins are not induced by gonadotropins, and indicates that other gonadotropin-responsive
factors may play a role in antrum formation in these externally-stimulated follicles. Together,
these results demonstrate that antrum formation is regulated by the stiffness of the matrix
surrounding the follicle in vitro, and that aquaporins 7 and 8, as well as additional factors are
responsible for antrum formation. Further studies using this system may provide insight into
ovarian disorders such as polycystic ovary syndrome, in which the ovarian stroma thickens
causing suspension of follicle growth in the early antral stage, often causing infertility.
Supported by NIH U54HD41857

                                                 35
                                                                                         Abstract # P9

Activation of Smoothened, a Hedgehog Signaling Transducer, Induces Ectopic Appearance
of Fetal Leydig Cells in Fetal Ovaries, Ivraym Barsoum1, Nathan Bingham2, Keith Parker2 and
Humphrey H-C Yao3, 1Department of Cell & Developmental Biology, University of Illinois,
Urbana, IL; 2Department of Internal Medicine, University of Texas Southwestern Medical
Center, Dallas, TX; 3Department of Veterinary Biosciences, University of Illinois, Urbana, IL

The fate of a gonad, in mammals, to become a testis or an ovary is genetically determined by the
Y-linked gene Sry. In fetal testis, Sry gene triggers certain events; one is the differentiation of
pre-Sertoli into Sertoli cells (SCs). Consequently, SCs regulate testis morphogenesis by
producing signaling molecules such as Desert hedgehog (Dhh). In Dhh null fetal mouse, testes
had lower numbers of fetal Leydig cells. Similarly, cyclopamine, an inhibitor that blocks the
intracellular hedgehog pathway, caused a complete loss of fetal Leydig cells, a more severe
effect than Dhh null testes. These findings have shown the essentiality of the hedgehog pathway
in fetal Leydig cell formation. To identify whether the activation of the hedgehog pathway is
sufficient to induce development, we designed a gain-of-function approach to activate the
hedgehog pathway in the fetal ovary, where the hedgehog pathway is normally inactive. We
argued that if the hedgehog pathway were essential for Leydig cell development, activating the
pathway in the ovary would lead to appearance of fetal Leydig cells. We developed in vitro and
in vivo models to activate Smoothened (Smo), an effector downstream of all hedgehog ligands.
In the in vitro model, we cultured fetal ovaries with or without purmorphamine, a chemical that
activates Smo. In the in vivo model, we developed a genetic approach in which a constitutively
active Smo transgene is stimulated under the control of Sf1-Cre, where Cre recombinase is
present in somatic cells of fetal ovaries. In both models, we found that induction of Smo led to
proper activation of the hedgehog pathway (expression of downstream targets such as Ptch1 and
Gli1) in contrast to absence of these molecules in the control. Furthermore, we observed clusters
of p450 side chain cleavage (Scc) positive cells in fetal ovaries in both models whereas in the
control ovaries, Scc-expressing cells were never present. Ectopic steroidogenic cells in the fetal
ovary exhibited androgen-production ability similar to fetal Leydig cells, as the ovaries from
Smo-activated embryos were descended to a position similar to fetal testes. Because appearance
of fetal Leydig cells is regulated by Sertoli cells, it was therefore possible that activation of Smo
induced Sertoli cells, which in turn caused Leydig cell appearance. We examined Sertoli cell
markers such as Sox9 and Amh, and found no evidence of Sertoli cell presence. In summary, we
demonstrated that hedgehog pathway is sufficient to initiate differentiation of androgen-
producing cells, possibly fetal Leydig cells, in fetal ovaries. Unlike testes, fetal ovaries do not
have Sry and Sertoli cells. Even when the hedgehog pathway is activated, Sertoli cells never
appeared in fetal ovaries. Our gain-of-function models for hedgehog provide evidence that
certain testis characteristics can be induced in fetal ovaries without Sry and Sertoli cells. The
hedgehog pathway is probably the sole mechanism responsible for induction of fetal Leydig cell
differentiation.

Supported by: NIH HD64841, March of Dimes, Billie Field Memorial Graduate Fellowship




                                                 36
                                                                                      Abstract # P10


Activin Signaling Influences the Stromal Cell Population in Neonatal Mouse Ovaries.
Daniel Trombly, Teresa Woodruff, and Kelly E. Mayo, Department of Biochemistry, Molecular
Biology & Cell Biology and Center for Reproductive Science, Northwestern University,
Evanston, IL 60208

Activin, a member of the TGF superfamily, has intraovarian actions which affect follicle
development. Recent studies suggest that activin is important in establishing the ovarian follicle
pool. We are further investigating how activin signaling influences early folliculogenesis. To
achieve this, we are using an ex vivo approach, where newborn mouse ovaries are cultured for
four days in the absence or presence of activin. The four-day interval is a period when germ
cells are encapsulated by somatic cells (pre-granulosa cells) within ovaries to generate primordial
follicles. Preliminary follicle counts from ovaries cultured in media containing 100ng/ml
recombinant activin A revealed a similar population of primordial follicles compared to vehicle-
treated ovaries. Surprisingly, activin treatment appeared to increase the number of stromal cells.
Histological examination revealed that these cells formed a layer in the cortical compartment
between the ovarian surface epithelium and primordial follicles. The stromal cell layer spanned
most of the cortex and displayed varying thicknesses among activin A-treated ovaries. The
expansion of stromal cells was abrogated when ovaries were treated with activin A plus the
antagonist follistatin. We employed immunolocalization experiments to determine the cell types
within the expanded zone of cells in activin A-treated ovaries. These cells were cytokeratin-8
(CK-8, epithelial marker) negative, but stained positively for vimentin, a mesenchymal cell
marker. We are exploring the expression of thecal cell markers in activin A-treated ovaries to
determine if the cells within the expanded zone are indeed thecal precursors. In addition to its
established role in regulating granulosa cell proliferation, our studies suggest that activin
signaling may play a part in the maintenance of stromal cells during early follicle development.

This work is supported by NIH grant PO1 HD021921, the Reproductive Biology Training
Grant (HD00768), and the ARCS Foundation.




                                                37
                                                                                    Abstract # P11

Activin/inhibin are Involved in the FSH-regulated Early Folliculogenesis in Mouse, Lei
Lei, Monica Antenos, Jei Zhu, Teresa K. Woodruff, Department of Obstetrics and Gynecology,
Northwestern University Feinberg School of Medicine, Evanston, IL 60208

Activin and inhibin belong to the transforming growth factor beta (TGFβ) family of cytokines
produced by the ovary of female mammalian. Besides regulating the secretion of pituitary
Follicle-stimulating hormone (FSH), activin and inhinbin are also involved in follicule
development through paracrine regulatory pathway. Based on its receptor distribution, FSH is
determined plays important stimulatory function in the mid and late follicular development in
mammalian ovary. In mouse, beyond17.5 day post ciotus (dpc) till 3 day post partum (dpp),
with the start of germ cellcyst breakdown, pre-granulosa cells enclose the primordial oocytes
and form the primordial follicle pool which provide the follicle resource for the whole life. To
explore the possible regulatory function of FSH during early folliculogensis, in this study,
mouse fetal ovaries of 17.5 dpc were isolated and cultured on membrane insert under several
different culture conditions. Medium was changed half amount every two days and collected
for activinA and total inhibin ELISA assay. At 6 days and 15 days culture, which corresponded
to day 3 and day 12 after birth respectively, cultured ovaries were fixed for histological
analysis. The number of germ cells in cyst, primordial follicle, primary follicle and secondary
follicle were counted respectively. The results showed that the addition of 1% FBS in culture
medium (DMEM/F12 with 1% FBS, group II) inhibited the process of germ cell cyst
breakdown significantly, comparing with the ovaries cultured in DMEM/F12 medium (group
I). While, with the number of germ cells in cyst decreased in group III (DMEM/F12 with 1%
FBS and 10mIU/ml FSH), the amount of primordial follicles increased dramatically. However,
when the ovaries were cultured in high level of FBS (DMEM/F12 with 10% FBS and
10mIU/ml FSH, group V), FSH could not induce the germ cell cyst breakdown evidently.
During the first 6 days culture, both the activinA and inhibin arrived at the peak level at day 4
of culture in group III which germ cell cyst breakdown was induced by FSH. While in the
group II, the decrease of activinA at day 4, and no obvious change of inhibin level during 6
days culture may be the potential reason for keeping germ cells in cyst. Meanwhile, the
presence of FSH in culture could stimulate the oocytes development obviously, the diameter of
oocytes at day 6 of culture were: 24.3 ± 2.6 μm (group I), 22.6 ± 2.6 μm (group II), 32.0 ± 2.2
μm (group III) and 26.4 ± 2.3μm (DMEM/F12 with 10mIU/ml FSH, group IV). But, the FSH
did not have obvious positive effects on the number of secondary follicles in later 9 days
culture. In conclusion, for the first time, our data suggested that activin, inhibin and FSH play
important regulatory role in mouse early folliculogenesis. Exploring the forming mechanisms
of primordial follicle pool and early folliculogenesis are significant for our understanding of
the ovarian physiological and pathological phenomena.


This research is supported by NIH grant P01HD021921




                                              38
                                                                                  Abstract # P12

ABNORMAL DEVELOPMENT IN FEMALE GONADS OF FUSED TOES MUTANT
MICE. B. Kim, Y. Kim, J.S. Jorgensen, Department of Veterinary Biosciences, University of
Illinois, Urbana, IL

The goal of the present study is to investigate the functional significance of Iroquois (Irx)
genes in developing gonads. The Iroquois homeobox family has been shown to be critical for
axis and pattern formation during the development of several organisms. We found that Irx3 is
expressed in developing female gonads at a time that coincides with critical milestones that
determine a female versus male gonad fate. However, the function of Irx3 during gonadal
development remains unknown. Because there is no Irx3 knock out mouse, we are using a
mutant mouse model, fused toes (Ft), to study the functional significance of Irx3. Ft mice lack
Irx3 along with 5 other genes including two other members of the IrxB cluster, Irx5 and 6,
Ftm, which is important for developmental processes such as the establishment of left-right
asymmetry and patterning of the neural tube and the limbs, and two other genes of unknown
function, Fts, and Fto. Homozygote mutants (Ft-/-) die in development (E10-E12) because of
severe malformation of the developing brain and loss of left-right asymmetry.
Semiquantitative PCR determined that only Irx3 and Irx5 were preferentially expressed in
female gonads while the other four genes exhibited no difference between sexed gonads.
Based on these findings, we hypothesize that the IrxB cluster, especially Irx3 and 5, will play
an important role in female gonad development. Fused toes embryos were dissected at
embryonic day 11.5 and gonads were cultured in vitro for 3 days or in vivo for 2 weeks under
the kidney capsule of a castrated wild type male recipient mouse. The 3-day cultured gonads
were assessed for morphology by histological sections and for apoptosis (Tunel) or
proliferation (PCNA staining). Morphology and cell proliferation were similar among gonads
of both sexes and of all genotypes. However, in female gonads from Ft -/- embryos, we
observed a significant decrease in cells positive for Tunel staining compared to control
samples. Embryonic gonads subject to 2 weeks of culture under the kidney capsule of
recipient mice approximates development at postnatal day 5. In the male gonads, significant
development of testicular cords was noted in both wild type and mutant samples. In contrast,
we observed abnormal follicle formation in the Ft-/- females. At this stage, control samples
contained expected primary and secondary follicles; however, Ft-/- follicles contained
multiple small oocytes. In addition, preliminary data from 1 week cultures suggest abnormal
cell division in ovaries of Ft-/- mice. Together, these results suggest that the absence of the
IrxB cluster, Fto, Fts, and Ftm induces abnormal gonad development in the female. We are
currently pursuing additional studies to address the impact of the Ft mutant on gonad
development.

Supported by the University of Illinois Campus Research Board and the University of
Illinois Veterinary Medical Research Fund.




                                             39
                                                                                      Abstract # P13

Conditional Knockout of Connexin 43 in Mouse Uterus Uncovers an Essential Role of
Stromal Cell Gap Junctions in Uterine Angiogenesis during Pregnancy, Mary Laws1,
Francesco DeMayo3, John Lydon3, Milan K Bagchi2, and Indrani C. Bagchi1. 1Department of
Veterinary Biosciences, 2Department of Molecular and Integrative Physiology, University of
Illinois at Urbana-Champaign, 3Baylor College of Medicine, Houston, TX

In most mammalian tissues, gap junction proteins connect cells by creating intercellular
cytoplasmic channels clustered in the plasma membrane. These channels allow for
communication between the cells by transferring ions and small molecules. Connexin 43 (Cx43)
is one such gap junction protein that is present in multiple cell types. Our studies indicated that
Cx43 is induced in the mouse uterus at the onset of implantation. The expression of Cx43
increases during the decidualization phase of pregnancy and is localized in the proliferating as
well as the decidualized stromal cells surrounding the implanted embryo. Because the Cx43
knockout mouse dies late in gestation or immediately following birth due to a developmental
heart defect, we have created a conditional knockout mouse to address the functional role of
Cx43 in the uterus during pregnancy. We created the conditional knockout mouse model by
crossing transgenic mice harboring a “floxed” Cx43 gene (Cx43fl/fl) with progesterone receptor
Cre knockin (PR-cre) mice. This mating generated female mice in which the Cx43 gene
undergoes Cre-mediated excision in the reproductive cell types expressing the PR. The PR-Cre X
Cx43fl/fl mice will be referred to as Cx43d/d mice. Our breeding studies revealed that Cx43d/d mice
exhibited more than 60% reduction in birth rate when compared to the Cx43fl/fl (control) mice.
To test whether or not this fertility defect is due to an impaired ovarian function, we subjected
the mice to gonadotropin-induced superovulation. We observed that the number of released
oocytes in Cx43d/d mice was comparable to that of Cx43fl/fl mice, suggesting that the reduced
fertility in Cx43d/d mice is not due to a functional defect of the ovary. Further reproductive
analyses indicated that the Cx43d/d mice were able to initiate embryo implantation and support
pregnancy up to day 7 of gestation. However, starting on day 8 of pregnancy, the implanted
embryos in Cx43d/d mice started to undergo resorption. Histological analysis of uterine sections
of the Cx43d/d mice on day 8 of gestation revealed a striking decrease in the number of
proliferating endothelial cells surrounding the embryo. Consistent with this observation,
immunohistochemical staining of uterine sections with PECAM, an endothelial marker, indicated
a severe reduction in angiogenesis in the absence of Cx43 leading to the resorption of embryos
on Day 8 of pregnancy. Further analysis showed that the expression of vascular endothelial
growth factor (VEGF) protein was markedly downregulated in Cx43d/d mice compared to the
Cx43fl/fl mice. Collectively, our studies indicated that Cx43 gap junctions in the stromal
compartment play an essential role in decidualization and the development of angiogenic
network during early pregnancy.

Supported by NIH grants




                                                40
                                                                                                Abstract # P14

The Adaptor Protein MyD88 is Essential for E. coli Induced Preterm Labor
Yana Filipovich, Shi-Jiang Lu, Shizuo Akira and Emmet Hirsch
Departments of Obstetrics and Gynecology, Evanston Northwestern Healthcare and Feinberg
School of Medicine, Northwestern University

OBJECTIVE: Preterm birth is the major cause of neonatal morbidity and mortality in the
developed world. Fifty percent or more of cases of preterm birth are associated with bacterial
colonization of the gestational compartment. Toll-like receptors (TLRs) are the critical
recognition factors for initiating innate immune responses to bacterial pathogens. We have
previously shown using mutant mice that TLR-4 (frequently referred to as the LPS receptor) is
essential for normal susceptibility to E. coli-induced preterm labor.
METHODS: We used a mouse model of infection-induced preterm delivery to examine the
roles of two adaptor proteins with central functions in toll-like receptor signaling: MyD88
(myeloid differentiation primary-response gene 88) and TRIF (Toll/IL-1 receptor (TIR)-domain-
containing adaptor protein inducing IFN-β). MyD88 knockout, TRIF knockout, MyD88/TRIF
double-knockout and B6129SF2/J wild-type control mice on day 14.5 of gestation underwent
intrauterine injection with varying quantities of killed E. coli. Mice were observed after surgery
for preterm delivery (delivery of at least one pup within 48 hours) or were euthanized at various
times after surgery for serum collection (for progesterone assays) and uterine tissue harvest (for
RT-PCR and immunohistochemistry).
RESULTS: E. coli-induced preterm delivery, intrauterine fetal survival and maintenance of
circulating progesterone levels depend upon MyD88, while induction of IL-1β and TNFα in
uterine tissues is a function of redundant              Preterm delivery (% of treated pregnancies)
actions of both MyD88 and TRIF.
CONCLUSIONS: Susceptibility to E. coli- 100%
induced preterm labor in the mouse is               80%
completely dependent upon MyD88 and not             60%
                                                                                                  WT
TRIF. Similar dependence on MyD88 occurs                                                          TRIF KO
                                                                                                  MyD88 KO
for fetal retention and survival in utero and       40%
                                                                                                  TRIF/MyD88 DKO
progesterone withdrawal. These results              20%
suggest that of the two alternate signaling          0%
pathways downstream of TLR-4, the                         5 x 108      109       5 x 109 1010
MyD88-dependent pathway is the critical                           # of E. coli organisms
one for bacterially induced preterm labor.
Given the important role played by MyD88 in signal transduction by toll-like receptors in
response to infection with various pathogens, these results identify MyD88 as a potentially
critical intermediary in infection-induced preterm labor due to a variety of organisms.

This study supported by March of Dimes Birth Defects Foundation, #FY06-573.




                                                     41
                                                                                     Abstract # P15

P450 Aromatase-Derived Intra-Uterine Estrogen is Critical for Decidualization,Amrita
Das1, Srinivasa R. Mantena1, Athilakshmi Kannan1, Milan K. Bagchi2, and Indrani C. Bagchi1,
1
  Department of Veterinary Biosciences. 2Department of Molecular and Integrative Physiology,
University of Illinois at Urbana-Champaign, Urbana, IL

In the mouse, implantation is initiated upon attachment of the embryo to the uterine luminal
epithelium on day 4 of pregnancy. This event is followed by proliferation and differentiation of
uterine stromal cells into morphologically distinct secretory decidual cells, which control
trophoblast invasion and embryonic growth until placentation ensues. In addition to the stromal
differentiation, development of an extensive angiogenic network in the uterine bed is critical for
the growth and development of the implanted embryo. Previous studies have shown that ovarian
steroids, estrogen (E) and progesterone (P), are essential for preparing the uterus for embryo
implantation. While P plays a critical role in decidualization and maintenance of pregnancy, E is
known to control epithelial receptivity and embryo attachment. The role of E in the
decidualization process, however, remains elusive. Interestingly, we have recently observed that
the entire steroid hormone biosynthetic machinery is present and functional in the decidual
uterus. More importantly, the expression of P450 aromatase, a key enzyme responsible for
conversion of androgens to E, is significantly enhanced in the decidualizing stromal cells. To
address the role of the aromatase-derived E in uterine stromal differentiation, we used
ovariectomized mice in which the decidualization process is experimentally induced by
administering a mechanical stimulation to the P-primed uterus. When we treated these animals
with letrozole, a specific inhibitor of aromatase, the uterine decidual response was severely
compromised. To identify the pathways regulated by intra-uterine generation of E, we performed
global gene expression profiling using RNA samples isolated from uteri treated with or without
letrozole. We identified several genes whose expression is significantly downregulated in the
uterus in response to this drug. These genes encoded several known markers of stromal
differentiation. Strikingly, a significant number of letrozole-regulated genes were found to be
regulators of endothelial cell proliferation and migration. These molecules are likely mediators
of uterine angiogenesis. Indeed, we observed a markedly reduced expression of endothelial cell
surface marker, PECAM, in the letrozole-treated uteri, indicating impaired formation of the
uterine vasculature. Our study, therefore, uncovered a novel role of aromatase-derived
intrauterine E in mediating stromal differentiation and angiogenesis during early pregnancy.

Supported by NIH grant




                                               42
                                                                                     Abstract # P16

Hyperphagia is Induced by the Steroid Milieu of Pregnancy: The Role of Estrogen-induced
Nuclear Progesterone Receptors, C. Park, Z. Zhao,T.H.Horton, J.E. Levine


Physiological elevations in serum progesterone (P4) concentrations, such as those observed
during pregnancy, are associated with hyperphagia, increased body mass, increased adiposity,
and leptin resistance. Progestins are also used clinically to increase appetite and food intake in
cancer and AIDS patients with anorexia-cachexia. Although these observations indicate that
endogenous or exogenous progestins can increase food intake, body mass and adiposity, the
cellular and molecular mechanisms by which they exert these effects remain largely unknown.
Recent studies in our laboratory have determined that P4 exerts such effects on energy
homeostasis through nuclear progesterone receptor (nPRA/B)-dependent mechanisms. In the
study, progesterone increased food intake, body mass, and visceral adiposity in gonadally-intact
wild-type (WT), but not in mifepristone-treated or PR null mutant (PRKO) mice. In this study,
the goal was to investigate the possible importance of estrogen in P4’s actions on energy
homeostasis. All mice were ovariectomized and implanted with either estrogen or vehicle
capsules. They were then treated with high doses of progesterone both with and without
mifepristone for 21d. Our findings demonstrate that 1) estrogen is necessary for progesterone to
generate increase in food intake, body mass and adiposity and 2) the absence of these effects in
mice lacking in estrogen and in the group treated with PR antagonist suggests that these actions
are mediated by estrogen induced progesterone receptors. These observations are also consistent
with the hypothesis that the hyperphagia of pregnancy results from the ability of high-
physiological P4 levels to activate estrogen induced nPRA/Bs, presumably those receptors
expressed in the feeding-related neural circuitries of the mediobasal hypothalamus, such as the
arcuate nucleus and ventral medial nucleus of the hypothalamus which have shown strong PR
induction in response to estrogen. RIA of serum confirmed that our hormonal treatment produced
serum progesterone and estrogen concentrations that closely mimicked those observed in
pregnancy.




                                               43
                                                                                    Abstract # P17

Neonatal Estrogen Exposure Results in Hyperplastic Obesity in Adult Mice. M.A.
Cimafranca, D. Meling, C.M. Beals, P.S. Cooke. Department of Veterinary Biosciences,
University of Illinois Urbana-Champaign, Urbana, IL.

Adipogenesis in mice occurs neonatally, but the precise role of estrogen in adipocyte
development is not clear. Exposure of neonatal mice to the synthetic estrogen diethylstilbestrol
(DES) has been reported to cause obesity in mature animals. As adipocyte number is established
neonatally, we hypothesized that neonatal DES exposure will induce adipocyte hyperplasia in
adults, and that this accounts for the observed adult obesity. C57BL/6 mice were injected i.p.
once daily with vehicle or 1 ng/g DES from postnatal day (PND) 1 – 5. Mice were weighed
every two weeks. At 4 months, DXA analysis was performed and fat pads were subsequently
collected to assess adipose tissue mass; adipocyte number and diameter were assessed via
osmium tetroxide fixation and Coulter counter measurement. In females (n=6) DES yielded an
almost two-fold increase in body fat percentage and weights of adipose depots, despite a modest
10-15% increase in body weight. Critically, the observed obesity in these mice resulted from
adipocyte hyperplasia, as there were no changes in average adipocyte size or the distribution of
adipocyte diameters, and these mice had a 2-fold increase in overall adipocyte number. In males,
neonatal DES treatment resulted in a 70% increase in body fat percentage and adipose depot
weights, accompanied by decreased percentages of large adipocytes and increased percentages of
small adipocytes. Thus, the magnitude of the overall adipocyte hyperplasia in these males was
comparable to the 2-fold increase seen in females. Despite the adipocyte hyperplasia, neither the
DES-treated males nor females showed impaired glucose tolerance. In summary, this is the first
report that a brief, early xenoestrogen exposure can result in a permanent and severe adipocyte
hyperplasia during adulthood. These results emphasize that neonatal estrogen stimulates
adipocyte number and that adipogenesis during development is vulnerable to disruption by
xenoestrogens, which can produce permanent changes in adipocyte number and adipose mass.


This work supported by NIH grants AG24387 and T32 ES07326 and the Billie A. Field
Endowment.yy




                                               44
                                                                                       Abstract # P18

Circadian and Diurnal Activity Rhythm Comparisons in ob/ob and db/db Mice,
D.M. Arble, M.R. Augustine, A.D. Laposky, & F.W. Turek,, Sleep & Circadian Biology,
Northwestern University, Evanston, IL


The satiety hormone leptin is critically involved in long term energy homeostasis and is
produced in adipose tissue in proportion to body fat stores. Leptin enters the brain and mediates
neuronal activity in the mediobasal hypothalamus (i.e. arcuate nucleus) and the circadian
pacemaker, located in the suprachiasmatic nuclei (SCN). Mice lacking leptin (ob/ob) or having a
mutation of the leptin receptor (db/db) develop obesity and diabetes at an early age. While
circadian regulation is an important component of energy metabolism, surprisingly few studies
have been conducted to examine circadian and diurnal regulation in animal models of obesity
and diabetes, and no published studies have directly compared circadian behavior of db/db and
ob/ob models. In this study, locomotor activity of 12-week old male ob/ob (N=8), db/db (N=6),
and C57Bl/6J (B6, N=8) mice was monitored (via infrared beams) for 100 days under entrained
12L:12D conditions, followed by 21 days in constant dark (DD) conditions. As expected,
C57Bl/6J mice exhibited significantly more activity in the dark phase compared to the light
phase throughout the 100 days, reflecting a robust diurnal rhythm of activity. During the first 3
time points analyzed (day 0, 50 and 100) db/db mice maintained a diurnal rhythm of activity,
however, the amplitude was notably decreased compared to C57Bl/6J mice. In sharp contrast,
ob/ob     mice     failed    to    exhibit    a    diurnal   rhythm at        any     time    point.
Activity was recorded in DD to determine the free-running period. We found that 100% of wild-
type mice had clear free-running rhythms with activity onsets averaging a period of 23.83 hours.
While clear activity onsets were not detectable in db/db and ob/ob mice, we were able to
calculate period length using Fast Fourier Transformation in a few mice. Four out of six db/db
mice (67%) showed a measurable rhythm in DD with an average period of 23.85 hours.
Remarkably, only one ob/ob mouse (13%) showed rhythmic behavior with a period of 23.92
hours. In summary, both db/db and ob/ob mice exhibit alterations in diurnal and circadian
rhythms of activity. The most striking feature of these results is the prominent difference
between db/db and ob/ob phenotypes. Given that both obesity models result from genetic defects
in leptin function, they may be expected to have similar circadian deficits; surprisingly, they do
not. Because the db/db mutation affects only one of the leptin receptor isoforms (Ob-Rb), it is
possible that leptin may still be functioning through another receptor to affect circadian and other
behaviors.

Supported by NIA-PPG Grant P01 AG 11412 and NIH/NHLBI Grant T32 HL007909




                                                45
                                                                                        Abstract # P19

Estrogen Exerts Rapid Effects on Thermoregulation in Ovariectomized Mice, J. L. White,
W. C. Levine, J.E. Levine, and T.H. Horton. Department of Neurobiology and Physiology and
the Center for Reproductive Science, Northwestern University, Evanston, IL.

Menopause occurs when a woman’s ovaries no longer produce estrogen. This loss of estrogen
coincides with a variety of physiological changes, including changes in body-temperature
regulating mechanisms resulting in hot flashes. Unfortunately, little is known about how
estrogen modulates this system. Estrogen is known to act by two distinct categories of signaling
mechanisms. One category of signaling mechanisms involves the binding of estrogen to nuclear
receptors, requires the induction of DNA transcription (“genomic effects”), and occurs over a
time from of hours or days. The other category is composed of non-genomic events and involves
the binding of estrogen to proteins associated with the plasma membrane. This category of
signaling does not require transcription of new genes produces a much more rapid response in a
cell, non-genomic events may occur within minutes. It is not yet known whether estrogen acts
by a genomic or non-genomic pathway in neurons that regulate body temperature. A recent study
has shown that administering estrogen the arcuate nucleus increases the activity POMC neurons
within 30 minutes (Gao, 2007). This indicates that estrogen is likely acting by a non-genomic
pathway in these neurons. Based on this study we tested the hypothesis that the hypothalamic
administration of estradiol could induce rapid changes in thermoregulation in mice.

Like humans, mice exhibit hot flashes subsequent to the loss of estrogen. These hot flashes can
be monitored by measuring tail skin temperature (TST). Ten adult female mice were
ovariectomized to induce hot flashes. Two weeks after ovariectomy they were implanted with a
cannula in the lateral ventricle of the brain. Beginning one week after cannulation, the mice
were infused three times with 0, 200, or 2000 ng of estradiol encapsulated in cyclodextrin
(Sigma E4389) diluted in artificial cerebrospinal fluid. The estradiol was infused in a volume of
2 µl over 2 minutes. Mice were treated in a randomized complete block repeated-measures
design on three separate occasions until each mouse had been infused with each dose. A
minimum of 24 hours rest was permitted between infusions. Prior to each infusion session, each
animal’s TST was recorded for 1 hour prior to infusion to establish a baseline TST for each day.
TST was recorded using a remote recording device (Minimitter) that recorded temperature
continuously at 1 minute intervals throughout each session.

There was a significant effect of the dose of estradiol on the variability in tail skin temperature (p
= 0.02) following infusion of estradiol. The effect is most apparent for the highest dose of
estradiol, which reduced the variance in tail skin temperature within 40 minutes of the onset of
the infusion. These results suggest that estradiol acts via a non-genomic mechanism to influence
thermoregulation.

Gao, Qian et al. Anorectic estrogen mimics leptin’s effect on the rewiring of
      Melanocortin cells and Stat3 signaling in obese animals. Nature Medicine
      13:89-94, 2007.
Supported by AG030181-01 from the NIA to JEL and THH and a Northwestern University
Undergraduate Research Fellowship to JLW

                                                 46
                                                                                  Abstract # P20

Ovarian Steroidogenic Regulation is Dependent upon ERE-mediated ERα Signaling. D.A.
Smith, C. Glidewell-Kenney, J.L. Jameson, Division of Endocrinology, Department of Medicine,
Feinberg School of Medicine, Northwestern University, Chicago, IL

The female estrogen receptor-α knock out (ERαKO) mouse demonstrates striking phenotypic
similarities to the human polycystic ovary syndrome (PCOS). The ERαKO phenotype is
characterized by chronic anovulation, elevated serum estradiol and testosterone, hemorrhagic
ovarian cysts, obesity and infertility. Classical estrogen receptor (ER) effects are mediated
through the direct interaction of ligand-bound ER with genomic estrogen response elements
(EREs). We have generated a knock-in mouse model expressing a mutant form of ERα (AA)
that is incapable of binding EREs. Due to the infertile phenotype of the female ER+/AA
heterozygote, we bred the AA allele onto the back ground of the ERαKO mouse. The ER-/AA
mouse is a model of non-ERE-only ERα signaling. Restoration of the AA allele rescues a major
portion of the aberrant ERαKO serum levels of luteinizing hormone (LH), however, high serum
androgens and the PCOS phenotype persist. Furthermore, the ER-/AA ovary demonstrates high
expression levels of the testis-specific enzyme, Hsd17b3. These data suggest that the capacity
for ERα to bind EREs is required for steroidogenic regulation in the ovary and suppression of
testosterone synthesis.




                                             47
                                                                                      Abstract # P21

Follicular Atresia During the Pre-pubertal Period in Mice is not Due to Classical
Apoptosis, Candace M Tingen1 and Dr. Teresa K Woodruff1,2,3, 1 Department of Biochemistry,
Molecular and Cell Biology, Northwestern University, Evanston, IL 60208 2 Department of
Neurobiology and Physiology, Northwestern, University, Evanston, IL 60208 3 Department of
Obstetrics and Gynecology, Feinberg, School of Medicine, Northwestern University, Chicago,
IL 60611, 4 Center for Reproductive Science, Northwestern University, Evanston, IL 60208


In mice and humans, a large portion of follicles created are lost before puberty. The vast majority
of follicles lost during this time are the small immature primordial and primary follicles. This
follicle loss has been attributed to classical apoptosis, but this has never been proven
experimentally. Our study seeks to investigate whether follicular atresia during the pre-pubertal
period can be wholly attributed to apoptosis. To study this, day 10 and day 26 ovaries from CD1
females were isolated and fixed for 24 hours. Tissue was either stained for TUNEL, cleaved
caspase-3, or hematoxylin and eosin. For each method, every tenth section was photographed at
20X magnification and counted. Positive follicles (containing even a single cell deemed positive
for TUNEL, cleaved caspase-3, or pyknosis) were counted at each day. The number of
primordial follicles within the epithelial layer and intrabursal space was also counted in day 10
ovaries at every tenth section. While large secondary and antral follicls showed signs of classical
apoptosis at day 26 as expected, virtually no primordial or primary follicles were positive at day
10 or 26. Additionally, no follicles were seen in the intrabursal space, despite many primordial
follicles existing close to or within the ovarian epithelium. The data suggests that classically
defined apoptosis is not the cause of the majority of primordial and primary follicle loss
witnessed during the pre-pubertal period. The theory of follicle shedding also seems inadequate
to explain the loss, as no follicles are ever seen extruded from the ovary. An alternate method of
cell death that does not exhibit the hallmarks of apoptosis (nuclear condensation and DNA
fragmentation) such as autophagy may be a candidate for the remaining follicle death.

Work is supported by NIH grant PO1 HD021921.




                                                48
                                                                                      Abstract # P22

Methoxychlor Alters the Expressionsof Selected Apoptotic Factors in the Ovary, Weck RL,
Gupta RK, Flaws JA, University of Illinois Urbana-Champaign, Urbana Illinois

Methoxychlor (MXC) is an organochlorine pesticide that is widely used in Africa, Asia, and
other developing countries to protect field crops, vegetables, fruits, stored grain, livestock, and
domestic pets against a range of pests, including houseflies, mosquitoes, cockroaches, chiggers,
and various arthropods. This chemical has been shown to bind to the estrogen receptor in the
mammalian ovary and target antral follicles, causing them to undergo atresia. The mechanism
by which MXC causes follicular atresia is not well understood. Previous studies have shown that
MXC causes atresia in part by altering highly conserved members of the Bcl-2 family such as
Bcl-2 (an anti-apoptotic factor) and Bax (a pro-apoptotic factor). Specifically, MXC treatment
decreases the mRNA and protein levels of Bcl-2 and increases the mRNA and protein levels of
Bax in antral follicles. Further, Bcl-2 overexpression and Bax deletion help protect antral
follicles from MXC-induced atresia. Currently, it is not known whether MXC alters the
expression of other Bcl-2 family members. Thus, this work was designed to test the hypothesis
that MXC alters the expression of other Bcl-2 members. This work specifically focused on Mcl-
1, Bok, Bad, and Bid because these factors are present in antral follicles and are known to be
either anti-apoptotic or pro-apoptotic factors. To test the hypothesis, adult cycling mice (39
days) were dosed with MXC (16, 32, or 64 mg/kg/day) or vehicle (sesame oil) for 20 days. After
dosing, ovaries were collected, antral follicles were mechanically isolated, and the follicles were
subjected to real time polymerase chain reaction for the measurement of mRNA levels of Mcl-1,
Bok, Bad, and Bid. The results indicate that antral follicles from MXC-treated ovaries had
significantly less mRNA expression of Mcl-1 compared to controls (control = 1.44 ± 0.26
genomic equivalents (ge); MXC 16 mg/kg/day = 0.33 ± 0.03 ge; MXC 32 mg/kg/day = 0.56 ±
0.16 ge; MXC 64 mg/kg/day = 0.41 ± 0.14 ge; n = 3; p≤ 0.005). The results also indicate that
antral follicles from MXC-treated ovaries had significantly more mRNA expression of Bok
compared to controls (control = 1.43 ± 0.08 genomic equivalents (ge); MXC 16 mg/kg/day =
1.94 ± 0.32 ge; MXC 32 mg/kg/day = 2.06 ± 0.17 ge; MXC 64 mg/kg/day = 2.97 ± 0.19 ge; n =
3; p≤ 0.05). In contrast, MXC treatment did not significantly affect the mRNA expression of
Bad or Bid in antral follicles (n = 3; p >0.05). Collectively, these data indicate that MXC
treatment decreases expression of Mcl-1 and increases the expression of Bok, but it does not alter
the expression levels of Bad or Bid in antral follicles. Thus, MXC may selectively target some
members of the Bcl-2 family in antral follicles, and this may drive them towards atresia.

Supported by NIH R01 ES012893 and the Interdisciplinary Environmental Toxicology
Program at the University of Illinois Urbana-Champaign.




                                                49
                                                                                      Abstract # P23

Cell Cycle Regulators in Mouse Ovarian Antral Follicles are Altered by Methoxychlor
Exposure, Gupta RK, Meachum SH, Yao HH, and Flaws JA, Department of Veterinary
Biosciences, School of Veterinary Medicine, University of Illinois at Urbana-Champaign

Methoxychlor (MXC) is an organochlorine pesticide used against insects that attack fruits,
vegetables, and home gardens. Humans and wildlife species are exposed to this chemical. This is
of concern because MXC has been shown to reduce fertility in female rodents by causing ovarian
atrophy and persistent estrous cyclicity. Further, MXC adversely affect antral follicles, which are
the functional units for maturation and release of oocytes and responsible for synthesis of sex
steroid hormones such as estrogen. Specifically, MXC exposure decreases antral follicle
numbers and increases the percentage of atretic antral follicles by inducing oxidative stress.
Furthermore, studies show that MXC inhibits antral follicle growth in vitro. The mechanism by
which MXC inhibits growth of antral follicles is unknown. Granulosa cell proliferation is
required for normal follicle growth and is controlled, in part, by cell cycle regulators such as
proliferating cell nuclear antigen (PCNA), cyclins, and cyclin dependant kinases. Thus, this
study tested the hypothesis that MXC inhibits follicle growth in vivo by reducing the levels of
cell cycle regulators. Adult cycling mice were dosed with MXC (16, 32, or 64 mg/kg/day) or
sesame oil (vehicle) for 20 days. Ovaries were collected and subjected to immunohistochemistry
for PCNA staining. In addition, ovaries from vehicle- and MXC-treated mice were used to
isolate antral follicles and subjected to real time polymerase chain reaction and western blot
analysis for measurement of mRNA and protein levels of cyclin D2 (CCND2) and cyclin
dependant kinase 4 (CDK4). The results indicate that antral follicles from MXC-treated ovaries
had significantly less staining for PCNA compared to controls (control = 71.2 ± 1.7% stained
area/follicle; MXC 16 mg/kg/day = 51.9 ± 3.2% stained area/per follicle; MXC 32 mg/kg/day =
46.0 ± 2.7% stained area/per follicle; and MXC 64 mg/kg/day = 26.2 ± 3.2% stained area/per
follicle; n = 3; p ≤ 0.05). MXC significantly reduced mRNA expression of Ccnd2 compared to
controls (control = 0.85 ± 0.06 genomic equivalents (ge); MXC 16 mg/kg/day = 0.44 ± 0.08 ge;
MXC 32 mg/kg/day = 0.46 ± 0.03 ge; MXC 64 mg/kg/day = 0.38 ± 0.09 ge; n = 3; p ≤ 0.05)
Similarly, MXC significantly reduced mRNA expression of Cdk4 compared to controls (control
= 0.89 ± 0.02 ge; MXC 16 mg/kg/day = 0.48 ± 0.02 ge; MXC 32 mg/kg/day = 0.56 ± 0.07 ge;
MXC 64 mg/kg/day = 0.45 ± 0.03 ge; n = 3; p ≤ 0.05). Furthermore, MXC at higher doses
significantly reduced protein expression of CCND2 compared to controls (control = 0.72 ± 0.01
ge; MXC 16 mg/kg/day = 1.07 ± 0.05 ge; MXC 32 mg/kg/day = 0.47 ± 0.02 ge; MXC 64
mg/kg/day = 0.52 ± 0.04 ge; n = 3; p ≤ 0.05). Similarly, MXC at higher doses significantly
reduced protein expression of CDK4 compared to controls (control = 0.84 ± 0.03 ge; MXC 16
mg/kg/day = 1.00 ± 0.04 ge; MXC 32 mg/kg/day = 0.54 ± 0.05 ge; MXC 64 mg/kg/day = 0.44 ±
0.06 ge; n = 3; p ≤ 0.05). Collectively, these data indicate that MXC exposure in vivo reduces the
expression of PCNA and the levels of CCND2 and CDK4 in antral follicles. Therefore, we
postulate that MXC may inhibit the growth of antral follicles by decreasing the levels of cell
cycle regulators in antral follicles.

Supported by NIH R21 ES13061, R01 ES012893, and NIH HD46861.




                                                50
                                                                                      Abstract # P24

Age-related changes in follicle growth in aryl hydrocarbon receptor-deficient mice, I.
Hernandez-Ochoa, R.K. Gupta, B.N. Karman, J.A. Flaws, Department of Veterinary
Biosciences, University of Illinois at Urbana-Champaign, Urbana IL.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor found in the
cytosol of most mammalian tissues and cells, including the ovarian follicles. A variety of
environmental contaminants such as polychlorinated biphenyls and polychlorinated dioxins are
known to bind to the AHR. The binding results in the induction of metabolic enzymes and
therefore, a variety of toxic effects. While there is extensive knowledge regarding the role of the
AHR in the regulation of toxicity of environmental contaminants, little is known regarding its
physiological role in the ovary. Therefore, the objective of this study was to compare the ovarian
follicular growth of AHR-deficient (AHRKO) mice and wild-type (WT) mice at different ages of
reproductive sexual maturity. Antral follicles were isolated and cultured from both AHRKO and
WT mice on postnatal days (PD) 30-35 (prior to puberty), on PD 52-56 (young recent-cycling
adults) and PD 92-98 (cycling adults). The follicular growth in culture was evaluated for 168 h
by measuring the diameters in two perpendicular axes. The results show that, the diameters of
AHRKO and WT follicles were similar at the beginning of culture (AHRKO = 329 ± 9.3 µm;
WT = 338 ± 12.9 µm; p = 0.082) from PD 30-35. However, after 168 h of culture, the AHRKO
follicles showed a significantly smaller diameter than the WT follicles (AHRKO = 537 ± 11.6
µm; WT = 684 ± 18.4 µm; p < 0.001). Interestingly, no differences were observed after 168 h of
culture when comparing the diameter of follicles from PD 52-56 (AHRKO = 550 ± 12.7 µm; WT
= 594 ± 16.3 µm; p = 0.093) or PD 92-98 (AHRKO = 530 ± 18.4 µm; WT = 531 ± 17.2 µm; p=
0.093). In addition, ovarian sections were prepared for histological evaluation of the number of
follicles that reached the preantral and antral stages at select ages of sexual maturity. At PD 30,
AHRKO and WT ovaries contained similar numbers of preantral and antral follicles. By PD 54,
however, AHRKO ovaries contained significantly fewer preantral and antral follicles than WT
ovaries (p < 0.05). Interestingly, by PD 90, AHRKO and WT ovaries contained similar number
of follicles in the preantral (AHRKO = 164 ± 24 preantral follicles; WT = 170 ± 28 preantral
follicles; p = 0.97) and antral stages (AHRKO = 160 ± 25 antral follicles; WT = 167 ± 20 antral
follicles; p = 0.56). Our results suggest that the AHR may regulate follicular growth before
puberty, but other related factors may be involved in regulating follicle growth once the mice
reach their sexual maturity. Although the reasons for this are unknown, it is possible that
differences observed at different ages are the result of alterations in hormones and their
corresponding receptors, and/or factors involved in the translocation of AHR in the cells.

Supported by NIH HD 047275 Grant.




                                                51
                                                                                        Abstract # P25

Epithelial Lineage of Ovarian Carcinoma in the Laying Hen, Gallus Domesticus, Kristine
Ansenberger, University of Illinois at Chicago,

More than 24,000 women in the United States are diagnosed with ovarian cancer each year and
half of these women die from their disease. Ninety percent of these cancers are epithelial in
origin, arising mainly from the ovarian surface epithelium (OSE). This is believed to be a
function of incessant ovulation, resulting in mutations and subsequent neoplasia due to the
constant remodeling in an inflammatory milieu. With the exception of the laying hen, there are
no other animal models of spontaneous OSE cancer that replicate the human disease. Ovarian
cancer in hens and humans share many features including tumor histotype, severe ascites and
peritoneal metastases. The objective of this study was to determine genes and factors important
in the development of OSE cancer and determine the mechanism through which OSE cells
acquire a metastatic and invasive phenotype. Tissue samples were collected from 3 and 4 year
old hens and subjected to immunohistochemical staining and quantitative gene analysis. Ascitic
cancer cells were also collected and used for gene analysis. Our findings indicate that in normal
ovary tissue, cytokeratin, E-cadherin, and N-cadherin are confined to OSE,
but tumors show mixed expression of these epithelial lineage markers. In solid tumors, there is
significant up-regulation of E-cadherin expression in the ovarian stroma, consistent with the
MET process resulting in re-epithelialization. This suggests that the inflammation that
accompanies ovulation may promote epithelial-mesenchymal transition (EMT) and subsequent
mesenchymal-epithelial transition (MET). In the OSE, both E-caherin and N-cadherin are
expressed, indicating that these cells are more pluripotent than strictly committed to the epithelial
lineage. The observation that cTwist is significantly upregulated in cancer, in parallel to
increased E-cadherin, is in marked contrast to what has been observed in other
epithelial carcinomas, and suggests that secondary events are mediating the dominant increase in
E-cadherin. In contrast, cSlug expression is not significantly changed in cancer, while N-
cadherin is most highly expressed in the OSE. The establishment of the ascitic cell line, COCA
65, will provide a homologous cell model to determine the mechanism through which OSE cells
acquire a metastatic and invasive phenotype. Similar to OSE, N-cadherin is highly expressed in
the COCA 65, whereas there is no detectable E-cadherin. However, these cells have cTwist
expression similar to the cancer phenotype. Analysis of surrogate cancer markers, such as ER
alpha/beta, PR, and COX2, in COCA 65 compared to human ovarian cancer cells will
demonstrate that these cells have a similar molecular phenotype and validates these cells as a
model for ovarian cancer. Analysis of specific transcription factors associated with EMT/MET
provides insight into the etiology of OSE cancer and may identify potential molecular targets for
dietary and/or therapeutic intervention.


CDMRP OC050091




                                                 52
                                                                                     Abstract # P26




From A to Zinc: The Inorganic Physiology of Mammalian Oocytes, Alison M. Kim1, Thomas
V. O’Halloran2, 3, Teresa K. Woodruff1, 3, 4 ,1Department of Obstetrics and Gynecology, Feinberg
School of Medicine, Northwestern University, Chicago, IL 60611; 2Department of Chemistry,
Northwestern University, Evanston, IL 60208; 3Department of Biochemistry, Molecular Biology
and Cell Biology, Northwestern University, Evanston, IL 60208; 4Center for Reproductive
Science, Northwestern University, Evanston, IL 60208

Nutritional studies continue to highlight the importance of the transition elements for the health
and metabolism of an organism. Zinc, in particular, is one of the most widely utilized metals in
biological systems. It is soluble at physiological pH and is redox inactive, unlike other
biologically relevant metals such as copper or iron. In addition, zinc is able to accommodate a
variety of coordination states, providing conformational flexibility. For these reasons, hundreds
of proteins, including transcription factors and metalloenzymes, utilize zinc as a structural or
catalytic cofactor. Despite its broad biological significance, little information is available
regarding zinc’s role in mammalian reproduction, particularly in the female. In the male,
concentrations of labile zinc in the sperm were shown to fluctuate during the maturation process.
The presence or absence of zinc also affects sperm motility and capacitation. We hypothesize
that the levels of zinc are similarly dynamic throughout the life cycle of the female gamete, the
oocyte. In accordance with these results, we have identified a subset of zinc transporter proteins
that are abundantly expressed in the oocyte in comparison to other cell types and tissues.
Additionally, manipulation of heavy metal concentrations during in vitro maturation of oocytes
was shown to affect both expansion of the surrounding cumulus cells and asymmetric division of
the oocyte. These aberrant phenotypes were rescued by the supplementation of the culture media
with an exogenous zinc source. Our results indicate that the maintenance of zinc homeostasis
during oocyte maturation may be critical to the successful progression of an immature oocyte to
one that is competent for fertilization.

This work was supported by the NIH U54 HD041857. A. M. K. was a fellow of the
Molecular Biophysics Training Grant GM008382.




                                               53
                           28th Annual Minisymposium on Reproductive Biology
                                                   List of Presenters

Deanna Arble                               Armita Das                                 Jaeyeon Kim
Northwestern University                    University of Illinois- Urbana Champaign   University of Illinois- Urbana Champaign
Neurobiology & Physiology                  Veterinary Bioscience                      Molecular & Integrative Physiology
2205 Tech Drive                            2001 South Lincoln Ave                     407 S. Goodwin Ave
Evanston, IL 60208                         Chicago, IL 61801                          Urbana, IL 61801
d-arble@northwestern.edu                   Das3@uiuc.edu                              Jkim15@uiuc.edu

Kristine Ansenberger                       Chongwen Duan                              Jijngjing Kipp
University of Illinois- Chicago            Northwestern University                    Northwestern University
Physiology and Biophysics                  Cell and Molecular Biology                 Cell and Molecular Biology
835 S. Wolcott                             2205 Tech Drive                            2205 Tech Drive
Chicago, IL 60612                          Evanston, IL 60208                         Evanston, IL 60208
ansenber@uic.edu                           c-duan@northwestern.edu                    j-kipp@northwestern.edu

Denise Archambeault                        Yana Filipovich                            Natalia Kostereva
University of Illinois- Urbana Champaign   Evanston Northwestern Healthcare           University of Illinois-Urbana Champaign
Veterinary Biosciences                     OB/GYN                                     Veterinary Bioscience
2001 South Lincoln Ave                     2650 Ridge Ave                             2001 South Lincoln Ave
Urbana, IL 61802                           Evanston, IL 60202                         Urbana, IL 61802
archambe@uiuc.edu                          yfilipovich@netscape.net                   jkosternv@uiuc.edu

Maria Barbolina                            Rupesh Gupta                               Monica Laronda
Northwestern University                    University of Illinois- Urbana Champaign   Northwestern University
Chemical & Biological Engineering          Veterinary Bioscience                      Medicine
2145 Sheridan Rd. Tech E136                2001 S. Lincoln Ave                        303 E. Superior St.
Evanston, IL 60208                         Urbana, IL 61802                           Chicago, IL 60611
m-barbolina@northwestern.edu               drrupesh@uiuc.edu                          m-laronda@northwestern.edu

Ivraym Barsoum                             Ivanna Ihnatovych                          Mary Laws
University of Illinois-Urbana Champaign    University of Illinois - Chicago           University of Illinois- Urbana Champaign
Veterinary Biosciences                     OB/GYN                                     Veterinary Biosciences
2001 South Lincoln Ave                     909 S. Wolcott, COMRB 1099                 2001 S. Lincoln Ave
Urbana, IL 61802                           Chicago, IL 60612                          Urbana, IL 61802
ijba73@gmail.com                           ivannai@uic.edu                            marylaws@northwestern.edu

Maria Bernhardt                            Vladimir Ilievski                          Lei Lei
Northwestern University                    Evanston Northwestern Hospital             Northwestern University
MED- Endocrinology                         OB/GYN                                     OB/GYN
303 E. Superior St                         2650 Ridge Avenue, SB640                   2205 Tech Drive
Chicago, IL 60611                          Evasnton, IL 60201                         Evanston, IL 60208-3520
m-bernhardt@northwestern.edu               Vilieski@enh.org                           lei-lei@northwestern.edu

Yanmei Chen                                Alison Kim
Northwestern University                    Northwestern University                    Chia-Feng Lui
OB/GYN                                     Chemical &Biological Engineering           University of Illinois- Urbana Champaign
303 E. Superior St                         2145 Sheridan Rd. Tech E136                Veterinary Biosciences
Chicago, IL 60611                          Evanston, IL 60208                         2001 South Lincoln Ave
Yanmei-chen@northwestern.edu               mkim@northwestern.edu                      Urbana, IL 61802
                                                                                      Cliu26@northwestern.edu
Melissa Cimafranca                         Bongki Kim
University of Illinois- Urbana Champaign   University of Illinois-Urbana Champaign    Benjamin Lucas
Veterinary Bioscience                      Veterinary Biosciences                     University of Illinois- Urbana Champaign
2001 South Lincoln Ave                     2001 South Lincoln Ave                     Veterinary Bioscience
Urbana, IL 61801                           Urbana, IL 61801                           2001 South Lincoln Ave
cimafran@uiuc.edu                          Bkim5@uiuc.edu                             Urbana, IL 61802
                                                                                      benlucas@uiuc.edu


                                                              54
                          28th Annual Minisymposium on Reproductive Biology
                                                   List of Presenters



Melissa Chamberlin McDevitt                Liz Simon                                  Michael VanGompel
Northwestern University                    University of Illinois- Urbana Champaign   OB/GYN
Neurobiology & Biology                     Veterinary Bioscience                      303 East Superior
2205 Tech Drive                            2001 S. Lincoln                            Chicago, IL 60611
Evanston, IL 60208                         Urbana, IL 61801                           g-van@northwestern.edu
m-chamberlin@northwestern.edu              lizsimon@northwestern.edu
                                                                                      Rebekah Weck
Kristin Meldi                              Aurora Shehu                               University of Illinois- Urbana Champaign
Northwestern University                    University of Illinois- Chicago            Veterinary Biosciences
Cell & Molecular Biology                   Physiology and Biophysics                  2001 South Lincoln Ave
2205 Tech Drive                            835 S. Wolcott Ave, M/C 901                Urbana, IL 61802
Evanston, IL 60208                         Chicago, IL 60612                          rweck2@uiuc.edu
k-meldi@northwestern.edu                   Ashehu1@uic.edu
                                                                                      Erin West
Maria Hernandez Ochoa                      Danielle Smith                             Northwestern University
University of Illinois-Urbana Champaign    Northwestern University                    Chemical and Biological Engineering
Veterinary Biosciences                     Medicine/Endocrinology                     2145 Sheridan Rd, Tech E136
2001 Lincoln Ave                           303 E Superior St, Lurie 7-220             Evanston, IL 60208
Urbana, IL 61802                           Chicago, IL 60611                          erwest@northwestern.edu
isabelho@uiuc.edu                          danielle@northwestern.edu
                                                                                      Anne Marie Wissman
Tessie Paulose                             Huanghui Tang                              Northwestern University
University of Illinois-Urbana Champaign    Northwestern University                    Neurobiology & Physiology
Veterinary Biosciences                     Cell & Molecular Biology                   2205 Tech Drive
2001 South Lincoln VMBSB                   2205 Tech Drive                            Evanston, IL 60208
Urbana, IL 61802                           Evanston, IL 60298                         a-wissman@northwestern.edu
tessie@uiuc.edu                            lh-tang2@northwestern.edu
                                                                                      Min Xu
Elizabeth Parrish                          Candace Tingen                             Northwestern University
Northwestern University                    Northwestern University                    OB/GYN
BMBCB                                      OB/GYN                                     2205 Tech Drive
2205 Tech Drive                            2205 Tech Drive                            Evanston, IL 60208
Evasnton, IL 60208                         Evanston, IL 60208                         m-xu3@northwestern.edu
e-parrish@northwestern.edu                 c-tingen@northwestern.edu
                                                                                      Zhen Zhao
Cyril Ramathal                             Daniel Trombly                             Northwestern University
University of Illinois- Urbana Champaign   Northwestern University                    Neurobiology & Physiology
Molecular and Integrative Physiology       Cell & Molecular Biology                   2205 Tech Drive
407 S. Goodwin Ave                         2205 Tech Drive                            Evanston, IL 60208
Urbana, IL 61801                           Evanston, IL 60208                         z-zhao@northwestern.edu
Cramat2@uiuc.edu                           d-trombley@northwestern.edu


Chirag Shah
Northwestern University
OB/GYN
303 E. Superior St
Chiacgo, IL 60611
c-shah@uiuc.edu




                                                              55
                                                       Organizing Committee for the
                                            28th Annual Minisymposium on Reproductive Biology:

Alison Kim                Monica Laronda             Kristin Meldi              Candace Tingen              Michael VanGompel         Anne Marie Wissman
Neurobiology &            MED-Endocrinology          Committee Chair            Neurobiology &              OB/GYN                    Neurobiology &
Physiology                Northwestern University    Biochemistry, Molecular    Physiology                  Northwestern University   Physiology
Northwestern University   303 E. Superior            Biology & Cell Biology     Northwestern University     303 E. Superior St        Northwestern University
2205 Tech Drive           Lurie Building 7-220       Northwestern University    2205 Tech Drive             Lurie Building 7-250      2205 Tech Drive
Hogan 4-160               Chicago, IL 60611          2205 Tech Drive            Hogan 4-160                 Chicago, IL 60611         Hogan 2-160
Evanston, IL 60208        312-503-2036               Hogan 4-110                Evanston, IL 60208          312-908-0842              Evanston, IL 60208
847-491-2511              m-                         Evanston, IL 60208         847-491-2511                g-van@northwesten.edu     847-491-3027
mkim@northwestern.edu     laronda@northwestern.edu   847-491-2872               c-tingen@northwestern.edu                             a-wissman@northwestern.edu
                                                     k-meldi@northwestern.edu




                           The Minisymposium on Reproductive Biology, held every year in October, is
                                                       hosted by:
                                                          Center for Reproductive Science
                                                                  Northwestern University
                                                              2205 Tech Drive (Hogan 2-171)
                                                              Evanston, IL 60208-3520, U.S.A.



                                                         Phone: 847-491-5767 Fax: 847-491-5211
                                                           www.northwestern.edu/research/crs/

                                                      Kelly Mayo, PhD                          Teresa K. Woodruff, PhD
                                                          Director                                Associate Director
                                                       847-491-8854                                 847-491-2666

                                     Susan Hall-Perdomo                         Ingrid Cox                          Liz Morris
                                       Center Manager                       Program Assistant                   Program Assistant
                                        847-491-4464                          847-467-5707                         847-491-5767

                                                            Special thanks to our sponsors:




                                                      Office of the President and Vice President for Research

				
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