Molecular Brain Research 142 (2005) 39 – 46 www.elsevier.com/locate/molbrainres
Neural steroid hormone receptor gene expression in pregnant rats
Phyllis E. Mann*, Jessica A. Babb
Department of Biomedical Sciences, Tufts University, Cummings School of Veterinary Medicine, N. Grafton, MA 01536, USA Accepted 5 September 2005 Available online 25 October 2005
Abstract Estrogen and progesterone play important roles during pregnancy in stimulating the onset of maternal behavior at parturition. The status of receptor expression of these hormones during pregnancy in neural regions that regulate maternal behavior is unclear. The objective of the present study is to characterize changes in neural gene expression of the estrogen receptors a and h (ERa and ERh) and the progesterone receptor (PR) during the latter part of pregnancy. Brains from primigravid Sprague – Dawley rats were collected on days 15 and 21 of pregnancy. Micropunches of the olfactory bulb (OB), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BnST), hypothalamus (HYP), medial amygdala (MeA), and the temporal cortex (TCx) were analyzed by real-time RT-PCR (Taqmani) for levels of gene expression. No changes in either ERa or ERh mRNA levels were detected in any brain region between days 15 and 21 of pregnancy: however, the MPOA had higher levels of both ERa and ERh than other brain regions. Progesterone receptor mRNA levels, in contrast, declined significantly in the MPOA, HYP, and TCx, between days 15 and 21 of pregnancy (P < 0.05). In addition, the levels of PR mRNA were significantly higher in the HYP and TCx compared to both the OB and MeA. These data indicate that there is a downregulation of PR prepartum and suggest that this decrease may play a role in the disinhibition of maternal behavior at parturition. D 2005 Elsevier B.V. All rights reserved.
Theme: Endocrine and autonomic regulation Topic: Neuroendocrine regulation: other Keywords: Taqman; Maternal behavior; MPOA; Progesterone receptor, estrogen receptor a, estrogen receptor h, hypothalamus, temporal cortex, olfactory bulb, medial amygdala; Bed nucleus of the stria terminalis; Gestation
1. Introduction Female mammals display a wide variety of behaviors in order to nurture their offspring. First-time pregnant (primigravid) rats will begin displaying maternal behavior only during the periparturitional period. The changes in the endocrine system during pregnancy are thought to underlie the display of maternal behavior at or around parturition [5,46,53]. The steroid hormones, estrogen (E2) and progesterone (P4), and the lactogenic hormones, prolactin and the placental lactogens, are involved in the onset of maternal behavior at parturition in the rat. E2 steadily increases during
* Corresponding author. Fax: +1 508 839 7091. E-mail address: Phyllis.Mann@Tufts.edu (P.E. Mann). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.09.001
pregnancy through parturition. P4, on the other hand, rapidly increases following mating and reaches two relative plateaus during gestation, one on days 7 to 10 and another between days 15 and 20 of pregnancy. Near term, P4 levels sharply decline, allowing parturition to occur and maternal behavior to be displayed. At the end of pregnancy, there is a shift in the ratio of E2 to P4, with P4 declining and E2 increasing which allows for a dramatic increase in serum prolactin at parturition. Together, these hormones are responsible for the immediate onset of maternal behavior at parturition . While maternal behavior is displayed immediately at parturition, maternal behavior does not occur spontaneously in virgin rats. However, if virgin females are housed with young pups for several days, they can be induced or ‘‘sensitized’’ to show maternal behavior with an average latency of 5 – 7 days [17,69]. Paradoxically,
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spontaneous maternal behavior does occur in both female and male 20– 24-day-old juvenile rats [11,53] and appears to be ‘‘hard-wired’’, and not dependent on the endocrine system [8,85]. The reason that juvenile maternal behavior disappears and adult virgin rats are not spontaneously maternal is due to the existence of a neural circuit that actively inhibits the display of maternal behavior. Early studies have shown that rendering the virgin female anosmic shortens the latencies to become maternal to 2– 3 days [18,19]. Further studies demonstrated that both the main and accessory (vomeronasal) olfactory systems are involved in the inhibition of maternal behavior , as well as the medial amygdala (MeA) and bed nucleus of the stria terminalis (BnST) [21,60,73,74]). Additional evidence indicates that the hypothalamus (HYP) also contributes to the maternal behavior inhibitory neural circuit [10,45,74]. Lesioning the anterior area of the HYP, and the dorsomedial (DMH) and ventromedial (VMH) nuclei of the HYP decrease the latency to display maternal behavior in steroid-primed, ovariectomized, virgin rats and in first-time pregnant rats [10,45]. The areas mentioned above are key for the inhibition of maternal behavior. Other regions of the CNS, such as the medial preoptic area (MPOA), are fundamental for the original onset and ongoing display of maternal behavior . Stimulation of the MPOA through kindling facilitates short-latency maternal behavior in virgin rats , while MPOA administration of the antiestrogen, 4hydroxytamoxifen, delays the onset of the behavior in postpartum, cesarean-delivered females . Estradiol administration to the MPOA induces short-latency maternal behavior in ovariectomized and hysterectomized pregnant rats  and facilitates retrieval behavior in ovariectomized virgin rats . Lesioning the MPOA either by using electrolytic stimulation or neurotoxins, disrupts postpartum maternal behavior [50,59]. Furthermore, knife cuts which functionally isolate the MPOA on the lateral aspect disrupt maternal behavior [50,52, 55,56,58]. Studies using a marker for neuronal activation, the Fos protein, have demonstrated an increase in Fos labeling in the MPOA of maternal animals (e.g., ). These findings support the concept that the MPOA plays a crucial role in both the onset and maintenance of maternal behavior in the female rat. Based on the abovementioned studies, specific areas of the brain were examined in the present study due to their involvement in either the inhibition or display of maternal behavior. Even though the anatomy of the inhibitory neural circuit has been partially revealed, little is known about the changes in the circuit’s hormone receptors in virgin and first-time pregnant rats. The objective of the present study was to determine if steroid hormone receptor gene expression changes between mid- and late-pregnancy when the female rat changes from long-latency to short-latency maternal behavior, respectively. Hormone receptor mRNA was quantified using real-time PCR for
the following hormone receptors at mid- and latepregnancy: estrogen receptor a (ERa), estrogen receptor h (ERh), and the progesterone receptor (PR).
2. Material and methods 2.1. Animals Nulliparous female Sprague – Dawley rats weighing 225– 250 g were obtained from Charles River Breeding Laboratories, Kingston, NY. Animals were housed in light- (on 0500– 1900 h) and temperature-controlled (21 – 24 -C) rooms and were provided food and water ad libitum. One week after the experimental animals arrived, they were housed with stud males. The day that sperm were present in the vaginal lavage was considered day 1 of pregnancy. The animals were then individually housed in 45 Â 25 Â 20 cm opaque polypropylene cages. All animals were maintained in accordance with the guidelines of the Division of Teaching and Research Resources at Tufts University, Cummings School of Veterinary Medicine, which are based on the Committee on Care and Use of Laboratory Animal Resources, National Research Council. 2.2. Tissue collection and preparation On days 15 and 21 of gestation (between 0900 and 1200 h), rats were briefly anesthetized with CO2 and brains were removed rapidly and flash-frozen in powdered dry ice under RNase-free conditions. Brains were then kept at À80 -C until micropunched in a cryostat (À20 -C) . Micropunches were collected from the anterior portion of the olfactory bulb (OB, å2 mm wide by 2 mm deep), BnST (1 mm in diameter by 1 mm deep, from åÀ0.26 to åÀ1.26, just lateral and dorsal to the anterior commissure), MPOA (1.5 mm in diameter by 1 mm deep, from åÀ0.26 to åÀ1.26 just ventral to the anterior commissure and on either side of the third ventricle), MeA (1 mm in diameter by 1.5 mm deep, from åÀ1.80 to åÀ2.80, just lateral and dorsal to the supraoptic nucleus), HYP (2 mm in diameter by 2 mm deep, from åÀ1.80 to åÀ3.80 on either side of the third ventricle), and temporal cortex (TCx, 1 mm in diameter by 1.5 mm deep). Coordinates are based on Bregma . Samples were immediately placed into 500 Al of 1Â lysis buffer (Applied Biosystems, Foster City, CA), stored at À20 -C and shipped on dry ice to AppliedGeneX (Davis, CA) for gene expression analysis. The tissue was homogenized with proteinase K and two grinding beads 4 mm in diameter (SpexCertiprep, Metuchen, NJ) using a GenoGrinder2000 (SpexCertiprep) for 2 min at 1000 strokes per minute. Protein digests were done at 56 -C for 30 min followed by a 30 min period at À20 -C to reduce foam. Total RNA was extracted using a 6700
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automated nucleic acid (ANA) workstation (Applied Biosystems). 2.3. Reverse transcriptase reaction Complementary DNA (cDNA) was synthesized using 100 units of SuperScript III (Life Technologies), 600 ng random hexadeoxyribonucleotide (pd(N)6) primers (random hexamer primer), 10 U RNaseOut (RNase inhibitor), and 1 mM dNTPs (all Invitrogen, Carlsbad, CA) in a final volume of 40 Al. The reverse transcription reaction proceeded for 120 min at 50 -C. After addition of 60 Al of water, the reaction was terminated by heating for 5 min at 95 -C and then cooling on ice. 2.4. Real-time PCR reaction Each PCR reaction contained 20Â Assay-on-Demand primer and probes for the respective TaqMan system and commercially available PCR mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems) containing 10 mM Tris –HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5 mM deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction, and 5 Al of the diluted cDNA sample in a final volume of 12 Al. The samples were placed in 96 well plates and amplified in an automated fluorometer (ABI PRISM 7700 Sequence Detection System, Applied Biosystems). Applied Biosystem’s standard amplification conditions were used: 2 min at 50 -C, 10 min at 95 -C, 40 cycles of 15 s at 95 -C, and 60 s at 60 -C. Fluorescent signals were collected during the annealing temperature and cycle threshold (CT) values extracted using a threshold of 0.04 and baseline values of 3 –15. The following gene accession numbers were used for the probes: ERa-NP _ 036821; ERh-NP _ 036886; PRNP_084038. The PR probe was not specific for either the ‘‘A’’ or ‘‘B’’ isoform. 2.5. Housekeeping gene validation experiment In order to determine the most stably transcribed housekeeping gene, a validation experiment was run on a representative number of samples. Three common housekeeping genes were used for this experiment: rat 18S rRNA (ssrRNA), rat glyseraldehyde-3-phosphate dehydrogenase (GAPDH), and rat hypoxanthine guanine phosphoribosyl transferase (HPRT1). Rat HPRT1 was used for subsequent data analysis. 2.6. Relative quantitation of gene transcription Final quantitation was done using the comparative CT method , which determines relative changes in gene expression based on fluorescence emission. In brief, the housekeeping gene for rat brain tissue, HPRT1, was used to
normalize the target raw signals (DCT). The DCT was then calibrated against the weakest signal of each target gene (calibrator). The linear amount of target molecules relative to the calibrator, was calculated by 2ÀDDCt. Therefore, all gene transcriptions are expressed as an n-fold difference relative to the calibrator. 2.7. Data analysis Linearized values were used for data analysis between the groups. Within each gene, two-way factorial ANOVAs were used (day of pregnancy [2 levels] Â region [6 levels]). When data were not normally distributed, Kruskal – Wallis One-Way Analysis of Variance on Ranks was used. Post hoc tests were performed on the parametric data using Fisher’s LSD and on the nonparametric data using Dunn’s Method. Differences were considered significant when P < 0.05.
3. Results 3.1. Housekeeping gene Analysis of the housekeeping gene, HPRT1, revealed a significant main effect of region (F 5,46 = 28.155, P < 0.001), but no main effect of day of pregnancy (F 1,46 = 0.480, P = 0.493) nor a significant interaction (F 5,46 = 1.836, P = 0.131). Therefore, ERa, ERh, and PR mRNA levels were adjusted proportionally based on the HPRT1 values. 3.2. Estrogen receptors a and b All brain areas examined demonstrated ERa and ERh mRNA (Fig. 1). For ERa, there was a significant main effect of region (F 5,46 = 11.634, P < 0.001), but no effect of day of pregnancy (F 1,46 = 0.0243, P = 0.877) nor a significant interaction (F 5,46 = 0.304, P = 0.907). The data were, therefore, collapsed across day of pregnancy and analyzed using a one-way ANOVA. Fisher’s LSD revealed that the MPOA had significantly higher ERa mRNA levels than all other regions examined (Fig. 1A; Ps all <0.001). In addition, the BnST had significantly higher ERa mRNA levels than the OB (P = 0.004) and TCx (P < 0.003). Similar results were obtained for ERh (Fig. 1B). There was a significant main effect of region (F 5,46 = 4.856, P = 0.002), but no effect of day of pregnancy (F 1,46 = 0.385, P = 0.540) or a significant interaction (F 5,46 = 0.280, P = 0.921). Fisher’s LSD on the collapsed ERh data demonstrated significantly higher levels in the MPOA compared to the OB (P = 0.017), BnST (P = 0.005), MeA (P < 0.001), and TCx (P < 0.001). In addition, the HYP had significantly higher ERh mRNA levels than the MeA (P = 0.021) and TCx (P < 0.004). The OB also had significantly higher levels than the TCx (P = 0.034).
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Fig. 1. Relative mRNA values of ERa (A) and ERh (B) in the brain during pregnancy. Gestation days 15 and 21 data were combined. OB—olfactory bulb; MPOA—medial preoptic area; MeA—medial amygdala; HYP— hypothalamus; TCx—temporal cortex. N = 6 – 8 per region. aSignificantly different from the MPOA (P < 0.05). bSignificantly different from the BnST (P < 0.05). cSignificantly different from the HYP (P < 0.05). d Significantly different from the TCx (P < 0.05).
3.3. Progesterone receptor Progesterone receptor mRNA was found in all areas of the brain examined. The BnST was not used for PR analyses because of small sample sizes. Analysis using a two-way factorial ANOVA revealed significant main effects of both day of pregnancy (F 1,38 = 23.798, P < 0.001) and region (F 4,38 = 3.649, P = 0.016), but no significant interaction of day and region (F 4,38 = 1.288, P = 0.298). Even though a significant interaction was not present, simple effects were tested using Fisher’s LSD . Gene expression for PR was significantly higher on day 15 of gestation in the MPOA (P = 0.03), HYP (P = 0.02), and TCx (P < 0.001) compared to day 21 (Fig. 2). Analyzed by region, PR gene expression was significantly higher in the HYP compared to both the OB (P = 0.01) and MeA (P = 0.014) on day 15 of gestation. In addition, PR gene expression was higher in the TCx compared to both the OB (P = 0.005) and MeA (P = 0.008). There were no differences between brain regions on day 21 of gestation.
83,86,88]. We also demonstrate that there were no changes in either ERa or ERh on day 15 of gestation compared to day 21 in any area of the brain examined. However, the levels of PR mRNA were found to be lower on day 21 of gestation compared to day 15. The role of estrogen in the hormonal regulation of maternal responsiveness in virgin and pregnant rats is wellestablished . Early studies using a hysterectomizedovariectomized (HO) model demonstrated that virgin rats respond maternally 0 – 2 days after HO and systemic estradiol benzoate treatment [79,80,82] as compared to 5– 8 days in untreated virgins . Other studies have supported a role for estradiol in the stimulation of maternal behavior after systemic administration in virgin [4,14,48] and pregnant rats [70 – 72,81]. Moreover, implants of estradiol directly into the MPOA stimulate short-latency maternal behavior in both virgin  and pregnant  rats. The mechanism of action of estradiol in the MPOA in stimulating maternal behavior is unknown. However, evidence from autoradiography, immunocytochemistry, and in situ hybridization histochemistry (ISHH) indicates that estrogen receptors are most likely involved. In pregnancy-terminated rats, levels of nuclear estrogen receptors rise in concert with short-latency maternal behavior . In intact pregnant rats, Giordano et al. [25,26] reported increases in nuclear estrogen receptor concentrations in the MPOA between days 8– 10 and day 16 of gestation, but found no difference between day 16 and day 22. In the present study, both ERa and ERh were examined, although similar results were found. Wagner and Morrell  examined estrogen receptor mRNA levels using ISHH in areas of the brain relevant to maternal behavior. They found a decrease in the number of silver grains per cell in the rostral medial preoptic nucleus between days 8 and 16 of gestation, but no difference between days 16 and 22. The number of labeled cells, however, increased
4. Discussion The present study characterizes levels of gene expression for ERa, ERh, and PR in the brain in primigravid rats. Confirming previous studies, we found ERa, ERh, and PR gene expression in the OB, MPOA, BnST (ER only), MeA, HYP, and cortex (in general) [29,35,37,39,44,63,75,77,78,
Fig. 2. Relative mRNA values of PR gene expression in various areas of the brain on days 15 and 21 of gestation. OB—olfactory bulb; MPOA—medial preoptic area; MeA—medial amygdala; HYP—hypothalamus; TCx— temporal cortex. N = 3 – 4 per region, per day of pregnancy. *Significantly different compared to the day 15 group (P < 0.05). cSignificantly different from the HYP (P < 0.05). dSignificantly different from the TCx (P < 0.05).
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between days 16 and 22 of gestation. Due to methodological reasons, it is difficult to compare the above results to the present study since the ER isoforms were not examined separately in that study and a quantitative technique, realtime PCR, was used in the present study. In addition, the present study used micropunches of particular regions, which do not have the same ‘‘spatial resolution’’ as studies which use ISHH. When ER protein was examined, again it was found that the number of cells expressing high levels of ER immunoreactivity was higher on days 16 and 22 of pregnancy compared to day 8 in the medial preoptic nucleus, but no change between days 16 and 22 . When ERa and ERh are examined separately, studies have shown that ERa is more likely to be involved in maternal behavior [13,42,62]. Maternal behavior is disrupted in ERa knockout mice  and a significant number of cells that are activated in response to pups in postpartum lactating females are also ERa-immunoreactive . Estrogen receptor h, on the other hand, while distributed in the forebrain and limbic regions and having an overlapping distribution with ERa [28,76,78], has not been shown to be involved in maternal behavior as yet. During pregnancy, however, the levels of ERh mRNA in the periventricular preoptic area are higher on day 22 of gestation compared to proestrus and day 10 of lactation , hinting at a possible relationship. Progesterone has two roles in the stimulation of maternal behavior. First, to prime the female to respond maternally at parturition by facilitating the effects of estrogen, and second, to regulate the timing of maternal behavior onset at parturition [5,53]. Even though progesterone exposure prior to estradiol treatment stimulates a faster onset of maternal behavior than with estrogen-priming alone , progesterone, itself, inhibits maternal behavior in rats [6,9,51,61]. If progesterone levels are artificially elevated at the end of pregnancy, the display of maternal behavior by postpartum females is prevented [6,9,51,81]. In addition, if progesterone is administered during maternal behavior testing in estrogen-primed, ovariectomized, virgin rats, the rapid onset of maternal behavior will not occur [79,80]. The PR appears to mediate the inhibitory actions of progesterone on maternal behavior in pregnant rats. The systemic administration of mifepristone, an intracellular PR antagonist, blocks the inhibitory actions of elevated progesterone levels on maternal behavior in pregnancy-terminated rats . Although the regulation of PR across the estrous cycle and in response to ovarian steroids has been well-studied (e.g., [36,67,78,84]), there are only two studies examining PR expression during pregnancy [23,61]. Francis et al.  report an increase in MPOA PR immunoreactivity in latepregnancy as compared to proestrous controls; however, there were no further time points reported during pregnancy. Numan et al.  characterized PR immunoreactivity at various time points during pregnancy in areas of the brain considered relevant to maternal behavior. They found an increase in PR immunoreactivity in the anteroventral
periventricular nucleus of the preoptic region when days 15 and 21 of gestation were compared. In all other areas examined (e.g., MPOA), there were no changes in PR immunoreactivity between days 15 and 21 of gestation. The fact that PR immunoreactivity did not decrease on day 21 in any brain region as did PR mRNA in the present study may be explained by the different techniques used and possibly the time course of progesterone release during pregnancy. Progesterone levels fall precipitously at the end of pregnancy and it is possible that this decline downregulates its own receptor, which is not yet evident from immunocytochemistry studies. In fact, Numan et al.  found a sharp decline in PR immunoreactivity in all areas of the brain examined on day 3 postpartum. Even though PRs are found throughout the brain , there are specific neural regions that subserve reproductive and maternal behaviors (e.g., MPOA, mediobasal hypothalamus; [54,66]) where the PR is induced by estradiol [43,78]. In terms of the present study, the fact that there is a reduction in PR mRNA in both estrogen sensitive (e.g., MPOA) and estrogen insensitive (e.g., cortex) areas of the brain on gestation day 21, suggests to us that the rise in estrogen that normally occurs in late-pregnancy is probably not involved in the decrease in PR mRNA. It is possible that both the high levels of circulating progesterone during most of pregnancy and that progesterone concentrations are in the process of declining prepartum downregulate PR expression [2,3,12]. The areas examined in the present study were chosen based on their known involvement in maternal behavior . The MPOA is a key area involved in the stimulation of maternal behavior. The changes in MPOA ERa, ERh, and PR were discussed above. The OB and MeA, on the other hand, appear to be involved in a neural circuit that inhibits maternal behavior in virgin rats  and rabbits . Early studies in rats demonstrated that rendering the virgin female anosmic shortens the latencies to become maternal to 2– 3 days [18,19] and later studies indicate that this phenomenon is regulated by the OB, MeA, and HYP [7,10,20 – 22,45,60,73]. In the present study, no changes were found in ERa and ERh gene expression in the OB during mid- to late-pregnancy, although there are reports that indicate that ERa and ERh mRNA as well as PR in the OB are sensitive to changes in estradiol levels [33,75]. The fact that there were no changes in ERa in the MeA during pregnancy was not surprising in light of a previous report that indicates that there are no changes in ER immunoreactivity on day 16 of gestation as compared to day 22 . In addition, MeA ERa gene expression is not altered by estradiol treatment . However, ERh gene expression in the MeA is reduced by estradiol treatment  although no change in ERh gene expression was observed at the end of pregnancy in the present study. In contrast to the ER, the present study demonstrated a decrease in PR gene expression in the MeA from mid- to late-pregnancy. However, a previous study reported no change in PR immunoreactivity from mid- to
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late-pregnancy . When the ER isoforms are examined separately, gene expression for ERh is not altered when comparing proestrous, pregnancy (day 22), and lactating rats . No studies have examined the changes in ERa, ERh, and PR gene expression in the cortex across pregnancy. In mice, both estradiol and exposure to pups increases ER immunoreactivity in the entorhinal and piriform cortex , while neither estradiol nor progesterone affects PR protein levels in the frontal cortex . Gene expression for the combined PR A and B isoforms does change across the estrous cycle with both isoforms highest on diestrus . These results indicate that PR expression in the cortex may be affected by pregnancy as the present study confirms. Several studies have compared steroid hormone gene expression between different regions of the rat brain by using ISHH [35,40,75,77]. In those studies, differences between regions are usually descriptive (e.g., ‘‘strong’’ vs. ‘‘moderate’’ vs. ‘‘weak’’ ) or semi-quantitative (e.g., using greater or fewer plus signs ). By utilizing a quantitative technique, real-time PCR, we are able to directly compare mRNA levels between regions of the brain during pregnancy in the present study, although the same spatial resolution does not exist. Since none of the abovementioned studies used the pregnancy model, a comparison of the present data is not helpful. However, Giordano et al. , using a nuclear estrogen receptor assay, found higher ER levels in the POA than in the HYP on days 16 and 22 of pregnancy, which agrees with the present data. There are no studies, to our knowledge, comparing PR mRNA levels in different areas of the brain across pregnancy. In summary, there were no changes in either ERa or ERh gene expression in any of the brain areas examined; however, levels of PR mRNA were lower at the end of pregnancy compared to levels at midgestation. The reduction in PR gene expression at the end of pregnancy may underlie the ‘‘release’’ or disinhibition of maternal behavior at parturition.
Acknowledgments We thank Eric Engelhard and Christian M. Leutenegger, AppliedGeneX, LLC, for expert real-time TaqMan PCR analysis. This project was supported by a grant to PEM (RO1 HD39668).
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