Meinhardt et al._ 2000

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					46     PROCEEDINGS

Macrophage migration inhibitory factor (MIF) as a paracrine
mediator in the interaction of testicular somatic cells
A. Meinhardt', M. Bacher2, G. Wennemuth, R. Eickhoff and M. Hedger3
'Department of Anatomy and Cell Biology and 'Institute of Immunology, Philipps-University,Marburg, Germany; 31nstituteof
Reproduction and Development, Monash University, Clayton, Victoria, Australia

Key words. Compensatory production-Leydig      cell-MIF-Sertoli     cells-testis

Summary. Originally the macrophage migra-                         growth factors and cytokines also play a vital role
tion inhibitory factor (MIF) was described as a                   in the local regulation of testicular function
classical T-cell cytokine. Recently, a much broader               (Pescovitz et al., 1994; Schlatt et al., 1997).
tissue distribution for MIF has been revealed. We                    Macrophage migration inhibitory factor (MIF)
demonstrated that MIF protein and mRNA are                        was originally identified as a protein secreted by
present in the Leydig cells of the normal adult rat               activated T lymphocytes (David, 1966).In addition
testis. Addition of recombinant MIF to cultures of                to its T-cell-associated functions, MIF was found
rat seminiferous tubules resulted in decreased                    to be a pituitary hormone and a macrophage-
secretion of inhibin, whereas follistatin and activin             derived cytokine that counter-regulates the gluco-
levels remained unchanged, suggesting a para-                     corticoid-mediated suppression of the immune
crine role for MIF in Sertoli cell regulation.                    response (Donnelly & Bucala, 1997). Furthermore,
Furthermore, MIF showed unique compensatory                       MIF was found to be a key regulator of LPS-
production in the rat testis. Depletion of the                    induced sepsis. Antibody neutralization of MIF or
original MIF source, the Leydig cells, by the                     targeted disruption of the MIF gene resulted in
specific toxin EDS prompted MIF expression by                     substantially increased survival rates of animals
the previously negative Sertoli cells. Leydig cell                treated with otherwise lethal doses of lipopolysac-
re-population of the interstitial tissue by precursor             caride (Bernhagen et al., 1993; Bozza et al., 1999).
cells resulted in a switch back to production by                  In the last few years, various additional properties
Leydig cells. Therefore, testicular MIF appears to                of MIF and a much broader tissue distribution
be under very tight paracrine control. MIF has                    have been revealed (Bacher et al., 1997; Nishihira,
thus been identified as a new mediator in the                     1998). MIF can function as an enzyme exhibiting
cross-talk between Leydig cells and the somatic                   tautomerase (Rosengren et al., 1997) and probably
cells of the seminiferous tubules of the rat testis.              thiol-protein oxidoreductase activity (Kleemann
                                                                  etal., 1998). This suggests that MIF has much
                                                                  broader roles beyond its function as a classical
Introduction                                                      cytokine.

The testis has to fulfil two major functions: the
generation of gametes and the production and                      MIF expression in the rat testis
controlled release of sex steroids. Spermatogenesis
occurs in the seminiferous tubules and steroido-                  Using immunohistochemistry, MIF was localized
genesis occurs in the Leydig cells of the interstit-              to the intertubular regions of the rat testis, and
ium. In order to coordinate these diverse functions,              specifically to the Leydig cells. Many cells with an
the hormone and gamete-producing compart-                         indented nucleus and morphological character-
ments of the testis need to communicate. Although                 istics of monocytes or macrophages were observed
the pituitary gonadotropins luteinizing hormone                   to be unlabelled. In addition, the seminiferous
(LH) and follicle-stimulating hormone (FSH), as                   epithelium was negative for MIF staining, except
well as testosterone, are the primary stimuli for                 for a very weak reaction around the heads of
the initiation and maintenance of testicular func-                elongated spermatids (Meinhardt et al., 1996). The
tion, there is substantial evidence that polypeptide              theory of a Leydig cell origin of MIF in the testis
                                                                  was supported by the fact that lysates of the Leydig
                                                                  cell line TM3 contained around 7 pg pg-' total
Correspondence: Andreas Meinhardt, Department of                  protein. These data are consistent with recent
Anatomy and Cell Biology, Philipps University of Marburg,
Robert-Koch-Str. 6, D-35037 Marburg, Germany. Tel.:
                                                                  findings that macrophages can display variable
49 6421 2864021; Fax: 49 6421 2865783; e-mail:                    degrees of immunoreactivity for MIF, depending
meinhar                                   on their tissue source and relative state of cellular

                                                                                              ANDROLOGIA 32,41-64 (2000)
activation (Bacher et al., 1997). It would therefore       pared with the gonadotropin-deficient rat does not
appear that in the testes somatic cells rather than        appear to be consistent with a role for Leydig cell-
resident tissue macrophages are the major source           derived MIF in maintaining the testicular macro-
of MIE'. In addition, MIF was found in normal              phage population, unless there are other potential
rat interstitial testicular fluid at a concentration of    sources of MIF in the testis. We therefore felt it
 14.7 f3.6 ng ml- Immunoblotting analysis with             was necessary to investigate testicular MIF mRNA
an anti-MIF antibody revealed a single band at             and protein levels and the cellular localization of
M,= 12 kDa in rat testis extract and in isolated           MIF in adult male rats after EDS treatment.
interstitial fluid, consistent with the relative              It was found that complete removal of the
mobility of rMIF (Meinhardt et al., 1996).                 Leydig cells, which had previously been identified
   A rat MIF cDNA probe was used to analyse                as the testicular source of MIF, had only a minor
mRNA expression in whole rat testes. MIF mRNA              impact on MIF protein and mRNA levels in the
was expressed as a single 0.6-kb species. There            adult rat testis. Immunohistochemical and Western
was no evidence for differential splicing in this          blot data established that this persistence of MIF
organ. To assess the potential role of circulat-           expression was due to a switch in the production
ing gonadotropins in testicular MIF mRNA                   of MIF protein from Leydig cells to the seminifer-
expression, we also examined testes obtained from          ous tubules, and in particular to the Sertoli cells
hypophysectomized rats. Hypophysectomy reduces             (Meinhardt et al., 1999), which previously had not
circulating gonadotropins levels and produces              been a detectable source of MIF (Meinhardt et al.,
Leydig cell atrophy. However, no difference in the         1996). Western blot and Northern blot analysis of
relative level of mRNA expression was observed             testicular samples, including the seminiferous
between the control and the hypophysectomized              tubule fraction collected from rats 7 days post-
animals. Also, at the protein level, immunoreactive        EDS treatment, confirmed the continuing synthesis
MIF was present at almost unchanged levels within          of authentic 12-kDa MIF in these Leydig
the atrophied Leydig cells of the hypophysectom-           cell-deficient animals. The up-regulated MIF
ized rats when compared to normal testes                   expression in the seminiferous tubule compartment
(Meinhardt et al., 1996). These data indicate that         of the testis declined with regeneration of a new
the expression of MIF mRNA and protein in the              population of MIF-positive Leydig cells from mes-
testis is not dependent upon gonadotropin support.
                                                           enchymal precursors at day 14 post-EDS appli-
This suggests that Leydig cells, regardless of their
                                                           cation. This latter observation suggests that MIF
functional status, are capable of producing MIF.
                                                           may be a very early product during Leydig cell
                                                           differentiation, and that MIF might be useful as a
Compensatov production of MIF                              marker for Leydig cells from the very earliest
                                                           stages of their development. These data establish
The macrophage-regulatory activities of MIF
during inflammation implicate MIF as a putative            that Leydig cell-depleted testes continue to pro-
regulator of macrophage numbers in the testis.             duce MIF and suggest the existence of a previously
Numerous studies have established that macro-              unknown mechanism of compensatory cytokine
phage numbers in the testis are maintained at a            production involving the Sertoli cells.
high level by the local regulatory influence of the           The production of MIF by a somatic cell type
adult Leydig cells (Hedger, 1997; Hutson, 1998).           in the normal testis, the Leydig cell, raises the
It has been shown that in the testes of rats which         question of whether MIF functions as a local,
have been depleted of Leydig cells by treatment            physiological regulator of cells that are in contact
with the specific Leydig cell-tolbn ethane dimeth-         with interstitial fluid. MIF could exert an autocrine
ane sulphonate (EDS), macrophage numbers                   effect on Leydig cells, or a paracrine effect on the
progressively decline below normal levels, only            seminiferous tubules. Exogenously added rMIF
returning to normal following the recovery of the          showed a dose-dependent suppression of inhibin
Leydig cell population several weeks later (Wang           production by isolated seminiferous tubules,
et al., 1994). However, approximately 50% of the           whereas activin and follistatin levels remained
normal macrophage population persists even in              unchanged (Meinhardt et al., 1996). These data
long-term Leydig cell-depleted rats. In contrast,          suggest that MIF has a specific effect on Sertoli
the testicular macrophage population declines to           cells, as these cells are the exclusive site of inhibin
around 30% of normal 3-1       hyophysectomized rats       production in the adult seminiferous epithelium
(Gaytan et al., 1994). Since Leydig cells are the          (Simpson et al., 1992). In contrast, no influence of
only si<gnificantsource of MIF in the normal adult         MIF on basal or LH-stimulated testosterone pro-
testis, the relative degree of persistence of testicular   duction of cultured rat Leydig cells was measurable
macrophages in the Leydig cell-depleted rat com-           (Meinhardt et al., 1996).

ANDROLOGIA 32, 41-64 (2000)

Acknowledgements                                                  Kleemann R, Kapurniotu A, Frank RW, Gessner A, Mischke
                                                                    R, Flieger 0,Jiittner S, Brunner H, Bernhagen J (1998)
                                                                    Disulfide analysis reveals a role of macrophage migration
The authors are supported by grants from the                        inhibitory factor (MIF) as thiol-protein oxidoreductase. J
Deutsche Forschungsgemeinschaft (Me 1323/2- 1)                      Mol Biol 280:85- 102.
and from the Fonds der Chemischen Industrie.                      Meinhardt A, Bacher M, McFarlaneJR, Metz CN, Seitz J,
                                                                    Hedger MP, de Kretser DM, Bucala R (1996) The macro-
                                                                    phage migration inhibitory factor (MIF) production by
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Inhibin and activin subunits and spermatogenesis
P. Tuohimaa, Y. -H. Zhuang, T. Ylikomi, H. Syvda and M. Blauer
Department of Anatomy, Medical School, University of Tampere, Tampere, Finland

Key words. Activin-inhibin-paracrine-testis-vitamin           A

Introduction                                                      (Mason et al., 1985). The precursor sequence (366
                                                                  amino acids) of the a-subunit is post-translationally
Inhibins and activins are TGF-P-related growth                    cleaved and glycosylated (Robertson et al., 1995).
and differentiation factors. Structurally, inhibins               The precursors of the PA- and PB-subunits are
(32 kDa) are heterodimers composed of an a-                       426 and 407 amino acids long, giving rise to
subunit (20kDa) and one of two related 14-kDa                     mature subunits of 1 16 and 115 amino acids,
P-subunits (PA and PB), while activins (24 kDa)                   respectively. Inhibins are known to inhibit FSH
are homodimers or heterodimers of two P-subunits                  secretion (Ling et aL, 1985), whereas activins
                                                                  induce FSH (Mason et al., 1989). In addition to
                                                                  dimeric hormones, monomeric subunits and pre-
Correspondence: Prof. Dr Pentti Tuohimaa, University of
Tampere, Department of Biomedical Sciences, PO Box 607,
                                                                  cursors are also known to have biological activity
FIN-33101 Tampere, Finland. Tel: 358 31 215 6726; Fax:            (Robertson et al., 1995). Recently, three new P-
358 3 1 2 15 6 170; e-mail:                subunits, PC, PD and PE, were cloned (Hotten

                                                                                                  ANDROLOGIA 32, 41-64 (2000)

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