Expression of Sox9 in granulosa cells lacking the estrogen receptors ...

DEVELOPMENTAL DYNAMICS 226:103–106, 2003

BRIEF COMMUNICATION

Expression of Sox9 in Granulosa Cells Lacking the Estrogen Receptors, ER␣ and ER␤ Sonia Dupont, Christine Dennefeld, Andre ´ e Krust, Pierre Chambon, and Manuel Mark*

Ovaries from adult mice lacking both estrogen receptors ER␣ and ER␤ (ER␣␤KO mice) contain abnormal cells sharing morphologic features with Sertoli cells, which are located mainly in the interstitial compartment. We show here that these cells express the Sertoli cell markers TIF1␤, TIF2, and Sox9. In ER␣␤KO ovaries, Sox9 is expressed by granulosa cells before the morphologic appearance of Sertoli cells, but neither by granulosa cell precursors nor by non-Sertolian interstitial cells. These findings suggest that functional Sertoli cells can transdifferentiate from mature granulosa cells devoid of estrogen receptors as a result of Sox9 expression. Developmental Dynamics 226:103–106, 2003. © 2002 Wiley-Liss, Inc. Key words: estrogen receptors; ovary; testis; granulosa cells; Sertoli cells; sex reversal; transdifferentiation; Sox9; mouse Received 5 August 2002; Accepted 9 September 2002

INTRODUCTION Estrogen binding to the nuclear receptors ER␣ (Green et al., 1986) and ER␤ (Kuiper et al., 1996; Mosselman et al., 1996; Tremblay et al., 1997) regulate reproductive functions, in particular ovarian physiology (reviewed in Couse and Korach, 1999). Both receptors are expressed in adult and immature ovary, the ER␣ protein being detected in interstitial and theca cells and the ER␤ protein in granulosa cells (Fitzpatrick et al., 1999; Hiroi et al., 1999; O’Brien et al., 1999; Sar and Welsch, 1999). Analysis of single and compound mutant mice for ER␣ and ER␤ has provided evidence that each receptor is required for specific ovarian functions, namely follicular development (ER␤) and ovulation (ER␣) (Dupont et al., 2000). Additionally, it was found that

abnormal cells, characterized as “Sertoli-like” on the basis of histologic and ultrastructural criteria, differentiate within ovaries lacking both estrogen receptors (ER␣␤KO ovaries; Couse et al., 1999; Dupont et al., 2000). Although showing very different morphologies, Sertoli cells and granulosa cells are difficult to distinguish from one another by molecular marker analysis. Indeed, because both cell types have a common embryologic origin from the genital ridge (reviewed in Swain and LovellBadge, 1999) and are similar with respect to their functions of nurse cells for the germ cells and to their main regulation by the gonadotropin FSH, they express similar sets of genes, e.g., genes encoding AMH (Mu¨nsterberg and Lovell-Badge, 1991; Hi-

robe et al., 1992, and references therein), inhibin/activin (Griswold, 1993; Richards, 1994), c-kit ligand (Manova et al., 1993; Richards, 1994; Vincent et al., 1998), aromatase (Richards, 1994; Levallet et al., 1998), and SGP-2 (Ahuja et al., 1994; Aronow et al., 1993). Sox9 is one of the very few genes whose expression profile allows to unambiguously distinguish Sertoli and their precursors from granulosa cells: Sox9 is an early and permanent marker of the Sertoli cell lineage, as it starts to be expressed in the male genital ridge and is subsequently strongly expressed in fetal, postnatal, and adult Sertoli cells (Morais da Silva et al., 1996). In addition, its expression in the bipotent precursor for granulosa and Sertoli cells is sufficient for male sexual development during normal

Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, Colle`ge de France, Illkirch Cedex, France Grant sponsor: Centre National de la Recherche Scientifique; Grant sponsor: Institut National de la Sante´ et de la Recherche Me´dicale; Grant sponsor: Colle`ge de France; Grant sponsor: Institut Universitaire de France; Grant sponsor: Hoˆpital Universitaire de Strasbourg; Grant sponsor: Association pour la Recherche sur le Cancer; Grant sponsor: Fondation pour la Recherche Me´dicale. *Correspondence to: Manuel Mark, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, Colle`ge de France, BP 10142, 67404 Illkirch Cedex, France. E-mail: [email protected] DOI 10.1002/dvdy.10202

© 2002 Wiley-Liss, Inc.

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embryogenesis (Vidal et al., 2001; Bishop et al., 2000). It was previously shown that ER␣␤KO ovaries display an overall increase of Sox9 expression (Couse et al., 1999), but the ovarian cell type overexpressing the gene was not determined. To further characterize the abnormal Sertolilike cells and to identify their precursors, we have analyzed here the expression of Sox9 and of two other recently characterized Sertoli cell molecular markers, TIF1␤ (Weber et al., 2002) and TIF2 (Gehin et al., 2002), in prepubertal and adult ER␣␤KO ovaries.

RESULTS AND DISCUSSION

Figure 1.

Figure 2.

In the adult testis, which serves here as a positive control of immunohistochemical assays, the transcription intermediary factor 1␤ (TIF1␤ ) is expressed in both Sertoli cells (Fig. 1a, S) and some germ cells (e.g., Fig. 1a, RS). Its germ cell expression is required for spermatogenesis (Weber

Fig. 1. The expression patterns of TIF1␤ and TIF2 are similar in wild-type (WT) testicular Sertoli cells and in ovarian estrogen receptors ER␣ and ER␤ knockout (ER␣␤KO) Sertoli cells. Immunohistochemical detection of TIF1␤ and TIF2 on frozen sections from adult testes and ovaries. Immunostaining with a Cy3-conjugated secondary antibody (red signal) and DAPI counterstain. The green and yellow spots in (b,c,g,h) correspond to autofluorescent cells. (d) same section as (e) stained with hematoxylin and eosin following immunostaining. G, granulosa cells; H, heterochromatin; I, interstitial cells; L, lumen of seminiferous tubules or of antral follicles; N, nucleolus; O, oocyte; RS, round spermatids; S, Sertoli cells. Scale bar in h ⫽ 15 ␮m in a,b, 55 ␮m in c, 40 ␮m in d,e, 22 ␮m in f– h. Fig. 2. Sox9 is expressed by the Sertoli cells and by a fraction of the granulosa cells in estrogen receptors ER␣ and ER␤ knockout (ER␣␤KO) ovaries. In situ hybridization for detection of Sox9 transcripts (blue signal) in sexually mature (adult) ovaries and testes and in prepubertal (postnatal day 22) ovaries. Note that differences in the degrees of follicular development in wild-type (WT) and mutant prepubertal ovaries (e, f), i.e., presence of antral follicles (A) in the ER␣␤KO ovary vs. primary follicles (P) in the WT ovary, have been documented in Dupont et al. (2000) and Couse et al. (1999). A, antral follicles; G, granulosa cells; I, interstitial cells; P, primary follicles; S, Sertoli cells; T, seminiferous tubule. Methyl green counterstain. Scale bar in f ⫽ 80 ␮m (applies to a–f).

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et al., 2002), but high levels of the TIF1␤ protein are also present in adult Sertoli cells where it is characteristically associated with perinucleolar heterochromatin (Fig. 1a, H). The transcription intermediary factor 2 (TIF2) is detected specifically in Sertoli cell nuclei (Fig. 1f, S) and is required for spermatogenesis (Gehin et al., 2002), Sox9 transcripts are detected at the periphery of the seminiferous tubules, which contains most of the Sertoli cell cytoplasm (Fig. 2a, S). In ovaries from wild-type (WT) adults, TIF1␤ is weakly and evenly expressed in nuclei of granulosa cells but not in interstitial cells, whereas TIF2 protein and Sox9 transcripts are undetectable (Fig. 2b, G and I, and data not shown). Cells within the interstitial compartment of ER␣␤KO ovaries, which were previously identified as Sertoli cells on the basis of morphologic criteria (Dupont et al., 2000), strongly express TIF1␤ (Fig. 1b,c, S), TIF2 (Fig. 1h, S), and Sox9 (Fig. 2c,d, S). These three markers are also occasionally detected in cells that are located within follicles at advanced stages of atresia and exhibit morphologic features of ovarian Sertoli cells (Fig. 1d,e, S, and data not shown). Ovarian Sertoli cells of ER␣␤KO mice display the association of TIF1␤ with perinucleolar heterochromatin (Fig. 1b, H, and data not shown), which is characteristic of testicular Sertoli cells (Fig. 1a, H). The expression patterns of TIF1␤, TIF2, and Sox9, in adult ER␣␤KO ovaries, therefore, provides additional evidence that the abnormal cell population found in ER␣␤KO ovaries consists in functional Sertoli cells. Expression of TIF2 and association of TIF1␤ with heterochromatin in ER␣␤KO ovaries are confined to cells displaying morphologic features of Sertoli cells such as large nuclei, prominent nucleoli, and abundant cytoplasm (Fig. 1b– e,h, and data not shown). TIF2 is absent from ER␣␤KO granulosa cells (Fig. 1g, G), where the nuclear immunostaining for TIF1␤ is weak and evenly distributed, similarly to what is observed in WT granulosa cells (Fig. 1c, G, and data not shown). In contrast, Sox9 transcripts are detected in granulosa cells from non-atretic antral fol-

licules of adult ER␣␤KO ovaries (Fig. 2d, G). Thus, during folliculogenesis, Sox9 expression in ER␣␤KO granulosa cells precedes the morphologic differentiation of Sertoli cells, which is only seen at advanced stages of follicular atresia (Dupont et al., 2000). To determine whether Sox9-expressing granulosa cells could be the precursors of ovarian ER␣␤KO Sertoli cells, we next analyzed Sox9 expression in prepubertal ovaries collected at postnatal day 22 (P22). Note that in ER␣␤KO ovaries, Sertoli cells can be morphologically identified for the first time at the onset of puberty (i.e., P30; Dupont et al., 2000). Sox9 transcripts are not detected in P22 WT ovaries (Fig. 2e, G and I). In contrast, in ER␣␤KO ovaries, Sox9 is strongly expressed by some granulosa cells (Fig. 2f, G), but not by interstitial cells (Fig. 2f, I). That expression of Sox9 by granulosa cells precedes the appearance of Sertoli cells during both ovarian development and ovarian folliculogenesis in ER␣␤KO mutants supports our previous proposal that Sertoli cells present in the interstitial compartment of adult ER␣␤KO ovaries are most probably derived from Sox9-expressing granulosa cells which, upon follicular atresia, are incorporated into the interstitial compartment (Dupont et al., 2000). Note that our present observation that interstitial cells of prepubertal ovaries do not express Sox9 makes it unlikely that these cells could be the source of Sertoli cells. Furthermore, this transformation into Sertoli cells appears to be a late event in the life cycle of ER␣␤KO granulosa cells, as Sox9 expression is never observed in primordial follicles, which contain the granulosa cell precursors (data not shown). Of interest, it was reported recently that a fraction of ovarian cells is transformed into Sertoli cells in mice lacking the aromatase enzyme that converts androgens to estrogens (ArKO mice; Britt et al., 2001), thus indicating that estrogen signaling is important for the maintenance of the differentiated state of ovarian cells. It is noteworthy that, similarly, only a fraction of the granulosa cells in ER␣␤KO mice express Sox9 and exhibit morphologic features of Sertoli cells. Thus, another event that is

distinct from the block in estrogen signalling and could be the loss of an oocyte-secreted factor (Vigier et al., 1989; Behringer et al., 1990; Taketo et al., 1993; Vainio et al., 1999) appears to be required to induce the transformation of granulosa cells into Sertoli cells. In this respect, it is noteworthy that oocytes in prepubertal ER␣␤KO ovaries appear histologically healthy and do not display DNA fragmentation, as assessed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL; data not shown). However, in contrast to their WT homologues, they are often separated from the follicular wall (Dupont et al., 2000), which could impair some of their interactions with granulosa cells. On the other hand, it is clear that follicular atresia, which may be increased in ER␣␤KO ovaries (Dupont et al., 2000) but also represents the normal fate of the vast majority of growing follicles in WT ovaries (reviewed in McGee and Hsueh, 2000), cannot, by itself, account for the ectopic differentiation of ovarian Sertoli cells. Sox9 has the capacity to influence local chromatin structure as well as to control mRNA splicing, and thus to induce stable, epigenetic, changes in cell phenotypes (Ohe et al., 2002, and references therein). Moreover, Sox9 is sufficient to trigger Sertoli cell differentiation in the embryo (Bishop et al., 2000; Vidal et al., 2001). We show here that, in ER␣␤KO ovaries, ectopic expression of Sox9 in some granulosa cells from antral follicles precedes the appearance of Sertoli cells. Altogether, these data strongly suggest that mature granulosa cells can transdifferentiate (Tosh and Slack, 2002) into Sertoli cells upon expression of Sox9 but that this potential is normally repressed through ERmediated estrogen signaling. On the other hand, maternal estrogens are unlikely to play a crucial role in the control of prenatal ovarian differentiation, which is known to occur normally in ER␣␤KO females (Couse et al., 1999; Dupont et al., 2000).

EXPERIMENTAL PROCEDURES Immunohistochemical detection of TIF1␤ and TIF2 were performed on

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frozen sections of ovaries and testis from adult (i.e., 16 weeks old) mice, as described (Weber et al., 2002; Gehin et al., 2002). Sox9 transcripts were detected on frozen sections from 4% paraformaldehyde-perfused cryoprotected tissues by in situ hybridisation with a digoxigenin-labeled probe corresponding to the complete open reading frame (ORF) of hSox9 (1530 bp) and transcribed from linearized pcDNA3-hSox9. The specificity of this probe was confirmed by using a shorter probe corresponding to region ⫹696 to ⫹1530 of hSox9 ORF that does not contain the HMG domain of Sox9,which is highly conserved in group E of Sox genes.

ACKNOWLEDGMENTS We thank Drs. M. Oulad-Abdelghani and F. Cammas (IGBMC, Strasbourg) for the anti-TIF1␤ antibody, T. Ylikomi (Tampere University, Finland) for the anti-TIF2 antibody, and B. Boizet (IGH, Montpellier, France) for the pcDNA-hSox9 plasmid.

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