JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 19:158-171 (1991 1
Testicular Differentiation in Mammals Under Normal and Experimental Conditions HORACIO MERCHANT-LARIOS AND TERUKO TAKETO Instituto de Investigaciones BiomCdicas, U N A M , Mexico D.F. 04510 (H.M.-L.); Urology Research L a b and Department of Biology, McGill University, Royal Victoria Hospital, Montreal, Canada H 3 A 1 A l (T.T.)
KEY WORDS
Testicular differentiation, Gonadal sex-reversal, Sexual differentiation
ABSTRACT Gonadal differentiation begins with the establishment of a sexually undifferentiated gonad, in which gonadal cords are formed by condensation of somatic cells and deposition of basal laminar components around the cluster of epithelial-like cells. The first event of sexual differentiation is the invasion of mesenchymal and endothelial cells into the genital ridge in the XY gonad. As a consequence of this event, the gonadal cords become conspicuous, recognized as seminiferous cords (or testis cords). Cytological differentiation of Sertoli cells follows these stromal changes. In the XX gonad, by contrast, the invasion of the mesenchyme is absent and gonadal cords remain associated with the surface epithelium. In the B6.YDoMXY ovotestis, seminiferous cords and ovarian gonadal cords are often enveloped by common basal laminae, confirming that both structures share the embryonic origin. It has been recently reported that seminiferous-like cords are formed after loss of oocytes in the rat XX ovary cultured in the presence of Mullerian inhibiting substance or after long-term culture in the basic medium alone. These results are comparable with our observation on the persistent gonadal cords in the ovary of busulphan-treated rats or W/Wv mutant mice, in which oogonia are absent or scarce. Ultrastructural evidence for Sertoli cell differentiation from XX cells has been presented, so far, only in the fetal mouse ovary that has been grafted beneath the kidney capsule of adult male mice. Possible mechanism of gonadal sex determination is discussed based on these morphological studies. INTRODUCTION
induced in the XX gonad, which otherwise develops into an ovary. In freemartins of cattle, for example, It has been widely accepted that testicular differen- testis-like structures develop in the XX gonad because tiation is determined by a gene or set of genes on the Y the female fetus is connected to a male twin through chromosome in mammals (TDF in human or Tdy in the the blood circulation (Lillie, 1917; Jost et al., 1972). mouse). Great efforts have been made to identify the Mullerian inhibiting substance (MIS), also known as Tdy gene or its gene product in the last two decades. antimullerian hormone (AMH), has been proposed as First, the male dominant histocompatibility Y-antigen the factor responsible for masculinization of the female (H-Y antigen) was proposed as the Tdy gene product gonad (Vigier et al., 1982). Supporting this hypothesis, based on the finding that individuals with testes carry the same authors have reported that exogenously apthe H-Y antigen regardless of their chromosomal sex plied MIS induces the development of seminiferous(Wachtel et al., 1975; Ohno et al., 1979). However, we like epithelial cords in the XX rat ovary in culture have learned by now that there are too many excep- (Vigier et al., 1987). Furthermore, chronic expression tions to this rule (Haseltine et al., 1981; McLaren et al., of MIS in female transgenic mice produces a phenotype 1984; Simpson et al., 1987). The recent finding of the similar to the freemartin (Behringer et al., 1990). Howhuman Y-specific ZFY sequence as a TDF canditate ever, it has recently been found that the rat ovarian appeared to be promising since its homologous se- explant develops epithelial cords after long term culquence can be found in most XX sex-reversed male ture even in the absence of MIS (Prepin and Hida, patients as well as the XX male mutant mouse (Page et 1989). Another example is the differentiation of testical., 1987; Nagamine et al., 1989). The ZFY hpothesis is ular components in the XX mouse ovary after transnow challenged by the finding that transcription of the plantation beneath the kidney capsule of adult mice Zfy gene (a ZFY homologous sequence in the mouse) is (Taketo-Hosotani et al., 1984, 1985). How can these absent in fetal testes of the We/We germ cell-free male findings be interpreted with regard to the mechanism mouse (Koopman et al., 1989). One can argue that testis determination may take place earlier than the developmental stage examined in these experiments. Then, how do we know the right stage in development to examine the Tdy gene function? Another approach to clarify the mechanism of testis Received June 8, 1990; accepted in revised form July 19, 1990. determination is to investigate the experimental conAddress reprint requests to Teruko Taketo, Urology Research Lab, Royal Vicditions under which testicular differentiation can be toria Hospital, 687 Pine Ave. W., Montreal, Canada H3A 1 A l .
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of sex determination? First of all, can these morphological events under abnormal conditions be considered to reflect true testicular differentiation? What criteria can be used to evaluate the testis-like structures? Gonadal differentiation begins with the establishment of a primordial gonad, followed by its sexual differentiation into a testis or an ovary. The latter event involves differentiation of germ cells and several somatic cell types, and their topological arrangement and interaction. We believe that deep insight into this morphological process, particularly at the early stage of gonadal differentiation, is essential for evaluation of hypotheses concerning the mechanism of gonadal sex determination. In this paper, we will discuss morphological and, to some extent, biochemical key events during the initial phase of testicular differentiation based on the results from our laboratories during the last 15 years. The studies on early stages of gonadal development by other investigators have previously been reviewed (Jost et al., 1973; Merchant-Larios, 1984; Byskov, 1986; Jost and Magre, 1988; Watenberg, 1989). ESTABLISHMENT O F A SEXUALLY UNDIFFERENTIATED GONAD Figure 1shows a cross-section of a typical rat genital ridge prior to sexual differentiation. It consists of somatic and primordial germ cells tightly packed and attached to the surface epithelium. At this early stage of development, three important morphogenetic characteristics can be recognized condensation of somatic cells along the genital ridge, gradual deposition of basal laminar components around the cluster of epithelial-like cells, and a low mitotic activity of the epithelial-like cells in the genital ridge. As a consequence of these changes, gonadal cords (previously called sex cords) are formed in the genital ridge of both XX and XY gonads. The origin of epithelial-like cells in the genital ridge has not been identified. However, conversion of the mesodermal mesenchyme into epithelial cells has been well documented during differentiation of the metanephros (Grobstein, 1956), which shares the embryonic origin with the gonad. Therefore, it is reasonable to assume that a similar process of cellular differentiation occurs during the establishment of a primordial gonad. During differentiation of the metanephros, two events have been described a t the molecular level: sequential changes of extracellular matrix components and cellcell adhesion molecules (Ekblom et al., 1985). In the sexually undifferentiated gonad, basal lamina is gradually formed around the cluster of epithelial-like cells, thus developing gonadal cords (Fig. 2) (Merchant, 1975;Pelliniemi, 1975). These ultrastructural observations have recently been confirmed by immunocytochemical staining for basement membrane components (Pelliniemi et al., 1984; Paranko, 1987; Gelly et al., 1989). Interestingly, the epithelial-like cells of gonadal cords show a low mitotic activity: less than 10% of epithelial-like cells incorporate [H3] thymidine while 35% of mesenchymal and mesothelial cells in the same region are labeled (Figs. 3, 4) (Merchant-Larios, 1979). This finding may indicate that the epithelial-
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like cells undergo an essential differentiation process in both XX and XY gonads prior to sexual differentiation. TESTICULAR DIFFERENTIATION Normal Development The first event of sexual differentiation is the gradual invasion of mesenchyme and endothelial cells into the genital ridge in the XY gonad (Fig. 5). As a result, gonadal cords become conspicuous, first in the medullary region, and then in the cortex region. In the mouse, this sequencial event is typically observed from the mid area to the cranial and caudal poles because the cortex in the mid portion is very thin. At the ultrastructural level, Sertoli cells are the first cell type to differentiate, as identified by the development of the rough endoplasmic reticulum (Fig. 7) (Merchant-Larios, 1976a). This cytological change may reflect active production of MIS. However, it must be pointed out that ultrastructural differentiation of Sertoli cells follows the invasion of stromal cells and segregation of gonadal cords from the surface epithelium. It is not clear whether physiological differentiation of Sertoli cells takes place without prominent morphological changes before the invasion of stromal cells. Sertoli precursor cells may stimulate the proliferation or migration of stromal cells. Differentiation of Leydig cells and peritubular myoid cells is usually seen a t later stages of development (Fig. 7). Development of Gonadal Cords in the XY Ovotestis When the Y chromosome of Mus musculus domesticus (YDoM) is placed onto the C57BU6J (B6) mouse background, the XY progeny (B6.YDoM)develop either ovaries or ovotestes but not normal testes during fetal life (Eicher et al., 1982; Eicher and Washburn, 1986; Nagamine et al., 1987a,b). This example of sexreversal provides strong evidence for the concept that sexual differentiation begins with an epithelial compartment in the primordial gonad. In the B6.YDoMfetal ovotestis, gonadal cords become conspicuous in the medullary region due to the invasion of stromal cells as seen in the normal XY fetal testis, whereas gonadal cords remain attached to the surface epithelium at the cranial and caudal poles as seen in the normal XX fetal ovary (Fig. 8) (Nagamine et al., 1987a; Taketo-Hosotani et al., 1989). Inside the gonadal cords of the ovarian region (ovarian gonadal cords), germ cells enter meiotic prophase as seen in the XX fetal ovary (Fig. 9). In contrast, inside the seminiferous cords, germ cells are arrested a t the prospermatogonia stage (Fig. 10). Leydig cells are seen only around the seminiferous cords, suggesting that fetal Sertoli cells may possess an inductive influence on stromal cell differentiation. Thus, gonadal cords develop into two distinct sexual structures despite the identical XY genetic background. Seminiferous cords and ovarian gonadal cords often remain enveloped by common basal laminae (Fig. 11). In the boundary region, occasional germ cells in meiosis are found in the gonadal cord that is surrounded by stromal cells and, therefore, resembles seminiferous cords. No ultrastructural differences were
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Fig. 1. Cross section of the genital ridge from a rat embryo at 12.5 day of gestation (d.g.1. Primordial germ cells (pgc) are embedded in the condensation of epithelial-like cells (EI). x 500.
Fig. 2. Electron micrograph of epithelial-like cells (EI) in a sexually undifferentiated rat gonad at 12.5 d.g. Deposit of basal laminar components (arrows) is demonstrated by staining with ruthenium red. x 7,000.
Fig. 3. Autoradiograph of a rat XY gonad at 13.5 d.g. labeled by incorporation of [H3] thymidine. [H3] thymidine was injected into the pregnant female 3 h before dissection of fetuses. The invasion and proliferation of stromal cells (st) delineates seminiferous cords (sc). Note that only a few epithelial cells inside the seminiferous cords are labeled. x 400.
Fig. 4. Autoradiograph of a rat XX gonad at 13.5 d.g. labeled by incorporation of [H31 thymidine. Most of the labeled cells inside gonadal cords (gc) are primordial germ cells (pgc) whereas only a few epithelial-like cells are labeled. Many mesenchymal cells (m)
and epithelial cells in the surface epithelium (se) are labeled. x 400. Fig. 5. Cross-section of a fetal mouse testis at 14 d.g. Seminiferous cords (sc) are clearly separated from the surface epithelium by stroma1 tissue (st).Dark cells around the seminiferous cords correspond to Leydig cells (L). x 200. Fig. 6. Cross-section of a fetal mouse ovary at 14 d.g. Gonadal cords (gc) remain attached to the surface epithelium. Note the scarce invasion or proliferation of mesenchymal cells.
Fig. 7. Part of a seminiferous cord in a rat testis at 15 d.g., which is composed of prospermatogonia (ps) and fetal Sertoli cells (S).In the cytoplasm of Sertoli cells, the endoplastic reticulum (er) is well developed. A Leydig cell (L) is seen among the stromal cells. x 7,500.
Fig. 8. Longitudinal section of a n ovotestis from a B6.YDoMmouse fetus at 14 d.g. Seminiferous cords (sc) in the medullary region appear interconnected through the rete ovarii (ro) with gonadal cords (gc) in the cranial and caudal regions. Note the abundant mesenchymal cells around the seminiferous cords. x 200.
Fig. 9. The ovarian region in a serial section of the ovotestis shown in Fig. 8. Most oocytes (0)have entered the meiotic prophase. Note that gonadal cords are mainly occupied by oocytes. x 600. Fig. 10. The testicular region in a serial section of the ovotestis shown in Fig. 8. Leydig cells (L) are seen around the seminiferous cords (sc). Inside the seminiferous cords, several germ cells are arrested a t the prospermatogonia stage (ps). x 600.
Fig. 11. Part of an ovotestis from a B6.YDOMmouse fetus a t 14 d.g. At the point indicated with a n asterisk, a seminiferous cord (sc) is continuous with ovarian gonadal cords (ogc), in which most oocytes have entered meiosis. Dark cells in the stroma are Leydig cells (L). x 300.
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found between the epithelial cells in the two regions. These observations strongly support the hypothesis that ovarian gonadal cords share the embryonic origin with seminiferous cords. The XY ovotestis also provides interesting information about the onset of testicular and ovarian differentiation. In the control B6 strain, seminiferous cords become conspicuous and fetal Sertoli cells initiate cytological differentiation, as demonstrated by immunocytochemical staining for MIS, in all XY gonads by 12 d.g. (Taketo et al., 1991) (Fig. 12). By contrast, in the B6.YDoMgonad, seminiferous cords are hardly recognizable at 12 d.g. Even at 13 or 14 d.g., some XY ovotestes, as identified by the invasion of stromal cells, show no or only faint staining for MIS. By 15 d.g., all XY ovotestes have developed seminiferous cords composed of fetal Sertoli cells, which are intensely stained for MIS (Fig. 13). Thus, the onset of testicular differentiation is severely delayed during the development of XY ovotestes. Further delay in the onset of testicular differentiation probably allows the ovarian differentiation pathway to be switched on, thus producing XY ovotestes or ovaries. In the boundary area between testicular and ovarian regions, a gradient of staining for MIS is often seen, with stronger staining on the side of gonadal cords in contact with stromal cells (Fig. 13). PERSISTENCE OF GONADAL CORDS IN THE XX OVARY During the early phase of ovarian differentiation, epithelial-like cells proliferate slowly in contrast to active proliferation of oogonia in the genital ridge (Figs. 4,6). The invasion of mesenchymal cells is absent and gonadal cords remain closely associated with the surface epithelium. Consequently, it becomes difficult to identify the epithelial cells inside the ovarian gonadal cords under the light microscope. Probably for these reasons, some authors have considered that gonadal cords are never formed in the XX gonadal primordium (Jost and Magre, 19881, or that gonadal cords are newly formed in the ovary after loss of oocytes (Vigier et al., 1987; Prepin and Hida, 1989. Behringer et al., 1990). In the XX gonad of W/Wir mutant mice or busulphan-treated rats, in which oogonia are absent or scarce, gonadal cords become conspicuous due to the growth of stromal cells (Figs. 14, 15) (Merchant, 1975; Merchant-Larios, 197613; Merchant-Larios and Centeno, 1981). By comparing the development of normal and oocyte-depleted ovaries, we concluded that oocytes are essential for the establishment of primordial follicles, which are formed by “splitting” of gonadal cords (Merchant-Larios, 1978; Merchant-Larios and ChimalMonroy, 1989). The invasion and proliferation of stroma1 cells is seen in the oocyte-free ovary around 3 days after birth, a t which stage the first primordial follicles begin to form in the normal ovary. It is interesting to compare the timings of stromal invasion between the two sexes-that is, at folliculogenesis in the neonatal ovary and a t the onset of testicular differentiation in the fetal testis. At later stages, gonadal cords that have been retained in the oocyte-depleted ovary resemble seminiferous cords at the light microscopic level because of the polarization of epithelial cells (Merchant,
1975). However, at the ultrastructural level, epithelial cells inside the ovarian gonadal cord are distinct from Sertoli cells inside the seminiferous cord (Figs. 16, 17) (Merchant, 1975; Merchant-Larios, 197613; MerchantLarios and Centeno, 1981). In this regard, previous reports on the development of seminiferous cord-like structures in the XX ovary under experimental conditions need to be re-evaluated since disappearance of oocytes is the first morphological event in all cases (Taketo-Hosotani et al., 1985; Vigier et al., 1987; Prepin and Hida, 1989; Behringer et al., 1990). So far, only in one case (i.e., transplantation of fetal mouse ovaries) ultrastructural evidence has been provided to indicate that XX cells can acquire the characteristics of Sertoli cells (Figs. 18,19) (Taketo-Hosotani et al., 1984,1985). The invasion of stromal cells is prominent in the testicular region of mouse ovotestes after transplantation (Fig. 20). We have previously demonstrated that direct contact with the host kidney is essential for the induction of testicular structures in the ovarian graft (Taketo-Hosotani, 1987). Thus, in all cases of testicular differentiation known so far, the establishment of epithelial-mesenchymal interaction appears to be closely associated with cytodifferentiation of Sertoli cells. Leydig cells and myoid-like cells are seen around the seminiferous cords, suggesting again the inductive activity of Sertoli cells on stromal cell differentiation (Taketo-Hosotani et al., 1984, 1985). MECHANISM OF GONADAL SEX DETERMINATION We will discuss in this section two previously proposed hypotheses concerning the mechanism of gonadal sex determination at the organization level, and propose modifications based on our recent findings.
Significance of the Growth Rate in Sex Determination Mittwoch has observed that the growth rate of the XY gonad is greater than the XX gonad around the time of sexual differentiation (Mittwoch et al., 1969). Based on this finding, she has postulated that differentiation of Sertoli cells depends on whether the genital ridge has reached a particular developmental stage at the right time, and that failure to do so allows the gonadal primordium to undergo the ovarian differentiation pathway. Hence, in addition to the Tdy gene on the Y chromosome, a factor(s1 which influences the growth rate can modulate the development of a gonad into a testis or an ovary (Mittwoch, 1989). This hypothesis agrees with our conclusion that the delay of testicular differentiation leads to ovarian differentiation despite the presence of the normal Y chromosome in the B6.YDoMovotestis or ovary. As shown in Figures 5 and 6, the normal XY testis is larger than the XX ovary mainly due to the invasion and proliferation of stromal cells a t the onset of sexual differentiation. Since epithelial cells proliferate slowly in both XX and XY gonads, the size of gonadal cords is similar between the two sexes. Therefore, according to the hypothesis by Mittwoch, we postulate that the mechanism controlling the invasion or proliferation of
Fig. 12. Part of a testis from a B6 mouse fetus a t 12 d.g., immunocytochemically stained for MIS. ABC staining (Vector Lab, CA) with 3,3'-diaminobenzidine tetrahydrochloride. Fetal Sertoli cells inside the seminiferous cords (sc) are intensely stained while other cell types remain at the background level. Note that seminiferous cords are not completely separated from each other near the mesonephros (ms). x400. Fig. 13. Part of an ovotestis from a B6.YDoMmouse fetus at 15 d.g., immunocytochemically stained for MIS. Seminiferous cords (sc) are well organized and intensely stained for MIS in the mid portion (md). Ovarian gonadal cords remain closely attached to each other in
Note the stronger staining (indicated by arrow the caudal region (cd). heads) on the side of gonadal cords in contact with stromal tissues, but not on the opposite side, which is continuous with ovarian gonadal cords. ~ 4 0 0 . Fig. 14. An ovary from a newborn Wlwv mutant mouse. Oocytes are absent inside the gonadal cords (gc). A few oocytes (*) are seen near the surface epithelium. x 160. Fig. 15. An ovary from a newborn rat which has been treated with busulphan on 12 d.g. Several gonadal cords (gc) devoid of oocytes are surrounded by stromal cells (st). x 200.
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Fig. 16. Electron micrograph of a gonadal cord in a busulphan-treated rat ovary at 15 days after birth. Epithelial cells (E) are well differentiated and polarized inside the ovarian gonadal cord. The junctional complexes with dense desmosomes (*), typical of epithelial cells, are seen at the luminal side (Lu) of plasma membranes. x 7,000.
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Fig. 17. Electron micrograph of a seminiferous cord in a mouse testis a t 10 days after birth. A prospematogonium (ps) and several Sertoli cells (S) are seen inside the seminiferous cords. Charaderistic intersertoli junctional specializations (*) are evident between Sertoli cells. x 7,500.
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Fig. 18. Electron micrograph of a seminiferous cord in a mouse ovarian graft. A fetal ovary at 12 d.g. was transplanted beneath the kidney capsule of an adult male mouse and fixed 14 days later. Intersertoli junctional specializations (*) are well developed between Sertoli cells. x 10,500.
Fig. 19. High magnification of the intersertoli junctional specialization (*) shown in Fig. 18, which is characterized by the presence of an intercellular space (about 7-9 nm) and a flat cisternum of endoplasmic reticulum parallel to plasma membranes. Some ribosomes are seen on the cytoplasmic side. x 21,000.
Fig. 20. A mouse ovarian graft, 14 days after transplantation. Seminiferous cords (sc) have developed over a large area next to follicles (f). Note the massive invasion of stromal cells (st) around the seminiferous cords whereas it is less abundant around follicles. bv, blood vessels. x 200.
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stromal cells must be responsible for the difference of gonadal size and, hence, may modulate gonadal sex determination. In the XX gonad, on the other hand, massive invasion and proliferation of stromal cells occurs a t the onset of folliculogenesis. This is the developmental stage at which the ovarian graft initiates differentiation of testicular components (Taketo-Hosotani et al., 1985). The timing of testicular differentiation appears to be closely associated with the rapid growth of stromal cells in both sexes.
Autonomous Expression of the Tdy Gene in Sertoli Cells Burgoyne et al. have demonstrated that in the adult testis of XX-XY chimeric mouse, Sertoli cells are exclusively XY while other testicular somatic cells are either XX or XY (Burgoyne et al., 1988).This finding has lead to a hypothesis that the Tdy gene is expressed in Sertoli precursor cells to trigger autonomous cytodifferentiation, which, in turn, regulates differentiation of other testicular cell types regardless of the chromosomal sex (Burgoyne, 1988). This hypothesis excludes the possibility that a diffusible factor may be involved in the primary testis determination. These observations have been made in the adult testis, and, therefore, we do not yet know what happens in fetal gonads of the XX-XY chimeric mouse. If XX cells can differentiate into Sertoli cells, as suggested by transplantation experiments, we can postulate a mechanism preventing XX cells from contributing to Sertoli cells at the onset of testicular differentiation. Alternatively, only XY cells may be able to reach the developmental stage to differentiate into Sertoli cells a t the right time according to the hypothesis by Mittwoch. This does not exclude the possibility that XX cells can follow a similar process a t a later stage of development under experimental conditions. Since the stromal change always precedes cytological differentiation of Sertoli cells, it is necessary to assume that XY epithelial cells in gonadal cords secrete a substance which induces the invasion and proliferation of stromal cells if one accepts the hypothesis by Burgoyne. The establishment of epithelial-mesenchymal interaction may, in turn, play an essential role in cytodifferentiation of Sertoli cells. CONCLUDING REMARKS Gonadal cords are formed in the genital ridge of both XX and XY primordial gonads. Testicular differentiation is characterized by the spatial separation of gonadal cords due to the growth of stromal cells, followed by cytodifferentiation of Sertoli cells and other testicular cell types. These events lead to the establishment of the characteristic structure of seminiferous cords. On the other hand, ovarian differentiation is characterized by the onset of meiosis in primordial germ cells into oocytes. Gonadal cords are not conspicuous a t the early phase of ovarian differentiation because of poor stromal growth. Consequently, follicles are formed by splitting of gonadal cords while stromal cells begin to proliferate actively in the interstitiurn. The absence or loss of oocytes results in the failure of folliculogenesis
and, therefore, the persistent presence of gonadal cords. These cords may resemble seminiferous cords at the light microscopic level, but do not necessarily represent true testicular differentiation.
ACKNOWLEDGMENTS We are grateful to Dr. Y. Nishioka for the mouse Y-specific DNA probes and Dr. P. Donahoe for the antiserum against MIS. We thank Jose G . Baltazar for preparing tissue sections, Jose Aviles for photomicrographs, and Jamilah Saeed for technical assistance. A part of this study is supported by MRC (Canada) grants to T.T. NOTE ADDED IN PROOF Since the acceptance of this manuscript, a single copy gene, named SRY in human and Sry in the mouse, has been identified and proposed as the testis determining gene on the Y chromosome (Sinclair et al., 1990; Gubbay et al., 19901, strongly supported by several lines of direct and indirect evidence (Koopman et al., 1990, 1991). Expression of the Sry gene appears to be the highest in the fetal gonad just prior to testicular organization and diminish afterward (Koopman et al., 1990). The gene product of Sry is expected to be a transcription factor according to the nucleotide sequence analysis, and, hence, the major interest in sex determination in mammals is shifting to the mechanism downstream of the Y-encoded gene. REFERENCES Behringer, R., Cate, R.L., Froelick, G.J., Palmiter, R.D., and Brinster, R.L. (1990) Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nature, 345:167-170. Burgoyne, P.S. (1988) Role of mammalian Y chromosome in sex determination. Phil. Trans. R. SOC.Lond., B322:63-72. Burgoyne, P.S., Buehr, M., Koopman, P., Rossant, J.,and McLaren, A. (1988) Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX-XY chimaeric mouse testes. Development, 102:443-450. Byskov, A.G. (1986) Differentiation of mammalian embryonic gonad. Physiol. Rev., 66:71-117. Eicher, E.M., and Washburn, L.L. (1986) Genetic control of primary sex determination in mice. Ann. Rev. Genet., 20:327-360. Eicher, E.M., Washburn, L.L., Whitney, J.B., and Morrow, K.E. (1982) Mus poschiavinus Y chromosome in the C57BLi6J murine genome causes sex reversal. Science, 217:535-537. Ekblom, P., Thesleff, I., and Sariola, H. (1985) The extracellular matrix in tissue morphogenesis and angiogenesis. In: The cell in Contact: Adhesion and Junctions a s Morphogenetic Determinants. G.M. Edelman and J.P.Thiery, eds. John Wiley & Sons, New York, pp. 365-392. Gelly, J.L., Richoux, J.P., Leheup, B.P., and Grignon, G. (1989) Immunolocalization of type IV collagen and laminin during rat gonadal morphogensis and postnatal development of the testis and epididymis. Histochemistry, 93:31-37. Grobstein, C. (1956) Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell. Res., 10424-440. Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou, A . , Munsterberg, A., Vivian, N., Goodfellow, P.,. and Lovell-Badge, R. (1990) A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature, 346245-250. Haseltine, F.P., Genel, M., Crawford, J.D., and Breg, W.R. (1981) H-Y antigen negative patients with testicular tissue and 46,XY karyotype. Hum. Genet., 57:265-268. Jost, A., and Magre, S. (1988) Control mechanisms of testicular differentiation. Phil Trans. R. SOC.Lond., B322:55-61.
TESTICULAR DIFFERENTIATION I N MAMMALS Jost, A., Vigier, B., and Prepin, J . (1972) Freemartins in cattle: The first steps of sexual organogenesis. J. Reprod. Fertil., 29:349-379. tJost, A., Vigier, B., Prepin, J . , and Perchellet, J.P. (1973) Studies on sex differentiation in mammals. Rec. Prog. Horm. Res., 29:l-41. Koopman, P., Gubbay, J., Collignon, J., and Lovell-Badge, R. (1989) Zfy gene expression patterns are not compatible with a primary role in mouse sex determination. Nature, 342:940-942. Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P., and LovellBadge, R. (1991) Male development of chromosomally female mice transgenic for Sry. Nature, 351:117-121. Koopman, P., Munsterberg, A., Capel, B., Vivian, N., and LovellBadge, R. (1990) Expression of a candidate sex-determining gene during mouse testis differentiation. Nature, 348:450-452. Lillie, F.R. (1917) The free-martin: A study of the action of sex hormones in the foetal life of cattle. J . Exp. Zool., 23:371-452. McLaren, A., Simpson, E., Tomonari, K., Chandler, P., and Hogg, H. (1984) Male sexual differentiation in mice lacking H-Y antigen. Nature, 312:552-555. Merchant, H. (1975) Rat gonadal and ovarian organogenesis with and without germ cells. An ultrastructural study. Dev. Biol., 44:l-21. Merchant-Larios, H. (1976a) The onset of testicular differentiation in the rat: An ultrastructural study. Am. J . Anat., 145:319-330. Merchant-Larios, H. (1976b3 The role of germ cells in the morphogenesis and cytodifferentiation of the rat ovary. In: Progress in Differentiation Research. Muller-Berat, ed. North Holland Pub. Co., Amsterdam, pp. 453-462. Merchant-Larios, H. (1978) Ovarian differentiation. In: The Vertebrate Ovary, R.E. Jones, ed. Plenum Press, New York, pp. 47-81. Merchant-Larios, H. (1979) Origin of the somatic cells in the rat gonad: An autoradiographic approach. Ann. Biol. Anim. Bioch. Biophys, 19(4B):1219-1229. Merchant-Larios, H. (1984)Germ and somatic cell interactions during gonadal morphogenesis. In: Ultrastructure of Reproduction. J. Van Blerkom and P.M. Motta, eds. Martinus Nijhoff Publ., Netherlands, pp. 19-30. Merchant-Larios, H., and Centeno, B. (1981) Morphogenesis of the ovary from the sterile W/Wv mouse. In: Advances in The Morphology of Cells and Tissues. A. Vidrio and C. Galina, eds. A.R. Liss, New York, pp. 383-392. Merchant-Larios, H., and Chimal-Monroy, J . (1989) The ontogeny of primodial follicles in the mouse ovary. In: Developments in Ultrastructure of Reproduction. P. Motta, ed. A.R. Liss, New York, pp. 55-63. Mittwoch, U. (1989) Sex differentiation in mammals and tempo of growth. J . Theor. Biol., 137:445-455. Mittwoch, U., Delhanty, J.D.A., and Beck, F. (1969) Growth of differentiating testes and ovaries. Nature, 224:1323-1325. Nagamine, C.M., Chan, K., Kozak, C.A., and Lau, Y.-F. (1989) Chromosome mapping and expression of a putative testis determining gene in mouse. Science, 243:SO-83. Nagamine, C.M., Taketo, T., and Koo, G.C. (1987a). Morphological development of the mouse gonad in tda-1 sex reversal. Differentiation, 33:214-222. Nagamine, C.M., Taketo, T., and Koo, G.C. (1987b). Studies on the genetics of tda-1 sex reversal in the mouse. Differentiation, 33: 223-231. Ohno, S., Nagai, Y., Ciccarese, S., and Iwata, H. (1979) Testis-orga-
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nizing H-Y antigen and the primary sex determining mechanism of mammals. Recent. Prog. Horm. Res., 35449-478. Page, P.C., Mosher, R., Simpson, E.M., Fisher, E.M.C., Mardon, G., Pollack, J., McGillivray, B., de la Chapelle, A., and Brown, L.G. (1987) The sex-determining region of the human Y chromosome encodes a finger protein. Cell, 51:1091-1104. Paranko, J . (1987) Expression of type I and 111 collagen during morphogenesis of fetal rat testis and ovary. Anat. Rec., 21991-101. Pelliniemi, L.J. (1975) Ultrastructure of the early ovary and testis in pig embryos. Am. J. Anat., 144239-112. Pelliniemi, L.J., Paranko, J., Grund, S.K., Frojdman, K., Foidard, J.-M., and Lakkala-Paranko, T. (1984) Extracellular matrix in testicular differentiation. Ann. New York Acad. Sci., 438:405-416. Prepin, J., and Hida, N. (1989) Influence of age and medium on formation of epithelial cords in the rat fetal ovary in vitro. J . Reprod. Fertil., 87:375-382. Simpson, E., Chandler, P., Goulmy, E., Disteche, C.M., FergusonSmith, M.A., and Page, D.C. (1987) Separation ofthe genetic loci for the H-Y antigen and for testis determination on human Y chromosome. Nature, 326 876-878. Sinclair, A.H., Berta, P., Palmer, M.S., Hawkins, J.R., Griffiths, B.L., Smith. M.J.. Foster. J.W.. Frischauf. A.-M.. Lovell-Badge. R.. and Goodfellow, P.N. (1990) A gene from the human sex-d&ermining region encodes a protein with homology to a conserved DNAbinding motif. Nature, 346:240-244. Taketo, T., Saeed, J., Nishioka, Y., and Donahoe, P.K. (1991) Delay of testicular differentiation in the B6.YDoMovotestis demonstrated by immunocytochemical staining for Mullerian inhibiting substance. Dev. Biol., 146 (in press). Taketo-Hosotani, T. (1987) Factors involved in the testicular development from fetal mouse ovaries following transplantation. J. Exp. Zool., 241:95-100. Taketo-Hosotani, T., Merchant-Larios, H., and Koide, S.S. (1984) Induction of testicular differentiation in the fetal mouse ovary by transplantation into adult male mice. Proc. Soc. Exp. Biol. Med., 176:148-153. Taketo-Hosotani, T., Merchant-Larios, H., Thau, R.B., and Koide, S.S. (1985) Testicular cell differentiation in fetal mouse ovaries following transplantation into adult male mice. J. Exp. Zool., 236229237. Taketo-Hosotani, T., Nishioka, Y ., Nagamine, M.C., Villalpando, I., and Merchant-Larios, H. (1989) Development and fertility of ovaries in the B6.YDoMsex-reversed female mouse. Development, 107: 95-105. Vigier, B., Legeai, L., Picard, J.Y., and Josso, N. (1982) A sensitive radioimmunoassay for bovine anti-Mullerian hormone, allowing its detection in male and freemartin fetal serum. Endocrinology, 111: 1409-141 1. Vigier, B., Watrin, F., Magre, S., "ran, D., and Josso, H. (1987) Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to i t in vitro. Development, 100 43-55. Wachtel, S.S., Ohno, S., Koo, G.C., and Boyse, E.A. (1975) Possible role for H-Y antigen in the primary determination of sex. Nature, 257:235-236. Watenberg, H. (1989) Differentiation and development of the testis. In: The Testis, 2nd edition. H. Burger and D. de Kretser, eds. Raven Press, New York, pp. 67-118.
Testicular Differentiation in Mammals Under Normal and Experimental Conditions HORACIO MERCHANT-LARIOS AND TERUKO TAKETO Instituto de Investigaciones BiomCdicas, U N A M , Mexico D.F. 04510 (H.M.-L.); Urology Research L a b and Department of Biology, McGill University, Royal Victoria Hospital, Montreal, Canada H 3 A 1 A l (T.T.)
KEY WORDS
Testicular differentiation, Gonadal sex-reversal, Sexual differentiation
ABSTRACT Gonadal differentiation begins with the establishment of a sexually undifferentiated gonad, in which gonadal cords are formed by condensation of somatic cells and deposition of basal laminar components around the cluster of epithelial-like cells. The first event of sexual differentiation is the invasion of mesenchymal and endothelial cells into the genital ridge in the XY gonad. As a consequence of this event, the gonadal cords become conspicuous, recognized as seminiferous cords (or testis cords). Cytological differentiation of Sertoli cells follows these stromal changes. In the XX gonad, by contrast, the invasion of the mesenchyme is absent and gonadal cords remain associated with the surface epithelium. In the B6.YDoMXY ovotestis, seminiferous cords and ovarian gonadal cords are often enveloped by common basal laminae, confirming that both structures share the embryonic origin. It has been recently reported that seminiferous-like cords are formed after loss of oocytes in the rat XX ovary cultured in the presence of Mullerian inhibiting substance or after long-term culture in the basic medium alone. These results are comparable with our observation on the persistent gonadal cords in the ovary of busulphan-treated rats or W/Wv mutant mice, in which oogonia are absent or scarce. Ultrastructural evidence for Sertoli cell differentiation from XX cells has been presented, so far, only in the fetal mouse ovary that has been grafted beneath the kidney capsule of adult male mice. Possible mechanism of gonadal sex determination is discussed based on these morphological studies. INTRODUCTION
induced in the XX gonad, which otherwise develops into an ovary. In freemartins of cattle, for example, It has been widely accepted that testicular differen- testis-like structures develop in the XX gonad because tiation is determined by a gene or set of genes on the Y the female fetus is connected to a male twin through chromosome in mammals (TDF in human or Tdy in the the blood circulation (Lillie, 1917; Jost et al., 1972). mouse). Great efforts have been made to identify the Mullerian inhibiting substance (MIS), also known as Tdy gene or its gene product in the last two decades. antimullerian hormone (AMH), has been proposed as First, the male dominant histocompatibility Y-antigen the factor responsible for masculinization of the female (H-Y antigen) was proposed as the Tdy gene product gonad (Vigier et al., 1982). Supporting this hypothesis, based on the finding that individuals with testes carry the same authors have reported that exogenously apthe H-Y antigen regardless of their chromosomal sex plied MIS induces the development of seminiferous(Wachtel et al., 1975; Ohno et al., 1979). However, we like epithelial cords in the XX rat ovary in culture have learned by now that there are too many excep- (Vigier et al., 1987). Furthermore, chronic expression tions to this rule (Haseltine et al., 1981; McLaren et al., of MIS in female transgenic mice produces a phenotype 1984; Simpson et al., 1987). The recent finding of the similar to the freemartin (Behringer et al., 1990). Howhuman Y-specific ZFY sequence as a TDF canditate ever, it has recently been found that the rat ovarian appeared to be promising since its homologous se- explant develops epithelial cords after long term culquence can be found in most XX sex-reversed male ture even in the absence of MIS (Prepin and Hida, patients as well as the XX male mutant mouse (Page et 1989). Another example is the differentiation of testical., 1987; Nagamine et al., 1989). The ZFY hpothesis is ular components in the XX mouse ovary after transnow challenged by the finding that transcription of the plantation beneath the kidney capsule of adult mice Zfy gene (a ZFY homologous sequence in the mouse) is (Taketo-Hosotani et al., 1984, 1985). How can these absent in fetal testes of the We/We germ cell-free male findings be interpreted with regard to the mechanism mouse (Koopman et al., 1989). One can argue that testis determination may take place earlier than the developmental stage examined in these experiments. Then, how do we know the right stage in development to examine the Tdy gene function? Another approach to clarify the mechanism of testis Received June 8, 1990; accepted in revised form July 19, 1990. determination is to investigate the experimental conAddress reprint requests to Teruko Taketo, Urology Research Lab, Royal Vicditions under which testicular differentiation can be toria Hospital, 687 Pine Ave. W., Montreal, Canada H3A 1 A l .
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of sex determination? First of all, can these morphological events under abnormal conditions be considered to reflect true testicular differentiation? What criteria can be used to evaluate the testis-like structures? Gonadal differentiation begins with the establishment of a primordial gonad, followed by its sexual differentiation into a testis or an ovary. The latter event involves differentiation of germ cells and several somatic cell types, and their topological arrangement and interaction. We believe that deep insight into this morphological process, particularly at the early stage of gonadal differentiation, is essential for evaluation of hypotheses concerning the mechanism of gonadal sex determination. In this paper, we will discuss morphological and, to some extent, biochemical key events during the initial phase of testicular differentiation based on the results from our laboratories during the last 15 years. The studies on early stages of gonadal development by other investigators have previously been reviewed (Jost et al., 1973; Merchant-Larios, 1984; Byskov, 1986; Jost and Magre, 1988; Watenberg, 1989). ESTABLISHMENT O F A SEXUALLY UNDIFFERENTIATED GONAD Figure 1shows a cross-section of a typical rat genital ridge prior to sexual differentiation. It consists of somatic and primordial germ cells tightly packed and attached to the surface epithelium. At this early stage of development, three important morphogenetic characteristics can be recognized condensation of somatic cells along the genital ridge, gradual deposition of basal laminar components around the cluster of epithelial-like cells, and a low mitotic activity of the epithelial-like cells in the genital ridge. As a consequence of these changes, gonadal cords (previously called sex cords) are formed in the genital ridge of both XX and XY gonads. The origin of epithelial-like cells in the genital ridge has not been identified. However, conversion of the mesodermal mesenchyme into epithelial cells has been well documented during differentiation of the metanephros (Grobstein, 1956), which shares the embryonic origin with the gonad. Therefore, it is reasonable to assume that a similar process of cellular differentiation occurs during the establishment of a primordial gonad. During differentiation of the metanephros, two events have been described a t the molecular level: sequential changes of extracellular matrix components and cellcell adhesion molecules (Ekblom et al., 1985). In the sexually undifferentiated gonad, basal lamina is gradually formed around the cluster of epithelial-like cells, thus developing gonadal cords (Fig. 2) (Merchant, 1975;Pelliniemi, 1975). These ultrastructural observations have recently been confirmed by immunocytochemical staining for basement membrane components (Pelliniemi et al., 1984; Paranko, 1987; Gelly et al., 1989). Interestingly, the epithelial-like cells of gonadal cords show a low mitotic activity: less than 10% of epithelial-like cells incorporate [H3] thymidine while 35% of mesenchymal and mesothelial cells in the same region are labeled (Figs. 3, 4) (Merchant-Larios, 1979). This finding may indicate that the epithelial-
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like cells undergo an essential differentiation process in both XX and XY gonads prior to sexual differentiation. TESTICULAR DIFFERENTIATION Normal Development The first event of sexual differentiation is the gradual invasion of mesenchyme and endothelial cells into the genital ridge in the XY gonad (Fig. 5). As a result, gonadal cords become conspicuous, first in the medullary region, and then in the cortex region. In the mouse, this sequencial event is typically observed from the mid area to the cranial and caudal poles because the cortex in the mid portion is very thin. At the ultrastructural level, Sertoli cells are the first cell type to differentiate, as identified by the development of the rough endoplasmic reticulum (Fig. 7) (Merchant-Larios, 1976a). This cytological change may reflect active production of MIS. However, it must be pointed out that ultrastructural differentiation of Sertoli cells follows the invasion of stromal cells and segregation of gonadal cords from the surface epithelium. It is not clear whether physiological differentiation of Sertoli cells takes place without prominent morphological changes before the invasion of stromal cells. Sertoli precursor cells may stimulate the proliferation or migration of stromal cells. Differentiation of Leydig cells and peritubular myoid cells is usually seen a t later stages of development (Fig. 7). Development of Gonadal Cords in the XY Ovotestis When the Y chromosome of Mus musculus domesticus (YDoM) is placed onto the C57BU6J (B6) mouse background, the XY progeny (B6.YDoM)develop either ovaries or ovotestes but not normal testes during fetal life (Eicher et al., 1982; Eicher and Washburn, 1986; Nagamine et al., 1987a,b). This example of sexreversal provides strong evidence for the concept that sexual differentiation begins with an epithelial compartment in the primordial gonad. In the B6.YDoMfetal ovotestis, gonadal cords become conspicuous in the medullary region due to the invasion of stromal cells as seen in the normal XY fetal testis, whereas gonadal cords remain attached to the surface epithelium at the cranial and caudal poles as seen in the normal XX fetal ovary (Fig. 8) (Nagamine et al., 1987a; Taketo-Hosotani et al., 1989). Inside the gonadal cords of the ovarian region (ovarian gonadal cords), germ cells enter meiotic prophase as seen in the XX fetal ovary (Fig. 9). In contrast, inside the seminiferous cords, germ cells are arrested a t the prospermatogonia stage (Fig. 10). Leydig cells are seen only around the seminiferous cords, suggesting that fetal Sertoli cells may possess an inductive influence on stromal cell differentiation. Thus, gonadal cords develop into two distinct sexual structures despite the identical XY genetic background. Seminiferous cords and ovarian gonadal cords often remain enveloped by common basal laminae (Fig. 11). In the boundary region, occasional germ cells in meiosis are found in the gonadal cord that is surrounded by stromal cells and, therefore, resembles seminiferous cords. No ultrastructural differences were
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Fig. 1. Cross section of the genital ridge from a rat embryo at 12.5 day of gestation (d.g.1. Primordial germ cells (pgc) are embedded in the condensation of epithelial-like cells (EI). x 500.
Fig. 2. Electron micrograph of epithelial-like cells (EI) in a sexually undifferentiated rat gonad at 12.5 d.g. Deposit of basal laminar components (arrows) is demonstrated by staining with ruthenium red. x 7,000.
Fig. 3. Autoradiograph of a rat XY gonad at 13.5 d.g. labeled by incorporation of [H3] thymidine. [H3] thymidine was injected into the pregnant female 3 h before dissection of fetuses. The invasion and proliferation of stromal cells (st) delineates seminiferous cords (sc). Note that only a few epithelial cells inside the seminiferous cords are labeled. x 400.
Fig. 4. Autoradiograph of a rat XX gonad at 13.5 d.g. labeled by incorporation of [H31 thymidine. Most of the labeled cells inside gonadal cords (gc) are primordial germ cells (pgc) whereas only a few epithelial-like cells are labeled. Many mesenchymal cells (m)
and epithelial cells in the surface epithelium (se) are labeled. x 400. Fig. 5. Cross-section of a fetal mouse testis at 14 d.g. Seminiferous cords (sc) are clearly separated from the surface epithelium by stroma1 tissue (st).Dark cells around the seminiferous cords correspond to Leydig cells (L). x 200. Fig. 6. Cross-section of a fetal mouse ovary at 14 d.g. Gonadal cords (gc) remain attached to the surface epithelium. Note the scarce invasion or proliferation of mesenchymal cells.
Fig. 7. Part of a seminiferous cord in a rat testis at 15 d.g., which is composed of prospermatogonia (ps) and fetal Sertoli cells (S).In the cytoplasm of Sertoli cells, the endoplastic reticulum (er) is well developed. A Leydig cell (L) is seen among the stromal cells. x 7,500.
Fig. 8. Longitudinal section of a n ovotestis from a B6.YDoMmouse fetus at 14 d.g. Seminiferous cords (sc) in the medullary region appear interconnected through the rete ovarii (ro) with gonadal cords (gc) in the cranial and caudal regions. Note the abundant mesenchymal cells around the seminiferous cords. x 200.
Fig. 9. The ovarian region in a serial section of the ovotestis shown in Fig. 8. Most oocytes (0)have entered the meiotic prophase. Note that gonadal cords are mainly occupied by oocytes. x 600. Fig. 10. The testicular region in a serial section of the ovotestis shown in Fig. 8. Leydig cells (L) are seen around the seminiferous cords (sc). Inside the seminiferous cords, several germ cells are arrested a t the prospermatogonia stage (ps). x 600.
Fig. 11. Part of an ovotestis from a B6.YDOMmouse fetus a t 14 d.g. At the point indicated with a n asterisk, a seminiferous cord (sc) is continuous with ovarian gonadal cords (ogc), in which most oocytes have entered meiosis. Dark cells in the stroma are Leydig cells (L). x 300.
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found between the epithelial cells in the two regions. These observations strongly support the hypothesis that ovarian gonadal cords share the embryonic origin with seminiferous cords. The XY ovotestis also provides interesting information about the onset of testicular and ovarian differentiation. In the control B6 strain, seminiferous cords become conspicuous and fetal Sertoli cells initiate cytological differentiation, as demonstrated by immunocytochemical staining for MIS, in all XY gonads by 12 d.g. (Taketo et al., 1991) (Fig. 12). By contrast, in the B6.YDoMgonad, seminiferous cords are hardly recognizable at 12 d.g. Even at 13 or 14 d.g., some XY ovotestes, as identified by the invasion of stromal cells, show no or only faint staining for MIS. By 15 d.g., all XY ovotestes have developed seminiferous cords composed of fetal Sertoli cells, which are intensely stained for MIS (Fig. 13). Thus, the onset of testicular differentiation is severely delayed during the development of XY ovotestes. Further delay in the onset of testicular differentiation probably allows the ovarian differentiation pathway to be switched on, thus producing XY ovotestes or ovaries. In the boundary area between testicular and ovarian regions, a gradient of staining for MIS is often seen, with stronger staining on the side of gonadal cords in contact with stromal cells (Fig. 13). PERSISTENCE OF GONADAL CORDS IN THE XX OVARY During the early phase of ovarian differentiation, epithelial-like cells proliferate slowly in contrast to active proliferation of oogonia in the genital ridge (Figs. 4,6). The invasion of mesenchymal cells is absent and gonadal cords remain closely associated with the surface epithelium. Consequently, it becomes difficult to identify the epithelial cells inside the ovarian gonadal cords under the light microscope. Probably for these reasons, some authors have considered that gonadal cords are never formed in the XX gonadal primordium (Jost and Magre, 19881, or that gonadal cords are newly formed in the ovary after loss of oocytes (Vigier et al., 1987; Prepin and Hida, 1989. Behringer et al., 1990). In the XX gonad of W/Wir mutant mice or busulphan-treated rats, in which oogonia are absent or scarce, gonadal cords become conspicuous due to the growth of stromal cells (Figs. 14, 15) (Merchant, 1975; Merchant-Larios, 197613; Merchant-Larios and Centeno, 1981). By comparing the development of normal and oocyte-depleted ovaries, we concluded that oocytes are essential for the establishment of primordial follicles, which are formed by “splitting” of gonadal cords (Merchant-Larios, 1978; Merchant-Larios and ChimalMonroy, 1989). The invasion and proliferation of stroma1 cells is seen in the oocyte-free ovary around 3 days after birth, a t which stage the first primordial follicles begin to form in the normal ovary. It is interesting to compare the timings of stromal invasion between the two sexes-that is, at folliculogenesis in the neonatal ovary and a t the onset of testicular differentiation in the fetal testis. At later stages, gonadal cords that have been retained in the oocyte-depleted ovary resemble seminiferous cords at the light microscopic level because of the polarization of epithelial cells (Merchant,
1975). However, at the ultrastructural level, epithelial cells inside the ovarian gonadal cord are distinct from Sertoli cells inside the seminiferous cord (Figs. 16, 17) (Merchant, 1975; Merchant-Larios, 197613; MerchantLarios and Centeno, 1981). In this regard, previous reports on the development of seminiferous cord-like structures in the XX ovary under experimental conditions need to be re-evaluated since disappearance of oocytes is the first morphological event in all cases (Taketo-Hosotani et al., 1985; Vigier et al., 1987; Prepin and Hida, 1989; Behringer et al., 1990). So far, only in one case (i.e., transplantation of fetal mouse ovaries) ultrastructural evidence has been provided to indicate that XX cells can acquire the characteristics of Sertoli cells (Figs. 18,19) (Taketo-Hosotani et al., 1984,1985). The invasion of stromal cells is prominent in the testicular region of mouse ovotestes after transplantation (Fig. 20). We have previously demonstrated that direct contact with the host kidney is essential for the induction of testicular structures in the ovarian graft (Taketo-Hosotani, 1987). Thus, in all cases of testicular differentiation known so far, the establishment of epithelial-mesenchymal interaction appears to be closely associated with cytodifferentiation of Sertoli cells. Leydig cells and myoid-like cells are seen around the seminiferous cords, suggesting again the inductive activity of Sertoli cells on stromal cell differentiation (Taketo-Hosotani et al., 1984, 1985). MECHANISM OF GONADAL SEX DETERMINATION We will discuss in this section two previously proposed hypotheses concerning the mechanism of gonadal sex determination at the organization level, and propose modifications based on our recent findings.
Significance of the Growth Rate in Sex Determination Mittwoch has observed that the growth rate of the XY gonad is greater than the XX gonad around the time of sexual differentiation (Mittwoch et al., 1969). Based on this finding, she has postulated that differentiation of Sertoli cells depends on whether the genital ridge has reached a particular developmental stage at the right time, and that failure to do so allows the gonadal primordium to undergo the ovarian differentiation pathway. Hence, in addition to the Tdy gene on the Y chromosome, a factor(s1 which influences the growth rate can modulate the development of a gonad into a testis or an ovary (Mittwoch, 1989). This hypothesis agrees with our conclusion that the delay of testicular differentiation leads to ovarian differentiation despite the presence of the normal Y chromosome in the B6.YDoMovotestis or ovary. As shown in Figures 5 and 6, the normal XY testis is larger than the XX ovary mainly due to the invasion and proliferation of stromal cells a t the onset of sexual differentiation. Since epithelial cells proliferate slowly in both XX and XY gonads, the size of gonadal cords is similar between the two sexes. Therefore, according to the hypothesis by Mittwoch, we postulate that the mechanism controlling the invasion or proliferation of
Fig. 12. Part of a testis from a B6 mouse fetus a t 12 d.g., immunocytochemically stained for MIS. ABC staining (Vector Lab, CA) with 3,3'-diaminobenzidine tetrahydrochloride. Fetal Sertoli cells inside the seminiferous cords (sc) are intensely stained while other cell types remain at the background level. Note that seminiferous cords are not completely separated from each other near the mesonephros (ms). x400. Fig. 13. Part of an ovotestis from a B6.YDoMmouse fetus at 15 d.g., immunocytochemically stained for MIS. Seminiferous cords (sc) are well organized and intensely stained for MIS in the mid portion (md). Ovarian gonadal cords remain closely attached to each other in
Note the stronger staining (indicated by arrow the caudal region (cd). heads) on the side of gonadal cords in contact with stromal tissues, but not on the opposite side, which is continuous with ovarian gonadal cords. ~ 4 0 0 . Fig. 14. An ovary from a newborn Wlwv mutant mouse. Oocytes are absent inside the gonadal cords (gc). A few oocytes (*) are seen near the surface epithelium. x 160. Fig. 15. An ovary from a newborn rat which has been treated with busulphan on 12 d.g. Several gonadal cords (gc) devoid of oocytes are surrounded by stromal cells (st). x 200.
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Fig. 16. Electron micrograph of a gonadal cord in a busulphan-treated rat ovary at 15 days after birth. Epithelial cells (E) are well differentiated and polarized inside the ovarian gonadal cord. The junctional complexes with dense desmosomes (*), typical of epithelial cells, are seen at the luminal side (Lu) of plasma membranes. x 7,000.
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Fig. 17. Electron micrograph of a seminiferous cord in a mouse testis a t 10 days after birth. A prospematogonium (ps) and several Sertoli cells (S) are seen inside the seminiferous cords. Charaderistic intersertoli junctional specializations (*) are evident between Sertoli cells. x 7,500.
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Fig. 18. Electron micrograph of a seminiferous cord in a mouse ovarian graft. A fetal ovary at 12 d.g. was transplanted beneath the kidney capsule of an adult male mouse and fixed 14 days later. Intersertoli junctional specializations (*) are well developed between Sertoli cells. x 10,500.
Fig. 19. High magnification of the intersertoli junctional specialization (*) shown in Fig. 18, which is characterized by the presence of an intercellular space (about 7-9 nm) and a flat cisternum of endoplasmic reticulum parallel to plasma membranes. Some ribosomes are seen on the cytoplasmic side. x 21,000.
Fig. 20. A mouse ovarian graft, 14 days after transplantation. Seminiferous cords (sc) have developed over a large area next to follicles (f). Note the massive invasion of stromal cells (st) around the seminiferous cords whereas it is less abundant around follicles. bv, blood vessels. x 200.
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stromal cells must be responsible for the difference of gonadal size and, hence, may modulate gonadal sex determination. In the XX gonad, on the other hand, massive invasion and proliferation of stromal cells occurs a t the onset of folliculogenesis. This is the developmental stage at which the ovarian graft initiates differentiation of testicular components (Taketo-Hosotani et al., 1985). The timing of testicular differentiation appears to be closely associated with the rapid growth of stromal cells in both sexes.
Autonomous Expression of the Tdy Gene in Sertoli Cells Burgoyne et al. have demonstrated that in the adult testis of XX-XY chimeric mouse, Sertoli cells are exclusively XY while other testicular somatic cells are either XX or XY (Burgoyne et al., 1988).This finding has lead to a hypothesis that the Tdy gene is expressed in Sertoli precursor cells to trigger autonomous cytodifferentiation, which, in turn, regulates differentiation of other testicular cell types regardless of the chromosomal sex (Burgoyne, 1988). This hypothesis excludes the possibility that a diffusible factor may be involved in the primary testis determination. These observations have been made in the adult testis, and, therefore, we do not yet know what happens in fetal gonads of the XX-XY chimeric mouse. If XX cells can differentiate into Sertoli cells, as suggested by transplantation experiments, we can postulate a mechanism preventing XX cells from contributing to Sertoli cells at the onset of testicular differentiation. Alternatively, only XY cells may be able to reach the developmental stage to differentiate into Sertoli cells a t the right time according to the hypothesis by Mittwoch. This does not exclude the possibility that XX cells can follow a similar process a t a later stage of development under experimental conditions. Since the stromal change always precedes cytological differentiation of Sertoli cells, it is necessary to assume that XY epithelial cells in gonadal cords secrete a substance which induces the invasion and proliferation of stromal cells if one accepts the hypothesis by Burgoyne. The establishment of epithelial-mesenchymal interaction may, in turn, play an essential role in cytodifferentiation of Sertoli cells. CONCLUDING REMARKS Gonadal cords are formed in the genital ridge of both XX and XY primordial gonads. Testicular differentiation is characterized by the spatial separation of gonadal cords due to the growth of stromal cells, followed by cytodifferentiation of Sertoli cells and other testicular cell types. These events lead to the establishment of the characteristic structure of seminiferous cords. On the other hand, ovarian differentiation is characterized by the onset of meiosis in primordial germ cells into oocytes. Gonadal cords are not conspicuous a t the early phase of ovarian differentiation because of poor stromal growth. Consequently, follicles are formed by splitting of gonadal cords while stromal cells begin to proliferate actively in the interstitiurn. The absence or loss of oocytes results in the failure of folliculogenesis
and, therefore, the persistent presence of gonadal cords. These cords may resemble seminiferous cords at the light microscopic level, but do not necessarily represent true testicular differentiation.
ACKNOWLEDGMENTS We are grateful to Dr. Y. Nishioka for the mouse Y-specific DNA probes and Dr. P. Donahoe for the antiserum against MIS. We thank Jose G . Baltazar for preparing tissue sections, Jose Aviles for photomicrographs, and Jamilah Saeed for technical assistance. A part of this study is supported by MRC (Canada) grants to T.T. NOTE ADDED IN PROOF Since the acceptance of this manuscript, a single copy gene, named SRY in human and Sry in the mouse, has been identified and proposed as the testis determining gene on the Y chromosome (Sinclair et al., 1990; Gubbay et al., 19901, strongly supported by several lines of direct and indirect evidence (Koopman et al., 1990, 1991). Expression of the Sry gene appears to be the highest in the fetal gonad just prior to testicular organization and diminish afterward (Koopman et al., 1990). The gene product of Sry is expected to be a transcription factor according to the nucleotide sequence analysis, and, hence, the major interest in sex determination in mammals is shifting to the mechanism downstream of the Y-encoded gene. REFERENCES Behringer, R., Cate, R.L., Froelick, G.J., Palmiter, R.D., and Brinster, R.L. (1990) Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nature, 345:167-170. Burgoyne, P.S. (1988) Role of mammalian Y chromosome in sex determination. Phil. Trans. R. SOC.Lond., B322:63-72. Burgoyne, P.S., Buehr, M., Koopman, P., Rossant, J.,and McLaren, A. (1988) Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX-XY chimaeric mouse testes. Development, 102:443-450. Byskov, A.G. (1986) Differentiation of mammalian embryonic gonad. Physiol. Rev., 66:71-117. Eicher, E.M., and Washburn, L.L. (1986) Genetic control of primary sex determination in mice. Ann. Rev. Genet., 20:327-360. Eicher, E.M., Washburn, L.L., Whitney, J.B., and Morrow, K.E. (1982) Mus poschiavinus Y chromosome in the C57BLi6J murine genome causes sex reversal. Science, 217:535-537. Ekblom, P., Thesleff, I., and Sariola, H. (1985) The extracellular matrix in tissue morphogenesis and angiogenesis. In: The cell in Contact: Adhesion and Junctions a s Morphogenetic Determinants. G.M. Edelman and J.P.Thiery, eds. John Wiley & Sons, New York, pp. 365-392. Gelly, J.L., Richoux, J.P., Leheup, B.P., and Grignon, G. (1989) Immunolocalization of type IV collagen and laminin during rat gonadal morphogensis and postnatal development of the testis and epididymis. Histochemistry, 93:31-37. Grobstein, C. (1956) Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell. Res., 10424-440. Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou, A . , Munsterberg, A., Vivian, N., Goodfellow, P.,. and Lovell-Badge, R. (1990) A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature, 346245-250. Haseltine, F.P., Genel, M., Crawford, J.D., and Breg, W.R. (1981) H-Y antigen negative patients with testicular tissue and 46,XY karyotype. Hum. Genet., 57:265-268. Jost, A., and Magre, S. (1988) Control mechanisms of testicular differentiation. Phil Trans. R. SOC.Lond., B322:55-61.
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