Sex-specific localization of laminin alpha 5 chain in the differentiating rat testis and ovary

The localization of laminin (Ln) alpha 5, beta 1 and beta 2 chains in the differentiating rat testis and ovary was studied by immunolabeling light and electron microscopy. The initial formation of the male and female gonadal blastemas included an emergence of Ln alpha 5 and beta 1 chains, but not of Ln beta 2 chain. The sexual differentiation of the embryonic male gonadal cords included rapid sex-specific disappearance of the incipient Ln alpha 5 chain. The rete testis cords, in contrast, remained positive for Ln alpha 5 chain. In the postnatal testis, the Ln alpha 5 chain reappeared in Ln beta 1 chain-positive cord basement membranes, which also became positive for Ln beta 2 chain. The differentiating myoid cells also gradually became positive for both Ln alpha 5 and Ln beta 1 chains. In the ovary Ln alpha 5 chain persisted in BMs of the cords throughout the fetal phase. Small and newly formed follicles in the early postnatal rat ovary were also positive for Ln alpha 5 chain, whereas growing and large follicles were negative. During the early postnatal phase, Ln beta 1-chain positive follicular BMs became also positive for the Ln beta 2 chain. Basement membranes of testicular and ovarian surface epithelia contained the Ln alpha 5 chain throughout the study. The blood vessels of the male and female gonad showed differentiation-dependent variation in their reactivity for the Ln alpha 5 and beta 2 chains. The present results show that the Ln alpha 5 chain is an early molecular marker for sexual differentiation, which therefore may be regulated by the testis-determining factors. The results also show that in the early postnatal rat ovary, the follicular basement membranes are heterogeneous in their Ln content, which may offer a means to distinguish different follicular populations from each other and to identify the different stages of follicular growth.     

Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in vitro

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal lesions observed in bovine genetic females united by placental anastomoses to male twins (freemartins), prospective ovaries of fetal rats were exposed to purified bovine AMH in vitro. In cultures initiated at 14 days p.c. and maintained 3 to 10 days, AMH consistently induced a characteristic 'freemartin effect', namely reduction of gonadal volume, germ cell depletion and differentiation, in the gonadal blastema, of epithelial cells with large clear cytoplasm linked by interdigitations, resembling rat fetal Sertoli cells. These cells tend to become polarized and form cords, delineated by a continuous basal membrane containing laminin and fibronectin. Such structures, resembling developing seminiferous cords, were not detected in control ovarian cultures. These data strongly suggest that AMH is the testicular factor responsible for triggering the morphological abnormalities of freemartin gonads.     

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-α and cytochrome P450aromatase genes in the rat ovary

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-α subunit and cytochrome P450 aromatase (P450_arom) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100–200 μm maximum diameter) and ten healthy antral ( > 300 μm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-α, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-α subunit and P450_arom were quantified by real-time polymerase chain reaction (PCR). While the amount of P450_arom mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-α mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-α subunit mRNA independently of antral formation in immature rat ovaries.     

Intermediate filaments and epithelial differentiation of male rat embryonic gonad

The presence and distribution of desmin, vimentin, cytokeratin, and laminin in the gonads of developing male rat embryos (11-17 days) were studied by immunocytochemistry. The findings were correlated with morphological changes of the cells and with the formation of basement membranes, as determined by electron microscopy. The surface epithelial and subepithelial cells of the meesonephros in the prospective gonadal region contained desmin. At the onset of gonadal development, vimentin appeared in the somatic cells of the thickening surface epithelium, which formed the gonadal ridge. Desmin disappeared and cytokeratins appeared in the Sertoli precursor cells at the inception of their epithelial differentiation. Simultaneously, the prospective Sertoli cells became polarized during their assembly into epithelial cell aggregates; the aggregates then fused and formed elongated testicular cords. The epithelial cell differentiation was accompanied by a deposition of basement membrane material around the cords and by an increase of desmin in the cells immediately around the cords. With further differentiation of the testicular cords, some cytokeratins from the Sertoli cells, but not from the cells of the rete cords, disappeared. On the other hand, other cytokeratin polypeptides and vimentin remained in the fetal Sertoli cells. The surface cell layer slowly differentiated towards a proper epithelium after the basic formation of the testicular cords and interstitium. Desmin and vimentin persisted in the interstitial cells throughout the entire study period. The early differentiation of the gonad is apparently under a general sex-independent initiation program. The developmental changes in intermediate filaments offer an opportunity for the further analysis of their general role in early organogenesis. In light of the genetic theory of testicular differentiation, the functions of the regulatory factor(s) include specific organization of cord cells, histological organization into looping cords rather than separated follicles, and male development of the interstitium, surface epithelium and tunica albuginea.     

Differential expression of acidic cytokeratins 18 and 19 during sexual differentiation of the rat gonad.

The expression of cytokeratins (CKs) 8, 18 and 19 was analyzed in male and female rat gonads from the undifferentiated stage (12.5 days of gestation) until two weeks after birth by indirect immunofluorescence, using specific monoclonal antibodies anti-CK 8 (LE41), anti-CK 19 (LP2K) and anti-CK 18 (LE65 and RGE53). In the undifferentiated blastema, the somatic cells were stained for CK 8 and CK 19, whereas no detectable immunoreactivity for CK 18 was obtained. The same staining CK pattern was observed in ovaries, in the somatic cells of ovigerous cords and in primary follicles. The staining was progressively decreasing in growing follicles after one week after birth. At the onset of testicular differentiation, when the first Sertoli cells differentiate in the gonad of 13.5-day old male fetuses, positive staining for CK 18 became evident, in addition to CK 8 and CK 19 expression. In the following days, CK 8, CK 18 and CK 19 were detected in Sertoli cells in the differentiating seminiferous cords, but progressively the reactivity for CK 19 decreased and was no longer observed after 18.5-19.5 days of gestation. In all cases, CKs were found to be coexpressed with vimentin, and germ cells were negative for both vimentin and CKs. The results reported here show first, that CKs are expressed before sexual differentiation in gonadal blastema in which no epithelial organization is observed, and second, that there is a CK 18/CK 19 shift in expression during morphogenesis of the testis which is not observed in the differentiating ovary. Future studies will have to determine whether these differences in CK expression are due to epitope-masking phenomena or to the regulation of CK synthesis.     

Marsupial anti-Mullerian hormone gene structure,regulatory elements,and expression

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.     

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.     

Inhibin may be involved in negative feedback in the prairie dog (Cynomys ludovicianus)

The changes in inhibin immunostaining in the gonads during the annual reproductive cycle of both sexes of the prairie dog are described. No inhibin immunostaining was found in primary or secondary follicles of the ovary. Theca and granulosa cells of preovulatory Graafian follicles found in January and February stained for inhibin. Corpora lutea of both pregnant and non-pregnant females stain more densely for inhibin than follicles. Inhibin staining is present in luteal cells for at least 4 months during regression, longer than detectable progesterone is secreted. Sertoli cells in the testes do not have inhibin immunostaining during recrudescence. These cells show light immunostain for inhibin during peak spermatogenic activity in January and February but stain more deeply during early regression of the testis. Stain is gradually lost in the next 4-5 months as the tubules close. Leydig cells and germ cells do not stain for inhibin at any stage of the annual cycle but interstitial cells and tunic cells stain during the breeding phase. The presence of immunochemical staining for inhibin in prairie dog gonads during regression suggests that inhibin is part of a negative feedback complex that includes progesterone in the female and testosterone or another androgen in the male. Negative feedback during regression may also cause gonadal inactivity.     

The presence of a common embryonic blastema for ovarian and testicular parenchymal (follicular,interstitial and tubular) cells in cattle,Bos taurus

Summary The early embryonic gonadal development in the cattle is characterized by the appearance of an alkaline phosphatase positive blastema. Its derivatives in gonads of both sexes, follicular cells in the female and interstitial cells in the male, also show positive alkaline phosphatase reaction. Primordial germ cells are equally alkaline phosphatase positive, but loose this activity when they later transform to oögonia and oöcytes, or to spermatogonia respectively. Using the enzyme activity as label to trace these constituents in the developmental steps of the bovine gonads, the following results were obtained.Differentiation processes leading to the appearance of the sex cords take place in situ within the gonadal blastema which occupies the main central part of the gonadal fold. It is essentially a segregation process of the follicular cell cords or of the interstitial cells and the tubular primordia from the undifferentiated common anlage.The so-called germinal epithelium is not involved in the differentiation of sex cords. Its participation — if any — in the gonadal development is restricted to a very short and rather early period. Secondary sex cords (Pflügers cords) do not occur. In the cattle there is no reason to assume a cortico-medullary antagonism in the sex determined gonadal development.It can be assumed that the follicular cells in the ovary and the interstitial cells in the testis are homologous. This applies possibly also to the tubular cells of the testis. Homology should be admitted also for the rete structures, which remain small and undeveloped in the ovary while in the male they show considerable development.In the ovary the follicular cell cords differentiating within the central blastema match in a junctional zone with the peripheral layer of oögonia. These are taken up by the most peripheral branches of the follicular cell cords, thus transforming to ovigerous cords. During the downward movement within these cords the germ cells transform to oöcytes which for their part proceed through first meiotic prophase and reach the dictyotene stage. The maturation of the germ cells seems to be controlled by the follicular cells and may even temporarily get out of control until an adequate number of follicular cells is found in vicinity of individual oöcytes to form primordial follicles.The alkaline phosphatase reaction reveals the presence of numerous persisting remnants of follicular cell cords in the developing and even adult ovary.It is suggested that the findings in the cattle gonads can be applied also to other mammals, mainly to those with longer gestation periods like man.Contribution No 58-66, Department of Biology, City of Hope Medical Center. This work was supported in part by a grant (CA 05138) from the National Cancer Institute, U.S. Public Health Service. The project was undertaken during a five-month visit to Dr. Ohno's laboratory by the senior author whose expenses were covered by the Deutsche Forschungsgemeinschaft.     

Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.     

Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice

Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare’s serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin α2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins α4 and α5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.     

Early stages of testicular differentiation in the rat

Summary The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells show an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords.The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.