Germinal cell ectopism in the strepsirhine prosimian Galago crassicaudatus crassicaudatus

The presence of germinal cells outside of the embryonal and fetal gonads of the strepsirhine prosimian Galago crassicaudatus crassicaudatus is described. Forty-three embryos and fetuses from day 26 or 27 of gestational age to near term were studied: more than 90% possessed germinal cells in ectopic sites situated either far from (extragonadal ectopism) or close to the gonads (perigonadal ectopism). The first sites were the walls of the aorta and mesenteric artery, the stroma between the aorta and the cardinal vein, and the retroperitoneal neuroganglia. The second were the mesenchyme dorsal to the gonads and around the vestigia of the mesonephric glomeruli and tubules, and the rete ovarii and testis. The ectopic cells were generally present in conscpicuous numbers, in some animals being more numerous than in the gonads. Those situated far from the gonads underwent degeneration and decreased significantly in numbers during post-embryonal stages of development, while the others remained numerous and functionally active up to near term. While the differentiation of the extragonadal germinal cells after day 60 of gestational age could not be studied due to technical difficulties, the XX and XY cells in perigonadal sites appeared to follow patterns of differentiation identical to those of their entopic counterparts.     

Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice

The sex-determining region of Chr Y (Sry) gene is sufficient to induce testis formation and the subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males, such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth because of germ cell-autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here, we demonstrate that the testicular somatic environment of XX/Sry males is defective in supporting the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, of entering meiosis and of differentiating to the round-spermatid stage. XY-donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, resulting in severe deficiency of elongated spermatid stages. By contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY-donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed the missing expression of several Y-linked genes and the alterations in the expression profile of genes associated with spermiogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, highlighting the idea that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.     

Development of the rete testis in the prenatal ontogeny of rodents and effect of prolactin and thyrotropin on its cellular differentiation

The development of rete testis in the rat, rabbit and guinea pig foetuses has been studied, as well as the influence of prolactin and thyrotropin on differentiation of its cells. It was shown that the rete testis tubules, as well as the seminiferous tubules develop from sex cords, which were derived from coelomic epithelium cells and gonocytes. The development of seminiferous tubules and rete testis was described at various stages of prenatal ontogenesis. Thyrotropin and prolactin exert different effects on differentiation of the rete testis cells: the former increases the mitotic activity of gonocytes and the latter increases that of epithelial cells and enhances degenerative processes in primary germ cells.     

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.     

Pathology of 46, XY pure gonadal dysgenesis: absence of testis differentiation associated with mutations in the testis-determining factor

Abstract. Individuals with 46, XY pure gonadal dysgenesis present with a completely female phenotype. These individuals develop bilateral streak gonads and have normal Müllerian structures. The apparent absence of testicular tissue in these individuals suggests a mutation in the initial steps of the male sex-determining pathway. A candidate gene for the primary signal in this pathway was recently cloned ( SRY ) which encodes a protein with a DNA-binding capacity. In a study of 14 XY females with pure gonadal dysgenesis harbouring SRY , we analysed the histology of the gonads and compared it to the presence or absence of mutations in the SRY open reading frame ( SRY -orf). The histological analysis revealed two distinct groups of streak gonads. In the first group, the gonad was composed of exclusively ovarian-like stroma, with sclero-hyaline nodules in some areas. No tubules were observed. The gonads in the second group were composed of undifferentiated stroma harbouring either tubules or a rete structure. This suggests that in the latter group some differentiation (towards testis formation) has occurred, whereas in the first group ovarian differentiation has been interrupted. Individuals with mutations in the SRY -orf were found to have streak gonads of the first group, whereas most of the remaining XY females without detectable mutation in the SRY -orf had streak gonads belonging to the second group.
On the basis of histology, it may be possible to distinguish between mutations in the sex-determining or sex-differentiation pathways. We suggest that SRY may play a role in rete testis formation. We also present arguments favouring the mesonephros as the origin of testicular somatic cells in humans.     

Morphological development and characterization of aromatase and estrogen receptors alpha and beta in fetal ovaries of cattle from days 110 to 250

To better understand the role of estradiol-17β in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERα) and beta (ERβ) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2–5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERα protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERα protein expression in stroma and rete masses. ERβ protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERβ protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17β, and estrogen receptors α and β to facilitate an estrogen response within the fetal ovary.     

Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX----XY chimaeric mouse testes

The distribution of XX and XY cells in XX----XY chimaeric mouse testes was analysed by enzyme marker analysis of separated testicular tissues and by in situ DNA marker analysis of air-dried testicular cells and testis sections. XX cells contributed to the Leydig cells, the peritubular cells and the vascularized connective tissue of the tunica albuginea. The Sertoli cells, on the other hand, appeared to be exclusively XY. These results indicate that during the development of the testis, Sertoli cell differentiation is triggered by cell-autonomous activity of the Y chromosomal testis-determining gene Tdy. Subsequent steps in testis differentiation may be a consequence of Sertoli cell activity.     

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.     

A sex cord‐like structure and some remarkable features in early gonadal sex differentiation in the marine teleost Siganus guttatus(Bloch)

Several notable features of early gonadal sex differentiation in the golden rabbitfish Siganus guttatus are described including the first report among teleosts of a distinctive dual structure, consisting of somatic cells directly enclosing germ cells (sex cord‐like structure, SCS) and outer somatic tissue surrounding the SCS, in both undifferentiated and early differentiated gonads. Germ cells occurred and proliferated exclusively in the SCS during the process of ovarian and testicular differentiation. A second remarkable characteristic was the delayed germinal cell proliferation for oogenesis in the ovary, that commenced simultaneously with that in the testis, a relatively long time after the onset of somatic development. These observations suggest the possibility that sex differentiation of germ cells is preceded by some sex specific changes in somatic components of the SCS that are light‐microscopically indistinguishable between the sexes. The third unique feature was the detachment of gonadal tissue, including both somatic and germ cells, into the ovarian cavity in the ovary and into the seminiferous lobules and main seminal duct in the testis. This phenomenon occurred in the testis, forming the efferent duct network after 73 days post‐hatch (DPH), and in the ovaries, forming the ovigerous lamellae and regulating the number of oocytes attaining full maturation at c . 129 DPH.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.     

Effect of an analog of proline (L-azetidine-2-carboxylic acid) on in vitro differentiation of the rat fetal testis

The initial stages of the development of the seminiferous cords involve the differentiation and the aggregation of primordial Sertoli cells opposite to cells which acquire a mesenchymal-like aspect. The hypothesis that the development of the seminiferous cords depends on epithelial-mesenchymal relations between the two cell types was submitted to experimental test. Male gonadal primordia of rat fetuses were cultured in vitro in a synthetic medium containing the proline competitor, L-Azetidine-2-Carboxylic Acid. This drug is known to disturb the synthesis and secretion of collagen and proline-containing proteins. It prevents testicular organogenesis or destroys it if it has begun. It suppresses the expression of laminin and fibronectin in the gonadal primordium. These observations are taken as evidence that cellular correlations of the epithelial-mesenchymal type play a role in the development of the testis as they do in that of other organs.     

Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice

Daily oral administration of ethinyl estradiol (0.02, 0.2, or 2.0 mg/kg of body weight) to pregnant Jc1:ICR mice resulted in ovotestis and intra-abdominal testis with persistent Müllerian duct and Wolffian duct in male fetuses and ovarian hypoplasia in female fetuses when it was given from day 11 through day 17 of gestation (before gonadal differentiation in the fetus). The ovotestis consisted of testicular and ovarian portions. In the testicular portion, a few solid seminiferous tubules containing spermatogonia, some with pachytene nuclei with Sertoli cells and compact interstitial tissue including Leydig cells, were seen. In the ovarian portion, pachytene nuclei were seen. The intra-abdominal testis was smaller and contained more spermatogonia per tubule in cross section than the control testis. These findings suggest that in male fetuses ethinyl estradiol affects Sertoli cell differentiation resulting in suppression of Müllerian inhibiting factor. On the other hand, in the ovarian hypoplasia, the primordial follicles and follicular cells in a primordial follicle were significantly decreased in number, and the number of the degenerated primordial follicles was significantly increased. It seems likely that ethinyl estradiol affects the intimate contact between follicular cells and oocytes to cause degeneration of primordial follicles.     

Composition of luminal fluid secreted by the seminiferous tubules and after reabsorption by the extratesticular ducts of the Japanese quail, Coturnix coturnix japonica

The present report examines the composition of luminal fluid in the seminiferous tubule (STF), rete testis (RTF), and ductus epididymidis of the Japanese quail (Coturnix coturnix japonica). This subject is of particular interest, both because the reproductive ducts are intra-abdominal and because sperm production is more rapid in birds than in mammals. It was interpreted that micropuncture samples of STF contain varying amounts of contamination with intracellular solute, particularly K and protein. The concentration of solute in samples was correlated with packed cell volume (spermatocrit), and when the latter was used to assess estimates of solute concentration in STF, the magnitude of the estimates were much the same as determinations in RTF. Consequently, it is concluded that the fluid entering the rete testis of the quail is the primary secretion of the seminiferous tubules. The composition of RTF in the quail was determined to be 148 mM Na, 126 mM Cl, 9.8 mM K, 2.7 mM Mg, 1.4 mM Ca, 2.1 mM glutamate, 3.4 mM glutamine, 20.2 mM bicarbonate, 1.8 microg microl(-1) of protein, pH 7.34, and 310 mmol kg(-1), and it is significantly different from the composition of blood plasma. Estimates of solute output by the testis and reabsorption by the extratesticular ducts indicate, first, that most of the solutes secreted into the seminiferous tubules are subsequently reabsorbed from the extratesticular ducts and, second, that sufficient solute of testicular origin (except for protein) exists to account for the concentrations of solutes throughout the lumen of the duct system. Changes in the concentration of solute in the extratesticular ducts probably result from different reabsorption rates of solute and water. The composition of fluid from the distal end of the ductus epididymidis was 133 mM Na, 125 mM Cl, 25 mM K, 1.0 mM Mg, 0.3 mM Ca, 6.7 mM glutamate, 4.0 mM glutamine, 19.5 mM bicarbonate, 6.0 microg microl(-1) of protein, pH 7.33, and 335 mmol kg(-1), and it is significantly different from those of RTF and blood.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

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.     

A successful second delivery outcome using refrozen thawed testicular sperm from an infertile male true hermaphrodite with a 46, XX/46, XY karyotype: case report

We report a successful second delivery of a healthy infant fathered using refrozen thawed testicular sperm from an infertile male chimera. We also examined sex chromosome distribution of the seminiferous tubule. Intracytoplasmic sperm injection (ICSI) was performed using the remaining refrozen testicular sperm, which had been stored during the first treatment. Biopsied testicular cells were examined by fluorescence in situ hybridization (FISH) and the peripheral lymphocyte karyotype was tested using a G-band. Following ICSI, a second pregnancy was established, and a healthy girl was successfully delivered at 40 gestational weeks without complications. Although the husband’s lymphocyte chromosomal analysis revealed a 46, XX [28]/46, XY [2] karyotype, the seminiferous tubule cells on histological examination by FISH were chimeric sex chromosome type XX [18]/XY [82]. In conclusion, this is a very rare case report of a successful subsequent delivery of a healthy infant (46, XX) from an infertile true hermaphrodite (46, XX/46, XY) using refrozen thawed testicular sperm. The seminiferous tubule cells’ karyotype ratio differed from that of the lymphocytes.     

The rete testis of the goat, a morphological study

The rete testis of the goat can be divided into three parts, septal, mediastinal and extratesticular. The septal rete is short, relatively straight and connects the terminal part of the seminiferous tubules with the mediastinal rete. The mediastinal rete is a labyrinth of intercommunicating channels that occupies about two-thirds of the central axis of the testis. The extratesticular rete is located outside the testis at its extremitas capitata and forms sac-like dilations up to 2 mm in diameter. The rete testis, regardless of its location, is lined by simple cuboidal to simple squamous epithelium that invariably contains a few intra-epithelial lymphocytes and macrophages. The epithelial cells possess few microvilli and a centrally located flagellum at the luminal border. With the exception of a few small vesicles in the apical cytoplasm, morphological features associated with absorption such as coated vesicles, canaliculi, vacuoles and multivesicular bodies are absent. However, basolateral interdigitations between adjacent cells, another feature of absorptive epithelium, are frequently noticed. Most cytoplasmic organelles except mitochondria and free ribosomes are poorly developed, suggesting that caprine rete epithelial cells are not associated with protein/glycoprotein secretion. There is no evidence of sperm phagocytosis by the rete epithelium, but luminal macrophages containing sperm fragments are occasionally encountered. The structural-functional relationships of the rete epithelial cells are discussed.