Regulation and perturbation of testicular functions by vitamin A

In addition to playing a fundamental role in very diverse processes such as vision and the growth and differentiation of numerous types of cell, vitamin A (retinol) and its principal biologically active derivative, retinoic acid, are clearly involved in the regulation of testicular functions in rodents. An excess of vitamin A leads to testicular lesions and spermatogenetic disorders, and a deficiency induces early cessation of spermatogenesis and adversely affects testosterone secretion. Furthermore, mice mutant for retinoic acid alpha receptors and retinoid X beta receptors are sterile. Retinoids appear to exert an action on the three main testicular types of cell (Sertoli, germinal and Leydig cells), as they act on the signalling pathways and Sertoli cell metabolism, and modify numerous factors secreted in Sertoli cells. Retinoids also appear to be necessary for the proliferation and differentiation of A spermatogonia, and for spermiogenesis. In addition, vitamin A deficiency leads to atrophy of the accessory sex organs after decreased testosterone production. Recent studies have shown that retinoids already affect these three types of cell in fetuses. Curiously, the effects of retinoids on fetal and adult testis seem opposed.     

NR5A1 is required for functional maturation of Sertoli cells during postnatal development

The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.     

Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.     

Stimulation of Sertoli cell proliferation: defining the response interval to an inhibitor of estrogen synthesis in the boar

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11-16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.     

Duplicated insertion mutation in the microtubule-associated protein Spag5 (astrin/MAP126) and defective proliferation of immature Sertoli cells in rat hypogonadic (hgn/hgn) testes

Male rats with hypogonadism (hgn/hgn) experience sterility from testicular dysplasia, which is controlled by a single recessive gene, hgn. The postnatal growth of the seminiferous tubules was severely affected. In this study, we localized the hgn locus to a 320 kb region on rat chromosome 10 and detected the insertion of a 25 bp duplication into the sixth exon of the sperm-associated antigen 5 (Spag5/astrin/MAP126) gene, which codes for a microtubule-associated protein. This mutation results in a truncated Spag5 protein lacking the primary spindle-targeting domain at the C terminus. Immunological staining with antibodies to markers for Sertoli and germ cells during the early postnatal period indicated that the abnormal mitosis with dispersed chromosomes in hgn/hgn testes occurs in proliferating Sertoli cells. Therefore, apoptotic Sertoli cell death would result from the disorganization of the spindle apparatus caused by defective Spag5. These findings suggested that the Spag5 is essential for testis development in rats and that the hgn/hgn rat is a unique animal model for studying the function of Spag5.     

Adiponectin and its receptors are expressed in the chicken testis: influence of sexual maturation on testicular ADIPOR1 and ADIPOR2 mRNA abundance

Adiponectin is an adipokine hormone that influences glucose utilization, insulin sensitivity, and energy homeostasis by signaling through two distinct receptors, ADIPOR1 and ADIPOR2. While adipose tissue is the primary site of adiponectin expression in the chicken, we previously reported that adiponectin and its receptors are expressed in several other tissues. The objectives of the present study are to characterize adiponectin, ADIPOR1, and ADIPOR2 expressions in the chicken testis and to determine whether sexual maturation affects the abundance of testicular adiponectin, ADIPOR1, and ADIPOR2 mRNAs. By RT-PCR and nucleotide sequencing, testicular adiponectin, ADIPOR1, and ADIPOR2 mRNAs were found to be identical to that expressed in the abdominal fat pad. Using anti-chicken adiponectin, ADIPOR1, or ADIPOR2 antibodies and immunohistochemistry, adiponectin-immunoreactive (ir) and ADIPOR1-ir cells were found exclusively in the peritubular cells as well as in Leydig cells. However, ADIPOR2-ir cells were found in the adluminal and luminal compartments of the seminiferous tubules as well as in interstitial cells. In particular, Sertoli cell syncytia, round spermatids, elongating spermatids, spermatozoa, and Leydig cells showed strong ADIPOR2 immunoreactivity. Using quantitative real-time PCR analyses, testicular ADIPOR1 and ADIPOR2 mRNA abundance were found to be 8.3- and 9-fold higher (P<0.01) in adult chickens compared with prepubertal chickens respectively, suggesting that sexual maturation is likely to be associated with an up-regulation of testicular ADIPOR1 and ADIPOR2 gene expressions. Collectively, our results indicate that adiponectin and its receptors are expressed in the chicken testis, where they are likely to influence steroidogenesis, spermatogenesis, Sertoli cell function as well as spermatozoa motility.     

Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36+/-7.34 microm in diameter) in a highly organized, hexagonal arrangement (376.95+/-21.93 microm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell-cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli-Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.     

Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice

The role of the gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development was examined using normal mice and hypogonadal (hpg) mice, which lack circulating gonadotrophins. The disector method was used to determine the number of cells from day 16 of gestation until adulthood. The numbers of Leydig cells did not change significantly between day 16 of gestation and day 5 after parturition in normal mice and were not significantly different from numbers in hpg mice at any age up to day 5 after parturition. There was a 16-fold increase in the number of Leydig cells in normal mice between day 5 and day 20 after parturition, followed by a further doubling of number of cells between day 20 and adulthood. The number of Leydig cells in hpg testes did not change between day 5 and day 20 after parturition but doubled between day 20 and adulthood so that the number of cells was about 10% of normal values from day 20 onwards. Leydig cell volume was constant in normal animals from birth up to day 20 and then showed a 2.5-fold increase in adult animals. Leydig cell volume was normal in hpg testes at birth but decreased thereafter and was about 20% of normal volume in adult mice. The number of Sertoli cells increased continuously from day 16 of gestation to day 20 after gestation in normal mice and then remained static until adulthood. The number of Sertoli cells in hpg testes was normal throughout fetal life but was reduced by about 30% on day 1 (day of parturition). Thereafter, Sertoli cells proliferated at a slower rate but over a longer period in the hpg testis so that on day 20 after parturition the number of Sertoli cells was about 50% of normal values, whereas in adult mice the number was 65% of normal. The number of gonocytes did not change between day 16 of gestation and day 1 and did not differ between normal and hpg testes. The number of gonocytes increased nine-fold in normal testes but only three-fold in hpg testes between day 1 and day 5 after parturition. Gonocytes differentiated into spermatogonia in both normal and hpg testes between day 5 and day 20 after parturition. These results show: (i) that fetal development of both Sertoli and Leydig cells is independent of gonadotrophins; (ii) that normal differentiation and proliferation of the adult Leydig cell population (starting about day 10 after parturition) is dependent on the presence of gonadotrophins; and (iii) that the number of Sertoli cells after birth is regulated by gonadotrophins, although proliferation will continue, at a lower rate and for longer, in the absence of gonadotrophins.     

Function and localization of oxytocin receptors in the reproductive tissue of rams

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.     

Comparison of androgen receptor and oestrogen receptor beta immunoexpression in the testes of the common marmoset (Callithrix jacchus) from birth to adulthood: low androgen receptor immunoexpression in Sertoli cells during the neonatal increase in testosterone concentrations

The aims of this study were: (i) to investigate the cellular immunoexpression of androgen receptor and oestrogen receptor beta in the testes of the common marmoset (Callithrix jacchus) during neonatal life compared with their expression at later ages; (ii) to establish whether neonatal marmoset Sertoli cells are targets for androgens or oestrogens or both; and (iii) to investigate the relationship between neonatal plasma testosterone concentrations and androgen receptor immunoexpression by abolishing the neonatal testosterone surge with a potent GnRH antagonist. Androgen receptor and oestrogen receptor beta immunoexpression were evaluated in neonatal animals aged 1-4 days, 4 weeks and 6 weeks, and compared with immunoexpression in animals aged 18-22 weeks (early infancy), 35 weeks (late infancy), 58-62 weeks (late pubertal) and > 100 weeks (adult). Immunoexpression of androgen receptor in the reproductive tract was also evaluated at each age. Sertoli cell immunoexpression of androgen receptor was weak or absent in neonatal animals, but increased substantially in infant animals, reaching adult levels by the end of infancy. In contrast, immunoexpression of androgen receptor during the neonatal period was strong in testicular interstitial cells and very strong in epithelial cell nuclei throughout the reproductive tract, and did not change greatly with age in these cells or tissues. Similarly, immunoexpression of oestrogen receptor beta was prominent in many Sertoli cells and in the germ cells of neonatal animals, and was relatively constant throughout life. Weak immunoexpression of androgen receptor in neonatal Sertoli cells was associated with high plasma testosterone concentrations (2.7-5.5 ng ml(-1)), whereas strong Sertoli cell immunoexpression was associated with baseline (approximately 0.12 ng ml(-1)) testosterone concentrations in infant animals and with > 10 ng ml(-1) in late pubertal and adult animals. Immunoexpression of androgen receptor and oestrogen receptor beta was also evaluated in co-twin males aged 4 and 35 weeks, after treatment from birth to 4 weeks or from week 25 to week 35, respectively, with either vehicle or with GnRH antagonist at a dose known to suppress the neonatal testosterone surge completely. Only GnRH antagonist treatment during weeks 25-35 reduced androgen receptor immunoexpression, whereas immunoexpression of oestrogen receptor beta was unaffected by treatment during either period. On the basis of these findings it is suggested that: (i) neonatal marmoset Sertoli cells may be targets primarily for oestrogens rather than androgens; (ii) androgen receptor expression in the testes of neonatal and infant marmosets is not regulated in a straightforward way by testosterone; and (iii) high neonatal concentrations of plasma testosterone are not absolutely necessary for expression of androgen receptor in marmoset testes at this time.     

Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.     

Expression of a blood-brain barrier-specific antigen in the reproductive tract of the male rat

The endothelial barrier antigen (EBA) is a protein expressed specifically by the endothelial cells of the rat brain barrier vessels. This antigen has been described as a 'barrier protein' and is used as a marker for the competent blood-brain barrier. A blood-testis barrier has also been described. However, unlike the blood-brain barrier, which is formed by endothelial cells, the blood-testis barrier is formed mainly by the Sertoli cells, which provide an isolated environment for spermatogenic cells within the seminiferous tubules. Testicular blood vessels express the erythroid glucose transporter protein and other markers, which are strongly expressed in brain blood vessels, and may contribute to the blood-testis barrier. This study was carried out to determine whether Sertoli cells or testicular blood vessels express EBA. Tissues of other organs were used as controls for EBA expression. EBA was expressed by the endothelial cells in most microvessels of the testis, and in a few vessels of the epididymis, seminal vesicle, prostate gland, vas deferens and bladder-neck region. Furthermore, EBA was strongly and consistently detected in epithelial cells of the rete testis and dorsolateral prostate gland, and in a few epithelial cells of the ventral prostate gland, the seminal vesicle and the coagulating gland. However, Sertoli cells, which are the main site of the blood-testis barrier, were negative for EBA. In conclusion, EBA may have a wider role in rat tissues than has been previously appreciated.     

Endocrinology of the mammalian fetal testis

The testes are essential endocrine regulators of fetal masculinization and male development and are, themselves, subject to hormonal regulation during gestation. This review focuses, primarily, on this latter control of testicular function. Data available suggest that, in most mammalian species, the testis goes through a period of independent function before the fetal hypothalamic-pituitary-gonadal axis develops at around 50% of gestation. This pituitary-independent phase coincides with the most critical period of fetal masculinization. Thereafter, the fetal testes appear to become pituitary hormone-dependent, concurrent with declining Leydig cell function, but increasing Sertoli cell numbers. The two orders of mammals most commonly used for these types of studies (rodents and primates) appear to represent special cases within this general hypothesis. In terms of testicular function, rodents are born 'early' before the pituitary-dependent phase of fetal development, while the primate testis is dependent upon placental gonadotropin released during the pituitary-independent phase of development.     

Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor

To better understand the role(s) of progestogens during early stages of spermatogenesis, we carried out studies on the nuclear progesterone receptor (Pgr) of the Atlantic salmon. Its open-reading frame shows the highest similarity with other piscine Pgr proteins. When expressed in mammalian cells, salmon Pgr exhibited progestogen-specific, dose-dependent induction of reporter gene expression, with 17α,20β-dihydroxy-4-pregnen-3-one (DHP) showing the highest potency. We then analyzed testicular pgr mRNA and DHP plasma levels in animals during the onset of spermatogenesis, which were exposed to natural light or to constant light, to induce significant differences in testis growth. Grouping of the animals according to their progress through spermatogenesis showed that testicular pgr mRNA levels as well as DHP plasma levels first increased when germ cells had reached the stage of late type B spermatogonia and further increased when entered meiosis, i.e. when spermatocytes were present. However, in situ hybridization studies revealed that pgr mRNA expression was restricted to Sertoli cells, with a strong signal in Sertoli cells contacting type A/early type B spermatogonia, while Sertoli cells contacting larger germ cell clones with further differentiated stages (e.g. late type B spermatogonia) were less intensely/not stained. We conclude that the increase in pgr mRNA levels per pair of testis reflects, at least in part, the increased number of Sertoli cells enveloping type A and early type B spermatogonia. We propose that Sertoli cell-expressed Pgr may mediate DHP-stimulated early steps in spermatogenesis in Atlantic salmon, such as an increase in the number of new spermatogonial cysts.     

Proliferation and apoptosis in bovine placentomes during pregnancy and around induced and spontaneous parturition as well as in cows retaining the fetal membranes

Placental growth can be achieved by either cellular proliferation or hypertrophy. Tissue regeneration and the nutrition of the fetus via embryotrophe require high rates of cellular turnover and the so-called pre-term 'maturation' of the placenta is correlated with a reduction of maternal crypt epithelial cells. Placentomes of 45 pregnant cows were collected from an abattoir to assess the role of proliferation and apoptosis in placental physiology and pathology. Placentomes were also taken from five cows undergoing premature Caesarean section and from ten naturally calving cows immediately after the expulsion of the fetus. Five of these animals had not released the fetal membranes after 12 h. Tissue sections of placentome were assessed for the Ki-67 protein; the TUNEL procedure was performed and verified by transmission electron microscopy. The maternal crypt epithelium and the fetal chorionic epithelium had a higher percentage of Ki-67-positive cells than the stroma. The percentage of Ki-67-positive cells increased significantly during pregnancy in fetal chorionic epithelium and was significantly decreased in fetal chorionic epithelium and maternal crypt epithelium after the expulsion of the fetus in comparison with tissue from month 9 of pregnancy. The number of apoptotic cells increased significantly during pregnancy in maternal crypt epithelium, maternal stroma and fetal chorionic epithelium as detected in slaughtered animals. Significantly more apoptotic fetal chorionic epithelial cells were found in animals retaining their fetal membranes in comparison with prepartum cattle during month 9 of pregnancy, at premature section and in animals releasing the fetal membranes completely. The results strongly indicate that bovine placentomes have cell type-specific rates of cellular turnover reflecting tissue growth, embryotrophe and placental maturation. Retention of fetal membranes is characterized by a large number of fetal chorionic epithelial cells undergoing apoptosis immediately after the expulsion of the fetus. This finding indicates that incomplete maturation of placentomes plays an important role in fetal membrane retention and that massive apoptosis after the expulsion of the fetus should be the consequence of diminished blood supply to the uterus, as verified in a recent study.     

FSH and testosterone signaling in Sertoli cells

Testosterone and follicle-stimulating hormone (FSH) are required to obtain full reproductive potential. In the testis, somatic Sertoli cells transduce signals from testosterone and FSH into the production of factors that are required by germ cells as they mature into spermatozoa. Recent advances in identifying new signaling pathways that are regulated by FSH and testosterone have allowed for refinement in the understanding of the independent, overlapping and synergistic actions of these hormones. In this review, we discuss the signaling pathways that are regulated by FSH and testosterone as well as the resulting metabolic and gene expression changes that occur as related to Sertoli cell proliferation, differentiation and the support of spermatogenesis.     

In vitro development of mouse fetal germ cells into mature oocytes

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish an in vitro experimental model that allows one to study such mechanisms. Mouse follicular development has been studied in vitro over the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were cultured in vitro for 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were matured in vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula-blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro.     

Endocrine differentiation of fetal ovaries and testes of the spotted hyena (Crocuta crocuta): timing of androgen-independent versus androgen-driven genital development

Female spotted hyenas (Crocuta crocuta) have an erectile peniform clitoris and a pseudoscrotum but no external vagina, all established by day 35 of a 110-day gestation. Recent studies indicate that these events are androgen-independent, although androgen secretion by fetal ovaries and testis was hypothesized previously to induce phallic development in both sexes. We present the first data relating to the capacity of the ovaries and testes of the spotted hyena to synthesize androgens at different stages of fetal life. Specifically, spotted hyena fetal gonads were examined by immunohistochemistry at GD 30, 45, 48, 65, and 95 for androgen-synthesizing enzymes, as related to the morphological development. Enzymes included 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5, 3beta-hydroxysteroid dehydrogenase (3betaHSD), and cholesterol side-chain cleavage cytochrome P450 (P450scc). Anti-Müllerian-hormone (AMH) expression was also examined. AMH was strongly expressed in fetal Sertoli cells from GD 30 and after. P450c17 expression was detected in Leydig cells of developing testes and surprisingly in Müllerian duct epithelium. Fetal ovaries began to organize and differentiate by GD 45, and medullary cells expressed P450c17, cytochrome b5, 3betaHSD, and P450scc. The findings support the hypothesis that external genital morphology is probably androgen-independent initially, but that fetal testicular androgens modify the secondary, male-specific phallic form and accessory organs. Fetal ovaries appear to develop substantial androgen-synthesizing capacity but not until phallic differentiation is complete, i.e. after GD 45 based on circulating androstenedione concentrations. During late gestation, fetal ovaries and testes synthesize androgens, possibly organizing the neural substrates of aggressive behaviors observed at birth in spotted hyenas. These data provide an endocrine rationale for sexual dimorphisms in phallic structure and reveal a potential source of androgenic support for neonatal aggression in female and male C. crocuta.