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.     

Differential expression of glucose transporter GLUT8 during mouse spermatogenesis

GLUT8 is a facilitative glucose transporter expressed at high levels in the testis. In this study, we analyzed the GLUT8 expression in mouse testis during spermatogenesis by RT-PCR, Western blot and immunohistochemistry methods. Our results show that GLUT8 expression is limited to spermatids and spermatozoa in the testis. Expression begins when round spermatids are formed at postnatal day 24. The expression persists throughout spermiogenesis, and it is also detected in spermatozoa, but it is absent in more immature germ cells, Sertoli cells and interstitial tissue. GLUT8 immunoreactivity is always restricted to the acrosomic system in a manner that matches the acrosome system formation. The GLUT8 expression is mainly associated with the acrosomic membrane in the acrosome, although significant immunoreactivity is also found inside the acrosomic lumen. The specific GLUT8 location suggests that this transporter plays a pivotal role in the fuel supply of spermatozoa, and in the traffic of sugars during the capacitation and fertilization processes.     

Proliferation and functional maturation of Sertoli cells,and their relevance to disorders of testis function in adulthood

Disorders of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of Sertoli cells. Failure of Sertoli cells to mature, with consequent inability to express functions capable of supporting spermatogenesis, is a prime example. In a similar way, failure of Sertoli cells to proliferate normally at the appropriate period in life will result in reduced production of spermatozoa in adulthood. This review focuses on the control of proliferation of Sertoli cells and functional maturation, and is motivated by concerns about 'testicular dysgenesis syndrome' in humans, a collection of common disorders (testicular germ-cell cancer, cryptorchidism, hypospadias and low sperm counts) which are hypothesized to have a common origin in fetal life and to reflect abnormal function of Sertoli (and Leydig) cells. The timing of proliferation of Sertoli cells in different species is reviewed, and the factors that govern the conversion of an immature, proliferating Sertoli cell to a mature, non-proliferating cell are discussed. Protein markers of maturity and immaturity of Sertoli cells in various species are reviewed and their usefulness in studies of human testicular pathology are discussed. These markers include anti-Mullerian hormone, aromatase, cytokeratin-18, GATA-1, laminin alpha5, M2A antigen, p27(kip1), sulphated glycoprotein 2, androgen receptor and Wilms' tumour gene. A scheme is presented for characterization of Sertoli-cell only tubules in the adult testis according to whether or not there is inherent failure of maturation of Sertoli cells or in which the Sertoli cells have matured but there is absence, or acquired loss, of germ cells. Functional 'de-differentiation' of Sertoli cells is considered. It is concluded that there is considerable evidence to indicate that disorders of maturation of Sertoli cells may be a common underlying cause of human male reproductive disorders that manifest at various life stages. This recognition emphasizes the important role that animal models must play to enable identification of the mechanisms via which failure of proliferation and maturation of Sertoli cells can arise, as this failure probably occurs in fetal life.     

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.     

The cholinergic system in rat testis is of non-neuronal origin

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three β subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.     

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.     

Relationship between seasonal changes in spermatogenesis in the juvenile ostrich (Stuthio camelus) and the presence of the LH receptor and 3beta-hydroxysteroid dehydrogenase

The immunohistochemical localization of the LH receptor and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied in the testis of the juvenile ostrich (Stuthio camelus) throughout a 1 year period. Spermatogenic activity of juvenile birds changed throughout the year, as has been reported previously for sexually mature birds. During the active stage of the testicular cycle, from September to January, spermatogenesis progressed up to the stage of formation of spermatozoa, although spermatozoa could not be detected in the epididymis. Leydig cells stained intensely with antibodies against the LH receptor and 3beta-HSD during the quiescent, recrudescent and active phases of the testicular cycle. During the regressive phase, there was a slight decrease in immunostaining for 3beta-HSD in these cells. These results indicate that Leydig cells in the testis of the juvenile ostrich are able to respond to LH and are capable of steroid synthesis. Furthermore, in juvenile (prepubertal) ostriches, spermatogeneic activity can be observed and, as in mature birds, spermatogenesis undergoes seasonal changes.     

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.     

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.     

The effects of oestrogen receptors alpha and beta on testicular cell number and steroidogenesis in mice

Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor alpha (ER alpha) or beta (ER beta) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ER alpha knockout mice (alpha ERKO) and ER beta knockout mice (beta ERKO) and wild-type littermates (n=5-8) were killed at 11 weeks post partum. One testis was fixed in Bouin's fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in beta ERKO mice but not in alpha ERKO mice. Plasma and testicular testosterone concentrations were increased in alpha ERKO mice but no changes were observed in beta ERKO mice. Hypertrophic Leydig cell changes were observed in alpha ERKO mice, and a decreased mean cell volume was seen in beta ERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in beta ERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in alpha ERKO mice but not beta ERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in alpha ERKO mice. This study suggests that ER beta may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.     

A deficiency of lunatic fringe is associated with cystic dilation of the rete testis

Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.     

Expression of IGF-II mRNA-binding proteins (IMPs) in gonads and testicular cancer

Insulin-like growth factor-II mRNA-binding proteins 1, 2 and 3 (IMP1, IMP2 and IMP3) belong to a family of RNA-binding proteins implicated in mRNA localization, turnover and translational control. We examined their expression pattern during development of murine and human testis and ovaries. In the mouse, IMPs were expressed in male and female gonadal cells at embryonic day 12.5 (E12.5). From E16.5, IMP1 and IMP3 became restricted to the developing germ cells, whereas IMP2 expression persisted in the interstitial cells. In mature mouse and human ovaries, IMP1, IMP2 and IMP3 were detected in resting and growing oocytes and in the granulosa cells. In testis, IMP1 and IMP3 were found mainly in the spermatogonia, whereas IMP2 was expressed in the immature Leydig cells. Moreover, all three IMPs were detected in human semen. The developmental expression pattern of IMP1 and IMP3 in the human testis prompted us to examine their possible involvement in testicular neoplasia. IMPs were detected primarily in germ-cell neoplasms, including preinvasive testicular carcinoma in situ, classical and spermatocytic seminoma, and nonseminomas, with particularly high expression in undifferentiated embryonal carcinoma. The relative expression of IMP1, IMP2 and IMP3 varied among tumor types and only IMP1 was detected in all carcinoma in situ cells. Thus IMPs, and in particular IMP1, may be useful auxiliary markers of testicular neoplasia.     

Expression and localization of inhibin alpha,inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.     

Endocrine changes associated with onset of spermatogenesis in Holstein bulls

After birth, a bull enters a period of infancy during which the reproductive organs are relatively quiescent. This is followed by the prepubertal period, which starts at 10 to 12 wk in well-fed Holstein bulls, characterized by profound changes of hypothalamic, pituitary, and gonadal function that culminate in puberty. The prepubertal sequence of events probably is: a) initiation of spontaneous discharge of luteinizing hormone; b) hormone induced differentiation of Leydig cells with increased secretion of androstenedione in response to luteinizing hormone stimulation; c) further differentiation of Leydig cells resulting in luteinizing hormone-stimulated secretion of testosterone; d) testosterone-induced differentiation of indifferent supporting cells to Sertoli cells concomitant with testosterone-induced differentiation of gonocytes to prespermatogonia and A-spermatogonia; e) increased sensitivity of the hypothalamus-anterior pituitary to negative feedback of gonadal steroids; f) diminished frequency and amplitude of luteinizing hormone discharge; g) formation of junctional complexes between Sertoli cells and establishment of the blood-testis barrier; h) formation of primary spermatocytes and ultimately spermatids and spermatozoa; and i) continued increase of efficiency of spermatogenesis until sufficient sperm are produced to provide the first ejaculum around 37 to 38 wk. Following puberty, the reproductive capacity of a bull increases for several years until the male is sexually mature.     

Expression of Cyp21a1 and Cyp11b1 in the fetal mouse testis

Fetal Leydig cells and fetal adrenocortical cells may share a common progenitor cell. Both cell types show several similarities, particularly in relation to their primary steroidogenic function. Differences in steroid secretion are largely due to the expression of 21-hydroxylase (CYP21A1) and 11beta-hydroxylase (CYP11B1) activity in the adrenal. To determine whether expression of these enzymes defines a clear difference between adrenocortical and Leydig cells, or is further evidence of a link between the cell types, we have measured Cyp21a1 and Cyp11b1 expression and related enzyme activity in the fetal testis. Expression of both Cyp21a1 and Cyp11b1 was clearly detectable in the fetal testis by RT-PCR and Southern blotting. Real-time PCR studies showed that Cyp11b1 was expressed only in the fetal/neonatal testis with no expression in the pubertal or post-pubertal animal. Cyp21a1 was also predominantly expressed in the fetal testis although some lower expression was also seen in the adult. Expression of Cyp21a1 and Cyp11b1 in neonatal testicular cells was unaffected by incubation in vitro with human chorionic gonadotrophin or ACTH. Using immunohistochemistry, CYP21A1 was localised to a subset of interstitial steroidogenic cells in the fetal testis although CYP11B1 was not detectable. Incubation studies showed that 21-hydroxylase activity was present in the tissue although 11beta-hydroxylase activity could not be detected. Results indicate that a subpopulation of steroidogenic cells in the fetal testis express Cyp21a1 and show 21-hydroxylase activity. This may provide further evidence of a link between fetal Leydig cells and adrenocortical cells but does not discount the possibility that these steroidogenic cells represent 'ectopic' adrenal cells.