A missense mutation of the Dhh gene is associated with male pseudohermaphroditic rats showing impaired Leydig cell development

Development of the male gonads is a complex process with interaction of various cells in the gonads including germ, Sertoli, Leydig, and myoid cells. TF is a mutant rat strain showing male pseudohermaphroditism, with agenesis of Leydig cells and androgen deficiency controlled by an autosomal single recessive gene (mp). The mp locus was mapped on the distal region of rat chromosome 7 by linkage analysis, but the gene responsible for the mp mutation has not been identified. In this study, we performed fine linkage mapping and sequence analysis to determine the causative gene of the mp mutation, and performed an immunohistochemical study using a Leydig cell-specific marker to investigate detailed phenotypes of the mutant rats during the testicular development. As a result, we found a missense mutation of the gene encoding Desert hedgehog (Dhh) in the mutant rat, which could result in loss of function of the DHH signaling pathway. Histochemical examination revealed remarkably reduced number of fetal Leydig cells and lack of typical spindle-shaped adult Leydig cell in the mp/mp rats. These phenotypes resembled those of the Dhh-null mice. Additionally, testosterone levels were significantly lower in the mp/mp fetus, indicating androgen deficiency during embryonic development. These results indicate that the mutation of the Dhh gene may be responsible for the pseudohermaphrodite phenotypes of the mutant rat, and that the Dhh gene is probably essential for the development of Leydig cells.     

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.     

Different expression and activity of the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

Two catalytic isoforms of the Na,K-ATPase, alpha1 and alpha4, are present in testis. While alpha1 is ubiquitously expressed in tissues, alpha4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase alpha isoforms in male germ cell biology, we have studied the expression and activity of alpha1 and alpha4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that alpha1 and alpha4 undergo differential regulation during development. Whereas alpha1 exhibits only modest changes, alpha4 increases with gamete differentiation. The most drastic changes for alpha4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of alpha4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the alpha1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase alpha1 and alpha4 isoforms are functional in diploid, meiotic and haploid male germ cells, alpha4 being significantly upregulated during spermatogenesis. These results support the importance of alpha4 in male gamete differentiation and function.     

Gonadal status of male recipient mice influences germ cell development in immature buffalo testis tissue xenograft

Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status.     

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.     

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.     

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.     

Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.     

Regucalcin is broadly expressed in male reproductive tissues and is a new androgen-target gene in mammalian testis

Regucalcin (RGN) is a calcium (Ca(2)(+))-binding protein which regulates intracellular Ca(2)(+) homeostasis by modulating the activity of enzymes regulating Ca(2)(+) concentration and enhancing Ca(2)(+)-pumping activity. Several studies have described the pivotal role of proper Ca(2)(+) homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of 5α-dihydrotestosterone (DHT) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen DHT in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis.     

Germ cell fate and seminiferous tubule development in bovine testis xenografts

Spermatogenesis can occur in testis tissue from immature bulls ectopically grafted into mouse hosts; however, efficiency of sperm production is lower than in other donor species. To elucidate a possible mechanism for the impaired spermatogenesis in bovine testis xenografts, germ cell fate and xenograft development were investigated at different time points and compared with testis tissue from age-matched calves as controls. Histologically, an initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting without an increase in germ cell apoptosis. From 2 months onward, the number of germ cells increased. In contrast, a continuous increase in germ cell number was seen in control tissue. Pachytene spermatocytes were observed in some grafts before 4 months, whereas in the control tissue they were not present until 5 months of age. Beyond 4 months post-grafting spermatogenesis appeared to be arrested at the pachytene spermatocyte stage in most grafts. Elongated spermatids were observed between 6 and 8 months post-grafting, similar to the controls, albeit in much lower numbers. Lumen formation started earlier in grafts compared with controls and by 6 months post-grafting tubules with extensively dilated lumen were observed. A donor effect on efficiency of spermatogenesis was also observed. These results indicate that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells and impaired meiotic and post-meiotic differentiation. The characterization of spermatogenic efficiency will provide the basis to understand the control of spermatogenesis in testis grafts.     

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.