Perturbation of spermatogenesis by androgen antagonists directly injected into seminiferous tubules of live mice

Natural and artificial substances present in the environment can affect our health. Testicular toxicants in particular are troublesome, because they disturb gonadal function of males. Translocation of substances into the seminiferous epithelium where sperm production proceeds is restricted due to the blood-testis barrier, but this permeability barrier temporarily disappears under physiological and sub-physiological conditions. This means that any substance could enter the seminiferous epithelium and disturb sperm production. To reduce the risk posed by such toxins, it is important to accurately determine which substances possess the toxicity. However, existing assay systems are not satisfactory in terms of both accuracy and sensitivity. Here, we report the establishment of such a system. We injected the androgen antagonists, flutamide and vinclozolin, directly into seminiferous tubules of live mice, which had been treated with busulfan for a temporal arrest of spermatogenesis, and the testes were histologically examined to see the effect of the injected materials on spermatogenesis that was in the process of recovery. The injection of either substance brought about a severe impairment of spermatogenesis at an amount over a million times smaller than that used in the previous assay systems where animals are administered with test substances outside of the testis. In contrast, these androgen antagonists at the same doses showed lesser effects when intratubularly or intraperitoneally administered into mice that had not been pretreated with busulfan. We propose that the method adopted in this study is a novel assay system to identify potential testicular toxicants.     

Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. We describe here a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems.     

Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.     

Reduction of long-term effects of local heating of the testis by treatment of rats with a GnRH agonist and an anti-androgen

Heating the testes of anaesthetized adult rats to 43 degrees C for 30 min in a waterbath was followed by a large decrease in testis and epididymis mass and number of spermatozoa 35 days later. These parameters had recovered to some extent, but not completely, by days 70 and 97 after heating, but had decreased again in rats examined on day 182. There were no consistent effects of heating on androgen status, as determined by the concentrations of testosterone in blood and testis fluids, or by seminal vesicle mass, and interstitial fluid volume was increased in the heated testes. Treatment of rats with an implant of a GnRH agonist and daily injections of an anti-androgen for 14 days (sufficient in itself to cause large temporary decreases in tissue mass, number of spermatozoa and androgen status) did not reduce the initial decrease in testis mass or number of spermatozoa seen after heating, but reduced the later decreases in mass and number of spermatozoa significantly. These findings indicate that, as well as causing damage to spermatocytes and spermatids, as previously reported, heating also reduces the ability of spermatogonia to repopulate the seminiferous tubules at longer intervals after heating. Furthermore, it appears that this effect on the spermatogonia can be reduced by treating the animals with a GnRH agonist and anti-androgen, a treatment similar to that shown by other authors to improve recovery of the testis from irradiation or drug treatment.     

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.     

An integrase facilitates long-lasting foreign gene expression in vivo in mouse spermatogenic cells

The objective of the present study was to attain long-lasting foreign gene expression in vivo in spermatogenic cells in the mouse testis for establishing spermatogenic-cell mediated gene transformation. Prior to in vivo gene transfer, surgical cryptorchidism was performed by retaining the testis into the abdominal cavity for 1 month to remove differentiated spermatogenic cells. Subsequently, in vivo gene transfer was conducted by electroporation with a lacZ reporter gene in combination with a retroviral integrase gene, and the testis was descended immediately to the scrotum to recover from the cryptorchidism, and restart spermatogenesis. At 1 month post-transfection in vivo, lacZ gene expression was detected in some spermatocyte-like cells in seminiferous tubules of the mouse testis. However, the recovery period of 1 month appeared to be too short, since no elongated and fully differentiated spermatids were found. At 2 months post-transfection, fully differentiated spermatogenic cells expressing the lacZ gene, albeit at low frequency, were detected when the integrase gene was co-transfected, while virtually no lacZ-positive cells were found in the absence of the integrase gene. It was concluded, therefore, that stable transformation of spermatogenic cells in vivo would be facilitated by integrase gene co-transfection.     

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.     

Autologous and homologous transplantation of bovine spermatogonial stem cells

The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.     

Postnatal testicular development in mouse species with different levels of sperm competition

Postcopulatory sexual selection leads to an increase in sperm numbers which is partly the result of an increase in relative testes mass and could also be the consequence of changes in testis architecture or function. Very little is known regarding developmental changes during the first spermatogenic wave that may lead to enhanced spermatogenic efficiency and increased sperm production. We examined testicular development after birth in four mouse species with different sperm competition levels to assess changes in testicular architecture and function. Differences in relative testes mass between species appeared soon after birth and were exacerbated thereafter. The volume of testes occupied by seminiferous tubules differed between species postnatally and were associated with sperm competition levels. Finally, changes over time in the proportions of tubules with different germ cell types were also associated with sperm competition levels, with the time taken for the transition between various cell stages being negatively associated with levels of sperm competition. We conclude that postnatal testis development differs between closely related species with different sperm competition levels influencing testis architecture and the rate of progression of spermatogenesis, leading to differences in testis function at reproductive maturity.     

Characterization of NADPH oxidase 5 in equine testis and spermatozoa

Reactive oxygen species (ROS) play an important role in normal sperm function, and spermatozoa possess specific mechanisms for ROS generation via an NAD(P)H-dependent oxidase. The aim of this study was to identify the presence of an NADPH oxidase 5 (NOX5) in equine testis and spermatozoa. The mRNA of NOX5 was expressed in equine testis as detected by northern blot probed with human NOX5 cDNA and by RT-PCR. Immunoblotting with affinity purified alpha-NOX5 revealed one major protein in equine testis and other tissues. Immunolocalization of NOX5 showed labeling over the rostral sperm head with some labeling in the equatorial and post-acrosomal regions. In the testis, there was abundant staining in the adluminal region of the seminiferous tubules associated with round and elongating spermatids. The RT-PCR and sequence analysis revealed a high homology with human NOX5. This study demonstrates that NOX5 is present in equine spermatozoa and testes and therefore represents a potential mechanism for ROS generation in equine spermatozoa.     

Successful transplantation of bovine testicular cells to heterologous recipients

While heterologous germ cell transplantation was successful in pigs and goats, autologous transplantation alone has been reported to result in donor-derived spermatogenesis in cattle. The objective of this study was to investigate whether the transplantation of heterologous germ cells could result in colonization of recipient testes in cattle of different breeds. Testicular cells were isolated from 8 Bos taurus donor bull calves and then transferred into 15 Bos indicus-cross bull calves. All animals were prepubertal, donors were aged 5-7 months and recipients 5-11 months, and scrotal circumferences ranged from 15 to 22 cm. Single cell suspensions of donor testicular cells, prepared by enzymatic digestion, were labelled with fluorescent dyes PKH26 or CFDA-SE, before transfer into the rete testis of recipients under ultrasonographic guidance. To assess the longevity of colonization by donor cells, recipients were castrated 2-30 weeks after cell transfer. Donor cells were observed in 15/25 (60%) of the testes that received PKH26-labelled cells, whereas no CFDA-SE-positive cell was identified in any recipients. The maturity of the donors or recipients (measured by scrotal circumference) did not affect colonization potential. In freshly isolated tubules, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the tubules. In frozen sections, PKH26-positive cells were identified on the seminiferous tubule basement membrane, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. We conclude that PKH26 was more suitable for labelling donor testis cells and donor cells can be identified up to 6 months following transfer. These results indicate that allogeneic transplantation of testicular cells can occur between Bos taurus and Bos indicus cattle. Further studies will investigate functionality of transferred testicular cells.     

Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats

In this study, female goats were orally exposed to PCB126 or PCB153, at 49 ng/kg body weight per day and 98 microg/kg body weight per day respectively, from gestational day 60 until delivery at approximately day 150. Exposure of the offspring continued via lactation until postnatal day 40. Reproductive toxicity in the male offspring was studied by the evaluation of conventional reproductive endpoints as well as flow cytometric analyses of spermatogenesis and sperm chromatin structure. PCB153-treated animals showed a significant smaller testis diameter in comparison to the control group. Neither of the treated groups showed differences for plasma FSH in comparison to controls. PCB153-treated animals differed significantly from the control group with respect to plasma LH and testosterone levels, whereas PCB126-treated animals only differed from the controls in plasma testosterone concentrations. Neither the PCB126 nor the PCB153 group differed from the controls with respect to the conventional sperm parameters or testis histology. A significant lower ratio of interstitium area to seminiferous tubules area and proportion of diploid testis cells were observed for the PCB153 group. Sperm from PCB153-treated animals showed a significantly higher percentage of sperm with damaged DNA. From the results of the present study it was concluded that PCB153 was able to induce alterations in reproductive endpoints related to the hypothalamic-pituitary-axis as well as to the testis. The effects observed in male kids after a long-term maternal exposure to PCB153 support the concept that exposure to endocrine-disrupting compounds during foetal development may lead to adverse reproductive effects in adult life.     

Spermatogenesis following syngeneic testicular transplantation in Balb/c mice

Transplantation of spermatogonial stem cells in cross-species has been widely used to study the function of Sertoli cells and the effect of phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation, whereas there have been only a few reports on the transplantation of testis tissue. The objective of the present study was to examine the development of grafted testes and the kinetics of spermatogenesis following syngeneic testicular transplantation in both male and female recipient Balb/c mice in an effort to establish an in vivo culture system and to compare the effects of host sex on spermatogenesis. The testes from 5-day-old Balb/c mice were transplanted under the dorsal skin of four-week-old mice. Twenty male and twenty female Balb/c mice were used as the hosts and each host received 4 grafts. The recipient mice were killed at 1, 2, 3, 5, 7, 9, 12 and 15 weeks after transplantation. The graft survival rate and graft size were measured. The status of spermatogenesis was assessed by histological analyses. The expression of the spermatid-specific Protamine-2 gene was examined by RT-PCR. Overall, 70.3% of the testicular grafts in male hosts and 67.2% in female hosts survived. All recovered grafts had increased in volume, some of them had increased by more than 30-fold. The architecture of the seminiferous tubules in female hosts appeared to be better than that in male hosts. The round spermatids were the most advanced germ cells until 15 weeks after transplantation, and no complete spermatozoon was observed in any of the grafts. The expression of protamine-2 was detected in grafts from 5 weeks posttransplantation in both male and female hosts, confirming that the spermatogenic cells differentiated into spermatids. In contrast to grafts, the testes of male hosts had a normal histological appearance. The results showed the schedule of spermatogenesis following syngeneic testicular transplantation in both male and female hosts. This model could be useful for further studies involving the endocrinology of the testis and the mechanisms of spermatogenesis.     

Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rhesus monkeys (Macaca mulatta)

Vasectomy reversal by vasovasostomy after long-term vasectomy in men results in lower sperm counts and pregnancy rates compared with controls, and severe damage to spermatogenesis has been observed in some animal models such as mice. The primary aim of this study was to evaluate, using sophisticated stereological methods, whether vasectomy of 6 and 12 months in a non-human primate would lead to, among other morphometric changes, reduced numbers of germ cells in testes and spermatozoa in epididymides. Five normal adult male rhesus monkeys (Macaca mulatta) underwent bilateral vasectomy, with another three aged-matched normal monkeys not undergoing vasectomy. One testis together with the ipsilateral epididymis was removed from each animal at 6 months, and the other testis and epididymis, the prostate gland and seminal vesicles were removed at 12 months. Various morphometric data were obtained using stereological methods and an unbiased and efficient stereological tool, the optical disector, was used to estimate nuclear numbers of all types of spermatogenic cells in testes and spermatozoa in epididymides using methacrylate-embedded sections 25 microm in thickness. As shown by a two-way repeated measures analysis of variance, vasectomy or hemicastration (removal of the organs at 6 months) had no significant effects on all quantitative parameters of stereology obtained from the testis, epididymis, prostate gland and seminal vesicle, except that (i) sperm granuloma was observed from three of five vasectomized animals both at 6 and 12 months, and (ii) hemicastration significantly reduced the diameter of the seminiferous tubules and increased the number of type A spermatogonia per testis. In conclusion, vasectomy in the non-human primate is a safe procedure in terms of effects on the structures of the reproductive organs.     

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.     

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.     

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.