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.     

A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.     

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.     

Adult-only exposure of male rats to a diet of high phytoestrogen content increases apoptosis of meiotic and post-meiotic germ cells

Apoptosis plays a critical role in regulating sperm production. Removal of androgens and gonadotropins, or estrogen administration induces germ cell apoptosis. It is hypothesized that dietary phytoestrogens increase apoptosis of developing germ cells, decreasing sperm production. This study aimed to test this in rats fed a high phytoestrogen diet only during adulthood. Male Wistar rats used in this study were offspring of females maintained on a low phytoestrogen diet prior to conception through to weaning. After weaning, juveniles were fed the same low phytoestrogen diet into adulthood. A cohort of males were transferred to a high phytoestrogen diet for 24 days and subsequently testes were collected from all animals. In the high phytoestrogen fed group, homogenization-resistant sperm counts were significantly decreased, as were epididymal sperm counts. Morphometric analysis determined round and elongated spermatid volumes to be significantly decreased, but seminiferous tubule lumen diameters to be significantly increased. TUNEL analysis determined that apoptosis of spermatocytes and round spermatids was significantly greater in the high phytoestrogen fed rats. Neither plasma gonadotropin concentrations nor testicular testosterone were altered. In conclusion, exposure of the adult male rat to a high phytoestrogen diet disrupts spermatogenesis, increasing germ cell apoptosis. This effect is independent of the hypothalamo-pituitary-testicular axis and is likely due to disruption of estrogen's actions in the testis.     

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.     

Gpr54-/- mice show more pronounced defects in spermatogenesis than Kiss1-/- mice and improved spermatogenesis with age when exposed to dietary phytoestrogens

Mice with mutations in the kisspeptin signaling pathway (Kiss1(-/-) or Gpr54(-/-)) have low gonadotrophic hormone levels, small testes, and impaired spermatogenesis. Between 2 and 7 months of age, however, the testes of the mutant mice increase in weight and in Gpr54(-/-) mice, the number of seminiferous tubules containing spermatids/spermatozoa increases from 17 to 78%. In contrast, the Kiss1(-/-) mice have a less severe defect in spermatogenesis and larger testes than Gpr54(-/-) mice at both 2 and 7 months of age. The reason for the improved spermatogenesis was investigated. Plasma testosterone and FSH levels did not increase with age in the mutant mice and remained much lower than in wild-type (WT) mice. In contrast, intratesticular testosterone levels were similar between mutant and WT mice. These data indicate that age-related spermatogenesis can be completed under conditions of low plasma testosterone and FSH and that intratesticular testosterone may contribute to this process. In addition, however, when the Gpr54(-/-) mice were fed a phytoestrogen-free diet, they showed no age-related increase in testes weight or improved spermatogenesis. Thus, both genetic and environmental factors are involved in the improved spermatogenesis in the mutant mice as they age although the mice still remain infertile. These data show that the possible impact of dietary phytoestrogens should be taken into account when studying the phenotype of mutant mice with defects in the reproductive axis.     

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.     

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.     

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.     

Effect of local heating of rat testes after suppression of spermatogenesis by pretreatment with a GnRH agonist and an anti-androgen

The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts.     

Chromatoid body and small RNAs in male germ cells

The chromatoid body (CB) is a germ granule in the cytoplasm of postmeiotic haploid round spermatids that is loaded with RNA and RNA-binding proteins. Following the discovery of small non-coding RNA-mediated gene regulation and the identification of PIWI-interacting RNAs (piRNAs) that have crucial roles in germ line development, the function of the CB has slowly begun to be revealed. Male germ cells utilise small RNAs to control the complex and specialised process of sperm production. Several microRNAs have been identified during spermatogenesis. In addition, a high number of piRNAs are present both in embryonic and postnatal male germ cells, with their expression being impressively induced in late meiotic cells and haploid round spermatids. At postmeiotic stage of germ cell differentiation, the CB accumulates piRNAs and proteins of piRNA machinery, as well as several other proteins involved in distinct RNA regulation pathways. All existing evidence suggests a role for the CB in mRNA regulation and small RNA-mediated gene control, but the mechanisms remain uncharacterised. In this review, we summarise the current knowledge of the CB and its association with small RNA pathways.     

Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated gamma-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.     

Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.     

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.     

Vezatin, a ubiquitous protein of adherens cell-cell junctions, is exclusively expressed in germ cells in mouse testis

In the male reproductive organs of mammals, the formation of spermatozoa takes place during two successive phases: differentiation (in the testis) and maturation (in the epididymis). The first phase, spermiogenesis, relies on a unique adherens junction, the apical ectoplasmic specialization linking the epithelial Sertoli cells to immature differentiating spermatids. Vezatin is a transmembrane protein associated with adherens junctions and the actin cytoskeleton in most epithelial cells. We report here the expression profile of vezatin during spermatogenesis. Vezatin is exclusively expressed in haploid germ cells. Immunocytochemical and ultrastructural analyses showed that vezatin intimately coincides, temporally and spatially, with acrosome formation. While vezatin is a transmembrane protein associated with adherens junctions in many epithelial cells, it is not seen at the ectoplasmic specializations, neither at the basal nor at the apical sites, in the seminiferous epithelium. In particular, vezatin does not colocalize with espin and myosin VIIa, two molecular markers of the ectoplasmic specialization. In differentiating spermatids, ultrastructural data indicate that vezatin localizes in the acrosome. In epididymal sperm, vezatin localizes also to the outer acrosomal membrane. Considering its developmental and molecular characteristics, vezatin may be involved in the assembly/stability of this spermatic membrane.     

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.     

Spermatogenesis in testes of Dazl null mice after transplantation of wild-type germ cells

Dazl knockout male mice are infertile because their germ cells are unable to complete the first meiotic prophase in the first wave of spermatogenesis and thereafter decrease in number due to a block at the A-aligned to A1 transition. The ability of the surviving somatic components of the testes to retain their function in the absence of mature germ cells was tested by injecting marked wild-type germ cell suspensions containing spermatogonial stem cells. Comparison of the frequency and extent of colonization of Dazl knockout testes with that of testes chemically depleted of germ cells showed little if any difference. It was concluded that Dazlko testes seem unimpaired in their ability to support spermatogenesis. Therefore, Dazlko testes provide a useful and reliable recipient in which to evaluate spermatogonial stem cells. The results furthermore demonstrate that the somatic compartment of the testis of these animals retains functionality.     

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.