Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes

Background and Aims

In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo.

Methodology and Principal Findings

Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules.

Conclusion/Significance

Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.     

Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.     

Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senescence-accelerated mice

Specific features of spermatogenesis were studied in senescence-accelerated mice of the strain SAMP1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with ³H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in histological sections of testes of experimental mice. These structures contained the cells morphologically similar to gonocytes and immature Sertoli cells.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Stability of spermatogenic synchronization achieved by depletion and restoration of vitamin A in rats

Studies of synchronization of spermatogenesis following vitamin A deficiency have suggested that this may provide an in vivo model for the study of stage-dependent changes in hormonal action and protein secretion within the seminiferous epithelium. However, until now, no information on the stability or durability of this condition has been available. In this study, 200 seminiferous tubules from each of 40 rats (including controls) were classified according to their spermatogenic stage after withdrawal and replenishment of vitamin A. Following 15 wk withdrawal and subsequent replenishment of vitamin A, spermatogenesis was initiated in a synchronous fashion. This synchrony remained stable for more than 10 cycles of the seminiferous epithelium (2.5 spermatogenic cycles). In association with the extended period of vitamin A deficiency, a proportion of tubules (30%) showed morphological characteristics of either Sertoli cells only or Sertoli cells plus spermatogonia with occasional pachytene spermatocytes. During the 11-wk period of observation in this study, no significant change in proportions of damaged tubules were observed. Testicular testosterone concentrations, although elevated with respect to controls, showed no correlation with the stage of the cycle of the seminiferous epithelium observed, whereas pituitary and serum follicle-stimulating hormone levels were elevated, probably due to the number of damaged tubules observed. The persistence of synchrony in spermatogenesis following vitamin A treatment suggests that this model is applicable for studies of paracrine actions within the testis. However, the decreased ratio of synchrony observed with time may provide evidence that duration of the individual stages of the cycle of the seminiferous epithelium might be subject to temporal variation, leading to a progressive desynchronization of spermatogenesis in this model system.     

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.     

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.     

Localization of the cytochrome p450 side-chain cleavage enzyme in the inactive testis of the naked mole-rat

Spermatogenesis was histologically examined in non-breeding male of the naked mole rat (Heterocephalus glaber) using a light microscopy. Spermatogonia, spermatocytes and spermatids were confirmed in the seminiferous tubules. However, the spermatogenesis was disordered, and many spermatocytes and spermatids were sloughing. Sperms could not be seen in the lumen of the tubules. The characteristic accumulation of interstitial cells was the most noteworthy. In the immunohistochemistry for cytochrome p450 side-chain cleavage enzyme, immunoreactions were not entirely distributed in each interstitial cell, although positive reactions were scattered in the interstitial cell-mass. The findings indicate that few interstitial cells act as a testosterone-synthesizing apparatus in the characteristic structure with accumulated cell-mass. From the immunohistochemical data we suggest the possibility that spermatogonia and Sertoli cells may secrete 17 beta-estradiol. We also suggest that 17 beta-estradiol from spermatogonia and Sertoli cells may inhibit the interstitial cells from synthesizing and secreting testosterone and may suppress the later stages of the spermatogenesis to induce apoptosis of germ cells. The TUNEL methods demonstrated that cell death occurred in some spermatocytes in non-breeding males.     

Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice

The effect of vitamin A deficiency and vitamin A replacement on spermatogenesis was studied in mice. Breeding pairs of Cpb-N mice were given a vitamin A-deficient diet for at least 4 wk. The born male mice received the same diet and developed signs of vitamin A deficiency at the age of 14-16 wk. At that time, only Sertoli cells and A spermatogonia were present in the seminiferous epithelium. These spermatogonia were topographically arranged as single and paired cells and as clones of 4, 8 and more cells. A few mitoses of single, paired, and clones of 4 A spermatogonia were found, which were randomly distributed over the seminiferous epithelium. When vitamin A-deficient mice were treated with retinol-acetate combined with a normal vitamin A-containing diet, spermatogenesis restarted again synchronously. Only a few successive stages of the cycle of the seminiferous epithelium were present up to at least 43 days after vitamin A replacement. After 20 days, 98.3% of the seminiferous tubules were synchronized, showing pachytene spermatocytes as the most advanced cell type, mostly being in epithelium stages IX-XII. After 35 and 43 days, spermatogenesis was complete in 99.6% of the tubular cross sections, and most tubular cross sections were in stages IV-VII of the cycle of the seminiferous epithelium. The degree of synchronization was comparable or even higher than found in rats. The rate of development of the spermatogenic cells between 8 and 43 days after vitamin A replacement seemed to be similar to that in normal mice. Assuming that the rate of development of the spermatogenic cells is also normal during the first 8 days after vitamin A replacement, it can be deduced that the preleptotene spermatocytes, present after 8 days, were A spermatogonia in the beginning of stage VIII at the moment of vitamin A replacement. These results indicate that the mouse can be used as a model to study epithelial stage-dependent processes in the testis.     

In vivo gene transfer to mouse spermatogenic cells using green fluorescent protein as a marker

Combination of the DNA injection into seminiferous tubules and the subsequent in vivo electroporation (EP) has become an efficient and convenient assay system for spermatogenic-specific gene expression during spermatogenesis of mice. In this study, we made methodological modifications to enhance the transfection efficiency, and evaluated the possibility of this technique to generate transgenic offspring using green fluorescent protein (GFP) as a marker. After the in vivo gene transfer, GFP expression could be monitored easily and repeatedly on the surface of the testis of live mice under fluorescent microscopy. The serial sections of the transfected testis revealed that transient expression of GFP was extended even in the innermost region of the testis uniformly, but confined to spermatogenic cells and Sertoli cells within the seminiferous tubules. Furthermore, long-lasting GFP expression could be detected in the spermatogenic cells even 2 months after EP. Natural mating with normal adult females revealed that 65% of the transfected males maintained fertilizable ability and could generate their offspring normally. Germ-line transmission of the GFP vector to the offspring was checked under fluorescent microscopy, but no transgenic offspring has been detected up to now. These results suggest that the application of additional techniques, such as cell sorting for GFP-positive germ cells followed by nuclear transfer to the oocytes, would make this method as a novel strategy for generating transgenic animals. J. Exp. Zool. 286:212-218, 2000.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Successful intra- and interspecific male germ cell transplantation in the rat

The lumen of the seminiferous tubules has hitherto been regarded as an immunologically privileged site. We report here the birth of young following transplantation of stem spermatogonia from Long-Evans rats to the seminiferous tubules of Sprague-Dawley rats after treatment with the immunosuppressive agent cyclosporin. Follicle-stimulating hormone was also given to stimulate Sertoli cell proliferation, and testosterone to stimulate the recovery of spermatogenesis. Donor germ cells underwent normal spermatogenesis, and progeny were repeatedly produced from the donor germ cells as demonstrated by microsatellite paternity analysis. In addition, donor germ cells from the cryptorchid testes of LacZ mice were also able to colonize the seminiferous tubules of Sprague-Dawley rats using this protocol. Morphologically normal rat and mouse spermatozoa were present in the epididymis and vas deferens of the recipient rats. This highlights the potential for transplantation of male germ cells between different species.