Three-dimensional morphological analysis of spermatogenesis in aged mouse testes.

Spermatogenesis, which is a continuous process from undifferentiated spermatogonia to spermatozoa in the seminiferous tubules, declines with age. To investigate changes in spermatogenesis with aging, we reconstructed the seminiferous tubules of 12 mice aged 12 to 30 months from serial sections and examined age-related and region-specific alterations in the seminiferous epithelium and spermatogenic waves in three dimensions. The basic structure of the seminiferous tubules, including the numbers of tubules, terminating points, branching points, and total tubule length, did not change with age. Age-related alterations in spermatogenesis, primarily assessed by the formation of vacuoles in Sertoli cells, were detected in the seminiferous tubules at 12 months. The proportion of altered tubule segments with impaired spermatogenesis further increased by 24 months, but remained unchanged thereafter. Altered tubule segments were preferentially distributed in tubule areas close to the rete testis and those in the center of the testis. Spermatogenic waves became shorter in length with age. These results provide a basis for examining the decline of spermatogenesis not only with aging, but also in male infertility.

Age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs.

There are age-related changes in testicular anatomy and physiology whereby there are modifications of sperm production and reproductive hormone functions. Effects of age on testicular microanatomy are well documented in humans, while there is limited understanding of these changes in dogs. The aim of this study was to evaluate age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs. Dogs (n = 32) were divided into four age groups: peripubertal (n = eight), relatively younger (n = seven), reproductively mature (n = seven) and relatively older (n = ten). Picrosirius Red stained sections were used for morphometrical analysis of testicular tissues, while the characteristics of seminiferous epithelium were assessed using a modified Johnsen scoring system for haematoxylin and eosin stained sections. Seminiferous epithelium and seminiferous tubule area increased from peripuberty to reproductive maturity, indicating there were changes during sexual maturation and subsequently there were decreases with further aging. There was a similar age-related trend for changes in seminiferous epithelium height with values being greatest in reproductively mature dogs; while there were no age-related differences in tubular diameter. Collagen content in the testicular interstitium gradually decreased from peripuberty to the age when dogs were reproductively mature and there were subsequent increases in relatively older dogs, thus, there was an association between the extent of testicular fibrosis and senescence. There was a decrease in spermatogenetic functions from relatively younger to older ages. Further investigations are warranted to establish mechanisms responsible for age-related changes of testicular morphology and related clinical implications.

Distribution of actin filaments in the seminiferous epithelium of the Habu, Trimeresurus flavoviridis.

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.

Influence of Sialadenectomy on the Development of Spermatogenesis in Rats: Ultrastructural Characteristics of Spermatogenic Epithelium and Leydig Cells.

Sialadenectomy in young rats modifies the development of the spermatogenic and steroidogenic functions of the testes. Sialadenectomy causes ultrastructural changes in spermatogenic cells, sustentocytes, and Leydig cells that disappear by week 8 of the experiment due to realization of compensatory and adaptive mechanisms. The effects of endocrine factors of the greater salivary glands on the spermatogenic cells are realized directly and indirectly via interstitial endocrinocytes and sustentocytes.

Transmission electron microscopy analysis of the origin and incidence of sperm intranuclear cytoplasmic retention in fertile and teratozoospermia men.

The human sperm nucleus contains cytoplasm. However, the origin and incidence of human sperm intranuclear cytoplasmic retention (INCR) remain unknown. The objectives of this study were to observe the morphological origin of INCR within the seminiferous epithelium and investigate the incidence of INCR in fertile and teratozoospermia men using transmission electron microscopy (TEM). By TEM, INCR initially appeared in elongating round spermatid nuclei and varied in size, number, shape, content, location and distribution within sperm nuclei. The teratozoospermia group (n = 16) demonstrated a higher incidence of INCR than did the fertile group (n = 16) (17.6 ± 5.2% vs. 9.7 ± 3.4%; p = 0.000). In the fertile group, no correlations were found between the incidence of INCR and abnormal sperm morphology, nuclear vacuole, acrosome integrity, motility or concentration (p > 0.05). However, the incidence of INCR exhibited a positive relationship with sperm abnormal morphology in the teratozoospermia group (r = 0.616, p = 0.011). These results demonstrate that INCR occurs in the early process of spermatogenesis and is an alteration found in the nucleus. Spermatozoa from teratozoospermia men contained more INCRs than those from fertile males. More attention should be paid to the possibility of spermatozoa containing INCR when using spermatozoa with abnormal head morphology for clinical or diagnostic purposes.

Histopathological and ultrastructural changes experimentally induced by bee venom in seminiferous epithelium via structural-functional alteration of Sertoli cells.

We tested here the ability of bee venom (BV) to interfere with spermatogenesis in rats in two experimental conditions. The histopathological changes were assessed with brightfield microscopy using a novel staining technique, based on methylene blue, orange G and ponceau xylidine. Transmission electron microscopy was also used to identify fine subcellular changes. BV injection for 30days in daily doses of 700µg BV/kg resulted in reducing testicular weight, along with significant larger diameters of seminiferous tubules and reduced number of Sertoli cells (SCs). SCs were vacuolated, detached from the basement membrane, many necrosed, leading to the basement membrane denudation. Germ cells layers were separated by empty spaces conferring a rarefied aspect to the tissue, and spermatids were detached into lumen. Thus, the seminiferous epithelium was significantly thinned. Many Leydig cells (LCs) were in a necrotic state, with disrupted plasma membrane and without smooth endoplasmic reticulum. The acute treatment with a single LD50 of 62mgBV/kg, was followed by focal disruptions of the basement membrane and localized areas of necrosis, mainly affecting the SCs. Most of the observed SCs as well as some spermatogonia were highly vacuoled, empty spaces being observed within the epithelium. The SCs count was significantly decreased. Spermatids had also the tendency of separation from the SCs, and the significant larger diameter of the tubules found was associated with a thicker epithelium. Many LCs were necrosed, with disrupted plasma membrane, swollen mitochondria, no endoplasmic reticulum and implicitly showing rarefied cytoplasm. We concluded that BV was a testicular toxicant affecting both the LCs and the seminiferous tubules. The SCs cells represented the primary target site of BV whose effects were next extended upon the germ cells. In all cells, BV triggered unspecific degenerative changes that could impaire spermatogenesis. The present study also proposes an alternative staining technique very useful in assessing the histopathological aspects of spermatogenesis.

Autophagy may play an important role in varicocele.

The present study aimed to determine the expression of autophagy and investigate whether the hypoxia­inducible factor 1α (HIF­1α)/BCL2 interacting protein (BNIP3)/Beclin­1 autophagy signaling pathway serves an important role in activating autophagy in varicocele (VC) rat testes cells. Furthermore, the current study aimed to explain the possible association between autophagy and apoptosis. A total of 48 adult male Sprague Dawley rats were divided into group A (control), group B (VC 15­day), group C (VC 30­day) and group D (VC 45­day), with 12 rats in each group. The rats in group A did not receive any interventions, and in groups B, C, and D the VC model was established simultaneously. At 0, 15, 30, and 45 days, an orchidectomy on the left testes was performed in groups A­D, each on its respective day. Transmission electron microscopy was used to investigate the expression of autophagy. Compared with groups A and B, it was demonstrated that the expression of autophagy in groups C, and D was significantly increased. Hematoxylin and eosin staining revealed that as the rats survived VC longer, the testicular tissue damage became more serious. Furthermore, the Johnson score revealed that VC impaired the spermeiogenesis function of the male rats. Additionally, it was demonstrated that the apoptosis index of the semini-ferous epithelia cells in VC rat testes increased over time, as measured using TUNEL staining. Immunohistochemical analysis revealed that as the VC was prolonged, the expression of HIF­1α gradually increased while the expression of (apoptosis regulator Bcl­2) Bcl­2 gradually decreased. Furthermore, western blot analysis revealed that the protein expression of Bcl­2 decreased and apoptosis regulator Bax increased. Furthermore, HIF­1α, BNIP3, Beclin1 and microtubule associated protein 1 light chain 3 α (LC3)II/LC3I expression gradually increased. However, significant increases in Beclin 1 and LC3II/LC3I were only observed between the day 0 and day 30 groups. In addition, the expression of p62 significantly increased between day 0 and day 15, but gradually decreased between day 15 and day 45. The results of the present study revealed that VC can lead to testicular tissue hypoxia, and that the HIF­1α/BNIP3/Beclin1 autophagy signaling pathway may upregulate autophagy in VC rats testes. Thus, the association between autophagy and apoptosis may serve an important role in male infertility caused by VC.

Revisiting the human seminiferous epithelium cycle.

STUDY QUESTION: Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? SUMMARY ANSWER: By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. WHAT IS ALREADY KNOWN: Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. STUDY DESIGN, SIZE, DURATION: Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levin's, Johnsen's and Bergmann's scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. LIMITATIONS, REASONS FOR CAUTION: Occasionally, germ cells can be absent in some tubular sections. In this situation, it has to be taken into account the germ cell association proposed in the present study to classify the stages. WIDER IMPLICATIONS OF THE FINDINGS: Our findings bring a new focus on the morphology and development of germ cells during the SEC in human. Application of HRLM may be a valuable tool for research studies and clinical andrology helping to understand some testicular diseases and infertility conditions which remain unsolved. STUDY FUNDING/COMPETING INTEREST: Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Vitamin B12-induced spermatogenesis recovery in cimetidine-treated rats: effect on the spermatogonia number and sperm concentration.

The H2-receptor antagonist cimetidine is an antiulcer drug also used for the treatment of cancer due to its antiangiogenic effect. However, this drug has caused structural changes in the seminiferous tubules. Vitamin B12 has been used as a therapeutic agent for the treatment of male infertility. The supplementation of rats with vitamin B12 during cimetidine treatment has recovered the damaged seminiferous tubules, but how this vitamin restores the seminiferous epithelium has not been clarified. In this study, we evaluated whether vitamin B12 improves the number of spermatogonia, spermatocytes, and sperm concentration in cimetidine-treated rats. Adult male rats were treated for 50 days as follows: cimetidine group received 100 mg kg-1 b.w. of cimetidine, cimetidine-B12 group received cimetidine and 3 µg of vitamin B12-hydroxocobalamin, B12 group received only 3 µg of vitamin, and control group received saline. Sperm concentration was calculated and historesin-embedded testes sections were used for the quantitative analyses of spermatogonia (A; In/B) and spermatocytes. TUNEL method and PCNA immunofluorescence were performed. Cimetidine caused a significant reduction in sperm concentration. TUNEL-positive spermatogonia and spermatocytes were correlated to a significant reduction in the number of these cells. In cimetidine-B12 group, sperm concentration was higher than cimetidine group and a significant increase in the number of spermatogonia (stages II-VI) was correlated to a high incidence of PCNA-immunolabeled spermatogonia and spermatocytes. The results show that the supplementation of rats with vitamin B12 during cimetidine treatment increases sperm concentration and exerts a potential effect in the recovery of spermatogonia and spermatocytes.

Malfunction of spermatogenesis in experimental ischemic mice.

An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.

Evaluation of the cell population of the seminiferous epithelium and spermatic indexes of the bat Sturnira lilium (Chiroptera: Phyllostomidae).

Due to the scarcity of information about patterns of spermatogenesis in bats, this study aimed to provide information on the testicular activity of the bat Sturnira lilium along the annual seasons. Thus, a series of morphometrical and stereological analyses were made using the testes of adult S. lilium in order to achieve a better understanding of the sperm production dynamics. Light and transmission electron microscopy analyses were performed in testicular fragments of animals captured during dry and rainy seasons. The testes followed the pattern of organization described for other mammals, and there were no morphological differences between organs collected either in dry or in rainy seasons. Each tubular cross-section in stage 1 was made of 0.5 type-A spermatogonia, 4.4 primary spermatocytes in preleptotene/leptotene, 3.7 in zygotene, 11.9 in pachytene, 35.6 round spermatids and 8.5 Sertoli cells. The mitotic and meiotic indexes were 15.4 and 2.9 cells, respectively, while the spermatogenesis yield was 68.7 cells. The testicular sperm reserves was 37.61×10(6) cells, and daily sperm production per gram of testis averaged 209.68×10(6) cells, both highest averages occurring in the rainy season. S. lilium male bats have a continuous reproductive pattern, high spermatogenesis yield and low support capacity by the Sertoli cells.

The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures.

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the "blood-testis barrier", formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or "desmosome-like" junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and "desmosome-like" junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and ß-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the "areae adhaerentes", i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5-7 nm internal diameter ("cribelliform junctions"). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.

Novel clathrin/actin-based endocytic machinery associated with junction turnover in the seminiferous epithelium.

Tubulobulbar complexes are elaborate clathrin/actin related structures that form at sites of intercellular attachment in the seminiferous epithelium of the mammalian testis. Here we summarize what is currently known about the morphology and molecular composition of these structures and review evidence that the structures internalize intercellular junctions both at apical sites of Sertoli cell attachment to spermatids, and at basal sites where Sertoli cells form the blood-testis barrier. We present updated models of the sperm release and spermatocyte translocation mechanisms that incorporate tubulobulbar complexes into their designs.

Bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats.

Whether environmental exposure to bisphenol A (BPA) may induce reproductive disorders is still controversial but certain studies have reported that BPA may cause meiotic abnormalities in C. elegans and female mice. However, little is known about the effect of BPA on meiosis in adult males. To determine whether BPA exposure at an environmentally relevant dose could induce meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 20 µg/kg body weight (bw)/day for 60 consecutive days. We found that BPA significantly increased the proportion of stage VII seminiferous epithelium and decreased the proportion of stage VIII. Consequently, spermiation was inhibited and spermatogenesis was disrupted. Further investigation revealed that BPA exposure delayed meiosis initiation in the early meiotic stage and induced the accumulation of chromosomal abnormalities and meiotic DNA double-strand breaks (DSBs) in the late meiotic stage. The latter event subsequently activated the phosphatidylinositol 3-kinase-related protein kinase (ATM). Our results suggest that long-term exposure to BPA may lead to continuous meiotic abnormalities and ultimately put mammalian reproductive health at risk.

Ultrastructural study of spermiogenesis in a rare desert amphisbaenian Diplometopon zarudnyi.

Spermiogenesis, in particular the head differentiation of Diplometopon zarudnyi, was studied at the ultrastructural level by Transmission Electron Microscope (TEM). The process includes acrosomal vesicle development, nuclear elongation, chromatin condensation and exclusion of excess cytoplasm. In stage I, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. This step commences with an acrosome vesicle forming from Golgi transport vesicles; simultaneously, the nucleus begins to move eccentrically. In stage II, the round proacrosomal vesicle is flattened by projection of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle as the fibrous layer forms the subacrosomal cone. Circular manchettes surrounded by mitochondria develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. The nucleus has uniform diffuse chromatin with small indices of heterochromatin. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. In stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. One endonuclear canal is present where the perforatorium resides. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are removed. In stage V, the sperm head matures.

What is the best cryopreservation protocol for human testicular tissue banking?

STUDY QUESTION: Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking? SUMMARY ANSWER: Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue. WHAT IS KNOWN ALREADY: Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue. STUDY DESIGN, SIZE, DURATION: Fragments (n = 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (-S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV). MATERIALS, SETTING, METHODS: Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types. MAIN RESULTS AND THE ROLE OF CHANCE: The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 ± 5.6 in control tissue to 4.9 ± 2.1, 8.2 ± 5.4, 11.6 ± 5.1, 8.8 ± 3.9, 12.6 ± 4.4 and 11.7 ± 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO -S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue. WIDER IMPLICATIONS OF THE FINDINGS: An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.

New insights into roles of tubulobulbar complexes in sperm release and turnover of blood-testis barrier.

Tubulobulbar complexes are actin-filament-related structures that form at intercellular junctions in the seminiferous epithelium of mammalian testis. The structures occur both at adhesion junctions between Sertoli cells and the maturing spermatids in apical regions of the epithelium, and at junction complexes between neighboring Sertoli cells near the base of the epithelium. Here, we review the general morphology and molecular composition of tubulobulbar complexes, and also include a description of tubulobulbar complex structure in the human seminiferous epithelium. Although tubulobulbar complexes are unique to the seminiferous epithelium, they have the molecular signature of clathrin-based endocytosis machinery present generally in cells. We review the evidence that tubulobulbar complexes internalize intact intercellular junctions and are significant components of the sperm-release mechanism and the process by which spermatocytes translocate from basal to adluminal compartments of the epithelium.

Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

Internalization of adhesion junction proteins and their association with recycling endosome marker proteins in rat seminiferous epithelium.

Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are formed in association with intercellular junctions in the seminiferous epithelium. They consist of a cylindrical double-membrane core composed of the plasma membranes of the two attached cells, cuffed by a dendritic network of actin filaments. TBCs are proposed to be subcellular machines that internalize intercellular junctions during the extensive junction remodeling that occurs during spermatogenesis. At the apical sites of attachment between Sertoli cells and spermatids, junction disassembly is part of the sperm release mechanism. In this study, we used immunological probes to explore junction internalization and recycling at apical TBCs in the rat seminiferous epithelium. We demonstrate that ß1-integrin and nectin 2 were concentrated at the ends of TBCs and for the first time show that the early endosome marker RAB5A was also distinctly localized at the ends of TBCs that appear to be the 'bulbar' regions of the complexes. Significantly, we also demonstrate that the 'long-loop' recycling endosome marker RAB11A was co-distributed with nectin 2 at junctions with early spermatids deeper in the epithelium. Our results are consistent with the hypothesis that TBCs associated with late spermatids internalize adhesion junctions and also indicate that some of the internalized junction proteins may be recycled to form junctions with the next generation of spermatids.

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.