Cycle Of Seminiferous Epithelium Research Articles

Sertoli-immature spermatids disengagement during testis regression in the armadillo

In nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and Western blotting we studied key proteins of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, B1-integrin, laminin G3, N-cadherin, B-catenin, P-B-catenin-Tyr654, FAK, P-FAK-Tyr397, SRC, P-SRC-Tyr416 proteins present a spermatogenetic cycle-dependent localisation pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, SRC and B-catenin contribute to the disassembly of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

Immunolocalization of RFamide-related peptide 3 in a desert rodent Gerbillus tarabuli during seminiferous epithelium cycle.

Gerbillus tarabuli is a nocturnal seasonal breeder desert rodent with a main breeding season in spring and summer, and sexual quiescence in winter. This species is an interesting model for studying testis function in rodents. Therefore, the present study was performed firstly to investigate the stages of seminiferous epithelium cycle of Gerbillus tarabuli with a histological, morphometric and statistical study. And secondly to investigate the expression and possible variations in cellular distribution of RFamide-related peptide-3 (RFRP-3) - the mammalian ortholog of avian gonadotropin-inhibitory hormone (GnIH) - during seminiferous epithelium cycle using immunohistochimestry. Our results showed for the first time that the seminiferous epithelium cycle in Gerbillus tarabuli comprises 14 well-defined stages according to the tubular morphology method. The seminiferous epithelium thickness showed a significant difference during the epithelium cycle, thus it was the only morphometric classification criterion of seminiferous epithelium cycle in Gerbillus tarabuli. The immunohistochemical study reveals, for the first time, the presence of RFRP-3 in Gerbillus tarabuli testes, in both testicular compartments: the tubular and the interstitial. RFRP-3 is expressed differently according to the seminiferous epithelium cycle, RFRP-3 seemed to be more expressed at the stages V-VII and XIII. RFRP-3 was detected in Sertoli cells (≈12%), spermatocytes I (≈19%), round and elongated spermatids (≈13%), and with a more important signal in Leydig cells (26.87%±0.07). These results indicated the importance of RFRP-3 in testicular function in Gerbillus tarabuli; its expression at the interstitial and germinal levels argues in favor of an involvement in androgens synthesis and in spermatogenesis specifically in meiosis and spermiogenesis. This action seems primordial from stages V-VII and XIII. Also, the study of the seminiferous epithelium cycle will enrich the histological identity of the species.

A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq

Spermatogenesis requires intricate interactions between the germline and somatic cells. Within a given cross section of a seminiferous tubule, multiple germand somatic cell types co-occur. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. To address this challenge, we collected single-cell RNA sequencing data from ∼35,000 cells from the adult mouse testis and identified all known germ and somatic cells, as well as two unexpected somatic cell types. Our analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to spermatids and identified candidate transcriptional regulators at several transition points during differentiation. Focused analyses delineated four subtypes of spermatogonia and nine subtypes of Sertoli cells; the latter linked to histologically defined developmental stages over the seminiferous epithelial cycle. Overall, this high-resolution cellular atlas represents a community resource and foundation of knowledge to study germ cell development and invivo gametogenesis.

Spermatogonial behavior in marmoset: a new generation, their kinetics and niche.

Could a more detailed evaluation of marmoset spermatogonial morphology, kinetics and niches using high-resolution light microscopy (HRLM) lead to new findings? Three subtypes of marmoset undifferentiated spermatogonia, which were not evenly distributed in terms of number and position along the basal membrane, and an extra premeiotic cell division not present in humans were identified using HRLM. The seminiferous epithelium cycle (SEC) of marmosets is divided into nine stages when based on the acrosome system, and several spermatogenic stages can usually be recognized within the same tubular cross-section. Three spermatogonial generations have been previously described in marmosets: types Adark, Apale and B spermatogonia. Testes from five adult Callithrix penicillata were fixed by glutaraldehyde perfusion via the cardiac route and embedded in Araldite plastic resin for HRLM evaluation. Semi-thin sections (1 μm) were analyzed morphologically and morphometrically to evaluate spermatogonial morphology and kinetics (number, mitosis and apoptosis), spermatogenesis efficiency and the spermatogonial niche. Shape and nuclear diameter, the presence and distribution of heterochromatin, the granularity of the euchromatin, as well as the number, morphology and degree of nucleolar compaction were observed for morphological characterization. Kinetics analyses were performed for all spermatogonial subtypes and preleptotene spermatocytes, and their mitosis and apoptosis indexes determined across all SEC stages. Spermatogenesis parameters (mitotic, meiotic, Sertoli cell workload and general spermatogenesis efficiency) were determined through the counting of Adark and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and Sertoli cells at stage IV of the SEC. This is the first time that a study in marmosets demonstrates: the existence of a new spermatogonial generation (B2); the presence of two subtypes of Adark spermatogonia with (AdVac) and without (AdNoVac) nuclear rarefaction zones; the peculiar behavior of AdVac spermatogonia across the stages of the SEC, suggesting that they are quiescent stem spermatogonia; and that AdVac spermatogonia are located close to areas in which blood vessels, Leydig cells and macrophages are concentrated, suggesting a niche area for these cells. Not applicable. The C. penicillata spermatogonial kinetics evaluated here consider spermatogonial number across the SEC and their mitotic and apoptotic figures identified in HRLM sections. Therefore, caution is required when comparing absolute values between species. Although morphometric evaluation has suggested that AdVac spermatogonia are stem cells, a functional proof of this is still missing. It is known that parameters of the spermatogenic process in C. penicillata have similarities with those of the common marmoset C. jacchus, however, a detailed study of spermatogonial morphology, kinetics and niche has not yet been performed in C. jacchus, and a full comparison of the two species is not possible. Our findings in C. penicillata contribute to a better understanding of the spermatogonial behavior and spermatogenesis efficiency in non-human primates. Given the phylogenetic closeness of the marmoset to the human species, similar processes might occur in humans. Therefore, marmosets may be an excellent model for studies regarding human testicular biology, fertility and related disorders. Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest.

Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats

Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5 mg/kg of DXR and were sacrificed at seven, 14, 21 and 28 days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14 days. At 21 and 28 days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.

Regionally distinct patterns of STAT3 phosphorylation in the seminiferous epithelia of mouse testes.

In mouse testes, Sertoli cells support the continuous process of spermatogenesis, which is dependent on seminiferous epithelial cycles along the longitudinal axis of the seminiferous tubule. Sertoli cell function is modulated partly by local cytokines and/or growth factors derived from adjacent tissues such as blood vessels, macrophages, rete testis, etc. However, the spatial activation patterns by local signals in vivo remain unclear. In this study, we focused on Signal Transducers and Activators of Transcription (STAT) signaling in Sertoli cells, because STAT is a major crucial cytokine transducer for somatic cyst cell regulation in Drosophila testis niches. In mouse testes, STAT3 was ubiquitously expressed in Sertoli cells throughout the seminiferous tubules. Phosphorylated STAT3 (p-STAT3) was predominantly observed in the Sertoli cells within the valve-like structure adjacent to the rete testis (i.e., the Sertoli valve [SV]) in the terminal segment of the proximal seminiferous tubules. In the distal seminiferous tubules with active spermatogenesis, most Sertoli cells were negative for anti-p-STAT3 staining. Albeit rarely, a small patch of several p-STAT3-positive Sertoli cells was detected frequently in seminiferous epithelial cycle stages I-VI. Such p-STAT3-positive ratios in the convoluted seminiferous epithelia were significantly increased in germ cell-less testes than in the wild-type testes, but with considerably lower ratios than in the SV region. These findings imply that regionally distinct patterns of STAT3 phosphorylation in the Sertoli cells depend on either location or spermatogenic activity in normal healthy testes in vivo, highlighting a novel entry point to understanding STAT signaling in mammalian spermatogenesis.

MiR-142-3p Inhibits TGF-β3-Induced Blood-Testis Barrier Impairment by Targeting Lethal Giant Larvae Homolog 2

Background/Aims: Transforming growth factor-β3 (TGF-β3) has been proved to perturb the blood-testis barrier (BTB) by accelerating junction protein endocytosis in Sertoli cells (SCs) to accommodate the traversing of preleptotene spermatocytes across the BTB around stage VIII in rat. Yet the molecular network underlying the impairment of TGF-β3 on BTB integrity is not fully elucidated. Our study herein was designed to investigate the participation of microRNA-142-3p (miR-142-3p), which has been reported to affect TGF-β3 signaling via different pathways, during BTB dynamics and the corresponding mechanisms. Methods: MiRNA mimic or agomiRNA was co-administered with or without TGF-β3 in the cultured SCs or in the rat testis. The SC permeability barrier function was reflected by measuring the transepithelial resistance (TER) and the permeability of the sodium fluorescein (Na-F). The BTB integrity was detected by the permeation of biotin. A luciferase reporter assay was used to testify the potential target of miR-142-3p, lethal giant larvae 2 (Lgl2). Laser capture microdissection (LCM) was applied to acquire cell components of different stages of seminiferious tubules, followed by detection of the expression levels of miR-142-3p, TGF-β3, and Lgl2 by qPCR. The SC barrier function was also detected as above in the presence of TGF-β3 after Lgl2 knockdown. Results: We revealed a reversion of TGF-β3-induced BTB impairment after miR-142-3p treatment both in vitro and in vivo. Meanwhile, the activation of Cdc42 and reduction in occludin aroused by TGF-β3 were also reversed by miR-142-3p. The predicted binding of miR-142-3p with 3’-untranslated region (3’-UTR) of Lgl2, was verified by the luciferase assay. Moreover, an increased Lgl2 level in TGF-β3-treated SCs was found and correlated stage-specific expressions of TGF-β3, miR-142-3p, and Lgl2 were revealed. Knockdown of Lgl2 in SCs was shown to partially antagonize the BTB disruption mediated by TGF-β3. Conclusions: Collectively, our results suggest a resistance of miR-142-3p on the BTB impairment caused by TGF-β3 during the seminiferous epithelial cycle by targeting Lgl2.

Influence of racial crossing on testicular structure and spermatogenesis in sheep Santa Inês and crossbred Santa Inês and Dorper.

The study aimed at evaluating the testicular structure and spermatogenesis of Santa Inês and Santa Inês and Dorper crossbred sheep. Sixteen testicles of the animals under study were used, in order to evaluate the volumetric proportion, the diameter of the tubules, the height of seminiferous epithelium, the frequency of stages of the seminiferous epithelium cycle and the spermatogenic yield. The data were submitted to Student-Newman-Keulstestat 5% significance. The tubular diameter was 173.12 ± 29.09 μm and 185.71 ± 29.7 μm, and the height of the seminiferous epithelium was 52.29 ± 9.98 μm and 56.68 ± 11.25 μm, for the crossbred and the Santa Inês, respectively. There was no difference between the testicular compartments and the frequency of the stages of the seminiferous epithelium cycle between the groups. Santa Inês sheep presented a number of 15.36 ± 4.49 spermatocytes in the pre-leptotene/leptotene stage and 27.42 ± 6.65 spermatocytes in the pachytene, whereas the crossbred presented 13.18 ± 5.19 and 23.48 ± 7.80, respectively. The crossbred showed higher meiotic yield (3.98 ± 1.28) and spermatogenesis yield (3.71 ± 1.02). It is concluded there are differences in testicular morphology and spermatogenesis between Santa Inês sheep and crossbred Dorper/Santa Inês, indicating that the crossbreeding between the Santa Inês and Dorper animals allows a gain in the reproductive potential.

Male Reproductive Patterns of Hibernating Korean Greater Horseshoe Bat,Rhinolophus ferrumequinum korai: I. Annual Cycle of the SeminiferousEpithelium and Morphological Changes of the Testes

Background: Morphological changes of testes and seminiferous epithelium cycle were observed by optical and transmission electron microscopy to determine male reproductive patterns of the hibernating Korean greater horseshoe bat, Rhinolophus ferrumequinum korai. Materials and methods: In this study, 40 male Rhinolophus ferrumequinum korai were collected from abandoned mines in Gyeongnam and Jeonnam provinces of South Korea from January 2014-December 2015. Differentiation process and cytological traits of seminiferous epithelium following monthly changes were examined with electron microscopic techniques and observed by optical and electron microscopy. Results and conclusion: Male reproductive pattern of hibernating Korean R. ferrumequinum korai consists of three main stages. The first is the spermatogenesis stage (from April-September), including spermatocytogenesis (which appears from April-May) and spermiogenesis (from June-September). The activity of spermatogenesis was the highest in August. The lumen of seminiferous tubules was open from mid-April to mid-October. It was closed from November to March of the following year. The second is the phagocytosis stage (from mid-Octobermid- November) that is a purification process to prepare for new spermatogenesis the following year. This period is called the cleansing period. The third is the dormant stage that is a state of holding only spermatogonia and Sertoli cells. It is an adaptation strategy to store energy for long periods of hibernation. Compared to previous studies [1], spermatogenesis period preceded one month earlier. This suggests that temperature increase can impact reproductive development and spermatogenesis.

Impaired expression of testicular androgen receptor and collagen fibers in the testis of diabetic rats under HAART: the role of Hypoxis hemerocallidea.

Wide spectrum of alterations associated with highly active antiretroviral therapy (HAART) has been reported. The current study aimed at evaluating the role of Hypoxis hemerocallidea (HH) aqueous extract on the testosterone levels, expression of androgen receptors and collagen fibers in the testes of streptozoto-cin-nicotinamide-induced diabetic rats under HAART regimen. Sixty two adult male Sprague-Dawley rats (189.0 ± 4.5 g) were divided into eight groups (8 animals in each treatment groups and 6 rats in the control group). Diabetes was induced by a single intraperi-toneal injection of nicotinamide (110 mg/kg bw) followed by streptozotocin (45 mg/kg bw) and the animals were then subjected to various treatments with HAART, HH extract or melatonin. At the end of the experiment, blood samples were collected to measure serum testosterone levels. Testes were fixed in buffered formaldehyde and paraffin processed. The expression of androgen receptor (AR) was assessed by immunohistochemistry and collagen fibers were visualized by Masson trichrome staining. Serum testosterone level was drastically (p < 0.0001) reduced in all rats with induced diabetes. In the testis of diabetic rats increased collagen fibers deposition with varying derangements in germinal epithelium of spermatogenic layers were observed. Intertubular hemorrhages and absence of spermatozoa were also noted in the testes of diabetic rats subjected to HAART. Reduced immunoexpression of ARs was found in the nuclei of Sertoli cells and the cytoplasm of spermatogonia and spermatocytes in III-IV stages of the seminiferous epithelium cycle of diabetic animals treated with different dosages of HH alone and those treated with HAART concomitantly with melatonin and HH. The expression of ARs was almost negative in the testes of rats treated with HAART alone. Concomitant treatment of rats with aqueous HH extract during the HAART did not change se-rum testosterone level nor mitigate the altered expression of collagen fibers and androgen receptor resulting from STZ-nicotinamide-induced diabetes. Therefore, anti-diabetic properties of Hypoxis extract require further investigation.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818)

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Morphofunctional evaluation of the testis, duration of spermatogenesis and spermatogenic efficiency in the Japanese fancy mouse (Mus musculus molossinus).

Japanese fancy mouse, mini mouse or pet mouse are common names used to refer to strains of mice that present with different colour varieties and coat types. Although many genetic studies that involve spotting phenotype based on the coat have been performed in these mice, there are no reports of quantitative data in the literature regarding testis structure and spermatogenic efficiency. Hence, in this study we researched testis function and spermatogenesis in the adult Japanese fancy mouse. The following values of 68 ± 6 mg and 0.94 ± 0.1% were obtained as mean testis weight and gonadosomatic index, respectively. In comparison with other investigated mice strains, the fancy mouse Leydig cell individual size was much smaller, resulting in higher numbers of these cells per gram of testis. As found for laboratory mice strains, as a result of the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle have been described in this study. The combined frequencies of pre-meiotic and post-meiotic stages were respectively 24% and 64% and very similar to the laboratory mice. The more differentiated germ cell types marked at 1 h or 9 days after tritiated thymidine administration were preleptotene/leptotene and pachytene spermatocytes at the same stage (VIII). The mean duration of one spermatogenic cycle was 8.8 ± 0.01 days and the total length of spermatogenesis lasted 37.8 ± 0.01 days (4.5 cycles). A high number of germ cell apoptosis was evident during meiosis, resulting in lower Sertoli cell and spermatogenic efficiencies, when compared with laboratory mice strains.

Revisiting the human seminiferous epithelium cycle

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)? 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. 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. 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. 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. 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. 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. 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. 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. Not applicable.

The spermatogenic process of the common vampire bat Desmodus rotundus under a histomorphometric view.

Among all bat species, Desmodus rotundus stands out as one of the most intriguing due to its exclusively haematophagous feeding habits. However, little is known about their spermatogenic cycle. This study aimed at describing the spermatogenic process of common vampire bats through testicular histomorphometric characterization of adult specimens, spermatogenic production indexes, description of stages of the seminiferous epithelium cycle and estimative of the spermatogenic process duration. Morphometrical and immunohistochemical analyzes for bromodeoxiuridine were conducted under light microscopy and ultrastructural analyzes were performed under transmission electron microscopy. Vampire bats showed higher investment in gonadal tissue (gonadosomatic index of 0.54%) and in seminiferous tubules (tubulesomatic index of 0.49%) when compared to larger mammals. They also showed a high tubular length per gram of testis (34.70 m). Approximately half of the intertubular compartment was found to be comprised by Leydig cells (51.20%), and an average of 23.77x106 of these cells was found per gram of testis. The germline cells showed 16.93% of mitotic index and 2.51% of meiotic index. The overall yield of spermatogenesis was 60% and the testicular spermatic reserve was 71.44x107 spermatozoa per gram of testis. With a total spermatogenesis duration estimated at 37.02 days, vampire bats showed a daily sperm production of 86.80x106 gametes per gram of testis. These findings demonstrate a high sperm production, which is commonly observed in species with promiscuous mating system.

Annexin A2 is critical for blood-testis barrier integrity and spermatid disengagement in the mammalian testis

Throughout spermatogenesis, two important processes occur at late stage VIII of the seminiferous epithelial cycle in the rat testis: preleptotene spermatocytes commence entry into the adluminal compartment and step 19 spermatids release from the seminiferous epithelium. Presently, it is not clear how these processes, which involve extensive restructuring of unique Sertoli–Sertoli and Sertoli–germ cell junctions, are mediated. We aimed to determine whether annexin A2 (ANXA2), a Ca2+-dependent and phospholipid-binding protein, participates in cell junction dynamics. To address this, in vitro and in vivo RNA interference studies were performed on prepubertal Sertoli cells and adult rat testes. The endpoints of Anxa2 knockdown were determined by immunoblotting, morphological analyses, fluorescent immunostaining, and barrier integrity assays. In the testis, ANXA2 localized to the Sertoli cell stalk, with specific staining at the blood–testis barrier and the concave (ventral) surface of elongated spermatids. ANXA2 also bound actin when testis lysates were used for immunoprecipitation. Anxa2 knockdown was found to disrupt the Sertoli cell/blood–testis barrier in vitro and in vivo. The disruption in barrier function was substantiated by changes in the localization of claudin-11, zona occludens-1, N-cadherin, and β-catenin. Furthermore, Anxa2 knockdown resulted in spermiation defects caused by a dysfunction of tubulobulbar complexes, testis-specific actin-rich ultrastructures that internalize remnant cell junction components prior to spermiation. Additionally, there were changes in the localization of several tubulobulbar complex component proteins, including actin-related protein 3, cortactin, and dynamin I/II. Our results indicate that ANXA2 is critical for the integrity of the blood–testis barrier and the timely release of spermatids.

Morphology and Morphometry of Seminiferous Tubules in Crioulo Horses

This study aimed to assess the biometrics of the testes and the morphology of the seminiferous tubules of Crioulo horses. We studied 10 sexually mature stallions (3–6 years of age). After orchiectomy, testes were perfused with Karnovsky's solution and then embedded in glycol methacrylate. Testis sections (4 μm) were cut and stained with toluidine blue and a solution of 1% sodium borate. The histological images were digitized, and the morphometric analysis was performed using ImageJ software. The average weight of the stallions was 377.5 kg, and the average weight of both testicles was 162.9 g. The percentage of testicular parenchyma occupied by the seminiferous tubules and the intertubular tissue was 77.97% ± 6.34% and 22.03% ± 6.34%, respectively. The average tubular diameter was 205.00 ± 36.91 μm, whereas the average height of the seminiferous epithelium was 70.56 ± 2.82 μm. Average tubular length per testicle and average tubular length per gram of testicle were 4,085.10 ± 1,170.68 m and 26.09 ± 10.63 m/g, respectively. The characteristics of the eight stages of the seminiferous epithelium cycle were similar to those reported in other horse breeds. We conclude that the morphometry of the seminiferous tubules of Crioulo horse resembles what has been reported in other horse breeds. The volumetric proportion of the seminiferous tubules and the Leydig cells of the Crioulo horse is one of the highest ever reported for stallions.

Rattus norvegicus Spermatogenesis Colony-Forming Assays.

Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cells during a seminiferous epithelial cycle. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia have not developed into meiosis, much less through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions will facilitate molecular biology studies on pre-meiotic steps in gamete development, and provide a soma-free bioassay to identify spermatogenic factors that promote meiotic progression in vitro.

Spermatogenesis and cellular associations in the seminiferous epithelium of Guinea cock (Numida meleagris)

The study describes the steps of spermiogenesis and stages of seminiferous epithelial cycle in breeding guinea cocks. Sections embedded in epoxy resin were employed for the determination of the sta...

Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP-a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts-activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca(2+)-activated large-conductance K(+) channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis.

Spermatogenesis in the Microminipig.

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.