Spermatogenic Process Research Articles

Genetic Landscape of Nonobstructive Azoospermia and New Perspectives for the Clinic.

Nonobstructive azoospermia (NOA) represents the most severe expression of male infertility, involving around 1% of the male population and 10% of infertile men. This condition is characterised by the inability of the testis to produce sperm cells, and it is considered to have an important genetic component. During the last two decades, different genetic anomalies, including microdeletions of the Y chromosome, karyotype defects, and missense mutations in genes involved in the reproductive function, have been described as the primary cause of NOA in many infertile men. However, these alterations only explain around 25% of azoospermic cases, with the remaining patients showing an idiopathic origin. Recent studies clearly suggest that the so-called idiopathic NOA has a complex aetiology with a polygenic inheritance, which may alter the spermatogenic process. Although we are far from a complete understanding of the molecular mechanisms underlying NOA, the use of the new technologies for genetic analysis has enabled a considerable increase in knowledge during the last years. In this review, we will provide a comprehensive and updated overview of the genetic basis of NOA, with a special focus on the possible application of the recent insights in clinical practice.

Contribution of Single-Cell Transcriptomics to the Characterization of Human Spermatogonial Stem Cells: Toward an Application in Male Fertility Regenerative Medicine?

Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.

Potential use of stem cells for fertility preservation.

Infertility and gonadal dysfunction can result from gonadotoxic therapies, environmental exposures, aging, or genetic conditions. In men, non-obstructive azoospermia (NOA) results from defects in the spermatogenic process that can be attributed to spermatogonial stem cells (SSC) or their niche, or both. While assisted reproductive technologies and sperm banking can enable fertility preservation (FP) in men of reproductive age who are at risk for infertility, FP for pre-pubertal patients remains experimental. Therapeutic options for NOA are limited. The rapid advance of stem cell research and of gene editing technologies could enable new FP options for these patients. Induced pluripotent stem cells (iPSC), SSC, and testicular niche cells, as well as mesenchymal stromal cells (aka medicinal signaling cells, MSCs), have been investigated for their potential use in male FP strategies. Here, we review the benefits and challenges for three types of stem cell-based approaches under investigation for male FP, focusing on the role that promising sources of MSC derived from human umbilical cord, specifically human umbilical cord perivascular cells (HUCPVC), could fulfill. These approaches are as follows: 1. isolation and ex vivo expansion of autologous SSC for in vivo transplantation or in vitro spermatogenesis; 2. in vitro differentiation toward germ cell and testicular somatic cell lineages using autologous SSC, or stem cells such iPSC or MSC; and 3. protection or regeneration of the spermatogenic niche after gonadotoxic insults in vivo. Our studies suggest that HUCPVC are promising sources of cells that could be utilized in multiple aspects of male FP strategies.

Exposure to hypoxia inhibits pituitary-testicular activity in the fish Oreochromis mossambicus

Fishes are exposed to natural hypoxic conditions during aquacultural practices. Although negative effects of hypoxia are documented on fish reproduction, the mechanism of hypoxia-induced alteration in spermatogenic process is not well understood in continuously breeding fishes. The aim of this investigation was to determine the influence of hypoxia on pituitary-testicular activity in the tilapia Oreochromis mossambicus, a cichlid fish that breeds throughout the year. The fish were exposed to moderate hypoxia (MH, Dissolved oxygen (DO) level, 3.24 ± 0.22 mg/L) and severe hypoxia (SH, DO level, 1.64 ± 0.38 mg/L) for 21 days. No significant differences were noticed in the mean numbers of spermatogonia (Sg), primary spermatocytes (Ps), secondary spermatocytes (Ss), early spermatids (Est) and late spermatids (Lst) between controls and fish subjected to MH, whereas the numbers of these cells were significantly lower in fish exposed to SH. Furthermore, weakly immunoreactive luteinizing hormone (LH) secreting cells in the proximal pars distalis (PPD) of the pituitary gland were concomitant with significant reduction in mean serum levels of LH and testosterone as well as androgen receptor immunoreactivity in the Sertoli cells in the testis of fish exposed to SH compared to other groups. On the other hand, the serum cortisol levels were significantly higher in fish exposed to SH compared with other experimental groups. Taken together, these results indicate that the tilapia is able to withstand the moderate hypoxic condition, whereas exposure to severe hypoxic condition suppresses the process of spermatogenesis through the inhibition of LH secretion, which in turn might suppress the testicular steroidogenesis and androgen receptor expression in the Sertoli cells. The results also suggest a possibility that this inhibition might be mediated through the activation of stress-axis in O. mossambicus.

Acute exposure to perfluorononanoic acid in prepubertal mice: Effect on germ cell dynamics and an insight into the possible mechanisms of its inhibitory action on testicular functions

Perfluoroalkyl acids (PFAAs) are anthropogenic compounds used globally in a variety of commercial products. Perfluorononanoic acid (PFNA), a member of PFAAs, is detected in human blood and this has been reported to cause hepatotoxic, immunotoxic, and developmental and testicular toxic effects in laboratory animals. We have recently shown that the acute exposure to PFNA in prepubertal Parkes (P) mice impairs spermatogenesis by inducing oxidative stress and inhibiting testosterone biosynthesis in the testis. The present study was aimed to examine the effect of acute exposure to PFNA in prepubertal P mice on germ cell dynamics and to understand the possible mechanisms of action of this compound on testicular functions. PFNA (2 and 5 mg/kg body weight) was orally administered to male mice for 14 days from postnatal day 25–38. The treatment caused a decrease in overall germ cell transformation. The results also reveal that impairment in testicular functions in treated mice is associated with alterations in cholesterol and glucose homeostasis; further, an inhibition in expressions of growth hormone receptor (GHR), insulin-like growth factor-1 (IGF-1), insulin-like growth factor-1 receptor (IGF-1R), androgen receptor (AR), phosphorylated mammalian target of rapamycin (p-mTOR) and peroxisome proliferator activated receptor α (PPAR α) in the testis is also implicated in this action. The findings thus suggest involvement of multiple factors which altogether contribute to the alterations in spermatogenic process and testosterone production following acute exposure to PFNA in prepubertal mice.

Influence of Wilms' tumor suppressor gene WT1 on bovine Sertoli cells polarity and tight junctions via non-canonical WNT signaling pathway

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.

Impact of maternal overweight on the sexual maturity of male offspring in rats

The aims of the present work were to study the effect of maternal overweight on both the count and quality of sperm of the offspring and to assess whether this maternal condition is able to alter testicular integrity and spermatogenic process. To this end, male offspring from rats fed a standard (OSD) or cafeteria (OCD) diet were used. Body and testis weight, length, preputial separation and ano-genital distance (AGD) were recorded and testes were removed at 60 days of age. In addition, the number of germ, Leydig and Sertoli cells, spermatogenesis and sperm integrity were examined. The OCD rats were divided into two groups: offspring from rats with 25% and≥35% of overweight (OCD25 and OCD35, respectively). Both OCD groups showed higher body and testis weight, higher length, and greater AGD than OSD rats. OCD25 also showed early preputial separation and OCD35 exhibited a high level of testosterone with normal glycemia. Both OCD25 and OCD35 rats had a lower number of spermatozoa and Leydig cells than OSD rats, and OCD35 also exhibited a lower number of spermatogonia and Sertoli cells than OSD rats. In addition, both OCD groups exhibited lower number of sperm cells with normal morphology and sperm motility, and OCD35 showed changes in both the seminiferous epithelium and spermatogenic process. These results suggest that maternal overweight severely affects the reproductive capacity of male offspring, likely leading to a subfertility condition and a premature reduction of the reproductive life span.

Transgenerational sperm DNA methylation epimutation developmental origins following ancestral vinclozolin exposure

ABSTRACTA number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

Bioinformatic identification of key genes and molecular pathways in the spermatogenic process of cryptorchidism

This study aims to determine key genes and pathways that could play important roles in the spermatogenic process of patients with cryptorchidism. The gene expression profile data of GSE25518 was obtained from the Gene Expression Omnibus (GEO) database. Microarray data were analyzed using BRB-Array Tools to identify differentially expressed genes (DEGs) between high azoospermia risk (HAZR) patients and controls. In addition, other analytical methods were deployed, including hierarchical clustering analysis, class comparison between patients with HAZR and the normal control group, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and the construction of a protein–protein interaction (PPI) network. In total, 1015 upregulated genes and 1650 downregulated genes were identified. GO and KEGG analysis revealed enrichment in terms of changes in the endoplasmic reticulum cellular component and the endoplasmic reticulum protein synthetic process in the HAZR group. Furthermore, the arachidonic acid pathway and mTOR pathway were also identified as important pathways, while RICTOR and GPX8 were indentified as key genes involved in the spermatogenic process of patients with cryptorchidism. In present study, we found that changes in the synthesis of endoplasmic reticulum proteins, arachidonic acid and the mTOR pathway are important in the incidence and spermatogenic process of cryptorchidism. GPX8 and RICTOR were also identified as key genes associated with cryptorchidism. Collectively, these data may provide novel clues with which to explore the precise etiology and mechanism underlying cryptorchidism and cryptorchidism-induced human infertility.

TorsinA Is Functionally Associated with Spermatogenesis.

TorsinA is a member of the AAA+ superfamily of adenosine triphosphatases. These AAA+ proteins have numerous biological functions, including vesicle fusion, cytoskeleton dynamics, intracellular trafficking, protein folding, and degradation as well as organelle biogenesis. Of particular interest is torsinA, which is mainly located in the endoplasmic reticulum (ER) and nuclear envelope (NE). Interestingly, mutations in the TOR1A gene (the gene encoding torsinA) are associated with DYT1 dystonia and with the preferential localization of mutated torsinA at the NE, where it is associated with lamina-associated polypeptide 1. A bioinformatics study of the torsinA interactome revealed reproductive processes to be highly relevant, as proteins in this class were found to interact with the former. Interestingly, the torsin protein family had never been previously described to be associated with the mammalian spermatogenic process. Histological staining of torsinA in human testis tissue revealed a granular cytoplasmic localization in mid- and late spermatocytes. We further sought to understand this newly discovered expression of torsinA in the meiotic phase of human spermatogenesis by studying its specific subcellular distribution. TorsinA is not present in the ER as commonly described. The proposal that torsinA might relocate to the pro-acrosomal vesicles in the Golgi apparatus is discussed.

Differential effects of depot formulations of GnRH agonist leuprorelin and antagonist degarelix on the seminiferous epithelium of the rat testis.

Despite their pharmacologically opposite actions, long-acting depot formulations of both GnRH agonists and antagonists have been clinically applied for treatment of androgen-sensitive prostate cancer. Sustained treatment with GnRH analogues commonly suppresses both the synthesis and release of gonadotropins, leading to depletion of testicular testosterone. To clarify the underlying differences in the effects of GnRH agonists and antagonists on spermatogenesis, we compared histological changes in the seminiferous epithelium after administration of depot formulations of GnRH agonist leuprorelin and antagonist degarelix to male rats. Testicular weight had markedly declined by 28 days after administration of both GnRH analogues, although the testicular weight was decreased more promptly by leuprorelin compared with degarelix. Shortly after administration, massive exfoliation of premature spermatids and anomalous multinucleated giant cells was observed in seminiferous tubules of leuprorelin-treated rats, probably via the initial hyperstimulatory effects on the hypothalamic-pituitary-testicular axis, whereas no discernible changes were found in those of degarelix-treated rats. Long term treatment with both types of GnRH analogues similarly induced a marked reduction in the height of the epithelium and deformation of apical cytoplasm in Sertoli cells, resulting in premature detachment of spermatids from the epithelium. Lipid droplets had accumulated progressively in Sertoli cells, especially in those of degarelix-treated rats. These findings clearly demonstrate the differences in the effects of GnRH agonists and antagonists on the spermatogenic process. This study suggests that an appropriate choice of GnRH analogues is necessary to minimize their adverse effects on spermatogenesis when reproductive functions should be preserved in patients.

Role of Sertoli Cell Proteins in Immunomodulation.

Sertoli cell, over the past 30 years, have been elevated from simple mechanical elements to the rank of a "sentinel" in spermatogenesis. By delivering potent immunomodulatory and trophic proteins, Sertoli cells are unique cell type with a pivotal role in maintaining testis immune privilege and the immune-protection of the antigenic germ cells. The findings from SC transplantation studies utilizing experimental animal models of disease, demonstrate the presence of the same immuno-modulation properties and mechanisms at tissue and organ sites far from testis. The complex pathways that generate and maintain the immune tolerance involve the production of several immunomodulatory or immune-related proteins such as cytokines, chemokines, growth factors, mediators of the inflammation, complement inhibitors or adhesion molecules. A better definition and understanding of these Sertoli cell proteins and the mechanisms of immunoprotection should help to elucidate their role in the spermatogenic process. The demonstration of their capabilities in transplantation experiments suggests that Sertoli cells may be good candidates in cell therapy for a number of cell-mediated chronic diseases.

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.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera

BackgroundRapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology.ResultsComparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects.ConclusionsOur results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph.

Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes

BackgroundRecently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development.MethodsTesticular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches.ResultsThe method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations.ConclusionsIn vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed that as long as cultures progress, the proportion of microRNAs declined respect to piwi-interactingRNAs levels that increased, showing a similar propensity than which happens in in vivo spermatogenesis. Our study allows to improve and potentially to control the ex vivo spermatogenesis development, opening new perspectives in the reproductive biology fields including male fertility.

Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis

Liver serine/threonine kinase B1 (LKB1) is a tumor suppressor associated with the pathogenesis of Peutz-Jeghers syndrome. Affected males are at increased risk of developing Sertoli cell tumors and display defective spermatogenesis. Male mice lacking the short isoform (Lkb1S) of Lkb1 were sterile and exhibited abnormal spermiogenesis. In addition to the short isoform, the long isoform of Lkb1 (Lkb1L) is also expressed in testis; however, the requirement of the long isoform for fertility and the functional difference between the isoforms remain unknown. Herein, different from the spermiation failure reported in Lkb1S knockout mice, conditional deletion (cKO) of both isoforms of Lkb1 in germ cells resulted in male sterility stemming from defects in acrosome formation, as well as nuclear elongation and condensation during spermatid differentiation. Additionally, cKO mice showed a progressive germ cell loss that was never reported in mice with Lkb1S deletion. Further experiments revealed that the defect resulted from the failure of spermatogonial stem/progenitor cells (SPCs) maintenance. Although increased mTORC1 activity in postnatal cKO testes was consistent with a tendency toward germline stem cell differentiation, in vivo inhibition of the pathway by rapamycin treatment failed to rescue the phenotype. Concurrently, we detected a significant reduction of mitochondrial activity in Lkb1deficient SPCs. The results suggest that the regulation of LKB1 on SPCs’ maintenance is associated with mitochondrial functions but not through the mTOR signaling pathway. In summary, our study supports different roles of Lkb1 isoforms in spermatogenesis with Lkb1L directing SPCs maintenance, and Lkb1L and Lkb1S coordinately regulating spermatid differentiation.

Arsenic trioxide exposure impairs testicular morphology in adult male mice and consequent fetus viability

ABSTRACTThe acute promyelocytic leukemia (APL) is a rare disease, affecting 0.1/100,000 individuals globally. Despite significant advances in APL therapy, some patients still experience relapsed disease. Currently, arsenic trioxide (As2O3) was found to be effective in relapsed APL treatment and considered as standard treatment for these cases. However, it has been shown that exposure to As2O3 may exert adverse effects on the male reproductive system since this substance might also induce apoptosis of other important cell types including stem cells. Studies demonstrated that treatment with this metallic substance decreased plasma levels of testosterone and interfered with sperm parameters such as concentration, motility, and viability. In addition, As2O3 was found to produce significant damage to spermatocytes, which may be associated with testicular toxicity and consequent inhibition of spermatogenesis. The aim of this study was to determine sub-chronic treatment effects of As2O3 on sperm and testicular morphology, androgen receptor (AR) immunoreactivity in testes and epididymis, in addition to evaluation of fertility parameters in adult male mice. Thirty adult Swiss mice were divided into three experimental groups: control; received distilled water (vehicle) while treated received 0.3 or 3 mg/kg/day As2O3 subcutaneously, for 5 days per week, followed by 2 days of interruption, for 5 weeks. Results showed that As2O3 (1) decreased spermatozoa number, (2) produced seminiferous epithelium degeneration and exfoliation of germ cells tubule lumen (3) altered nucleus/cytoplasm proportion of Leydig cells and (4) reduced AR immunoreactivity in both Leydig and epithelial epididymal cells. Further, fetal viability tests demonstrated an increase in post-implantation loss in females that were mated with As2O3-treated males. Data indicate that As2O3 exposure altered the spermatogenic process and subsequently fetal viability.

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.

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.

Seasonal reproductive cycle and variability in plasma testosterone levels in the domestic duck (Anas platyrhynchos)

Morphofunctional gonadal alterations constitute an annual cycle in birds during which four phases can be distinguished in the testes based on histological modifications in the seminiferous epithelium, called quiescence, recrudescence, reproduction and regression. The aim of the present investigation was to conduct a morphometric and morphological study of the seminiferous epithelium on the testis of domestic duck and to evaluate the plasma testosterone levels in order to determine the possible presence of a reproductive cycle in the tropical region. The study was conducted on 60 adult domestic ducks ( Anas platyrhynchos ), where five animals were sacrificed per month and the testes were removed and histologically and morphometrically studied. Blood samples of the five animals were collected monthly in the morning period and the testosterone levels were analyzed. Annual reproductive cycle of the domestic duck presented by four distinct phases, starting with the reproductive phase at the beginning of winter (July) and peak of reproduction in spring (October), accompanied by an increase in tubule seminiferous diameter, epithelium height, weight and testicular volume, followed by a regressive phase at the end of spring (November) and the beginning of summer (December). Testicular quiescence was observed during summer (January and February) and was followed by a phase of spermatogenic recrudescence, which lasted throughout autumn (March to June), corresponding to the longest phase of the cycle. Complete spermiogenesis in terms of spermatozoa maturation and spermiation was observed during the reproductive phase, coinciding with the plasma testosterone peak. At regressive phase an exfoliation of the seminiferous epithelium was observed, accompanied by a great amount of fibrillar collagen. A decrease in tubular seminiferous diameter and epithelium height was observed during testicular quiescence, with predominance of spermatogonia and Sertoli cells and a second plasma testosterone peak was also observed during this phase (January), probably representing the preparation for a gradual return to the spermatogenic process during recrudescence. The latter phase was characterized by gradual recovery of the seminiferous epithelium, accompanied by the increase of spermatogonia, onset of meiosis, and the presence of primary spermatocytes and maturing spermatids. In conclusion the domestic duck presented the testicular cycle divided in four distinct phases in the tropical region with testosterone peaks in the testicular quiescence and reproductive phases.