In recent years, the incidence of teratospermia has been increasing, and it has become a very important factor leading to male infertility. The research on the molecular mechanism of teratospermia is also progressing rapidly. This article briefly summarizes the clinical incidence of teratozoospermia, and makes a retrospective summary of related studies reported in recent years. Specifically discussing the relationship between gene status and spermatozoa, the review aims to provide the basis for the genetic diagnosis and gene therapy of teratozoospermia.

The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate ‘tuning’ of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial–temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

Butterflyfish Chaetodon striatus is highly sought after in the marine ornamental aquarium, although studies about its reproductive biology are scarce. Therefore, to contribute to a better understanding of the reproductive aspects of C. striatus, we describe in detail with the use of high resolution histology the cellular dynamics of the germinal epithelium during the reproductive life history of this species. Based on the activity of the germinal epithelium, this study describes different stages of the gonadal development, similar to the reproductive phases found in other fish, to determine the reproductive period of C. striatus. In characterization of gonadal development, the following germ cells are described for males: spermatogonia, spermatocytes, spermatids and spermatozoa. Oogonia, early, primary, secondary, full-grown and maturing oocytes are described for females. Female germinal epithelium of C. striatus showed substantial changes over the study period, indicating that there was an active spawning period. Male germinal epithelium also presented relevant alterations, indicating reproductive activity in the testicular lobules. Morphological data confirm how informative was the cellular dynamics of the germinal epithelium for understanding gonadal development during adult reproductive life of fish in general. Although Chaetodon are a popular species, previous studies have only produced superficial and rough histological analyses. Therefore, this study demonstrates important information on germinal epithelium of Chaetodon. This knowledge could be a fundamental tool for development of new strategies for breeding of several species in captivity, especially butterflyfishes.

Studies have indicated that psychological stress impairs human fertility and that various stressors can induce apoptosis of testicular cells. However, the mechanisms by which psychological stress on males reduces semen quality and stressors induce apoptosis in testicular cells are largely unclear. Using a psychological (restraint) stress mouse model, we tested whether male psychological stress triggers apoptosis of spermatozoa and spermatogenic cells through activating tumour necrosis factor (TNF)-α signalling. Wild-type or TNF-α−/− male mice were restrained for 48 h before examination for apoptosis and expression of TNF-α and TNF receptor 1 (TNFR1) in spermatozoa, epididymis, seminiferous tubules and spermatogenic cells. The results showed that male restraint significantly decreased fertilization rate and mitochondrial membrane potential, while increasing levels of malondialdehyde, active caspase-3, TNF-α and TNFR1 in spermatozoa. Male restraint also increased apoptosis and expression of TNF-α and TNFR1 in caudae epididymides, seminiferous tubules and spermatogenic cells. Sperm quality was also significantly impaired when spermatozoa were recovered 35 days after male restraint. The restraint-induced damage to spermatozoa, epididymis and seminiferous tubules was significantly ameliorated in TNF-α−/− mice. Furthermore, incubation with soluble TNF-α significantly reduced sperm motility and fertilizing potential. Taken together, the results demonstrated that male psychological stress induces apoptosis in spermatozoa and spermatogenic cells through activating the TNF-α system and that the stress-induced apoptosis in spermatogenic cells can be translated into impaired quality in future spermatozoa.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

A histological characterization of gonadal development in the tetra Astyanax bimaculatus was performed, aimed at determining its reproductive cycle in streams localized inside the Amazonian forest. Collections were carried out monthly from August 2017 to July 2018 at the Zoobotânica Foundation of Marabá, PA. Collected specimens were weighed and measured, and their gonads and liver were removed and weighed to calculate gonadosomatic and hepatosomatic indexes. Gonads were fixed and treated for routine histology for light microscopy. Materials were stained with toluidine blue and haematoxylin and eosin. The Amazonian A. bimaculatus species presented two reproductive periods in the year, one at the end of the winter season and another during the summer. Females showed an asynchronous development of their oocytes and only two reproductive phases of development were observed during the whole period ‘developing’ and ‘spawning capable’. Males presented cystic spermatogenesis, with an anastomosing tubular testis containing spermatogonia spread along the germinal epithelium (unrestricted spermatogonial). These morphological characteristics are considered phylogenetically more primitive. Male specimens were observed to have five different phases during the period: immature, initial maturation, mid maturation, final maturation and regression. The huge fluctuations in Amazonian streams was observed, in which water volumes varied considerably across seasons, culminating even in total drought. In spite of this, A. bimaculatus could be found during all seasons, showing its impressive reproductive adaptation to its conditions.

Germ plasm, a cytoplasmic factor of germline cell differentiation, is suggested to be a perspective tool for in vitro meiotic differentiation. To discriminate between the: (1) germ plasm-related structures (GPRS) involved in meiosis triggering; and (2) GPRS involved in the germ plasm storage phase, we investigated gametogenesis in the marine medaka Oryzias melastigma. The GPRS of the mitosis-to-meiosis period are similar in males and females. In both sexes, five events typically occur: (1) turning of the primary Vasa-positive germ plasm granules into the Vasa-positive intermitochondrial cement (IMC); (2) aggregation of some mitochondria by IMC followed by arising of mitochondrial clusters; (3) intramitochondrial localization of IMC-originated Vasa; followed by (4) mitochondrial cluster degradation; and (5) intranuclear localization of Vasa followed by this protein entering the nuclei (gonial cells) and synaptonemal complexes (zygotene–pachytene meiotic cells). In post-zygotene/pachytene gametogenesis, the GPRS are sex specific; the Vasa-positive chromatoid bodies are found during spermatogenesis, but oogenesis is characterized by secondary arising of Vasa-positive germ plasm granules followed by secondary formation and degradation of mitochondrial clusters. A complex type of germ plasm generation, ‘the follicle cell assigned germ plasm formation’, was found in late oogenesis. The mechanisms discovered are recommended to be taken into account for possible reconstruction of those under in vitro conditions.

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone–protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

Cryptorchidism, characterized by the presence of one (unilateral) or both (bilateral) undescended testes, is a common male urogenital defect. Cryptorchidism can lead to male infertility, testicular cancer being the most extreme clinical symptom, as well as psychological issues of the inflicted individual. Despite this, both knowledge about the aetiology of cryptorchidism and the mechanism for cryptorchidism-induced male infertility remain limited. In this present study, by using an artificial cryptorchid mouse model, we investigated the effects of surgery-induced cryptorchidism on spermatogenic cells and seminiferous epithelial cycles. We found that surgery-induced cryptorchidism led to a reduced testicular weight, aberrant seminiferous epithelial cycles and impaired spermatogenesis characterized by degenerating spermatogenic cells. We also observed multinucleated giant cells after surgery-induced cryptorchidism. Transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot assays demonstrated cryptorchidism-induced apoptosis of spermatogenic cells. Moreover, we identified the occurrence of autophagy in germ cells after surgery-induced cryptorchidism. Interestingly, apoptosis and autophagy were synchronous, suggestive of their synergetic roles in promoting germ cell death. Our results provide novel insights into the cryptorchidism-induced male infertility, thereby contributing to the development of male contraceptive strategies as well as treatment options for male infertility caused by cryptorchidism.

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.

This review attempts to explain why consuming extra myoinositol (Ins), an essential component of membrane phospholipids, is often beneficial for patients with conditions characterised by insulin resistance, non-alcoholic fatty liver disease and endoplasmic reticulum (ER) stress. For decades we assumed that most human diets provide an adequate Ins supply, but newer evidence suggests that increasing Ins intake ameliorates several disorders, including polycystic ovary syndrome, gestational diabetes, metabolic syndrome, poor sperm development and retinopathy of prematurity. Proposed explanations often suggest functional enhancement of minor facets of Ins Biology such as insulin signalling through putative inositol-containing ‘mediators’, but offer no explanation for this selectivity. It is more likely that eating extra Ins corrects a deficiency of an abundant Ins-containing cell constituent, probably phosphatidylinositol (PtdIns). Much of a cell’s PtdIns is in ER membranes, and an increase in ER membrane synthesis, enhancing the ER’s functional capacity, is often an important part of cell responses to ER stress. This review: (a) reinterprets historical information on Ins deficiency as describing a set of events involving a failure of cells adequately to adapt to ER stress; (b) proposes that in the conditions that respond to dietary Ins there is an overstretching of Ins reserves that limits the stressed ER’s ability to make the ‘extra’ PtdIns needed for ER membrane expansion; and (c) suggests that eating Ins supplements increases the Ins supply to Ins-deficient and ER-stressed cells, allowing them to make more PtdIns and to expand the ER membrane system and sustain ER functions.

Spermatogenesis is a finely regulated process of germ cell multiplication and differentiation leading to the production of spermatozoa in the seminiferous tubules. Spermatogenesis can be divided into three parts: spermatocytogenesis, meiosis and spermiogenesis. During spermatocytogenesis, germ cells engage in a cycle of several mitotic divisions that increases the yield of spermatogenesis and to renew stem cells and produce spermatogonia and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number and yield four haploid round spermatids. Spermiogenesis involves the differentiation of round spermatids into fully mature spermatozoa released into the lumin of seminiferous tubules. The seminiferous epithelium is composed of several generations of germ cells due to the fact that new generations of sperm cells engage in the spermatogenic process without waiting for the preceding generations to have completed their evolution and to have disappeared as spermatozoa into the lumen of the tubules. In bulls, the duration of the seminiferous epithelium cycle is 13.5 days. The total duration of spermatogenesis is 61 days, that is 4.5 times the duration of the cycle of the seminiferous epithelium. The spermatogenetic wave is used to describe the spatial arrangement of cell associations along the tubules. Several theories have been described to explain the renewal of spermatogonia. Depending on the model, there are five or six spermatogonial mitoses explaining the renewal of stem cells and the proliferation of spermatogonia. Daily sperm production and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis. Bulls have a lower efficiency of spermatogenesis than most species examined, but higher than that of humans.

Prothymosin alpha (PTMA) is a highly acidic, intrinsically disordered protein that was first extracted from rat thymus and characterized as an immunogenic factor but soon detected in a variety of mammalian tissues. The presence of a nuclear localization signal and the adoption of a peculiar random-coil conformation are among the reasons behind its interaction with several molecular partners, hence at this time PTMA is known to be a very conserved and widely expressed molecule, involved in numerous and diverse biological processes. Only few studies have tried to weigh its possible involvement in reproduction, specifically in male gametogenesis: first reports have suggested that PTMA might be associated with the proliferative and early-meiotic phases of mammal spermatogenesis. Some years later, a comparative project on vertebrate spermatogenesis reported the isolation, for the first time, of prothymosin in a non-mammalian species, the amphibian Pelophylax esculentus. PTMA transcript and protein are localized in the germinal compartment, from spermatocytes to spermatozoa. A congruent pattern has been highlighted in studies on the fish Torpedo marmorata and Danio rerio, and in the mammal Rattus norvegicus, in which the expression of PTMA has been found in meiotic and post-meiotic germ cells inside testicular cysts and tubules. Moreover, its presence has been confirmed in rat and human spermatozoa (associated with the acrosome); its retention in the apical region of the head after the acrosome reaction revealed a striking conservation of the pattern during phylogenesis and suggested a possible role for the protein in gametogenesis and in fertilization.

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.

Lead poisoning is a stealthy threat to human physiological systems as chronic exposure can remain asymptomatic for long periods of time before symptoms manifest. We presently review the biophysical mechanisms of lead poisoning that contribute to male infertility. Environmental and occupational exposure of lead may adversely affect the hypothalamic−pituitary−testicular axis, impairing the induction of spermatogenesis. Dysfunction at the reproductive axis, namely testosterone suppression, is most susceptible and irreversible during pubertal development. Lead poisoning also appears to directly impair the process of spermatogenesis itself as well as sperm function. Spermatogenesis issues may manifest as low sperm count and stem from reproductive axis dysfunction or testicular degeneration. Generation of excessive reactive oxygen species due to lead-associated oxidative stress can potentially affect sperm viability, motility, DNA fragmentation, membrane lipid peroxidation, capacitation, hyperactivation, acrosome reaction, and chemotaxis for sperm-oocyte fusion, all of which can contribute to deter fertilization. Reproductive toxicity has been tested through cross-sectional analysis studies in humans as well as in vivo and in vitro studies in animals.

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17–19%), whilst stages II to V showed the lowest frequencies (~2–4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.