Spermatogenic Process Research Articles

The association of CFAP65 with male infertility in Vietnamese individuals

Cilia- and flagella-associated protein (CFAP) is a well-known protein family that plays a vital role in the spermatogenic process. Recently, the gene CFAP65, which encodes the cilia- and flagella- associated protein 65, has been focused on as a new candidate for male infertility. Mutations in this gene are frequently detected in patients with primary infertility, especially among cases with combined phenotypes of acrosome abnormalities and multiple morphological abnormalities of the flagella (MMAF). In addition, mice carrying both a complete knock-out of the CFAP65 gene and a CFAP65 homozygous frameshift mutation exhibited sterility with the typical phenotypes of MMAF. However, no case-control study has been performed on the relationship between polymorphisms in CFAP65 and male infertility in any population. Hence, our study aimed to investigate the correlation between the polymorphism CFAP65 rs117885048 and male infertility in a Vietnamese population comprising 207 infertile men and 217 healthy controls. As a result, the studied population followed Hardy-Weinberg equilibrium (HWE) (p> 0.05) and the frequencies of genotypes CC/CT/TT were 0.875, 0.12, and 0.003, respectively. The Chi-square test revealed no association between the polymorphism CFAP65 rs117885048 and the disease in this population (p > 0.05). To further interpret the correlation between single nucleotide polymorphisms in the CFAP65 gene and male infertility, a more comprehensive study with other polymorphisms needs to be considered.

Testicular Localization and Potential Function of Vimentin Positive Cells during Spermatogonial Differentiation Stages.

Simple SummarySpermatogonia stem cells (SSCs) in the testis are responsible for transmitting genetic information to subsequent generations. During the differentiation of SSCs, various cytoskeletons are involved in giving the cell shape and internal organization. One of the essential cytoskeletons that play functions in these spermatogenic processes is vimentin. This study examined the vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm real-time polymerase chain reaction, and bioinformatics analysis. IMH analysis demonstrated that the high vimentin expression was localized in the middle and central testicular cords cells of seminiferous tubules and low expression in the basal region under in vivo conditions. To evaluate the expression of vimentin in vitro, we first isolated SSCs and then cultured cells from the testis. Our results showed that vimentin plays important roles in the differentiation of testicular germ cells.Vimentin is a type of intermediate filament (IF) and one of the first filaments expressed in spermatogenesis. Vimentin plays numerous roles, consisting of the determination of cell shape, differentiation, cell motility, the maintenance of cell junctions, intracellular trafficking, and assisting in keeping normal differentiating germ cell morphology. This study investigated the vimentin expression in two populations of undifferentiated and differentiated spermatogonia. We examined vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), and Fluidigm real-time polymerase chain reaction. IMH data showed that the high vimentin expression was localized in the middle of seminiferous tubules, and low expression was in the basal membrane. ICC analysis of the colonies by isolated differentiated spermatogonia indicated the positive expression for the vimentin antibody, but vimentin’s expression level in the undifferentiated population was negative under in vitro conditions. Fluidigm real-time PCR analysis showed significant vimentin expression in differentiated spermatogonia compared to undifferentiated spermatogonia (p < 0.05). Our results showed that vimentin is upregulated in the differentiation stages of spermatogenesis, proving that vimentin is an intermediate filament with crucial roles in the differentiation stages of testicular germ cells. These results support the advanced investigations of the spermatogenic process, both in vitro and in vivo.

The Rete Testis: Development and Role in Testis Function

The rete testis connects seminiferous tubules in which germ cells develop to the efferent ducts and the epididymis, where gametes mature and gain mobility. Several recent studies have thoroughly explored the morphogenesis of this structure in mice during embryonic and postnatal periods. A part of the rete testis has been shown to derive from the precursors of gonad somatic cells before sex determination. The other part forms from embryonal Sertoli cells of testis cords adjacent to the mesonephros. The transformation of Sertoli cells into rete testis cells is apparently not limited to the embryonic stage of development and continues during postnatal testis development. Recently, it was found that the rete testis participates in the formation and maintenance of specialized Sertoli cells in terminal segments of seminiferous tubules, transitional zones. Current views suggest that the transitional zones of the seminiferous tubules may represent a niche for spermatogonial stem cells, the site of the prolonged proliferation of Sertoli cells in the pubertal and postpubertal periods of testis development, and also could be a generator of spermatogenic waves. To sum up, the rete testis transports gametes from the testis to the epididymis, maintains pressure within seminiferous tubules, regulates the composition of the testicular fluid, and impacts the spermatogenic process itself.

Sperm production and seminal analyses in a Neotropical sperm-storing vespertilionid bat yellowish myotis (Myotis levis)

Yellowish myotis is a Neotropical vespertilionid bat that presents a seasonal reproduction. The sperm is produced in the Mature stage, stored in the Regressed stage and released in the Rest stage (mating period). Aiming to understand, for the first time, the relationship between testis and epididymis physiology in yellowish myotis reproduction, the spermatogenesis length, sperm production, and seminal parameters were herein evaluated. Fifty-one adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil. The gonads were collected in the Maturing and Mature stages for histomorphometric and immunohistochemical analyses, whereas the epididymis was evaluated in all reproductive stages for seminal studies. Our results demonstrated that the yellowish myotis spermatogenic process is fast, lasting 31.70 ± 0.15 days. Despite the low Sertoli cell efficiency (6.60 ± 1.23), the high numbers of Sertoli cells per testis enable an elevated sperm production in the Mature stage. The sperm concentration, vitality, and motility presented the highest values in the Regressed stage; however, in this period, an increased incidence of sperm morphological defects was detected. In the following period (Rest stage), a drastic reduction of defective sperm was observed, suggesting quality control of sperm before the mating period. Furthermore, the epididymis ability to maintain a long-term sperm-storage was observed in 26.7% of the bats in the Maturing stage. In summary, yellowish myotis presented a fast and high sperm production during the Mature stage. These sperms are stored and selected before mating period.

P–142 Comparison of the IVF outcome in the course of ICSI vs PICSI treatment continuation in exactly the same group of HBA abnormal patients

Abstract Study question Does the PICSI have a beneficial effect for men with abnormal HBA on the fertilization rate, blastocysts number and clinical pregnancies in the next attempt? Summary answer Patients with HBA &amp;lt;80% choosing to undergo PICSI after ICSI failure see an increase in blastocyst and pregnancy rates. What is known already Hyaluronic acid (HA) is a main component of cervical mucus and the extracellular matrix of cumulus cells. The formation of HA-binding sites in sperm cell membranes is one of the markers of sperm maturation indicating completion the spermatogenic process of remodelling the plasmatic membrane, cytoplasmic extrusion and nuclear maturity. Spermatozoa selected by the HA-binding technique (the physiologically selected intracytoplasmic sperm injection – PICSI) have a potentially reduced risk of chromosomal aneuploidy or DNA fragmentation. Recent evidence do not show significant benefits in using PICSI. However, it has not been analysed in the course of treatment continuation in the same patients. Study design, size, duration This was a retrospective case-control study. It included exactly the same 58 patients with abnormal HBA, who underwent IVF treatment with ICSI initially and later with PICSI, between January 2014 and October 2020 at INVICTA Fertility Centre, Poland. Median female partner age in PICSI group was 36,2±5,34, without PICSI 35,8 ±5,28. Participants/materials, setting, methods 275 cycles (130 ICSI and 145 PICSI) resulted in 793 and 897 MII respectively. Patients were also divided into two groups &amp;lt;80% and ≥80% depending on the obtained HBA score expressed as the percentage of sperm bound with hyaluronan. The analysis covered the fertilization rate (FR), TQ and total blastocyst rate on day 5 and clinical pregnancy rate. Patients with poor response to stimulation were excluded from the study. Main results and the role of chance FR in ICSI and PICSI groups was not significantly difference (57.00%±31.2 vs 59.87%±30.8) even when taking into account the division of patients according to the obtained HBA score. In the &amp;lt;80% group the FR was 57.04%±29.3 vs 59.54%±30.8 in ICSI vs PICSI group respectively. There were no significant differences when comparing the under HBA ≥80% subgroups for all analysed outcomes. Fertilization rate ​​was 56.88% in the ICSI group vs 61.03% in the PICSI group. The percentage of blastocysts was 28.61% vs 34.45% and the percentage of TQ blastocysts on day 5 was 15.32% vs 16.81% with ICSI and PICSI respectively, in the group consisting of the same patients. In the HBA &amp;lt;80% group significant differences were observed in the percentage of obtained blastocysts 37.81% vs 47.61% by comparing the ICSI and PICSI approaches (p &amp;lt; 0.05). Also, percentage of TQ blastocyst on day 5 also was higher in patients with &amp;lt;80% HBA score after PICSI and was statistically significant (17.07% ICSI vs 23.92% PICSI, p &amp;lt; 0.05). We saw statistically significant (p &amp;lt; 0.01) increase in percentage of clinical pregnancies from 29.03% without PICSI to 69.44% in patient’s subsequent procedures involving PICSI. Limitations, reasons for caution More data is required to confirm that improved results of PICSI procedure are consistent and possible to reproduce in a larger group – and as a result could be included as part of the standard treatment process. Wider implications of the findings: The presented results show that in patients with normal HBA score, PICSI does not bring a measurable benefit and this may be important factor to consider in decision-making for couples seeking assistance. Trial registration number Not applicable

Comparison of the spermatogenic process in three different mice strains: Swiss, Balb/C and C57BL/6

Many studies have been trying to establish standard protocols for animal experimentation, especially for animal species or strains, to master research variables with high precision. The main mouse strains used in the field of the biology of reproduction are Swiss, Balb/c, and C57BL/6. Since some of the strains show reproduction limitations, such as the size of the litter, the present study aimed to compare their spermatogenic processes to verify differences regarding the testicular parenchyma and germ cell populations, which could explain low offspring production. In addition, the present study provides additional information concerning the testicular parenchyma of such strains, which consequently would help researchers to choose the most suitable strain for reproductive studies. Six adult male mice were used for each of the strains. After euthanasia, the testes were weighed, fixated with Karnovsky fixative, embedded in methacrylate, sectioned, and stained with toluidine blue/sodium borate 1%. Morphometrical analyses from the testicular parenchyma (seminiferous tubules and interstitium) were made using the software ImageJ. Germ and Sertoli cells populations were counted in seminiferous tubules cross-sections at stage I of the seminiferous epithelium cycle. The lowest body and testicular weights were observed in C57BL/6 animals, followed by Balb/c and Swiss, however, the relative testes, parenchyma, and albuginea weights were significantly lower only in C57BL/6. Despite the seminiferous tubules and seminiferous epithelium proportions were lower in Swiss animals, their relative amount related to the body weight was the same among strains. The total number of germ cells was higher in Swiss animals, reflecting higher spermatogenic yield and daily sperm production. Due to the lower relative number of Sertoli cells, the Swiss animals showed the highest Sertoli cell index and support capacity. On the other hand, the lowest pathological indexes regarding the germ cells were observed in Balb/c animals, followed by Swiss and C57BL/6. In the interstitium, the proportion of blood vessels was lower in Swiss mice, while the lymphatic cell proportion was lower in C57BL/6 animals. Moreover, the highest proportions of Leydig cells and macrophages were noticed in Swiss mice, which may indicate increased testosterone levels. Altogether, such observations must be taken into account when choosing any of the studied strains for reproduction studies.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

MiR-181a Promotes Spermatogenesis by Targeting the S6K1 Pathway.

Approximately 15% of couples suffer from infertility worldwide, and male factors contribute to about 30% of total sterility cases. However, there is little progress in treatments due to the obscured understanding of underlying mechanisms. Recently microRNAs have emerged as a key player in the process of spermatogenesis. Expression profiling of miR-181a was carried out in murine testes and spermatocyte culture system. In vitro cellular and biochemical assays were used to examine the effect of miR-181a and identify its target S6K1, as well as elucidate the function with chemical inhibitor of S6K1. Human testis samples analysis was employed to validate the findings. miR-181a level was upregulated during mouse spermatogenesis and knockdown of miR-181a attenuated the cell proliferation and G1/S arrest and increased the level of S6K1, which was identified as a downstream target of miR-181a. Overexpression of S6K1 also led to growth arrest of spermatocytes while inhibitor of S6K1 rescued the miR-181a knockdown-mediated cell proliferation defect. In human testis samples of azoospermia patients, low level of miR-181a was correlated with defects in the spermatogenic process. miR-181a is identified as a new regulator and high level of miR-181a contributes to spermatogenesis via targeting S6K1.

Telomere Length and Male Fertility.

Over the past decade, telomeres have attracted increasing attention due to the role they play in human fertility. However, conflicting results have been reported on the possible association between sperm telomere length (STL) and leukocyte telomere length (LTL) and the quality of the sperm parameters. The aim of this study was to run a comprehensive study to investigate the role of STL and LTL in male spermatogenesis and infertility. Moreover, the association between the sperm parameters and 11 candidate single nucleotide polymorphisms (SNPs), identified in the literature for their association with telomere length (TL), was investigated. We observed no associations between sperm parameters and STL nor LTL. For the individual SNPs, we observed five statistically significant associations with sperm parameters: considering a p < 0.05. Namely, ACYP2˗rs11125529 and decreased sperm motility (p = 0.03); PXK˗rs6772228 with a lower sperm count (p = 0.02); NAF1˗rs7675998 with increased probability of having abnormal acrosomes (p = 0.03) and abnormal flagellum (p = 0.04); ZNF208˗rs8105767 and reduction of sperms with normal heads (p = 0.009). This study suggests a moderate involvement of telomere length in male fertility; however, in our analyses four SNPs were weakly associated with sperm variables, suggesting the SNPs to be pleiotropic and involved in other regulatory mechanisms independent of telomere homeostasis, but involved in the spermatogenic process.

Destruction or Reconstruction: A Subtle Liaison between the Proteolytic and Signaling Role of Protein Ubiquitination in Spermatogenesis.

Ubiquitination is one of the most diverse forms of protein post-translational modification that changes the function of the landscape of substrate proteins in response to stimuli, without the need for "de novo" protein synthesis. Ubiquitination is involved in almost all aspects of eukaryotic cell biology, from the best-studied role in promoting the removal of faulty or unnecessary proteins by the way of the ubiquitin proteasome system and autophagy-lysosome pathway to the recruitment of proteins in specific non-proteolytic signaling pathways, as emerged by the more recent discoveries about the protein signature with peculiar types of ubiquitin chains. Spermatogenesis, on its own, is a complex cellular developmental process in which mitosis, meiosis, and cell differentiation coexist so to result in the continuous formation of haploid spermatozoa. Successful spermatogenesis is thus at the same time a mixed result of the precise expression and correct intracellular destination of structural proteins and enzymes, from one hand, and the fine removal by targeted degradation of unfolded or damaged proteins as well as of obsolete, outlived proteins, from the other hand. In this minireview, I will focus on the importance of the ubiquitin system all over the spermatogenic process, discussing both proteolytic and non-proteolytic functions of protein ubiquitination. Alterations in the ubiquitin system have been in fact implicated in pathologies leading to male infertility. Notwithstanding several aspects of the multifaceted world of the ubiquitin system have been clarified, the physiological meaning of the so-called ubiquitin code remains still partially elusive. The studies reviewed in this chapter provide information that could aid the investigators to pursue new promising discoveries in the understanding of human and animal reproductive potential.

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of USP8-rs7174015 was observed under the recessive model between the NOA group and both the control group (p = 0.0226, OR = 1.33) and the SO group (p = 0.0048, OR = 1.78). Other genetic associations for EPSTI1-rs12870438 and PSAT1-rs7867029 with SO and between TUSC1-rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.

Seasonal evaluation of spermatogenesis of the hematophagous bat Desmodus rotundus in the Caatinga biome.

This study was aimed to characterize the spermatogenic process and its seasonal variation in Desmodus rotundus, in the Caatinga biome, a water-limited ecosystem, with marked water restriction during most of the year. Collections of adult animals were performed during the dry and rainy seasons, and after euthanasia, their testes were processed histologically to perform morphological, morphometric, ultrastructural and immunohistochemical analyzes. The percentage of seminiferous epithelium, number of Leydig cells per gram of testis, and population of Sertoli cells and A-type spermatogonia presented by D. rotundus were significantly higher in the rainy season, while the percentage of lumen, mitotic index, support capacity performed by Sertoli cells, and overall yield of spermatogenesis were higher in the dry season. The ultrastructure of spermatogenesis was similar to that described in other mammals, and the immunohistochemical analysis revealed activity of the aromatase enzyme in Sertoli cells, Leydig cells, spermatocytes and spermatids, as well as the presence of androgen receptors in Sertoli cells and Leydig cells. FGF2 activity was detected in primary spermatocytes in zygotene and pachytene, as well as secondary spermatocytes and rounded and elongated spermatids, while the BCL-2 protein was expressed in primary spermatocytes in zygotene and pachytene, secondary spermatocytes, and rounded spermatids. The activity of these molecules was similar in both seasons, and associated with the morphometric findings, indicates maintenance in the integrity of the seminiferous epithelium throughout the year. The seasonal study of D. rotundus spermatogenesis indicates a continuous spermatogenesis pattern and suggests a greater production of spermatozoa in the rainy season in the Caatinga biome.

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2′-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.

Testis-specific peroxiredoxin 4 variant is not absolutely required for spermatogenesis and fertility in mice

PRDX4, a member of peroxiredoxin family, is largely concentrated in the endoplasmic reticulum (ER) and plays a pivotal role in the redox relay during oxidative protein folding as well as in peroxidase reactions. A testis-specific PRDX4 variant transcript (PRDX4t) lacks the conventional exon 1, which encodes the signal peptide that is required for entry into the ER lumen, but instead carries alternative exon 1, which is transcribed from the upstream promoter in a testis-specific manner and results in the PRDX4t protein being localized in the cytosol. However, the potential roles of PRDX4t in male genital action remain unknown. Using a CRISPR/Cas9 system, we first disrupted the testis-specific promoter/exon 1 and generated mice that were specifically deficient in PRDX4t. The resulting PRDX4t knockout (KO) mice underwent normal spermatogenesis and showed no overt abnormalities in the testis. Mating PRDX4t KO male mice with wild-type (WT) female mice produced normal numbers of offspring, indicating that a PRDX4t deficiency alone had no effect on fertility in the male mice. We then generated mice lacking both PRDX4 and PRDX4t by disrupting exon 2, which is communal to these variants. The resulting double knockout (DKO) mice were again fertile, and mature sperm isolated from the epididymis of DKO mice exhibited a normal fertilizing ability in vitro. In the meantime, the protein levels of glutathione peroxidase 4 (GPX4), which plays an essential role in the disulfide bond formation during spermatogenesis, were significantly increased in the testis and caput epididymis of the DKO mice compared with the WT mice. Based on these results, we conclude that the disruption of the function of PRDX4t in the spermatogenic process appears to be compensated by other factors including GPX4.

Computerized spermatogenesis staging (CSS) of mouse testis sections via quantitative histomorphological analysis.

Spermatogenesis in mammals is a cyclic process of spermatogenic cell development in the seminiferous epithelium that can be subdivided into 12 subsequent stages. Histological staging analysis of testis sections, specifically of seminiferous tubule cross-sections, is the only effective method to evaluate the quality of the spermatogenic process and to determine developmental defects leading to infertility. Such staging analysis, however, is tedious and time-consuming, and it may take a long time to become proficient. We now have developed a Computerized Staging system of Spermatogenesis (CSS) for mouse testis sections through learning of an expert with decades of experience in mouse testis staging. The development of the CSS system comprised three major parts: 1) Developing computational image analysis models for mouse testis sections; 2) Automated classification of each seminiferous tubule cross-section into three stage groups: Early Stages (ES: stages I-V), Middle Stages (MS: stages VI-VIII), and Late Stages (LS: stages IV-XII); 3) Automated classification of MS into distinct stages VI, VII-mVIII, and late VIII based on newly developed histomorphological features. A cohort of 40 H&E stained normal mouse testis sections was built according to three modules where 28 cross-sections were leveraged for developing tubule region segmentation, spermatogenic cells types and multi-concentric-layers segmentation models. The rest of 12 testis cross-sections, approximately 2314 tubules whose stages were manually annotated by two expert testis histologists, served as the basis for developing the CSS system. The CSS system's accuracy of mean and standard deviation (MSD) in identifying ES, MS, and LS were 0.93±0.03, 0.94±0.11, and 0.89±0.05 and 0.85±0.12, 0.88±0.07, and 0.96±0.04 for one with 5 years of experience, respectively. The CSS system's accuracy of MSD in identifying stages VI, VII-mVIII, and late VIII are 0.74±0.03, 0.85±0.04, and 0.78±0.06 and 0.34±0.18, 0.78±0.16, and 0.44±0.25 for one with 5 years of experience, respectively. In terms of time it takes to collect these data, it takes on average 3 hours for a histologist and 1.87 hours for the CSS system to finish evaluating an entire testis section (computed with a PC (I7-6800k 4.0 GHzwith 32GB of RAM & 256G SSD) and a Titan 1080Ti GPU). Therefore, the CSS system is more accurate and faster compared to a human histologist in staging, and further optimization and development will not only lead to a complete staging of all 12 stages of mouse spermatogenesis but also could aid in the future diagnosis of human infertility. Moreover, the top-ranking histomorphological features identified by the CSS classifier are consistent with the primary features used by histologists in discriminating stages VI, VII-mVIII, and late VIII.

Altered gene expression profiles of testicular tissues from azoospermic patients with maturation arrest.

Maturation arrest is a common cause of male infertility which has caused worldwide concern, and its pathophysiological process remains further elucidation. Our study aimed to identify genetic characteristics of maturation arrest by comparing gene expression between maturation arrest and normal samples using microarray technology. A total of 6,373 genes were identified differentially expressed (p<.05, fold change>2.0 or <-2.0) and 1,594 genes were selected as statistically significant after Bonferroni correction, including 419 up-regulated and 1,175 down-regulated genes. Microarray data were validated by quantitative reverse transcriptase-polymerase chain reaction. Bioinformation analysis was performed to explore genetic function of statistically significant genes. Gene Ontology results showed the statistically significant genes enriched in sexual reproduction, spermatogenesis and male gamete generation. Reactome pathway analysis highlighted the olfactory signalling pathway, fertilisation, developmental biology, etc. One module and eight hub genes were found to be involved in ubiquitin-mediated proteolysis and may affect as indicators of spermatogenic process through protein-protein interaction analysis. Our study provided a comprehensive genetic characteristic of differential expressed genes in testicular tissues from maturation arrest patients and speculated several genes as potential indicators of disease.

Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14-/- mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.

Retinoic acid receptor antagonists for male contraception: current status†

Retinoic acid receptor alpha (RARA), a nuclear receptor protein, has been validated as a target for male contraception by gene knockout studies and also pharmacologically using a pan-retinoic acid receptor antagonist. Retinoic acid receptor alpha activity is indispensable for the spermatogenic process, and therefore its antagonists have potential as male contraceptive agents. This review discusses the effects of systematic dosing regimen modifications of the orally bioavailable and reversible pan-antagonist BMS-189453 as well as studies with the alpha-selective antagonists BMS-189532 and BMS-189614 in a murine model. We also provide an overview of structure–activity studies of retinoic acid receptor alpha antagonists that provide insight for the design of novel alpha-selective ligands.

Chronic exposure to diesel exhaust particulate matter impairs meiotic progression during spermatogenesis in a mouse model

Exposure to ambient PM2.5 may correlate with the decline of semen quality, and the underlying biological mechanism has not been fully understood. In the present study, mice were intratracheally instilled with diesel exhaust PM2.5 (DEP), and its effects on the spermatogenic process as well as the alterations of testicular gene expression profile were assessed. Our results showed that chronic exposure to DEP impaired the fertility of male mice without influencing their libido. Compared with Vehicle-exposed group, the sperm count and motility from DEP-exposed mice were significantly decreased. In addition, immunohistological staining of γH2AX and DMC1, biomarkers for meiotic double strand breaks (DSBs), demonstrated that chronic exposure to DEP comprised the repair of meiotic DSBs, thus disrupting the spermatogenesis. Deep RNA sequencing test showed altered expressions of testicular genes including the GnRH signaling pathway. In summary, our research demonstrated that chronic exposure to DEP may disrupt spermatogenesis through targeting the meiotic recombination, providing a new perspective for the research on the male reproductive system damage caused by air pollution.

Maternal CBZ exposure impairs testicular development, spermatogenesis and sperm parameters in male offspring at puberty.

Carbamazepine (CBZ), an anticonvulsant drug, is used by pregnant women and crosses the placental barrier, reaching the embryo/foetus. CBZ inhibits testicular steroidogenesis and may lead to alterations in testicular development, spermatogenesis and male fertility. The purpose of this study was to evaluate the CBZ effects on testicular parameters in the neonatal and pubertal phases, as well as the spermatic parameters of pubertal rats, originated from dams treated during different periods of the pregnancy. Pregnant rats were treated with CBZ (20mg/kg/day; intraperitoneal route), from 12-20 gestation day (GD) (CBZ12 group) and 15-20 GD (CBZ15 group). The testicular morphometric and stereological analysis of rats aged 4 and 63days was performed. The oestradiol and testosterone plasmatic levels, as well as spermatic parameters, were achieved at 63days. CBZ12 group showed a reduction in testicular weight and volume at 4days post-partum (dpp); however, there was an increase in the seminiferous cords' length of the CBZ12 and CBZ15 groups. At 63days, the CBZ12 group showed increases of the daily sperm production and damage in the seminiferous epithelium. The results suggest that CBZ interferes with the testis development and the establishment of the spermatogenic process, which can be detected in the puberty phase.