Germ Cell Arrest Research Articles

A review of testicular histopathology in nonobstructive azoospermia.

One major challenge in male factor infertility is nonobstructive azoospermia (NOA), which is characterized by spermatozoa-deficient semen without physical duct blockage. This review offers a thorough overview of the histopathology of the testes in NOA cases, clarifying its complex etiology and emphasizing the possible value of histopathology inspection for both diagnosis and treatment. Variable histopathological findings have been linked to NOA, such as tubular hyalinization, Sertoli cell-only syndrome, hypospermatogenesis, and germ cell arrest. Understanding the pathophysiology and forecasting the effectiveness of treatment are further enhanced by both morphometric and ultrastructural analyses. The potential significance of testicular biopsy in forecasting reproductive outcomes is assessed, especially concerning assisted reproductive technologies like intracytoplasmic sperm injection (ICSI). Besides, testicular microlithiasis, serum hormone profiles, and testicular size are investigated concerning NOA histopathology. It is concluded that understanding the histopathological patterns in NOA is crucial for its accurate diagnosis and appropriate management. Further research is still warranted to improve understanding of the complex pathophysiology underlying NOA.

P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure

Abstract Study question Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate? Summary answer There was no significant difference between these 2 groups of patients. What is known already Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients. Study design, size, duration This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed. Participants/materials, setting, methods Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program. Main results and the role of chance Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls. For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P < 0.05, we found that there were significantly more patients with cryptorchism in the NOA group. As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage. Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients. Limitations, reasons for caution The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest. Wider implications of the findings This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes. Trial registration number not applicable

MIWI N-terminal RG motif promotes efficient pachytene piRNA production and spermatogenesis independent of LINE1 transposon silencing.

PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.

DNA damage signals from somatic uterine tissue arrest oogenesis through activated DAF-16.

Germ line integrity is crucial for progeny fitness. Organisms deploy the DNA damage response (DDR) signaling to protect the germ line from genotoxic stress, facilitating the cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germ line quality in response to DNA damage is well studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin/IGF-1 signaling (IIS) is low, arrests oogenesis in the pachytene stage of meiosis I, in a FOXO/DAF-16 transcription factor-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape the arrest to produce poor-quality oocytes, showing that the transcription factor imposes strict quality control during low IIS. Activated FOXO/DAF-16 senses DDR perturbations during low IIS to lower ERK/MPK-1 signaling below a threshold to promote germ line arrest. Altogether, we elucidate a new surveillance role for activated FOXO/DAF-16 that ensures optimal germ cell quality and progeny fitness in response to somatic DNA damage.

Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects

Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance−/− mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription–replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance−/− PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance−/− PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance−/− PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance−/− PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.

Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Mitotic arrest is necessary for the embryonic development of germ cells, and thus, it is important to understand the signaling pathways that regulate mitotic arrest. Here, we investigated the signaling pathway dynamics of male embryonic chicken germ cells during mitotic arrest by single-cell transcriptome analysis using germ-cell tracing models. We identified signaling pathways that change at the transcriptional level during chicken male germ-cell development after sex determination. We found that several components of the BMP, Notch, and JAK-STAT signaling pathways were downregulated at the mitotic-arrest stage and were reactivated 1 week after hatching when all germ cells are quiescent after entering mitotic arrest. In addition, the transcriptional levels of components of the MAPK, Hedgehog, and thyroid-hormone signaling pathways were steadily upregulated after mitotic arrest. This suggests the cooperation of multiple signaling pathways during entry into mitotic arrest and subsequent quiescence of chicken male germ cells.

Cell-fate transition and determination analysis of mouse male germ cells throughout development

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells’ identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

The Zebrafish Meiotic Cohesin Complex Protein Smc1b Is Required for Key Events in Meiotic Prophase I.

The eukaryotic structural maintenance of chromosomes (SMC) proteins are involved in key processes of chromosome structure and dynamics. SMC1β was identified as a component of the meiotic cohesin complex in vertebrates, which aids in keeping sister chromatids together prior to segregation in meiosis II and is involved in association of homologous chromosomes in meiosis I. The role of SMC1β in meiosis has primarily been studied in mice, where mutant male and female mice are infertile due to germ cell arrest at pachytene and metaphase II stages, respectively. Here, we investigate the function of zebrafish Smc1b to understand the role of this protein more broadly in vertebrates. We found that zebrafish smc1b is necessary for fertility and has important roles in meiosis, yet has no other apparent roles in development. Therefore, smc1b functions primarily in meiosis in both fish and mammals. In zebrafish, we showed that smc1b mutant spermatocytes initiated telomere clustering in leptotene, but failed to complete this process and progress into zygotene. Furthermore, mutant spermatocytes displayed a complete failure of synapsis between homologous chromosomes and homolog pairing only occurred at chromosome ends. Interestingly, meiotic DNA double strand breaks occurred in the absence of Smc1b despite failed pairing and synapsis. Overall, our findings point to an essential role of Smc1b in the leptotene to zygotene transition during zebrafish spermatogenesis. In addition, ovarian follicles failed to form in smc1b mutants, suggesting an essential role in female meiosis as well. Our results indicate that there are some key differences in Smc1b requirement in meiosis among vertebrates: while Smc1b is not required for homolog pairing and synapsis in mice, it is essential for these processes in zebrafish.

P–116 A 9-year monocentric retrospective analysis of glutaraldehyde-fixed and semithin section of testicular biopsies and TESE in azoospermic patients

Abstract Study question What is the outcome of testicular sperm extraction (TESE) after microinjection of frozen–thawed sperm and the correlation with histological analysis in azoospermic patients? Summary answer In our cohort of 240 azoospermic patients, sperm could be retrieved in 167 patients (69.6%). What is known already Testicular biopsy is a crucial assessment in reproductive practice with diagnostic and prognostic importance for ICSI. Divers histological procedures are used throughout the centres. There is increasing need to accurately analyse histological biopsies in order to characterise different type of spermatogenic failure and allows data storage of value in clinical practice and research. Compared with Bouin’s and formalin, glutaraldehyde fixed and semithin section of testicular biopsies have the advantage of yielding not only good cellular morphology but also it allows the possibility of performing electron microscopy. Study design, size, duration This is a monocentric retrospective study of TESE practice in azoospermic patients in Strasbourg University Hospital from February 2011 to December 2019. Participants/materials, setting, methods A total of 240 azoospermic patients underwent TESE followed by sperm cryopreservation when sperm were present and data of histological analysis and clinical outcome of ICSI were analysed. The analysis include initial hormonal status, type of azoospermia, body max index, classification of histological findings, freezing rate and outcome of ICSI-IVF procedure. Main results and the role of chance The mean age of 240 patients was 34.5 years. Out of all patients, 42% were diagnosed with obstructive azoospermia (OA) and 58% patients with non-obstructive azoospermia (NOA). There was no correlation of sperm retrieval with the body mass index. Overall, sperm could be retrieved in 69.6% patients. Spermatozoa were always successfully recovered in patients with normal testicular histological findings, 41.7% patients (n 100). Histological analysis revealed a Sertoli cell-only (SCO) syndrome in 27.5% cases (n 66), hypospermatogenesis in 14.2% (n 34), germ cell arrest in 7.9 (n 19) and mixed pattern in 8.3% (n 20). In patients with serum FSH concentrations >12 IU/l, 46% of patients (n 42) sperm were present at TESE. In patients with no sperm retrieval, 31.5% had normal FSH levels. Out of all men with elevated FSH, 63% had SCO pattern, 4% germ cell arrest, 21% hypospermatogenesis and 12% mixt patterns. In the group of patients with no sperm retrieved at TESE, histological analysis showed SCO in 69.9% cases, germ cell arrest in 15% and hypospermatogenesis in 9.6% of cases. Out of 167 patients with TESE and sperm cryopreservation, 126 patients undergone ICSI-IVF procedure and 80 babies were born. Limitations, reasons for caution This study is a retrospective analysis in a single centre. The cohort was not compared with groups of patients with different histological and fixation techniques. Wider implications of the findings: Accurate histological diagnosis is a prerequisite for research and clinical data collection and open the possibilities to include patients with NOA in subsequent detailed genetic screen. Trial registration number NA

Male fertility in mice requires classical and nonclassical androgen signaling.

Molecular mechanisms by which androgens signal through the androgen receptor (AR) to maintain male fertility are poorly understood. Transgenic mice were produced expressing mutant ARs that can only (1) alter gene transcription through the classical response pathway (AR-C) or (2) activate kinase signaling cascades via the nonclassical pathway (AR-NC). AR-C is sufficient to produce sperm and fertility. Haploid germ cell production, the blood-testis barrier, and spermatid migration are supported by AR-NC. Gene expression essential for chromosome synapsis during meiosis requires AR-C. We identify targets of androgen signaling required for male fertility and provide a mechanistic explanation for meiotic germ cell arrest in the absence of androgen signaling. Prostate differentiation occurs with AR-C alone, but full development requires synergistic nonclassical signaling. Both AR signaling pathways are necessary for normal male reproductive tract development and function, validating our mouse models for studies of AR functions in other target tissues.

Knockout Gene-Based Evidence for PIWI-Interacting RNA Pathway in Mammals.

The PIWI-interacting RNA (piRNA) pathway mainly consists of evolutionarily conserved protein factors. Intriguingly, many mutations of piRNA pathway factors lead to meiotic arrest during spermatogenesis. The majority of piRNA factor-knockout animals show arrested meiosis in spermatogenesis, and only a few show post-meiosis male germ cell arrest. It is still unclear whether the majority of piRNA factors expressed in spermatids are involved in long interspersed nuclear element-1 repression after meiosis, but future conditional knockout research is expected to resolve this. In addition, recent hamster knockout studies showed that a piRNA factor is necessary for oocytes—in complete contrast to the findings in mice. This species discrepancy allows researchers to reexamine the function of piRNA in female germ cells. This mini-review focuses on the current knowledge of protein factors derived from mammalian knockout studies and summarizes their roles in the biogenesis and function of piRNAs.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. ABLIM1-rs7099208 was associated with SpF under both additive (OR=0.86, p=0.036) and dominant models (OR=0.78, p=0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR=4.45, p=0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR=1.32, p=0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

SAT-031 Linking Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination Network and Acetylation During Spermiogenesis

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), a testis specific RNA helicase essential for the completion of spermatogenesis. Our early studies discovered a missense mutation (R242H) of GRTH gene in 5.8 % of a Japanese population with azoospermia. This mutation in COS-1 cells showed loss of the 61 kDa cytoplasmic phospho-species with preservation of the 56Kda nuclear non-phospho form of GRTH. Mice with knock-in (KI) of the human GRTH mutation, lack the phospho-form of GRTH, are sterile and lack sperm, with spermatogenic arrest at stage 8 of round spermatids. To determine the impact of phospho-GRTH on gene expression comparative studies of germ cells transcriptome profiles from WT and KI mice were conducted using Illumina RNA sequencing. RNA-Seq analysis revealed 1614 differentially expressed genes of which 886 down-regulated and 728 genes up-regulated genes. Gene Ontology analysis revealed several genes relevant to spermatogenesis, spermatid development and differentiation significantly downregulated. KEGG analysis showed genes related to Ubiquitin mediated proteolysis, protein processing in ER, RNA transport, Glycolysis pathways are down-regulated, and genes related to Focal adhesion and ECM interaction are up-regulated. RealTime-PCR analysis confirmed dramatic reduction in mRNA expression of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA’s in KI mice compared to WT. Western blot analysis revealed marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase) and their modified Histone substrates (H2AUb, H2BUb), as well as H4Ac, H4K16Ac in KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice compared to WT. In KI mice absence of phospho-GRTH impairs RNF8 and MOF dependent ubiquitination and acetylation of histones required for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis which finally results in germ cell arrest in step 8 of round spermatids. Thus, we conclude that phospho-GRTH affects the network which is critical for the replacement of histones during spermiogenesis.

ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.

Deletion of the mouse X-linked Prame gene causes germ cell reduction in spermatogenesis.

Preferentially expressed antigen in melanoma (PRAME) is cancer/testis antigen and a transcriptional repressor, inhibiting the signaling of retinoic acid through the retinoic acid receptor (RAR) for promoting cell proliferation and preventing cell apoptosis in cancer cells. The role of PRAME in testis and germline is unknown. We report here the generation and characterization of an X-linked Prame conditional knockout (cKO) mouse. Although fertile, the testis size (p < .01) and sperm count (p < .05) of the Prame cKO mice were significantly reduced by 12% at 4 months of age compared with the Prame floxed mice. Histological, immunofluorescence with germ cell-specific markers and terminal deoxynucleotidyl transferase dUTP nick end labeling analyses of testis cross-sections at postnatal day 7 (P7), P14, P21, P35, P120, and P365 indicated a significant increase in apoptotic germ cells at P7 and P14 and an increase in abnormal seminiferous tubules at P21 and P35. Germ cells were gradually lost resulting in two different phenotypes in the Prame cKO testes: Sertoli-cell-only for some of the affected tubules in young mice (at P35) and germ cell arrest at spermatogonia stage for other affected tubules in mature mice. Both phenotypes were a consequence of disruption in RAR signaling pathway by the depletion of Prame at a different time point during the first and subsequent rounds of spermatogenesis. The results suggest that Prame plays a minor, but important role in spermatogenesis and different paralogs in the Prame gene family may be functionally and partially redundant.

The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrestand elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

Mechanisms of Stress-Induced Spermatogenesis Impairment in Male Rats Following Unpredictable Chronic Mild Stress (uCMS).

The negative association between psychological stress and male fertility has been known for many years. This study was aimed at (i) identifying spermatogenesis impairment induced by psychological stress in rats and (ii) exploring the role of glucocorticoid receptor (GR) signaling in these adverse effects (if they exist). Male Sprague Dawley rats were exposed to a six-week period of unpredictable chronic mild stress (uCMS) along with cotreatment of GR antagonist RU486 (1 mg/kg/day). Testicular damage was assessed by testicular pathological evaluation, epididymal sperm concentration, serum testosterone levels, testicular apoptotic cell measurements, and cell cycle progression analyses. Rats in the uCMS group had decreased levels of serum testosterone and decreased epididymal sperm concentration. The uCMS-treated rats also had decreased numbers of spermatids and increased levels of apoptotic seminiferous tubules; additionally, cell cycle progression of spermatogonia was arrested at the G0/G1 phase. Furthermore, uCMS exposure caused an increase in serum corticosterone level and activated GR signaling in the testes including upregulated GR expression. RU486 treatment suppressed GR signaling and alleviated the damaging effects of stress, resulting in an increased epididymal sperm concentration. Overall, this work demonstrated for the first time that the activation of GR signaling mediates stress-induced spermatogenesis impairment and that this outcome is related to cell apoptosis and cell cycle arrest in germ cells.

Hybrid Sterility in Fish Caused by Mitotic Arrest of Primordial Germ Cells.

Sterility in hybrid animals is widely known to be due to a cytological mechanism of aberrant homologous chromosome pairing during meiosis in hybrid germ cells. In this study, the gametes of four marine fish species belonging to the Sciaenid family were artificially fertilized, and germ cell development was examined at the cellular and molecular levels. One of the intergeneric hybrids had gonads that were testis-like in structure, small in size, and lacked germ cells. Specification of primordial germ cells (PGCs) and their migration toward genital ridges occurred normally in hybrid embryos, but these PGCs did not proliferate in the hybrid gonads. By germ cell transplantation assay, we showed that the gonadal microenvironment in hybrid recipients produced functional donor-derived gametes, suggesting that the germ cell-less phenotype was caused by cell autonomous proliferative defects of hybrid PGCs. This is the first evidence of mitotic arrest of germ cells causing hybrid sterility in animals.

Melatonin attenuates radiofrequency radiation (900 MHz)-induced oxidative stress, DNA damage and cell cycle arrest in germ cells of male Swiss albino mice

Increasing male infertility of unknown aetiology can be associated with environmental factors. Extensive use of mobile phones has exposed the general population to unprecedented levels of radiofrequency radiations (RFRs) that may adversely affect male reproductive health. Therefore, the present study investigated the effect of RFR Global System for Mobile communication (GSM) type, 900 MHz and melatonin supplementation on germ cell development during spermatogenesis. Swiss albino mice were divided into four groups. One group received RFR exposure for 3 h twice/day for 35 days and the other group received the same exposure but with melatonin ( N-acetyl-5-methoxytryptamine) (MEL; 5 mg/kg bw/day). Two other groups received only MEL or remain unexposed. Sperm head abnormality, total sperm count, biochemical assay for lipid peroxides, reduced glutathione, superoxide dismutase activity and testis histology were evaluated. Additionally, flow cytometric evaluation of germ cell subtypes and comet assay were performed in testis. Extensive DNA damage in germ cells of RFR-exposed animals along with arrest in pre-meiotic stages of spermatogenesis eventually leading to low sperm count and sperm head abnormalities were observed. Furthermore, biochemical assays revealed excess free radical generation resulting in histological and morphological changes in testis and germ cells morphology, respectively. However, these effects were either diminished or absent in RFR-exposed animals supplemented with melatonin. Hence, it can be concluded that melatonin inhibits pre-meiotic spermatogenesis arrest in male germ cells through its anti-oxidative potential and ability to improve DNA reparative pathways, leading to normal sperm count and sperm morphology in RFR-exposed animals.

Molecular Pathogenesis of Testicular Germ Cell Tumors

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men and their incidence has been increasing over the past decades. Several factors may combine and play a role in the TGCT etiology, including environmental factors and genetic predispositions at multiple genomic loci that affect both testicular germ cells and stromal cells, and their interactions within the testicular microenvironment. The pathogenesis of TGCT starts prenatally with primordial germ cell arrest, and further proceeds postnatally, giving rise to in situ germ cell neoplasia and, finally, to invasive TGCT with the characteristic 12p chromosome amplification. Apart from the genes localized here, further molecular mechanisms have been linked to TGCT pathogenesis, such as the activation of the KIT/KITL signaling pathway, and aberrations in genes involved in DNA reparation, regulation of cellular differentiation, proliferation, and survival. Despite the relatively good prognosis and known etiopathogenesis of these tumors, neither targeted therapy nor molecular prognostic/predictive factors have yet been implemented in the management of TGCT, because there is not enough information about the molecular pathways or molecules involved in TGCT development that could be used for patient stratification and treatment. Current high-throughput technologies, such as next generation sequencing at the exome or transcriptome level could provide this missing information on genetic predispositions and other factors influencing the clinical course of the disease and treatment response in TGCT. In this review, we summarize the main molecular characteristics of TGCT and the probable mechanisms participating in tumor initiation and progression.Key words: testicular germ cell tumors - signaling pathways - molecular aberrations - predictive factors - prognostic factors The work was supported by the Czech Ministry of Education, Youth and Sports NPU I nr.LO 1604. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 20. 3. 2017Accepted: 23. 7. 2017.