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Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

Heavy metals like cadmium (Cd) are one of the main environmental pollutants, with no biological role in the human body. Cd has been well-documented to have disastrous effects on both plants and animals. It is known to accumulate in kidneys, lungs, liver, and testes and is thought to affect these organs' function over time, which is linked to a very long biological half-life and a very poor rate of elimination. According to recent researches, the testes are extremely vulnerable to cadmium. The disruption of the blood–testis barrier, seminiferous tubules, Sertoli cells, and Leydig cells caused by cadmium leads to the loss of sperm through various mechanisms, such as oxidative stress, spermatogenic cell death, testicular swelling, dysfunction in androgen-producing cells, interference with gene regulation, disruption of ionic homeostasis, and damage to the vascular endothelium. Additionally, through epigenetic control, cadmium disrupts the function of germ cells and somatic cells, resulting in infertile or subfertile males. A full grasp of the mechanisms underlying testicular toxicity caused by Cd is very important to develop suitable strategies to ameliorate male fertility. Therefore, this review article outlines cadmium's impact on growth and functions of the testicles, reviews therapeutic approaches and protective mechanisms, considers recent research findings, and identifies future research directions. [ABSTRACT FROM AUTHOR]

This article reports on two cases of testicular sclerosing stromal tumors (SSTs) that show GLI1 gene alterations. SSTs are neoplasms that were first described in the ovary in 1973 and are characterized by lobulated microscopic appearance. The patients in these cases were aged 35 and 65 years and presented with testicular masses. The tumors demonstrated large areas of hypocellular myxoid/oedematous stroma with scattered hypercellular areas. Both tumors expressed nuclear GLI1. The article suggests that these findings support the existence of rare SSTs in the testis and their potential pathogenesis related to ovarian SSTs. [Extracted from the article]

Non‐obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype‐testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole‐genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562‐2A>T). TDRKH encodes a conserved mitochondrial membrane‐anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de‐repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval. [ABSTRACT FROM AUTHOR]

Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41–42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis. [ABSTRACT FROM AUTHOR]

Switching from mitotic spermatogonia to meiotic spermatocytes is critical to producing haploid sperms during male germ cell differentiation. However, the underlying mechanisms of this switch remain largely unexplored. In Drosophila melanogaster, the gene RpL38 encodes the ribosomal protein L38, one component of the 60S subunit of ribosomes. We found that its depletion in spermatogonia severely diminished the production of mature sperms and thus led to the infertility of male flies. By examining the germ cell differentiation in testes, we found that RpL38-knockdown blocked the transition from spermatogonia to spermatocytes and accumulated spermatogonia in the testis. To understand the intrinsic reason for this blockage, we conducted proteomic analysis for these spermatogonia populations. Differing from the control spermatogonia, the accumulated spermatogonia in RpL38-knockdown testes already expressed many spermatocyte markers but lacked many meiosis-related proteins, suggesting that spermatogonia need to prepare some important proteins for meiosis to complete their switch into spermatocytes. Mechanistically, we found that the expression of bag of marbles (bam), a crucial determinant in the transition from spermatogonia to spermatocytes, was inhibited at both the mRNA and protein levels upon RpL38 depletion. We also confirmed that the bam loss phenocopied RpL38 RNAi in the testis phenotype and transcriptomic profiling. Strikingly, overexpressing bam was able to fully rescue the testis abnormality and infertility of RpL38-knockdown flies, indicating that bam is the key effector downstream of RpL38 to regulate spermatogonia differentiation. Overall, our data suggested that germ cells start to prepare meiosis-related proteins as early as the spermatogonial stage, and RpL38 in spermatogonia is required to regulate their transition toward spermatocytes in a bam-dependent manner, providing new knowledge for our understanding of the transition process from spermatogonia to spermatocytes in Drosophila spermatogenesis. [ABSTRACT FROM AUTHOR]

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is known to cause teratogenesis. Environmental exposure of BaP has led to wide public concerns due to their potential risk of reproductive toxicity. However, the exact mechanism is still not clear. We aimed to explore the alterations of oxidative stress and DNA hydroxymethylation during BaP‐impaired reproductive function. BALB/c mice were intragastrically administered with different doses of BaP (0.01, 0.1, and 1 mg/kg/day, once a day), while control mice were administered with corn coil. Then, the reproductive function, alterations of oxidative stress, DNA methylation, and DNA hydroxymethylation of testis tissues were evaluated. We found that BaP caused obvious histopathological damages of testis tissues. As for sperm parameters after BaP administration, testis weight and the rate of teratosperm were increased, as well as sperm count and motility were decreased. In mechanism, BaP upregulated HO‐1 and MDA levels and downregulated SOD and CAT activity and GSH content in testis tissues, indicating that oxidative stress was induced by BaP. Furthermore, a significant induction of hydroxymethylation and inhibition of methylation were observed in testis tissues after BaP exposure. Collectively, BaP‐induced oxidative stress and hydroxymethylation were involved in impairing reproductive function, which may be the mechanism of the male infertility. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 is a transcription factor involved in a variety of crucial cellular functions, including cell cycle arrest, DNA repair and apoptosis. Still, a growing number of studies indicate that p53 plays multiple roles in spermatogenesis, as well as in the occurrence and development of male infertility. The representative functions of p53 in spermatogenesis include the proliferation of spermatogonial stem cells (SSCs), spermatogonial differentiation, spontaneous apoptosis, and DNA damage repair. p53 is involved in various male infertility-related diseases. Innovative therapeutic strategies targeting p53 have emerged in recent years. This review focuses on the role of p53 in spermatogenesis and male infertility and analyses the possible underlying mechanism involved. All these conclusions may provide a new perspective on drug intervention targeting p53 for male infertility treatment. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Materials and Methods Results Conclusions Hypogonadotropic hypogonadism (HH) is a treatable cause of nonobstructive azoospermic male infertility. Gonadotropin treatment can successfully induce spermatogenesis in most patients, although comprehensive quantitative summary data on spermatogenic outcomes like those required to induce pregnancy is lacking in the literature.Systematic review and meta‐analysis of outcomes related to male reproductive function following gonadotropin treatment.Our search strategy identified 41 studies encompassing 1673 patients with a mean age of 25 (± 5) years. Average sperm concentration achieved after a median of 18 months of gonadotropin treatment was 11.6 M/mL of ejaculate (95% CI 8.4–14.9). Sperm concentrations > 0, > 1, > 5, > 10 and > 20 M/mL were achieved by 78%, 55%, 36%, 24% and 15% of patients, respectively. Mean sperm output and the proportion of patients achieving all sperm thresholds were significantly greater following combined hCG/FSH treatment compared with hCG monotherapy. When compared by diagnosis, patients with congenital HH (CHH) had significantly lower mean sperm output compared with patients with hypopituitarism or mixed patient cohorts that did not differentiate between CHH and hypopituitarism. Treatment‐related increases in testosterone and testicular volume (TV) were not different between hCG and combined hCG/FSH treated patients, although increases in TV were lower in men with CHH compared with those with hypopituitarism.Gonadotropin treatment successfully induced spermatogenesis in most men with pathological gonadotropin deficiency. Sperm outputs more consistent with those typically needed to induce a natural pregnancy were less commonly achieved. Despite similar effects on serum testosterone and TV, combined hCG/FSH appeared more efficacious than hCG alone at inducing spermatogenesis. [ABSTRACT FROM AUTHOR]

Infertility affects approximately 15 % of couples globally, with 50 % cases of male factor infertility. Precise assessment of spermatogenesis is essential for evaluating male infertility. Recent studies suggest serum inhibin B as a promising biomarker for testicular function. This study aims to evaluate the diagnostic utility of serum inhibin B in predicting male infertility, particularly focusing on its relationship with sperm count.A cross-sectional study was conducted on 80 adult men (mean age 31.4 ± 6.89 years) presenting with infertility at gynecology and urology outpatient departments. Semen analysis was performed following WHO (2010) guidelines, and serum inhibin B levels were quantified. The correlation between serum inhibin B levels and sperm parameters was assessed using Pearson’s correlation test. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic accuracy of serum inhibin B and the inhibin B/FSH ratio for non-obstructive azoospermia (NOA) and oligozoospermia.A significant positive correlation was observed between serum inhibin B and sperm count (r=0.94, p<0.001). ROC analysis demonstrated that the inhibin B/FSH ratio had the highest diagnostic accuracy for NOA and oligozoospermia (AUC=0.986), with sensitivity of 100 % and specificity of 91.67 %. Serum inhibin B alone also showed high diagnostic value (AUC=0.965 for NOA and 0.969 for oligozoospermia).Serum inhibin B is a reliable biomarker for assessing male infertility, particularly in evaluating spermatogenic function. The inhibin B/FSH ratio provides superior diagnostic accuracy for NOA and oligozoospermia, offering valuable clinical utility in male infertility diagnosis. [ABSTRACT FROM AUTHOR]

The intermitochondrial cement (IMC) is a prominent germ granule that locates among clustered mitochondria in mammalian germ cells. Serving as a key platform for Piwi-interacting RNA (piRNA) biogenesis; however, how the IMC assembles among mitochondria remains elusive. Here, we identify that Tudor domain-containing 1 (TDRD1) triggers IMC assembly via phase separation. TDRD1 phase separation is driven by the cooperation of its tetramerized coiled-coil domain and dimethylarginine-binding Tudor domains but is independent of its intrinsically disordered region. TDRD1 is recruited to mitochondria by MILI and sequentially enhances mitochondrial clustering and triggers IMC assembly via phase separation to promote piRNA processing. TDRD1 phase separation deficiency in mice disrupts IMC assembly and piRNA biogenesis, leading to transposon de-repression and spermatogenic arrest. Moreover, TDRD1 phase separation is conserved in vertebrates but not in invertebrates. Collectively, our findings demonstrate a role of phase separation in germ granule formation and establish a link between membrane-bound organelles and membrane-less organelles. [Display omitted] • TDRD1 undergoes phase separation via oligomerization of coiled-coil domain • Tudor domains facilitate TDRD1 phase separation by recognizing sDMA • TDRD1 phase separation deficiency disrupts IMC assembly and piRNA biogenesis in mice • TDRD1 phase separation is conserved in vertebrates but not in invertebrates Gao et al. address the mechanism governing the assembly of mitochondrial-associated germ granules (IMCs) in mammalian germ cells. The IMC component TDRD1 undergoes phase separation to trigger IMC assembly, which is essential for piRNA biogenesis, transposon silencing, and male fertility in mice. [ABSTRACT FROM AUTHOR]

Background: The management of Non-Obstructive (NOA) Azoospermia or Obstructive Azoospermia (OA) patients relies on testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI). In NOA patients the sperm recovery is successful in only 50% of cases and therefore the ability to predict those patients with a high probability of achieving a successful sperm retrieval would be a great value in counselling the patient and his partner. Several studies tried to suggest predictors of a positive TESE (e.g. FSH concentration), but most concluded that diagnostic testicular biopsy (histology) is best. Methods: This is a retrospective analysis of 526 TESE patients. After the extraction of the testis, the resulting sample was immediately given to the embryologist, who examined the tubules for sperm cryopreservation. During the same procedure, a different specimen was destined to the histological analysis. The comparison between the two methodological approaches was carried out through a score. Results: Concordance between TESE and testicular histology outcomes was found in 70,7% of patients; discordance was found in 29,3% of patients. Among the discordance outcomes, in approximately 95% we found at least 1 sperm in the TESE retrieval, while the histology report did not find any spermatozoa or found not enough compared to our evaluation; in only 5% of cases we did not find any spermatozoa or found not enough compared to what was detected in the testicular histology. Conclusion: Based on our experience, to increase diagnostic accuracy, a larger biopsy should be sent to the histopathology laboratory; another option may be to use TESE cell suspension (the same embryologists employ for cryopreservation) for cytological evaluation of spermatogenesis. [ABSTRACT FROM AUTHOR]

As the global male infertility rate continues to rise, there is an urgent imperative to investigate the underlying causes of sustained deterioration in sperm quality. The gut microbiota emerges as a pivotal factor in host health regulation, with mounting evidence highlighting its dual influence on semen. This review underscores the interplay between the Testis-Gut microbiota axis and its consequential effects on sperm. Potential mechanisms driving the dual impact of gut microbiota on sperm encompass immune modulation, inflammatory responses mediated by endotoxins, oxidative stress, antioxidant defenses, gut microbiota-derived metabolites, epigenetic modifications, regulatory sex hormone signaling. Interventions such as probiotics, prebiotics, synbiotics, fecal microbiota transplantation, and Traditional natural herbal extracts are hypothesized to rectify dysbiosis, offering avenues to modulate gut microbiota and enhance Spermatogenesis and motility. Future investigations should delve into elucidating the mechanisms and foundational principles governing the interaction between gut microbiota and sperm within the Testis-Gut microbiota Axis. Understanding and modulating the Testis-Gut microbiota Axis may yield novel therapeutic strategies to enhance male fertility and combat the global decline in sperm quality. [ABSTRACT FROM AUTHOR]

Introduction: The increasing prevalence of infertility in male dogs in clinical practice mirrors current trends seen in human medicine. Acquired infertility is notably more common in dogs compared to congenital causes, with conditions such as testicular degeneration leading to irreversible loss of fertility. Current diagnostic methods for testicular degeneration, such as histopathological and cytological examinations, rely on testicular biopsy or fine needle aspiration, making them less feasible for routine use. Anti-Müllerian hormone (AMH), produced by Sertoli cells, has emerged as a potential alternative biomarker for testicular health, which can be measured in serum. This study evaluates AMH as a potential marker for testicular degeneration, using cryptorchid dogs as models for impaired fertility and altered testicular histology. Methods: The relationship between serum AMH levels and AMH tissue expression with impaired spermatogenesis and altered histology was investigated. Serum AMH levels were determined in intact, cryptorchid, and castrated individuals using an immuno-enzymatic ELISA kit and compared between subgroups based on testicular location. Tissue AMH immuno-expression was differentially quantified in two regions of interest (ROIs), the interstitial space and the seminiferous tubule, in both descended and retained gonads. Furthermore, testicles were analyzed using histomorphometric analysis in seminiferous tubules, while spermatogenesis was evaluated using the Johnsen score. Results: Serum AMH levels were positively correlated with AMH expression assessed in both interstitial space (ρ = 0.494, p ≤ 0.01) and seminiferous tubules (ρ = 0.610, p ≤ 0.001). Conversely, serum AMH levels showed a negative correlation with the seminiferous tubule area (ρ = -0.435, p ≤ 0.05). Smaller seminiferous tubule areas were linked to increased AMH reactivity in both seminiferous tubules (ρ = -0.774, p ≤ 0.001) and interstitial space (ρ = -0.725, p ≤ 0.001). Additionally, lower Johnsen scores were associated with higher serum AMH levels (ρ = -0.537, p ≤ 0.01) and elevated AMH expression in both seminiferous tubules (ρ = -0.756, p ≤ 0.001) and interstitial space (ρ = -0.679, p ≤ 0.001). Discussion: Our results suggest that higher serum levels and tissue expression of AMH are linked to smaller seminiferous tubules and poorer Johnsen scores, reflecting degenerative changes and Sertoli cell dysfunction in retained testicles. Given the similarities in the mechanisms that increase AMH levels in both cryptorchid and non-cryptorchid testicles affected by testicular degeneration, this study recommends using AMH as a marker for diagnosing testicular degeneration in dogs. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results Conclusions Imatinib and dasatinib are tyrosine kinase inhibitors (TKIs) increasingly used to treat several diseases in both children and adults at fertile age. We have previously shown that imatinib has adverse effects on developing testis, and imatinib‐treated male patients have been reported to have reduced sperm counts. However, the cellular level effects of imatinib and dasatinib on adult male germ cells and germline stem cells (mGSCs) have not been thoroughly investigated.To analyze whether imatinib or dasatinib exposure ex vivo and in vitro is harmful to adult male rodent germ cells and mGSCs.Seminiferous tubule segments of adult male mouse or rat were cultured in the presence or the absence of imatinib or dasatinib. Stage‐specific effects were monitored by 3H‐thymidine incorporation assay (DNA synthesis), immunohistochemistry (cleaved Caspase‐3; apoptosis), immunofluorescence (KI67, GFRα1, STRA8, c‐KIT, LIN28A; proliferation and spermatogonial differentiation) and flow cytometry (Hoechst). Mouse mGSCs were exposed to imatinib and dasatinib to study proliferation, apoptosis, and differentiation.Imatinib decreased stage‐specific DNA synthesis, and induced apoptosis in cultured rat seminiferous tubule segments. Imatinib also had an adverse effect on mGSC proliferation both in vitro and ex vivo, but did not induce cell death in cultured mGSCs. Imatinib did not impinge on induction of spermatogonial differentiation but suppressed c‐KIT expression in nascent differentiating spermatogonia, providing a plausible mechanism for its pro‐apoptotic function in spermatogenic cells. Clinically relevant doses of dasatinib did not induce apoptosis in seminiferous tubules but decreased mGSC colony growth in vitro.Imatinib exposure ex vivo and in vitro impinges on male rodent germ cell proliferation and survival. The plausible mechanism in spermatogenic cells is the inhibition of SCF/c‐KIT signaling, and reduced expression of c‐KIT. Dasatinib did not show significant adverse effects with clinical doses ex vivo but inhibited mGSC colony growth in vitro. [ABSTRACT FROM AUTHOR]

The transcription factors NANOG and POU5F1 (OCT4) play crucial roles in maintaining pluripotency in embryonic stem (ES) cells. While their functions have been well-studied, the specific interactions between NANOG and POU5F1 and their combined effects on pluripotency in ES-like and Epiblast cells remain less understood. Understanding these associations is vital for refining pluripotent stem cell characterization and advancing regenerative medicine. In this matter, we investigated the associations between NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells and how these interactions contribute to the distinct pluripotent states of these cells. In the present paper, we examined the pattern of NANOG expression by the immunocytochemical method in embryonic stem-like (ES-like) cells and compared it with its expression pattern in embryonic stem cells (ESCs). Similarly, we examined the expression pattern of POU5F1 in ES-like cells, ESCs, and epiblast cells and compared the expression pattern of these two genes with each other. On the other hand, using Fluidigm Biomark system analysis, we compared the amount of NANOG mRNA in these three cell lines and differentiated and undifferentiated Spermatogonial stem cells in several passages. Microscopic observations indicated the cytoplasmic expression of NANOG in the considered cells; moreover, they showed a similar expression pattern of NANOG with POU5F1 in the experimented cells. It has also been suggested that the more limited the cell’s pluripotency, the lower the expression of these two genes. However, the decrease in NANOG expression is less than that of POU5F1. Fluidigm real-time RT-PCR analysis also confirmed these results. During the experimental process, protein-protein (PPI) network analysis shows a significant association of NANOG with other stem cell proteins, such as POU5F1. Our findings reveal distinct yet overlapping roles of NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells. The differential binding patterns and functional interactions between these factors underscore the complexity of pluripotency regulation in different stem cell states. This study provides new insights into the molecular mechanisms governing pluripotency and highlights potential targets for enhancing stem cell-based therapies. [ABSTRACT FROM AUTHOR]

Background: The deep-sea acorn worm Quatuoralisia malakhovi belongs to the phylum Hemichordata, class Enteropneusta, family Torquaratoridae, which was described in 2005. Owing to their epibenthic lifestyle and deep-sea habitat features, torquaratorids differ anatomically from shallow-water acorn worms; however, their morphology and fine structure are poorly studied. We have the opportunity to make three complete detailed series of histological sections of Q. malakhovi and to study the microscopic anatomy, histology and fine structure of the reproductive system of this acorn worm using scanning and transmission electron microscopy. Results: The sexes of Q. malakhovi are separate and indistinguishable externally. The lobed testes occupy the dorsal side of the genital wings and distinctly bulge into the peribranchial cavity by their mature lobes. The central part of the testis is always submerged into the genital wing and opens via a single gonad pore. The monociliary muscle cells stretch along the external wall of the testis and surround the gonad pore, probably taking part in the contraction of the testis lobes for spawning. The germinative epithelium of the testis contains spermatogenic cells at different stages of development and interstitial cells. Yolk cells are not found. Interstitial cells embrace the spermatogonia and spermatogenic columns, providing horizontal compartmentalization of the germinative epithelium, and contain numerous phagosomes with remnants of degenerating spermatogenic cells. The testis wall contains haemal lacunae, which are usually located on the side opposite the gonad pore. We describe the fine structure of spermatogonia, spermatocytes clustered in spermatogenic columns, spermatids, and spermatozoa. Spermatozoa are of the ectaquasperm type and consist of an acorn-shaped head and a flagellum 18–25 µm long. The sperm head includes a beak-shaped acrosomal part, a spherical nucleus and a midpiece containing a ring of 5 or rarely 6 mitochondria. Conclusions: The male reproductive system and sperm structure of Q. malakhovi, a representative of the family Torquaratoridae, have a number of differences from shallow-water acorn worms; however, the spermatogenesis and sperm structure of Q. malakhovi generally follow the pattern of the other three enteropneust families, and the phylogenetic significance of these deviations should be the subject of further research. [ABSTRACT FROM AUTHOR]

The current study focused on identifying the potential of irisin in mammalian reproduction. The established role of irisin, a proteolytic product of FNDC5, in adipose tissue browning, energy metabolism, and thermogenesis suggests its role in reproductive health, often disturbed by metabolic imbalances. Various studies on mice demonstrated irisin′s role in improving spermatogenesis, sperm count, and testosterone levels by influencing the hypothalamus–pituitary–gonadal axis. Moreover, in females, there is a fluctuation in levels of irisin during critical reproductive stages, including menstrual cycles, puberty, and pregnancy. Conditions like pregnancy complications, precocious puberty, and polycystic ovary syndrome (PCOS) are found to have an association with abnormal irisin levels. The potential role of irisin in endometrial receptivity and preventing endometritis is also discussed in this review. Overall, the influence of irisin on female and male reproduction is evident from various studies. However, further research is needed to elucidate irisin mechanism in reproduction and its potential as a therapeutic or diagnostic tool for reproductive dysfunctions and infertility. [ABSTRACT FROM AUTHOR]

Simple Summary: Surgical castration is a common practice for controlling animal reproduction. However, surgical castration can cause pain and stress and may even lead to psychological harm. As an alternative, GnRH vaccines can lower androgen levels without surgery. In this study, we compared the effects of a newly developed GnRH vaccine and surgical castration in rats. Both methods effectively suppressed sexual behavior, but surgical castration resulted in significant depression and social withdrawal, while the vaccinated rats did not exhibit these behavioral issues. This suggests that GnRH immunocastration may be a more humane method that better aligns with animal welfare standards. Castration is often employed in animal management for reproductive control. However, it is important to evaluate its impact on animal welfare. In this study, we developed rat models for both surgical (n = 6) and GnRH immunocastration (n = 6) to assess the effects of these castration methods on physiological and behavioral characteristics. The novel GnRH-based vaccine significantly increased serum GnRH antibody levels and drastically reduced testosterone, with the testes shrinking to one-fifth the size of those in the control group, thereby halting spermatogenesis at the secondary spermatocyte stage. Behavioral evaluations demonstrated that sexual behavior was significantly suppressed in both surgically and immunologically castrated groups compared to the control, confirming the effectiveness of both methods. However, psychological tests revealed significant signs of depression and social deficits in the surgically castrated group, whereas the behavior of the GnRH-immunocastrated group did not significantly differ from the control. Furthermore, no significant differences in learning and memory were observed among the three groups in the water maze test. Compared to surgical castration, GnRH immunocastration offers effective results and better animal welfare, providing a more humane alternative for livestock management. [ABSTRACT FROM AUTHOR]

Simple Summary: The testis plays a crucial role in spermatogenesis and sperm production for the male sheep; the focus of this study was to use transcriptome to analyze the key developmental periods of an F1 hybrid of Southdown × Hu sheep, a cross between Southdown and Hu sheep. Through M0, M3 and M6 and Y1, four key-stage-two comparative screening differentially genes, and GO and KEGG enrichment analysis, we found the cAMP signaling pathway, MAPK signaling pathway, ECM–receptor interaction, PI3K-Akt signaling pathway, FOXO signaling pathway and other signaling pathways by enrichment, and south lake sheep testicular-development-related key genes have been found. These findings provide new insights into the development of sheep and help to improve sheep productivity. Numerous genes govern male reproduction, modulating testicular development and spermatogenesis. Our study leveraged RNA-Seq to explore candidate genes and pivotal pathways influencing fecundity in an F1 hybrid of Southdown × Hu sheep testes across four developmental milestones: M0 (0 months old, newborn), M3 (3 months old, sexually immature), M6 (6 months old, sexually mature), and Y1 (1 years old, adult). Histological examination using hematoxylins and eosin staining revealed that the cross-sectional area of the spermatid tubules and the number of supportive cells increased in the other groups, as compared to the M0 group. The cross-sectional area of the vasculature and the number of supporting cells were found to be significantly increased in all other groups in comparison to the M0 group. We conducted GO and KEGG analyses of the differentially expressed genes (DEGs) in the three comparison groups and identified key pathways, including cAMP, MAPK, ECM–receptor interactions, PI3K-Akt, and FOXO signaling, which are closely related to testicular development and spermatogenesis. Notably, alternative splicing (AS) events were markedly elevated in M6 and Y1 stages. Key genes like GATA4, GATA6, SMAD4, SOX9, YAP1, ITGB1 and MAPK1 emerged as significantly enriched in these pathways, potentially orchestrating the transition from immature to mature testes in sheep. These findings offer valuable insights into male reproductive potential and can inform strategies for optimizing animal breeding. [ABSTRACT FROM AUTHOR]

Oxidative stress triggered by testicular torsion and detorsion in young males could negatively impact future fertility. Using a rat animal model for testicular IRI (tIRI), we aim to study the induction of autophagy (ATG) during testicular ischemia and tIRI and the role of oxidative-stress-induced c-Jun N-terminal Kinase (JNK) as a cytoprotective mechanism. Sixty male Sprague-Dawley rats were divided into five groups: sham, ischemia only, ischemia+SP600125 (a JNK inhibitor), tIRI only, and tIRI+SP600125. The tIRI rats underwent an ischemic injury for 1 h followed by 4 h of reperfusion, while ischemic rats were subjected to 1 h of ischemia only without reperfusion. Testicular-ischemia-induced Beclin 1 and LC3B expression was associated with decreased p62/SQSTM1 expression, increased ATP and alkaline phosphatase (AP) activity, and slightly impaired spermatogenesis. SP600125 treatment improved p62 expression and reduced the levels of Beclin 1 and LC3B but did not affect ATP or AP levels. The tIRI-induced apoptosis lowered the expression of the three ATG proteins and AP activity, activated caspase 3, and caused spermatogenic arrest. SP600125-inhibited JNK during tIRI restored sham levels to all investigated parameters. This study emphasizes the regulatory role of JNK in balancing autophagy and apoptosis during testicular oxidative injuries. [ABSTRACT FROM AUTHOR]

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder caused by deficient secretion or action of gonadotropin‐releasing hormone (GnRH) and is a hormonally treatable form of male infertility. Both pulsatile GnRH treatment and combined gonadotropin therapy effectively induce spermatogenesis in 75%–80% of males with CHH, albeit the ejaculate does not usually approach normal semen parameters by WHO criteria. This is in some contrast to the cumulative fertility outcomes in females with CHH on gonadotropin treatment that are indistinguishable from those of reproductively normal females. Emerging data provide insights into early life determinants of male fertility (i.e., minipuberty), and research has identified key predictors of outcomes for fertility‐inducing treatment in men with CHH. Such developments provide mounting evidence for tailoring approaches to maximize fertility potential in CHH, although there is no clear consensus to date on the optimal approach to fertility‐inducing treatment. This review provides an up‐to‐date review on the current evidence underpinning therapeutic approaches for inducing spermatogenesis in males with CHH. In the absence of evidence‐based clinical guidelines, this synthesis of current evidence provides guidance for clinicians working with males with CHH seeking fertility. [ABSTRACT FROM AUTHOR]

Diabetes leads to testicular damage and infertility. Mesenchymal stem cells and their secretory trophic factors have shown potential as regenerative therapies for diabetes and its associated complications. This study examined the effects of conditioned medium derived from Wharton's jelly mesenchymal stem cells (WJMSCs‐CM) on sperm parameters, reproductive hormones, biochemical parameters, and histological changes in the testes of diabetic rats. Fifty‐six male Sprague–Dawley rats (250–300 g) were assigned to eight groups: control, diabetes, and six diabetic groups receiving early or late treatments with WJMSCs‐CM (D‐CME, D‐CML), insulin (D‐INSE, D‐INSL), or DMEM (D‐DME, D‐DML). In the early treatment groups, insulin (3 U/day, subcutaneously) and WJMSCs‐CM (10 mg/week, intraperitoneally) were administered immediately after diabetes induction; in the late treatment groups, these interventions began 30 days postinduction. Blood glucose and insulin levels, along with sperm parameters, were assessed. Sex hormones, testicular antioxidant enzyme activity, malondialdehyde (MDA), and glutathione (GSH) concentrations were measured using colorimetric methods. Real‐time PCR detected Bax, Bcl‐2, and tumor necrosis factor‐alpha (TNF‐α) gene expression. Our results showed that diabetes increased blood glucose levels, decreased insulin and sex hormone levels, induced testicular oxidative stress and apoptosis, and reduced sperm parameters compared to the control. WJMSCs‐CM significantly ameliorated hyperglycemia, increased insulin and sex hormone levels, and improved sperm quality. In WJMSCs‐CM‐treated diabetic rats, MDA levels were reduced, while GSH and antioxidant enzyme activity increased. Furthermore, WJMSCs‐CM decreased the testicular Bax/Bcl‐2 ratio and TNF‐α expression, as well as enhanced spermatogenic, Sertoli, and Leydig cells. In conclusion, WJMSC‐CM administration effectively mitigated diabetes‐induced testicular damage by reducing oxidative stress, inflammation, and apoptosis. Early treatment with WJMSCs‐CM was more effective than late treatment for diabetes‐induced reproductive dysfunction. [ABSTRACT FROM AUTHOR]

Background information: Extracellular matrix (ECM)‐derived hydrogels are frequently used in three‐dimensional (3D) cell culture and organoid formation in several tissues. However, in the 3D cultivation of testicular cells, the hyaluronic acid (HA) hydrogel has not received as much attention. This study examined the effects of three distinct composites, including HA‐alginate (HA‐Alg), HA‐alginate‐collagen (HA‐Alg‐Col), and HA‐alginate‐decellularized ECM (HA‐Alg‐dECM), on mouse testicular cell culture and in vitro spermatogenesis. Methods: For the creation of composites, the concentration of biomaterials used was 0.5% HA, 1% alginate, 2.5 mg/mL collagen, and 25 mg/mL dECM derived from the testicles of Rams. After 3D culture of 5 days post‐partum (dpp) mouse testicular cells for 14 days, HA‐Alg was selected as a superior composite due to the greater number and size of the produced organoids. Then, cell culture was rerun by HA‐Alg for 14 days, which was later extended for an additional 28 days. In addition, the 3D culture of 10 dpp mouse testicular cells was used to compare with 5 dpp mice on day 14. The morphology and gene expression were analyzed using appropriate techniques. Results: On day 14, the HA‐Alg hydrogel showed significantly more organoids in terms of size and number than the other two groups (p < 0.05); nevertheless, none of the groups showed the expected signs of testis organoids. Remarkably, on day 14, the histology and immunostaining tests revealed features of hepatocyte‐like cells (HLCs) and albumin production as a marker of HLC functionality. Furthermore, the analysis of gene expression verified the significant expression of angiogenesis markers (p < 0.01). After the extended culture to 28 days, 5 dpp testicular cells once more differentiated into erythrocytes and HLCs, while a small number of organoids showed the characteristic of renal cells. Cell culture of 10 dpp mice for 14 days showed a wide range of cell lineages, including renal, glandular, chondrocyte, and hepatocyte‐like cells in comparison to the 5 dpp mice. Conclusion and significance: While the HA‐Alg composite did not support spermatogenesis in the 3D culture of mouse testicular cells, it demonstrated an unpredicted potential for promoting the differentiation of neonate mouse testicular cells into HLC, erythrocytes, and other cell lineages. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell–cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does RXFP2 disruption impair male fertility? SUMMARY ANSWER We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation. WHAT IS KNOWN ALREADY Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown. STUDY DESIGN, SIZE, DURATION A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations. PARTICIPANTS/MATERIALS, SETTING, METHODS The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay. MAIN RESULTS AND THE ROLE OF CHANCE An intragenic deletion of exon 1–5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility. LIMITATIONS, REASONS FOR CAUTION Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B. 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C. FIKO-IV institutional fund). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Atrazine (ATZ, C8H14ClN5) is a widely used synthetic herbicide that contaminates drinking water. It is a known endocrine disruptor that disrupts various molecular pathways involved in hormone signaling, and DNA damage, and can cause reproductive disorders, including decreased fertility, and abnormal development of reproductive organs, as revealed in animal model studies. However, the effect of ATZ on steroidogenesis in the male reproductive system, especially reduction of ketosteroids to hydroxysteroids, remains unclear. This study investigated the toxicity of ATZ on the male reproductive system in the Wistar rat model, with an emphasis on its adverse effect on aldo-ketoreductase family 1 member C2 (AKR1C2). Male Wistar rats were administered ATZ for 56 days (duration of one spermatogenic cycle) through oral route, at 20, 40 and 60 mg/kg body weight (bw) doses. The results indicate that ATZ exposure affects the body weight, impairs sperm production, and decrease FSH, LH, and testosterone levels. Additionally, the down-regulation of key steroidogenic enzymes by ATZ disrupted the synthesis of testosterone, leading to decreased levels of this essential male hormone. On the other hand, the expression of AKR1C2 (mRNA and protein) in the testis was upregulated. The findings suggest that AKR1C2 plays a role in androgen metabolism. Furthermore, its overexpression may lead to alteration in the expression of genes in the connected pathway, causing an increase in the breakdown or inactivation of androgens, which would result in lower androgen levels and, thereby, lead to hypoandrogenism, as the combined effects of down-regulation of steroidogenic genes and up-regulation of AKR1C2. These findings reveal direct implication of disrupted AKR1C2 in male reproductive health and highlight the need for further research on the impact of environmental toxins on human fertility, ultimately providing for better patient care. [ABSTRACT FROM AUTHOR]

Purpose: The leaves of Perilla frutescens var. acuta (PFA) are generally reported to have antioxidant, anti-allergic, anti-inflammatory, and antitumor effects and commonly used as a traditional medicine in East Asia. This study aimed to investigate the protective effect and antioxidant activity of PFA on busulfan-induced testicular dysfunction, histological damage, oxidative stress (OS), sperm quality, and hormone levels using a mouse model. Materials and Methods: C57BL/6 male mice were divided into four groups: control, busulfan-only treated, and varying concentrations of PFA (100 and 200 mg/kg) with busulfan. In the busulfan group, 40 mg/kg of busulfan was intraperitoneally injected to induce azoospermia. Mice were orally administered PFA for 35 consecutive days after busulfan administration. Samples were collected and assessed for testis/body weight, testicular histopathology, sperm quality, serum hormone levels, and OS to evaluate the effects of PFA treatment on spermatogenesis dysfunction induced by busulfan. Results: The busulfan-induced testicular dysfunction model showed reduced testis weight, adverse histological changes, significantly decreased sex hormones and sperm quality, and attenuated OS. These results indicate that PFA treatment significantly increased testis weight, testis/body weight, epididymal sperm count, motility, and testosterone level compared with busulfan alone. PFA treatment also attenuated the busulfan-induced histological changes. Furthermore, compared with mice treated with busulfan alone, PFA supplementation upregulated the testicular mRNA expression of the antioxidant enzymes superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), with a decrease in malondialdehyde (MDA) production and an increase in SOD and GPx activities. Conclusions: This study shows that PFA exerts a protective effect against testicular damage by attenuating OS induced by busulfan. Our results suggest that PFA is a potentially relevant drug used to decrease the side effects induced by busulfan on testicular function and sperm during cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Genes encoding Toll-like receptors (TLRs) are expressed by germ cells in the mouse testis. Nevertheless, the expression of TLRs by germ cells has only been demonstrated for TLR-3, TLR-9, and TLR-11. Furthermore, the expression of each TLR in relation to the stage of spermatogenesis remains uncertain. We aimed in the present study to examine the expression pattern of all TLRs in germ cells throughout the cycle of seminiferous epithelium in the adult mouse testis. Immunohistochemistry was used to evaluate the expression of TLRs. Results of the present study reveal the expression of TLRs by specific populations of germ cells. Expression of TLRs, except for TLR-7, at endosomal compartments, acrosomes, and/or residual bodies was another interesting and novel finding of the present study. We further demonstrate that the expression of TLR-1, -2, -3, -4, -5, -7, -11, -12, and -13 follows a distinct spatiotemporal pattern throughout the cycle of seminiferous epithelium. While TLR-1, -3, -5, -11, and -12 are expressed in all stages, TLR-4 is expressed only in early and middle stages of spermatogenic cycle. On the other hand, TLR-2, -7, and -13 are expressed only in early stage of spermatogenic cycle. Evidence demonstrating the expression of TLRs in a stage specific manner throughout spermatogenesis strengthen the hypothesis that the expression of various TLRs by germ cells is a developmentally regulated process. However, if TLRs play a role in the regulation of proliferation, growth, maturation, and differentiation of germ cells throughout the cycle of the seminiferous epithelium warrants further investigations. [ABSTRACT FROM AUTHOR]

Long-term radiofrequency radiation (RFR) exposure, which adversely affects organisms, deteriorates testicular functions. Misfolding or unfolding protein accumulation in the endoplasmic reticulum (ER) initiates an intracellular reaction known as ER stress (ERS), which activates the unfolded protein response (UPR) for proteostasis. Since both RFR exposure and ERS can cause male infertility, we hypothesized that RFR exposure causes ERS to adversely affect testicular functions in rats. To investigate role of ERS in mediating RFR effects on rat testis, we established five experimental groups in male rats: control, short-term 2100-megahertz (MHz) RFR (1-week), short-term sham (sham/1-week), long-term 2100-MHz RFR (10-week), and long-term sham (sham/10-week). ERS markers Grp78 and phosphorylated PERK (p-Perk) levels and ERS-related apoptosis markers Chop and caspase 12 were investigated by immunohistochemistry, immunoblotting, and quantitative real-time polymerase chain reaction (qPCR). Long-term RFR exposure increased Grp78, p-Perk, and Chop levels, while short-term RFR exposure elevated Chop and caspase 12 levels. Chop expression was not observed in spermatogonia and primary spermatocytes, which may protect spermatogonia and primary spermatocytes against RFR-induced ERS-mediated apoptosis, thereby allowing transmission of genetic material to next generations. While short and long-term RFR exposures trigger ERS and ERS-related apoptotic pathways, further functional analyses are needed to elucidate whether this RFR-induced apoptosis has long-term male infertility effects. Article Highlights: Long-term radiofrequency radiation in testis triggers endoplasmic reticulum stress. Short-term radiofrequency radiation causes apoptosis in testis. Spermatogonia and spermatocytes are not affected by short-term radiofrequency radiation. [ABSTRACT FROM AUTHOR]

The decrease in fertility in aging roosters is related to the reduced quality of ejaculated sperm. This study aimed to investigate the effect of dietary supplementation with ginseng extract at various concentrations (0–150 mg/kg) on testicular function, semen preservation, and fertility at different stages of sexual maturity (mature and aging roosters) in Thai native roosters. Pradu Hang Dum roosters at 32 (mature; n = 24) and 75 (aging; n = 24) weeks of age were fed diets with non-supplemented or supplemented ginseng extracts (50, 100, and 150 mg/kg) until the end of the experiment. In experiment 1, fresh semen samples were examined for the quality parameters of semen volume, sperm concentration, sperm motility, sperm viability, lipid peroxidation, and enzymatic activities. In experiment 2, semen was preserved at 5 °C for up to 48 h, and the semen quality and fertility potential were determined. In experiment 3, testicular function and testosterone concentrations were evaluated. The results showed that ginseng extract supplementation in the diets of both mature and aging roosters at 50 and 100 mg/kg improved fresh semen quality (P < 0.05). A decrease in malondialdehyde levels in fresh semen was observed with increasing enzyme activities. In mature roosters, the progressive motility of cold-stored semen and fertility rates were higher in the G50 and G100 groups compared to the control and G150 groups after 24 h of storage (P < 0.05). In aging roosters, the highest significant differences in progressive motility, viability, and fertility rates were observed in the G50 and G100 groups at all storage times (P < 0.01). These improvements might be attributed to good testicular function in spermatogenesis, as revealed by the results of histological examination and testosterone concentrations. However, higher doses of ginseng extract supplementation negatively affected sperm quality. In summary, the recommended dose of ginseng extract supplementation in diets is 50 mg/kg. Fertility results indicated that insemination with semen preserved for 24 h was satisfactory in both mature and aging roosters. • Ginseng extract improved sperm production and semen preservation in mature and aging roosters. • Semen quality was highest in G50 and G100 groups, confirmed by histological and testosterone results. • High doses (150 mg/kg) negatively impacted sperm quality. • 24 hour preserved semen showed satisfactory fertility rates in both mature and aging roosters. [ABSTRACT FROM AUTHOR]

Although bitter receptors, known as Tas2Rs, have been identified in the testes and mature sperm, their expression in testicular Sertoli cells (SCs) and their role in recognizing harmful substances to maintain the immune microenvironment remain unknown. To explore their potential function in spermatogenesis, this study utilized TM4 cells and discovered the high expression of the bitter receptor Tas2R143 in the cells. Interestingly, when the Tas2R143 gene was knocked down for 24 and 48 h, there was a significant downregulation (P < 0.05) in the expression of tight junction proteins (occludin and ZO-1) and NF-κB. Additionally, Western blot results demonstrated that the siRNA-133+NF-κB co-treatment group displayed a significant downregulation (P < 0.05) in the expression of occludin and ZO-1 compared to both the siRNA-133 transfection group and the NF-κB inhibitors treatment group. These findings suggest that Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway and provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system, aiming to attract attention to the chemical perception mechanism of spermatogenesis. • Tas2R143 affected to varying degrees the expression of BTB tight junction proteins (occludin, ZO-1) and NF-κB. • Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway in vitro. • This study proposes for the first time that the bitter receptor Tas2R143 is expressed in mouse Sertoli cells (TM4). • This study provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system. [ABSTRACT FROM AUTHOR]

Melatonin plays a major role in regulating the sleep-wake cycle and enhancing testosterone production. We investigated the short-term effects of melatonin treatment for 14 consecutive days in the cryptorchidism model. We categorized experimental mice into Sham (S), Orchiopexy (O), Melatonin (Mel), and Orchiopexy + Melatonin (OMel) groups. Surgery involved inducing cryptorchidism in the left testis for seven days, followed by orchiopexy. The Mel group's testes did not descend, but they received melatonin injections after seven days of cryptorchidism. The OMel group underwent both orchiopexy and melatonin treatment. Both O and Mel groups exhibited decreased sperm and round-headed sperm in the epididymis. Significant increases were observed in the numbers of giant cells and negative Nectin-3 cells at p -value<0.05. The pattern of Cadm1 expression changed, and Nectin-2 and Nectin-3 co-expression was lacking in abnormal spermatids. Sertoli cell cytoplasm in both O and Mel groups exhibited autophagosomes and multivesicular bodies, which correlated with increased cyclooxygenase-2 expression. However, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cell numbers increased significantly in all treatment groups compared to the S group. Our study found that the combination of orchiopexy and melatonin positively influenced the expression of cell adhesion molecules (Cadm1, Nectin-2, and Nectin-3) involved in spermatogenesis, while reducing giant cells, autophagosomes, and apoptosis. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is one of the most prevalent endocrine disrupting chemicals (EDCs) and there is widespread concern about the adverse effects of EDCs on human health. However, the exact mechanism of these toxicities has still not been fully deciphered. Additionally, studies have reported the toxicological effects at far low doses to the generally considered no‐observed‐adverse‐effect level (NOAEL) dose. The present study investigates the effects of a sub‐acute (28 days) exposure to BPA (10, 50 and 100 mg/kg/day) in adult male mice on various hormones levels, sperm motility, sperm count, functional integrity of sperm plasma membrane, testicular histological changes, oxidative stress markers and DNA damage. The key proteome signatures were quantified by LC‐MS/MS analysis using Orbitrap Fusion Lumos Tribrid Mass Spectrometer equipped with nano‐LC Easy‐nLC 1200. Data suggest that the BPA exposure in all doses (below/above NOAEL dose) have greatly impacted the hormone levels, sperm parameters (sperm count, motility and membrane integrity) and testicular histology. Mass spectrometry‐based proteomics data suggested for 1352 differentially expressed proteins (DEPs; 368 upregulated, 984 downregulated) affecting biological process, cellular component, and molecular functions. Specifically searched male reproductive function related proteins suggested a complex network where 46 potential proteins regulating spermatogenesis, sperm structure, activity and membrane integrity while tackling oxidative stress responses were downregulated. These potential biomarkers could shed some more light on our current understanding of the reproductive toxicological effects of BPA and may lead to exploration of novel interventions strategies against these targets for male infertility. Highlights: Bisphenol A (BPA), a potent endocrine disruptor has known reproductive toxicitiesIt shows varied degree of responses and toxicities in doses well below no‐observed‐adverse‐effect level.Sub‐acute BPA exposure leads sperm count, motility and structure alterations46 potential proteins relevant to spermatogenesis and testes tissue were downregulated by BPARedox reaction, hormonal changes, cell cycle and structural proteins affected by BPA [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is widely exposed in populations worldwide and has negative effects on spermatogenesis both in animals and humans. The homeostasis of the actin cytoskeleton in the spermatogenic epithelium is crucial for spermatogenesis. Actin cytoskeleton destruction in the seminiferous epithelium is one of the important reasons for BPA‐induced spermatogenesis disorder. However, the underlying molecular mechanisms remain largely unexplored. Herein, we explored the role and mechanism of Rsad2, an interferon‐stimulated gene in BPA‐induced actin cytoskeleton disorder in mouse GC‐2 spermatocyte cell lines. After BPA exposure, the actin cytoskeleton was dramatically disrupted and the cell morphology was markedly altered accompanied by a significant increase in Rsad2 expression both in mRNA and protein levels in GC‐2 cells. Furthermore, the phalloidin intensities and cell morphology were restored obviously when interfering with the expression of Rsad2 in BPA‐treated GC‐2 cells. In addition, we observed a significant decrease in intracellular ATP levels after BPA treatment, while the ATP level was obviously upregulated when knocking down the expression of Rsad2 in BPA‐treated cells compared to cells treated with BPA alone. Moreover, Rsad2 relocated to mitochondria after BPA exposure in GC‐2 cells. BPA promoted Rsad2 expression by activating type I IFN‐signaling in GC‐2 cells. In summary, Rsad2 mediated BPA‐induced actin cytoskeletal disruption in GC‐2 cells, which provided data to reveal the mechanism of BPA‐induced male reproductive toxicity. BPA induces actin cytoskeleton disruption in the seminiferous epithelium. However, the underlying molecular mechanisms remain unclear. Herein, we found that Rsad2 was required for BPA‐induced cytoskeletal abnormality in GC‐2 cells. Mechanistically, BPA promoted Rsad2 expression by activating type I IFN‐signaling. Afterward, Rsad2 relocated to mitochondria, leading to a decrease in intracellular ATP levels and subsequently resulting in actin cytoskeletal disruption. Taken together, Rsad2 mediated BPA‐induced actin cytoskeleton disorder in GC‐2 cells. [ABSTRACT FROM AUTHOR]

Purpose: Adropin is an emerging metabolic hormone that has a role in regulating energy homeostasis. The present study aimed to explore the impact of adropin on redox homeostasis and its possible role in testicular functions in adult mouse testis. Methods: Western blot, flow-cytometry, and TUNEL assay were performed to explore the impact of intra-testicular treatment of adropin (0.5 μg/testis) on testicular functions of adult mice. Hormonal assay was done by ELISA. Further, antioxidant enzyme activities were measured. Results: Adropin treatment significantly increased the sperm count and testicular testosterone by increasing the expression of GPR19 and steroidogenic proteins. Also, adropin treatment reduced the oxidative/nitrosative stress by facilitating the translocation of NRF2 and inhibiting NF-κB into the nucleus of germ cells. Enhanced nuclear translocation of NRF2 leads to elevated biosynthesis of antioxidant enzymes, evident by increased HO-1, SOD, and catalase activity that ultimately resulted into declined LPO levels in adropin-treated mice testes. Furthermore, adropin decreased nuclear translocation of NF-κB in germ cells, that resulted into decreased NO production leading to decreased nitrosative stress. Adropin/GPR19 signaling significantly increased its differentiation, proliferation, and survival of germ cells by elevating the expression of PCNA and declining caspase 3, cleaved caspase 3 expression, Bax/Bcl2 ratio, and TUNEL-positive cells. FACS analysis revealed that adropin treatment enhances overall turnover of testicular cells leading to rise in production of advanced germ cells, notably spermatids. Conclusion: The present study indicated that adropin improves testicular steroidogenesis, spermatogenesis via modulating redox potential and could be a promising target for treating testicular dysfunctions. [ABSTRACT FROM AUTHOR]

Male postsmolt maturation ("jacking") is undesired in Atlantic salmon aquaculture due to economic and welfare impacts. Unfortunately, incidence of jacking has increased linked to intensive rearing conditions. This study subjected 1000 salmon (52.1 ± 5.2 g) to one of two temperatures (15, 12.5°C) and one of two photoperiods (constant light‐LL, a 5‐week LD12:12 winter signal regime‐WS) to assess their effects on spermatogenesis regulation. Indicators included testis histology images, mRNA transcription of gonadotropin receptors for follicle‐stimulating hormone (fshr) and luteinizing hormone (lhr), and of factors regulating spermatogenesis like anti‐Müllerian hormone (amh), gonadal‐soma‐derived factors 1 and 2 (gsdf1 and gsdf2), and insulin‐like growth factor‐3 (igf3). High temperature (15°C) induced early testis development processes irrespective of photoperiod, evidenced by presence of type B spermatogonia before clear increases in testis size or any transcriptional changes. The winter signal at 15°C caused a synchronized onset of spermatogenesis not present under constant light. This was evidenced by a pronounced downregulation of the spermatogenesis‐inhibiting factor amh and a clear upregulation in the stimulating factor igf3. Consequently, combining high temperatures with winter signal regimes pose risk; high temperature can stimulate early cellular/endocrine processes of spermatogenesis, which can later be synchronized in the population by an increase in daylength. [ABSTRACT FROM AUTHOR]

Identifying factors required for spermatogenesis is important for understanding mechanisms of male fertility. Inactivation of either the Mgat1 or Man2a2 gene leads to a block in spermatogenesis causing infertility in male mice. MGAT1 GlcNAc-transferase initiates complex N-glycan synthesis and MAN2A2 mannosidase generates the substrate for MGAT2 GlcNAc-transferase to form a biantennary complex N-glycan. In this paper, we show that conditional deletion of Mgat2 in spermatogonia via Stra8-iCre caused a novel block in spermatogenesis, largely prior to the formation of round spermatids. Mgat2 [-/-] germ cells did not bind the lectins Phaseolus vulgaris leucoagglutinin (L-PHA) or Griffonia simplicifolia II (GSA-II), similar to germ cells lacking MGAT1 and complex N-glycans. However, overall spermatogenic defects were distinct in germ cells with deleted Mgat2 versus Mgat1. In addition, RNAseq analysis at 15 days after birth revealed a unique transcriptomic landscape in Mgat2[-/-] germ cells with genes required for sperm formation and functions being most downregulated. Bioinformatic analyses using the ingenuity pathway analysis (IPA) algorithm identified ERK and AKT as central activities. Western blot analyses of 15-day germ cell lysates confirmed that both AKT and ERK1/2 signaling were increased by loss of MGAT2 in germ cells. By contrast, Mgat1 [-/-] germ cells were previously shown to have reduced ERK signaling and unchanged AKT activity. Therefore, since the loss of all complex N-glycans is common to each mutant model, the different immature N-glycans that accumulate in Mgat2[-/-] versus Mgat1[-/-] germ cells are proposed to be the basis of their unique spermatogenic phenotypes. KEYWORDS complex N-glycans, MGAT2, conditional knockout, spermatogenesis, AKT and Identifying factors required for spermatogenesis is important for understanding mechanisms of male fertility. Inactivation of either the Mgat1 or Man2a2 gene leads to a block in spermatogenesis causing infertility in male mice. MGAT1 GlcNAc-transferase initiates complex N-glycan synthesis and MAN2A2 mannosidase generates the substrate for MGAT2 GlcNAc-transferase to form a biantennary complex N-glycan. In this paper, we show that conditional deletion of Mgat2 in spermatogonia via Stra8-iCre caused a novel block in spermatogenesis, largely prior to the formation of round spermatids. Mgat2 [-/-] germ cells did not bind the lectins Phaseolus vulgaris leucoagglutinin (L-PHA) or Griffonia simplicifolia II (GSA-II), similar to germ cells lacking MGAT1 and complex N-glycans. However, overall spermatogenic defects were distinct in germ cells with deleted Mgat2 versus Mgat1. In addition, RNAseq analysis at 15 days after birth revealed a unique transcriptomic landscape in Mgat2[-/-] germ cells with genes required for sperm formation and functions being most downregulated. Bioinformatic analyses using the ingenuity pathway analysis (IPA) algorithm identified ERK and AKT as central activities. Western blot analyses of 15-day germ cell lysates confirmed that both AKT and ERK1/2 signaling were increased by loss of MGAT2 in germ cells. By contrast, Mgat1 [-/-] germ cells were previously shown to have reduced ERK signaling and unchanged AKT activity. Therefore, since the loss of all complex N-glycans is common to each mutant model, the different immature N-glycans that accumulate in Mgat2[-/-] versus Mgat1[-/-] germ cells are proposed to be the basis of their unique spermatogenic phenotypes. [ABSTRACT FROM AUTHOR]

Arecoline hydrobromide has been demonstrated to regulate cellular survival and male reproduction. However, the specific roles and regulated genes of arecoline hydrobromide in spermatogenesis are poorly understood. This study aimed to investigate the effects of arecoline hydrobromide on spermatogenesis and associated transcriptome changes. The spermatogenic cell line GC-1 cells were treated with arecoline hydrobromide to determine the effects of proliferation and apoptosis for arecoline hydrobromide in vitro. The ICR mice were treated with arecoline hydrobromide by intragastric administration. The testes and epididymis were used for sperm count, HE staining, apoptosis detection and transcriptome analysis. Cellular morphology was significantly altered and the number of apoptotic cells increased with increasing concentration and duration of arecoline hydrobromide treatment, and cell proliferation was inhibited. Animal experiments showed that the spermatozoa in the experimental groups were significantly reduced compared with that in the control group. Transcriptome sequencing was executed for testes of four-week arecoline hydrobromide treated mice, which identified 181 significant up-regulated genes and 159 significant down-regulated genes which were then analyzed with gene ontology (GO) and revealed some testicular development and hormone-related pathways. The results verified that arecoline hydrobromide inhibited the proliferation and promoted the apoptosis of GC-1 cells, and caused a decrease in the number of spermatozoa and an increase in level of testosterone in mice. [ABSTRACT FROM AUTHOR]

Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin. [ABSTRACT FROM AUTHOR]

In this study, we have explored the role of the KATNB1 gene, a microtubule‐severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co‐localize with α‐tubulin showing a unique stage‐specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ‐barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium‐induced blood–testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin‐ and microtubule‐based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin‐ and microtubule‐based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results Discussion and conclusion HENMT1 encodes a small RNA methyltransferase that plays a crucial role in mouse spermatogenesis through the methylation of the 3′ end of PIWI‐interacting RNAs.Our study aims to elucidate the relationship between HENMT1 and male infertility in humans.A consanguineous family, having a single non‐obstructive azoospermia patient was recruited for pathogenic variants screening. The research includes genetic analysis and experimental validation using mouse models. The patient was diagnosed with non‐obstructive azoospermia. Whole‐exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of the identified variant was assessed and studied in vivo using a mouse model that mimicked the patient's mutation.Through whole‐exome sequencing, we identified a homozygous nonsense variant (c.555G > A, p.Trp185*) in HENMT1 in the patient. The presence of the mutant HENMT1 mRNA was detected in the patient's blood, and the truncated HENMT1 protein was observed in transfected HEK293T cells. The mutant mice modeling this HENMT1 variant displayed an infertile phenotype similar to that of the patient, characterized by spermiogenesis arrest. Further analysis revealed a significant derepression of retrotransposon LINE1 in the testes of the Henmt1 mutant mice, and increased apoptosis of spermatids.Our findings provide the evidence of pathogenicity of the identified HENMT1 variant, thus shedding light on the indispensable role of HENMT1 in human spermatogenesis. [ABSTRACT FROM AUTHOR]

PIWI-interacting RNAs (piRNAs) play critical and conserved roles in transposon silencing and gene regulation in the animal germline. Three distinct piRNA populations are present during mouse spermatogenesis: fetal piRNAs in fetal/perinatal testes, pre-pachytene and pachytene piRNAs in postnatal testes. PNLDC1 is required for piRNA 3' end maturation in multiple species. However, whether PNLDC1 is the bona fide piRNA trimmer and the physiological role of 3' trimming of different piRNA populations in spermatogenesis in mammals remain unclear. Here, by inactivating Pnldc1 exonuclease activity in vitro and in mice, we reveal that the PNLDC1 trimmer activity is essential for spermatogenesis and male fertility. PNLDC1 catalytic activity is required for both fetal and postnatal piRNA 3' end trimming. Despite this, postnatal piRNA trimming but not fetal piRNA trimming is critical for LINE1 transposon silencing. Furthermore, conditional inactivation of Pnldc1 in postnatal germ cells causes LINE1 transposon de-repression and spermatogenic arrest in mice, indicating that germline-specific postnatal piRNA trimming is essential for transposon silencing and germ cell development. Our findings highlight the germ cell-intrinsic role of PNLDC1 and piRNA trimming in mammals to safeguard the germline genome and promote fertility. Author summary: PNLDC1 is required for piRNA trimming and is essential for spermatogenesis and male fertility in mice. Loss-of-function mutations in human PNLDC1 cause azoospermia in men. However, the precise functions of PNLDC1 exonuclease activity in piRNA trimming and germ cell development remain unknown. Here we genetically inactivate the trimmer activity of PNLDC1 in mice and show that the catalytic activity of PNLDC1 is critical for male fertility. Strikingly, postnatal germline conditional deletion of Pnldc1 in mice that disrupt pre-pachytene and pachytene piRNA trimming causes transposon activation and male infertility. Collectively, these new genetic models provide the proof-of-principle evidence that ablating the catalytic activity of a key piRNA pathway factor in adult males as a novel strategy for non-hormonal male contraception. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that belongs to the group of gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO). H2S plays a pivotal role in male reproductive processes. It is produced in various tissues and cells of the male reproductive system, including testicular tissue, Leydig and Sertoli cells, epididymis, seminal plasma, prostate, penile tissues, and sperm cells. This review aims to summarize the knowledge about the presence and effects of H2S in male reproductive tissues and outline possible therapeutic strategies in pathological conditions related to male fertility, e. g. spermatogenetic disorders and erectile dysfunction (ED). For instance, H2S supports spermatogenesis by maintaining the integrity of the blood-testicular barrier (BTB), stimulating testosterone production, and providing cytoprotective effects. In spermatozoa, H2S modulates sperm motility, promotes sperm maturation, capacitation, and acrosome reaction, and has significant cytoprotective effects. Given its vasorelaxant effects, it supports the erection of penile tissue. These findings suggest the importance and therapeutic potential of H2S in male reproduction, paving the way for further research and potential clinical applications. [ABSTRACT FROM AUTHOR]

Modification of guanosine to N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by the METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) causes no major effect on the development of non-gonadal tissues, but abolishes the production of elongated spermatids and mature sperm, which is fully rescued by expression of a Mettl1-transgene, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO results in a loss of m7G modification on a subset of tRNAs and decreased tRNA abundance. Ribosome profiling shows that Mettl1-KO led to ribosomes stalling at codons decoded by tRNAs that were reduced in abundance. Mettl1-KO also significantly reduces the translation efficiency of genes involved in elongated spermatid formation and sperm stability. Germ cell-specific expression of Mettl1 rescues disrupted m7G tRNA modification and tRNA abundance in Mettl1-KO testes but not in non-gonadal tissues. Ribosome stalling is much less detectable in non-gonadal tissues than in Mettl1-KO testes. These findings reveal a developmental role for m7G tRNA modification and indicate that m7G modification-dependent tRNA abundance differs among tissues. Here the authors reveal essential functions of Mettl1, catalyzing N7-guanosine methylation (m7G) of tRNAs, in Drosophila male fertility through a regulation of the steady-state level of tRNAs and the translational efficiency of genes essential for spermatogenesis. [ABSTRACT FROM AUTHOR]

Germ cell development in mammals is a complex physiological process that involves the proliferation of primordial germ cells, meiosis, and the formation of male and female gametes. Long non-coding RNA (lncRNA) is a type of RNA with more than 200 nucleotides that does not code for proteins. A small number of lncRNAs have been shown to participate in spermatogenesis in the testes and in follicular development in the ovaries, but the role of the vast majority of lncRNAs and their molecular mechanisms still need further study. LncRNA Gm2044 was identified as a differentially expressed lncRNA in mouse spermatogenesis by microarray technology. In mouse testis, lncRNA Gm2044 can act as competing endogenous RNA to regulate SYCP1 expression in GC-2 cells derived from mouse spermatocyte cells, and it can also act as a host gene for miR-202 to regulate RBFOX2 protein expression. In female mouse ovaries, lncRNA Gm2044 regulates 17ß-estradiol synthesis through the miRNA-138-5p-Nr5a1 pathway or by interacting with EEF2. In addition, studies suggest that lncRNA Gm2044 is also involved in the progression of reproductive system diseases such as male nonobstructive azoospermia. Here, we summarize the roles and molecular mechanisms of lncRNA Gm2044 in male and female gametogenesis and its potential role in some infertility disorders. [ABSTRACT FROM AUTHOR]