Essential Role of COPII Proteins in Maintaining the Contractile Ring Anchoring to the Plasma Membrane during Cytokinesis in Drosophila Male Meiosis.

Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Here, we report that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. We explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila.

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions.

Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identify HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyzes and single-cell RNA-sequencing demonstrate that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggest that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that is required for the gene expression program for pachytene transition during meiotic prophase in males.

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Lycopene inhibits apoptosis of mouse spermatocytes in varicocele via miR-23a/b-induced downregulation of PROK2.

Context The varicocele is the leading cause of male infertility and can impair sperm quality and testicular function through various mechanisms. In our previous study, we found that lycopene could attenuate hypoxia-induced testicular injury. Aims To illustrate the detailed mechanism of lycopene on spermatocytes. Methods The effect of lycopene on GC-2 cells under hypoxia were detected by flow cytometry and western blot assay. miR-seq was used to determine miRNA expression in varicocele rat model testes. The function of miR-23a/b were determined by flow cytometry and western blot assay. Key results We demonstrate that lycopene could alleviate hypoxia-induced GC-2 cell apoptosis and could elevate miR-23a/b expression of the hypoxia model in vivo and in vitro . The miR-23a and -23b mimics could reduce the hypoxia-induced GC-2 cell apoptosis. Both miR-23a and -23b could directly bind with prokineticin 2 (PROK2) mRNA and downregulate its expression. Conclusions Lycopene could attenuate hypoxia-induced spermatocyte injury through the miR-23a/b-PROK2 pathway. Implications Lycopene may be an effective treatment for varicocele to improve testicular impairment.

Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining.

In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.

Tris(1,3-dichloro-2-propyl) phosphate induces endoplasmic reticulum stress and mitochondrial-dependent apoptosis in mouse spermatocyte GC-2 cells.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a frequently detected organophosphorus flame retardants (OPFRs) in various environmental media, and has been evidenced as reproductive toxicity. However, its adverse effects on spermatogenic cells are unknown. In this study, mouse spermatocyte GC-2spd (GC-2) cells were selected as an in vitro model, and the impact of mitochondrial structure and function, endoplasmic reticulum (ER) stress, cell apoptosis and the related molecular mechanisms were investigated. Our study indicated that cell viability was decreased significantly in a dose-dependent manner after TDCIPP treatment with the half lethal concentration (LC50) at 82.8 µM, 50.0 µM and 39.6 µM for 24 h, 48 h and 72 h, respectively. An apoptosis was observed by Annexin V-FITC/PI stain. In addition, fragmentation of mitochondrial structure, an increase of mitochondrial membrane potential (MMP), reduction of cellular adenosine triphosphate (ATP) content, release of cytochrome c and activation of Caspase-3 and Caspase-9 activity implicated that Caspase-3 dependent mitochondrial pathway might play a key role in the process of GC-2 cell apoptosis. Furthermore, ER stress induction was convinced by altered morphology of ER and up-regulation of ER targeting genes, including (Bip, eIF2α, ATF4, XBP1, CHOP, ATF6 and Caspase-12). Taken together, these results demonstrate that both mitochondrial apoptotic pathways and ER stress apoptotic pathways might play important roles in the process of apoptosis in GC-2 cells induced by TDCIPP treatment. Therefore, the potential reproductive toxicity of TDCIPP should not be ignored.

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.

ARF6, a component of intercellular bridges, is essential for spermatogenesis in mice.

Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.

Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20 mg/kg·bw) once every 5 days via intratracheal instillation for 35 days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20 µg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15 days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

Balanced spatiotemporal arrangements of histone H3 and H4 posttranslational modifications are necessary for meiotic prophase I chromosome organization.

Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate (heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.

The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo.

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

Exploring testicular miRNA profiles during developmental stages in Dezhou donkeys: A preliminary insight.

Testicular development and spermatogenesis are complex phenomena controlled by various genetic factors, including miRNA-based post-transcriptional gene expression regulation. Exploring the miRNA expression patterns during testicular development in Dezhou donkeys would enhance our understanding of equine fertility and spermatogenesis. In this investigation, we examined the testicular miRNA profiles at various stages of development. The experimental animals were divided into three groups based on their developmental stages: 2 months old (juvenile: n = 3), 12 months old (adolescent; n = 3) and 24 months old (adult; n = 3) donkeys. Total RNA was extracted from dissected testicles for miRNA sequencing and analysis. In total, 586 miRNAs, including 451 known miRNAs and 135 novel miRNAs, were identified. Among identified miRNAs, 315 displayed age-dependent expression differences. The levels of miRNA expression in the juvenile group were significantly higher than in the adolescent or adult groups. The MiR-483 exhibited the maximum fold change between juvenile and adolescent groups. Several screened genes, including SLC45A4 and TFCP2L1, have been linked to male reproductive pathways in donkeys. In addition, miR-744 was predicted to regulate SPIN2B, a gene implicated in spermatocyte cell cycle progression and genomic integrity of spermatozoa. These results contribute to our comprehension of microRNA regulation during testicular development and spermatogenesis in Dezhou donkeys. The identified microRNAs and their target genes have the potential to serve as biomarkers for evaluating the reproductive capacity of stud donkeys.

TAF7L regulates early stages of male germ cell development in the rat.

Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at late zygotene/early pachynema stages, with defects in sex body formation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Perturbation of IP3R-dependent endoplasmic reticulum calcium homeostasis by PPARδ-activated metabolic stress leads to mouse spermatocyte apoptosis: A direct mechanism for perfluorooctane sulfonic acid-induced spermatogenic disorders.

Perfluorooctane sulfonic acid (PFOS) as an archetypal representative of per- and polyfluoroalkyl substances (PFAS) is ubiquitously distributed in the environment and extensively detected in human bodies. Although accumulating evidence is suggestive of the deleterious effects of PFOS on male reproduction, the direct toxicity of PFOS towards spermatogenic cells and the relevant mechanisms remain poorly understood. The aims of the present study were to explore the direct effects and underlying molecular mechanisms of PFOS on spermatogenesis. Through integrating animal study, transcriptome profiling, in silico toxicological approaches, and in vitro validation study, we identified the molecular initiating event and key events contributing to PFOS-induced spermatogenic impairments. The mouse experiments revealed that spermatocytes were involved in PFOS-induced spermatogenic disorders and the activation of peroxisome proliferator-activated receptor delta (PPARδ) was linked to spermatocyte loss in PFOS-administrated mice. GC-2spd(ts) cells were treated with an increased gradient of PFOS, which was relevant to environmental and occupational exposure levels of PFOS in populations. Following 72-h treatment, cells was harvested for RNA sequencing. The transcriptome profiling and benchmark dose (BMD) modeling identified endoplasmic reticulum (ER) stress as the key event for PFOS-mediated spermatocyte apoptosis and determined the point-of-departure (PoD) for perturbations of ER stress signaling. Based on the calculated PoD value, further bioinformatics analyses combined with in vitro and in vivo validations showed that PFOS caused metabolic stress by activating PPARδ in mouse spermatocytes, which was responsible for Beclin 1-involved inositol 1,4,5-trisphosphate receptor (IP3R) sensitization. The disruption of IP3R-mediated ER calcium homeostasis triggered ER calcium depletion, leading to ER stress and apoptosis in mouse spermatocytes exposed to PFOS. This study systematically investigated the direct impacts of PFOS on spermatogenesis and unveiled the relevant molecular mechanism of PFOS-induced spermatogenic disorders, providing novel insights and potential preventive/therapeutic targets for PFAS-associated male reproductive toxicity.

PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene.

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities.

Gss deficiency causes age-related fertility impairment via ROS-triggered ferroptosis in the testes of mice.

Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss-/- male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss-/- mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.

Chromosome-specific maturation of the epigenome in the Drosophila male germline.

Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.