Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility.

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.

C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line.

In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.

Sperm bauplan and function and underlying processes of sperm formation and selection.

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.

DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.

Spermatogonial stem cell (SSC) self-renewal and differentiation provide foundational support for long-term, steady-state spermatogenesis in mammals. Here, we have investigated the essential role of RNA exosome associated DIS3 ribonuclease in maintaining spermatogonial homeostasis and facilitating germ cell differentiation. We have established male germ-cell Dis3 conditional knockout (cKO) mice in which the first and subsequent waves of spermatogenesis are disrupted. This leads to a Sertoli cell-only phenotype and sterility in adult male mice. Bulk RNA-seq documents that Dis3 deficiency partially abolishes RNA degradation and causes significant increases in the abundance of transcripts. This also includes pervasively transcribed PROMoter uPstream Transcripts (PROMPTs), which accumulate robustly in Dis3 cKO testes. In addition, scRNA-seq analysis indicates that Dis3 deficiency in spermatogonia significantly disrupts RNA metabolism and gene expression, and impairs early germline cell development. Overall, we document that exosome-associated DIS3 ribonuclease plays crucial roles in maintaining early male germ cell lineage in mice.

Development of functional spermatozoa in mammalian spermiogenesis.

Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models.

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

CAMSAP1 role in orchestrating structure and dynamics of manchette microtubule minus-ends impacts male fertility during spermiogenesis.

The manchette is a crucial transient structure involved in sperm development, with its composition and regulation still not fully understood. This study focused on investigating the roles of CAMSAP1 and CAMSAP2, microtubule (MT) minus-end binding proteins, in regulating manchette MTs, spermiogenesis, and male fertility. The loss of CAMSAP1, but not CAMSAP2, disrupts the well-orchestrated process of spermiogenesis, leading to abnormal manchette elongation and delayed removal, resulting in deformed sperm nuclei and tails resembling oligoasthenozoospermia symptoms. We investigated the underlying molecular mechanisms by purifying manchette assemblies and comparing them through proteomic analysis, and results showed that the absence of CAMSAP1 disrupted the proper localization of key proteins (CEP170 and KIF2A) at the manchette minus end, compromising its structural integrity and hindering MT depolymerization. These findings highlight the significance of maintaining homeostasis in manchette MT minus-ends for shaping manchette morphology during late spermiogenesis, offering insights into the molecular mechanisms underlying infertility and sperm abnormalities.

TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis.

Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. We observed similar premeiotic mitochondrial clusters in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, we identified that Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. We suggest that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.

Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ.

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

Therapeutic potential of exosomes in spermatogenesis regulation and male infertility.

BACKGROUND: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication. OBJECTIVE: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell-to-cell communication. METHODS: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis. RESULTS: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells. CONCLUSION: The emerging understanding of exosome-mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice.

Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12-/-) mice. These Zmynd12-/- mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12-/- mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.

Prolonged exposure to dexamethasone alters the proteome and cellular phenotype of human testicular peritubular cells.

Human testicular peritubular cells (HTPCs) are smooth muscle cells, which in the testis form a small compartment surrounding the seminiferous tubules. Contractions of HTPCs are responsible for sperm transport, HTPCs contribute to spermatogenesis, have immunological roles and are a site of glucocorticoid receptor expression. Importantly, HTPCs maintain their characteristics in vitro, and thus can serve as an experimental window into the male gonad. Previously we reported consequences of 3-day treatment with Dexamethasone (Dex), a synthetic glucocorticoid and multi-purpose anti-inflammatory drug. However, as glucocorticoid therapies in man often last longer, we now studied consequences of a prolonged 7-day exposure to 1 µM Dex. Combining live cell imaging with quantative proteomics of samples taken from men, we confirmed our recent findings but more importantly, found numerous novel proteomic alterations induced by prolonged Dex treatment. The comparison of the 7-day treatment with the 3-day treatment dataset revealed that extracellular matrix- and focal adhesion-related proteins become more prominent after 7 days of treatment. In contrast, extended stimulation is, for example, associated with a decrease of proteins related to cholesterol and steroid metabolism. Our dataset, which describes phenotypic and proteomic alterations, is a valuable resource for further research projects investigating effects of Dex on human testicular cells.

The elusive male microbiome: revealing the link between the genital microbiota and fertility. Critical review and future perspectives.

There is a growing focus on understanding the role of the male microbiome in fertility issues. Although research on the bacterial communities within the male reproductive system is in its initial phases, recent discoveries highlight notable variations in the microbiome's composition and abundance across distinct anatomical regions like the skin, foreskin, urethra, and coronary sulcus. To assess the relationship between male genitourinary microbiome and reproduction, we queried various databases, including MEDLINE (available via PubMed), SCOPUS, and Web of Science to obtain evidence-based data. The literature search was conducted using the following terms "gut/intestines microbiome," "genitourinary system microbiome," "microbiome and female/male infertility," "external genital tract microbiome," "internal genital tract microbiome," and "semen microbiome." Fifty-one relevant papers were analyzed, and eleven were strictly semen quality or male fertility related. The male microbiome, especially in the accessory glands like the prostate, seminal vesicles, and bulbourethral glands, has garnered significant interest because of its potential link to male fertility and reproduction. Studies have also found differences in bacterial diversity present in the testicular tissue of normozoospermic men compared to azoospermic suggesting a possible role of bacterial dysbiosis and reproduction. Correlation between the bacterial taxa in the genital microbiota of sexual partners has also been found, and sexual activity can influence the composition of the urogenital microbiota. Exploring the microbial world within the male reproductive system and its influence on fertility opens doors to developing ways to prevent, diagnose, and treat infertility. The present work emphasizes the importance of using consistent methods, conducting long-term studies, and deepening our understanding of how the reproductive tract microbiome works. This helps make research comparable, pinpoint potential interventions, and smoothly apply microbiome insights to real-world clinical practices.

Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells.

BACKGROUND: Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution. METHODS: Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas. RESULTS: Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area. CONCLUSIONS: This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.

Transcription factor-like 5 is a potential DNA- and RNA-binding protein essential for maintaining male fertility in mice.

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion as the heterozygous males (Tcfl5+/-) were infertile. However, we did observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RNA-binding protein, FXR1, as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis. This article has an associated First Person interview with the first author of the paper.

Kpna6 deficiency causes infertility in male mice by disrupting spermatogenesis.

Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The karyopherin α family of proteins mediates movement of specific cargo proteins when bound to karyopherin ß. Karyopherin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here, we use a loss-of-function approach to determine specifically the role of Kpna6 in spermatogenesis and male fertility. We show that ablation of Kpna6 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Kpna6-deficient mice exhibit impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear localization of the androgen receptor. Furthermore, our data demonstrate devastating defects on spermiogenesis, including incomplete sperm maturation and a massive reduction in sperm number, accompanied by disturbed histone-protamine exchange, differential localization of the transcriptional regulator BRWD1 and altered expression of RFX2 target genes. Our work uncovers an essential role of Kpna6 in spermatogenesis and, hence, in male fertility.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Male infertility and Perfluoroalkyl and poly-fluoroalkyl substances: Evidence for alterations in phosphorylation of proteins and fertility-related functional attributes in bull spermatozoa.

BACKGROUND: Perfluoroalkyl and poly-fluoroalkyl substances (PFAS) are pervasive environmental pollutants and emerging risk factors for reproductive health. Although epidemiological evidence supports the link between these substances and male infertility, their specific effects on male fertility remain poorly understood. OBJECTIVES: Investigate the effect of perfluorooctane sulfonic acid (PFOS), the most prevalent and prominent PFAS, on bull sperm protein phosphorylation, a post-translational modification process governing sperm functionality and fertility. METHODS: We exposed bull sperm to PFOS at 10 µM (average population level) and 100 µM (high-exposure level), and analyzed global proteome and phosphoproteome profile by TMT labeling and NanoLC-MS/MS. We also measured sperm fertility functions by flow cytometry. RESULTS: PFOS at 10 µM altered sperm proteins linked to spermatogenesis and chromatin condensation, while at 100 µM, PFOS affected proteins associated with motility and fertility. We detected 299 phosphopeptides from 116 proteins, with 45 exhibiting differential expression between control and PFOS groups. PFOS dysregulated phosphorylation of key proteins (ACRBP, PRKAR2A, RAB2B, SPAG8, TUBB4B, ZPBP, and C2CD6) involved in sperm capacitation, acrosome reaction, sperm-egg interaction, and fertilization. PFOS also affected phosphorylation of other proteins (AQP7, HSBP9, IL4I1, PRKAR1A, and CCT8L2) related to sperm stress resistance and cryotolerance. Notably, 4 proteins (PRM1, ACRBP, TSSK1B, and CFAP45) exhibited differential regulation at both the proteomic and phosphoproteomic levels. Flow cytometric analysis confirmed that PFOS increased protein phosphorylation in sperm as well as reduced sperm motility, viability, calcium, and membrane potential and increased mitochondrial ROS in a dose-dependent manner. CONCLUSIONS: This study shows that PFOS exposure adversely impacts phosphorylation of proteins critical for bull sperm function and fertilization. Moreover, the concentration of PFOS influences the severity of these effects. The comprehensive bull sperm phosphoproteomics data from this study can help us understand the molecular mechanisms of environmental exposure-related male infertility.