Mitochondrial defects triggered by amg-1 mutation elicit UPRmt and phagocytic clearance during spermatogenesis in C. elegans.

Mitochondria are the powerhouses of many biological processes. During spermatogenesis, post-transcriptional regulation of mitochondrial gene expression is mediated by nuclear-encoded mitochondrial RNA-binding proteins (mtRBPs). We identified AMG-1 as an mtRBP required for reproductive success in Caenorhabditis elegans. amg-1 mutation led to defects in mitochondrial structure and sperm budding, resulting in mitochondria being discarded into residual bodies, which ultimately delayed spermatogenesis in the proximal gonad. In addition, mitochondrial defects triggered the gonadal mitochondrial unfolded protein response and phagocytic clearance to ensure spermatogenesis but ultimately failed to rescue hermaphroditic fertility. These findings reveal a previously undiscovered role for AMG-1 in regulating C. elegans spermatogenesis, in which mitochondrial-damaged sperm prevented the transmission of defective mitochondria to mature sperm by budding and phagocytic clearance, a process which may also exist in the reproductive systems of higher organisms.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

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.

Discovery of a novel miRNA involved in the regulation of male infertility in zebrafish.

Azoospermia and asthenospermia are common manifestations of male infertility, but it needs further studies to understand the intrinsic regulation mechanism. As a popular model organism, zebrafish is often used to assess reproductive complications. In this study, by analyzing miRNA transcriptome of the mature triploid zebrafish testis afflicted with spermatogenic dysfunctions, leading to the identification of 36 miRNAs that are differentially expressed in comparison with diploid, which are predicted to target 2737 genes. Subsequent functional annotation of these genes pinpointed two miRNAs might association with spermatogenesis. Inhibitory experiments showed that NC_007115.7.7_998413 inhibited conducts a substantial decline in sperm density, and conducted lower embryo fertilization rate than control. And putative target genes qRT-PCR evaluation showed that spata2 was significant down-regulate upon inhibited NC_007115.7.7_998413. In summary, this research positions newly identified miRNA NC_007115.7.998413 as a regulatory factor in male zebrafish reproductive development, enhancing our comprehension of the molecular regulated pathways involved in spermatogenesis.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

Site-specific N-glycoproteomic analysis reveals up-regulated fucosylation in seminal plasma of asthenozoospermia.

N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N-glycopeptides in seminal plasma, we identified 92 intact N-glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility.

LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA.

BACKGROUND: The unmet challenge in prostate cancer (PCa) management is to discriminate it from benign prostate hyperplasia (BPH) due to the lack of specific diagnostic biomarkers. Contemporary research on potential PCa biomarkers is directed toward methylated cell-free DNA (cfDNA) from liquid biopsies since epigenetic mechanisms are strongly involved in PCa development. METHODS: In the present research, cfDNA methylation of the LGALS3 gene in blood and seminal plasma of PCa and BPH patients was assessed using pyrosequencing, as well as LGALS3 DNA methylation in tissue biopsies. Liquid biopsy samples were taken from patients with clinical suspicion of PCa, who were subsequently divided into two groups, that is, 42 with PCa and 55 with BPH, according to the histopathological analysis. RESULTS: Statistically significant higher cfDNA methylation of LGALS3 in seminal plasma of BPH than in PCa patients was detected by pyrosequencing. ROC curve analysis showed that it could distinguish PCa and BPH patients with 56.4% sensitivity and 70.4% specificity, while PSA did not differ between the two patient groups. In contrast, there was no statistically significant difference in LGALS3 cfDNA methylation in blood plasma between the two patient groups. In prostate tumor tissue, there was a statistically significant DNA hypermethylation of LGALS3 compared to surrounding nontumor tissue and BPH tissue. CONCLUSIONS: The DNA hypermethylation of the LGALS3 gene represents an event specific to PCa development. In conclusion, LGALS3 cfDNA methylation in seminal fluid discriminates early PCa and BPH presenting itself as a powerful novel PCa biomarker highly outperforming PSA.

Purification, identification and Cryo-EM structure of prostatic acid phosphatase in human semen.

Prostatic acid phosphatase (PAP) is a glycoprotein that plays a crucial role in the hydrolysis of phosphate ester present in prostatic exudates. It is a well-established indicator for prostate cancer due to its elevated serum levels in disease progression. Despite its abundance in semen, PAP's influence on male fertility has not been extensively studied. In our study, we report a significantly optimized method for purifying human endogenous PAP, achieving remarkably high efficiency and active protein recovery rate. This achievement allowed us to better analyze and understand the PAP protein. We determined the cryo-electron microscopic (Cryo-EM) structure of prostatic acid phosphatase in its physiological state for the first time. Our structural and gel filtration analysis confirmed the formation of a tight homodimer structure of human PAP. This functional homodimer displayed an elongated conformation in the cryo-EM structure compared to the previously reported crystal structure. Additionally, there was a notable 5-degree rotation in the angle between the α domain and α/ß domain of each monomer. Through structural analysis, we revealed three potential glycosylation sites: Asn94, Asn220, and Asn333. These sites contained varying numbers and forms of glycosyl units, suggesting sugar moieties influence PAP function. Furthermore, we found that the active sites of PAP, His44 and Asp290, are located between the two protein domains. Overall, our study not only provide an optimized approach for PAP purification, but also offer crucial insights into its structural characteristics. These findings lay the groundwork for further investigations into the physiological function and potential therapeutic applications of this important protein.

RpS25 is required for sperm elongation and individualization during Drosophila spermatogenesis.

Ribosomal protein 25 (RPS25) has been related to male fertility diseases in humans. However, the role of RPS25 in spermatogenesis has yet to be well understood. RpS25 is evolutionarily highly conserved from flies to humans through sequence alignment and phylogenetic tree construction. In this study, we found that RpS25 plays a critical role in Drosophila spermatogenesis and its knockdown leads to male sterility. Examination of each stage of spermatogenesis from RpS25-knockdown flies showed that RpS25 was not required for initial germline cell divisions, but was required for spermatid elongation and individualization. In RpS25-knockdown testes, the average length of cyst elongation was shortened, the spermatid nuclei bundling was disrupted, and the assembly of individualization complex from actin cones failed, resulting in the failure of mature sperm production. Our data revealed an essential role of RpS25 during Drosophila spermatogenesis through regulating spermatid elongation and individualization.

Abundance of selected genes implicated in testicular functions in Camelus dromedarius with high and low epididymal semen quality.

Studying testicular genes' expression may give key insights into precise regulation of its functions that influence epididymal sperm quality. The current study aimed to investigate the abundance of candidate genes involved in the regulation of testicular functions specially those regulate sperm function (PLA2G4D, SPP1, and CLUAP1), testicular steroidogenic function (ESR1 and AR), materials transport (AQP12B and LCN15), and defense mechanisms (DEFB110, GPX5, SOCS3, and IL6). Therefore, blood samples and testes with epididymis were collected from mature middle-aged (5-10 years) dromedary camels (n = 45) directly prior and after their slaughtering, respectively, during breeding season. Sera were evaluated for testosterone level and testicular biometry was measured with caliper. The epididymal tail semen was evaluated manually. Samples were distinguished based on testosterone level, testicular biometry, as well as epididymal semen features into high and low fertile groups. Total RNA was isolated from testicular tissues and gene expression was done using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Results revealed that testosterone levels were significantly (P < 0.005) higher in camels with good semen quality than those of low quality. There was a significant (P < 0.0001) increase in testicular weight, length, width, thickness, and volume in high fertile than low fertile camels. PLA2G4D, SPP1, CLUAP1, ESR1, AR, AQP12B, LCN15, DEFB110, GPX5, and SOCS3 genes were upregulated (P < 0.001), and IL6 gene was downregulated (P < 0.01) in the testes of high fertile camels compared to the low fertile one. Thus, it could be concluded that examined genes might be valuable monitors of testicular functional status and fertility in dromedary camels.

LRRC52 is likely a functional component of human KSper†.

Completion of fertilization is orchestrated by various ion channels in sperm membrane. Hyperpolarization of membrane potential, an indispensable event during the capacitation process, is dominated by sperm potassium channel (KSper). In addition to sperm-specific SLO3, which forms the channel pore, the auxiliary subunit leucine-rich-repeat-containing protein 52 (LRRC52) is required to form mKSper to function under physiological conditions. However, in human sperm, although most evidence supports that hSLO3 is the pore-forming subunit, whether hLRRC52 contributes to hKSper conductance and modulates sperm function remains to be understood. Here, using an extracellular segment that is homologous between mice and humans as an antigen, we developed a polyclonal antibody designed as LID1 that specifically detected mLRRC52 and performed co-immunoprecipitation with mSLO3. Additionally, patch-clamp recordings of mouse sperm showed that, physiological activation of mKSper and sperm functions were dramatically attenuated after treatment with LID1, indicating that LID1 functionally disrupted the regulation of mLRRC52 on mKSper. Next, LID1 was used to investigate the significance of hLRRC52 for hKSper activation. As a result, hLRRC52 was expressed in human sperm and might be assembled with hSLO3. More importantly, LID1 inhibited hKSper currents and depolarized sperm membrane potential, supporting essential modulation of hLRRC52 in hKSper. Ca2+ signaling of human sperm was also compromised in the presence of LID1, which impaired sperm motility and acrosome reaction. Because LID1 specifically inhibited both mKSper and hKSper but not mCatSper or hCatSper, our results suggest that hLRRC52 functions as an important component of hKSper and regulates sperm physiological functions.

AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

In vivo effect of vaginal seminal plasma application on the human endometrial transcriptome: a randomized controlled trial.

Studies in humans and animals suggest that seminal plasma, the acellular seminal fluid component, stimulates the endometrium to promote immune tolerance and facilitate implantation. We designed a randomized, double-blinded, placebo-controlled trial to investigate changes in the endometrial transcriptomic profile after vaginal application of seminal plasma. The study participants were randomized into two groups. Five women received a vaginal application of seminal plasma, and four received a placebo application with saline solution. The application was performed 2 days after HCG-triggered ovulation in an unstimulated cycle. After 5-8 days, an endometrial biopsy was collected to analyze differences in the endometrial transcriptomic profile using microarray analyses. A differential gene expression analysis and a gene set analysis were performed. The gene set enrichment analysis showed a positive enrichment of pathways associated with the immune response, cell viability, proliferation, and cellular movement. Moreover, pathways involved in implantation, embryo development, oocyte maturation, and angiogenesis were positively enriched. The differential gene expression analysis, after adjusting for multiple testing, showed no significantly differentially expressed genes between the two groups. A comparative analysis was also performed with similar studies conducted in other animals or in vitro using human endometrial cells. The comparative analysis showed that the effect of seminal plasma effect on the endometrium is similar in pigs, mice, and in vitro human endometrial cells. The present study provides evidence that seminal plasma might impact the endometrium during the implantation window, with potential to affect endometrial receptivity and embryo development.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient's exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Lipidomics random forest algorithm of seminal plasma is a promising method for enhancing the diagnosis of necrozoospermia.

BACKGROUND: Despite the clear clinical diagnostic criteria for necrozoospermia in andrology, the fundamental mechanisms underlying it remain elusive. This study aims to profile the lipid composition in seminal plasma systematically and to ascertain the potential of lipid biomarkers in the accurate diagnosis of necrozoospermia. It also evaluates the efficacy of a lipidomics-based random forest algorithm model in identifying necrozoospermia. METHODS: Seminal plasma samples were collected from patients diagnosed with necrozoospermia (n = 28) and normozoospermia (n = 28). Liquid chromatography-mass spectrometry (LC-MS) was used to perform lipidomic analysis and identify the underlying biomarkers. A lipid functional enrichment analysis was conducted using the LION lipid ontology database. The top 100 differentially significant lipids were subjected to lipid biomarker examination through random forest machine learning model. RESULTS: Lipidomic analysis identified 46 lipid classes comprising 1267 lipid metabolites in seminal plasma. The top five enriched lipid functions as follows: fatty acid (FA) with ≤ 18 carbons, FA with 16-18 carbons, monounsaturated FA, FA with 18 carbons, and FA with 16 carbons. The top 100 differentially significant lipids were subjected to machine learning analysis and identified 20 feature lipids. The random forest model identified lipids with an area under the curve > 0.8, including LPE(20:4) and TG(4:0_14:1_16:0). CONCLUSIONS: LPE(20:4) and TG(4:0_14:1_16:0), were identified as differential lipids for necrozoospermia. Seminal plasma lipidomic analysis could provide valuable biochemical information for the diagnosis of necrozoospermia, and its combination with conventional sperm analysis may improve the accuracy and reliability of the diagnosis.