Epidemiological studies have linked ambient ozone (O₃) and residential greenness to semen quality, but their interaction has rarely been explored. We conducted a nationwide cross-sectional analysis of 26,997 semen donors from 33 Chinese provinces (from 2019 to 2022), with retrospective window-specific exposure estimates across the spermatogenic cycle. O₃ and normalized difference vegetation index (NDVI) exposures were estimated using inverse distance weighting (IDW) across the full spermatogenic cycle (0-90 days) and key developmental windows. Each interquartile range (IQR, 34.46 μg/m³) increase in O₃ was associated with higher odds ratios (OR, 95 % CI) of abnormal semen volume (1.592, 1.409-1.799), progressive motility (1.478, 1.344-1.626), sperm concentration (1.568, 1.402-1.755), and total sperm count (1.716, 1.547-1.903). Consistent patterns were observed in continuous outcome models, with semen volume (β= -0.004), progressive motility (β= -0.002), sperm concentration (β= -0.005), and total sperm count (β= -0.009). In the combined exposure model, participants with high O₃ and low NDVI exposure had the highest risk of abnormal semen quality compared with those with low O₃ and high NDVI exposure. Additive interaction analyses based on binary outcomes suggested an antagonistic interaction pattern between O₃ and NDVI for progressive motility and total sperm count, with consistently negative estimates on the additive scale. By incorporating exposures at different developmental stages, we found that O₃ and NDVI exposures during the sperm development period (70-90 days before semen collection) showed the strongest associations with abnormal semen quality. Overall, O₃ exposure was associated with impaired semen quality, while higher residential greenness appeared to attenuate these associations on the additive scale and during sensitive exposure windows.

Primate-specific genes (PSGs), important contributors to the origin of adaptive evolutionary novelties, are abundantly expressed in the testis. However, the specific roles of PSGs in the male reproductive system of humans and other primates are largely unknown. Here, we employed whole-exome sequencing and identified deleterious variants of TFDP3, an X-linked PSG, in eight infertile men with oligoasthenoteratozoospermia. All of the male subjects harboring TFDP3 variants presented dramatic reductions in sperm concentration, motility, and abnormal sperm morphology. Furthermore, Tfdp3-knockdown (KD) in the testes of cynomolgus monkeys confirmed the important role of TFDP3 in normal spermatogenesis in primates. Consistently, dramatic decreases in sperm concentration, motility, and abnormal sperm morphology were also observed in Tfdp3-KD male cynomolgus monkeys. More importantly, further functional studies revealed that TFDP3 deficiency activated E2F1 induced apoptosis and thus led to decreased sperm count and motility. In addition, five of the eight couples underwent intra-cytoplasmic sperm injection treatment and achieved a successful pregnancy, indicating a potentially good outcome of assisted reproduction for those with TFDP3 deficiency-mediated oligoasthenoteratozoospermia. Collectively, our genetic analyses and experimental observations in humans and cynomolgus monkeys highlight the crucial role of TFDP3, an inhibitor of apoptosis, in normal spermatogenesis. These findings expand the spectrum of pathogenic variants for oligoasthenoteratozoospermia-associated male infertility and also reveal the special significance of primate-specific TFDP3 for the human male reproductive system, thus providing important guidance for genetic counseling and the clinical diagnosis of male infertility.

Our recent large-scale population study demonstrated that several per- and polyfluoroalkyl substances (PFAS) are potential risk factors for sperm quality-with perfluorohexane sulfonate (PFHxS) and 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) emerging as the strongest effect contributors to asthenospermia, yet their causal relationships remain unclear. Here, we provide in vivo experimental evidence and evaluation of the combined effects of 6:2 Cl-PFESA and PFHxS under human-relevant exposure. Male ICR mice were orally exposed to each compound alone or in combination for 35 days, covering one full spermatogenic cycle. Both single and combined exposures were associated with alterations of the male reproductive axis, including reduced serum testosterone and elevations in the gonadotropins luteinising hormone (LH) and follicle-stimulating hormone (FSH), and in sex hormone-binding globulin (SHBG). Testicular injury included disturbed seminiferous architecture, decreased sperm motility, and increased germ-cell apoptosis. Transcriptomics highlighted pathways related to cell adhesion, junctional integrity, and apoptosis, and Western blotting and TUNEL confirmed blood-testis barrier (BTB) protein disruption and increased germ-cell apoptosis. Toxic potency differed by endpoint, with endpoint-specific patterns across endocrine, BTB, and motility outcomes. Combined-effect analysis identified a positive non-additive interaction for LH, whereas most BTB-associated proteins showed no statistically significant interaction despite endpoint-specific deviations from the concentration-addition expectation. Collectively, these findings indicate that human-relevant co-exposure to 6:2 Cl-PFESA and PFHxS is associated with asthenospermia-related sperm and testicular alterations. The converging phenotypic, molecular, and functional findings support BTB impairment and germ-cell apoptosis as plausible contributing processes, yet the precise mechanisms still need further investigation.

Mutations in the Cfap251 gene have been identified as causative for morphology and motility abnormalities in spermatozoa of infertile males, manifesting as multiple morphological abnormalities of the sperm flagella (MMAF). However, the mechanism underlying CFAP251-associated MMAF remains poorly understood, and the role of CFAP251 deficiency in PCD remains unclear. This study aimed to elucidate the pathogenic mechanism linking CFAP251 deficiency to human male infertility and to determine whether CFAP251 plays an active role in both flagella and cilia across species. A Cfap251 knockout mouse line was generated on a C57Bl/6J strain. CFAP251 mutations were identified by whole-exome sequencing on probands from Han Chinese families with primary infertility and MMAF. The Cfap251 knockout mouse model replicated the MMAF and male infertility phenotypes observed in humans for the first time. Moreover, the Cfap251 knockout mouse and one patient showed PCD-like symptoms, including tinnitus, seasonal cough, and radiological findings of coarsened lung markings and solitary pulmonary bulla associated with chronic bronchitic inflammation. Mechanistically, CFAP251 was confirmed to interact with TUBB4B, and its disruption led to the downregulation of TUBB4B, impairing spermiogenesis and ciliary function. Furthermore, CFAP251 was found to interact with the mitochondrial protein SLC25A4, suggesting a role in regulating energy transport. Favorable outcomes following ICSI were observed, with the successful birth of healthy offspring in four patients caused by CFAP251 variants. These included two cases with two novel homozygous splice-altering variants (c.1535+1G>C and c.1269+2T>C) and two previously reported MMAF cases. Our findings highlight the underlying risk of male infertility and PCD-like ciliary defects associated with CFAP251 and provide a valuable reference for personalized genetic counselling and clinical treatment of affected individuals.

Post-transcriptional regulation is pivotal for cellular differentiation, yet how translationally silent mRNAs are selectively reactivated remains elusive. Here, we identify the MEX3D-HIP1 (MX-H) pathway and its associated organelle, the MEX3D-associated lysosomal vesicle (MXLV), as a shared system governing mRNA activation during spermiogenesis. Our data support a model in which MEX3D acts as an RNA-associated E3 ligase that selectively promotes ubiquitination of RBPs within RBP-mRNA complexes. This ubiquitination signal recruits HIP1, triggering the formation of MXLV, an autophagic vesicle that degrades translationally silent mRNP complexes. Genetic ablation of MX-H components in male germ cells disrupts spermiogenesis, leading to the accumulation of mRNP aggregates and male infertility. Intriguingly, we discovered that this germline-restricted pathway is aberrantly activated in gastric cancer cells, where MXLV biogenesis promotes tumor progression. The strict restriction of MXLV to male germ cells under physiological conditions may provide a unique therapeutic window, suggesting that targeting this pathway could suppress tumor progression while minimizing adverse effects on normal physiological functions. Our work establishes MXLV as a specialized vesicular structure essential for cellular remodeling during development and reveals how a germline-specific membrane trafficking system is co-opted in pathological proteome remodeling in gastric cancer.

RESEARCH QUESTION:Given the association between abnormal PIWI-interacting RNA (piRNA) biogenesis and human male infertility, are there novel variants of genes involved in the piRNA pathway responsible for human azoospermia or oligozoospermia? DESIGN:Whole-exome sequencing and Sanger sequencing were performed to identify and validate potential deleterious variants associated with spermatogenesis in a cohort of 592 idiopathic infertile men. The impact of variants on protein expression was validated using in-vitro experiments. The functional effects of the candidate gene variants were evaluated by haematoxylin and eosin staining, immunofluorescence and small RNA sequencing. Intracytoplasmic sperm injection (ICSI) was performed in the EXD1-affected individual. RESULTS:Homozygous variants in three genes crucial for piRNA biogenesis were identified in infertile men from four unrelated families. Notably, a homozygous missense variant (c.3587T>C, p.L1196P) and a homozygous frameshift variant (c.179_186del, p.Q61Gfs*22) in TDRD9 were identified in two patients. The patient carrying the frameshift variant in TDRD9 exhibited incomplete arrest of spermatogenesis and partial meiotic defects. In addition, a homozygous HENMT1 frameshift variant (c.47_75del, p.F16Sfs*3) was identified in one patient whose testicular tissue showed reduced abundance of piRNA. These three patients were diagnosed with non-obstructive azoospermia. Finally, the fourth patient harboured the first reported loss-of-function variant of EXD1 (c.550C>T, p.R184*), and exhibited oligoasthenoteratozoospermia characterized by acrosomal hypoplasia; ICSI treatment in this case resulted in a live birth, suggesting its potential utility for further investigation in larger cohorts. CONCLUSIONS:Novel homozygous variants were identified in TDRD9, HENMT1 and EXD1, broadening the piRNA-related genetic spectrum in male infertility. EXD1 stop-gain variant was linked to male infertility for the first time, with ICSI showing potential efficacy. This provides a theoretical basis for genetic counselling and potential clinical treatment of these patients.

OBJECTIVES:Acetylated tubulin is a hallmark of flagellar stability in spermatozoa, and studies have demonstrated the ability of CDYL to function as a tubulin acetyltransferase in spermatozoa. Of note, germline conditional knockout of Cdyl can lead to asthenoteratozoospermia and infertility in male mice. However, the role of CDYL gene in human fertility remains uncharacterized. MATERIALS AND METHODS:Data were collected through in silico analysis for an infertile man with asthenoteratozoospermia of Han Chinese descent by performing whole-exome sequencing. Light and electron microscopy were used to characterize the sperm cells of the proband, and the pathogenicity of the genetic factors was determined by functional experiments. To overcome fertility problems, intracytoplasmic sperm injections were performed in the couple. MAIN RESULTS:Here, we recruited an infertile proband, born to first-cousin parents, displaying idiopathic asthenoteratozoospermia. Whole-exome sequencing identified a splicing mutation (c.103+1G>A) in CDYL, recessively cosegregating in the family. In vitro minigene assays demonstrated that the mutation resulted in aberrant alternative splicing. We found that CDYL co-localizes with Ac-tubulin along the flagella of human spermatozoa. In addition, the expression of Ac-tubulin was severely reduced in spermatozoa from the patient with CDYL mutation. Disruption in CDYL results in thin mid-piece related abnormal flagella morphology and decreased sperm motility. The primary manifestation of sperm ultrastructural abnormalities under the electron microscope is primarily characterized by disorder of axonemal protein complex and anulus. DISCUSSION AND CONCLUSION:We demonstrated that a homozygous CDYL splicing mutation specifically induces a decrease in microtubule acetylation, resulting in thin mid-piece related asthenoteratozoospermia, providing a novel marker for genetic counseling and diagnosis of male infertility.

Premature ovarian insufficiency (POI) is the main cause of infertility in women. Some cases of POI are thought to be caused by genetic defects and the clinical outcomes of these patients are unknown. Here, we performed whole-exome sequencing of the peripheral blood of a cohort of 55 subjects with POI and identified one heterozygous missense variant in FOXL2 (c.1045G>C; p.Arg349Gly) and two heterozygous missense variants in ERCC6 (c.379G>A; p.Val127Ile and c.4223A>C; p.Glu1408 Ala) in four POI patients. All of these heterozygous mutations were predicted to be deleterious and were parentally inherited from their heterozygous fathers. The mRNA and protein expression of FOXL2 and ERCC6 were absent or decreased in the patients. The patients carrying the variants of FOXL2 (c.1045G>C; p.Arg349Gly) and ERCC6 (c.379G>A; p.Val127Ile) failed to conceive in two and four assisted reproductive cycles, respectively. Another patient and her sister carrying the ERCC6 (c.4223A>C; p.Glu1408 Ala) variant achieved good clinical outcomes after assisted reproductive therapy. Our findings support the possible roles of FOXL2 and ERCC6 in POI and might contribute to the genetic counseling of POI patients.

Canonical splicing variants (±2) contribute significantly to genetic disorders, yet the clinical significance of non-canonical splicing variants (NCSVs) that occur outside of canonical splicing sites remains unknown in male infertility. A comprehensive evaluation of reported studies on hereditary male infertility revealed that the 2,404 pathogenic variants contained 120 canonical splicing variants and 32 NCSVs. Among the remaining 2,252 variants, the splicing variant analytical strategy identified 17 novel NCSVs that disrupt normal mRNA splicing from previously classified missense variants. This expands the contribution of NCSVs by 53.13% (17/32), with NCSVs accounting for 28.99% (49/169) of all the splicing variants. Moreover, thirteen positively validated NCSVs are identified in 12 of 718 idiopathic male infertility patients with negative results by conventional genetic analysis. The first pathogenic variant in the TATA element modulatory factor 1 (TMF1: c.2859+4A>G) results in TMF1 exon 14 skipping and decreased progressive sperm motility and morphological abnormalities in a patient with male infertility. Tmf1 NCSV knock-in mice recapitulated human phenotype, showing significantly decreased sperm count, motility, ultrastructural head defects, and subfertility. This study provides the first comprehensive landscape of NCSVs in male infertility, suggesting that NCSVs may constitute a hidden etiological factor for male infertility.

Non-obstructive azoospermia (NOA) represents the most severe form of male infertility; however, its genetic etiology remains largely elusive. MCM9 is crucial for DNA damage repair in mammalian somatic cells, playing a key role in regulating both homologous recombination (HR) and mismatch repair (MMR) pathways. In mice, MCM9 deficiency leads to spermatogenic failure characterized by progressive germ cell depletion and impaired HR repair. However, the underlying mechanism remains unclear in humans. Our study identified two novel homozygous loss-of-function (LoF) mutations in MCM9 in two unrelated NOA patients presenting with Sertoli cell-only syndrome (SCOS). The absence of testicular MCM9 confirmed the pathogenicity of these LoF mutations. Furthermore, diminished HR-mediated DNA repair capacity observed in HEK293T cells, either lacking MCM9 or overexpressing mutant MCM9 plasmids, highlighted the deleterious impact of these LoF mutations on HR repair. Additionally, the confirmed interaction between human testicular MCM9 and both MSH2 and MLH1, alongside findings that human MCM9 is predominantly expressed in spermatogonial stem cells and spermatogonia, provides compelling evidence for the involvement of the MCM9-mediated MMR pathway in maintaining genomic integrity and supporting the viability and proliferation of spermatogonia in humans. Given the poor outcomes of microdissection testicular sperm extraction (micro-TESE) observed in both probands, we propose that biallelic LoF mutations in MCM9 may serve as non-invasive molecular biomarkers for predicting micro-TESE failure. These findings enhance our understanding of the genetic basis of human NOA, particularly SCOS, and provide valuable insights for genetic counseling and fertility guidance tailored to these patients.

Etomidate (ETO), a hypnotic agent used for anesthesia induction, has been shown to induce long-lasting cognitive deficits. In the present study, we investigated whether ETO could activate the HIF1A/PGK1 pathway to antagonize oxidative damage in mice with postoperative cognitive dysfunction (POCD). A mouse model of ETO-mediated POCD was established, and pathological changes, apoptosis, and inflammatory factors in mouse hippocampal tissues were analyzed by HE staining, TUNEL assay, and ELISA. ETO was revealed to cause cognitive dysfunction in mice. Integrated database mining was conducted to screen out transcription factors that are both related to ETO and POCD. Hypoxia-inducible factor 1-alpha (HIF1A) was overexpressed in mice with POCD, and downregulation of HIF1A alleviated cognitive dysfunction in mice. HIF1A downregulation inhibited the transcription of phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 abated the alleviating effects of HIF1A knockdown on oxidative stress in mice with POCD. In addition, HIF1A activation of PGK1 induced oxidative stress and apoptosis in HT-22 cells while inhibiting cell viability. Taken together, we demonstrated that HIF1A activation of PGK1 induced oxidative stress in ETO-mediated POCD.

Non-obstructive azoospermia (NOA) is the most severe form of human male infertility, and the genetic causes of NOA with meiotic arrest remain largely unclear. In this study, we identified novel compound heterozygous MEIOB variants (c.814C > T: p.R272X and c.976G > A: p.A326T) and a previously undescribed homozygous non-canonical splicing variant of MEIOB (c.528 + 3A > C) in two NOA-affected individuals from two irrelevant Chinese families. MEIOB missense variant (p.A326T) significantly reduced protein abundance and nonsense variant (p.R272X) produced a truncated protein. Both of two variants impaired the MEIOB-SPATA22 interaction. The MEIOB non-canonical splicing variant resulted in whole Exon 6 skipping by minigene assay, which was predicted to produce a frameshift truncated protein (p.S111Rfs*32). Histological and immunostaining analysis indicated that both patients exhibited a similar phenotype as we previously reported in Meiob mutant mice, that is, absence of spermatids in seminiferous tubules and meiotic arrest. Our study identified three novel pathogenic variants of MEIOB in NOA patients, extending the mutation spectrum of the MEIOB and highlighting the contribution of meiotic recombination related genes in human fertility.

BACKGROUND:The association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT). METHODS:Whole-exome sequencing and Sanger sequencing were employed to identify potential pathogenic variants of TDRD6 in infertile men. Histology, immunofluorescence, immunoblotting and ultrastructural analyses were conducted to clarify the structural and functional abnormalities of sperm in mutated patients. Tdrd6-knockout mice were generated using the CRISPR-Cas9 system. Total RNA-seq and single-cell RNA-seq (scRNA-seq) analyses were used to elucidate the underlying molecular mechanisms, followed by validation through quantitative RT-PCR and immunostaining. Intracytoplasmic sperm injection (ICSI) was also used to assess the efficacy of clinical treatment. RESULTS:Bi-allelic TDRD6 variants were identified in five unrelated Chinese individuals with OAT, including homozygous loss-of-function variants in two consanguineous families. Notably, besides reduced concentrations and impaired motility, a significant occurrence of acrosomal hypoplasia was detected in multiple spermatozoa among five patients. Using the Tdrd6-deficient mice, we further elucidate the pivotal role of TDRD6 in spermiogenesis and acrosome identified. In addition, the mislocalisation of crucial chromatoid body components DDX4 (MVH) and UPF1 was also observed in round spermatids from patients harbouring TDRD6 variants. ScRNA-seq analysis of germ cells from a patient with TDRD6 variants revealed that TDRD6 regulates mRNA metabolism processes involved in spermatid differentiation and cytoplasmic translation. CONCLUSION:Our findings strongly suggest that TDRD6 plays a conserved role in spermiogenesis and confirms the causal relationship between TDRD6 variants and human OAT. Additionally, this study highlights the unfavourable ICSI outcomes in individuals with bi-allelic TDRD6 variants, providing insights for potential clinical treatment strategies.

Male infertility is a major concern affecting reproductive health. Biallelic deleterious variants of most DNAH gene family members have been linked to male infertility, with intracytoplasmic sperm injection (ICSI) being an efficacious way to achieve offspring. However, the association between DNAH12 and male infertility is still limited. Here, we identified one homozygous variant and two compound heterozygous variants in DNAH12 from three infertile Chinese men. Semen analysis revealed severe asthenozoospermia, abnormal morphology, and structure of sperm flagella. Furthermore, the Dnah12 knock-out mouse revealed severe spermatogenesis failure and validated the same male infertility phenotype. Favorable fertility outcomes were achieved through ICSI in three human individuals and Dnah12 knock-out mice. Collectively, our study indicated that biallelic variants of DNAH12 can induce male infertility in both human beings and mice. Notably, evidence from DNAH12 enhanced that ICSI was an optimal intervention to achieve favorable fertility outcomes for infertile males with DNAH gene family variants.

STUDY QUESTION Do biallelic deleterious variants of Calreticulin 3 (CALR3) cause fertilization failure (FF), resulting in male infertility in humans? SUMMARY ANSWER Biallelic mutations in CALR3 were identified in two infertile men from unrelated families and were shown to cause FF associated with failed sperm-zona pellucida (ZP) binding. WHAT IS KNOWN ALREADY In male mice, the Calr3-knockout has been reported to cause male infertility and FF. However, the mechanism behind this remains unclear in humans. STUDY DESIGN, SIZE, DURATION Sequencing studies were conducted in a research hospital on samples from Han Chinese families with primary infertility and sperm head deformations to identify the underlying genetic causes. PARTICIPANTS/MATERIALS, SETTING, METHODS Data from two infertile probands characterized by sperm head deformation were collected through in silico analysis. Sperm cells from the probands were characterized using light and electron microscopy and used to verify the pathogenicity of genetic factors through functional assays. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of FF. ICSI were administered to overcome CALR3-affected male infertility. MAIN RESULTS AND THE ROLE OF CHANCE Novel biallelic deleterious mutations in CALR3 were identified in two infertile men from unrelated families. We found one homozygous frameshift CALR3 mutation (M1: c.17_27del, p.V6Gfs*34) and one compound heterozygous CALR3 mutation (M2: c.943A>G, p.N315D; M3: c.544T>C, p.Y182H). These mutations are rare in the general population and cause acrosomal ultrastructural defects in affected sperm. Furthermore, spermatozoa from patients harbouring the CALR3 mutations were unable to bind to the sperm-ZP or they disrupted gamete fusion or prevented oocyte activation. Molecular assays have revealed that CALR3 is crucial for the maturation of the ZP binding protein in humans. Notably, the successful fertilization via SUZI and ICSI attempts for two patients, as well as the normal expression of PLC zeta in the mutant sperm, suggests that ICSI is an optimal treatment for CALR3-deficient FF. LIMITATIONS, REASONS FOR CAUTION The results are based on sperm-related findings from two patients. Further studies are required to gain insight into the developmental stage and function of CALR3 in human testis. WIDER IMPLICATIONS OF THE FINDINGS Our findings highlight the underlying risk of FF associated with sperm defects and provide a valuable reference for personalized genetic counselling and clinical treatment of these patients.

OBJECTIVES:In male mice, adgb-knockout has been reported to cause male infertility with spermatogenesis defects involving flagella and acrosome. However, this remains unclear for humans. MATERIALS AND METHODS:Sequencing studies were conducted in a research hospital on samples from three unrelated infertile men with severe asthenoteratozoospermia from Han Chinese families. Data were collected through rigorous in silico analysis. Sanger sequencing were performed to identify pathogenic mutations. Sperm cells from patients were characterized using electron microscopy and used to verify the pathogenicity of the genetic factors through functional assays. Intracytoplasmic sperm injections (ICSI) assays were performed in ADGB-affected males. MAIN RESULTS:Herein, in a cohort of 105 Han Chinese men with idiopathic asthenoteratozoospermia, we reported the identification of bi-allelic deleterious variants of ADGB in three infertile men from unrelated families using whole-exome sequencing. We found one homozygous frameshift ADGB variant (NM_024694.4: c.2801_2802del:p.K934Rfs*33), one homozygous missense ADGB variant (NM_024694.4: c.C3167T:p.T1056I), and one compound heterozygous ADGB variant (NM_024694.4: c.C3167T:p.T1056I; c.C3197T:p.A1066V). These variants were rare in general population and were predicted to be damaging by multiple bioinformatics tools. Further, the spermatozoa from patients harboring ADGB variants showed multiple acrosome and flagellum malformations under light and electron microscopy. Functional assays revealed the structural defects associated with dysregulation of ADGB and multiple spermatogenesis proteins. Notably, the fertilization success via ICSI treatment in all three patients, as well as the normal expression of PLCζ but CaM deficiency in the spermatozoa, suggesting that ICSI other than in vitro fertilization (IVF) is an optimal treatment for ADGB-deficient patients. DISCUSSION AND CONCLUSION:Our findings provide new information for the molecular diagnosis of asthenoteratozoospermia and valuable reference for personalized genetic counselling and clinical treatment for these patients. The underlying risk of IVF failure behind sperm defects was highlighted.

The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species. Enzymatic shuttles, particularly adenylate kinase (AK) and creatine kinase (CK), are pivotal in the efficient transfer of intracellular ATP, showing distinct tissue- and species-specificity. Here, the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups, of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates. Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort. Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility. Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke (RS) of the axoneme. Examination of various human and mouse sperm samples with substructural damage, including the presence of multiple RS subunits, showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme. Using an ATP probe together with metabolomic analysis, it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme, and were concentrated at sites associated with energy consumption in the flagellum. These findings indicate a novel function for RS beyond its structural role, namely, the regulation of ATP transfer. In conclusion, the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.

Given the physiological function and anatomical location of the reproductive tract, studying the upper reproductive tract microbiota may be essential for studying male infertility and other male diseases. This study aimed to characterize the microbiota of the upper reproductive tract male rats and investigate whether specific microbial compositions are associated with sperm parameters. 16S rRNA gene sequencing was used to characterize the microbial composition in the testis, epididymis, seminal vesicles, vas deferens and prostate tissues of the rats. The results showed significant enrichment of Methyloperoxococcus spp. in testicular tissues, Jeotgalicoccus spp. in epididymal tissues. Spearman’s correlation analysis revealed that the abundance of several bacterial genera in epididymal, testicular, and seminal vesicle gland tissues correlated with several sperm activity parameters. Our findings provide detailed information on characterizing the upper reproductive tract microbiome in male rats, as well as a potentially crucial link between the reproductive system microbiota and sperm quality.

BACKGROUND:Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS:A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS:In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.