Defective Spermatogenesis Research Articles

Berberine alters the gut microbiota metabolism and impairs spermatogenesis.

Berberine (BBR) is used to treat diarrhea clinically. However, its reproductive toxicity is unclear. This study aims to investigate the impact of BBR on the male reproductive system. Intragastric BBR administration for 14 consecutive days results in a significant decrease in the serum testosterone concentration, epididymal sperm concentration, mating rate and fecundity of male mice. Testicular treatment with testosterone propionate (TP) partially reverses the damage caused by BBR to the male reproductive system. Mechanistically, the decrease in Muribaculaceae abundance in the gut microbiota of mice is the principal cause of the BBR-induced decrease in the sperm concentration. Both fecal microbiota transplantation (FMT) and polyethylene glycol (PEG) treatment demonstrate that Muribaculaceae is necessary for spermatogenesis. The intragastric administration of Muribaculaceae intestinale to BBR-treated mice restores the sperm concentration and testosterone levels. Metabolomic analysis reveals that BBR affects arginine and proline metabolism, of which ornithine level is downregulated. Combined analysis via 16S rRNA metagenomics sequencing and metabolomics shows that Muribaculaceae regulates ornithine level. The transcriptomic results of the testes indicate that the expressions of genes related to the low-density lipoprotein receptor (LDLR)-mediated testosterone synthesis pathway decrease after BBR administration. The transcriptional activity of the Ldlr gene in TM3 cells is increased with increased ornithine supplementation in the culture media, leading to increased testosterone synthesis. Overall, this study reveals an association between a BBR-induced decrease in Muribaculaceae abundance and defective spermatogenesis, providing a prospective therapeutic approach for addressing infertility-related decreases in serum testosterone triggered by changes in the gut microbiota composition.

Oogenesis and Spermatogenesis of the Triploid Black Tiger Shrimp, Penaeus monodon

Triploidy (3n) induction of Penaeus monodon was performed separately by Australian and Thai groups of researchers 14 years ago, incidentally at the same period. The Australian group employed the chemical induction method, while the Thai group the cold shock one, and both groups obtained 3n P. monodon in their attempts. The success has led to several studies on the physiology of the 3n P. monodon, including growth, survival and reproductive functions. Both groups reported sterility of the 3n shrimp. The Australian group reported defected oogenesis and spermatogenesis of the 3n shrimp examined at subadult stage. Since the evaluation of the reproductive functions should be performed at the adult stage of the animals, we, therefore, performed in-depth studies on oogenesis and spermatogenesis of adult 3n P. monodon induced by cold shock. The studies include gross observation of the ovary, light (LM) and transmission electron (TEM) microscopy and image analysis of sex cells. The ovary of the 3n P. monodon females could not develop to full-maturation and their gonadosomatic index (GSI) was significantly lower than that of the 2n shrimp. Oogenesis of the 3n females proceeded from oogonia to cortical rod oocytes, but with significantly lower percentage than that of the 2n shrimp. The GSI of the 3n males was significantly lower than that of the 2n shrimp. Their spermatogenesis, however, proceeded normally from spermatogonia to spermatozoa, but with low density of spermatozoa in the seminiferous tubule and vas deferens, and none in the terminal ampoule. The average nuclear area of the 3n spermatozoa was significantly lower than that of the 2n shrimp as well. Altogether, these results suggest that 3n P. monodon induced by cold shock had defect in both oogenesis and spermatogenesis, probably more in quantity than in quality. HIGHLIGHTS Oogenesis and spermatogenesis of the triploid black tiger shrimp, monodon, induced by cold shock were studied. The study found abnormalities in the spermatogenesis, but not oogenesis, of the triploid P. monodon induced by cold shock. It may be concluded that triploidy condition of P. monodon induced by cold shock affects meiotic, but not mitotic activity of the sex cells. GRAPHICAL ABSTRACT

Steroid hormone-deprived sex reversal in cyp11a1 mutant XX tilapia experiences an ovary-like stage at molecular level

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish.

FBXO38 is dispensable for PD-1 regulation

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

DNAJA1 regulates protein ubiquitination and is essential for spermatogenesis in the testes of mice and rats

DNAJA1 is a member of type I DnaJ proteins, which is essential for spermatogenesis and male fertility. However, its expression pattern in the testes and its impact on spermatogenesis remains unclear. Our study aimed to elucidate the mechanism of action of DNAJA1. We employed DNAJA1 knockout mice in this study. Western blotting and immunofluorescence analysis were conducted to determine the protein abundance of DNAJA1 in testes at various developmental stages. Our results revealed that DNAJA1 is predominantly expressed in the testes, and its knockout leads to complete infertility in male mice. We observed that DNAJA1 protein levels increased on postnatal days 14, 21, and 28, peaking on postnatal day 35 in mice. Immunofluorescence staining indicated that DNAJA1 expression varies across different stages of the spermatogenesis cycle. Additionally, DNAJA1 was absent in epididymal sperm. In early- and mid-stage tubules, DNAJA1 protein distribution was co-localized with residual bodies in elongating spermatids. Furthermore, we found that DNAJA1 knockout significantly reduced protein polyubiquitination in the testis. Analysis of the GEO database showed that DNAJA1 levels were significantly decreased in semen samples from subjects with teratozoospermia, asthenozoospermia, and impaired spermatogenesis. Our findings suggest that DNAJA1 is an essential protein for spermatogenesis, and its deletion reduces protein polyubiquitination in the testis, ultimately resulting in infertility and spermatogenesis defects.

Molecular characterization of DNAH6 and ATPase6 (Mitochondrial DNA) genes in asthenozoospermia patients in the northern region of India

BackgroundMale infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. The present study was undertaken to assess the association of DNAH6 and ATPase6 genes in asthenozoospermia patients in the northern region of India.MethodsA total of 60 semen samples were collected for the study, of which 30 were from the case group and 30 were from the control group. The semen samples for the case group (asthenozoospermia) and control groups were collected from IVF and Reproductive Biology Centre, Maulana Azad Medical College, New Delhi. Sperm count and motility were classified as per World Health Organization (WHO 2021) protocol. A total genomic DNA was extracted as per the stranded TRIZOL method with little modification.ResultsIn-vitro molecular characterizations of DNAH6 and ATPase6 genes in both groups were checked by Polymerase Chain Reaction (PCR). The 675 bp and 375 bp amplicons were amplified using PCR for ATPase6 and DNAH6 genes. Our study results showed a significant (P ≤ 0.05) null deletion of DNAH6 and ATPase6 genes in asthenozoospermia patients as compared to the control. We found the significant null deletion of DNAH6 in case 45.0%, and the control group was 11.7%. However, in the case of APTase6, it was 26.7% and 10.0%, respectively.ConclusionsOur study concluded that the presence of DHAH6 and ATPase6 genes had a significant impact on male infertility.

TRIM59 is required for mouse GC-1 cell maintenance through modulating the ubiquitination of AXIN1

Tripartite motif-containing protein 59 (TRIM59) is a biomarker for multiple tumors with crucial roles. However, the specific role of TRIM59 in germ cells remains largely unknown. Here, we investigated the effects and underlying regulatory mechanisms of TRIM59 on germ cells using the mouse spermatogonial cell line GC-1. Our results demonstrated that TRIM59 promoted proliferation and inhibited apoptosis of GC-1 cells. Mechanistically, TRIM59 maintained GC-1 cell behaviors through ubiquitination of AXIN1 to activate β-catenin signaling. Furthermore, activation of β-catenin signaling reversed the effects mediated by Trim59 knockdown in GC-1 cells. Collectively, our study revealed a major role and regulatory mechanism of TRIM59 in GC-1 cells, which sheds new light on the molecular pathogenesis of defects in spermatogenesis and may provide therapeutic targets for treatment of male infertility.

Study on the mechanisms of defective spermatogenesis induced by TiO2 NPs based on 3D blood−testis barrier microfluidic chip

Titanium dioxide nanoparticles (TiO2 NPs) can reduce sperm number, but the mechanisms of defective spermatogenesis induced by TiO2 NPs have not been studied through cell-cell interactions at present. A kind of biomimetic three-dimensional blood−testis barrier microfluidic chip capable of intercellular communication was constructed with soft lithography techniques, including Sertoli cell (TM4), spermatogonia (GC-1) and vascular endothelial cell units, to study the mechanisms of TiO2 NPs-induced defective spermatogenesis. TM4 and GC-1 cells cultured in TiO2 NPs exposure and control chips were collected for transcriptomics and metabonomics analysis, and key proteins and metabolites in changed biological processes were validated. In TM4 cells, TiO2 NPs suppressed glucose metabolism, especially lactate production, which reduced energy substrate supply for spermatogenesis. TiO2 NPs also decreased the levels of key proteins and metabolites of lactate production. In GC-1 cells, TiO2 NPs disturbed chemokine signaling pathways regulating cell proliferation and interfered with glutathione metabolism. The Cxcl13, Stat3 and p-Stat3 levels and cell proliferation rate were decreased, and the GSR, GPX4 and GSH contents were increased in GC-1 cells in chips under TiO2 NPs treatment. The decrease in energy substrate supply for spermatogenesis and inhibition of spermatogonia proliferation could be the main mechanisms of defective spermatogenesis induced by TiO2 NPs.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality

Abstract Study question Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance? Summary answer This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status. What is known already We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology. Study design, size, duration In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR. Participants/materials, setting, methods Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change>2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results. Main results and the role of chance We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis. Limitations, reasons for caution This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing. Wider implications of the findings The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions. Trial registration number not applicable

Triptolide induced spermatogenesis dysfunction via ferroptosis activation by promoting K63-linked GPX4 polyubiquitination in spermatocytes

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.

Protein disulfide isomerase is essential for spermatogenesis in mice.

Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.

The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice.

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left-right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3+/- knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3+/- males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations.

Impact of coenzyme Q10 as an adjuvant therapy to letrozole on spermiogram results and sex hormone levels in Iraqi men with infertility; randomized open label comparative study.

Background: Worldwide, infertility affects about 15% of reproductive-age couples. In many cases, infertility can't be treated, however new treatment options with promising value have been involved in recent clinical trials. The aim of this clinical trial was to evaluate the impacts of adding coenzyme Q10 (CoQ10) to letrozole on the results of spermiogram and sex hormone tests in men diagnosed with idiopathic oligoasthenoteratozoospermia (iOAT) syndrome, which is a type of male defective spermatogenesis of unknown etiology. Methods: This randomized, open-label, parallel two-arm interventional study included 67 adult male patients aged 18-60 years with a confirmed diagnosis of iOAT syndrome recruited from The High Institute for Infertility Diagnosis & Assisted Reproduction Technologies/Nahrain University. Patients were randomly separated into two groups, Group A included 29 patients treated with letrozole 2.5 mg tablet orally twice a week, Group B included 38 patients treated with a combination of letrozole 2.5 mg tablet orally twice a week plus CoQ10 400 mg per day. Both groups completed treatment for three months. Semen samples, serum follicle-stimulating hormone (FSH), estradiol (E 2), and testosterone (T) were analyzed at day one, and at the end of month one, two and three. Results: Both groups showed that sperm concentration, normal morphology, total sperm count and motility, serum testosterone and FSH levels, and T/E 2 ratio were significantly increased, while estradiol levels significantly decreased after three months of treatment. Seminal fluid volume changed significantly in group A only. In comparing between the two groups, all measured parameters, apart from sperm motility and FSH level, demonstrated a significant difference after three months of treatment, while sperm volume reached significant value after only two months of therapy. Conclusions: CoQ10 as adjuvant treatment to letrozole effectively improved most of the tested sperm parameters in Iraqi men with iOAT. Registration: ClinicalTrials.gov ( NCT05847257, May 6, 2023).

Potential Protective Effects of Tribulus terrestris Extract (TTE) against Reproductive Damage Induced by Cytarabine (Ara-C) Chemotherapy in Male Rats: Histopathological Study

The cytotoxic effects of many cancer medications cause different general side effects and defects in spermatogenesis, so still a concern that requires additional enhancement. The study was performed to assess the preventive benefits of Tribulus terrestris extract (TTE) against reproductive damage induced by cytarabine (Ara-C) chemotherapy. Twenty mature male rats will be divided into four groups. Each group contains 5 rats. A control group is included in the first group, T. terrestris extract (250 mg/kg) is given to the second group, Ara-C (25 mg/kg BW) is given to the third group, and the fourth group receives both an oral dose of TTE (250 mg/kg BW) and an intraperitoneal dosage of Ara-C (25 mg/kg BW) for a total of 28 days. After the end of the experiment tissue samples in testes and epididymis are taken for the necessary histopathological changes that revealed detectable spermatogenesis degeneration. Treatment caused spermatocytes disruption, significant histopathological alterations, rounded, shrunken seminiferous tubules with less cellular epithelium, degeneration in germinal epithelium represented by depletion with damaged Leydig cells, and this may result in reduced rat fertility. The results of the study indicate that Tribulus terrestris exhibits significant histological alterations, as evidenced by an increase in sperm growth, in contrast to Ara C group, which displayed some detrimental effects.

Identification of a novel mutation in chibby family member 2 in a non-obstructive azoospermic patient

Azoospermia constitutes a significant factor in male infertility, defined by the absence of spermatozoa in the ejaculate, afflicting 15% of infertile men. However, a subset of azoospermic cases remains unattributed to known genetic variants. Prior investigations have identified the chibby family member 2 (CBY2) as prominently and specifically expressed in the testes of both humans and mice, implicating its potential involvement in spermatogenesis. In this study, we conducted whole exome sequencing (WES) on an infertile family to uncover novel genetic factors contributing to azoospermia. Our analysis revealed a homozygous c .355 C>A variant of CBY2 in a non-obstructive azoospermic patient. This deleterious variant significantly diminished the protein expression of CBY2 both in vivo and in vitro, leading to a pronounced disruption of spermatogenesis at the early round spermatid stage post-meiosis. This disruption was characterized by a nearly complete loss of elongating and elongated spermatids. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation assays demonstrated the interaction between CBY2 and Piwi-like protein 1 (PIWIL1). Immunofluorescence staining further confirmed the co-localization of CBY2 and PIWIL1 in the testes during the spermatogenic process in both humans and mice. Additionally, diminished PIWIL1 expression was observed in the testicular tissue from the affected patient. Our findings suggest that the homozygous c .355 C>A variant of CBY2 compromises CBY2 function, contributing to defective spermatogenesis at the round spermiogenic stage and implicating its role in the pathogenesis of azoospermia.

Study of Correlation between Serum Vitamin B12 Level and Aberrant DNA Methylation in Infertile Males.

Altered DNA methylation pattern in sperms has been associated with infertility in males demonstrating defective spermatogenesis or low semen quality. Vitamin B-12, by affecting 1-carbon metabolism pathways, might alter the DNA methylation pattern. We aimed to study the correlation of serum vitamin B12 levels with aberrant DNA methylation in infertile male patients. A cross-sectional study was conducted on 17 oligozoospermic infertile males (WHO criteria, 2010) and 10 healthy fertile males. Serum vitamin B12 levels were estimated using the chemiluminescence method. Global methylation was determined using the ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich). The levels of global DNA methylation were calculated and compared relative to the methylated (100%) control DNA provided with the kit. Mean serum vitamin B12 concentration in the control group was higher than that of the case group. This difference in serum vitamin B12 concentration in both groups was found statistically significant. Although the results of this study show that oligozoospermic men have relatively lower global DNA methylation as compared to normozoospermic control, the values could not reach a statistically significant level. A small positive correlation was found between serum vitamin B12 levels and percent methylation defect (r = 0.14) but was statistically insignificant. Our study concludes that oligozoospermic infertile males have a significant deficiency of vitamin B12 as compared to normozoospermic fertile males. This study did not find any significant difference in global DNA methylation between the two groups. The present study does not suggest any correlation between serum vitamin B12 level and percent DNA methylation.

Mitochondrial Fission in Nickel Nanoparticle-Induced Reproductive Toxicity: An In Vitro GC-1 Cell Study.

Reproductive disorders and declining fertility rates are significant public health concerns affecting birth rates and future populations. Male infertility, often due to spermatogenesis defects, may be linked to environmental pollutants like nickel nanoparticles (Ni NPs). Ni NPs are extensively utilized across different industries. Nevertheless, their potential adverse effects cannot be overlooked. Previous studies have linked the reproductive toxicity induced by Ni NPs with disturbances in mitochondrial function. Mitochondrial division/fusion dynamics are crucial to their proper function, yet little is known about how Ni NPs perturb these dynamics and whether such perturbation contributes to the impairment of the male reproductive system. Herein, we demonstrated that the exposure of Ni NPs to the mouse-derived spermatogonia cell line (GC-1 cells) triggered DRP1-mediated mitochondrial division and the enhanced impairment of mitochondria, consequently promoting mitochondria-dependent cell apoptosis. Notably, both the mitochondrial division inhibitor (Mdivi-1) and lentiviral-transfected cells with low expression of Dnm1l-DK in these cells could mitigate the toxic effects induced by Ni NPs, pointing to the potential role of mitochondrial dynamics in Ni NP-induced reproductive toxicity. Collectively, our work contributes to the understanding of the mechanisms by which Ni NPs can impact male reproductive function and identifies mitochondrial division as a potential target for intervention.

Thymoquinone effects on autophagy, apoptosis, and oxidative stress in cisplatin-induced testicular damage in mice.

In this study, the effect of thymoquinone (TQ) on CP-induced spermatogenesis defects in mice has been investigated. Sperm parameters, serum testosterone concentration, histology, Bax/Bcl-2 ratio, and expression of autophagy-related biomarkers have been assessed. Total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) in testicular tissue were examined for the evaluation of oxidative stress levels. CPhas induced histological changes and significantlyincreased the Bax/Bcl-2 ratio, decreased testosterone concentration, testicular weight, and sperm quality. CP induced oxidative stress by elevating OSIin the testicular tissue(p < 0.05).Expression of theautophagy-inducer genes (ATG7, ATG5, and Beclin-1) andratio ofLC3B/LC3Aproteinswere significantly decreased, while mTOR expression was increased in the CP group.TQ pretreatment dose-dependently decreased the Bax/Bcl-2 ratio and mTOR gene expression while increasing the expression of ATG5 and ATG7 genes, LC3B/LC3A ratio, and Beclin-1 proteins.TQ could also dose-dependently reverse the histology, testosterone level, and sperm quality of the CP-intoxicated mice. These findings show that TQ pretreatment can enhance sperm production by inducing autophagy and reducing apoptosis and oxidative stress in the CP-intoxicated mouse testicles.

PiRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment. The regulatory mechanism underpinning MIWI translocation, however, has remained elusive. We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB. Mechanistically, piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH. This, in turn, enables arginine methylation of MIWI, augmenting its binding affinity for TDRD6 and ensuring its integration within the CB. Notably, loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells, leading to defective spermatogenesis and male infertility in mice. Collectively, our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis, offering new insights into piRNA biology in mammals.