Regulation Of Spermatogenesis Research Articles

Oxidative-Stress-Mediated Epigenetic Dysregulation in Spermatogenesis: Implications for Male Infertility and Offspring Health.

Male reproductive health is governed by an intricate interplay of genetic, epigenetic, and environmental factors. Epigenetic mechanisms-encompassing DNA methylation, histone modifications, and non-coding RNA activity-are crucial both for spermatogenesis and sperm maturation. However, oxidative stress, driven by excessive reactive oxygen species, disrupts these processes, leading to impaired sperm function and male infertility. This disruption extends to epigenetic modifications, resulting in abnormal gene expression and chromatin remodeling that compromise genomic integrity and fertilization potential. Importantly, oxidative-stress-induced epigenetic alterations can be inherited, affecting the health and fertility of offspring and future generations. This review investigates how oxidative stress influences epigenetic regulation in male reproduction by modifying DNA methylation, histone modifications, and non-coding RNAs, ultimately compromising spermatogenesis. Additionally, it discusses the transgenerational implications of these epigenetic disruptions and their potential role in hereditary infertility and disease predisposition. Understanding these mechanisms is vital for developing therapeutic strategies that mitigate oxidative damage and restore epigenetic homeostasis in the male germline. By integrating insights from molecular, clinical, and transgenerational research, this work emphasizes the need for targeted interventions to enhance male reproductive health and prevent adverse outcomes in progeny. Furthermore, elucidating the dose-response relationships between oxidative stress and epigenetic changes remains a critical research priority, informing personalized diagnostics and therapeutic interventions. In this context, future studies should adopt standardized markers of oxidative damage, robust clinical trials, and multi-omic approaches to capture the complexity of epigenetic regulation in spermatogenesis. Such rigorous investigations will ultimately reduce the risk of transgenerational disorders and optimize reproductive health outcomes.

Integrative Analysis of Whole-Genome and Transcriptomic Data Reveals Novel Variants in Differentially Expressed Long Noncoding RNAs Associated with Asthenozoospermia.

Background/Objectives: Asthenozoospermia, characterized by reduced sperm motility, is a common cause of male infertility. Emerging evidence suggests that noncoding RNAs, particularly long noncoding RNAs (lncRNAs), play a critical role in the regulation of spermatogenesis and sperm function. Coding regions have a well-characterized role and established predictive value in asthenozoospermia. However, this study was designed to complement previous findings and provide a more holistic understanding of asthenozoospermia, this time focusing on noncoding regions. This study aimed to identify and prioritize variants in differentially expressed (DE) lncRNAs found exclusively in asthenozoospermic men, focusing on their impact on lncRNA structure and lncRNA-miRNA-mRNA interactions. Methods: Whole-genome sequencing (WGS) was performed on samples from asthenozoospermic and normozoospermic men. Additionally, an RNA-seq dataset from normozoospermic and asthenozoospermic individuals was analyzed to identify DE lncRNAs. Bioinformatics analyses were conducted to map unique variants on DE lncRNAs, followed by prioritization based on predicted functional impact. The structural impact of the variants and their effects on lncRNA-miRNA interactions were assessed using computational tools. Gene ontology (GO) and KEGG pathway analyses were employed to investigate the affected biological processes and pathways. Results: We identified 4173 unique variants mapped to 258 DE lncRNAs. After prioritization, 5 unique variants in 5 lncRNAs were found to affect lncRNA structure, while 20 variants in 17 lncRNAs were predicted to disrupt miRNA-lncRNA interactions. Enriched pathways included Wnt signaling, phosphatase binding, and cell proliferation, all previously implicated in reproductive health. Conclusions: This study identifies specific variants in DE lncRNAs that may play a role in asthenozoospermia. Given the limited research utilizing WGS to explore the role of noncoding RNAs in male infertility, our findings provide valuable insights and a foundation for future studies.

Argonaute proteins regulate the timing of the spermatogenic transcriptional program.

Argonaute proteins are best known for their role in microRNA-mediated post-transcriptional gene silencing. Here, we show that AGO3 and AGO4, but not AGO2, localize to the sex chromatin of pachytene spermatocytes where they are required for transcriptional silencing of XY-linked genes, known as Meiotic Sex Chromosome Inactivation (MSCI). Using an Ago413 -/- mouse, we show that AGO3 and AGO4 are key regulators of spermatogenesis, orchestrating expression of meiosis-related genes during prophase I while maintaining silencing of spermiogenesis genes. Premature overexpression of spermiogenesis genes during prophase I in Ago413 -/- mice results in subfertility, altered sperm morphology and reduced fertilization capability. We also identify BRG1, a BAF complex subunit, as an AGO3 interactor. Loss of AGO3 and AGO4 results in increased BRG1 in spermatocytes, suggesting that AGO3 aids in removing BRG1 from the XY chromatin to achieve MSCI and demonstrating a meiotic role for AGO3 in transcriptional control through the chromatin remodeling machinery.

Identification of crucial LncRNAs associated with testicular development and LOC108635509 as a potential regulator in black goat spermatogenesis

The establishment and maintenance of spermatogenesis is a complex process involving a vast of regulatory pathways. There is growing evidence revealing that long noncoding RNAs (lncRNA) play important roles in regulating testicular development and spermatogenesis in a stage-specific way. However, our understanding of how lncRNA regulates testicular development and spermatogenesis in black goats is quite limited. In the current study, we screened the transcriptomes (lncRNA and mRNA) of testicular from Guangxi black goats before puberty (3 days old, D3; 30 days old, D30), puberty (90 days old, D90) and postpuberty (180 days old, D180), in order to identify the lncRNA interaction with mRNAs contributes to goat spermatogenesis. The RNA-sequencing (RNA-seq) analysis showed that there were 1211, 12,180, 834 differential lncRNAs and 1196, 8838,269 differential mRNAs at the ages of D30 vs. D3, D90 vs. D30, and D180 vs. D90. The lncRNAs showed the most significantly changes from D30 to D90, which indicated that D90 was a key node of lncRNAs participated in the regulation of testicular development and spermatogenesis in black goat. According to functional enrichment analysis of GO and KEGG, we found that differentially expressed lncRNAs (DE lncRNAs) and their target genes regulated spermatogenesis through signal pathways including MAPK, Ras, and PI3K-Akt. Using cis- and trans-acting, 39 DE lncRNAs-targeted genes were found to be enriched for male reproduction. Of these, LOC108635509, which specific expressed in testis and upregulated the expression levels at D90, was found participated in the regulation of testicular development through promoting the proliferation of Sertoli cells (SCs). Overall, this study provides new insight into the regulatory mechanisms that support spermatogenesis and testicular development in black goats.

Effects of Acute Alcohol Intoxication on Testicular Dna Stability, Gene Expression of Cytochromes CYP3A and CYP2E1, and Serum Pool of Free Amino Acids in Rats

Background. Alcohol's toxic effects on the organism is a long-known medical problem. Alcohol's damaging effect is the end result of the complex interplay between ethanol metabolism, inflammation and innate immunity. Previously, we studied the long-term consequences of chronic alcoholism and demonstrated that especially profound changes were in testes on the level of proteome and genome. Objective. This work aimed to study short-term acute alcohol intoxication (AAI) effects for rat testis DNA fragmentation, cytochromes CYP3А and CYP2Е1 genes expression, and serum pool of free amino acids in rats. Methods. Wistar albino male rats were divided into 2 groups (8 animals in each group): 1 – Control (intact rats), and 2 – AAI (rats with short-term acute alcohol intoxication). AAI was induced by repeated administration per os 40% ethanol solution in a dose 7 ml/kg body weight, for 7 days. Contents of amino acids in serum, testes mRNA CYP2E1 and CYP3A2 expression, and DNA fragmentation were evaluated. Results. In our experiments, the development of acute alcohol intoxication (AAI) led to increased DNA fragmentation processes in the testes of adult rats compared to the control group. Additionally, in the serum of ethanol-treated rats, the levels of histidine increased by 1.67 times and glutamine by 1.13 times in correlation with this pathology. Conversely, the levels of valine, phenylalanine, as well as non-essential and essential amino acids, decreased. Furthermore, there was a statistically significant increase in the expression of CYP2E1 and CYP3A2 genes in rat testes under the conditions of AAI. Conclusions. In conclusion, investigation of rats' short-term alcohol administration effects permitted us to obtain the picture of complex metabolomic changes at the different levels. The main outcome of rats short-term ethanol administration in our experiments seems to be to some extent similar to changes described for rats with chronic alcohol consumption. Our results demonstrated profound changes in testes affecting the state of the genome, transcription processes and the exchange of amino acids and proteins. We suggest that the revealed testicular metabolic disorders could have negative implications on cellular regulation of spermatogenesis even under short-term ethanol exposure.

Genome-Wide Association Study and Phenotype Prediction of Reproductive Traits in Large White Pigs.

In a study involving 385 Large White pigs, a genome-wide association study (GWAS) was conducted to investigate reproductive traits, specifically the number of healthy litters (NHs) and the number of weaned litters (NWs). Several SNP loci, including ALGA0098819, ALGA0037969, and H3GA0032302, were significantly associated with these traits. In the combined-parity analysis, candidate genes, such as BLVRA, STK17A, PSMA2, and C7orf25, were identified. GO and KEGG pathway enrichment analyses revealed that these genes are involved in key biological processes, including organic synthesis, the regulation of sperm activity, spermatogenesis, and meiosis. In the by-parity analysis, the PLCXD3 gene was significantly associated with the NW trait in the second and fourth parities, while RNASEH1, PYM1, and SEPTIN9 were linked to cell proliferation, DNA repair, and metabolism, suggesting their potential role in regulating reproductive traits. These findings provide new molecular markers for the genetic study of reproductive traits in Large White pigs. For the phenotypic prediction of NH and NW traits, several machine learning models (GBDT, RF, LightGBM, and Adaboost.R2), as well as traditional models (GBLUP, BRR, and BL), were evaluated using SNP data in varying proportions. After PCA processing, the GBDT model achieved the highest PCC for NH (0.141), while LightGBM reached the highest PCC for NW (0.146). The MAE, MSE, and RMSE results showed that the traditional models exhibited stable error rates, while the machine learning models performed comparatively better across the different SNP ratios. Overall, PCA processing provided some improvement in the predictive performance of all of the models, though the overall increase in accuracy was limited.

Systemic alpha-1 adrenergic receptor inhibition reduces sperm damage in adult and aging spontaneously hypertensive rats

Decreased sperm quality has been reported in men with different clinical conditions, including aging and hypertension. In the male reproductive tract, it has been suggested that the α1-adrenergic receptor influences fertility and spermatogenesis, and important functions are also attributed to the renin–angiotensin axis, such as regulation of steroidogenesis, spermatogenesis, and sperm function. Previously, our group demonstrated impaired testicular vasomotion via α1-adrenergic receptor activation and increased hypoxia-related proteins in the testes of spontaneously hypertensive rats (SHRs) compared to Wistar normotensive rats. In this study, we aimed to investigate the effect of hypertension and inhibition of systemic α1-adrenergic receptor or angiotensin II AT1 receptor on sperm quality, sperm functional characteristics, and testicular microcirculation in rats from three different ages: young (8–10-week-old), adult (20–24-week-old) and older adult (60–66-week-old). We observed higher blood pressure in SHRs of all ages compared to age-matched Wistar rats. Lower blood pressure was observed either in prazosin or losartan-treated adult or aged SHRs. Additionally, lower sperm concentration, impaired motility and higher acrosome damage were demonstrated in SHRs. Prazosin treatment alleviated the effects of hypertension on sperm concentration and motility but not acrosome damage. Higher vasomotion was noticed in testicular blood vessels of adult and aged SHRs compared to Wistar rats. Thus, impaired sperm quality was observed in SHRs of different ages and was improved by sub-chronically blocking the α1-adrenergic receptor.

Germ cell progression through zebrafish spermatogenesis declines with age.

Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal, but fertility declines with age. Although morphological studies have informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying the maintenance and decline of spermatogenesis are not yet understood. We used single-cell RNA sequencing to generate a developmental atlas of the aging zebrafish testis. All testes contained spermatogonia, but we observed a progressive decline in spermatogenesis that correlated with age. Testes from some older males only contained spermatogonia and a reduced population of spermatocytes. Spermatogonia in older males were transcriptionally distinct from spermatogonia in testes capable of robust spermatogenesis. Immune cells including macrophages and lymphocytes drastically increased in abundance in testes that could not complete spermatogenesis. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of spermatogenesis.

Whole transcriptome sequencing of testis and epididymis reveals genes associated with sperm development in roosters

BackgroundChickens play a crucial role as the primary global source of eggs and poultry, and the quality of rooster semen significantly impacts poultry reproductive efficiency. Therefore, it is imperative to comprehend the regulatory mechanisms underlying sperm development.ResultsIn this study, we established transcriptome profiles of lncRNAs, miRNAs, and mRNAs in 3 testis tissues and 3 epididymis tissues from “Jing Hong No.1” roosters at 24, 35, and 64 weeks of age. Using the data, we conducted whole transcriptome analysis and constructed a ceRNA network. We detected 10 differentially expressed mRNAs (DEmRNAs), 33 differentially expressed lncRNAs (DElncRNAs), and 10 differentially expressed miRNAs (DEmiRNAs) in the testis, as well as 149 DEmRNAs, 12 DElncRNAs, and 10 DEmiRNAs in the epididymis. These genes were found to be involved in cell differentiation and development, as well as various signaling pathways such as GnRH, MAPK, TGF-β, mTOR, VEGF, and calcium ion pathways. Subsequently, we constructed two competing endogenous RNA (ceRNA) networks comprising DEmRNAs, DElncRNAs, and DEmiRNAs. Furthermore, we identified four crucial lncRNA-mRNA-miRNA interactions that govern specific biological processes in the chicken reproductive system: MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 regulating sperm motility in the testis; MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 governing immunomodulation in the epididymis; and MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 controlling epididymal sperm maturation and motility.ConclusionsWhole transcriptome sequencing of chicken testis and epididymis screened several key genes and ceRNA regulatory networks, which may be involved in the regulation of epididymal immunity, spermatogenesis and sperm viability through the pathways of MAPK, TGF-β, mTOR, and calcium ion. These findings contribute to our comprehensive understanding of the intricate molecular processes underlying rooster spermatogenesis, maturation and motility.

Dissecting the dynamic cellular transcriptional atlas of adult teleost testis development throughout the annual reproductive cycle.

Teleost testis development during the annual cycle involves dramatic changes in cellular compositions and molecular events. In this study, the testicular cells derived from adult black rockfish at distinct stages - regressed, regenerating and differentiating - were meticulously dissected via single-cell transcriptome sequencing. A continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, was delineated, elucidating the molecular events involved in spermatogenesis. Subsequently, the dynamic regulation of gene expression associated with spermatogonia proliferation and differentiation was observed across spermatogonia subgroups and developmental stages. A bioenergetic transition from glycolysis to mitochondrial respiration of spermatogonia during the annual developmental cycle was demonstrated, and a deeper level of heterogeneity and molecular characteristics was revealed by re-clustering analysis. Additionally, the developmental trajectory of Sertoli cells was delineated, alongside the divergence of Leydig cells and macrophages. Moreover, the interaction network between testicular micro-environment somatic cells and spermatogenic cells was established. Overall, our study provides detailed information on both germ and somatic cells within teleost testes during the annual reproductive cycle, which lays the foundation for spermatogenesis regulation and germplasm preservation of endangered species.

The conserved genetic program of male germ cells uncovers ancient regulators of human spermatogenesis

The conserved genetic program of male germ cells uncovers ancient regulators of human spermatogenesis

The conserved genetic program of male germ cells uncovers ancient regulators of human spermatogenesis.

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.

Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

Busulfan Chemotherapy Downregulates TAF7/TNF-α Signaling in Male Germ Cell Dysfunction.

Background: Busulfan is an FDA-approved alkylating drug used in the chemotherapy of advanced acute myeloid leukemia. The precise mechanisms by which Busulfan kills spermatogonia stem cells (SSCs) are not yet completely understood. Methods: Using a murine model, we evaluated Busulfan-induced apoptosis and DNA damage signaling between testis and ovary tissues. We executed RT-qPCR, analyzed single-nuclei RNA sequencing data and performed in situ hybridization for the localization of the gene expression in the tissues. Results: The results indicate that, in contrast to female germ cells, haploid male germ cells undergo significant apoptosis following Busulfan chemotherapy. Moreover, a gene enrichment analysis revealed that reactive oxygen species may activate the inflammatory response in part through the TNF-α/NF-κB signaling pathway. Interestingly, in the testis, the mRNA levels of TNF-α and TAF7 (TATA box-binding protein-associated factor 7) are downregulated, and testosterone levels suppressed. Mechanistically, the promoter of TNF-α has a conserved motif for binding TAF7, which is necessary for its transcriptional activation and may require further in-depth study. We next analyzed the tumorigenic function of TAF7 and revealed that it is highly overexpressed in several types of human cancers, particularly testicular germ cell tumors, and associated with poor patient survival. Therefore, we executed in situ hybridization and single-nuclei RNA sequencing, finding that less TAF7 mRNA is present in SSCs after chemotherapy. Conclusions: Thus, our data indicate a possible function of TAF7 in the regulation of SSCs and spermatogenesis following downregulation by Busulfan. These findings may account for the therapeutic effects of Busulfan and underlie its potential impact on cancer chemotherapy prognosis.

Влияние послеоперационного гипотиреоза на гормональную регуляцию сперматогенеза и функциональное состояние семенников у экспериментальных животных

Abstract: the aim of the study was to determine the effect of postoperative hypothyroidism on spermatogenesis in experimental dogs. Materials and Methods: experimental studies were performed on 20 mongrel male dogs. The study was randomized, 3 groups were identified. The first group was a control group, 4 dogs served as controls. Hemithyroidectomy was performed in the second group, subtotal thyroidectomy was performed in the third group. The operations were performed under general anesthesia. The testes, thyroid gland and blood were the objects of the study. In the postoperative period, biochemical and histological analyses of the removed thyroid gland tissue and testicles of the examined dogs were performed. The determination of hormones in the blood before and after surgery was carried out using a chemiluminescent analyzer Roche Codas E601 (Switzerland). Results: in the course of the study, in the postoperative period, experimental dogs had a decrease in testosterone in serum by 15% on day 5-8, and from 15% to 30.1% on day 20; in dogs after subtotal thyroidectomy, this indicator was 41.1% on day 20 (p < 0.005). Changes in FSH and prolactin in the blood were different in all groups. The FSH index had a significant increase on day 5-8, and a decrease on day 20, and the prolactin index on the contrary: a decrease on day 5-8 and an increase on day 20. The hormonal activity of the thyroid gland was reduced, especially after subtotal thyroidectomy. Morphological studies have shown a marked decrease in the activity of the spermatogenic layer and interstitial edema of the testes on 5-8 days in both groups. On the 30th day, in both groups after surgery, there was a noticeable decrease in both the spermatogenic layer and the interstitial cells of the testes. Conclusion: the results of experimental studies have shown that as a result of hemithyroidectomy and subtotal thyroidectomy, there is a noticeable change in the regulation of spermatogenesis and structural and functional changes in the testes of experimental animals, especially in the iodine-deficient region. Keywords: postoperative hypothyroidism, spermatogenesis, semens, hormones, hemitireoidectomy.

PSIV-5 Prioritizing candidate genes for seminal traits and residual feed intake in cattle: Integrating QTLome Analysis and RNAseq Datasets

Abstract The QTLome represents the collection of QTLs (quantitative trait loci) documented for each trait within a specific species, detailing the position, effect, and mode of inheritance of each locus. Despite the abundance of known QTLs for productive traits in cattle, only a few genes have been identified as selection markers. This study aimed to prioritize genes within validated QTLs associated with crucial traits in cattle by integrating this information with RNAseq data. Initially, bovine QTLs (n = 161,730) were sourced from the AnimalQTLdb database (version 44), and both graphical and quantitative analyses were conducted using R software. Only annotations within the UMD 3.1 genome were considered, with overlapping QTLs from the same scientific report grouped together. Following quality control measures, the number of QTLs was reduced to 37,146. Validated QTLs were determined by identifying overlapping annotations from various studies or those within a distance of < 1 Mb for the same phenotypic trait, resulting in a total of 3,856 validated QTLs. For gene prioritization, validated QTLs were selected based on semen traits and residual feed intake (RFI). Transcriptomes corresponding to these traits were sourced from NCBI (PRJNA516089 and PRJEB7696). Sequence filtering was performed using the fastp program, and transcript counts were obtained with SALMON software. Sequences were aligned to the Ensembl UMD 3.1 reference genome (version 94), and differential expression analysis was conducted using the R package DESeq2, considering only the group factor (high/low) for each trait (fertility/RFI). Integration of QTLs and transcriptomes utilized the methodology of the cageminer R package, which prioritizes candidate genes within validated QTLs, identifies those grouped in co-expression modules enriched by reference genes, and correlates with the study phenotype (biserial r > 0.2). Reference genes were obtained from Ignatieva et al. (2021 PMID 34290736) and Chen et al. (2021; PMID 33664767). Among the 18 validated QTLs for semen traits, four candidate genes (TSPAN6, QSER1, ADRB2, AKAP4) were identified. Except for ADRB2, these genes exhibited significant differential expression (P < 0.05) in contrasting groups of Holstein cattle. They are associated with semen traits and perform functions such as regulation of spermatogenesis, development, activation, and cell growth. Regarding RFI, 127 validated QTLs yielded 205 prioritized genes, of which 123 showed significant differential expression (P < 0.05) in divergent groups of the Nelore breed. These genes are involved in the immune response and the polymerization and organization of actin filaments, processes that have been reported as biologically relevant to RFI. The implemented strategy was efficient for gene prioritization through the integration of QTLs and RNAseq.

Expression of exosomal microRNAs miR-34a and miR-210 in male infertility: relationship with morphokinetic parameters and sperm DNA fragmentation

Introduction. Infertility affects tens of millions of men and women across the globe. In approximately half of cases, male factors are the cause of infertility. In recent decades, there has been a significant decline in the quality of male ejaculate, which is characterized by reduced sperm concentration and motility. The insufficient diagnostic and prognostic value of routine semen analysis results highlights the challenge of developing effective diagnostic tools and searching for reliable biomarkers of male infertility. One of the most promising approaches may be assessing sperm microRNA expression.Objective. To study the role of sperm exosomal microRNAs miR-34a and miR-210 in the development of male infertility.Materials & methods. The retrospective study included 150 men aged 25 – 49 years; of these, 96 patients were diagnosed with idiopathic infertility. The comparison group consisted of 54 fertile men. To assess the structure and motility of sperm, the results of a standard ejaculate study (WHO, 2021) and computer data analysis using MMC Sperm (MMCSoft, St. Petersburg, Russia) software were used. The degree of DNA fragmentation was assessed using the TUNEL method. To analyze the expression of miR-34a and miR-210, quantitative real-time PCR was performed using the miRCURY LNA SYBR Green PCR Kit («Qiagen» GmbH, Hilden, Germany) and the Rotor-Gene Q PCR product detection system («Qiagen» GmbH, Hilden, Germany).Results. The study of ejaculate using the Computer Assisted Sperm Analysis System (CASA) method revealed a statistically significant relationship between the level of DNA fragmentation of sperm and indicators of the speed of their movement: rectilinear (VSL) (r = -0.522726; p < 0.01), curvilinear (VCL) (r = -0.499096; p < 0.01), along the middle path (VAP) (r = -0.429533; p < 0.01), as well as with the amplitude of lateral head displacement (ALH) (r = -0.294779, p < 0.01), the linearity of their curvilinear path (LIN) (r = -0.385796; p < 0.01), the degree of straight-line movements (STR) (r = -0.268248; p < 0.05) and their progressive mobility (r = -0.411547; p < 0.01). A study of the level of microRNA expression in sperm exosomes revealed a statistically significant decrease in its miRNA-34a pool (p = 0.0116). According to the Chaddock scale, the strength of the correlation between miR-210 expression and the effectiveness of ART programs was moderate (0.437993). The inverse relationship between miR-34a expression and IVF and ICSI results was weak (0.135314).Conclusion. The analysis of exosomal microRNA-34a and microRNA-210, which are involved in the regulation of spermatogenesis, reveals a direct correlation between their variations and changes in the kinetic and morphological parameters of gametes. It also indicates a relationship with the state of DNA fragmentation. These findings suggest varying levels of gene expression among infertile patients, men with proven fertility, and those undergoing assisted reproductive technology (ART) treatments, both with successful and repeated unsuccessful outcomes.

Gene regulation and signaling transduction in mediating the self-renewal, differentiation, and apoptosis of spermatogonial stem cells.

Infertility has become one of the most serious diseases worldwide, and 50% of this disease can be attributed to male-related factors. Spermatogenesis, by definition, is a complex process by which spermatogonial stem cells (SSCs) self-renew to maintain stem cell population within the testes and differentiate into mature spermatids. It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility. Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs. In this review, we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal, differentiation, and apoptosis of SSCs, and we illustrate the networks of genes and signaling pathways in SSC fate determinations. We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways. This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.

Elucidating the Transcriptional States of Spermatogenesis-Joint Analysis of Germline and Supporting Cell, Mice and Human, Normal and Perturbed, Bulk and Single-Cell RNA-Seq.

In studying the molecular underpinning of spermatogenesis, we expect to understand the fundamental biological processes better and potentially identify genes that may lead to novel diagnostic and therapeutic strategies toward precision medicine in male infertility. In this review, we emphasized our perspective that the path forward necessitates integrative studies that rely on complementary approaches and types of data. To comprehensively analyze spermatogenesis, this review proposes four axes of integration. First, spanning the analysis of spermatogenesis in the healthy state alongside pathologies. Second, the experimental analysis of model systems (in which we can deploy treatments and perturbations) alongside human data. Third, the phenotype is measured alongside its underlying molecular profiles using known markers augmented with unbiased profiles. Finally, the testicular cells are studied as ecosystems, analyzing the germ cells alongside the states observed in the supporting somatic cells. Recently, the study of spermatogenesis has been advancing using single-cell RNA sequencing, where scientists have uncovered the unique stages of germ cell development in mice, revealing new regulators of spermatogenesis and previously unknown cell subtypes in the testis. An in-depth analysis of meiotic and postmeiotic stages led to the discovery of marker genes for spermatogonia, Sertoli and Leydig cells and further elucidated all the other germline and somatic cells in the testis microenvironment in normal and pathogenic conditions. The outcome of an integrative analysis of spermatogenesis using advanced molecular profiling technologies such as scRNA-seq has already propelled our biological understanding, with additional studies expected to have clinical implications for the study of male fertility. By uncovering new genes and pathways involved in abnormal spermatogenesis, we may gain insights into subfertility or sterility.

Characterization of expression patterns and dynamic relocation of Notch proteins during acrosome reaction of bull spermatozoa

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.