Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth.

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.

CFTR Exon 10 deleterious mutations in patients with congenital bilateral absence of vas deferens in a cohort of Pakistani patients.

Congenital bilateral absence of vas deferens (CBAVD) is a urological syndrome of Wolffian ducts and is responsible for male infertility and obstructive azoospermia. This study is designed to explore the integrity of exon 10 of CFTR and its role in male infertility in a cohort of CBVAD patients in Pakistan. Genomic DNA was extracted from 17 male patients with CBAVD having clinical symptoms, and 10 healthy controls via phenol-chloroform method. Exon 10 of the CFTR gene was amplified, using PCR with specific primers and DNA screening was done by Sanger sequencing. Sequencing results were analyzed using freeware Serial Cloner, SnapGene, BioEdit and FinchTV. Furthermore, bioinformatics tools were used to analyze the mutations and their impact on the protein function and stability. We have identified 4 mutations on exon 10 of CFTR in 6 out of 17 patients. Two of the mutations were missense variants V456A, K464E, and the other two were silent mutations G437G, S431S. The identified variant V456A was present in 4 of the studied patients. Whereas, the presence of K464E in our patients further weighs on the crucial importance for its strategic location to influence the gene function at post-transcriptional and protein level. Furthermore, Polyphen-2 and SIFT analyze the mutations as harmful and deleterious. The recurrence of V456A and tactically conserved locality of K464E are evidence of their potential role in CBAVD patients and in male infertility. The data can contribute in developing genetic testing and treatment of CBAVD.

A partial deletion within the meiosis-specific sporulation domain SPO22 of Tex11 is not associated with infertility in mice.

Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.

Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice.

Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.

A homozygous missense variant in YTHDC2 induces azoospermia in two siblings.

Male infertility is a complex multifactorial reproductive disorder with highly heterogeneous phenotypic presentations. Azoospermia is a medically non-manageable cause of male infertility affecting ∼1% of men. Precise etiology of azoospermia is not known in approximately three-fourth of the cases. To explore the genetic basis of azoospermia, we performed whole exome sequencing in two non-obstructive azoospermia affected siblings from a consanguineous Pakistani family. Bioinformatic filtering and segregation analysis of whole exome sequencing data resulted in the identification of a rare homozygous missense variant (c.962G>C, p. Arg321Thr) in YTHDC2, segregating with disease in the family. Structural analysis of the missense variant identified in our study and two previously reported functionally characterized missense changes (p. Glu332Gln and p. His327Arg) in mice showed that all these three variants may affect Mg2+ binding ability and helicase activity of YTHDC2. Collectively, our genetic analyses and experimental observations revealed that missense variant of YTHDC2 can induce azoospermia in humans. These findings indicate the important role of YTHDC2 deficiency for azoospermia and will provide important guidance for genetic counseling of male infertility.

The non-canonical bivalent gene Wfdc15a controls spermatogenic protease and immune homeostasis.

Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility.

Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility.

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.

Whole exome sequencing analysis of 167 men with primary infertility.

BACKGROUND: Spermatogenic failure is one of the leading causes of male infertility and its genetic etiology has not yet been fully understood. METHODS: The study screened a cohort of patients (n = 167) with primary male infertility in contrast to 210 normally fertile men using whole exome sequencing (WES). The expression analysis of the candidate genes based on public single cell sequencing data was performed using the R language Seurat package. RESULTS: No pathogenic copy number variations (CNVs) related to male infertility were identified using the the GATK-gCNV tool. Accordingly, variants of 17 known causative (five X-linked and twelve autosomal) genes, including ACTRT1, ADAD2, AR, BCORL1, CFAP47, CFAP54, DNAH17, DNAH6, DNAH7, DNAH8, DNAH9, FSIP2, MSH4, SLC9C1, TDRD9, TTC21A, and WNK3, were identified in 23 patients. Variants of 12 candidate (seven X-linked and five autosomal) genes were identified, among which CHTF18, DDB1, DNAH12, FANCB, GALNT3, OPHN1, SCML2, UPF3A, and ZMYM3 had altered fertility and semen characteristics in previously described knockout mouse models, whereas MAGEC1,RBMXL3, and ZNF185 were recurrently detected in patients with male factor infertility. The human testis single cell-sequencing database reveals that CHTF18, DDB1 and MAGEC1 are preferentially expressed in spermatogonial stem cells. DNAH12 and GALNT3 are found primarily in spermatocytes and early spermatids. UPF3A is present at a high level throughout spermatogenesis except in elongating spermatids. The testicular expression profiles of these candidate genes underlie their potential roles in spermatogenesis and the pathogenesis of male infertility. CONCLUSION: WES is an effective tool in the genetic diagnosis of primary male infertility. Our findings provide useful information on precise treatment, genetic counseling, and birth defect prevention for male factor infertility.

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

TUBB4B is essential for the expansion of differentiating spermatogonia.

Microtubules, polymers of αß-tubulin heterodimers, are essential for various cellular processes. The incorporation of different tubulin isotypes, each encoded by distinct genes, is proposed to contribute to the functional diversity observed in microtubules. However, the functional roles of each tubulin isotype are not completely understood. In this study, we investigated the role of the ß4B-tubulin isotype (Tubb4b) in spermatogenesis, utilizing a Tubb4b knockout mouse model. We showed that ß4B-tubulin is expressed in the germ cells throughout spermatogenesis. ß4B-tubulin was localized to cytoplasmic microtubules, mitotic spindles, manchette, and axonemes of sperm flagella. We found that the absence of ß4B-tubulin resulted in male infertility and failure to produce sperm cells. Our findings demonstrate that a lack of ß4B-tubulin leads to defects in the initial stages of spermatogenesis. Specifically, ß4B-tubulin is needed for the expansion of differentiating spermatogonia, which is essential for the subsequent progression of spermatogenesis.

A homozygous ARMC3 splicing variant causes asthenozoospermia and flagellar disorganization in a consanguineous family.

Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.

The functions and mechanisms of piRNAs in mediating mammalian spermatogenesis and their applications in reproductive medicine.

As the most abundant small RNAs, piwi-interacting RNAs (piRNAs) have been identified as a new class of non-coding RNAs with 24-32 nucleotides in length, and they are expressed at high levels in male germ cells. PiRNAs have been implicated in the regulation of several biological processes, including cell differentiation, development, and male reproduction. In this review, we focused on the functions and molecular mechanisms of piRNAs in controlling spermatogenesis, including genome stability, regulation of gene expression, and male germ cell development. The piRNA pathways include two major pathways, namely the pre-pachytene piRNA pathway and the pachytene piRNA pathway. In the pre-pachytene stage, piRNAs are involved in chromosome remodeling and gene expression regulation to maintain genome stability by inhibiting transposon activity. In the pachytene stage, piRNAs mediate the development of male germ cells via regulating gene expression by binding to mRNA and RNA cleavage. We further discussed the correlations between the abnormalities of piRNAs and male infertility and the prospective of piRNAs' applications in reproductive medicine and future studies. This review provides novel insights into mechanisms underlying mammalian spermatogenesis and offers new targets for diagnosing and treating male infertility.

A novel homozygous mutation in the DNAAF3 gene leads to severe asthenozoospermia and teratospermia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder characterized by ultrastructural defects in the cilia or flagella of cells, causing respiratory abnormalities, sinusitis, visceral transposition, and male infertility. DNAAF3 plays an important role in the assembly and transportation of axonemal dynein complexes in cilia or flagella and has been shown to be associated with PCD. To date, only two cases of PCD with infertility associated with DNAAF3 mutations have been reported, and no mouse models for this gene have been successfully constructed. This study was conducted on an infertile Chinese male patient with a history of bronchitis. Examination of the patient's semen revealed severe asthenozoospermia and teratospermia. Whole exome sequencing revealed a new homozygous loss-of-function DNAAF3 mutation. CRISPR-Cas9 gene-editing technology was used to construct the same mutation in C57/B6 mice, revealing that homozygous C57/B6 mice were characterized by severe hydrocephalus and early death. The results of this study expand the mutation spectrum of DNAAF3 and confirm its correlation with PCD pathogenesis. This study provides new insights on the mechanisms underlying male infertility related to DNAAF3 mutation and PCD.

Decoding the pathogenesis of spermatogenic failure in cryptorchidism through single-cell transcriptomic profiling.

Cryptorchidism, commonly known as undescended testis, affects 1%-9% of male newborns, posing infertility and testis tumor risks. Despite its prevalence, the detailed pathophysiology underlying male infertility within cryptorchidism remains unclear. Here, we profile and analyze 46,644 single-cell transcriptomes from individual testicular cells obtained from adult males diagnosed with cryptorchidism and healthy controls. Spermatogenesis compromise in cryptorchidism links primarily to spermatogonium self-renewal and differentiation dysfunctions. We illuminate the involvement of testicular somatic cells, including immune cells, thereby unveiling the activation and degranulation of mast cells in cryptorchidism. Mast cells are identified as contributors to interstitial fibrosis via transforming growth factor ß1 (TGF-ß1) and cathepsin G secretion. Furthermore, significantly increased levels of secretory proteins indicate mast cell activation and testicular fibrosis in the seminal plasma of individuals with cryptorchidism compared to controls. These insights serve as valuable translational references, enriching our comprehension of testicular pathogenesis and informing more precise diagnosis and targeted therapeutic strategies for cryptorchidism.

Sperm DNA Fragmentation: Unraveling Its Imperative Impact on Male Infertility Based on Recent Evidence.

Male factors may be present in up to 50-70% of infertile couples and the prevalence of male infertility accounts for 20-30% of infertility cases. Understanding the mechanisms and causes behind male infertility remains a challenge, but new diagnostic tools such as DNA fragmentation might aid in cases where the routine semen analysis is insufficient. DNA fragmentation, which refers to damages or breaks of the genetic material of the spermatozoa, is considered one of the main causes of male infertility due to impaired functional capability of sperm. The aim of the present narrative review is to investigate and enlighten the potential correlation between DNA fragmentation and male infertility parameters such as the seminal profile and the reproductive outcomes. Comprehensive research in PubMed/Medline and Scopus databases was conducted and 28 studies were included in the present review. Fourteen studies provided data regarding the impact of DNA fragmentation and seminal parameters and showed a correlation of significantly lower sperm count, lower concentration, motility, and abnormal morphology with an increased DNA fragmentation index (DFI). Similarly, 15 studies provided data regarding the impact of DFI on reproductive outcomes. Two studies showed higher aneuploidy rates with higher DFI values, and seven studies showed significantly lower pregnancy rates and live birth rates with higher DFI values. Ultimately, the studies included in this review highlight, collectively, the importance of measuring sperm DFI in the assessment of male infertility. Further studies are needed to explore the effectiveness of interventions aiming to reduce DFI levels.

Loss-of-function variant in TDRD6 cause male infertility with severe oligo-astheno-teratozoospermia in human and mice.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, but the genetic basis of most OAT cases is still unknown. Here, one homozygous loss-of-function (LOF) variant in TDRD6, c.G1825T/p.Gly609X, was identified in an infertile patient with severe OAT by whole-exome sequencing (WES) and Sanger confirmation. Furthermore, Tdrd6-mutant mice (p.Gly615X; equivalent to p.Gly609X in human TDRD6) were generated. Remarkably, the Tdrd6-mutated mice mimicked the severe OAT symptoms of the patient. In addition, the architecture of chromatoid bodies (CBs) were disrupted in round spermatids from Tdrd6-mutant mice, leading to blocked spermatogenesis in the round spermatids. The assembly of PIWIL1, TDRD1, TDRD7 and DDX25 in CBs was disturbed in the Tdrd6-mutant mice. Applying immunoprecipitation-mass spectrometry (IP-MS), we identified some TDRD6-interacting partners, including CB proteins TDRD7, MAEL and PCBP1. Moreover, we described the assisted reproductive technology (ART) outcomes of the infertile patient and his partner. Altogether, our findings provide necessary evidences to support the idea that the homozygous LOF variant in TDRD6 induces male infertility with severe OAT, suggesting that TDRD6 could be a useful genetic diagnostic target for male infertility.

The Protective Role of L-Cysteine in the Regulation of Blood-Testis Barrier Functions-A Brief Review.

Blood-testis barrier (BTB) genes are crucial for the cellular mechanisms of spermatogenesis as they protect against detrimental cytotoxic agents, chemicals, and pathogens, thereby maintaining a sterile environment necessary for sperm development. BTB proteins predominantly consist of extensive tight and gap junctions formed between Sertoli cells. These junctions form a crucial immunological barrier restricting the intercellular movement of substances and molecules within the adluminal compartment. Epithelial tight junctions are complex membrane structures composed of various integral membrane proteins, including claudins, zonula occludens-1, and occludin. Inter-testicular cell junction proteins undergo a constant process of degradation and renewal. In addition, the downregulation of genes crucial to the development and preservation of cell junctions could disrupt the functionality of the BTB, potentially leading to male infertility. Oxidative stress and inflammation may contribute to disrupted spermatogenesis, resulting in male infertility. L-cysteine is a precursor to glutathione, a crucial antioxidant that helps mitigate damage and inflammation resulting from oxidative stress. Preclinical research indicates that L-cysteine may offer protective benefits against testicular injury and promote the expression of BTB genes. This review emphasizes various BTB genes essential for preserving its structural integrity and facilitating spermatogenesis and male fertility. Furthermore, it consolidates various research findings suggesting that L-cysteine may promote the expression of BTB-associated genes, thereby aiding in the maintenance of testicular functions.

Screening of the Combined Risk of Genetics and Epidemiology on Infertility Among Indian Men: Synergistic Effect of AZFc Partial Deletions and Habits of Smokeless Chewing Tobacco.

The AZFc partial deletions of Y chromosome and lifestyle/epidemiological factors such as the use of smokeless chewing tobacco (SCT) exhibit intriguing variations in their association with male infertility across the population, ethnicity, and genetic background. Here, a pioneering attempt has been made to elucidate the interactions of such deletions with the habits of SCT consumption among the participating individuals, using their large epidemiological data. This screening program was conducted among Bengali-speaking men in West Bengal, India. We screened the prevalence and association of distinct partial deletions (gr/gr, b1/b3, and b2/b3) of the AZFc region using locus-specific sequence-tagged site (STS) markers among 728 case subjects and compared them with 264 ethnicity- and age-matched proven-fertile control men. The recorded epidemiological data of the study group and the outcome of partial deletion analysis were compiled to frame the plausible Gene × Epidemiological factor (G × E) interactions. The gr/gr deletion was reported to be significantly associated with azoospermic (p = .0015, odds ratio [OR] = 3.413) and oligozoospermic (p = .0382, OR = 3.012) case subgroups, and b1/b3 deletions were also detected among the infertile persons only. The G × E model revealed that men who carried microdeletions as well as were SCT users had an elevated risk of infertility (p = .002, OR = 6.38). The study highlights the fact that AZFc partial deletions and SCT, when co-occurred, synergistically increase the risk of infertility among men. This work helps to get more insight into the etiology of male infertility in the light of gene-environmental interaction.

Conditional ablation of DIS3L2 ribonuclease in pre-meiotic germ cells causes defective spermatogenesis and infertility in male mice.

Rationale: Spermatogenesis is a highly organized cell differentiation process in mammals, involving mitosis, meiosis, and spermiogenesis. DIS3L2, which is primarily expressed in the cytoplasm, is an RNA exosome-independent ribonuclease. In female mice, Dis3l2-deficient oocytes fail to resume meiosis, resulting in arrest at the germinal vesicle stage and complete infertility. However, the role of DIS3L2 in germ cell development in males has remained largely unexplored. Methods: We established a pre-meiotic germ cell conditional knockout mouse model and investigated the biological function of DIS3L2 in spermatogenesis and male fertility through bulk RNA-seq and scRNA-seq analyses. Results: This study unveils that conditional ablation of Dis3l2 in pre-meiotic germ cells with Stra8-Cre mice impairs spermatogonial differentiation and hinders spermatocyte meiotic progression coupled with cell apoptosis. Such conditional ablation leads to defective spermatogenesis and sterility in adults. Bulk RNA-seq analysis revealed that Dis3l2 deficiency significantly disrupted the transcriptional expression pattern of genes related to the cell cycle, spermatogonial differentiation, and meiosis in Dis3l2 conditional knockout testes. Additionally, scRNA-seq analysis indicated that absence of DIS3L2 in pre-meiotic germ cells causes disrupted RNA metabolism, downregulated expression of cell cycle genes, and aberrant expression of spermatogonial differentiation genes, impeding spermatogonial differentiation. In meiotic spermatocytes, loss of DIS3L2 results in disturbed RNA metabolism, abnormal translation, and disrupted meiotic genes that perturb meiotic progression and induce cell apoptosis, leading to subsequent failure of spermatogenesis and male infertility. Conclusions: Collectively, these findings highlight the critical role of DIS3L2 ribonuclease-mediated RNA degradation in safeguarding the correct transcriptome during spermatogonial differentiation and spermatocyte meiotic progression, thus ensuring normal spermatogenesis and male fertility.