The essential and conserved role of Cbx3a/HP1γ in Nile tilapia spermatogenesis is clarified, thereby advancing the field of vertebrate reproductive epigenetics and providing a valuable theoretical basis for potential applications in reproductive management, such as improving sperm quality.
Abstract
Heterochromatin Protein 1γ, encoded by the Cbx3 gene, is a crucial epigenetic regulator that plays an essential role in mammalian meiotic progression. However, the functional divergence and conservation of this protein in teleosts—organisms possessing duplicated Cbx3 paralogs due to whole-genome duplication—remain to be elucidated. Building on previous research, we focused on cbx3a in Nile tilapia (Oreochromis niloticus), a significant aquaculture species and an excellent model for teleost reproductive studies, emphasizing its role in spermatogenesis. Expression analysis revealed that Cbx3a is localized to primordial germ cells and is sustained in spermatogonia, spermatocytes, and spermatids during spermatogenesis. CRISPR/Cas9-mediated knockout of cbx3a demonstrated that Cbx3a deficiency induces germ cell apoptosis, meiotic arrest, and sperm defects, including shortened tails and impaired motility, resulting in profound defects in sperm quantity and quality, strongly implying compromised male fertility. Transcriptomic analysis further identified dysregulated molecular pathways, including cytokine signaling and neuroactive ligand–receptor interactions. This provides novel mechanistic insights into HP1γ-mediated epigenetic regulation of meiosis. Notably, cbx3a mutants exhibited phenotypic bifurcation: a subset showed meiotic defects accompanied by sporadic germ cell apoptosis, whereas others underwent full meiotic arrest with pervasive germ cell apoptosis in adult gonads. Collectively, these findings clarify the essential and conserved role of Cbx3a/HP1γ in Nile tilapia spermatogenesis, thereby advancing the field of vertebrate reproductive epigenetics and providing a valuable theoretical basis for potential applications in reproductive management, such as improving sperm quality.
N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes. ALKBH5 is an m6A demethylase that is essential for spermatogenesis. However, its function in meiotic prophase remains elusive. Here, we report that Alkbh5-knockout spermatocytes exhibit normal double-strand break (DSB) formation, homologous recombination and synapsis on autosomes but impaired synapsis between sex chromosomes. ALKBH5 depletion causes a significantly upregulated transcriptome on sex chromosomes and meiotic sex chromosome inactivation (MSCI) defects in spermatocytes at the pachytene stage. Mechanistically, ALKBH5 regulates SETDB1 expression in a post-transcriptional manner. Alkbh5 knockout inhibits SETDB1 protein expression, leading to the decreased SETDB1 signals on sex chromosomes and subsequently inhibiting H3K9me3 to form heterochromatin and silence transcriptional activity. Taken together, our findings identify ALKBH5 as a critical regulator of male meiosis by demonstrating that it is required to maintain proper SETDB1 protein levels, which in turn ensures the faithful execution of MSCI.
Qingqing Chen, Yu Xiang, Yang Song et al.· Communications Biology· 0 citations
Serine proteases are essential for diverse physiological functions, including digestion, blood clotting, immune response, fertilization, and cancer metastasis. Here, we report that CG31200, a predicted serine protease-related protein in Drosophila, plays an essential role in spermatogenesis. The depletion of CG31200 severely impaired male fertility, characterized by the complete absence of mature sperm and disrupted differentiation of spermatogonia. Single-cell RNA sequencing (scRNA-seq) analysis revealed extensive transcriptional dysregulation in both the late spermatogonia cluster and the spermatogonia-to-spermatocyte transitional cluster of CG31200-depleted testes. These changes were characterized by reduced expression of genes involved in flagellar assembly and axoneme formation, together with increased expression of mitochondrial and oxidative phosphorylation-related programs. Functional assays further showed reduced ATP content together with increased ROS levels and LPO in CG31200-knockdown testes, supporting mitochondrial dysfunction and oxidative stress. Additionally, pseudotime analysis indicated that CG31200-knockdown germ cells accumulated in early inferred developmental states. Knockdown Not1, a poly(A)-specific ribonuclease, partially phenocopied the CG31200 RNAi phenotype, and CG31200 depletion was associated with altered Not1 transcript features. Together, these findings indicate that CG31200 depletion disrupts germ-cell differentiation and sperm morphogenesis and is associated with mitochondrial dysfunction, oxidative stress, altered transcriptional states, and defective axoneme formation. This study provides a phenotypic and scRNA-seq framework for understanding the role of SPH-related proteins in Drosophila spermatogenesis.
Yang Fang, Fengchao Zhang, Xiuling Zhang et al.· Insect Science· 0 citations
A homozygous mutation in
RNF220
, encoding a RING-type E3 ubiquitin ligase, is associated with male infertility featuring small-headed sperm, but the precise roles and mechanisms of
RNF220
in spermatogenesis remain elusive. Here, we explored the function of
RNF220
in spermatogenesis using a Stra8-Cre-mediated germ cell-specific conditional knockout mouse model.
RNF220
was highly expressed in mouse spermatocytes, and its germ cell-specific deletion caused male subfertility, accompanied by testicular atrophy, oligozoospermia, and abnormal sperm morphology. Spermatocyte nuclear spreading assay revealed severe defects in homologous chromosome synapsis and DNA damage repair, as well as elevated apoptosis in pachytene spermatocytes. Mechanistically, SYCE1, a core component of the synaptonemal complex central element essential for synapsis, was identified as a testicular target of RNF220. Notably, RNF220 stabilized SYCE1 by antagonizing its ubiquitination mediated by other ubiquitin ligases.
RNF220
deficiency led to reduced SYCE1 protein level and marked increase in its K48-linked polyubiquitination. Furthermore, RNF220-mediated SYCE1 stabilization was independent of its intrinsic ubiquitin ligase activity. Our findings define the essential role of
RNF220
in spermatogenesis and provide novel molecular insights into the pathogenesis of male infertility.
Jing-Mei Cha, Huijuan Lin, Yingqian Xia et al.· Cell Death & Disease· 0 citations
It is shown that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood, and it is proposed that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly.
Dylan M. Lynch, Anastasia A. Labudina, Sarada Ketharnathan et al.· bioRxiv· 0 citations
Infertility frequently arises from defects in germ cells and early embryonic transition. Successful fertilization depends on the developmental competence and molecular integrity of mature gametes from both parents, which are established through tightly coordinated programs of RNA regulation, metabolism, and genome maintenance. In our previous work, we identified Cth1 as a maternally regulated RNA-decay factor essential for early embryonic development, acting through spatiotemporal control of maternal transcript clearance. Interestingly, Cth1 loss of function in adults also resulted in infertility, suggesting an additional and unexplored role during gametogenesis. Here, we extend these findings by defining the gametogenic function of Cth1 in zebrafish. Through detailed phenotypic, cytological and molecular characterization of Cth1 loss-of-function mutants, we show that Cth1 is highly enriched in germ cells and early embryos and is spatio-temporally localized across oogenesis and early development. Loss of Cth1 causes severe defects in early oogenesis and spermatogenesis, resulting in complete infertility in males and females. Mutant germ cells display transcriptomic changes consistent with metabolic and translational dysregulation, increased DNA damage, and striking abnormalities in gamete morphology. Together, these findings identify Cth1 as essential for gamete quality and fertility. Our study suggests a link between RNA decay–mediated regulation of metabolism and genome integrity during germ cell development and reveals disruption of post-transcriptional control as a potential mechanism underlying infertility. Highlights xxxx
Gopal Kushawah, Stephanie H. Nowotarski, Carmichael Carrie et al.· bioRxiv· 0 citations
It is found that knockdown of Sec61β in either germ cells or somatic cells of ovaries causes complete female sterility, with a small size of ovaries from larval to adult stages, significant reduction in germ cell numbers beginning in the late third-instar larval stage and a complete absence of germ cells in pupal and adult ovaries.
Zhi-Xian Cao, Xiao-Min Xiao, Yi-Ming Ji et al.· International Journal of Mol...· 0 citations
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