Primate-specific sperm lnc-CLCN7 reveals embryo quality in IVF

Abstract

Long non-coding RNAs (lncRNAs) are an essential class of regulatory molecules that participate in diverse biological processes. However, whether sperm-derived lncRNAs from infertile men contribute to impaired embryo development during in vitro fertilization (IVF) remains unclear. In this study, we investigate the lncRNA expression profiles in sperm from asthenozoospermic patients with poor embryo development and explore their potential roles in early embryo development. Microarray analyses identify 993 differentially expressed lncRNAs in sperm samples from these patients, including 626 downregulated and 367 upregulated probes. Among them, an antisense transcript, lnc-CLCN7, is validated as the most significantly dysregulated lncRNA in an expanded cohort. In situ hybridization demonstrates that lnc-CLCN7 is localized in spermatogenic cells of primate testes and within the nuclei of HTR-8 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses reveal that lnc-CLCN7 is associated with the regulation of ion transport, ion homeostasis and related signaling pathways. Further experiments demonstrate that Lnc-CLCN7 directly binds to the histone modification H3K9me2/3 in HTR-8 cells and that its expression in sperm is modulated by oxidative stress induced by H2O2 treatment. Additionally, dysregulation of the glycolysis/gluconeogenesis and pyrimidine metabolism pathways in sperm is found to contribute to poor embryo development. Collectively, our findings identify Lnc-CLCN7 as an H3K9me2/3-binding, oxidative stress–responsive lncRNA that may serve as a potential biomarker for predicting poor embryo development in IVF and provide new insights into the molecular mechanisms linking sperm RNA regulation to embryo quality.

1. Introduction

Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are more than 200 nucleotides in length. Increasing evidence has underscored the pivotal roles of lncRNAs in a variety of biological processes, including carcinogenesis, neurodevelopment, and spermatogenesis [1]. Emerging studies have further elucidated the extensive involvement of lncRNAs in reproductive biology, with particular emphasis on their regulatory functions in spermatogenesis and early embryonic development [2–4]. In the context of spermatogenesis, the expression of lncRNAs is strongly correlated with mRNA expression during the transition from spermatogonial stem cells (SSCs) to type A spermatogonia. These findings suggest that lncRNAs play a dynamic role in regulating their associated genes, thereby contributing to the precise orchestration of spermatogenesis [5].

LncRNAs regulate gene expression through a variety of mechanisms [4,6,7]. For example, lncRNAs can function as molecular decoys by binding to DNA-binding proteins, thereby obstructing the access of transcription factors to specific genomic regions and suppressing transcription. Alternatively, lncRNAs may act as scaffolds, promoting the assembly of protein complexes that modulate their functional activities. Additionally, lncRNAs can serve as guides, directing histone-modifying enzymes to particular genomic loci, thereby influencing chromatin states and regulating gene expression [7,8]. Furthermore, lncRNAs interact directly with other RNA molecules, thereby participating in RNA processing and maturation [1]. These diverse and multifaceted regulatory mechanisms highlight the functional versatility of lncRNAs across a broad range of biological processes [1].

Human preimplantation embryo development is a pivotal biological process that lays the groundwork for successful implantation and subsequent fetal development. Increasing evidence underscores the significant role of paternal factors, particularly sperm quality, in early embryogenesis, as demonstrated in both murine and human models [9,10]. Studies utilizing intracytoplasmic sperm injection (ICSI) have further revealed that oxidative stress in sperm can severely impair early embryonic development [10]. Mechanistic studies have revealed that phospholipase C zeta (PLCζ), a testis-specific enzyme involved in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), is critical for sperm-oocyte fusion via calcium-mediated signaling pathways [11]. Additionally, emerging research indicates that both gametes and their associated follicular or a variety of non-coding RNAs (ncRNAs) may play essential regulatory roles in early embryogenesis. For example, epididymal-acquired...