Rnf32 is not essential for spermatogenesis and male fertility in mice

Introduction

Recent epidemiological studies indicate that infertility affects nearly 15% of reproductive-age couples in China, with a persistent upward trend observed over the past two decades (De Kretser & Baker, 1999; Hackstein, Hochstenbach & Pearson, 2000). Male-related factors account for over 50% of these cases, predominantly linked to disrupted spermatogenic processes (Barratt et al., 2017). Clinically, such dysregulation manifests as compromised sperm parameters, including reduced counts, impaired motility, and morphological defects (McLachlan et al., 2007). Therefore, understanding the molecular mechanisms governing germ cell development represents a critical frontier in addressing male reproductive disorders. Spermatogenesis in mammals such as mice is a highly coordinated process of germ cell development, predominantly occurs in the seminiferous tubules through three key phases: spermatogonial stem cell self-renewal and differentiation, meiotic division of spermatocytes, and post-meiotic spermiogenesis involving cytoplasmic remodeling (Chen et al., 2018). Mature sperm released into the tubular lumen undergo functional maturation during epididymal transit, acquiring progressive motility and fertilization competence (Cooper, 2007; Dacheux & Dacheux, 2014). Final activation events, including capacitation and acrosome reaction, occur in the female reproductive tract to enable successful oocyte fusion (Ickowicz, Finkelstein & Breitbart, 2012; Saint-Dizier et al., 2020).

RPL39L, a germline-specific ribosomal protein, exhibits stage-specific expression in pachytene spermatocytes and post-meiotic germ cells (round/elongated spermatids). Phenotypic analysis of RPL39L-deficient mice revealed impaired sperm quality parameters (morphology, count, and motility), confirming its essential role in male reproductive competence. Proteomic analysis further revealed that the silencing of RPL39L induces the coordinated downregulation of 276 cellular proteins (Zou et al., 2021; Li et al., 2022). Among these downregulated proteins, we specifically focused on Rnf32, hypothesizing that it may also be a component of the ribosomal or translational regulatory network and is specifically expressed in the testis. Given its potential role and testis-specific expression, we propose that Rnf32, like RPL39L, may play a critical role in male reproduction. This chromosome 7q36-located gene encodes a 362-amino acid protein containing two RING-H2 zinc-binding domains flanking a central IQ calmodulin-binding motif. Notably, Rnf32 represents the first reported gene encoding a dual RING-H2 domain protein in mammals (Van Baren et al., 2002), positioning it as a unique model for investigating ubiquitination-related regulatory networks in reproductive biology.

Transcript analysis of Rnf32 splice variants revealed distinct functional impacts: Exon 1 variations maintain the open reading frame (ORF) due to the initiation codon’s exclusive localization in exon 2. However, a premature termination codon in exon 3a causes protein truncation upstream of the first RING domain. Furthermore, extended exon 4 transcripts contain a 16-base termination signal immediately downstream of the canonical splice site, resulting in ORF truncation within the first RING-H2 domain and producing a 145-amino acid polypeptide (Van Baren et al., 2002; Chasapis & Spyroulias, 2009). Tissue-specific expression profiling demonstrated Rnf32’s predominant transcription in gonadal tissues, with stage-specific upregulation during spermatogenesis. This spatiotemporal expression pattern, particularly in meiotic spermatocytes and post-meiotic germ cells, strongly suggests its functional involvement in gamete maturation (Yu et al., 2003; Guo et al., 2004). Despite our existing understanding of Rnf32, its specific role in spermatogenesis remains enigmatic. To further explore this issue, this study adopted the advanced CRISPR/Cas9 technology and successfully constructed a Rnf32 gene knockout mouse model. With the help of this model, we hope to unambiguously reveal the physiological role played by Rnf32 in the complex mechanism of spermatogenesis.

Materials & Methods

Results

The expression of Rnf32 and the generation of Rnf32^−/− mice

Rnf32 is highly conserved in mammals (Van Baren et al., 2002). Therefore, studying its reproductive effects on mice provides critical insights into human reproductive pathologies. To explore its biological roles, we first performed quantitative PCR (qPCR) to analyze tissue-specific Rnf32 expression profiles in mice. This analysis identified predominant Rnf32 expression within the male reproductive system, with testicular tissue showing significantly higher transcript levels compared to other examined organs (Fig. 1A). Developmental time-course analysis revealed stage-specific upregulation, with Rnf32 expression peaking at postnatal week 3 (Fig. 1B), a critical period corresponding to spermatid differentiation during murine testicular maturation (Gong et al., 2013; Nishimura & L’Hernault, 2017; Chen et al., 2024). To mechanistically investigate Rnf32’s role in gametogenesis, we engineered a CRISPR/Cas9-mediated exon 7 in-frame deletion (Fig. 1C) through microinjection of sgRNA/Cas9 complexes into superovulated zygotes. Exon 7 was selected as the target site because it encodes a critical structure motif within the zinc finger domain in the Rnf32. By specifically targeting this domain for disruption, we have effectively compromised the structural integrity and consequently maximally impaired the functional capacity of this protein. Sanger sequencing confirmed successful generation of Rnf32^−/− mice, revealing a precise 35-bp deletion within exon 7 (Fig. 1D). Transcript analysis via RT-PCR confirmed efficient Rnf32 knockout, demonstrating significantly reduced mRNA levels in testicular tissue from Rnf32^−/− animals compared to wild-type controls (Fig. 1E).

Rnf32^−/− mice are fertile

We conducted an examination of testicular morphology and testicular/body weight ratios in Rnf32-KO male mice, finding no statistically significant difference compared to the control group (Figs. 2E–2D). Following this, we carried out fertility tests to investigate the impact of Rnf32 on mouse fertility, counting the pups and litters in both the experimental and control groups. The findings indicated that there was no statistically significant disparity in fertility between the WT and KO mice (Fig. 2E). In addition, we performed H&E staining on the testis, revealing normal spermatogenic cells of all stages of adult (postnatal 8w) Rnf32^−/− mice (Fig. 3A).

The knockout of Rnf32 does not impair spermatogenesis

Spermatogenesis, the fundamental biological process of germ cell differentiation, proceeds through precisely coordinated developmental stages in murine testes. The seminiferous epithelial cycle is classically categorized into 12 distinct phases based on spermatid maturation (Abe, Shen & Takano, 1991; Hess & Renato De Franca, 2008; Griswold, 2016), as defined by Bouin’s fixative (picric acid/formaldehyde/acetic acid)-preserved tissue analysis coupled with periodic acid-Schiff (PAS)-hematoxylin staining for precise staging of seminiferous epithelial cycles. To evaluate potential spermatogenic disruptions in Rnf32^+/+ and Rnf32^−/− mice, we systematically analyzed germ cell cytoarchitecture across all 12 epithelial stages. Histological assessment revealed preserved seminiferous tubule organization in knockout testes, displaying complete germ cell lineages—including spermatogonia, spermatocytes, and elongating spermatids—with normal spatiotemporal progression of germ cell maturation indistinguishable from wild-type controls (Fig. 3B). Immunofluorescence quantification demonstrated comparable expression of SOX9, a Sertoli cell-specific transcription factor essential for spermatogonial niche maintenance, between adult Rnf32^+/+ and Rnf32^−/− mice (Figs. 4A–4B). Quantitative data can be found in the Figs. S1A–S1B. Furthermore, TUNEL assay revealed statistically comparable rates of luminal germ cell apoptosis between adult Rnf32 ^+/+ and Rnf32^−/− mice (Figs. 4C–4D). Quantitative data can be found in the Figs. S1C–S1D.

The knockout of Rnf32 has no impact on sperm counts, morphology and motility

To investigate the potential impact of Rnf32 deficiency on sperm maturation in the epididymis, histopathological evaluation of the cauda epididymidis was performed. Comparative analysis demonstrated conserved sperm concentration in Rnf32^−/− epididymal luminal contents relative to wild-type controls (Fig. 5A). Ultrastructural assessment via differential interference contrast microscopy revealed preserved spermatozoal morphology in mutant mice, including typical head curvature and flagellar architecture (Fig. 5B). Quantitative data can be found in the (Fig. S1E). Computer-assisted semen analysis (CASA) was employed to quantify sperm kinematic parameters, such as count, motility, and progressive motility. The results indicated that there were no significant disparities in these metrics between Rnf32 ^+/+ and Rnf32^−/− mice (Figs. 5C–5E).

Discussion

The utilization of genetically engineered mouse models (GEMMs) has profoundly enhanced our mechanistic understanding of reproductive developmental disorders associated with human infertility. Systematic interrogation of germline-specific gene ablation models has provided critical insights into the regulatory networks governing stem cell self-renewal, meiotic progression, and spermatogenic differentiation (Cooke & Saunders, 2002; Gilbert, Roof & Rajendra Kumar, 2018; Azhar et al., 2021). In this study, the generation of a CRISPR/Cas9-mediated Rnf32 knockout murine model was achieved, enabling comprehensive phenotypic characterization. Comparative analysis revealed no significant genotypic differences in reproductive competence between Rnf32^−/− and wild-type (WT) males. These findings demonstrate that Rnf32 is dispensable for murine male fertility, aligning with emerging evidence that numerous testis-enriched genes exhibit functional redundancy in reproductive contexts (Lv et al., 2022; Chen et al., 2022; Meng et al., 2023). Importantly, this study precludes RNF32 as a viable contraceptive target or monogenic infertility factor, thereby optimizing research resource allocation by eliminating nonproductive investigative pathways.

While Rnf32 appears dispensable for baseline spermatogenesis, we hypothesize that Rnf32 may have a potential cytoprotective role in maintaining male fertility, particularly under conditions of environmental toxicity. Notably, certain genetic regulators, exemplified by the melanoma antigen (MAGE) family genes, demonstrate functional specialization in xenobiotic detoxification pathways. Although MAGE proteins are nonessential for physiological gametogenesis, they confer resilience against toxicant-induced spermatogenic impairment by modulating oxidative stress response cascades (Fon Tacer et al., 2019; Florke Gee et al., 2020; Li et al., 2023). Intriguingly, Rnf32 shares evolutionary conserved structural motifs with these xenobiotic-responsive factors, suggesting possible involvement in analogous molecular mechanisms. We postulate that Rnf32 may activate compensatory proteostatic networks under specific pathological conditions, thereby maintaining genomic integrity during spermatogonial stem cell proliferation and meiotic recombination. This hypothetical safeguard mechanism could explain the preserved fertility observed in Rnf32-deficient mice under controlled laboratory conditions while highlighting its potential clinical relevance in environmentally compromised reproductive scenarios. Another possibility is that functionally related genes may compensate for the loss of Rnf32 upon its knockout. As Rnf32 is a member of the extensive RING finger protein family, characterized by conserved E3 ubiquitin ligase activity, it is plausible that structurally homologous family members exhibiting spatiotemporal expression overlap with Rnf32 may functionally compensate for the absence of Rnf32 through redundant or complementary enzymatic mechanisms. While our study employed a multi-modal approach to evaluate reproductive function—including fertility metrics (litter size) and spermatogenesis endpoints (sperm count, motility, histopathology)—the limited cohort size (n = 3 per group) constrains statistical power and generalizability. Additionally, our observations were confined to male mice.

Conclusions

Despite robust Rnf32 expression in murine testicular tissue, genetic ablation of this locus failed to induce discernible morphological abnormalities in testicular dimensions or spermatogenic cell populations. Crucially, comparative analysis of breeding performance revealed preserved reproductive capacity in Rnf32-KO males relative to wild-type counterparts.

Supplemental Information