Clk3 but not Clk4 is essential for post-meiotic transcriptome and splicing remodelling during spermatid maturation and male fertility — ASN Events
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Clk3 but not Clk4 is essential for post-meiotic transcriptome and splicing remodelling during spermatid maturation and male fertility (#541)

Qing Zhang 1 , Rachel A. Ogle 1 , Grace Williams 1 , Georgia Tennant 2 , John Aitken 2 , Song Chen 3 , Rick F. Thorne 3 , Geoffry De Iuliis 2 , Benjamin R. Robinson 1
  1. School of Biomedical Sciences and Pharmacy, College of Health Medicine and Wellbeing, The University of Newcastle, Callaghan, NSW, Australia
  2. School of Science, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, NSW, Australia
  3. Translational Research Institute, Henan Provincial People's Hospital, Zhengzhou University, Zhengzhou, Henan, China

Background: Spermatogenesis requires testicular temperatures 2–4°C below body core. The CLK kinase family regulates alternative splicing and exhibits temperature-sensitive activity, yet their tissue-specific functions remain incompletely defined. Notably, CLK3 and CLK4 are selectively expressed in heat-sensitive meiotic spermatocytes and post-meiotic spermatids, suggesting specialised roles in germ cell differentiation and maturation. However, the functions of individual CLK family members in mammalian spermatogenesis remain unknown.

Aims: We hypothesized that CLK3 and CLK4 are essential for spermatocyte maturation and haploid differentiation, and that their loss disrupts splicing programs required for sperm development, leading to male infertility. To test this, we generated conditional knockouts targeting Clk3 and Clk4 in the male germline and assessed their impacts on fertility, testicular histology, germ cell development, and global splicing.

Methods: We generated global knockout models targeting Clk3 and Clk4. Testes from adult wild-type and knockout mice were examined by histology. Round spermatids were FACS-isolated from wild-type and Clk3 knockout testes and subjected to RNA-seq (WT n=4, KO n=3), followed by differential expression and alternative splicing analyses.

Results: Histology revealed that Clk3 knockout, but not Clk4 knockout, causes severe post-meiotic defects, including impaired spermatid differentiation and germ cell sloughing, consistent with CLK3 expression in post-meiotic germ cells. RNA-seq of round spermatids from Clk3 knockout testes identified 1,209 differentially expressed genes (389 upregulated, 820 downregulated) and 3,280 significant alternative splicing events across 1,969 genes. Pathway analyses enriched for spermatid differentiation, ciliary function, RNA processing, and cytoskeletal organization.

Conclusion: CLK3 is essential for spermatogenesis and regulates post-meiotic germ cell development through widespread effects on expression and splicing, whereas CLK4 is dispensable. These findings identify CLK3 as a critical regulator of male fertility.