Too hot to function: insights into the role of thermosensitive Doa kinase in spermatogenesis and testicular heat stress — ASN Events
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  3. Too hot to function: insights into the role of thermosensitive Doa kinase in spermatogenesis and testicular heat stress

Too hot to function: insights into the role of thermosensitive Doa kinase in spermatogenesis and testicular heat stress (#88)

Grace E Williams 1 2 , Rippei Hayashi 3 , Fernando Wijaya 4 , Rachel A Ogle 1 2 , Heather C Murray 1 , Georgia M Tennant 2 5 , Gary R Hime 4 , Mark A Baker 6 , Robert J Aitken 2 5 , Benjamin R Robinson 1 2
  1. School of Biomedical Sciences and Pharmacy , The University of Newcastle, Callaghan , NSW , Australia
  2. Hunter Medical Research Institute Reproductive and Family Health Research Program , New Lambton Heights , NSW, Australia
  3. John Curtin School of Medical Research, Australian National University , Acton, ACT, Australia
  4. School of Biomedical Sciences, University of Melbourne, Parkville, VIC, Australia
  5. School of Science, The University of Newcastle , Callaghan, NSW, Australia
  6. Independent Researcher (no current affiliation) , Newcastle, NSW, Australia

Background: Male infertility is a growing concern, with environmental, genetic and physiological factors impairing sperm production and function [1, 2]. Spermatogenesis is highly sensitive to elevated temperature, with heat stress disrupting fertility across species [3-6]. However, the molecular pathways underlying this conserved sensitivity remain poorly understood.

Cdc2-like kinases (CLKs) are conserved dual-specificity kinases that regulate RNA processing by phosphorylating serine/arginine-rich proteins [7]. In Drosophila melanogaster, the CLK orthologue Darkener of apricot (DOA) is essential for spermatogenesis, with loss-of-function mutations disrupting germ-cell development  [8]. Importantly, DOA activity decreases at elevated physiological temperatures, with its thermosensitive enzymatic profile paralleling the temperature sensitivity of spermatogenesis [4, 9]. Here, we investigated whether reduced germline DOA function contributes to heat-induced spermatogenic dysfunction.

Methods: Germline-specific doa knockdown was generated using a nos-GAL4/UAS-RNAi system. Control and doa-knockdown males were raised at 25°C, 27°C or 29°C. Reproductive output and reproductive lifespan were assessed by sequentially mating individual males with virgin females for the entire reproductive lifespan (n=7/genotype/temperature), with average output compared using Welch’s t-test. Immunohistochemical characterisation was conducted using DAPI and phalloidin (n=5 testes//genotype/temperature). TMT-based whole-testis proteomes from control, doa-knockdown and 31°C heat-stressed males (n=4/group) were analysed using limma with Benjamini–Hochberg correction (adjusted P<0.1; |log₂FC|>0.5).

Results: Germline doa knockdown significantly reduced reproductive output at 25°C (P<0.05), 27°C (P<0.0001),  29°C (P<0.01). DOA-knockdown testes exhibited post-meiotic defects and reduced seminal-vesicle filling, resembling heat-stress phenotypes. Proteomic analysis identified 22 differentially abundant proteins following doa depletion and 156 following heat stress, with 11 commonly dysregulated proteins associated with spermiogenesis, cytoskeletal organisation and oxidative-stress defence.

Conclusion: DOA is required for normal spermiogenesis and sustained male fertility, with elevated temperature exacerbating the effects of doa depletion. The thermosensitive activity and evolutionary conservation of CLK-family kinases identify DOA signalling as a candidate mechanism contributing to the conserved thermal sensitivity of spermatogenesis.

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