Mammalian heat shock protein A4 family ortholog Hsc70Cb is required for two phases of spermatogenesis in D. melanogaster.

In brief: Hsc70Cb is essential for spermatogonia survival and sperm individualisation in Drosophila. This study highlights the conserved roles of the HSPA4 family across animals and the utility of flies as a model organism for male fertility research.

Abstract: Heat shock proteins play essential roles as molecular chaperones, enacting protein folding and preventing protein aggregation. In a previous study, a homozygous non-synonymous genetic variant that was predicted to be damaging was detected in an infertile man in the heat shock protein gene HSPA4L. Here, we used RNA interference in Drosophila melanogaster to explore the role of the heat shock protein A member 4 family (HSPA4) family in male fertility. Expression of the fly orthologue of the mammalian HSPA4 and HSPA4L genes, Hsc70Cb, was reduced in the male germline using two RNAi lines driven by Nanos-Gal4. Robust knockdown of Hsc70Cb in male germ cells resulted in male sterility, characterised by the absence of germ cells in testes and the overproliferation of the testis soma. A less robust knockdown of Hsc70Cb in the male germline resulted in a sperm individualisation defect and a failure of sperm release into the seminal vesicle (analogous to the epididymis). When human HSPA4 or HSPA4L cDNA was introduced into infertile Hsc70Cb mutant flies, a partial rescue was observed, whereby in the robust Hsc70Cb knockdown setting, germ cells progressed to the spermatocyte stage before undergoing cell death. The absence of sperm in Hsc70Cb mutant (line 1) is consistent with the clinical presentation of the infertile man harbouring a homozygous HSPA4L genetic variant, supporting the hypothesis that HSPA4L is required for male fertility in humans and flies. This study also highlights the utility of the fly as a model of human spermatogenesis.