Investigating the roles of steroid signalling in gonadal development, maintenance and function through use of androgen and cortisol deficient mutant zebrafish lines

The roles of androgen signalling in the differentiation, maintenance and function of the zebrafish testis are poorly understood. This project aimed to explore this topic by characterising the phenotypes of androgen and cortisol deficient fdx1b and cyp11c1 mutant zebrafish. Homozygous mutant adult zebrafish were found to exhibit female type pigmentation patterns, however these fish could possess either differentiated ovaries or testes. This demonstrates that androgens are dispensable for testis differentiation in zebrafish, as has also been found in androgen receptor mutant zebrafish. Androgen deficient male zebrafish were infertile in standard breeding scenarios, but their sperm could fertilise eggs collected from wild-type female zebrafish in IVF experiments. Various phenotypes in these fish are likely to contribute to their infertility, including perturbed breeding behaviours, disorganised testis organisation, reduced spermatogenesis and impaired sperm release. Targeted gene expression analysis by qPCR revealed downregulation of igf3 and insl3 in the testes of mutant fish, these genes encode important factors for spermatogonial differentiation. Histological examination of the testes and analysis of germ cell marker expression by qPCR revealed an accumulation of early germ cells. Taken together these findings indicate that spermatogonial differentiation is impaired, and that this process is highly dependent on androgens in zebrafish. Further investigations into the molecular mechanisms underlying the assorted phenotypes of these fish were undertaken by characterisation of the impacts of fdx1b mutation on the testis transcriptome. Several miRNAs with roles in repressing spermatogonial differentiation in mammals were upregulated in the testes of Fdx1b deficient zebrafish, indicating a potentially conserved role for these miRNAs in zebrafish and mammalian spermatogenesis. In addition to this, dysregulation of several structural elements of the testes was identified, including the basement membrane and Sertoli cell barrier. This is the first investigation into the mechanisms underlying disorganisation of testicular structure in androgen deficient or resistant zebrafish.