Understanding the molecular mechanism of male infertility using flies, mice and humans

Abstract

Male infertility is an extremely complex disorder that is contributed to by genetics, the environment, or a combination of both. Infertility variants are large in number, but low in incidence, meaning there are a large number of variants which can cause infertility, but the number of each is relatively low. Therefore, studying these mutations in large cohorts is difficult. For this reason, model organisms are required to screen large numbers of genes to determine their role in male reproduction. This study used the Drosophila melanogaster and mouse model organisms in order to screen a large number of genes to determine if they play a role in male fertility. While previous research had significantly outlined and compared spermatogenesis between organisms at many points in the process, there has yet to be a review which broke down the individual steps of spermatogenesis. This is important to determine whether genes expressed at specific points during spermatogenesis can be studied using Drosophila. For this reason, the first step was to write a literature review specifically outlining the steps of spermatogenesis where Drosophila could be used to study male infertility. In collaboration with the International Male Infertility Genomics Consortium, a list of gene variants discovered through exome analysis of infertile men was designed. From this list, 10 genes with Drosophila orthologues were further analysed. Zn72D with the patient orthologue ZFR2 and DCAF12 with the patient orthologue DCAF12L1 were found to cause infertility when knocked down in the testis. Analysis in human testicular tissue confirmed that both ZFR2 and DCAF12L1 were expressed in the testis. As ZFR2 was a promising candidate, a Zfr2-/- knockout mouse was generated. These knockout mice were healthy and able to produce litter of a size comparable to the control. There were no noticeable defects in the testicular or epididymal histology, nor in their sperm motility or morphology. For this reason, it was determined that Zfr2 is not an absolute requirement for male fertility in the mouse. The effect of zinc and zinc transport on fertility was also analysed. Previous research has determined that zinc is important in fertility, as decreased zinc in the seminal plasma was associated with subfertility. Zinc transport, through the Zrt-, Irt-like protein (ZIP) and Zinc Transporter (ZnT) protein families, which are responsible for zinc influx and efflux6 respectively, was manipulated to determine its effect on fertility. In particular, Zip42C.1, Zip42C.2, Zip89B and Zip71B were found to cause a significant reduction in fertility when misexpressed in the testis. The expression of the human orthologues of these were then analysed in human tissue. It was found that ZIP1 was expressed primarily in germ cells, and ZIP5 and ZnT9 were expressed primarily in Sertoli cells. In order to determine the environmental role of zinc, by omitting it from the Drosophila diet, a chemically defined diet was used. Removing neither zinc nor copper caused a noticeable reduction in fertility compared to the control. In conclusion, this research has demonstrated how Drosophila and mice can be used to evaluate infertility candidate genes.