The importance of balanced canonical Wnt signalling and analysis of its pathway components in spermatogenesis

Understanding fundamental reproductive biology is essential for generating improved contraceptive options, resolving fertility problems, and preserving fertility in individuals choosing to reproduce later in life. Disruptions in the development of male germ cells and their supporting somatic cells are considered to underpin many cases of idiopathic disease. Wnt signalling is a highly conserved pathway that plays essential roles in many tissue types and organs. Reports of Wnt signalling in the testis demonstrating that it is functionally important are limited, so further research is required to understand the contribution of this pathway to male fertility. Wnt signalling is tightly controlled in normal development. This thesis investigates the importance of appropriate levels of Wnt signalling for spermatogenic development. These studies employed AhCre mouse models with acute testis-wide perturbations in β-catenin and the negative regulator, Adenomatous Polyposis Coli, to examine outcomes of down- and up-regulation of Wnt signalling respectively. Both mutant models displayed germ cell loss, increased levels of TUNEL-positive cells, maintained PLZF-positive spermatogonia numbers, displayed normal Sertoli cell SOX9 expression, abnormal localisation of the blood-testis-barrier marker CNX43, and increased levels of immature Sertoli cell marker AMH. These results demonstrated that balanced levels of Wnt signalling are essential for normal adult spermatogenesis. Sites of active Wnt signalling were determined in the postnatal and adult mouse testis using several detection methods. The first demonstration of Wnt signalling activation in spermatogonia in vivo was presented through Axin2 expression, whilst active Wnt signalling in post-mitotic germ cells was confirmed though BATGAL expression and nuclear β-catenin localisation. In addition, Wnt signalling activation was identified in early postnatal Sertoli cells, but terminates once they terminally differentiate at puberty. Transcriptome analysis and protein localisation studies identified sites of several Wnt ligands, receptors, and other components that may mediate Wnt signalling in the testis. These data have demonstrated that while Wnt signalling is active in both mitotic and post-mitotic germ cells, each spermatogenic cell type has different signalling component expression profiles and Wnt signalling outcomes are driven by distinct transcription factors. To investigate the requirement of Wnt signalling within spermatogenic cells, VasaCre Ctnnb1fl/fl mice were employed. This demonstrated that β-catenin signalling controls the speed of the first wave of spermatogenesis, and is required for the maintenance of spermatogonia in adults. Transcriptome analysis identified several potential Wnt signalling downstream targets in the adult testis. Additionally, characterisation of the novel application of the AhCre mouse line for use in acute genetic recombination studies in the testis was demonstrated. Recombination efficiency differences between distinct floxed β-catenin alleles and the complexities of using different Cre drivers to examine gene function in the context of the testis have also been highlighted. Data presented in this thesis and by others have demonstrated that tight regulation of Wnt signalling, controlled by compartmentalised expression of different Wnt ligands, receptors, antagonists and enhancers is essential for normal spermatogenesis. These findings may contribute to a better understanding of fertility issues in men.