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A detailed understanding of the genes and mechanisms that regulate oocyte growth and maturation underpins the development of improved methods of assisted conception. Gametocyte specific factor 1 (GTSF1)-a putative marker of gamete developmental competence, is highly conserved across species but in mammals demonstrates a sexual dimorphism in its function. In mice, male mutants for Gtsf1 have an infertile phenotype, whereas female mutants appear to have normal ovarian function. It is hypothesised that GTSF1 regulates oocyte development in monovular species such as the sheep. Initial studies characterised the expression and cellular distribution of GTSF1 across cDNA libraries spanning ovine oogenesis and embryogenesis and by using in situ hybridisation of fixed tissue. GTSF1 expression was confined to gonadal and embryonic tissues with highest expression in the ooplasm of germinal vesicle (GV)-staged secondary oocytes. The gene sequence of GTSF1 was obtained and the gene and predicted protein sequences revealed close homology across many species with two conserved CHHC zinc finger domains known to bind RNA. Functional analysis of the role of GTSF1 during sheep oocyte maturation was conducted using short interference RNA (siRNA injection) in conjunction with oocyte in vitro maturation (IVM) and oocytectomised cumulus shell co-culture. This system was validated using siRNA knockdown (kd) for a known oocyte-specific gene, Growth differentiation factor 9 (GDF9). The effect on GTSF1 kd was evaluated following the microinjection of 770 GV oocytes with siRNA target against the sixth exon of ovine …