Several lines of evidence from budding yeast, plants and flies suggest that TOP3A is critical for quality control in oocytes, with roles in the repair of meiotic DNA double strand breaks and DNA damage arising from exogenous stressors, as well as in the maintenance of mitochondrial DNA (mtDNA). Whilst these studies are compelling, the role of TOP3A in the oocytes of higher vertebrates has never been established. Hence, this study aimed to use novel mouse models to define the functional impact of the conditional loss of TOP3A in oocytes (cKO: TOP3Afl/fl;GDF9cre/+).
In situ hybridisation using ovarian tissue sections from WT (TOP3Afl/fl;GDF9+/+) animals revealed that Top3a mRNA is expressed by oocytes within primordial, primary, secondary and antral follicles. Furthermore, analyses of WT oocytes injected with GFP-tagged TOP3A protein demonstrated that TOP3A co-localises with mitochondria during oocyte maturation.
Ovarian follicles were subsequently quantified in prepubertal (postnatal day 20) and adult (postnatal day 60) WT and TOP3A-cKO mice (n=6 mice/genotype/age). Follicle numbers were similar in prepubertal WT and TOP3A-cKO mice, but loss of TOP3A resulted in the dramatic depletion of follicles in adults, indicative of premature ovarian aging (follicle number: WT 2367±463 vs TOP3A-cKO 0, p=0.007).
To gain further insight into the requirement for TOP3A for oocyte quality, in vitro fertilisation (IVF) and embryo culture were performed using mature oocytes obtained from WT and TOP3A-cKO mice at PN20 (n=6 mice/genotype). Strikingly, almost all oocytes from TOP3A-cKO mice failed to progress beyond fertilisation, whilst fertilisation and embryonic development progressed normally when oocytes from WT mice were used.
Collectively, these data demonstrate that TOP3A is essential for the maintenance of the ovarian follicular reserve and oocyte quality. Although the precise roles played by TOP3A are not yet known, future studies will investigate the hypothesis that TOP3A is a central mediator of mtDNA homeostasis in oocytes.