Bcl2-modifying factor (Bmf) regulates germ cell death during ovarian development

Abstract

The number and quality of oocytes stored within the ovary as primordial follicles influence the length of the fertile lifespan, the age at which menopause begins, and the health of offspring. Primordial germ cells and oogonia, which are the embryonic precursors of oocytes, multiply dramatically during the early stages of fetal development. Germ cell numbers then undergo a dramatic decline as a consequence of the permanent cessation of mitosis and the loss of approximately two-thirds of the newly made oogonia and oocytes. The loss of germ cells is largely due to apoptosis and this leaves a reduced number of oocytes within the ovary at birth, and, because new germ cells cannot be made after this point, it limits female fertility and reproductive lifespan. Despite the critical role of apoptosis in regulating the number of available oocytes, and hence in determining the length of a women's fertile lifespan and the timing of menopause, the proteins that regulate the death of germ cells are largely unknown and the reasons underlying their attrition are unclear. The overall aim of this project was to investigate the role of the Bci2-Modifying Factor (BMF), a pro-apoptotic protein belonging to the BH3-only sub-group of the Bcl-2 superfamily, in the death of germ cells during oogenesis in mice. BMF protein was immunohistochemically localised to germ cells at embryonic days (E) 15.5, 17.5 and PN1, coincident with entry into the meiotic prophase and the beginning of germ cell nest breakdown, but was undetectable at E13.5 and postnatal days {PN) 3 and 5. Targeted deletion of the Bmf gene in female mice resulted in a transient increase in germ cell number compared to wild type (WT) at (E) 15.5, and between PN1 and PN5. However, germ cell numbers were comparable in WT and Bmf1- ovaries at E13.5, E17.5 and PN10. Loss of BMF was also associated with a decrease in apoptosis, as determined by Terminal deoxynucleotidyl-transferase dUTP nick-end labelling (TUNEL) at ElS.S and E17.5. lmmunostaining for yH2AX, a meiotic prophase maker, and TAp63, a marker of diplotene arrest, indicated that germs cells cease proliferation and enter meiotic arrest at a similar time in ovaries from WT and Bmf1- mice. Additionally, immunostaining for PH3 (a marker of mitotic cells) and MVH (a marker of germ cells) showed no differences in the percentage of germ cells labelled with PH3 between Bm[1- and WT ovaries at E13.5. Thus, altered proliferation could not explain the observed increases in germ cell number in ovaries from Bmf1- compared to WT mice. Notably, BMF was dispensable for the massive germ cell loss that occurs during nest breakdown, which was compared in ovaries from WT and Bm[1- mice using morphological analyses as well as by immunostaining for MVH and the basal laminar marker laminin. Collectively, these data indicate that BMF is expressed in oocytes during the meiotic prophase and likely mediates their apoptosis at that time, but does not influence the number of primordial follicles initially making up the ovarian reserve at PNlO.