BH3-only proteins are pro-apoptotic members of the BCL2 family that play pivotal roles in embryonic development, tissue homeostasis and immunity by triggering cell death through the intrinsic apoptosis pathway. Recent in vitro and in vivo studies have demonstrated that BH3-only proteins are also essential mediators of apoptosis within the ovary and are responsible for the initiation of the cell death signalling cascade in a cell type and stimulus-specific fashion. This review gives a brief overview of the intrinsic apoptosis pathway and summarise the roles of individual BH3-only proteins in the promotion of apoptosis in embryonic germ cells, oocytes, follicular granulosa cells and luteal cells. The role of these proteins in activating apoptosis in response to developmental cues and cell stressors, such as exposure to chemotherapy, radiation and environmental toxicants, is described. Studies on the function of BH3-only proteins in the ovary are providing valuable insights into the regulation of oocyte number and quality, as well as ovarian endocrine function, which collectively influence the female reproductive lifespan and health.
R John Aitken, Jock K Findlay, Karla J Hutt, and Jeff B Kerr
Apoptosis is a critical process for regulating both the size and the quality of the male and female germ lines. In this review, we examine the importance of this process during embryonic development in establishing the pool of spermatogonial stem cells and primordial follicles that will ultimately define male and female fertility. We also consider the importance of apoptosis in controlling the number and quality of germ cells that eventually determine reproductive success. The biochemical details of the apoptotic process as it affects germ cells in the mature gonad still await resolution, as do the stimuli that persuade these cells to commit to a pathway that leads to cell death. Our ability to understand and ultimately control the reproductive potential of male and female mammals depends upon a deeper understanding of these fundamental processes.
Amy L Winship, Sarah E Gazzard, Luise A Cullen-McEwen, John F Bertram, and Karla J Hutt
The ovarian reserve of primordial follicle oocytes is formed during in utero development and represents the entire supply of oocytes available to sustain female fertility. Maternal undernutrition during pregnancy and lactation diminishes offspring ovarian reserve in rats. In mice, maternal oocyte maturation is also susceptible to undernutrition, causing impaired offspring cardiovascular function. We aimed to determine whether programming of the ovarian reserve is impacted in offspring when maternal undernutrition extends from preconception oocyte development through to weaning. C57BL6/J female mice were fed normal protein (20%) or low-protein (8%) diet during preconception, pregnancy and lactation periods. Maternal ovaries were harvested at weaning and offspring ovaries were collected at postnatal day (PN)21 and 24 weeks of age. Total follicle estimates were obtained by histologically sampling one ovary per animal (n = 5/group). There was no impact of diet on maternal follicle numbers. However, in offspring, maternal protein restriction significantly depleted primordial follicles by 37% at PN21 and 51% at 24 weeks (P < 0.05). There were no effects of diet on other follicle classes. Histological analysis showed no differences in the proportion of proliferative follicles (pH3 positive), but increased atresia (cleaved caspase-3-positive, or TUNEL-positive) was detected in ovaries of protein-restricted offspring at both ages (P < 0.05). Our data show that maternal diet during the preconception period, in utero development and early life has significant impacts on follicle endowment and markers of follicle health later in life. This highlights the need for further investigation into the importance of maternal preconception diet for offspring reproductive development and health.
Saranya Giridharan, Karla J Hutt, and Amy L Winship
Human genome-wide association studies and evidence from animal models link ovarian ageing to double-strand (ds)DNA break repair capacity. Is there a connection between single-strand (ss)DNA repair mechanisms and ovarian function? We hypothesize that endogenous cellular processes subject oocytes to ssDNA lesions, and thus, ssDNA repair capacity is fundamental to their survival and maintenance.
Kavitha Vaithiyanathan, Seng H Liew, Nadeen Zerafa, Thilini Gamage, Michele Cook, Lorraine A O’Reilly, Philippe Bouillet, Clare L Scott, Andreas Strasser, Jock K Findlay, and Karla J Hutt
Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf −/− mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf −/− and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.
Elizabeth K McReight, Seng H Liew, Sarah E Steane, Karla J Hutt, Karen M Moritz, and Lisa K Akison
Prenatal alcohol exposure (PAE) has been associated with reproductive dysfunction in offspring. However, studies in females, particularly examining long-term infertility or impacts on ovarian reserve, are lacking. The current study utilised a moderate, episodic exposure model in rats to mimic ‘special occasion’ drinking, which is reported to be common during pregnancy. Our objective was to examine the consequences of this prenatal alcohol exposure on reproductive parameters in female offspring. Pregnant Sprague–Dawley rats were treated with either an ethanol gavage (1 g EtOH/kg body weight), or an equivalent volume of saline, on embryonic days 13.5 and 14.5 of pregnancy, resulting in a peak blood alcohol concentration of ~0.04%. Neonatal female offspring were examined for molecular markers regulating early follicle numbers in the ovary, and unbiased stereology was used to quantify primordial and early growing follicle numbers. Puberty onset (age at vaginal opening and first estrous) was measured post-weaning, and estrous cycles, reproductive hormones (progesterone and estradiol) and pregnancy success was measured in adults (5–6 months of age). We found no evidence that any of these reproductive parameters were significantly altered by PAE in this model. This animal study provides some reassurance for women who may have consumed a small amount of alcohol during their pregnancy. However, previously published effects on offspring metabolism using this model reinforce avoidance of alcohol during pregnancy.
Michelle Myers, F Hamish Morgan, Seng H Liew, Nadeen Zerafa, Thilini Upeksha Gamage, Mai Sarraj, Michele Cook, Ileana Kapic, Antony Sutherland, Clare L Scott, Andreas Strasser, Jock K Findlay, Jeffrey B Kerr, and Karla J Hutt
The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3 −/− female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3 −/− mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.