Measuring ovarian toxicity in clinical trials: an American Society of Clinical Oncology research statement.

Anticancer agents can impair ovarian function, resulting in premature menopause and associated long-term health effects. Ovarian toxicity is not usually adequately assessed in trials of anticancer agents, leaving an important information gap for patients facing therapy choices. This American Society of Clinical Oncology (ASCO) statement provides information about the incorporation of ovarian toxicity measures in trial design. ASCO recommends: (1) measurement of ovarian toxicity in relevant clinical trials of anticancer agents that enrol post-pubertal, pre-menopausal patients; (2) collection of ovarian function measures at baseline and at 12-24 months after anticancer agent cessation, as a minimum, and later in line with the trial schedule; and (3) assessment of both clinical measures and biomarkers of ovarian function. ASCO recognises that routine measurement of ovarian toxicity and function in cancer clinical trials will add additional complexity and burden to trial resources but asserts that this issue is of such importance to patients that it cannot continue to be overlooked.

Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health.

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation-induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.

Does single-strand DNA break repair capacity influence oocyte maintenance and quality?

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.

HENMT1 is involved in the maintenance of normal female fertility in the mouse.

PIWI-interacting small RNAs (piRNAs) maintain genome stability in animal germ cells, with a predominant role in silencing transposable elements. Mutations in the piRNA pathway in the mouse uniformly lead to failed spermatogenesis and male sterility. By contrast, mutant females are fertile. In keeping with this paradigm, we previously reported male sterility and female fertility associated with loss of the enzyme HENMT1, which is responsible for stabilising piRNAs through the catalysation of 3'-terminal 2'-O-methylation. However, the Henmt1 mutant females were poor breeders, suggesting they could be subfertile. Therefore, we investigated oogenesis and female fertility in these mice in greater detail. Here, we show that mutant females indeed have a 3- to 4-fold reduction in follicle number and reduced litter sizes. In addition, meiosis-II mutant oocytes display various spindle abnormalities and have a dramatically altered transcriptome which includes a down-regulation of transcripts required for microtubule function. This down-regulation could explain the spindle defects observed with consequent reductions in litter size. We suggest these various effects on oogenesis could be exacerbated by asynapsis, an apparently universal feature of piRNA mutants of both sexes. Our findings reveal that loss of the piRNA pathway in females has significant functional consequences.

DNA repair in primordial follicle oocytes following cisplatin treatment.

PURPOSE: Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma-/- mice in response to DSBs caused by the chemotherapy agent cisplatin. METHODS: Adult C57BL/6 WT and Puma-/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised. RESULTS: Puma-/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma-/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma-/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role. CONCLUSION: PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.

The PARP inhibitor, olaparib, depletes the ovarian reserve in mice: implications for fertility preservation.

STUDY QUESTION: What is the impact of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, alone or in combination with chemotherapy on the ovary in mice? SUMMARY ANSWER: Olaparib treatment, when administered alone, depletes primordial follicle oocytes, but olaparib does not exacerbate chemotherapy-mediated ovarian follicle loss in mice. WHAT IS KNOWN ALREADY: The ovary contains a finite number of oocytes stored within primordial follicles, which give rise to all mature ovulatory oocytes. Unfortunately, they are highly sensitive to exogenous DNA damaging insults, such as cytotoxic cancer treatments. Members of the PARP family of enzymes are central to the repair of single-strand DNA breaks. PARP inhibitors have shown promising clinical efficacy in reducing tumour burden, by blocking DNA repair capacity. Olaparib is a PARP1/2 inhibitor recently FDA-approved for treatment of BRCA1 and BRCA2 mutation carriers with metastatic breast cancer. It is currently being investigated as an adjunct to standard treatment at an earlier stage, potentially curable, BRCA1- and BRCA2-associated breast cancer which affects reproductive age women. Despite this, there is no preclinical or clinical information regarding the potential impacts of olaparib on the ovary or on female fertility. Unfortunately, it may be many years before clinical data on fertility outcomes for women treated with PARP inhibitors becomes available, highlighting the importance of rigorous preclinical research using animal models to establish the potential for new cancer therapies to affect the ovary in humans. We aimed to comprehensively determine the impact of olaparib alone, or following chemotherapy, on the ovary in mice. STUDY DESIGN, SIZE, DURATION: On Day 0, mice (n = 5/treatment group) were administered a single intraperitoneal dose of cyclophosphamide (75 mg/kg/body weight), doxorubicin (10 mg/kg), carboplatin (80 mg/kg), paclitaxel (7.5 mg/kg) or vehicle control. From Days 1 to 28, mice were administered subcutaneous olaparib (50 mg/kg) or vehicle control. This regimen is proven to reduce tumour burden in preclinical mouse studies and is also physiologically relevant for women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult female wild-type C57BL6/J mice at peak fertility (8 weeks) were administered a single intraperitoneal dose of chemotherapy, or vehicle, then either subcutaneous olaparib or vehicle for 28 days. Vaginal smears were performed on each animal for 14 consecutive days from Days 15 to 28 to monitor oestrous cycling. At 24 h after final treatment, ovaries were harvested for follicle enumeration and immunohistochemical analysis of primordial follicle remnants (FOXL2 expressing granulosa cells), DNA damage (γH2AX) and analysis of apoptosis by TUNEL assay. Serum was collected to measure circulating anti-Müllerian hormone (AMH) concentrations by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Olaparib significantly depleted primordial follicles by 36% compared to the control (P < 0.05) but had no impact on other follicle classes, serum AMH, corpora lutea number (indicative of ovulation) or oestrous cycling. Primordial follicle remnants were rarely detected in control ovaries but were significantly elevated in ovaries from mice treated with olaparib alone (P < 0.05). Similarly, DNA damage denoted by γH2AX foci was completely undetectable in primordial follicles of control animals but was observed in ∼10% of surviving primordial follicle oocytes in mice treated with olaparib alone. These observations suggest that functional PARPs are essential for primordial follicle oocyte maintenance and survival. Olaparib did not exacerbate chemotherapy-mediated follicle depletion in the wild-type mouse ovary. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed in mice, so the findings may not translate to women and further studies utilizing human ovarian tissue and sera samples should be performed in the future. Only one long-term time point was analysed, therefore olaparib-mediated follicle damage should be assessed at more immediate time points in the future to support our mechanistic findings. WIDER IMPLICATIONS OF THE FINDINGS: Olaparib dramatically depleted primordial follicles and this could be attributed to loss of intrinsic PARP-mediated DNA repair mechanisms. Importantly, diminished ovarian reserve can result in premature ovarian insufficiency and infertility. Notably, the extent of follicle depletion might be enhanced in BRCA1 and BRCA2 mutation carriers, and this is the subject of current investigations. Together, our data suggest that fertility preservation options should be considered for young women prior to olaparib treatment, and that human studies of this issue should be prioritized. STUDY FUNDING/COMPETING INTEREST(S): This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This work was supported by funding from the National Health and Medical Research Council (NHMRC); (K.J.H. #1050130) (A.L.W. #1120300). K.A.P. is a National Breast Cancer Foundation Fellow (Australia-PRAC-17-004). K.A.P. is the Breast Cancer Trials (Australia) Study Chair for the OlympiA clinical trial sponsored by AstraZeneca, the manufacturer of olaparib. All other authors declare no competing financial or other interests.

NMN does not protect the ovarian reserve from cancer treatments.

Primordial follicle oocytes are extremely vulnerable to DNA damage caused by exogenous agents, such as those commonly used to treat cancer. Consequently, female cancer patients often have diminished ovarian reserve, which if severe enough, can cause premature ovarian failure and early menopause. Advances in cancer therapies have resulted in significantly improved cancer survival rates; therefore, it is becoming increasingly important to devise strategies to protect the ovarian reserve from cancer treatments, to avoid loss of fertility and endocrine dysfunction. In this study, we aimed to determine whether supplementation with nicotinamide mononucleotide (NMN) could preserve the ovarian reserve following exposure to DNA-damaging cancer treatments. Adult female mice (n = 5-6/group) received saline or NMN (500 mg/kg/day) for 8 days. Mice were left untreated or exposed to γ-irradiation (0.1 Gy) or cyclophosphamide (150 mg/kg) on day 7 and ovaries and serum collected for analysis on day 12. We report that γ-irradiation treatment significantly reduced the number of primordial follicles, but supplementation with NMN did not prevent the observed follicle loss. Similarly, cyclophosphamide treatment significantly reduced primordial follicle numbers, but these losses were not prevented by NMN supplementation. In conclusion, depletion of the ovarian reserve following γ-irradiation or cyclophosphamide was not protected by NMN supplementation under the conditions employed in this study.

Moderate episodic prenatal alcohol does not impact female offspring fertility in rats.

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.

DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa.

Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocyte nuclei and is essential for their apoptosis triggered by DNA damage in vivo. After γ-irradiation, induction of the proapoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from WT and Trp53(-/-) mice but not in those from TAp63-deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women's health.

The capacity of oocytes for DNA repair.

Female fertility and offspring health are critically dependent on the maintenance of an adequate supply of high-quality oocytes. Like somatic cells, oocytes are subject to a variety of different types of DNA damage arising from endogenous cellular processes and exposure to exogenous genotoxic stressors. While the repair of intentionally induced DNA double strand breaks in gametes during meiotic recombination is well characterised, less is known about the ability of oocytes to repair pathological DNA damage and the relative contribution of DNA repair to oocyte quality is not well defined. This review will discuss emerging data suggesting that oocytes are in fact capable of efficient DNA repair and that DNA repair may be an important mechanism for ensuring female fertility, as well as the transmission of high-quality genetic material to subsequent generations.

Maternal low-protein diet programmes low ovarian reserve in offspring

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.

Examination of the ovotoxicity of 5-fluorouracil in mice.

PURPOSE: Undesirable side effects of cancer treatments are common and include damage to the ovary, and depletion of the follicle reserve, which if severe enough, can lead to infertility and early menopause. Antimetabolite drugs, such as 5-fluorouracil (5-FU), are not considered to be detrimental to the ovary, but the ovotoxicity of 5-FU has not been evaluated in any detail. The purpose of this study was to evaluate the effects of 5-FU on follicle number. METHODS: In this study, adult female C57Bl6 mice (n = 4-6 animals/group) received a single dose of saline or 5-FU (150 mg/kg) and markers of ovarian damage and follicle depletion were assessed 12 h and 7 days later. RESULTS: Exposure to 5-FU did not alter primordial and primary follicle numbers. Atresia of secondary and antral follicles was increased significantly 12 h after 5-FU treatment, but atresia rates returned to levels similar to that of saline treated controls at 7 days. The number of corpora lutea were reduced 7 days after exposure to 5-FU, possibly as a consequence of earlier follicular atresia. CONCLUSIONS: These findings suggest that a single dose of 5-FU is mildly ovotoxic, but any effects on ovarian function are likely transient because the primordial follicle population is not depleted. Collectively, these data support the notion that 5-FU is unlikely to impact on the long-term fertility of women.

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.

BCL2-modifying factor promotes germ cell loss during murine oogenesis.

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.

Taking control of the female fertile lifespan: a key role for Bcl-2 family proteins.

Precisely how the length of the female fertile lifespan is regulated is poorly understood and it is likely to involve complex factors, one of which is follicle number. Indeed, the duration of female fertility appears to be intimately linked to the number of available oocytes, which are stored in the ovary as primordial follicles. There is mounting evidence implicating the intrinsic apoptosis pathway, which is controlled by members of the B-cell lymphoma-2 (BCL-2) family, as a key regulator of the number of primordial follicles established in the ovary at birth and maintained throughout reproductive life. Consequently, the pro- and anti-apoptotic BCL-2 family proteins are emerging as key determinants of the length of the female fertile lifespan. This review discusses the relationship between the intrinsic apoptosis pathway, follicle number and length of the female fertile lifespan.

How Is the Number of Primordial Follicles in the Ovarian Reserve Established?

The number of primordial follicles in the ovarian reserve is an important determinant of the length of the ovarian lifespan, and therefore the fertility of an individual. This reserve contains all of the oocytes potentially available for fertilization throughout the fertile lifespan. The maximum number is set during pregnancy or just after birth in most mammalian species; current evidence does not support neofolliculogenesis after the ovarian reserve is established, although this is increasingly being reexamined. Under physiological circumstances, this number will be influenced by the number of primordial germ cells initially specified in the epiblast of the developing embryo, their proliferation during and after migration to the developing gonads, and their death during oogenesis and formation of primordial follicles at nest breakdown. Death of germ cells during the establishment of the ovarian reserve occurs principally by autophagy or apoptosis, although the triggers that initiate these remain elusive. This review outlines the regulatory steps that determine the number of primordial follicles and thus the number of oocytes in the ovarian reserve at birth, using the mouse as the model, interspersed with human data where available. This information has application for understanding the variability in duration of fertility that occurs between normal individuals and with age, in premature ovarian insufficiency, and after chemotherapy or radiotherapy.

The role of BH3-only proteins in apoptosis within the ovary.

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.

Loss of the proapoptotic BH3-only protein BCL-2 modifying factor prolongs the fertile life span in female mice.

The duration of the female fertile life span is influenced by the number of oocytes stored in the ovary as primordial follicles. Cell death, both during ovarian development in the embryo and in the postnatal ovary, plays a critical role in determining how many primordial follicles are established and maintained within the ovary. However, the roles of individual apoptotic regulators in mediating cell death within the ovary have not yet been characterized. In this study, gene targeted mice were used to investigate the role of BCL-2-modifying factor (BMF), a proapoptotic protein belonging to the BH3-only subgroup of the BCL-2 family, in determining the number of primordial follicles maintained in the adult ovary and the length of the fertile life span. Stereological analysis of ovaries showed that Bmf(-/-) mice had significantly more primordial follicles than wild-type (WT) control animals at Postnatal Days 100, 200, 300, and 400 but not at Day 20. No differences were observed between WT and Bmf(-/-) mice in the number of ova shed following ovulatory stimulation with exogenous gonadotropins. Bmf(-/-) females were fertile and produced the same number pups/litter as WT females, but Bmf(-/-) females produced litters more frequently and consequently more offspring than WT females over a 6-mo period. Furthermore, the fertile life span of Bmf(-/-) females was significantly extended compared to WT females. Our findings support an important role for BMF in determining the number of primordial follicles maintained in the ovary throughout adult reproductive life and thus indicate that the length of female fertility may be extended by increasing the number of primordial follicles maintained within the ovary through inhibition of BMF.

PUMA regulates germ cell loss and primordial follicle endowment in mice.

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.

Exome-informed formulations of food proteins enhance body growth and feed conversion efficiency in ad libitum-fed mice.

Meeting requirements for dietary proteins, especially of essential amino acids (EAAs), is critical for the life-long health of living organisms. However, defining EAA targets for preparing biologically-matched nutrition that satisfies metabolic requirements for protein remains challenging. Previous research has shown the advantages of 'exome matching' in representing the specific requirement of dietary AAs, where the target dietary AA profile was derived from in silico translation of the genome of an organism, specifically responsible for protein expression (the 'exome'). However, past studies have assessed these effects in only one sex, for few parameters (body mass and composition), and have used purified diets in which protein is supplied as a mixture of individual AAs. Here, for the first time, we utilise a computational method to guide the formulation of custom protein blends and test if exome matching can be achieved at the intact protein level, through blending standard protein ingredients, ultimately leading to optimal growth, longevity and reproductive function. Mice were provided ad libitum (ad lib) access to one of the four iso-energetic protein-limited diets, two matched and two mis-matched to the mouse exome target, and fed at a fixed protein energy level of 6.2%. During or following 13-weeks of feeding, the food intake, body growth, composition and reproductive functions were measured. Compared to the two mis-matched diets, male and female animals on the exome-matched diet with protein digestibility correction applied, exhibited significantly improved growth rates and final body mass. The feed conversion efficiency in the same diet was also increased by 62% and 40% over the worst diets for males and females, respectively. Male, not female, exhibited higher accretion of lean body mass with the matched, digestibility-corrected diet. All reproductive function measures in both sexes were comparable among diets, with the exception of testicular daily sperm production in males, which was higher in the two matched diets versus the mis-matched diets. The results collectively demonstrate the pronounced advantages of exome-matching in supporting body growth and improving feed conversion efficiency in both sexes. However, the potential impact of this approach in enhancing fertility needs further investigation.