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.

Conditional loss of Brca1 in oocytes causes reduced litter size, ovarian reserve depletion and impaired oocyte in vitro maturation with advanced reproductive age in mice.

BACKGROUND: An estimated 1 in 350 women carry germline BRCA1/2 mutations, which confer an increased risk of developing breast and ovarian cancer, and may also contribute to subfertility. All mature, sex steroid-producing ovarian follicles are drawn from the pool of non-renewable primordial follicles, termed the 'ovarian reserve'. The clinical implications of early ovarian reserve exhaustion extend beyond infertility, to include the long-term adverse health consequences of loss of endocrine function and premature menopause. We aimed to determine whether conditional loss of Brca1 in oocytes impacts ovarian follicle numbers, oocyte quality and fertility in mice with advancing maternal age. We also aimed to determine the utility of AMH as a marker of ovarian function, by assessing circulating AMH levels in mice and women with BRCA1/2 mutations, and correlating this with ovarian follicle counts. METHODS: In this study, we addressed a longstanding question in the field regarding the functional consequences of BRCA1 inactivation in oocytes. To recapitulate loss of BRCA1 protein function in oocytes, we generated mice with conditional gene deletion of Brca1 in oocytes using Gdf9-Cre recombinase (WT: Brca1fl/flGdf9+/+; cKO: Brca1fl/flGdf9cre/+). FINDINGS: While the length of the fertile lifespan was not altered between groups after a comprehensive breeding trial, conditional loss of Brca1 in oocytes led to reduced litter size in female mice. Brca1 cKO animals had a reduced ovarian reserve and oocyte maturation was impaired with advanced maternal age at postnatal day (PN)300, compared to WT animals. Serum anti-Müllerian hormone (AMH) concentrations (the gold-standard indirect marker of the ovarian reserve used in clinical practice) were not predictive of reduced primordial follicle number in Brca1 cKO mice versus WT. Furthermore, we found no correlation between follicle number or density and serum AMH concentrations in matched samples from a small cohort of premenopausal women with BRCA1/2 mutations. INTERPRETATION: Together, our data demonstrate that BRCA1 is a key regulator of oocyte number and quality in females and suggest that caution should be used in relying on AMH as a reliable marker of the ovarian reserve in this context. FUNDING: 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 Australian Research Council (ALW - DE21010037 and KJH - FT190100265), as well as the National Breast Cancer Foundation (IIRS-22-092) awarded to ALW and KJH. LRA, YML, LT, EOKS and MG were supported by Australian Government Research Training Program Scholarships. LRA, YML and LT were also supported by a Monash Graduate Excellence Scholarship. YC, SG and XC were supported by Monash Biomedicine Discovery Institute PhD Scholarships. LRA was also supported by a Monash University ECPF24-6809920940 Fellowship. JMS was supported by NHMRC funding (2011299). MH was supported by an NHMRC Investigator Grant (1193838).

Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

Structure of a 14-3-3ε:FOXO3apS253 Phosphopeptide Complex Reveals 14-3-3 Isoform-Specific Binding of Forkhead Box Class O Transcription Factor (FOXO) Phosphoproteins.

The transcriptional activity of Forkhead Box O3 (FOXO3a) is inactivated by AKT-mediated phosphorylation on Serine 253 (S253), which enables FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3 facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation. The FOXO3a/14-3-3 interaction has, therefore, emerged as an important therapeutic target. Here, we report a comprehensive analysis using fluorescence polarization, isothermal titration calorimetry, small-angle X-ray scattering, X-ray crystallography, and molecular dynamics simulations to gain molecular-level insights into the interaction of FOXO3apS253 phosphopeptide with 14-3-3ε. A high-resolution structure of the fluorophore-labeled FOXO3apS253:14-3-3ε complex revealed a distinct mode of interaction compared to other 14-3-3 phosphopeptide complexes. FOXO3apS253 phosphopeptide showed significant structural difference in the positions of the -3 and -4 Arg residues relative to pSer, compared to that of a similar phosphopeptide, FOXO1pS256 bound to 14-3-3σ. Moreover, molecular dynamics studies show that the significant structural changes and molecular interactions noticed in the crystal structure of FOXO3apS253:14-3-3ε are preserved over the course of the simulation. Thus, this study reveals structural differences between the binding to 14-3-3 isoforms of FOXO1pS256 versus FOXO3apS253, providing a framework for the rational design of isoform-specific FOXO/14-3-3 protein-protein interaction inhibitors for therapy.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain.

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.