AMPK regulates phagophore-to-autophagosome maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

AMPK Regulates Phagophore-to-Autophagosome Maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK's regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

TCAB1 prevents nucleolar accumulation of the telomerase RNA to facilitate telomerase assembly.

Localization of a variety of RNAs to non-membrane-bound cellular compartments such as nucleoli and Cajal bodies is critical for their stability and function. The molecular mechanisms that underly the recruitment and exclusion of RNAs from these phase-separated organelles is incompletely understood. Telomerase is a ribonucleoprotein composed of the reverse transcriptase protein telomerase reverse transcriptase (TERT), the telomerase RNA (TR), and several auxiliary proteins, including TCAB1. Here we show that in the absence of TCAB1, a large fraction of TR is tightly bound to the nucleolus, while TERT is largely excluded from the nucleolus, reducing telomerase assembly. This suggests that nuclear compartmentalization by the non-membrane-bound nucleolus counteracts telomerase assembly, and TCAB1 is required to retain TR in the nucleoplasm. Our work provides insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase separation and demonstrates that TCAB1 plays an important role in telomerase assembly.

In Vivo Analysis of Heterogeneous Extracellular Vesicles Using a Red-Shifted Bioluminescence Resonance Energy Transfer Reporter Protein.

Current methods for characterizing the biodistribution of extracellular vesicles (EVs) are not sensitive enough to track EVs in vivo, despite significant advances over the past decade. Commonly used lipophilic fluorescent dyes are convenient, but lack specificity and yield inaccurate spatiotemporal images in the long-term tracking of EVs. In contrast, protein-based fluorescent or bioluminescent EV reporters have more accurately revealed their distribution in cells and mouse models. Here, we describe a red-shifted bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL, to analyze the trafficking of small EVs (<200 nm; sEVs) and medium/large EVs (>200 nm; m/lEVs) in mice. Its advantages are that (i) background signals in bioluminescence imaging (BLI) are negligible and (ii) the photons PalmReNL emits have spectral wavelengths longer than 600 nm and can more efficiently penetrate tissues than reporters emitting shorter wavelength light.

Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

In Vitro and In Vivo Analysis of Extracellular Vesicle-Mediated Metastasis Using a Bright, Red-Shifted Bioluminescent Reporter Protein.

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo. PalmReNL enables the authors to study the common surface molecules across EV subtypes that determine EV organotropism and their functional differences in cancer progression. Regardless of injection routes, whether retro-orbital or intraperitoneal, PalmReNL positive EVs, isolated from murine mammary carcinoma cells, localized to the lungs. The early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple intraperitoneal injections of the medium and large EV (m/lEV)-enriched fraction derived from mammary carcinoma cells is demonstrated. In addition, the results presented here show that tumor cell-derived m/lEVs act on distant tissues through upregulating LC3 expression within the lung.

NCOA5 Haplo-insufficiency Results in Male Mouse Infertility through Increased IL-6 Expression in the Epididymis.

Male infertility might be caused by genetic and/or environmental factors that impair spermatogenesis and epididymal sperm maturation. Here we report that heterozygous deletion of the nuclear receptor coactivator-5 (Ncoa5) gene resulted in decreased motility and progression of spermatozoa in the cauda epididymis, leading to infertility in male mice. Light microscopic and ultrastructural analysis revealed morphological defects in the spermatozoa collected from the cauda epididymis of Ncoa5+/- mice. Immunohistochemistry showed that interleukin-6 (IL-6) expression in epithelial cells of Ncoa5+/- epididymis was higher than wild type counterparts. Furthermore, heterozygous deletion of Il-6 gene in Ncoa5+/- male mice partially improved spermatozoa motility and moderately rescued infertility phenotype. Our results uncover a previously unknown physiological role of NCOA5 in the regulation of epididymal sperm maturation and suggest that NCOA5 deficiency could cause male infertility through increased IL-6 expression in epididymis.

Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy.

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

Use of soluble sperm extract to improve cloning efficiency in zebrafish.

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.

Conservation of avian germplasm by xenogeneic transplantation of spermatogonia from sexually mature donors.

Approximately 12.5% of all 9,920 extant bird species in the world are threatened with extinction, and yet conservation efforts through natural breeding of captive species continue to encounter difficulties. However, sperm cryopreservation and artificial insemination offer potential benefits over natural breeding, but their applicability is still limited in nondomestic species. In this study, we aimed to exploit the potential of germ cell xenotransplantation as an alternative tool for preserving germplasm of endangered birds. The study was designed to investigate whether transfer of either spermatogonia-enriched cell fraction (SEF) or crude testicular cell fraction (CTF) from adult Japanese quails (as a model for wild species) would result in recolonization of gamma-irradiated gonads of adult recipient chickens. One month after transplantation, 75% of recipients injected with SEF and 25% of recipients injected with CTF resumed spermatogenesis. However, it took more than 3 months for 33% of the negative controls to resume marginal production of sperm. Some SEF recipients produced more spermatozoa bearing head morphology compared with donor controls. DNA analysis using quail-specific primers did not detect donor's DNA in these recipients' semen. However, 6 months after xenotransplantation, presence of quail germ cells was demonstrated by polymerase chain reaction and by immunohistochemistry in 1 rooster injected with SEF. These findings indicate that spermatogonia from adult quails were capable of colonizing immunocompetent testis of adult chickens but failed to produce sufficient sperm. Despite this limitation, the present approach represents a potential conservation tool that may be used to rescue germ cells of endangered adult male birds.

Ceramide and its transport protein (CERT) contribute to deterioration of mitochondrial structure and function in aging oocytes.

In women as well as in mice, oocytes exhibit decreased developmental potential (oocyte quality) with advanced age. Our current data implicate alterations in the levels of oocyte ceramide and associated changes in mitochondrial function and structure as being prominent elements contributing to reduced oocyte quality. Both ROS levels and ATP content were significantly reduced in aged oocytes. The decreased in ROS levels are of intrigue because it is contrary to what has been previously reported. Lowered levels of both ROS and ATP indicate diminished mitochondrial function that was accompanied by alterations in mitochondrial structure. Interestingly, developmental potential of old oocytes was improved by microinjection of mitochondria isolated from young oocytes. Co-treatment of aged oocytes with ceramide and a cytoplasmic lipid carrier (l-carnitine) improved both mitochondrial morphology and function, and totally rescued spontaneous in vitro fragmentation. In addition, ceramide localization was altered in old oocytes possibly due to downregulation of the ceramide transport protein (CERT). However, knockdown of CERT alone was not sufficient to increase young oocyte's susceptibility to death, because the sequential manipulation of ceramide levels (its chronic decrease, followed by downregulation of CERT, and finally a ceramide spike) were all necessary to replicate the aging phenotype. These results indicate that oocyte aging is due to a multiplicity of events; and that with increasing biological age, changes in levels of both ceramide and its transport protein contribute to deterioration of oocyte mitochondrial structure and function. Hence, those changes may represent potential targets to manipulate when attempting to ameliorate aging phenotypes in germ cells.

RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

Ceramide and mitochondrial function in aging oocytes: joggling a new hypothesis and old players.

Maternal aging adversely affects oocyte quality (function and developmental potential) and consequently lowers pregnancy rates while increasing spontaneous abortions. Substantial evidence, especially from egg donation studies, implicates the decreased quality of an aging oocyte as a major factor in the etiology of female infertility. Nevertheless, the cellular and molecular mechanisms responsible for the decreased oocyte quality with advanced maternal aging are not fully characterized. Herein we present information in the published literature and our own data to support the hypothesis that during aging induced decreases in mitochondrial ceramide levels and associated alterations in mitochondrial structure and function are prominent elements contributing to reduced oocyte quality. Hence, by examining the molecular determinants that underlie impairments in oocyte mitochondria, we expect to sieve to a better understanding of the mechanistic anatomy of oocyte aging.

The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models.

OBJECTIVE: Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS: Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS: We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α-induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM(-/-) mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS: This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

Caspase-2 deficiency protects mice from diabetes-induced marrow adiposity.

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Bone formation and density are decreased in T1-diabetic mice. Correspondingly, the number of TUNEL positive, dying osteoblasts increases in bones of T1-diabetic mice. Moreover, two known mediators of osteoblast death, TNFα and ROS, are increased in T1-diabetic bone. TNFα and oxidative stress are known to activate caspase-2, a factor involved in the extrinsic apoptotic pathway. Therefore, we investigated the requirement of caspase-2 for diabetes-induced osteoblast death and bone loss. Diabetes was induced in 16-week old C57BL/6 caspase-2 deficient mice and their wild type littermates and markers of osteoblast death, bone formation and resorption, and marrow adiposity were examined. Despite its involvement in extrinsic cell death, deficiency of caspase-2 did not prevent or reduce diabetes-induced osteoblast death as evidenced by a twofold increase in TUNEL positive osteoblasts in both mouse genotypes. Similarly, deficiency of caspase-2 did not prevent T1-diabetes induced bone loss in trabecular bone (BV/TV decreased by 30 and 50%, respectively) and cortical bone (decreased cortical thickness and area with increased marrow area). Interestingly, at this age, differences in bone parameters were not seen between genotypes. However, caspase-2 deficiency attenuated diabetes-induced bone marrow adiposity and adipocyte gene expression. Taken together, our data suggest that caspase-2 deficiency may play a role in promoting marrow adiposity under stress or disease conditions, but it is not required for T1-diabetes induced bone loss.

Chemotherapy-induced late transgenerational effects in mice.

To our knowledge, there is no report on long-term reproductive and developmental side effects in the offspring of mothers treated with a widely used chemotherapeutic drug such as doxorubicin (DXR), and neither is there information on transmission of any detrimental effects to several filial generations. Therefore, the purpose of the present paper was to examine the long-term effects of a single intraperitoneal injection of DXR on the reproductive and behavioral performance of adult female mice and their progeny. C57BL/6 female mice (generation zero; G0) were treated with either a single intraperitoneal injection of DXR (G0-DXR) or saline (G0-CON). Data were collected on multiple reproductive parameters and behavioral analysis for anxiety, despair and depression. In addition, the reproductive capacity and health of the subsequent six generations were evaluated. G0-DXR females developed despair-like behaviors; delivery complications; decreased primordial follicle pool; and early lost of reproductive capacity. Surprisingly, the DXR-induced effects in oocytes were transmitted transgenerationally; the most striking effects being observed in G4 and G6, constituting: increased rates of neonatal death; physical malformations; chromosomal abnormalities (particularly deletions on chromosome 10); and death of mothers due to delivery complications. None of these effects were seen in control females of the same generations. Long-term effects of DXR in female mice and their offspring can be attributed to genetic alterations or cell-killing events in oocytes or, presumably, to toxicosis in non-ovarian tissues. Results from the rodent model emphasize the need for retrospective and long-term prospective studies of survivors of cancer treatment and their offspring.

Mitochondrial rejuvenation after induced pluripotency.

BACKGROUND: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal. CONCLUSIONS/SIGNIFICANCE: These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.

Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i) DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii) oocytes possess the machinery and capability for repairing such DNA damage; (iii) Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv) in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in toxicity. The use of Rad51 and Bax modulating compounds could offer women the opportunity to maintain fully functional germ cells despite cancer treatments or aging.

Absence of the proapoptotic Bax protein extends fertility and alleviates age-related health complications in female mice.

The menopausal transition in human females, which is driven by a loss of cyclic ovarian function, occurs around age 50 and is thought to underlie the emergence of an array of health problems in aging women. Although mice do not undergo a true menopause, female mice exhibit ovarian failure long before death because of chronological age and subsequently develop many of the same age-associated health complications observed in postmenopausal women. Here we show in mice that inactivation of the proapoptotic Bax gene, which sustains ovarian lifespan into advanced age, extends fertile potential and minimizes many age-related health problems, including bone and muscle loss, excess fat deposition, alopecia, cataracts, deafness, increased anxiety, and selective attention deficit. Further, ovariectomy studies show that the health benefits gained by aged females from Bax deficiency reflect a complex interplay between ovary-dependent and -independent pathways. Importantly, and contrary to popular belief, prolongation of ovarian function into advanced age by Bax deficiency did not lead to an increase in tumor incidence. Thus, the development of methods for postponing ovarian failure at menopause may represent an attractive option for improving the quality of life in aging females.