Search

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A > G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A > G levels decrease in blood with age, and an age correction representing ~ 2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate that the dynamics of m.3243A > G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate ‘on average’, creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A > G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A > G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A > G. Germline selection of m.3243A > G shows an ‘arching’ profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post–natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non–human primates and humans, a recent study rooted in single–cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non–overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. Here we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation via transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3-20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier which promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence datasets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. Additionally, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology20: 144–51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between pooled early-stage and late-stage PGCs. We re-analyzed Floros’ et al. pooled PGC data and noticed that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by cross-specific PCR primers. Importantly,when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to pooled PGCs, Floros et al. reported the analysis ofsinglelate-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, we found no NUMT-derived SNVs among single PGCs mutations. Interestingly, single PGC mutations show adecreaseof synonymity with increased intracellular mutant fraction. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously and potentially may be evolutionarily advantageous.

Using low-coverage, whole-genome sequences of trophectoderm biopsies from 11,610 human blastocyst-stage embryos, we analyzed the relationship between chromosomal abnormalities and mitochondrial (mt) DNA dynamics. Comparing 6,208 aneuploid and 5,402 euploid embryos in cohort studies, we found that mtDNA content in aneuploid embryos was significantly higher than that in euploid embryos. This outcome was confirmed through intrafamilial analyses of embryos with matched parents andin vitrofertilization cycles, and it occurred independent of maternal age. Additional human population-based studies uncovered a higher abundance of ultra-rare mtDNA variants located in never-altered positions in the human population in aneuploid compared to euploid embryos in both cohort- and family-based analyses. This maternal age-independent association of increased mtDNA content and aneuploidy in human embryos may reflect a novel mechanism of purifying selection against potentially deleterious mtDNA variants, which arise from germline or early developmental mtDNA damaging events, that occurs in human embryos prior to implantation.

Cells within tissues are routinely subjected to physiological stress and strain, arising from direct interactions with neighboring cells as well as with extracellular matrix components. Accordingly, there is tremendous interest in deciphering how cells sense, and respond to, changes in biomechanical forces. In this study, we explored the effects of mechanostimulation on the differentiation of mouse female germline or oogonial stem cells (OSCs) as a model for adult stem cell function. We report that increasing levels, or repeated application of a subthreshold fixed level, of radial strain to OSCs in culture significantly increased rates of in vitro oocyte formation as a measure of stem cell differentiation. These responses involved changes in F-actin-mediated cytoskeletal tension as well as in activation of intracellular signaling by Rho-associated protein kinase (ROCK) and Yes-associated protein (YAP) phosphorylation. In addition, application of mechanical strain to OSCs enhanced association of YAP with muscle-specific cytidine-adenosine-thymidine (MCAT) response elements in the promoter stimulated by retinoic acid gene 8 (Stra8), the transcriptional activation of which is required for germline meiotic commitment. These data indicate that biomechanical strain directly promotes the differentiation of adult female germline stem cells through a signaling pathway involving F-actin, ROCK, YAP, and Stra8.

Progressive loss of ovarian estrogen (E2) production is a hallmark feature of, if not a driving force behind, reproductive aging and the menopause. Recent genetic studies in mice have shown that female germline or oogonial stem cells (OSCs) contribute to maintenance of adult ovarian function and fertility under physiological conditions through support of de-novo oogenesis. Here we show that mouse OSCs express E2 receptor-α (ERα). In the presence of E2, ERα interacts with the stimulated by retinoic acid gene 8 (Stra8) promoter to drive Stra8 expression followed by oogenesis. Treatment of mice with E2 in vivo increases Stra8 expression and oogenesis, and these effects are nullified by ERα (Esr1), but not ERβ (Esr2), gene disruption. Although mice lacking ERα are born with a normal quota of oocytes, ERα-deficient females develop premature ovarian insufficiency in adulthood due to impaired oogenesis. Lastly, mice treated with reversible ER antagonists show a loss of Stra8 expression and oocyte numbers; however, both endpoints rebound to control levels after ceasing drug treatment. These findings establish a key physiological role for E2-ERα signaling in promoting OSC differentiation as a potential mechanism to maintain adequate numbers of ovarian follicles during reproductive life.

The resilience of the mitochondrial genome to a high mutational pressure depends, in part, on purifying selection against detrimental mutations in the germline. It is crucial to understand the mechanisms of this process. Recently, Floros et al. concluded that much of the purifying selection takes place during the proliferation of primordial germ cells (PGCs) because, according to their analysis, the synonymity of mutations in late PGCs was seemingly increased compared to those in early PGCs. We re-analyzed the Floros et al. mutational data and discovered a high proportion of sequence variants that are not true mutations, but originate from NUMTs, the latter of which are segments of mitochondrial DNA (mtDNA) inserted into nuclear DNA, up to millions of years ago. This is a well-known artifact in mtDNA mutational analysis. Removal of these artifacts from the Floros et al. dataset abolishes the reported effect of purifying selection in PGCs. We therefore conclude that the mechanism of germline selection of mtDNA mutations remains open for debate, and more research is needed to fully elucidate the timing and nature of this process.

Background Third-generation sequencing offers some advantages over next-generation sequencing predecessors, but with the caveat of harboring a much higher error rate. Clustering-related sequences is an essential task in modern biology. To accurately cluster sequences rich in errors, error type and frequency need to be accounted for. Levenshtein distance is a well-established mathematical algorithm for measuring the edit distance between words and can specifically weight insertions, deletions and substitutions. However, there are drawbacks to using Levenshtein distance in a biological context and hence has rarely been used for this purpose. We present novel modifications to the Levenshtein distance algorithm to optimize it for clustering error-rich biological sequencing data. Results We successfully introduced a bidirectional frameshift allowance with end-user determined accommodation caps combined with weighted error discrimination. Furthermore, our modifications dramatically improved the computational speed of Levenstein distance. For simulated ONT MinION and PacBio Sequel datasets, the average clustering sensitivity for 3GOLD was 41.45% (S.D. 10.39) higher than Sequence-Levenstein distance, 52.14% (S.D. 9.43) higher than Levenshtein distance, 55.93% (S.D. 8.67) higher than Starcode, 42.68% (S.D. 8.09) higher than CD-HIT-EST and 61.49% (S.D. 7.81) higher than DNACLUST. For biological ONT MinION data, 3GOLD clustering sensitivity was 27.99% higher than Sequence-Levenstein distance, 52.76% higher than Levenshtein distance, 56.39% higher than Starcode, 48% higher than CD-HIT-EST and 70.4% higher than DNACLUST. Conclusion Our modifications to Levenshtein distance have improved its speed and accuracy compared to the classic Levenshtein distance, Sequence-Levenshtein distance and other commonly used clustering approaches on simulated and biological third-generation sequenced datasets. Our clustering approach is appropriate for datasets of unknown cluster centroids, such as those generated with unique molecular identifiers as well as known centroids such as barcoded datasets. A strength of our approach is high accuracy in resolving small clusters and mitigating the number of singletons.

Nucleic acid sequence analyses are fundamental to all aspects of biological research, spanning aging, mitochondrial DNA (mtDNA) and cancer, as well as microbial and viral evolution. Over the past several years, significant improvements in DNA sequencing, including consensus sequence analysis, have proven invaluable for high-throughput studies. However, all current DNA sequencing platforms have limited utility for studies of complex mixtures or of individual long molecules, the latter of which is crucial to understanding evolution and consequences of single nucleotide variants and their combinations. Here we report a new technology termed LUCS (Long-molecule UMI-driven Consensus Sequencing), in which reads from third-generation sequencing are aggregated by unique molecular identifiers (UMIs) specific for each individual DNA molecule. This enables in-silico reconstruction of highly accurate consensus reads of each DNA molecule independent of other molecules in the sample. Additionally, use of two UMIs enables detection of artificial recombinants (chimeras). As proof of concept, we show that application of LUCS to assessment of mitochondrial genomes in complex mixtures from single cells was associated with an error rate of 1X10-4 errors/nucleotide. Thus, LUCS represents a major step forward in DNA sequencing that offers high-throughput capacity and high-accuracy reads in studies of long DNA templates and nucleotide variants in heterogenous samples.

The endocrine function of the ovary is dependent upon the ovarian follicle, which on a cellular basis consists of an oocyte surrounded by adjacent somatic cells responsible for generating sex steroid hormones and maintenance of hormonal stasis with the hypothalamic-pituitary axis. As females age, both fertility and the endocrine function of the ovary decline due to waning follicle numbers as well as aging-related cellular dysfunction. Although there is currently no cure for ovarian failure and endocrine disruption, recent advances in ovarian biology centered on ovarian stem cell and progenitor cell populations have brought the prospects of cell- or tissue-based therapeutic strategies closer to fruition. Herein, we review the relative contributions of ovarian stem cells to ovarian function during the reproductive lifespan, and postulate steps toward the development of ovarian stem cell-based approaches to advance fertility treatments, and also importantly to provide a physiological long-term means of endocrine support.

Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi–parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell., Julie MacDonald et al. present a method for mitochondrial isolation using fluorescence-activated mitochondria sorting to facilitate analysis from individual organelles. They validate their platform using a number of downstream assays, including electron microscopy, proteomic profiling, and mtDNA analysis.

A now large body of work has solidified the central role that mitochondria play in oocyte development, fertilization, and embryogenesis. From these studies, a new technology termed autologous germline mitochondrial energy transfer was developed for improving pregnancy success rates in assisted reproduction. Unlike prior clinical studies that relied on the use of donor, or nonautologous, mitochondria for microinjection into eggs of women with a history of repeated in vitro fertilization failure to enhance pregnancy success, autologous germline mitochondrial energy transfer uses autologous mitochondria collected from oogonial stem cells of the same woman undergoing the fertility treatment. Initial trials of autologous germline mitochondrial energy transfer during - in vitro fertilization at three different sites with a total of 104 patients indicated a benefit of the procedure for improving pregnancy success rates, with the birth of children conceived through the inclusion of autologous germline mitochondrial energy transfer during in vitro fertilization. However, a fourth clinical study, consisting of 57 patients, failed to show a benefit of autologous germline mitochondrial energy transfer– in vitro fertilization versus in vitro fertilization alone for improving cumulative live birth rates. Complicating this area of work further, a recent mouse study, which claimed to test the long-term safety of autologous mitochondrial supplementation during in vitro fertilization, raised concerns over the use of the procedure for reproduction. However, autologous mitochondria were not actually used for preclinical testing in this mouse study. The unwarranted fears that this new study’s erroneous conclusions could cause in women who have become pregnant through the use of autologous germline mitochondrial energy transfer during- in vitro fertilization highlight the critical need for accurate reporting of preclinical work that has immediate bearing on human clinical studies.

The in vivo gene networks involved in coordinating human fetal ovarian development remain obscure. In this study, quantitative mass spectrometry was conducted on ovarian tissue collected at key stages during the first two trimesters of human gestational development, confirming the expression profiling data using immunofluorescence, as well as in vitro modeling with human oogonial stem cells (OSCs) and human embryonic stem cells (ESCs). A total of 3,837 proteins were identified in samples spanning developmental days 47-137. Bioinformatics clustering and Ingenuity Pathway Analysis identified DNA mismatch repair and base excision repair as major pathways upregulated during this time. In addition, MAEL and TEX11, two key meiosis-related proteins, were identified as highly expressed during the developmental window associated with fetal oogenesis. These findings were confirmed and extended using in vitro differentiation of OSCs into in vitro derived oocytes and of ESCs into primordial germ cell-like cells and oocyte-like cells, as models. In conclusion, the global protein expression profiling data generated by this study have provided novel insights into human fetal ovarian development in vivo and will serve as a valuable new resource for future studies of the signaling pathways used to orchestrate human oogenesis and folliculogenesis.

Ovarian aging is characterized by a decline in both the total number and overall quality of oocytes, the latter of which has been experimentally tied to mitochondrial dysfunction. Clinical studies in the late 1990s demonstrated that transfer of cytoplasm aspirated from eggs of young female donors into eggs of infertile women at the time of intracytoplasmic sperm injection improved pregnancy success rates. However, donor mitochondria were identified in offspring, and the United States Food and Drug Administration raised questions about delivery of foreign genetic material into human eggs at the time of fertilization. Accordingly, heterologous cytoplasmic transfer, while promising, was in effect shut down as a clinical protocol. The recent discovery of adult oogonial (oocyte-generating) stem cells in mice, and subsequently in women, has since re-opened the prospects of delivering a rich source of pristine and patient-matched germline mitochondria to boost egg health and embryonic developmental potential without the need for young donor eggs to obtain cytoplasm. Herein we overview the science behind this new protocol, which has been patented and termed autologous germline mitochondrial energy transfer, and its use to date in clinical studies for improving pregnancy success in women with a prior history of assisted reproduction failure.

Multiple labs have reported that mammalian ovaries contain oogonial stem cells (OSCs), which can differentiate into oocytes that fertilize to produce offspring. However, the physiological relevance of these observations to adult ovarian function is unknown. Here we performed targeted and reversible ablation of premeiotic germ cells undergoing differentiation into oocytes in transgenic mice expressing the suicide gene, herpes simplex virus thymidine kinase (HSVtk), driven by the promoter of stimulated by retinoic acid gene 8 (Stra8), a germ cell-specific gene activated during meiotic commitment. Over a 21-day ablation phase induced by the HSVtk pro-drug, ganciclovir (GCV), oocyte numbers declined due to a disruption of new oocyte input. However, germ cell differentiation resumed after ceasing the ablation protocol, enabling complete regeneration of the oocyte pool. We next employed inducible lineage tracing to fate map, through Cre recombinase-mediated fluorescent reporter gene activation only in Stra8-expressing cells, newly-formed oocytes. Induction of the system during adulthood yielded a mosaic pool of unmarked (pre-existing) and marked (newly-formed) oocytes. Marked oocytes matured and fertilized to produce offspring, which grew normally to adulthood and transmitted the reporter to second-generation offspring. These findings establish that oocytes generated during adulthood contribute directly to ovarian function and natural fertility in mammals.

Historically, approaches designed to offer women diagnosed with cancer the prospects of having a genetically matched child after completion of their cytotoxic treatments focused on the existing oocyte population as the sole resource available for clinical management of infertility. In this regard, elective oocyte and embryo cryopreservation, as well as autologous ovarian cortical tissue grafting posttreatment, have gained widespread support as options for young girls and reproductive-age women who are faced with cancer to consider. In addition, the use of ovarian protective therapies, including gonadotropin-releasing hormone agonists and sphingosine-1-phosphate analogs, has been put forth as an alternative way to preserve fertility by shielding existing oocytes in the ovaries in vivo from the side-effect damage caused by radiotherapy and many chemotherapeutic regimens. This viewpoint changed with the publication of now numerous reports that adult ovaries of many mammalian species, including humans, contain a rare population of oocyte-producing germ cells—referred to as female germline or oogonial stem cells (OSCs). This new line of study has fueled research into the prospects of generating new oocytes, rather than working with existing oocytes, as a novel approach to sustain or restore fertility in female cancer survivors. Here, we overview the history of work from laboratories around the world focused on improving our understanding of the biology of OSCs and how these cells may be used to reconstitute “artificial” ovarian tissue in vitro or to regenerate damaged ovarian tissue in vivo as future fertility-preservation options.

The concept that oogenesis continues into reproductive life has been well established in nonmammalian species. Recent studies of mice and women indicate that oocyte formation is also not, as traditionally believed, restricted to the fetal or perinatal periods. Analogous to de novo oocyte formation in flies and fish, newly formed oocytes in adult mammalian ovaries arise from germline stem cells (GSCs) or, more specifically, oogonial stem cells (OSCs). Studies of mice have confirmed that isolated OSCs, once delivered back into adult ovaries, are capable of generating fully functional eggs that fertilize to produce healthy embryos and offspring. Parallel studies of OSCs recently purified from ovaries of reproductive-age women indicate that these cells closely resemble their mouse ovary-derived counterparts, although the fertilization competency of oocytes generated by human OSCs awaits clarification. Despite the ability of OSCs to produce new oocytes during adulthood, oogenesis will still ultimately cease with age, contributing to ovarian failure. The causal mechanisms behind these events in mammals are unknown, but studies of flies have revealed that GSC niche dysfunction plays a critical role in age-related oogenic failure. Such insights derived from evaluation of nonmammalian species, in which postnatal oogenesis has been studied in depth, may aid in development of new strategies to alleviate ovarian failure and infertility in mammals.

The mtDNA mutator mouse lacks the proofreading capacity of the sole mtDNA polymerase, leading to accumulation of somatic mtDNA mutations, and a profound premature aging phenotype including elevated oxidative stress and apoptosis, and reduced mitochondrial function. We have previously reported that endurance exercise alleviates the aging phenotype in the mutator mice, reduces oxidative stress, and enhances mitochondrial biogenesis. Here we summarize our findings, with the emphasis on the central role of p53 in these adaptations. We demonstrate that mtDNA in sedentary and exercised PolG mice carry similar amounts of mutations in muscle, but in addition to that sedentary mice have more non-mutational damage, which is mitigated by exercise. It follows therefore that the profound alleviation of the mtDNA mutator phenotype in muscle by exercise may not require a reduction in mtDNA mutational load, but rather a decrease of mtDNA damage and/or oxidative stress. We further hypothesize that the observed 'alleviation without a reduction of mutational load' implies that the oxidative stress in PolG muscle is maintained, at least in part, by the 'malicious cycle', a hypothetical positive feedback potentially driven by the 'transcriptional mutagenesis', that is the conversion of chemically modified nucleotides into mutant RNA bases by the mitochondrial RNA polymerase.

The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry.

Women approaching advanced maternal age have extremely poor outcomes with both natural and assisted fertility. Moreover, the incidence of chromosomal abnormalities and birth defects increases with age. As of yet, there is no effective and practical strategy for delaying ovarian aging or improving oocyte quality. We demonstrate that the lifelong consumption of a diet rich in omega-3 fatty acids prolongs murine reproductive function into advanced maternal age, while a diet rich in omega-6 fatty acids is associated with very poor reproductive success at advanced maternal age. Furthermore, even short-term dietary treatment with a diet rich in omega-3 fatty acids initiated at the time of the normal age-related rapid decline in murine reproductive function is associated with improved oocyte quality, while short-term dietary treatment with omega-6 fatty acids results in very poor oocyte quality. Thus, omega-3 fatty acids may provide an effective and practical avenue for delaying ovarian aging and improving oocyte quality at advanced maternal age.

Through the use of parabiosis in mice, aging-related deterioration of skeletal muscle and liver has been linked to a loss of systemic factors that support adult stem or progenitor cell activity. Since aging-related ovarian failure has recently been attributed, at least in part, to a loss of de-novo oocyte-containing follicle formation associated with declining oogonial stem cell activity, herein we tested in mice if aging-related changes in systemic factors influence the size of the ovarian follicle reserve. Ovaries of young (2-month-old) females parabiotically joined with young females for 5 weeks possess comparable numbers of healthy and degenerative (atretic) oocyte-containing follicles in their ovaries as those detected in non-parabiotic young females. Joining of young females with young males significantly increases follicle atresia without a net change healthy follicle numbers. Surprisingly, young females joined with aged (24-month-old) males exhibit a significant increase in the number of primordial follicles comprising the ovarian reserve, and this occurs without changes in follicle growth activation or atresia. Blood of aged males also induces ovarian expression of the germ cell-specific meiosis gene, Stimulated by retinoic acid gene 8 (Stra8), in ovaries of female parabionts, further supporting the conclusion that the observed changes in the follicle reserve of females joined with aged males reflect increased oocyte formation. Thus, factors in male blood exert dramatic effects on ovarian follicle dynamics, and aging males possess a beneficial systemic factor that significantly expands the ovarian follicle reserve in females through enhanced oogenesis.

Cables1 protects p63 from proteasomal degradation to ensure deletion of cells after genotoxic stress Cables1 interacts with TAp63 to protect it from ubiquitin-mediated degradation after exposure to genotoxic stress. Oocytes lacking Cables1 accumulate less phosphorylated TAp63 in response to genotoxic stress, which enhances the survival of these cells., The p63 gene product regulates epithelial morphogenesis and female germline integrity. In this study, we show that cyclin-dependent kinase 5 and Abl enzyme substrate 1 (Cables1) interacts with the trans-activating (TA) p63α isoform to protect it from proteasomal degradation. Using the female germline of Cables1-null mice as an in vivo model, we demonstrate further that oocytes lacking Cables1 exhibit lower basal levels of TAp63α and reduced accumulation of phosphorylated TAp63α in response to genotoxic stress. This in turn enhances the survival of these cells after ionizing radiation exposure. Thus, Cables1 modulates p63 protein stability and function during genotoxic stress.

Contrasting the equal contribution of nuclear genetic material from maternal and paternal sources to offspring, passage of mitochondria, and thus mitochondrial DNA (mtDNA), is uniparental through the egg. Since mitochondria in eggs are ancestral to all somatic mitochondria of the next generation and to all cells of future generations, oocytes must prepare for the high energetic demands of maturation, fertilization and embryogenesis while simultaneously ensuring that their mitochondrial genomes are inherited in an undamaged state. Although significant effort has been made to understand how the mtDNA bottleneck and purifying selection act coordinately to prevent silent and unchecked spreading of invisible mtDNA mutations through the female germ line across successive generations, it is unknown if and how somatic cells of the immediate next generation are spared from inheritance of detrimental mtDNA molecules. Here, we review unique aspects of mitochondrial activity and segregation in eggs and early embryos, and how these events play into embryonic developmental competency in the face of advancing maternal age.

The meiotic cell cycle is required for production of fertilization-competent gametes. Germ cell meiotic commitment requires expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). Meiotic suppression in embryonic male germ cells is believed to result from sex-specific differences in CYP26B1-catalyzed RA metabolism in the developing gonads. Here we show in mice that RA-induced Stra8 transcription is epigenetically controlled and requires a co-activator that binds proximal to the RA response elements (RAREs) in the Stra8 promoter. Embryonic male germ cells exposed in utero to the class I/II histone deacetylase (HDAC) inhibitor, trichostatin-A (TSA), show premature Stra8 activation and meiotic entry without altered Cyp26b1 expression. We also show that Stra8 expression is detectable and physiologically regulated in adult mouse ovaries. Further, oogenesis induction in adult females using TSA is associated with Stra8 activation, and these events are absent in mice deficient in the RA precursor vitamin A. Finally, all of the actions of TSA in premeiotic germ cells in vitro and in mouse ovaries in vivo can be reproduced with the small molecule HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). Thus, the ability of RA to transcriptionally induce expression of the meiosis-commitment gene, Stra8, is epigenetically controlled and this process involves a novel co-activator that functions upstream of the RAREs. These events not only coordinate the sex-specific timing of meiotic entry during embryogenesis, but also contribute to the regulation of oogenesis in adult female mammals.

Of all the major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Until recently, traditional thinking was that female mammals are provided with a non-renewable pool of oocyte-containing follicles at birth that are depleted during postnatal life to exhaustion, driving ovarian failure. However, a growing body of evidence, including the isolation of germline stem cells (GSC) from adult mouse ovaries that produce developmentally-competent oocytes, has challenged this belief. In addition, rare germline stem-like cells capable of generating oocytes in vitro that undergo parthenogenesis to form blastocyst-like structures have recently been identified in postmenopausal human ovaries. Here we show that the germline-specific meiosis-commitment genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted in azoospermia-like (Dazl), are highly expressed in aged mouse ovaries. However, histological and marker analyses fail to demonstrate the presence of oocytes, supporting that Stra8 and Dazl are expressed in premeiotic germ cells that do not undergo further differentiation. Through the use of aged germline-specific GFP-expressing transgenic mice, we further show that these germ cells can generate GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and form follicles when grafted into young adult wild-type female hosts. Thus, aged mouse ovaries possess a rare population of premeiotic germ cells that retain the capacity to form oocytes if exposed to a young host environment.

Activation of the aryl hydrocarbon receptor (AHR) by polycyclic aromatic hydrocarbons (PAH), a ubiquitous class of environmental and occupational biohazards, accelerates germ cell depletion in female mice during prenatal and postnatal life. Like AHR, BAX is also functionally required for PAH to kill oocytes. Here, we show that PAH upregulates ovarian expression of not just Bax but a large cassette of proapoptotic genes that function at multiple steps of the cell death signaling pathway. We further show that ovarian expression of p53 and several proapoptotic genes that are known transcriptional targets of p53 are increased by PAH treatment, and that mice lacking functional p53 are resistant to the ovotoxic effects of in vivo PAH exposure. This study provides further mechanistic insights into how PAH accelerate oocyte depletion in females and adds p53 to the list of genes whose functional importance to PAH-induced ovotoxicity has been demonstrated by gene knockout technology.

Whether or not oogenesis continues in the ovaries of mammalian females during postnatal life was heavily debated from the late 1800s through the mid-1900s. However, in 1951 Lord Solomon Zuckerman published what many consider to be a landmark paper summarizing his personal views of data existing at the time for and against the possibility of postnatal oogenesis. In Zuckerman's opinion, none of the evidence he considered was inconsistent with Waldeyer's initial proposal in 1870 that female mammals cease production of oocytes at or shortly after birth. This conclusion rapidly became dogma, and remained essentially unchallenged until just recently, despite the fact that Zuckerman did not offer a single experiment proving that adult female mammals are incapable of oogenesis. Instead, 20 years later he reemphasized that his conclusion was based solely on an absence of data he felt would be inconsistent with the idea of a nonrenewable oocyte pool provided at birth. However, in the immortal words of Carl Sagan, an "absence of evidence is not evidence of absence." Indeed, building on the efforts of a few scientists who continued to question this dogma after Zuckerman's treatise in 1951, we reported several data sets in 2004 that were very much inconsistent with the widely held belief that germ cell production in female mammals ceases at birth. Perhaps not surprisingly, given the magnitude of the paradigm shift being proposed, this work reignited a vigorous debate that first began more than a century ago. Our purpose here is to review the experimental evidence offered in recent studies arguing support for and against the possibility that adult mammalian females replenish their oocyte reserve. "Never discourage anyone who continually makes progress, no matter how slow."-Plato (427-347 BC).

The female reproductive axis is the first major organ system of the body to fail with advancing age. In addition to a permanent cessation of fertile potential, the loss of cyclic ovarian function in humans heralds the onset of menopause, which in turn underlies the emergence of a diverse spectrum of health issues in aging women. Recently, it was reported that bone marrow (BM) transplantation (BMT) into adult female mice conditioned a week earlier with highly cytotoxic drugs rescues ovarian function and fertility. Herein we show in mice receiving no prior conditioning regimen that once-monthly infusions of BM-derived cells retrieved from young adult female donors bearing an enhanced green fluorescent protein (EGFP) transgene sustain the fertile potential of aging wild-type females long past their time of normal reproductive senescence. The fertility-promoting effects of female donor BM are observed regardless whether the infusions are initiated in young adult or middle-aged females. Although the mechanism by which BM infusions benefit the reproductive performance of aging females remains to be elucidated, the absence of EGFP-expressing offspring suggests that it does not depend on development of mature eggs derived from germline-committed cells in the donor marrow. However, donor BM-derived somatic cells accumulate in the recipients, indicating efficient donor cell engraftment without prior conditioning. These findings provide a strong impetus to further explore development of adult stem cell-based technologies to safely extend function of the female reproductive axis into advanced age without the need for toxic pre-conditioning protocols routinely used in other models of stem cell delivery.

By virtue of the fact that oocytes not only serve to produce embryos after fertilization but also can effectively reprogram adult somatic cell nuclei to a pluripotent state, much of the interest in the role of stem cells in ovarian biology has been focused on the germline. However, very recent studies have revealed that somatic stem cells may also be of considerable relevance to the study of normal ovarian function. Furthermore, stem cell dysfunction may underlie or contribute to disease states such as ovarian cancer and polycystic ovary syndrome. Our objective is to explore these concepts in greater detail, with the hope of stimulating further research efforts into understanding what role stem cells may play in the physiology and pathology of the mammalian female gonads.

Age-related ovarian failure in women heralds the transition into postmenopausal life, which is characterized by a loss of fertility and increased risk for cardiovascular disease, osteoporosis and cognitive dysfunction. Unfortunately, there are no options available for delaying loss of ovarian function with age in humans. Rodent studies have shown that caloric restriction (CR) can extend female fertile lifespan; however, much of this work initiated CR at weaning, which causes stunted adolescent growth and a delayed onset of sexual maturation. Herein we tested in mice if CR initiated in adulthood could delay reproductive aging. After 4 months of CR, the ovarian follicle reserve was doubled compared to ad libitum (AL)-fed age-matched controls, which in mating trials exhibited a loss of fertility by 15.5 months of age. In CR females returned to AL feeding at 15.5 months of age, approximately one-half remained fertile for 6 additional months and one-third continued to deliver offspring through 23 months of age. Notably, fecundity of CR-then-AL-fed females and postnatal offspring survival rates were dramatically improved compared with aging AL-fed controls. For example, between 10 and 23 months of age, only 22% of the 54 offspring delivered by AL-fed females survived. In contrast, over 73% of the 94 pups delivered by 15.5- to 23-month-old CR-then-AL-fed mice survived without any overt complications. These data indicate that in mice adult-onset CR maintains function of the female reproductive axis into advanced age and dramatically improves postnatal survival of offspring delivered by aged females.

In 2004, a study from our lab published in the journal Nature reignited a worldwide debate over the validity of the dogma that mammalian females are incapable of oocyte and follicle production during postnatal life. Amidst widespread skepticism, we forged ahead and published a second study in 2005 in the journal Cell, which not only reaffirmed with different experimental approaches that this dogma is invalid but also identified cells in bone marrow (BM) and blood of adult female mice that could generate oocytes contained within immature follicles in the ovaries of recipient females following transplantation. Although this work has been the subject of extensive critical commentary as well, two recent reports from others have confirmed the germline potential of adult BM-derived cells in mice. Further, independent corroboration of the results and conclusions presented in our earlier Nature paper is also now available. However, three papers have been published that purportedly question our work and conclusions. The first is a paper by Eggan et al. published in the journal Nature, which attempts to draw conclusions about the germline potential of BM-derived cells after focusing solely on ovulated eggs while ignoring what may be occurring at the level of oogenesis in the ovaries. The second, from Veitia and colleagues, attempts to draw the same conclusions as Eggan et al. from a single clinical case report of a female Fanconi anemia patient who conceived a genetically-related daughter after allogeneic bone marrow transplantation. The third is a report from Liu et al. just released in the journal Developmental Biology that claims to provide evidence refuting the possibility that adult female mammals produce new oocytes. However, all of the data presented in this latter report are derived from gene expression studies that the authors say fail to show the occurrence of meiosis or germ cell mitosis in adult human ovaries. Given that more than three years have passed since our initial study challenging the dogma was published, it is our belief that continuing arguments against the possibility of postnatal oogenesis in mammals should be based on more rigorous experimental approaches than simply an absence of evidence, especially from gene expression analyses. Further, the interpretations offered by Liu et al. of their results are not as straightforward as they contend since some of their data can also be viewed as supportive of postnatal oogenesis in reproductive age women.

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.

Previously published studies have identified the presence of a rare population of very small cells (< 5.0 microns in diameter) in the bone marrow of mice (Leukemia 2006 May;20(5):857-69). In additi...

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

We previously published evidence that oocytes exposed to doxorubicin (DXR), a widely used chemotherapeutic agent, rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis. In this report, we elucidated the molecular requirements for actions of this drug in oocytes. Our results indicate that within 1 h of exposure DXR causes rapid DNA damage, and commits the oocyte to cytoplasmic fragmentation by the fourth hour, followed by delayed oocyte activation and execution of cytoplasmic fragmentation. Inhibitors that interfere with oocyte activation consistently rescue cytoplasmic fragmentation, but fail to suppress DNA damage. There was evidence of depletion of Bax, Caspase-2, MA-3 and Bcl-x transcripts, suggesting that modulations by DXR caused recruitment of these maternal transcripts into the translation process. Furthermore, sphingolipids such as sphingosine-1-phosphate and ceramide modulate DXR actions by, respectively, altering its intracellular trafficking, or by sustaining the drug's contact with DNA.

Theca-interstitial (T-I) cells play a fundamental role in the control of ovarian function. Steroidogenic activity and growth of the T-I cells are regulated by many paracrine and endocrine factors. However, little is known about the mechanisms controlling T-I death. In an in vitro model of apoptosis, purified rat T-I cells were cultured for 24 h with serum and subsequently for up to an additional 24 h with serum or in serum-free medium with or without insulin, insulin-like growth factors (IGF-I and IGF-II) and LH or 8-bromo-cyclic AMP (8Br-cAMP). Apoptosis was identified by histological assessment of nuclear morphology and by detection of internucleosomal cleavage and quantified by determination of [alpha32P]-dideoxy-ATP 3'-end labeling of low molecular weight DNA. Serum withdrawal resulted in nuclear condensation and fragmentation into apoptotic bodies of T-I cells and led to pronounced DNA cleavage. Insulin (10 nM) protected T-I cells from apoptosis, reducing DNA fragmentation by 39 +/- 8% compared to serum-free controls. IGF-I (10 nM) and IGF-II (10 nM) had comparable antiapoptotic effects, decreasing DNA fragmentation by 55 +/- 9% and 37 +/- 14%, respectively. In contrast, LH (100 ng/ml) and 8Br-cAMP (1 mM) augmented the pro-apoptotic effect of serum withdrawal, increasing DNA fragmentation by 85 +/- 55% and 72 +/- 42%, respectively. The antiapoptotic effects of insulin and IGFs and the pro-apoptotic effect of LH, acting via the cAMP system, may be important in the maintenance of T-I homeostasis. Moreover, excessive levels of insulin and free IGFs may lead to T-I cell hyperplasia characteristic of conditions such as polycystic ovary syndrome.