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Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

Using a protocol that recapitulates both meiosis and oocyte growth in vitro , the authors induce mouse pluripotent stem cells to differentiate into fully functional oocytes that can be fertilized and generate viable offspring, thereby recapitulating the full mammalian female germline cycle in a dish. A mouse female germline in vitro This study reports the successful production of functional mouse eggs entirely in culture, a long-standing goal in the fields of stem cell biology and regenerative medicine. From mouse pluripotent stem cells, the authors generated functional oocytes in vitro that can be fertilized and generate fully viable offspring. Their protocol recapitulates both meiosis and oocyte growth, and the authors are also able to isolate pluripotent stem cell lines from the in-vitro -generated eggs after fertilization, thereby recapitulating the full mammalian female germline cycle in a dish. The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg 1 , 2 . The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro , thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

Key Points Aneuploidy is extraordinarily common in humans, occurring in an estimated 20–40% of all conceptions. It is the most common cause of miscarriages and congenital defects in our species and is a leading impediment to the treatment of infertility. Most aneuploidy results from maternal meiotic nondisjunctional errors. However, there is remarkable variation among chromosomes in the way in which these errors originate, indicating that there are multiple mechanisms by which human aneuploidy occurs. Studies of human fetal oocytes indicate a high level of recombination errors, indicating that some oocytes are predisposed to nondisjoin because of events that occurred before birth. Cell cycle control checkpoints that operate in meiotic prophase and at the metaphase–anaphase transition are less stringent in females than in males. Consequently, abnormal cells that are eliminated in spermatogenesis may escape detection in the female, ultimately leading to aneuploid eggs. Studies from mice suggest that loss of cohesin proteins over the reproductive life of the female contribute to the maternal age effect on human trisomy. Exposure to endocrine disruptors (for example, bisphenol A) disrupts oogenesis at multiple stages and predisposes the oocyte to aneuploidy. Aneuploidy is the leading cause of congenital defects in humans and nearly always results from errors occurring in oocytes. Here, the authors review the evidence pointing towards the mechanistic basis of meiotic defects leading to aneuploidy and discuss the potential role of environmental factors. Trisomic and monosomic (aneuploid) embryos account for at least 10% of human pregnancies and, for women nearing the end of their reproductive lifespan, the incidence may exceed 50%. The errors that lead to aneuploidy almost always occur in the oocyte but, despite intensive investigation, the underlying molecular basis has remained elusive. Recent studies of humans and model organisms have shed new light on the complexity of meiotic defects, providing evidence that the age-related increase in errors in the human female is not attributable to a single factor but to an interplay between unique features of oogenesis and a host of endogenous and exogenous factors.

During mitosis, cells undergo symmetrical cell division, while oocyte meiotic maturation undergoes two consecutive, asymmetric divisions that generate a totipotent haploid oocyte and two small polar bodies not involved in DNA replication. This specialized division allows most maternal components to be maintained in the oocytes for early embryo development. Nuclear positioning, germinal vesicle breakdown, spindle migration, spindle rotation, chromosome segregation, and polar body extrusion are the most critical cellular processes during oocyte meiosis I and II, and a growing number of studies primarily using the mouse oocyte model revealed that actin filaments were critical for these processes, especially for spindle migration. Several important molecules have been reported to be involved in these processes. One family of molecules are the small GTPases, such as Rho GTPases, Ran GTPases, and Rab GTPases and another are the actin nucleators, such as the formin family and the Arp2/3 complex. The present review summarizes recent progress made regarding the roles of actin filaments in the asymmetric oocyte division. Summary Sentence Actin filaments widely involve into multiple cellular processes such as nuclear positioning, germinal vesicle breakdown, spindle migration, chromosome segregation, spindle rotation and polar body extrusion in oocyte mammalian meiosis.

Hong Xu and colleagues demonstrate reduced germline replication and selection against the transmission of mitochondria encoding a temperature-sensitive cytochrome c oxidase subunit. Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist 1 , crippling mitochondrial mutations are exceedingly rare 2 . Recent studies have demonstrated strong purifying selection in the mouse female germline 3 , 4 . However, the mechanisms underlying positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila melanogaster oogenesis, finding that mtDNA replication commenced before oocyte determination during the late germarium stage and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA allele, mt:CoI T300I , which resulted in reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of the mt:CoI T300I allele in heteroplasmic flies was decreased, both during oogenesis and over multiple generations, at the restrictive temperature. Furthermore, we determined that selection against mt:CoI T300I overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for the selective amplification of wild-type mtDNA, which may be evolutionarily conserved to limit the transmission of deleterious mutations.

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.

PurposeMammalian oogenesis and folliculogenesis share a dynamic connection that is critical for gamete development. For maintenance of quiescence or follicular activation, follicles must respond to soluble signals (growth factors and hormones) and physical stresses, including mechanical forces and osmotic shifts. Likewise, mechanical processes are involved in cortical tension and cell polarity in oocytes. Our objective was to examine the contribution and influence of biomechanical signaling in female mammalian gametogenesis.MethodsWe performed a systematic review to assess and summarize the effects of mechanical signaling and mechanotransduction in oocyte maturation and folliculogenesis and to explore possible clinical applications. The review identified 2568 publications of which 122 met the inclusion criteria.ResultsThe integration of mechanical and cell signaling pathways in gametogenesis is complex. Follicular activation or quiescence are influenced by mechanical signaling through the Hippo and Akt pathways involving the yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), phosphatase and tensin homolog deleted from chromosome 10 (PTEN) gene, the mammalian target of rapamycin (mTOR), and forkhead box O3 (FOXO3) gene.ConclusionsThere is overwhelming evidence that mechanical signaling plays a crucial role in development of the ovary, follicle, and oocyte throughout gametogenesis. Emerging data suggest the complexities of mechanotransduction and the biomechanics of oocytes and follicles are integral to understanding of primary ovarian insufficiency, ovarian aging, polycystic ovary syndrome, and applications of fertility preservation.

Cell death plays a major role during C. elegans oogenesis, where over half of the oogenic germ cells die in a process termed physiological apoptosis. How germ cells are selected for physiological apoptosis, or instead become oocytes, is not understood. Most oocytes produce viable embryos when apoptosis is blocked, suggesting that physiological apoptosis does not function to cull defective germ cells. Instead, cells targeted for apoptosis may function as nurse cells; the germline is syncytial, and all germ cells appear to contribute cytoplasm to developing oocytes. C. elegans has been a leading model for the genetics and molecular biology of apoptosis and phagocytosis, but comparatively few studies have examined the cell biology of apoptotic cells. We used live imaging to identify and examine pre-apoptotic germ cells in the adult gonad. After initiating apoptosis, germ cells selectively export their mitochondria into the shared pool of syncytial cytoplasm; this transport appears to use the microtubule motor kinesin. The apoptotic cells then shrink as they expel most of their remaining cytoplasm, and close off from the syncytium. Shortly thereafter the apoptotic cells restructure their microtubule and actin cytoskeletons, possibly to maintain cell integrity; the microtubules form a novel, cortical array of stabilized microtubules, and actin and cofilin organize into giant cofilin-actin rods. We discovered that some apoptotic germ cells are binucleate; the binucleate germ cells can develop into binucleate oocytes in apoptosis-defective strains, and appear capable of producing triploid offspring. Our results suggest that the nuclear layer of the germline syncytium becomes folded during mitosis and growth, and that binucleate cells arise as the layer unfolds or everts; all of the binucleate cells are subsequently removed by apoptosis. These results show that physiological apoptosis targets at least two distinct populations of germ cells, and that the apoptosis machinery efficiently recognizes cells with two nuclei.

Epigenetic reprogramming of the zygote involves dynamic incorporation of histone variant H3.3. However, the genome-wide distribution and dynamics of H3.3 during early development remain unknown. Here, we delineate the H3.3 landscapes in mouse oocytes and early embryos. We unexpectedly identify a non-canonical H3.3 pattern in mature oocytes and zygotes, in which local enrichment of H3.3 at active chromatin is suppressed and H3.3 is relatively evenly distributed across the genome. Interestingly, although the non-canonical H3.3 pattern forms gradually during oogenesis, it quickly switches to a canonical pattern at the two-cell stage in a transcription-independent and replication-dependent manner. We find that incorporation of H3.1/H3.2 mediated by chromatin assembly factor CAF-1 is a key process for the de novo establishment of the canonical pattern. Our data suggest that the presence of the non-canonical pattern and its timely transition toward a canonical pattern support the developmental program of early embryos. Ultra-low-input native ChIP-seq analyses of genome-wide histone H3.3 distribution during early mouse development reveal a CAF-1-dependent transition from a non-canonical pattern in the oocyte and zygote to a canonical distribution at the two-cell stage.

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial–mesenchymal Notch signaling interactions. Depletion of Nestin⁺ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.