Sanjiv Risal

Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions

In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreERT2 and Sohlh1-CreERT2 inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ∼3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo, we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan.

Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

BACKGROUND The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. RESULTS By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. CONCLUSIONS Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals.

The Safe Use of a PTEN Inhibitor for the Activation of Dormant Mouse Primordial Follicles and Generation of Fertilizable Eggs

Background Primordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation. Methodology/Principal Findings We have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer. Results and Conclusions Long-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques.

Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells.

The generally accepted viewpoint for more than 50 years has been that the number of oocytes is fixed in fetal or neonatal ovaries, and therefore, oocytes cannot renew themselves in postnatal or adult life. Over the past decade, however, the traditional viewpoint has been challenged by a number of investigators who have presented evidence that postnatal follicular renewal occurs in mammals, and that mitotically active oogonial stem cells (OSCs) exist in postnatal mouse ovaries. Health, Inc. All rights reserved. 30 Obstetrical and Gynecological Survey This letter to the editor presents experimental evidence that disputes the existence of mitotically active OSC in postnatal mouse ovaries. The results presented here are the summary of research conducted independently in 4 laboratories. A previous study (White et al.Nat Med. 2012;18:413–421) reported that OSCs could be purified from adult human and mouse ovaries by use of DEAD box polypeptide 4 (DDX4) antibody–based fluorescence-activated cell sorting (FACS), and that after in vitro manipulation, these isolated OSCs could form oocytes. Based on the well-established cytoplasmic location of DDX4, the use of this protein as a cell surface marker is controversial. Using the same DDX4 antibody–based FACS approach as in the White et al study, the investigators isolated a population of cells from human ovarian cortical tissue biopsied from 16 fertile reproductive-age women who had had at least 1 previous live birth. No DDX4 messenger RNA (mRNA) expression was detected using quantitative polymerase chain reaction in these cells or by a more sensitive single-cell mRNA sequencing analysis that could detect low expression of DDX4. In additional experiments, the sorted human ovarian cells were cultured as described in the previous study. Although no DDX4 expression was detected in the cultured DDX4-positive cells (cultured-POS) or cultured DDX4-negative cells (cultured-NEG) by immunofluorescence staining, the cultured-POS cells and cultured-NEG cells both bound tightly to the DDX4-specific antibody in FACS and became DDX4-positive after culture. The previous study had reported that oocytes enclosed in follicles regenerated 1 week after the DDX4-positive human OSCs were injected into human ovarian cortical tissues that were subsequently xenografted into female severe combined immunodeficient mice. The investigators repeated this experiment and labeled the cultured-POS cells with stable enhanced green fluorescent protein (EGFP) expression. After culturing and expanding this cell population, EGFP-expressing cultured-POS cells were injected into human ovarian cortical tissue biopsies, and these cortical tissues were then xenografted into female severe combined immunodeficient mice for further growth. Grafts were analyzed 1 week, 2 weeks, and 4 weeks after transplantation of the EGFP–cultured-POS cells into the human cortical tissues. The results of this experiment showed that EGFP-positive cells could be observed in the vicinity of the injection sites, but the absence of any EGFP-positive oocytes demonstrated that the DDX4-positive human cells obtained with the DDX4 antibody are not functional stem cells and cannot regenerate oocytes. To confirm these findings that the DDX4-specific antibody–based FACS does not select for a specific cell population expressing DDX4, the same FACS was performed with mouse cells from several organs (including adult liver, spleen, and kidney) that do not express DDX4. DDX4-positive cell populations were obtained from cells of these organs, which provide additional evidence that use of the DDX4-specific antibody using the FACS protocol (critical in purifying the reported OSCs) does not select for DDX4-expressing cells. These findings provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active DDX4-expressing female germline progenitors exist in postnatal mouse ovaries.

Pharmacological Inhibition of mTORC1 Prevents Over-Activation of the Primordial Follicle Pool in Response to Elevated PI3K Signaling

The majority of ovarian primordial follicles must be preserved in a quiescent state to allow for the regular production of gametes over the female reproductive lifespan. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Under certain pathological conditions, the entire pool of primordial follicles matures simultaneously leading to an accelerated loss of primordial follicles and to premature ovarian failure (POF). We have previously shown that loss of Pten (phosphatase and tensin homolog deleted on chromosome ten) in mouse oocytes leads to premature activation of the entire pool of primordial follicles, subsequent follicular depletion in early adulthood, and the onset of POF. Lack of PTEN leads to increased phosphatidylinositol 3-kinase (PI3K)–Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in the oocytes. To study the functional and pathological roles of elevated mTORC1 signaling in the oocytes, we treated the Pten-mutant mice with the specific mTORC1 inhibitor rapamycin. When administered to Pten-deficient mice prior to the activation of the primordial follicles, rapamycin effectively prevented global follicular activation and preserved the ovarian reserve. These results provide a rationale for exploring the possible use of rapamycin as a drug for the preservation of the primordial follicle pool, and the possible prevention of POF.

Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan

A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals.

Speedy A–Cdk2 binding mediates initial telomere–nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation

Significance In meiotic prophase I, telomere attachment to the nuclear envelope is a prerequisite for subsequent prophase events, such as homologous pairing and recombination. In this study, we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is essential for telomere attachment to the nuclear envelope in mice. We have identified a telomere localization domain in Speedy A, which covers the protein’s distal N-terminus and Cdk2-binding Ringo domain but excludes its Cdk-activation domain. Furthermore, we found that the binding of Cdk2 to Speedy A is essential for Cdk2’s localization to telomeres. Our results suggest that Speedy A–Cdk2 binding might mediate the initial assembly of the meiotic telomere complex, a process that is independent of Cdk2 activation. Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere–NE attachment. Deletion of Spdya in mice disrupts telomere–NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2’s localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2–mediated telomere–NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere–NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.

MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice

In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75–8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (α subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis.

Animal Models for Studying the In Vivo Functions of Cell Cycle CDKs.

Multiple Cdks (Cdk4, Cdk6, and Cdk2) and a mitotic Cdk (Cdk1) are involved in cell cycle progression in mammals. Cyclins, Cdk inhibitors, and phosphorylations (both activating and inhibitory) at different cellular levels tightly modulate the activities of these kinases. Based on the results of biochemical studies, it was long believed that different Cdks functioned at specific stages during cell cycle progression. However, deletion of all three interphase Cdks in mice affected cell cycle entry and progression only in certain specialized cells such as hematopoietic cells, beta cells of the pancreas, pituitary lactotrophs, and cardiomyocytes. These genetic experiments challenged the prevailing biochemical model and established that Cdks function in a cell-specific, but not a stage-specific, manner during cell cycle entry and the progression of mitosis. Recent in vivo studies have further established that Cdk1 is the only Cdk that is both essential and sufficient for driving the resumption of meiosis during mouse oocyte maturation. These genetic studies suggest a minimal-essential cell cycle model in which Cdk1 is the central regulator of cell cycle progression. Cdk1 can compensate for the loss of the interphase Cdks by forming active complexes with A-, B-, E-, and D-type Cyclins in a stepwise manner. Thus, Cdk1 plays an essential role in both mitosis and meiosis in mammals, whereas interphase Cdks are dispensable.