Yin-Li Zhang

CRL4 complex regulates mammalian oocyte survival and reprogramming by activation of TET proteins.

Ubiquitin Fertility Insurance The female mammals reproductive lifespan is determined by a pool of ovarian primordial follicles that are generated early in life. Yu et al. (p. 1518) found that in mice, the ubiquitin E3 ligase complex CRL4 is essential for oocyte survival within primordial follicles and for development after fertilization. CRL4 binds to and activates an adaptor protein that mediates ubiquitination, but if any component is deleted, the genes required for oocyte maintenance and early embryo development are silenced and the female mice become infertile. Continuing female fertility in mammals requires the function of a ubiquitin ligase complex in oocytes. The duration of a woman’s reproductive period is determined by the size and persistence of a dormant oocyte pool. Specific oocyte genes are essential for follicle maintenance and female fertility. The mechanisms that regulate the expression of these genes are poorly understood. We found that a cullin-ring finger ligase-4 (CRL4) complex was crucial in this process. Oocyte-specific deletion of the CRL4 linker protein DDB1 or its substrate adaptor VPRBP (also known as DCAF1) caused rapid oocyte loss, premature ovarian insufficiency, and silencing of fertility maintaining genes. CRL4VPRBP activates the TET methylcytosine dioxygenases, which are involved in female germ cell development and zygote genome reprogramming. Hence, CRL4VPRBP ubiquitin ligase is a guardian of female reproductive life in germ cells and a maternal reprogramming factor after fertilization.

BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes

The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4–NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3′ untranslated region, thereby coupling CCR4–NOT deadenylase–mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

Selective Smad4 Knockout in Ovarian Preovulatory Follicles Results in Multiple Defects in Ovulation

The TGF-β signaling pathway is involved with multiple processes in the mammalian ovary, including primordial follicle formation, granulosa cell (GC) proliferation, follicle atresia, ovulation, and feedback regulation between the pituitary and ovary. The transcriptional factor SMAD4 (Sma- and Mad-related protein 4) is the central component of the canonical TGF-β signaling pathway. Smad4 knockout (KO) using Amhr2-Cre, which is expressed in GCs of immature developing follicles, causes premature luteinization. In this study, we specifically depleted Smad4 in GCs of preovulatory follicles using Cyp19-Cre mice. As different from results with Smad4(fl/fl);Amhr2-Cre mice, Smad4 depletion in preovulatory follicles did not cause premature luteinization or suppress GC proliferation; rather, it increased follicle atresia. In addition, Nppc and Npr2 expressions were reduced by Smad4 depletion; thus, their effect of maintaining oocyte meiotic arrest was weakened in Smad4 conditional KO mice. Smad4(fl/fl);Cyp19-Cre female mice were subfertile and had irregular estrous cycles and ovulation defects. Smad4 KO also blocked LH-induced cumulus expansion and follicle rupture, but not oocyte meiotic resumption. Our results also indicated that SMAD4 was required for LH-stimulated activation of ERK1/2 and the expressions of ovulation-related genes. The defects arising from SMAD4 depletion could not be rescued by intraovarian mediators of LH actions, such as epidermal growth factor-like factors and prostaglandin E2. Furthermore, corpus lutea did not form in Smad4(fl/fl);Cyp19-Cre female mice, indicating that SMAD4 was crucial for GCs terminal differentiation. Thus, by characterizing the ovarian phenotypes of preovulatory follicle-specific Smad4 KO mice, we identified the developmental stage-specific functions of the canonical TGF-β signaling pathway in ovulation and luteinization.

Phosphoinositide 3-Kinase p110δ Mediates Estrogen- and FSH-Stimulated Ovarian Follicle Growth

In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

DNA Topoisomerase II Is Dispensable for Oocyte Meiotic Resumption but Is Essential for Meiotic Chromosome Condensation and Separation in Mice

ABSTRACT During mitosis, DNA topoisomerase II (TOP2) is required for sister chromatid separation. When TOP2 activity is inhibited, a decatenation checkpoint is activated by entangled chromatin. However, the functions of TOP2 in oocyte meiosis, particularly for homologous chromosome segregation during meiosis I, have not been investigated. In addition, it remains unknown if TOP2 inhibition activates a decatenation checkpoint at the G2/M transition in oocytes. In this study, we used mouse oocytes and specific inhibitors of TOP2 (ICRF-193 and etoposide) to investigate the role of TOP2 in meiosis. Our results indicated that an effective decatenation checkpoint did not exist in fully grown oocytes, as oocytes underwent the G2/M transition and reinitiated meiosis even when TOP2 activity was inhibited. However, oocytes treated with ICRF-193 had severe defects in chromosome condensation and homologous chromosome separation. Furthermore, condensed chromosomes failed to maintain their normal configurations in matured oocytes that were treated with ICRF-193. However, sister chromatid separation and subsequent chromosome decondensation during the exit from meiosis were not blocked by TOP2 inhibitors. These results indicated that TOP2 had a specific, crucial function in meiosis I. Thus, we identified important functions of TOP2 during oocyte maturation and provided novel insights into the decatenation checkpoint during meiosis.

TOP2β Is Essential for Ovarian Follicles That Are Hypersensitive to Chemotherapeutic Drugs

The mechanisms underlying chemotherapy-induced acceleration of ovarian insufficiency are not fully understood, particularly for ovarian granulosa cells (GCs). We used two widely used cancer chemotherapeutic reagents, bleomycin and VP-16, and an in vivo GC-specific DNA topoisomerase II-β (TOP2β) (Top2b) knockout mouse model to investigate the effects of chemotherapy-induced DNA damage on growing mouse follicles. Bleomycin and VP-16 caused massive double-strand DNA breaks in the GCs of growing follicles in a time-dependent manner as shown by DNA-damage checkpoint activation. This damage was associated with apoptotic GC death and resulted in follicle atresia and ovulation failure. However, FSH-regulated ovarian functions, including estrogen biosynthesis and estrogen target gene expression, were not significantly affected by these genotoxins. TOP2β, a target of several chemotherapeutic drugs including VP-16, was abundantly expressed in the GCs of growing follicles. GC-specific deletion of Top2b using Cyp19-Cre caused DNA damage accumulations in these cells, follicle atresia, and decreased ovulation in response to exogenous gonadotropins. The ovaries of Top2b conditional knockout mice were also more sensitive to low-dose genotoxin treatment than wild-type mice ovaries. Thus, our results indicate that GCs are hypersensitive to genotoxic chemotherapeutic drugs and can activate the canonical DNA-damage checkpoint and the p53-dependent apoptotic pathway in response to insults that damage DNA. We also newly identified TOP2β as a factor involved in regulating GC genomic integrity and follicle atresia. This study has clinical implications for ovarian functional defects both for premenopausal cancer survivors and healthy women.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-β subunit

STUDY QUESTION Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHβ-Fc/Fc, offer a potential FSH alternative? SUMMARY ANSWER KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. WHAT IS KNOWN ALREADY rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. STUDY DESIGN, SIZE, DURATION Plasmids containing FSHα, FSHβ-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. PARTICIPANTS/MATERIALS, SETTING, METHODS The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. MAIN RESULTS AND THE ROLE OF CHANCE KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSHs activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. LIMITATIONS, REASONS FOR CAUTION All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. STUDY FUNDING/COMPETING INTERESTS This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development

Mammalian oocytes and zygotes have the unique ability to reprogram a somatic cell nucleus into a totipotent state. SUV39H1/2‐mediated histone H3 lysine‐9 trimethylation (H3K9me3) is a major barrier to efficient reprogramming. How SUV39H1/2 activities are regulated in early embryos and during generation of induced pluripotent stem cells (iPSCs) remains unclear. Since expression of the CRL4 E3 ubiquitin ligase in oocytes is crucial for female fertility, we analyzed putative CRL4 adaptors (DCAFs) and identified DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is expressed from eight‐cell to morula stages in both murine and human embryos, and Dcaf13 knockout in mice causes preimplantation‐stage mortality. Dcaf13 knockout embryos are arrested at the eight‐ to sixteen‐cell stage before compaction, and this arrest is accompanied by high levels of H3K9me3. Mechanistically, CRL4‐DCAF13 targets SUV39H1 for polyubiquitination and proteasomal degradation and therefore facilitates H3K9me3 removal and zygotic gene expression. Taken together, CRL4‐DCAF13‐mediated SUV39H1 degradation is an essential step for progressive genome reprogramming during preimplantation embryonic development.

The polycystic ovary syndrome-associated gene Yap1 is regulated by gonadotropins and sex steroid hormones in hyperandrogenism-induced oligo-ovulation in mouse

STUDY QUESTION What is the physiological function of Yes-associated protein-1 (Yap1), a susceptibility gene for polycystic ovary syndrome (PCOS), in ovarian granulosa cells (GCs)? SUMMARY ANSWER Physiologically, steroid sex hormones stimulate follicle growth by activating YAP1; however, the preovulatory inhibition of YAP1 activity in GCs is a prerequisite of LH actions. WHAT IS KNOWN ALREADY PCOS is a common gynecologic and endocrine disease with multiple short and long-term consequences. Many PCOS patients suffer anovulation caused by hyperandrogenism, but its etiology remains unclear. STUDY DESIGN, SIZE, DURATION To study the effect of acute hyperandrogenism on ovulation, we injected pregnant mare serum gonadotrophin (PMSG)-primed (44 h) pubertal mice with dihydrotestosterone (DHT), the major biologically active form of androgen, in a superovulation assay. We investigated if YAP1 is regulated by testosterone and if it is potentially involved in follicle development and ovulation. Cultured primary GCs were subjected to Yap1 depletion by RNA interference and Yap1 overexpression by adenoviral infections. PARTICIPANTS/MATERIALS, SETTING, METHODS Female mice at postnatal day (PD)-21~23 were analyzed to avoid the complexity of ovarian functions associated with estrous cycles and endogenous surges of gonadotropins. Immature mice were injected intraperitoneally with five IU PMSG to stimulate preovulatory follicle development followed 44 h later with five IU hCG to stimulate ovulation. For DHT treatments, female mice at PD23 were injected intraperitoneally with five IU PMSG followed 44 h later with five IU hCG alone (as control) or five IU hCG plus 100 μg DHT, which was dissolved in 0.1 ml DMSO. Methods of gene expression detection used include immunohistochemistry, immunofluorescence, Western blotting and quantitative PCR. More than three biological and technical replicates were included in each experiments. MAIN RESULTS AND THE ROLE OF CHANCE we provide novel evidence in a mouse model that YAP1 is required for proliferation of ovarian GCs, but is down-regulated by LH through the extracellular-regulated kinase-1/2 (ERK1/2) cascade. Acute hyperandrogenism blocks LH actions and causes oligo-ovulation by activating YAP1. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Results shown were obtained only in mouse, and need to be further confirmed in human samples. WIDER IMPLICATIONS OF THE FINDINGS These findings not only elucidated the role of YAP1 in maintaining normal ovarian functions, but also link the YAP1 deregulation to the pathogenesis of PCOS. STUDY FUNDING AND COMPETING INTEREST(S) This study is funded by the National Key Research and Development Program of China (2016YFC1000600 and 2017YFSF1001500) and National Natural Science Foundation of China (31528016, 31371449 and 31671558). The authors have no competing interests.