CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to prevent DNA hypermethylation and ensure normal transcription in growing oocytes.

The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.

The EZH2-H3K27me3 axis modulates aberrant transcription and apoptosis in cyclophosphamide-induced ovarian granulosa cell injury.

Chemotherapy-induced ovarian damage and infertility are significant concerns for women of childbearing age with cancer; however, the underlying mechanisms are still not fully understood. Our study has revealed a close association between epigenetic regulation and cyclophosphamide (CTX)-induced ovarian damage. Specifically, CTX and its active metabolite 4-hydroperoxy cyclophosphamide (4-HC) were found to increase the apoptosis of granulosa cells (GCs) by reducing EZH2 and H3K27me3 levels, both in vivo and in vitro. Furthermore, RNA-seq and CUT&Tag analyses revealed that the loss of H3K27me3 peaks on promoters led to the overactivation of genes associated with transcriptional regulation and apoptosis, indicating that stable H3K27me3 status could help to provide a safeguard against CTX-induced ovarian damage. Administration of the H3K27me3-demethylase inhibitor, GSK-J4, prior to CTX treatment could partially mitigate GC apoptosis by reversing the reduction of H3K27me3 and the aberrant upregulation of specific genes involved in transcriptional regulation and apoptosis. GSK-J4 could thus potentially be a protective agent for female fertility when undergoing chemotherapy. The results provide new insights into the mechanisms for chemotherapy injury and future clinical interventions for fertility preservation.

Ovulatory signal-triggered chromatin remodeling in ovarian granulosa cells by HDAC2 phosphorylation activation-mediated histone deacetylation.

BACKGROUND: Epigenetic reprogramming is involved in luteinizing hormone (LH)-induced ovulation; however, the underlying mechanisms are largely unknown. RESULTS: We here observed a rapid histone deacetylation process between two waves of active transcription mediated by the follicle-stimulating hormone (FSH) and the LH congener human chorionic gonadotropin (hCG), respectively. Analysis of the genome-wide H3K27Ac distribution in hCG-treated granulosa cells revealed that a rapid wave of genome-wide histone deacetylation remodels the chromatin, followed by the establishment of specific histone acetylation for ovulation. HDAC2 phosphorylation activation coincides with histone deacetylation in mouse preovulatory follicles. When HDAC2 was silenced or inhibited, histone acetylation was retained, leading to reduced gene transcription, retarded cumulus expansion, and ovulation defect. HDAC2 phosphorylation was associated with CK2α nuclear translocation, and inhibition of CK2α attenuated HDAC2 phosphorylation, retarded H3K27 deacetylation, and inactivated the ERK1/2 signaling cascade. CONCLUSIONS: This study demonstrates that the ovulatory signal erases histone acetylation through activation of CK2α-mediated HDAC2 phosphorylation in granulosa cells, which is an essential prerequisite for subsequent successful ovulation.

HucMSC-EVs Facilitate In Vitro Development of Maternally Aged Preantral Follicles and Oocytes.

Follicle developmental capacity and oocyte quality decline with advanced maternal age. Extracellular vesicles from human umbilical cord mesenchymal stem cells (HucMSC-EVs) act as a potential therapeutic product in the treatment of age-related ovarian dysfunction. In vitro culture (IVC) of preantral follicles is a useful method for understanding the mechanism of follicle development and is a promising means for improving female fertility. However, whether HucMSC-EVs have beneficial effects on aged follicle development during IVC has not yet been reported. Our research demonstrated that follicular development with single-addition withdrawal of HucMSC-EVs was better than that with continuous treatment with HucMSC-EVs. HucMSC-EVs facilitated the survival and growth of follicles, promoted the proliferation of granulosa cells (GCs), and improved the steroid hormone secretion of GCs during IVC of aged follicles. Both GCs and oocytes could uptake HucMSC-EVs. Moreover, we observed elevated cellular transcription in GCs and oocytes after treatment with HucMSC-EVs. The RNA sequencing (RNA-seq) results further validated that the differentially expressed genes are related to the promotion of GC proliferation, cell communication, and oocyte spindle organization. Additionally, the aged oocytes displayed a higher maturation rate, presented less aberrant spindle morphology, and expressed a higher level of the antioxidant protein Sirtuin 1 (SIRT1) after treatment with HucMSC-EVs. Our findings suggested that HucMSC-EVs can improve the growth and quality of aged follicles and oocytes in vitro through the regulation of gene transcription, which provides evidence for HucMSC-EVs as potential therapeutic reagents to restore female fertility with advanced age.

Decreased histidine-rich glycoprotein and increased complement C4-B protein levels in follicular fluid predict the IVF outcomes of recurrent spontaneous abortion.

BACKGROUND: Recurrent spontaneous abortion (RSA) is a common and complicated pregnancy-related disease that lacks a suitable biomarker to predict its recrudescence. METHODS: Tandem mass tag (TMT) analysis was conducted to obtain quantitative proteomic profiles in follicular fluid from patients with a history of RSA and from control group. ELISA validation of candidate differentially expressed proteins was conducted in a larger group of patients. RESULTS: A total of 836 proteins were identified by TMT analysis; 51 were upregulated and 47 were downregulated in follicular fluid from cases of RSA versus control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed several important pathways were enriched, involving a dysregulated immunoglobulin Fc receptor signaling pathway and overactivated complement cascade pathways. ELISA validated the differential expression of two proteins, histidine-rich globulin (HRG) and complement C4-B (C4B), which were downregulated and upregulated, respectively, in follicular fluid of patients with RSA. We performed receiver operating characteristic curve analysis of the ELISA results with the outcomes of current IVF cycles as classification variables. The area under the curve results for HRG alone, C4B alone and HRG-C4B combined were 0.785, 0.710 and 0.895, respectively. CONCLUSIONS: TMT analysis identified 98 differentially expressed proteins in follicular fluid from patients with RSA, indicating follicle factors that act as early warning factors for the occurrence of RSA. Among them, HRG and C4B provide candidate markers to predict the clinical outcomes of IVF/ICSI cycles, and the potential for modeling an early detection system for RSA.

Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation.

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F-actin assembly. Using oocyte-specific Erk1/2 knockout mice, we verified that MOS-ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS-ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization.

PURPOSE: To study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization. METHODS: Thirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells. RESULTS: Three of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle. CONCLUSION: Three novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

Cyclophosphamide Exposure Causes Long-Term Detrimental Effect of Oocytes Developmental Competence Through Affecting the Epigenetic Modification and Maternal Factors' Transcription During Oocyte Growth.

Cyclophosphamide (CTX) is widely used in various cancer therapies and in immunosuppression, and patients can still have babies after CTX chemotherapy. CTX directly causes primordial follicle loss with overactivation and DNA damage-induced apoptosis. Previous studies have shown that maternal exposure to CTX before conception increases the incidence of birth abnormalities and alters the methylation of genes in the oocytes of offspring. Mice were treated with a single dose of CTX (100 mg/kg) at post-natal day 21 and sacrificed 47 days later when primordial follicles surviving chemotherapy developed to the antral stage. Acute DNA damage and acceleration of the activation of primordial follicles after CTX treatment were repaired within several days, but the remaining follicle numbers remarkably decrease. Although partial surviving primordial follicle were developed to mature oocyte, oocyte quality hemostasis was impaired exhibiting aberrant meiosis progression, abnormal spindle and aneuploidy, mitochondrial dysfunction and increased endoplasmic reticulum stress. Thereafter, embryo development competency significantly decreased with fewer blastocyst formation after CTX exposure. CTX treatment resulted in alteration of DNA methylations and histone modifications in fully grown GV oocytes. Single-cell RNA-seq revealed CTX treatment suppressed multiple maternal genes' transcription including many methyltransferases and maternal factor YAP1, which probably accounts for low quality of CTX-repaired oocyte. In vitro addition of lysophosphatidic acid (LPA) to embryo culture media to promote YAP1 nuclear localization improved CTX-repaired embryo developmental competence. This study provides evidence for the consistent toxic effect of CTX exposure during follicle development, and provide a new mechanism and new insights into future clinical interventions for fertility preservation.

Lysophosphatidic acid improves oocyte quality during IVM by activating the ERK1/2 pathway in cumulus cells and oocytes.

Oocyte IVM technology is an option for fertility preservation in some groups of patients, such as those with polycystic ovary syndrome, patients with ovarian hyperstimulation syndrome, and for patients with cancer. However, the developmental potential of oocytes from IVM still needs to improve. Several previous studies have reported that lysophosphatidic acid (LPA) promotes glucose metabolism, cumulus cell (CC) expansion, and oocyte nuclear maturation. However, the effect of LPA on oocyte cytoplasmic maturation, particularly mitochondrial function, has rarely been studied and the underlying mechanism is largely unknown, which impedes (pre)clinical applications of LPA. In this study, cumulus-oocyte complexes (COCs) and cumulus-denuded germinal vesicle oocytes (DOs) were treated with various concentrations of LPA during IVM, in the presence or absence of the oxidative stressor cyclophosphamide (CTX). In both normal and CTX-damaged COCs, the 25 µM LPA group exhibited improved CC expansion capacity, a higher nuclear maturation rate, and superior mitochondrial function, compared to no LPA treatment. When the concentration of LPA was over 40 µM, detrimental effects of LPA on oocyte maturation occurred. Compared with COCs, the addition of LPA slightly improved oocyte nuclear and cytoplasmic maturation of DOs, but this was not statistically significant. We observed that LPA promotes the activation of extracellular signal-regulated kinase (ERK)1/2, although this was not statistically significant in DOs. Furthermore, LPA could not reverse the negative effect of CC expansion and mitochondrial function after inactivation of ERK1/2 by U0126. RNA-sequencing and RT-PCR results showed that LPA upregulated several ERK1/2 downstream genes related to CC expansion, such as Areg, Cited4, and Ptgs2. This study demonstrates that LPA improves oocyte quality during IVM through the activation of ERK1/2 pathway CCs and oocytes, which provides evidence for the potential addition of LPA to IVM medium.

Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

STUDY QUESTION: Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? SUMMARY ANSWER: Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. WHAT IS KNOWN ALREADY: Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. STUDY DESIGN, SIZE, DURATION: Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. PARTICIPANTS/MATERIALS, SETTING, METHODS: The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. MAIN RESULTS AND THE ROLE OF CHANCE: The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. LIMITATIONS, REASONS FOR CAUTION: The number of PCOS patients in this study was small. WIDER IMPLICATIONS OF THE FINDINGS: This study provides further evidence for metformin as a PCOS treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

ADAMTS1 and HSPG2 mRNA levels in cumulus cells are related to human oocyte quality and controlled ovarian hyperstimulation outcomes.

PURPOSE: The study investigated potential correlations between the expression levels of ADAMTS1 and HSPG2 in cumulus cells (CCs) and controlled ovarian hyperstimulation (COH) outcomes. METHODS: RT-PCR was used to determine ADAMTS1 and HSPG2 mRNA levels in mice CCs at different timepoints (0, 4, 8, 12, and 16 h) after human chorionic gonadotropin (hCG) injection, and in CCs after RNAi treatment. Women with polycystic ovary syndrome (PCOS) (n = 45) and normal ovulatory controls (n = 103) undergoing IVF/ICSI were recruited. Relative ADAMTS1 and HSPG2 mRNA levels were measured by RT-PCR. Moreover, correlations of ADAMTS1 and HSPG2 levels with COH outcomes were analyzed. RESULTS: At different timepoints after hCG treatment, ADAMTS1 mRNA had the highest level at 12 h, whereas HSPG2 showed opposite profiles to ADAMTS1 with the lowest level at 12 h. HSPG2 expression was upregulated after ADAMTS1 RNAi treatment The PCOS group had higher HSPG2 and lower ADAMTS1 expression levels than controls. In normal ovulatory women (control group), a higher expression of ADAMTS1 and lower expression of HSPG2 were associated with more mature oocytes, transplantable embryos, and good quality embryos, whereas higher transplantable embryo rates and good quality embryo rates were obtained only with lower HSPG2 expression. ROC curves showed the co-measurement of ADAMTS1 and HSPG2 had a better predictive power than separate analyses. CONCLUSION: The dynamic profiles of ADAMTS1 and HSPG2 were inversely correlated in CCs. In PCOS and normal ovulatory patients, higher ADAMTS1 and lower HSPG2 expression levels in CCs were related to better COH outcomes.