NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Toxic effects of AZD1208 on mouse oocytes and its possible mechanisms.

AZD1208, a pan-inhibitor that can effectively inhibit PIM kinase, is used for the treatment of advanced solid tumors and malignant lymphomas. Numerous studies have proved its curative effects while its potential cellular toxicity on reproduction was still little known. In this study, we investigated the toxic effects of AZD1208 on mouse oocytes. The results showed that AZD1208 treatment did not affect meiotic resumption, but postponed oocyte maturation as indicated by delayed first polar body extrusion. Further mechanistic study showed that AZD1208 treatment delayed spindle assembly. In addition, we found that oocytes treated with AZD1208 showed mitochondrial dysfunction. Abnormal mitochondrial clusters with decreased mitochondrial membrane potential were observed in oocytes during incubation in vitro. Moreover, increased oxidative stress was observed by testing the level of reactive oxygen species. In summary, our results suggest that AZD1208 treatment influences oocyte meiotic progression by causing mitochondrial dysfunctions and subsequent delayed spindle assembly.

OTSSP167 leads to follicular dysplasia and negatively affects oocyte quality in mice.

OTSSP167 is an anti-tumor drug significantly inhibiting tumor growth in xenotransplantation studies using mouse breast, lung, prostate, and pancreatic cancer cell lines. Its phase I clinical trial has been completed, indicating its great potential for future treatment of solid tumors. However, its drug-related adverse effects on reproductive systems have not yet been reported. In this study, we evaluated the effects of OTSSP167 on reproduction of female mice by determining oocyte quality and follicular development. We selected four-week-old female ICR mice for a 21-day intraperitoneal injection of OTSSP167 at a dose of 5 mg/kg/d. We found that OTSSP167 could block the meiotic process of oocytes, leading to a decrease in oocyte maturation and ovulated oocyte numbers, as well as a decrease in the quality of oocytes. The results showed that OTSSP167 treatment caused disordered spindle assembly, decreased mitochondria membrane potential, and increased accumulation of reactive oxygen species in oocytes. Further investigation showed that OTSSP167 induced DNA double-strand breaks, as indicated by increased levels of γH2AX in oocytes of primordial follicles and granulosa cells of growing follicles, which induced follicular atresia and decreased the numbers of follicles at various growing stages. Our study suggests that OTSSP167 treatment may have serious effects on the ovary and consequences for female cancer patients, providing strong evidence for the necessity of protecting female fertility in clinical OTSSP167 trials.

Gefitinib reduces oocyte quality by disturbing meiotic progression.

Gefitinib is a first-line anti-cancer drug for the treatment of advanced non-small cell lung cancer (NSCLC). It has been reported that gefitinib can generate several drug-related adverse effects, including nausea, peripheral edema, decreased appetite and rash. However, the reproductive toxicity of gefitinib has not been clearly defined until now. Here we assessed the effects of gefitinib on oocyte quality by examining the critical events and molecular changes of oocyte maturation. Gefitinib at 1, 2, 5 or 10 µM concentration was added to culture medium (M2). We found that gefitinib at its median peak concentration of 1 µM did not affect oocyte maturation, but 5 µM gefitinib severely blocked oocyte meiotic progression as indicated by decreased rates of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE). We further showed that gefitinib treatment increased phosphorylation of CDK1 at the site of Try15, inhibited cyclin B1 entry into the nucleus, and disrupted normal spindle assembly, chromosome alignment and mitochondria dynamics, finally leading to the generation of aneuploidy and early apoptosis of oocytes. Our study reported here provides valuable evidence for reproductive toxicity of gefitinib administration employed for the treatment of cancer patients.

Single-cell RNA sequencing reveals regulation of fetal ovary development in the monkey (Macaca fascicularis).

Germ cells are vital for reproduction and heredity. However, the mechanisms underlying female germ cell development in primates, especially in late embryonic stages, remain elusive. Here, we performed single-cell RNA sequencing of 12,471 cells from whole fetal ovaries, and explored the communications between germ cells and niche cells. We depicted the two waves of oogenesis at single-cell resolution and demonstrated that progenitor theca cells exhibit similar characteristics to Leydig cells in fetal monkey ovaries. Notably, we found that ZGLP1 displays differentially expressed patterns between mouse and monkey, which is not overlapped with NANOG in monkey germ cells, suggesting its role in meiosis entry but not in activating oogenic program in primates. Furthermore, the majority of germ cell clusters that sharply express PRDM9 and SPO11 might undergo apoptosis after cyst breakdown, leading to germ cell attrition. Overall, our work provides new insights into the molecular and cellular basis of primate fetal ovary development at single-cell resolution.

Deletion of BAF250a affects oocyte epigenetic modifications and embryonic development.

BRG1-associated factor 250a (BAF250a) is a component of the SWI/SNF adenosine triphosphate-dependent chromatin remodeling complex, which has been shown to control chromatin structure and transcription. BAF250a was reported to be a key component of the gene regulatory machinery in embryonic stem cells controlling self-renewal, differentiation, and cell lineage decisions. Here we constructed Baf250aF/F ;Gdf9-cre (Baf250aCKO ) mice to specifically delete BAF250a in oocytes to investigate the role of maternal BAF250a in female germ cells and embryo development. Our results showed that BAF250a deletion did not affect folliculogenesis, ovulation, and fertilization, but it caused late embryonic death. RNA sequencing analysis showed that the expression of genes involved in cell proliferation and differentiation, tissue morphogenesis, histone modification, and nucleosome remodeling were perturbed in Baf250aCKO MII oocytes. We showed that covalent histone modifications such as H3K27me3 and H3K27ac were also significantly affected in oocytes, which may reduce oocyte quality and lead to birth defects. In addition, the DNA methylation level of Igf2r, Snrpn, and Peg3 differentially methylated regions was decreased in Baf250aCKO oocytes. Quantitative real-time polymerase chain reaction analysis showed that the relative messenger RNA (mRNA) expression levels of Igf2r and Snrpn were significantly increased. The mRNA expression level of Dnmt1, Dnmt3a, Dnmt3l, and Uhrf1 was decreased, and the protein expression in these genes was also reduced, which might be the cause for impaired imprinting establishment. In conclusion, our results demonstrate that BAF250a plays an important role in oocyte transcription regulation, epigenetic modifications, and embryo development.

Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the methyltransferase-like 3-methyltransferase-like 14 (METTL14)-Wilms tumor 1-associated protein complex dynamically regulates m6A modification and plays important roles in diverse biologic processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation, and their effects on different biologic pathways are still limited. By using both in vivo and in vitro models, we here report that METTL14 is indispensable for postimplantation embryonic development by facilitating the conversion from naive to primed state of the epiblast. Depletion of Mettl14 leads to conspicuous embryonic growth retardation from embryonic d 6.5, mainly as a result of resistance to differentiation, which further leads to embryonic lethality early in gestation. Our data highlight the critical function of METTL14 as an m6A modification regulator in orchestrating early mouse embryogenesis.-Meng, T.-G., Lu, X., Guo, L., Hou, G.-M., Ma, X.-S., Li, Q.-N., Huang, L., Fan, L.-H., Zhao, Z.-H., Ou, X.-H., OuYang, Y.-C., Schatten, H., Li, L., Wang, Z.-B., Sun, Q.-Y. Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.

Transfer of autologous mitochondria from adipose tissue-derived stem cells rescues oocyte quality and infertility in aged mice.

Elder women suffer from low or loss of fertility because of decreasing oocyte quality as maternal aging. As energy resource, mitochondria play pivotal roles in oocyte development, determining oocyte quality. With advanced maternal age, increased dysfunctions emerge in oocyte mitochondria, which decrease oocyte quality and its developmental potential. Mitochondria supplement as a possible strategy for improving egg quality has been in debate due to ethnic problems. Heterogeneity is an intractable problem even transfer of germinal vesicle, spindle, pronuclei or polar body is employed. We proposed that the autologous adipose tissue-derived stem cell (ADSC) mitochondria could improve the fertility in aged mice. We found that autologous ADSC mitochondria could promote oocyte quality, embryo development and fertility in aged mice, which may provide a promising strategy for treatment of low fertility or infertility in elder women.

Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice.

The process of follicular development involves communications between oocyte and surrounding granulosa cells. FURIN is a member of the family of proprotein convertases that is involved in the activation of a large number of zymogens and proproteins by cleavage at its recognition motif. To investigate the functions of FURIN in female fertility, furinflox/flox (furfl/fl) mice were crossed with Zp3-Cre mice and Gdf9-Cre, respectively, to achieve oocyte-specific disruption of FURIN. Here we report for the first time that FURIN is dispensable for primordial follicle maintenance and activation but important for early secondary follicular development, as ablation of FURIN in oocytes caused failure of follicle development beyond the type 4 and/or 5a follicles in mutant mice, resulting in increased number of early secondary follicles and the severely decreased number of mature follicles, thus anovulation and infertility. We also found that the developmental arrest of early secondary follicles might be rooted in the loss of the mature form of ADAMTS1 (85-kDa prodomain truncated) and compromised proliferation of granulosa cells in mutant mice. Taken together, our data highlight the importance of FURIN in follicle development beyond the early secondary follicle stage and indicate that compromised FURIN function leads to follicular dysplasia and female infertility in mice.