Background Nicotinamide (NAM) is an important antioxidant, which is closely related to female fertility, but its role has not been clearly elucidated. The purpose of the present study was to investigate the effects of NAM on follicular development at different stages and the quality of oocytes. Methods The concentration of NAM in follicular fluid (FF) of 236 women undergoing in vitro fertilization (IVF) was ascertained by enzyme-linked immunosorbent assay (ELISA), and the correlation between NAM and clinical indexes was analyzed. During the in vitro maturation (IVM) of mice cumulus-oocyte complexes (COCs), different concentrations of NAM were added to check the maturation rate and fertilization rate. The reactive oxygen species (ROS) levels in the oocytes treated with different hydrogen peroxide (H2O2) and NAM were assessed. Immunofluorescence staining was performed to measure the proportion of abnormal spindles. Results The level of NAM in large follicles was significantly higher than that in small follicles. In mature FF, the NAM concentration was positively correlated with the rates of oocyte maturation and fertilization. Five mM NAM treatment during IVM increased maturation rate and fertilization rate in the oxidative stress model, and significantly reduced the increase of ROS levels induced by H2O2 in mice oocytes. Conclusions Higher levels of NAM in FF are associated with larger follicle development. The supplement of 5 mM NAM during IVM may improve mice oocyte quality, reducing damage caused by oxidative stress.

Mammalian blastocyst hatching is an essential prerequisite for successful embryo implantation. As the rate-limiting step of current assisted reproductive technology, understanding the key factors regulating blastocyst hatching would be significantly helpful to improve the performance of the assisted reproductive practice. In early embryo development, the fine-tuned elimination of maternal materials and the balanced protein turnover are inevitable for the competent to hatch and implant into endometrium. Neddylation, a ubiquitination-like protein modification, has been shown to be involved in oocyte maturation and early embryo development. In this study, aiming to discover an unknown role of neddylation in the blastocyst hatching process, we provided functional evidence of neddylation in mammalian embryo quality and blastocyst hatching. Treatment with MLN4924, a specific neddylation inhibitor, lowered the embryo quality and dramatically reduced the hatching rate in mouse blastocysts. The transcriptional profile showed the upregulation of oxidative stress-related genes and aberrant expression of immune-related genes. The elevated oxidative stress was validated by qPCR and markers of apoptosis, DNA damage, reactive oxygen species, and cytoskeleton. Moreover, we found the secreted IL-1β level was reduced in an NF-κB-independent manner, leading to the final poor embryo quality and blastocyst hatching failure. This is the first report of neddylation being of great importance in the mammalian blastocyst hatching process. Further investigations uncovering more detailed molecular mechanisms of neddylation regulation in blastocyst hatching would greatly promote not only the understanding of this crucial biological process but also the clinical application in reproductive centers.

Background Maternal protein degradation in mammalian preimplantation embryos has yet to be fully understood. One major pathway is the ubiquitin-proteasome system. Recently, new forms of ubiquitination such as neddylation have been found to play important roles in a wide variety of biological processes, including reproduction. However, the molecular mechanism of neddylation in the early embryonic development of mammals is mostly unknown. Methods The zygotes were collected through in vitro fertilization and the expression of marker genes during embryonic development and zygotic genome activation (ZGA) was monitored after 24,48,72,96 hours of culture with MLN4924 (specific inhibition of neddylation) using real-time quantitative PCR. Single-cell RNA sequencing and quantitative PCR were applied to monitor and validate the changes in the downstream transcriptome. We utilized immunofluorescence and Western blotting to detect the expression and localization of proteins in mouse embryos. Results Blocking neddylation in mouse zygotes led to a statistically significant decrease in the cleavage rate to the 2-cell stage. Transcriptional profiling showed genes differentially expressed in pathways involving cell fate determination, cell differentiation, and cytoskeletal proteins. The expressions of zygotic ZGA markers were significantly reducejiand, indicating a significant downstream alteration in relevant pathways leading to the 2-cell stage arrest phenotype. A decrease in the level of RNA polymerase II in the nucleus was detected, showing impaired gene transcription in the embryo. We also identified a decrease in methyltransferase expression and concomitant reduction in histone H3K4 trimethylation, which may be the molecular mechanism of early embryonic developmental arrest caused by neddylation inhibition. Reduction of Yap1 was detected, suggesting aberrant downstream reactions of the Hippo signaling pathway. It also addressed the problem of the neddylation inhibition caused early embryonic arrest. Our study shed light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology. Conclusions Our data suggest that blocking neddylation leads to ZGA failure, possibly due to a decrease in H3K4me3 caused by a decrease in methyltransferase (KMT2D).

Genetic screening is an important approach for etiology determination and helps to optimize administration protocols in reproductive centers. After the first pathogenic gene of female infertility was reported in 2016, more and more new pathogenic genes were discovered, and we sought to develop an efficient and cost-effective method for genetic screening in patients. In this study, we designed a target-sequencing panel with 22 female infertility-related genes, namely, TUBB8, PATL2, WEE2, and PANX1 and sequenced 68 primary infertility (PI) and recurrent pregnancy loss (RPL) patients. We sequenced 68 samples reaching an average depth of 1559× and detected 3,134 variants. Among them, 62.2% were synonymous single-nucleotide variants (SNVs) and 36.3% were non-synonymous SNVs. The remaining 1.5% are indels (insertions and deletions) and stop-gains. DNAH11 and TUBB8 are the two genes that mutated most frequently. We also found a novel TUBB8 variant (c.898_900del; p.300_300del), proved its loss-of-function mechanism, and profiled the interactome of the wild-type (WT) and mutant TUBB8 proteins. Overall, this target-sequencing method provides an efficient and cost-effective approach for screening in IVF clinics and will support researchers for the discovery of new pathogenic variants.

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