secondary oocyte: an oocyte in which the first meiotic division is completed; the second meiotic division usually stops short of completion unless fertilization occurs. Using isolated GV oocytes, both western blotting and qPCR verified the markedly reduced protein and mRNA levels of Nat10 in Nat10-ScKO oocytes compared with WT oocytes (Fig. As shown in Fig. Co-staining of the nuclear chromosome spreads by SYCP1 and SYCP3 markers discovered the accumulation of aberrant pachytene-like cells with partially synapsed homologous chromosomes in the perinatal Nat10-ScKO ovaries (Fig. The only normal human type of secondary oocyte has the 23rd (sex) chromosome as 23,X (female-determining), whereas sperm can have 23,X (female-determining) or 23,Y (male-determining). The box indicates the upper and lower quantiles, the thick line in the box indicates the median and whiskers indicate 2.5th and 97.5th percentiles. 5b). In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and post-transcriptional levels. Reciprocally, rescue by Nat10 overexpression in Nat10-inducible KO MEF cells recovered and enhanced the cell division (Fig. n=3 biologically independent samples were included in each group (ac). Source data are provided as a source data file. 9, 704341 (2021). Oogenesis, in contrast to spermatogenesis, is the creation of a female gamete (also known as oocyte) from a PGC. Data are presented as meanSEM, n=3. Allelic reprogramming of the histone modification H3K4me3 in early mammalian development. Oogenesis, ovogenesis, or ogenesis / o.dnss / [1] is the differentiation of the ovum (egg cell) into a cell competent to further develop when fertilized. n.s., non-significant by two-tailed Students t-test. 9ce) and the RPFs enrichment levels (Supplementary Fig. 50, 51295144 (2022). 24, 917927 (2022). The super tiny one is called a polar body, and will never support fertilization. The Spin1 interactor, Spindoc, is dispensable for meiotic division, but essential for haploid spermatid development in mice. In contrast, H3K9me3 staining revealed dispersed and elevated chromatin signals in both NSN and SN oocytes in Nat10-ZcKO oocytes (Fig. Before ovulation, the cumulus complex goes through a structural change known as cumulus expansion. This evidence altogether implies that NAT10 likely has important physiological roles through mouse oocyte development in vivo. 11, e1005462 (2015). Biol. 286, 493506 (2005). The final DNA library was separated and visualized in a 4% agarose gel. In brief, ~100 oocytes were collected in a pre-warmed M2 medium supplemented with cycloheximide (CHX) at a final concentration of 100g/ml. Oocyte Development, Maturation & Function | What is an Oocyte CAS Secondary oocytes are the immature ovum shortly after ovulation, to fertilization, where it turns into an ootid. To determine the accurate stage and how meiotic divisions were impacted in Nat10-ZcKO oocytes, we collected the superovulated oocytes in vivo after PMSG/hCG injection and performed the co-staining with DAPI and a cytoskeleton marker, tubulin, in the formaldehyde-fixed oocytes. 4b, c, Supplementary Fig. Q4, Viable cells, p=0.2983; Q3, Early apoptosis, p=0.2147; Q2, Late apoptosis, p=0.3122; Q1, Necrotic cells, p=0.7863. p-value are analyzed by two-tailed Students t-test. On the other hand, the permissive sterile phenotype in all the Nat10-ZcKO females implies that there exists a functional deficiency in Nat10-ZcKO oocytes despite their morphological similarity in WT ovaries at the age of 1 month. Samples were detected by the BD Accuri C6 flow cytometry, and the results were analyzed with FlowJo V10 software. Indeed, this phenomenon for the transdifferentiation of ovarian cells to Sertoli-like cells has been previously reported, wherein deletion of Mtor in the primordial oocytes induced the conversion of granulosa cells to Sertoli-like cells, displaying a seminiferous tubule-like testicular structure in the oocyte-specific Mtor-null ovaries29. Briefly, ~2000GV oocytes were collected and lysed with 100l lysis buffer (20mM TrisHCl at PH 7.4, 150mM NaCl, 2mM EDTA, 0.5% Triton X-100, 0.5% NP-40, 0.5mM DTT, 40U murine RNase inhibitor (APExBIO, K1046), 1 Protease inhibitors and 25U/ml Turbo DNase). Ovary samples were cut into 8m slides. 9 chapters | In any one human generation, the egg's development starts before the female that carries it is even born; 8 to 20 weeks after the fetus has started to grow, cells that are to become mature ova have been multiplying, and by the time that the female is born, all of . Reproductive and Genetic Hospital, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, Xue Jiang,Caoling Xu,Wenqing Li,Jiaqi Zou,Lan Meng,Muhammad Azhar&Jianqiang Bao, School of Information Science and Technology, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, Division of Life Sciences and Medicine, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, Laboratory animal center, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, Reproductive and Genetic Hospital, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, Xuemei Xing,Yuzhu Cao,Xianhong Tong&Xiaoli Zhu, NHC Key Laboratory of Male Reproduction and Genetics, Guangdong Provincial Reproductive Science Institute (Guangdong Provincial Fertility Hospital), 510600, Guangzhou, China, Hefei National Research Center for Physical Sciences at the Microscale, Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China (USTC), 230001, Hefei, Anhui, China, You can also search for this author in Because the fate of an oocyte is to become fertilized and ultimately grow into a fully functioning organism, it must be ready to regulate multiple cellular and developmental processes. EMBO Mol. 23, 387394 (2016). Cell Death Differ. Lan, Z. J., Xu, X. Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Antibodies used were as follows: mouse anti-SYCP3 (Abcam, ab97672, 1:1000), rabbit anti-SYCP1 (Abcam, ab15090, 1:1000), rabbit anti-SYCP3 (Proteintech, 23024-1-AP, 1:200), mouse anti-H2AX (Millipore, 05-636, 1:1000), rabbit anti-RPA2 (Proteintech, 10412-1-AP, 1:400). 3c, d), suggesting defective DSB repair in Nat10-null pachytene oocytes33. 8d), suggesting that Nat10 has a profound impact on both the transcriptional and translational regulation. The transcriptional activity peaks in the early growing oocytes, but decreases thereafter and is considered silent in the fully grown GV oocyte10. 7j). ****p<0.0001 by two-tailed Students t-test. The smart-seq2 in this study has been deposited in the Sequence Read Archive database under accession code SRP392832. We verified the validity of our mini-bulk SMART-seq2 method by comparing our data with published bulk RNA-seq results in WT oocytes. This revealed that NAT10 protein diffuses from the nucleolus to the nucleoplasm in NSN and SN oocytes, and fills in the whole cytoplasm in the MI and MII oocytes (Supplementary Fig. METTL3-mediated m(6)A is required for murine oocyte maturation and maternal-to-zygotic transition. Statistical analyses were performed using R software (http://www.rproject.org). Nevertheless, after the first wave of folliculogenesis, Nat10-ZcKO oocytes appeared to quickly degenerate, resulting in developmental arrest at secondary follicles in Nat10-ZcKO ovaries, as compared with WT ovaries (Fig. A female oocyte is a cell that develops at various times from an oogonium, which is a cell that was developed during female embryonic development. h qPCR analyses of the relative expression levels for a cohort of genes showing specific or characteristic expression in ovarian granulosa cells (Left) or testicular Sertoli/Leydig cells (Right) in 1-month-old WT and Nat10-ScKO ovaries. As shown in Fig. ****p<0.0001 by two-tailed Students t-test. Importantly, as a positive control, they successfully identified the previously known ac4C sites in both rRNA and tRNAs, as well as low levels of a few hundred ac4C acetylation sites in Nat10-overexpressing human cells, implying that their adopted method is sensitive and feasible for detecting ac4C modification59,60. Mol. Dev. Inducible Nat10 KO was achieved by 2M OHT treatment for three consecutive days. The genetic material is portioned to each daughter cell so the final, large cell (now a mature oocyte) and the remaining polar body have only one copy of DNA (2n>>>n). Oocytes are immature eggs of sexually reproducing females. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 3). Xiang, Y. et al. The larger cell undergoes meiosis II, once again producing a large cell and a polar body. Secondary oocytes have completed meiosis I, but are halted in meiosis II until fertilization, when they become matured oocytes prior to the fusing of egg and sperm DNA in the new organism. After incubation for 10min, the oocytes were directly lysed in 50l ribosome lysis buffer (20mM TrisHCl at PH 7.4, 150mM NaCl, 5mM MgCl2, 1% Triton X-100, 1mM DTT, 100g/ml CHX and 25U/ml Turbo DNase). However, the proportion of four-cell stage embryos markedly declined in the Nat10 KO group. Thus it appears that a process evolved to avoid this vulnerability of germline DNA. We next performed H&E staining and counted the average number of follicles at various stages in the postnatal ovary sections. Exp. 23, 13771384 (2008). The purified RNA fragments were subjected to library construction using the identical procedures as described in Ribo-seq protocol that incorporated end repair, 3 polyadenylation, reverse transcription, and barcode PCR amplification. In other words, it is an immature ovum, or egg cell. To determine which transcripts were susceptible to degradation in the presence of Nat10, we defined a total of 2011 transcripts down-regulated in WT oocytes during the GV-MII transition and 1206 transcripts down-regulated in Nat10-ZcKO oocytes through GV-MII transition. 136, 113138 (2020). Thomas, J. M., Bryson, K. M. & Meier, J. L. Nucleotide resolution sequencing of N4-acetylcytidine in RNA. Half of the resultant cDNA product (25l) was combined with 25l of 2Ultra II Q5 master mix, 0.5M index i5 and i7 primers (50l in total), for barcoded library PCR amplification for 18 cycles. Typically, only one oocyte each cycle will become a mature egg and be ovulated from its follicle. 79, 4 (2021). cyte. - Statistics, Types & Causes, What Is Syphilis? Nucleic Acids Res. 9b, c), indicative of the successful establishment of two stable cell lines. Language links are at the top of the page across from the title. 3). Sci. Role of Cnot6l in maternal mRNA turnover. an oocyte in which the first meiotic division is completed; the second meiotic division usually stops short of completion unless fertilization occurs. The lysate was cleared by centrifugation at 12,000g for 10min at 4C, and the supernatant was loaded onto a 20~50% density gradient of sucrose cushion [30mmol/l TrisHCl (pH 7.5), 100mmol/l NaCl, 10mmol/l MgCl2, protease inhibitor cocktail (EDTA-free), and 100 units/ml RNase inhibitor (APExBIO, K1046)], ensued by ultracentrifugation in a rotor at 36,800g for 3h at 4C. & Beilharz, T. H. ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3 RACE applications. Further examination by staining with RPA2, a marker that exclusively labels unrepaired DSBs, unveiled that more RPA2 foci were present in the Nat10-ScKO oocytes at pachytene stage, rather than at diplotene stage (Fig. Liu, H. et al. Natl Acad. Unlike the male germline, the female germ cells initiate meiotic prophase I division early following sex determination in the embryonic gonad, and sequentially undergo leptotene, zygotene, and pachytene, but are fully arrested at the diplotene stage perinatally (Fig. d Quantitative RTPCR (qPCR) assay showing the relative expression levels of Nat10 mRNA in adult WT and Nat10-ScKO mouse ovary. Biol. The infected cells were positively selected with puromycin (12.5g/ml) (Solarbio, P8230) for 48h. The 21-day-old female (P21) mice were injected with 5 IU of pregnant mares serum gonadotropin (PMSG) (Ningbo Sansheng Pharmaceutical). Early tertiary DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). Google Scholar. Protein concentrations were determined using a BCA protein assay kit. Mitosis is a process that makes daughter cells with equal amounts of cytoplasm, and the same amount of DNA as the parent cells. & Lu, F. Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq. The box indicates the upper and lower quantiles, the thick line in the box indicates the median and whiskers indicate 2.5th and 97.5th percentiles. For extension, Poly(A) Test (ePAT), oocyte poly(A)-tail RNAs were 3-prime extended using ePAT anchor primer as a template with Klenow enzyme (New England Biolabs, K0210) at 25C for 1h and 80C for 10min. Development 133, 45274537 (2006). Inoue, A., Nakajima, R., Nagata, M. & Aoki, F. Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice. (Most of the time.). Most recently, Bryan has been creating content and writing curriculum in the educational technology field. f IF images of 21-day-old WT ovarian sections co-stained with NAT10 antibody (Red), Nucleophosmin (NPM, Green), and DAPI (Blue) in the follicles as indicated. For the Ligation-Mediated Poly(A) Test (LM-PAT), oocyte samples were hybridized with oligo(dT)20 at 42C for 30min and then ligated with dT anchor primer by T4 DNA ligase (Sangon, B600511) at 12C for 2h. Reverse transcription was performed with P5TSO and 1mM dCTP for 1h, ensued by PCR pre-amplification for 8 or 16 cycles using dT anchor and P5TSO. Primary oocytes are formed prior to birth, so a female is born with as many primary oocytes as she will ever have. c Quantitative RT-PCR results showing the relative expression levels of mouse Nat10 mRNA in oocytes, and preimplantation embryos.