secondary oocyte is also known as

PubMed collected equal amounts of mouse oocytes used in this study and western blot analysis. By comparison, the down-regulated genes were enriched in transcription-related GO terms (Supplementary Fig. This revealed a higher number of genes (1196) up-regulated than down-regulated (555) in Nat10-ZcKO MII oocytes (Fig. 10df). b Quantification of collected average numbers of GV oocytes. Any new organism has to have the right amount of both genetic and cellular material to be healthy, and the eggs that survive to join with a sperm are unique, unlike any other body cells, and have gone through a process that millions of other eggs have died for. 8e). For immunofluorescent staining, cells were permeabilized with 0.3% Triton X-100, blocked with 5% normal goat serum (Solarbio, SL038) in PBST, and incubated with primary antibodies diluted in blocking solution at 4C overnight. These data suggest that NAT10 is functional through oocyte maturation, at least in part, through the deposition of ac4C modification at both the transcriptional and translational levels. 5, 10511066 (2013). After filtering through a 70m cell strainer, the single cells were cultured in MEF medium (DMEM medium supplemented with 10% FBS (VivaCell, C04001) and 1% Penicillin-Streptomycin (Biosharp, BL505A)). 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). Fertilization is only possible for the one daughter cell that gets the most cytoplasm when meiosis has finished - the others are called polar bodies and eventually disintegrate. 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. We selected Bst2, Fus, and Nomo as the positive control (ac4C+), and Eef1a1 and Gapdh as the negative control (ac4C). The degradation trend profiling verified that the 1416 transcripts indeed displayed a decreased degradation speed in the absence of Nat10 (Fig. 5a, b). Nucleic Acids Res. 1c). As the oocytes develop, they get enclosed in structures known as follicles. Nature 548, 347351 (2017). We collected five hormone-primed oocytes at GV and MII stages for each sample and executed 16 cycles of PCR amplification. 22.7: Functions of the Female Reproductive System MacQueen, A. J. : an oocyte that is produced by division of a primary oocyte in the first meiotic division. An oocyte in which the first meiotic division is completed; the second meiotic division usually stops short of completion unless fertilization occurs. & Jaffrey, S. R. Mapping m(6)A at individual-nucleotide resolution using crosslinking and immunoprecipitation (miCLIP). Female reproductive system: The Histology Guide - University of Leeds Polar bodies are also created during oogenesis, and differ from oocytes in that they have different amounts of cytoplasm. 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. 300, 249258 (2007). After washing in 1XPBS (0.1% Tween) for three times, the samples were incubated with TRITC-conjugated Goat Anti-Rabbit IgG (Proteintech, SA00007-2) or 488-conjugated secondary antibodies (Proteintech, gb2AF488). IF staining by H2AX revealed the elevated DSB signals in oocytes at the pachytene stage, but not at the diplotene stage, in embryonic Nat10-ScKO ovaries (Fig. Upon meiosis resumes, the GV maternal transcriptome undergoes a global but selective degradation of ~20% poly(A) mRNAs, culminating in a characteristic maternal transcriptome in MII oocytes that substantially differs from that in GV oocytes7,15,42,43. To unlock this lesson you must be a Study.com Member. The oocyte mini-bulk SMART-seq2 protocol was based on the well-established SMART-seq2 protocol with in-house optimized modifications as indicated below. 7c). 3e, f), which presumably point to oocytes with elevated H2AX staining resulting from DSB repair deficiency in Nat10-ScKO ovaries33. & Richter, J. D. CPEB controls oocyte growth and follicle development in the mouse. Reprod. f Quantification of the numbers of RPA2 foci (representative of the unrepaired DSBs) in WT and Nat10-ScKO mouse oocytes at birth. Vector-OHT, p=0.0026; OHT-Nat10, p=0.0340. i, j The polysome profiling displaying the translational efficiency and ribosome assembly in MEF cells analyzed by sucrose density gradient centrifugation. Shen, Q. et al. https://doi.org/10.1038/s41467-023-39256-0, DOI: https://doi.org/10.1038/s41467-023-39256-0. 5b). Biology Ch 27 A&P 5.0 (1 review) fertilization Click the card to flip The fusion of a sperm cell and secondary oocyte is known as __________. Mole, M. A., Weberling, A. PO, Preovulatory Follicle. Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development. After birth, individual oocytes arrested at the diplotene of prophase I are encircled by a flattened layer of granulosa cells, which together constitute the primordial follicles. 1b, c)27. Biochem. Y.Ch. Scale bar, 50m. Statistical analyses were performed using R software (http://www.rproject.org). Noteworthily, we consistently observed that longer induction of Nat10-inducible KO MEF cells by 4-OHT treatment led to complete cell cycle arrest and resultant cell death, suggesting that Nat10 is essential for cell survival. a The gross morphology of oocytes at Germinal vesicle (GV) stage collected from PMSG-primed WT and Nat10-ZcKO females at P21. General Studies Health Science: Help & Review, What Is a Blastocyst? Together, these studies suggest that Nat10 is required for oocyte meiotic maturation (Fig. The transcriptional activity peaks in the early growing oocytes, but decreases thereafter and is considered silent in the fully grown GV oocyte10. b Dynamic mRNA expression levels of Nat10 from RNA-seq analyses in oocytes and preimplantation embryos in mice (GSE71434). Accessed 30 Jun. As shown in Fig. The oogonia grow and divide by mitosis like other body cells, and have 2 DNA copies (2n). In brief, freshly collected or frozen tissues were homogenized in TRIzol reagent. Source data are provided as a source data file. Therefore, this single hairpin adaptor not only provides an anchor primer for reverse transcription but also harbors the full reverse primer sequence for subsequent cDNA PCR amplification. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Scale bar, 50 m. Cell Biol. 4d, e). 10j, k). The divisions of meiosis produce one large mature oocyte and 2 polar bodies with little cytoplasm. 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In brief, ovary samples were freshly collected, and fixed in Bouins solution at room temperature overnight. 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. Data are presented as the meanSEM, n=3. PLoS Genet. PubMed Central 88, 11 (2013). Of note, compared with SN oocytes, NSN oocytes exhibit higher transcriptional activity and lower developmental competence, with most derived zygotes arresting at the 2-cell stage. Sui, X. et al. We reveal that Nat10 plays a profound role that is indispensable for oocyte meiotic cell-cycle progression. 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. Provost, the UC Davis Library, the California State University Affordable . Biol. Values of p<0.05 were deemed statistically significant. Next, ovaries were transferred to 100mM sucrose, and single cells were released into sucrose solution using syringe needles. 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. 8a). [27] Currently under investigation is the possibility of other cytoplasmic contributions made to the embryo by the spermatozoon. The following antibodies were used: rabbit anti-NAT10 (ZENBIO, 389412, 1:1000), mouse anti-PCNA (Proteintech, 60097-1-Ig, 1:2000), mouse anti-GAPDH (Proteintech, 60004-1-Ig, 1:10,000), rabbit anti-Tubulin (Proteintech, 11224-1-AP, 1:5000). Cryoconservation of animal genetic resources, "The human oocyte and cumulus cells relationship: new insights from the cumulus cell transcriptome", "Vegetal localization of maternal mRNAs is disrupted by VegT depletion", "Xenopus X-box binding protein 1, a leucine zipper transcription factor, is involved in the BMP signaling pathway", "The role of maternal CREB in early embryogenesis of Xenopus laevis", "New roles for FoxH1 in patterning the early embryo", "Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus", "Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase", "XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development", "Cell fate specification and competence by Coco, a maternal BMP, TGFbeta and Wnt inhibitor", "The maternally localized RNA fatvg is required for cortical rotation and germ cell formation", "Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo", "Oocytes can efficiently repair DNA double-strand breaks to restore genetic integrity and protect offspring health", "Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes", Micrograph of a primary oocyte and follicle of a monkey, https://en.wikipedia.org/w/index.php?title=Oocyte&oldid=1137668856, Short description is different from Wikidata, Creative Commons Attribution-ShareAlike License 4.0, Halted in metaphase II until fertilization, Germ cell formation and cortical rotation, This page was last edited on 5 February 2023, at 21:34. HA-PAT utilizes an exquisitely designed hairpin adaptor that can self-hybridize plus an extended oligo (dT)8 with two additional degenerate W nucleotides at the 3 end (Fig. 29, 846860 (2022). e Heatmap of representative genes from three major functional GO categories showing up-regulated RPFs in Nat10-ZcKO MII oocytes. Maternal NAT10 orchestrates oocyte meiotic cell-cycle progression and maturation in mice, https://doi.org/10.1038/s41467-023-39256-0. Indeed, further counting of NSN versus SN oocytes uncovered that the ratio of NSN to SN was distorted between Nat10-ZcKO and WT ovaries (Fig. In growing oocytes, cytoplasmic maturation is accompanied by the non-surrounded nucleolus (NSN)-to-surrounded nucleolus (SN) DNA configuration transition11. The oocyte is arrested in Meiosis II at the stage of metaphase II and is considered a secondary oocyte. Finally, another cycle of division begins (this is meiosis 2), and the few cells which reach this stage are called secondary oocytes. For the cell proliferation assay, 2000 MEF and OHT-treated MEF cells were plated in 96-well plates with 200l of fresh complete DMEM medium supplemented with 10% FBS. An oocyte is produced in the ovary during female gametogenesis. Through acRIP-sequencing, we have found that ac4C modification is abundantly present in the maternal oocyte transcripts, which are functionally involved in translation and RNA processing (Supplementary Fig. The primary oocyte is a cell formed from an oogonium, which was created when a female was developing during pregnancy, prior that that individual's birth. Dashed circle indicates cellular membrane of oocytes. 50, 1089610913 (2022). During oogenesis, the oogonia become primary oocytes. Since there exists global poly(A) mRNA degradation during GV-MII transition in WT oocytes, the expressed transcripts in WT MII oocytes were allocated into three types: Up- [FC(MII/GV)2, p<0.05], Down- [FC(MII/GV) 2, p<0.05], and Stable-type (remaining transcripts). Compelling studies have shown evidence related to Nat10s function in cell-cycle control in somatic cells19,20,21. Mol. At this point, these cells grow and divide like all the other cells in the body: through mitosis. 8f, g, all the poly(A) tails of the seven genes were lengthened in MII Nat10-null oocytes, as judged by the elevated, smear PCR bands. 43, 22422258 (2015). Y.Z. It has been documented that the dys-regulated chromatin signature associates closely with defective NSN to SN transition as evidenced in Zfp36l2- and Rps26-null oocytes, which presumably resulted from the insufficient expression of two key paracrine growth factors Gdf9 and Bmp1514,35. At post-hCG 16h, the cumulusoocyte complex (COC) was retrieved from the oviducts, and the oocytes were counted after digestion with 0.3% hyaluronidase (Sigma, H4272). Zheng, J. et al. During this prophase I arrested stage (dictyate), which may last for many years, four copies of the genome are present in the oocytes. Immature ovum - Wikipedia Google Scholar. GO analyses of ac4C+ transcripts implied that they were mainly related to translation, RNA splicing and chromosome separation (Supplementary Fig. 8b). The oocyte definition is a female gametocyte that is either in primary or secondary stages of development. Protein concentrations were determined using a BCA protein assay kit. There is one other way that meiosis is peculiar for the development of the oocyte. Cnot6l, p=0.0906; Cnot7 p=0.0020; Btg4 p=0.0031. d Western blot displaying the NAT10, CNOT6L, CNOT7 and BTG4 protein levels in MII oocytes of WT and Nat10-ZcKO mice. 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. The daughter cells produced by mitosis are genetically identical. This revealed that a total of 6188 transcripts likely harbor the ac4C modification (ac4C/IgG>2) in the oocytes, although this approach cannot pinpoint the accurate positions of cytosine acetylation due to the technical limitation of ~200bp mRNA fragment resolution. & Beilharz, T. H. ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3 RACE applications. Data are presented as the meanSEM, n=3, *p<0.05 by two-tailed Students t-test. Mol. 49, 20512062 (2021). Rev. Follicles were counted on serial ovarian sections after H&E staining. using 500 oocytes37, our Ribo-seq protocol detected comparable numbers of translated genes and global translational patterns (Supplementary Fig. Equal amounts of total RNA were loaded to synthesize cDNAs using the Hiscript III Reverse Transcriptase (Vazyme, R302-1). Wiley Interdiscip. Cloutier, J. M. et al. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Asp. PLoS Biol. Meiosis is similar to mitosis, but it results in the right amount of DNA and cellular material being present in the mature oocyte. Creation of Oocytes - Unacademy List the steps of oogenesis Describe the hormonal changes that occur during the ovarian and menstrual cycles Trace the path of an oocyte from ovary to fertilization Guan, Y. et al. 9 chapters | Rinaldi, V. D., Bolcun-Filas, E., Kogo, H., Kurahashi, H. & Schimenti, J. C. The DNA damage checkpoint eliminates mouse oocytes with chromosome synapsis failure. For the Hairpin-Adaptor PAT (HA-PAT) assay, 10 denuded oocytes were freshly lysed in 2l lysis buffer (0.2% Triton X-100 and 2IU/l RNase inhibitor). Early oocytes are also classified as immature (germinal vesicle (GV) or metaphase I (MI) stage). The destruction of paternal mitochondria ensures the strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA). Differentially expressed genes (DEGs) were assessed with the DESeq2 package with a cutoff: Padj <0.05 and fold change (FC)2. Google Scholar. Ivanova, I. et al. Source data are provided as a source data file. Data are presented as the meanSEM, n=3. collected mouse oocytes used for RNA-seq. PubMed and Q.L. Jiang, X., Cheng, Y., Zhu, Y. et al. Histologically, the preantral follicle is marked by a fully grown oocyte surrounded by a zona pellucida, approximately nine layers of granulosa cells, a basal lamina, a theca interna, a capillary net, and a theca externa. To dissect the factors underlying defective GV oocytes in Nat10-ZcKO ovaries, we carried out RNA-seq analyses with GV oocytes retrieved from WT and Nat10-ZcKO ovaries. BIO 140 - Human Biology I - Textbook - City University of New York Nucleic Acids Res. All rights reserved. BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes. Two stable MEF lines were achieved after passage 23 following recovery of the MEF cells from the crisis around passage ~1014 (total time is ~2 months). Scale bar, 20m. Article Cell 44, 392402.e397 (2018). a Schematic illustration showing the procedures for the generation of two stable, TMX-inducible MEF cell lines (Nat10+/Cre, lox/lox) from the Ubc-cre; Nat10lox/lox embryos following a 3T3 protocol (see the Methods section). Ovary samples were cut into 8m slides. On day 14, in response to the LH surge, the mature follicle (now termed a. gi Bar plots showing the relative mRNA expression levels in g, RPF in h and TE in i for a panel of up-regulated gene transcripts. The oogonia, then, begins meiosis I, which is the type of cell division that prepares cells for sexual reproduction. Scale bar, 10m. We showed that this defect was likely caused by the deficient DSB repair as judged by the high persistent H2AX intensity and enhanced RPA2 remnant (Fig. Sharma, S. et al. 1a). Nat10 depletion in growing oocytes of primary follicles disrupted the NSN-SN transition of GV oocytes and damaged meiotic maturation, as evidenced by the aberrant GV to MII transition11. For in vitro fertilization (IVF), superovulated female mice were euthanized 15h after hCG injection, and the ampulla parts of the oviducts were collected in an HTF medium covered by mineral oil. 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). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Indeed, we discovered that ~65% (782/1196) of transcripts up-regulated in Nat10-ZcKO MII oocytes were also accumulated in the Cnot6l (CCR4-NOT subunit) KO MII oocytes (Fig. Data were presented as meanSEM, n=3. - Statistics, Types & Causes, What Is Syphilis? 5e, f). Conversely, the secondary oocyte is a cell that develops after meiosis I is complete, which has the diploid number (2n) with just 1 set of chromosomes. Article For each sample, the expressed genes were defined with cutoff: TPM1. 6a, b, Supplementary Fig. Biotechnol. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Oocytes begin the process of meiosis to become differentiated from millions of oogonia, or early oocytes, in the female fetus before 20 weeks of life. did HE staining of mouse ovaries with the help of W.L. Source data are provided as a source data file. In addition, we discovered that the key components of CCR4NOT complex, including Cnot6l, Cnot7, and Btg4, all harbor the ac4C modification, and were subjected to transcriptional and translational repression in the Nat10-null oocytes, causing the delayed maternal transcript degradation owing to reduced activity of CCR4NOT complex and the resultant dys-regulated maternal transcriptome. To trace at which stage the Nat10 abrogation impacted follicle development, we next examined the histology of Hematoxylin&Eosin (HE)-stained sections using paraffin-embedded ovaries during the first wave of postnatal follicle development. Our interest in exploring the role of Nat10 in oocyte development was initially piqued for two reasons. Because each of these reproductive cells is a haploid cell containing half of the genetic material needed to form a human being, their combination forms a diploid cell. The stages of oocyte maturation are as follows: The oocyte development is very intricate and important process that can lead to the fertilization process that creates new life. The oocyte definition is a female gametocyte that is either in primary or secondary stages of development. The lysate was sheared 4 with a 26-gauge needle, and treated with 0.5l RNase I (100U/l from Invitrogen AM2294) for 1.5h at room temperature. The granulosa cells transform from tightly compacted to an expanded mucoid matrix. 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. Stra8-GFPCre is specifically activated around embryonic day 12 (E12) in the primordial germ cells (PGCs) prior to meiosis in the female embryonic gonad (Fig.

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