261 IDENTIFICATION OF OOCYTE- AND CUMULUS-DERIVED CO-REGULATED AND DIFFERENTIALLY REGULATED TRANSCRIPTS ASSOCIATED WITH BOVINE MEIOTIC MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 238
Author(s):  
O. V. Patel ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Oocyte Maturation ◽  
Cdna Array ◽  
Ring Finger ◽  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Leukotriene B4 ◽  
Mrna Abundance ◽  
Meiotic Maturation

Understanding the process of oocyte maturation is critical for efficient application of biotechnologies such as in vitro embryo production and nuclear transfer/cloning. Intercellular communication between the oocyte and the encompassing somatic (cumulus) cells is pivotal for successful growth of ovarian follicles and oocyte maturation. Therefore, we utilized global gene expression profiling to determine changes in the transcriptome of oocytes and their adjacent cumulus cells during meiotic maturation in vitro to identify both co-regulated and differentially regulated transcripts within the two cell compartments of the cumulus oocyte complex (COC). Germinal vesicle (GV) and in vitro matured metaphase II (MII) COC (n = 5 pools of 5 COC per group) were denuded and separated into oocytes and cumulus cells. RNA was extracted from the oocytes and cumulus cells and subjected separately to microarray analysis using a bovine cDNA array containing expressed sequence tags (ESTs) representing 15 500 unique genes. A combined total of 1045 genes displaying greater mRNA abundance in GV oocytes and associated cumulus cells compared to MII samples were detected (P < 0.05; false discovery rate (FDR) = 5%). A combined total of 711 genes displaying greater mRNA abundance in MII oocytes and enclosing cumulus cells compared to GV samples were detected (P < 0.05; FDR = 5%). Fourteen transcripts were identified that were co-regulated and of greater abundance in GV or MII oocytes and in their matching cumulus cells (P < 0.05; FDR = 5%). The co-regulated transcripts identified are implicated in metabolism (e.g. heme oxygenase-2, leukotriene B4 12-hydroxydehydrogenase), signal transduction (e.g. caveolin 1, ring finger protein 31), and cell growth (e.g. BTG family member 2, myosin regulatory light chain 2). In contrast, thirteen transcripts differentially regulated in the GV oocyte versus MII cumulus cells were identified (P < 0.05; FDR = 5%). Similarly, nine transcripts differentially regulated in the MII oocyte versus GV cumulus cells were identified (P < 0.05; FDR = 5%). Some of the identified differentially regulated transcripts encode for genes associated with the cytoskeleton (e.g. tropomyosin 1), apoptotic activity (e.g. death effector domain containing protein 2) and DNA replication (e.g. epsilon polymerase). The results provide novel insights into the identity of transcripts whose abundance is co-regulated or differentially regulated between the oocyte and cumulus cells during the transition of a COC from the GV to the MII stage. Characterization of the signaling pathways driving changes in transcript abundance for co-regulated and differentially regulated genes in oocytes versus associated cumulus cells may lead to a better understanding of regulation of meiotic maturation and potential cross-talk between germ cells and somatic cells during the oocyte maturation cascade. This work was supported by the Rackham Foundation and the MI Agriculture Experiment Station.

scholarly journals MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

2015 ◽  
Vol 308 (6) ◽  
pp. E525-E534 ◽  
Author(s):  
Bo Pan ◽  
Derek Toms ◽  
Wei Shen ◽  
Julang Li
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Specific Effect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
And Function

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion ( HAS2, PTGS2) and oocyte maturation ( CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), zona pellucida 3 ( ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

46 Presence of porcine TET3L isoform in oocytes: Potential involvement in the DNA demethylation process

2020 ◽  
Vol 32 (2) ◽  
pp. 148
Author(s):  
K. Uh ◽  
N. Wax ◽  
K. Farrell ◽  
K. Lee
Keyword(s):  
Oocyte Maturation ◽  
Internal Control ◽  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Dna Demethylation ◽  
Expression Level ◽  
Rt Pcr ◽  
Genome Wide ◽  
Rapid Amplification

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.

172 THE APOPTOTIC EFFECTS OF BISPHENOL A-INDUCED MITOCHONDRIAL-DERIVED REACTIVE OXYGEN SPECIES ON MATURATION OF PORCINE OOCYTES IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 194
Author(s):  
S.-Y. Park ◽  
H.-J. Park ◽  
J.-W. Kim ◽  
J.-Y. Park ◽  
S.-G. Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Critical Role ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Ros Production ◽  
Maturation In Vitro ◽  
Porcine Oocyte

Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P < 0.05). Next, we confirmed the secretion functions of oocyte and cumulus cell of cumulus-oocyte complex (COC) and ROS production. Cumulus cell secretion factors (has2, tnfaip6, and cx37) mRNA expression in COC were decreased in the BPA-treated (75 µM) group. In addition, mRNA expressions of mitochondrial-specific antioxidant enzymes (sod2, P < 0.001; prdx3, P < 0.01; prdx5, P < 0.001) and mitochondrial apoptosis genes (bax and caspase-3, P < 0.01) were significantly increased in COC of the BPA-treated (75 µM) group. We measured mitochondrial membrane potential and mito-ROS production using JC-1 analysis and Mito-SOX staining, respectively. The BPA treatment caused a rapid decrease of mitochondrial membrane potential maintenance and increase of mito-ROS production in porcine COC. Moreover, mitochondrial-specific ROS scavenger, Mito-Tempo (0.1 µM) treatment was significantly increased the meiotic maturation of porcine oocytes compared with control group (78.5 ± 3.5 v. 65.8 ± 5.0%; P < 0.05). Based on these results, we first confirmed that BPA exposure reduces the meiotic maturation and cumulus cells expansion of COC by increasing mito-ROS production during porcine oocyte maturation in vitro. Therefore, controlling of mito-ROS for mitochondrial function maintenance and apoptosis plays a critical role in improving porcine oocyte maturation in vitro. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

scholarly journals Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 163
Author(s):  
Payungsuk Intawicha ◽  
Li-Kuang Tsai ◽  
Shih-Ying Yen ◽  
Neng-Wen Lo ◽  
Jyh-Cherng Ju
Keyword(s):  
Oocyte Maturation ◽  
Mammalian Cells ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitogen Activated Protein ◽  
Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

scholarly journals Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

2014 ◽  
Vol 28 (9) ◽  
pp. 1502-1521 ◽  
Author(s):  
Laura Sanchez-Lazo ◽  
Daphné Brisard ◽  
Sébastien Elis ◽  
Virginie Maillard ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Oocyte Maturation ◽  
Binding Protein ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitochondrial Fatty Acid ◽  
Before And After

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes.

Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI)

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 277-282 ◽  
Author(s):  
Yukiko Yamazaki ◽  
Teruhiko Wakayama ◽  
Ryuzo Yanagimachi
Keyword(s):  
Oocyte Maturation ◽  
Intracytoplasmic Sperm Injection ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Serum Free ◽  
Nuclear Maturation ◽  
Normal Term ◽  
Developmental Capacity ◽  
Oocyte Cytoplasm

The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.

274 LIPOLYSIS IN CUMULUS CELLS ACCOMPANIES OOCYTE MATURATION IN BOVINE

2015 ◽  
Vol 27 (1) ◽  
pp. 226 ◽  
Author(s):  
S. Uzbekova ◽  
L. Sanchez-Lazo ◽  
A. Desmachais ◽  
V. Maillard ◽  
S. Elis
Keyword(s):  
Oocyte Maturation ◽  
Energy Homeostasis ◽  
Binding Proteins ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hormone Sensitive Lipase ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

Oocyte maturation relies on energy from different nutrients, including fatty acids (FA). Cumulus cells (CC) are metabolically coupled with enclosed oocyte and active FA metabolism occurs in both compartments. Excess of lipids in oocyte environment alters its developmental competence. Lipid droplets (LD), mainly composed of triacylglycerides (TG), are formed inside of CC and in oocyte to store lipids. Liberation of free FA from TG requires lipolysis, which is catalyzed by lipases and involves FA-binding proteins (FABP) and perilipins (PLIN), which interact at the surface of LD as shown in lipogenic tissues. The objective was to elucidate the main factors involved in lipolysis in bovine cumulus-oocyte complex (COC) during oocyte maturation. Gene expression before and after maturation was analysed in CC by microarray hybridization and validated by real time RT-PCR; proteins were detected by Western blot and immunofluorescence. For statistics, ANOVA and Mann-Whitney (M-W) tests were used. In CC, adipose triglyceride lipase PNPLA2, lipoprotein lipase LPL, and monoacylglycerol lipase ABHD6 showed the highest mRNA expression level among 7 detected lipases. Both PLIN5 and PLIN2 were the most abundant perilipins, and among 8 FA-binding proteins, FABP3 and FABP5 were predominant. During in vitro maturation (IVM), expression of most of these genes increased at 6 h of IVM (P < 0.05, ANOVA) in CC. At that time, germinal vesicle breakdown occurred in enclosed oocytes and hyaluronan synthase HAS2, involved in the extra-cellular matrix formation, was upregulated in CC. The most upregulated genes after 18 h of IVM in CC were ABDH6 (48.5-fold as compared to immature, P < 0.01, M-W), FABP3 (16.6-fold, P < 0.01, M-W), and PLIN2 (5.5-fold, P < 0.05, M-W). Expression of all of these lipolysis-related genes was also detected in the oocytes. At the protein level, PLIN2 was mainly localised in the cytoplasmic LD, both in CC and in the oocyte. In CC, FABP3 was detected in the cytoplasm, whereas in oocyte it was also localised to the germinal vesicle of immature oocytes and closely to the chromosomes during the first meiotic division. In addition, active phosphorylated hormone sensitive lipase HSL was always detected in CC and in mature oocytes, but not in immature oocytes. All these data demonstrate that lipolysis occurs both in CC and in the oocyte during maturation. Lipolysis may be necessary to maintain cell energy homeostasis by regulating intracellular concentration of free FA. Moreover, CC were already described to store the excess FA from follicular fluid in order to protect the oocyte. Our data corroborate the essential role of CC in oocyte survival through controlling FA metabolism inside the COC. Active lipolysis may therefore be required to reduce lipid storages as well as to produce energy necessary for oocyte meiosis progression and extracellular matrix secretion by CC in order to prepare COC for further fertilization.This work was supported by INRA, ANR (OSCILE project) and European subvention FP7-KBBE-2012–6 (FECUND project).

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

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