scholarly journals NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuting Xiang ◽  
Chuanchuan Zhou ◽  
Yanyan Zeng ◽  
Qi Guo ◽  
Jiana Huang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
High Throughput Sequencing ◽  
Epigenetic Modification ◽  
Binding Protein ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Translation Efficiency ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Heparin effect on in vitro nuclear maturation of bovine oocytes

Zygote ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Juan Carlos Flores-Alonso ◽  
Leticia Lezama-Monfil ◽  
María Luisa Sánchez-Vázquez ◽  
Rosalina Reyes ◽  
Néstor M. Delgado
Keyword(s):  
Oocyte Maturation ◽  
Morphological Changes ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Salt Medium ◽  
Germinal Vesicle Breakdown ◽  
Maturation Factor ◽  
First Polar Body ◽  
Bovine Oocytes

SummaryOocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized.Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 °C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7 ± 1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0 ± 1.1 h to 9.0–11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

scholarly journals Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Svetlana Uzbekova ◽  
Mohamad Salhab ◽  
Christine Perreau ◽  
Pascal Mermillod ◽  
Joëlle Dupont
Keyword(s):  
Granulosa Cells ◽  
Map Kinases ◽  
Glycogen Synthase ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Glycogen Synthase Kinase ◽  
Aurora A Kinase ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

Glycogen synthase kinase 3 (GSK3) regulates cellular metabolism and cell cycle via different signalling pathways. In response to insulin and growth factors GSK3 is serine-phosphorylated and inactivated. We analysed GSK3B expression and activation in bovine cumulus cells (CC) and oocytes at different meiotic stagesin vitroin parallel with MAP kinases ERK (MAPK3/MAPK1) and p38 (MAPK14). GSK3B localised to cytoplasm in granulosa cells and in oocytes throughout folliculogenesis. In mature metaphase-II (MII) oocytes, GSK3B was concentrated to the region of midzone between the oocyte and the first polar body, as well as active phospho-Thr Aurora A kinase (AURKA). Duringin vitromaturation (IVM), in oocytes, phospho-Ser9-GSK3B level increased as well as phospho-MAPK3/MAPK1, while phospho-MAPK14 decreased. In CC, phospho-MAPK14 increased upon germinal vesicle breakdown (GVBD)/metaphase-I (MI) and then decreased during transition to MII. Administration of inhibitors of GSK3 activity (lithium chloride or 2′Z,3′E -6-bromoindirubin-3′-oxime) rapidly increased phospho-Ser9-GSK3B, and led to transient decrease of phospho-MAPK3/MAPK1 and to durable enhancing of phospho-MAPK14 in granulosa primary cell culture. GSK3 inhibitors during IVM diminished cumulus expansion and delayed meiotic progression. In cumulus, phospho-MAPK14 level was significantly higher in the presence of inhibitors, comparing with control, through the time of MI/MII transition. In oocytes, phospho-GSK3B was increased and phospho-MAPK3/MAPK1 was decreased before GVBD and oocytes were mainly arrested at MI. Therefore, GSK3B might regulate oocyte meiosis, notably MI/MII transition being the part of MAPK3/1 and MAPK14 pathways in oocytes and CC. GSK3B might be also involved in the local activation of AURKA that controls this transition.

Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration

2008 ◽  
Vol 24 (4) ◽  
pp. 227-234 ◽  
Author(s):  
SY Hou ◽  
L Zhang ◽  
K Wu ◽  
L Xia
Keyword(s):  
Thioglycolic Acid ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Chromosomal Arrangement ◽  
First Polar Body ◽  
Inverted Microscope ◽  
Polar Body Formation ◽  
Spindle Elongation

Previous studies have shown that thioglycolic acid (TGA) leads to potential reproductive toxicology. To clarify the exact effects of this compound on reproduction, mice oocytes were treated with different TGA doses. At the end of the culture period, the nuclear status of mice oocytes was assessed under an inverted microscope. After immunofluorescence staining, the chromosomal arrangement and spindle configuration of oocytes were evaluated. The results indicated that TGA decreases the percentage of first polar body formation but does not influence that of germinal vesicle breakdown. TGA induces abnormal chromosomal arrangement and spindle elongation. In conclusion, TGA inhibits in-vitro maturation of mice oocytes and affects chromosomal arrangement and spindle configuration. Furthermore, it probably interferes with biochemical changes that occur during meiosis, resulting in aberrant development.

263 REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES

2011 ◽  
Vol 23 (1) ◽  
pp. 229
Author(s):  
M. J. Izquierdo-Rico ◽  
R. Romar ◽  
C. Kohata ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Medium Size ◽  
Total Rna ◽  
Follicle Diameter ◽  
First Polar Body

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (<2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P < 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles). Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression

1976 ◽  
Vol 22 (3) ◽  
pp. 531-545
Author(s):  
P.M. Wassarman ◽  
W.J. Josefowicz ◽  
G.E. Letourneau
Keyword(s):  
Cyclic Amp ◽  
Cytochalasin B ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Dibutyryl Cyclic Amp ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.

scholarly journals Folic acid facilitates in vitro maturation of mouse and Xenopus laevis oocytes

2012 ◽  
Vol 109 (8) ◽  
pp. 1389-1395 ◽  
Author(s):  
Xiaoli Huang ◽  
Shu Gao ◽  
Wei Xia ◽  
Shaoying Hou ◽  
Kun Wu
Keyword(s):  
Folic Acid ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Water Soluble ◽  
Mitogen Activated Protein Kinase ◽  
Xenopus Laevis Oocytes ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

The water-soluble B vitamins, folate and folic acid, play an important role in reproductive health, but little is known about the effects of folic acid on infertility. The present study tested the hypothesis that folic acid affects oocyte maturation, a possible cause of female infertility. We have studied the in vitro maturation of mouse and Xenopus oocytes. Hypoxanthine (Hx) was used as an inhibitor of mouse oocyte maturation to mimic in vivo conditions by maintaining high levels of cyclic-AMP. The frequency of first polar body (PB1) formation and germinal vesicle breakdown (GVBD) in mouse oocytes was decreased by Hx. This effect was counteracted by folic acid added to the medium. PB1 extrusion and GVBD percentages rose to 27·7 and 40·0 % from 12·8 and 19·9 %, respectively, by exposure to 500 μm-folic acid. Folic acid also restored the spindle configuration, which had been elongated by Hx, as well as normalising the distribution of cortical granules (CG). In folic acid-treated Xenopus eggs, extracellular signal-regulated kinase 1 was phosphorylated, cyclin B2 and Mos were up-regulated and the frequency of GVBD was accelerated. Taken together, the findings suggest that folic acid facilitates oocyte maturation by altering the expression and phosphorylation of proteins involved in M-phase-promoting factor and mitogen-activated protein kinase pathways, as well as causing changes in spindle configuration and CG migration.

Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3′,5′-monophosphate.

1990 ◽  
Vol 68 (4) ◽  
pp. 1182-1187 ◽  
Author(s):  
E. Sato ◽  
M. Matsuo ◽  
H. Miyamoto
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Razor Blade ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Polar Body Formation ◽  
Bovine Oocytes ◽  
Meiotic Competence

Abstract The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in bovine oocytes. Oocytes of various sizes were isolated from ovaries by mechanical dissection using an 18-gauge needle followed by a razor blade. This method yielded an average of 26.2 ± 7.4 growing and fully grown oocytes from an ovary. Cumulus-enclosed oocytes were cultured in vitro in tissue culture medium 199 containing 10% fetal calf serum. Oocytes ≤ 90 µm in diameter did not resume meiosis. However, germinal vesicle breakdown was observed in oocytes whose diameters exceeded 91 µm. Polar body formation was observed in oocytes with diameters exceeding 101 µm. About 80% of the oocytes with diameters ≥ 121 µm were able to extrude the polar body. The percentage of large oocytes (101 to 120 µm) with first polar body increased when incubated in medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate; however, oocytes 90 to 101 µm did not extrude the first polar body even when cultured in a medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate. These observations indicate that the capability to resume meiosis is acquired gradually during development of oocytes and that dibutyryl cyclic adenosine 3′,5′-monophosphate can improve the meiotic competence of bovine oocytes in culture.

Effect of oestrogen on mouse follicle growth and meiotic resumption

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Heng Chi ◽  
Zuowu Cao
Keyword(s):  
Polar Body ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Membrane Receptor ◽  
Follicle Growth ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Two Phases

Summary Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus–oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 μm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 μM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 μM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

Oocyte maturation in rabbits: effects of calmodulin inhibitors

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 255-260 ◽  
Author(s):  
Michael A. Henry ◽  
Richard G. Rawlins ◽  
Ewa Radwanska ◽  
Mary M. Fahy
Keyword(s):  
Oocyte Maturation ◽  
Calcium Binding ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Polar Bodies ◽  
Downstream Effectors ◽  
First Polar Body ◽  
Metaphase I

SummaryOocyte maturation in mammals follows a highly conserved pattern of release from arrest through to the extrusion of the first polar body and formation of the second metaphase spindle. Oscillations in cytoplasmic calcium concentration precede the events of maturation in many species. These calcium ions interact with and activate calcium-binding proteins, including calmodulin, within the cell. Thus, it was of interest to us to examine whether calcium acted through calmodulin in the initial stages of maturation in rabbit oocytes or whether calmodulin was required for continuation through metaphase I on to metaphase II. Using the calmodulin inhibitor W-7 we found a significant (p < 0.05) decrease in the percentage of oocytes that underwent germinal vesicle breakdown. Calmidazolium did not prevent germinal vesicle breakdown; however, it caused a significant (p < 0.05) decrease in the proportion of oocytes with fully elaborated spindles and taxol-induced cytoplasmic asters. Both inhibitors caused a significant (p < 0.05) reduction in the proportion of oocytes that extruded their first polar bodies. The kinase inhibitor 6-DMAP caused a significant reduction in the proportion of oocytes with spindles and condensed chromatin, indicating the necessity for phosphorylation events in the resumption of meiosis. In rabbit oocytes calmodulin may play a role in the release from prophase arrest, and it is necessary for spindle preservation and continuation through metaphase I to metaphase II. The varying effects of the two inhibitor stems from their different binding sites on the calmodulin molecule thus causing a differential effect on its downstream effectors.

Platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) is required for the formation of the meiotic spindle during in vitro oocyte maturation

2018 ◽  
Vol 30 (12) ◽  
pp. 1739 ◽  
Author(s):  
L. T. M. Vandenberghe ◽  
B. Heindryckx ◽  
K. Smits ◽  
K. Szymanska ◽  
N. Ortiz-Escribano ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Close Association ◽  
Polar Body ◽  
Platelet Activating Factor ◽  
Germinal Vesicle ◽  
Meiotic Spindle ◽  
Intracellular Enzyme ◽  
Human Oocytes ◽  
First Polar Body

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.

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