Mechanisms of Estradiol-induced EGF-like Factor Expression and Oocyte Maturation via G Protein-coupled Estrogen Receptor

Endocrinology ◽  
2020 ◽  
Vol 161 (12) ◽  
Author(s):  
Hui Zhang ◽  
Sihai Lu ◽  
Rui Xu ◽  
Yaju Tang ◽  
Jie Liu ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
G Protein ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
Mitochondrial Activity ◽  
Creb Phosphorylation ◽  
First Polar Body ◽  
Factor Expression ◽  
G Protein Coupled

Abstract Estrogen is an important modulator of reproductive activity through nuclear receptors and G protein–coupled estrogen receptor (GPER). Here, we observed that both estradiol and the GPER-specific agonist G1 rapidly induced cyclic adenosine monophosphate (cAMP) production in cumulus cells, leading to transient stimulation of phosphorylated cAMP response element binding protein (CREB), which was conducive to the transcription of epidermal growth factor (EGF)-like factors, amphiregulin, epiregulin, and betacellulin. Inhibition of GPER by G15 significantly reduced estradiol-induced CREB phosphorylation and EGF-like factor gene expression. Consistently, the silencing of GPER expression in cultured cumulus cells abrogated the estradiol-induced CREB phosphorylation and EGF-like factor transcription. In addition, the increase in EGF-like factor expression in the cumulus cells is associated with EGF receptor (EFGR) tyrosine kinase phosphorylation and extracellular signal–regulated kinase 1/2 (ERK1/2) activation. Furthermore, we demonstrated that GPER-mediated phosphorylation of EGFR and ERK1/2 was involved in reduced gap junction communication, cumulus expansion, increased oocyte mitochondrial activity and first polar body extrusion. Overall, our study identified a novel function for estrogen in regulating EGFR activation via GPER in cumulus cells during oocyte maturation.

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

scholarly journals Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 791-799 ◽  
Author(s):  
Lisa M Mehlmann
Keyword(s):  
G Protein ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Follicle Cells ◽  
Meiotic Maturation ◽  
Meiotic Arrest ◽  
Epidermal Growth ◽  
High Level ◽  
G Protein Coupled ◽  
Stimulation Of

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Oocytes are arrested at the first meiotic prophase, held in meiotic arrest by the surrounding follicle cells until a surge of LH from the pituitary stimulates the immature oocyte to resume meiosis. Meiotic arrest depends on a high level of cAMP within the oocyte. This cAMP is generated by the oocyte, through the stimulation of the GsG-protein by the G-protein-coupled receptor, GPR3. Stimulation of meiotic maturation by LH occurs via its action on the surrounding somatic cells rather than on the oocyte itself. LH induces the expression of epidermal growth factor-like proteins in the mural granulosa cells that act on the cumulus cells to trigger oocyte maturation. The signaling pathway between the cumulus cells and the oocyte, however, remains unknown. This review focuses on recent studies highlighting the importance of the oocyte in producing cAMP to maintain arrest, and discusses possible targets at the level of the oocyte on which LH could act to stimulate meiotic resumption.

scholarly journals G protein-coupled estrogen receptor is not required for sex determination or ovary function in zebrafish

2018 ◽  
Author(s):  
Camerron M. Crowder ◽  
Shannon N. Romano ◽  
Daniel A. Gorelick
Keyword(s):  
Estrogen Receptor ◽  
Sex Determination ◽  
G Protein ◽  
Oocyte Maturation ◽  
Ovary Development ◽  
Wild Type ◽  
Ovary Function ◽  
And Function ◽  
G Protein Coupled

ABSTRACTEstrogens regulate vertebrate development and function through binding to nuclear estrogen receptors alpha and beta (ERα, ERβ) and the G protein-coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERβ are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo, we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios and fertility. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function were normal in gper mutant ovaries compared to wild type. Our findings suggest that GPER is not required for sex determination, ovary development or fertility in zebrafish.

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

359 THE EFFICIENT MAINTENANCE OF OXYGEN CONSUMPTION BY PORCINE OOCYTES RELATES TO THE HIGH COMPETENCE OF OOCYTE MATURATION

2007 ◽  
Vol 19 (1) ◽  
pp. 295 ◽  
Author(s):  
M. Yokoo ◽  
T. Ito-Sasaki ◽  
H. Shiku ◽  
T. Matsue ◽  
S. Aoyagi ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
Atp Content ◽  
Small Areas ◽  
Morphological Criteria ◽  
First Polar Body ◽  
Peripheral Type ◽  
High Competence

We succeeded in noninvasively and quantitatively determining the oxygen consumption rate of single bovine oocytes and embryos by scanning electrochemical microscopy (SECM). Results from our earlier studies with bovine embryos established a positive relationship between oxygen consumption and embryo quality by using SECM (Abe et al. 2004 J. Mamm. Ova Res. 21, 22–30). However, the oxygen consumption of porcine oocytes and the relationship between oxygen consumption and oocytes quality were still unclear. The aims of this study were: (1) to assess the oxygen consumption of single porcine cumulus–oocyte complexes (COCs) and oocytes; and (2) to examine the change of oxygen consumption of oocytes during IVM. The COCs collected from 2–5 mm follicles of a porcine ovary were classified into four grades according to morphological criteria (A, compact multilayered cumulus; B, 3 to 5 layers of compact cumulus; C, less than 3 layers of cumulus, including small areas of denuded zonae pellucidae; and D, completely or mostly denuded). Oxygen consumption of these COCs was measured using by the SECM system (HV-403; Research Institute for Functional Peptides, Yamagata, Japan). After the measurement, cumulus cells were removed from COCs mechanically, and oxygen consumption of the oocytes was evaluated. The COCs were cultured in TCM-199 supplemented with 10% porcine follicular fluid and 10 IU mL-1 eCG. After IVM, oxygen consumption and ATP content of the oocytes were determined. ATP content was measured using a commercial assay kit (Promega K.K. Japan, Chuo-Ku, Tokyo, Japan) based on the luciferin-luciferase reaction. Moreover, distribution of active mitochondria in the oocytes during IVM was investigated using MitoTracker Orange staining (Invitrogen Japan K.K., Tokyo, Japan). The rates of oxygen consumption and oocyte maturation of higher-graded COCs and oocytes tended to be higher. In matured oocytes with a first polar body, oxygen consumption (F × 10-14 mol s-1) was maintained at a high level during IVM (GV oocytes: 0.60 ± 0.03; matured oocytes: 0.57 ± 0.01). ATP content (pmol/oocyte) of matured oocytes was significantly increased by IVM culture (GV oocytes: 1.16 ± 0.11; matured oocytes: 2.06 ± 0.06). On the other hand, in non-matured oocytes without a first polar body, oxygen consumption (0.31 ± 0.04) and ATP content (1.51 ± 0.21) were significantly lower than in matured oocytes. The mitochondrial distribution pattern of the oocytes during IVM was changed into an aggregated type (mitochondria distributed to the inner region of the cytoplasm with aggregation of larger fluorescent areas) from a peripheral type (mitochondria evenly distributed to the periphery of the cytoplasm) through a semi-peripheral type (mitochondria distributed to the periphery of the cytoplasm with small spots of fluorescence intensity). However, most of non-matured oocytes showed a semi-peripheral type. These results demonstrated that the oocytes with high oxygen consumption and ATP levels showed higher competence of oocyte maturation. Furthermore, these findings suggest that mitochondrial reorganization may be partly related to the oxygen consumption and consequently the quality of porcine oocytes.

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