265 HYALURONAN SYNTHESIS ABILITY OF PORCINE CUMULUS - OOCYTE COMPLEXES DERIVED FROM SMALL FOLLICLES

2013 ◽  
Vol 25 (1) ◽  
pp. 280
Author(s):  
M. Nakakoji ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cumulus Expansion ◽  
Elisa Kit ◽  
Porcine Oocyte ◽  
Post Hoc ◽  
Metaphase Ii Oocytes

The degree of cumulus expansion, an important step in oocyte maturation, of porcine cumulus–oocyte complexes (COC) derived from small follicles (SF: 1 to 2 mm in diameter) is known to be lower than those derived from middle follicles (MF: 3 to 6 mm in diameter). The objective of this study was to compare the abilities of hyaluronan (HA) synthesis of COC from SF and MF. Furthermore, the effect of oestradiol during pre-incubation of COC on proliferation of the cumulus cells was examined. Cumulus–oocyte complexes from SF and MF of porcine ovaries were cultured for in vitro maturation [IVM, in modified porcine oocyte medium (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) supplemented with 50 µM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dbcAMP for 20 h and then in the fresh medium without those supplements for another 24 h]. Hyaluronan production was quantified at 20 h after the start of IVM with a commercial HA-ELISA kit (20 COC/tube × 4 times). The number of cumulus cells was assessed 0 and 20 h after the start of IVM (50 COC × 4 times). Furthermore, proliferation of cumulus cells was examined after pre-culture of COC (n = 40 COC × 5 times) in modified porcine oocyte medium with various concentrations of oestradiol (0, 0.1, 1, and 10 ng mL–1) for 6 h. Statistical analyses of results from 4 to 5 replicated trials were performed by ANOVA with a Bonferroni-Dunn post-hoc test (significance, P < 0.05). The degree of cumulus expansion of COC from MF (n = 152) was higher than that of COC from SF (n = 156). The incidence of metaphase-II oocytes was significantly lower in COC from SF (n = 133; 48.9%) than in COC from MF (n = 148; 74.7%). The HA content of COC was higher in those from MF (20.8 µg/COC) than in those from SF (10.8 µg/COC), whereas the content per cumulus cell was not different because the numbers of cumulus cells at 0 and 20 h were also higher in COC (n = 200 in each group) from MF (3.0 × 103 and 3.3 × 103 cells, respectively) than from SF (2.0 × 103 and 2.5 × 103 cells, respectively). Cumulus cells proliferated significantly in the presence of oestradiol, regardless of the concentration, during pre-incubation for 6 h (2.5 to 2.8 × 103 cells), as compared with the oestradiol-free controls (2.2 × 103 cells). These results demonstrate that the different abilities of cumulus expansion between COC (n = 200 in each group) from SF and MF may be due to the number of cumulus cells per COC. Pre-incubation in the presence of oestradiol stimulates the proliferation of cumulus cells and may improve the oocyte maturation of COC derived from SF.

71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

2017 ◽  
Vol 29 (1) ◽  
pp. 143
Author(s):  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
G. A. Kim ◽  
B. C. Lee
Keyword(s):  
Embryo Development ◽  
Sonic Hedgehog ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Melatonin Treatment ◽  
Cumulus Expansion ◽  
Hedgehog Signalling

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6 mm in diameter were recovered by aspiration with an 18 G needle. Cumulus–oocyte complexes were pooled and cultured in TCM-199 medium for 44 h. The aim of this study was to evaluate the effects of melatonin (10−9 M) with or without cyclopamine (2 μM) on cumulus cell expansion (a total of 432 cumulus–oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75 ± 0.02 v. 3.51 ± 0.03 and 3.59 ± 0.05, respectively; P < 0.05) and blastocyst formation rates (30.4 ± 2.4 v. 21.9 ± 2.2 and 20.0 ± 2.2, respectively; P < 0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncan’s multiple range test (when the variances were assumed to be equal) or Dunnet’s T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means ± SEM; P-values < 0.05 were considered to be statistically significant. This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (#311011–05–4-SB010, #114059–03–2-SB010), National Research Foundation (2016M3A9B6903410), China Scholarship Council (CSC, No. 2015–3022), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 322
Author(s):  
D. D. Bücher ◽  
M. A. Castro ◽  
M. E. Silva ◽  
M. A. Berland ◽  
I. I. Concha ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Control Group ◽  
Cumulus Expansion ◽  
Gm Csf ◽  
Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

2014 ◽  
Vol 26 (1) ◽  
pp. 200 ◽  
Author(s):  
C. de Frutos ◽  
R. Vicente-Perez ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Mixed Logit ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

scholarly journals Exposure of Triclosan in Porcine Oocyte Leads to Superoxide Production and Mitochondrial-Mediated Apoptosis during In Vitro Maturation

2020 ◽  
Vol 21 (9) ◽  
pp. 3050 ◽  
Author(s):  
Hyo-Jin Park ◽  
Bong-Seok Song ◽  
Jin-Woo Kim ◽  
Seul-Gi Yang ◽  
Sun-Uk Kim ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Superoxide Production ◽  
Mrna Levels ◽  
Female Reproduction ◽  
Mitochondrial Superoxide ◽  
Porcine Oocyte

While triclosan (TCS) exerts detrimental effects on female reproduction, the effect of TCS-derived toxins on porcine oocytes during in vitro maturation (IVM) is unclear. This study investigated the effects of TCS on mitochondrion-derived reactive oxygen species (ROS) production and apoptosis pathways during porcine oocyte maturation. Porcine oocytes were treated with TCS (1, 10, and 100 μM) and triphenylphosphonium chloride (Mito-TEMPO; 0.1 μM), and matured cumulus oocyte complexes (COCs) were stained with orcein, dichlorofluorescein diacetate (DCF-DA), and Mito-SOX. Proteins and mRNA levels of factors related to cumulus expansion and mitochondrion-mediated apoptosis and antioxidant enzymes were analyzed by western blotting and reverse-transcription polymerase chain reaction (RT-PCR), respectively. Meiotic maturation and cumulus cell expansion significantly decreased for COCs after TCS treatment along with an increase in mitochondrial superoxide levels at 44 h of IVM. Further, mitochondrion-related antioxidant enzymes and apoptosis markers were significantly elevated in porcine COCs following TCS-mediated oxidative damage. The protective effect of Mito-TEMPO as a specific superoxide scavenger from TCS toxin improved the maturation capacity of porcine COCs. Mito-TEMPO downregulated the mitochondrial apoptosis of TCS-exposed porcine COCs by reducing superoxide level. In conclusion, our data demonstrate that TCS mediates toxicity during porcine oocyte maturation through superoxide production and mitochondrion-mediated apoptosis.

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.

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.

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 309-314 ◽  
Author(s):  
Masaki Yokoo ◽  
Naoko Kimura ◽  
Hiroyuki Abe ◽  
Eimei Sato
Keyword(s):  
Confocal Microscopy ◽  
Oocyte Maturation ◽  
Extracellular Space ◽  
Cumulus Cells ◽  
Maturation Process ◽  
Cumulus Expansion ◽  
Total Inhibition ◽  
Maturation Medium ◽  
Porcine Oocyte

SummaryDuring oocyte maturation, the cumulus–oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.

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