• Development and Reproduction
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• v.5 no.2
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• pp.123-129
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• 2001

When mammalian oocytes undergo maturation, cumulus cells surrounding the oocyte exhibit remodeling of their structure known as cumulus expansion. Many molecules including hyaluronic acid participate in this remodeling. The present study aimed to investigate a possible existence of matrix metalloproteinases(MMPs) in the extracellular matrix(ECM) of human oocyte-cumulus complex. ECM was extracted from the human oocyte-cumulus complex. Gelatin gel zymogram of ECM exhibited 7 gelatinases having molecular weight of 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, and 84kDa. This gelatinase profile was very different from that of ovarian mural granulosa cell extract or white blood cell extract, indicating that the oocyte-cumulus complex donating ECM was free from other than cumulus cells. When ethylenediaminetetraacetic acid or 1', 10'-phenanthroline was added to the reaction buffer during zymographic development, almost gelatinase activities were abolished, suggesting that they were MMPs. Following incubation of ECM in the presence of aminophenylmercuric acetate, an activator of proMMPs, 4 gelatinases of 240kDa, 180kDa, 97kDa, and 84kDa disappeared with the concomitant appearance of 80kDa, 65kDa, and 60kDa gelatinases. Based upon these observation, it is suggested that ECM of the human oocyte-cumulus complex consists of gelatinases, presumed to be MMP-2 and MMP-9 isoforms.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.9
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.222-231
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• 2019

Among fatty acid families, the polyunsaturated fatty acids were demonstrated to be mediators in various reproductive processes as precursor of steroid hormone (via cholesterol) and prostaglandins (via arachidonic acid), and in the last decade, major research was focused on the effects of omega-6 and especially omega-3 fatty acid. Eicosapentaenoic acid, the longest members of omega-3 fatty acid family, can be produced by a series of desaturation and elongation reactions from shorter member such as α-Linolenic acid. However, very few studies have provided detailed descriptions of Eicosapentaenoic acid effects and mechanisms of action in mammalian oocytes. The purpose of this study was to evaluate the effect of Eicosapentaenoic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of Eicosapentaenoic acid was added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, nuclear maturation rate, blastocysts quality, and levels of prostaglandin E2, 17β-estradiol, progesterone in the spent medium. High doses (100 μM) of Eicosapentaenoic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM Eicosapentaenoic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of E2/P4 also significantly increased compared with control group (p < 0.05). However, Supplementation of 100 μM Eicosapentaenoic acid showed high apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17β-estradiol/progesterone also significantly decreased compared with control group (p < 0.05). Our results indicated that supplementation with appropriate levels of Eicosapentaenoic acid beneficially affects the change of hormone synthesis for controlling oocyte maturation, leading to improved embryo quality. However, high doses of Eicosapentaenoic acid treatment results in detrimental effects.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.75-85
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• 2019

Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E₂, 17β-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E₂ synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17β-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17β-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.1-11
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• 1997

It is well known that the zona pellucidae of mouse oocytes become "hardened" when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent zona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to $ZP2_{f}$ was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to $ZP2_{f}$. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit zona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.11
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• pp.1557-1563
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• 2005

Ovarian follicular fluid contains both stimulatory and inhibitory agents that influence the growth and maturation of oocyte. In the present study, an attempt was made to isolate and study the biological properties of ovarian follicular fluid peptide(s) in buffaloes. Bubaline ovarian follicular was made steroid- and cell-free. A protein fraction was obtained by saturation (30-35% level) of the follicular fluid with ammonium sulfate. The protein fraction was purified with Sephadex-G 50 gel filtration chromatography and a single peak was obtained in the eluant volume, which was lyophilized. SDS-PAGE of the lyophilized fraction revealed a single band and the molecular weight of the peptide was 26.6 kDa. The peptide stimulated the cumulus cell expansion and in vitro maturation rate of oocytes in buffaloes in a dose dependent manner when it was incorporated at different dose levels (0, 10, 25, 50, 100 and 1,000 ng $ml^{-1}$ of maturation medium). The basic culture medium consisted of TCM 199 with Bovine serum albumin (0.3%). The in vitro maturation rates were comparable to those obtained with a positive control medium (TCM 199+20 ng EGF $ml^{-1}$+steer serum (20%)). Further purification and biological assays may throw more light on the nature and functions of this peptide.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.361-371
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• 2013

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.