• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.223-227
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• 2005

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture $43.8\%$ of oocytes cultured in only TCM 199 remained at GV stage whereas $56.2\%$ of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than $90\%$ of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.7
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• pp.972-979
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• 2008

It has been recently shown that expression of the LH receptor (LHR) in cumulus cells is related with FSH-induced meiotic resumption of mouse cumulus enclosed oocytes (CEOs). However, to date, it is still unclear whether LHR expression in cumulus cells plays a key role during FSH-induced oocyte maturation. The purpose of this study was to characterize the functional role of LHRs in cumulus cells. CEOs were isolated from eCG-primed preovulatory follicles and cultured in hypoxanthine (HX) arrested medium. LHR protein expression in cumulus cells was time-dependent increasing during the process of FSH-induced oocyte maturation. While the sense oligodeoxynucleotide (ODN) had no effect, antisense ODN inhibited FSH-induced LHR expression and meiotic resumption. Moreover, this antisense ODN against LHR could inhibit FSH-induced mitogen-activated protein kinase (MAPK) phosphorylation. This study suggested that LHR expression in cumulus cells is involved in FSH-induced oocyte meiotic resumption, which process is possibly regulated by MAPK cascade.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.105-116
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• 1992

The present study was examined to clarify the role of calcium ion as a factor for the maturation of mouse oocytes. Follicles and cumulus-enclosed oocytes were isolated with two sharp needles under a stereomicroscope from female mouse (ICR) ovaries which were treated PMSG 5 IU 45-46 hours previously. Isolated follicles and cumulus-enclosed oocytes were cultured for 14-16 hours in an organ culture system at $37^{\circ}C$, 5% $CO_2$ in air and 100% humudified in incubator. MHBS was the basic medium used from which A23187, verapamil, $NiCl_{2.}$ $6H_2O$ and $LaCl_{3.}$ $7H_2O$ were added depending on the experimental groups. In follicle- or cumulus-enclosed oocytes wre cultured in these differently treated media. Following results were obtained from the present study. 1. The calcium ionophore A23187 directly or indirectly seems to stimulate GVBD of follicle-enclosed mouse oocytes. Increasing concentration of ionophore A23187 1ed to an increase in oocytes degeneration from the cumulus-enclosed mouse oocytes. 2. The organic $Ca^{++}$ channel blocker, verapamil does not induce GVBD of follicle-enclosed mouse oocytes. Specially, higher dose of 1 mM verapamil induced GVBD of follicle-enclosed mouse oocytes. However, cytoplasm of GVBD oocytes in 1 mM verapamil treated groups appeared shrunk. In the cumulus-enclosed oocytes, polar body formation was reduced in verapamil treated groups and degeneration increased. Verapamil inhibit oocyte maturation (polar body formation). 3. The $Ca^{++}$ inhibitor, Nickel ($NiCl_{2.}$ $6H_2O$) inhibits maturation of the follicle-enclosed oocytes. In the cumulus-enclosed oocytes the progression to MII (PB formation) was reduced and degeneration of mouse oocytes increased as the concentration of $Ni^{++}$ increase. The results indicates that nickel act as an inhibitor of calcium. 4. The $Ca^{++}$ inhibitors, Lanthanum ($LaCl_{3.}$ $7H_2O$) has shown different effect from that of nickel. In follicle-enclosed oocytes, 0.01mM lanthanum induced maturation of mouse oocytes. Polar body formation was reduced in the cumulus-enclosed oocytes all lanthanum treated group.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.191-200
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• 1994

The influence of hypoxanthine and ovarian steroids on the meiotic maturation process of mouse oocytes was investigated for the qualified application of culture medium in in vitro fertilization(IVF). Mouse oocytes were cultured in hypoxanthine and various ovarian steroids(progesterone, estradiol-17${\beta}$ and testosterone) and their effects on the oocyte maturation had been observed. When mouse oocytes were cultured in the various concentration(1-4mM) of hypoxanthine, meiotic maturation of cumulus cell-enclosed oocytes was inhibited by presence itself, which was a dose-dependent effect in meiotic arrest of mouse oocytes. The presence of progesterone, estradiol-17${\beta}$ and testosterone have made the mouse oocyte mature properly. Meanwhile maturation of cumulus cell-enclosed oocyte was severely inhibited by 3 hoursculture in the media of progesterone supplemented with hypoxanthine. However the continuous presence lasting 24 hours of progesterone even supplemented with hypoxanthine had got rid of the inhibition of oocytes maturation. Not only estradiol-17${\beta}$ supplemented with hypoxanthine but also testosterone supplemented with hypoxanthine exert the severe inhibition of the maturation of cumulus cell-enclosed oocytes for 3-hours culture. However the continuous presence lasting 24 hours of estradiol-17${\beta}$ and testosterone even supplemented with· hypoxanthine had relieved the inhibition of oocytes maturation. These results make us suggest that hypoxanthine inhibits the mouse oocyte maturation, particularly markedly in conjunction with ovarian steroids for short period, which indicated some sort of the synergistic inhibitory retationship between the ovarian steroids and hypoxanthine.

• Development and Reproduction
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• v.20 no.4
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• pp.359-365
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• 2016

Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

• Development and Reproduction
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• v.17 no.2
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• pp.141-147
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• 2013

Keeping the intact germinal vesicle (GV) is essential for maintaining the capacity of mammals including human. It is maintained by very complex procedures along with folliculogenesis and is a critical step for getting competent oocyte. So far, a few mechanisms involved in folliculogenesis are known but GV arrest mechanisms are largely unrevealed. Cyclic AMP, a adenosine derived substance, have been used as inhibitor of germinal vesicle breakdown as a putative oocyte maturation inhibitor. In this study, we examined the potency of adenosine as GV maintainer and a possible signaling mediator for that. A1, A2b, and A3 were detected in cumulus cells of cumulus enclosed-oocyte (CEO). Intact of germinal vesicle was not kept like in follicle but the spontaneous maturation was inhibited by exogenous adenosine. It is inhibited with concentration dependent manners. Intracellular calcium level of cumulus was extensively increased after adenosine treatment. Based on these results it is suggested that one of the pathway for GV arrest by adenosine and its receptors is calcium mediated signaling pathway in CEO.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.235-240
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• 2004

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.95-99
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• 2004

This study was carried out to assess whether the addition of myo-inositol to maturation medium could improve porcine oocyte maturation in vitro. Oocytes were cultured for the first 22 h in Witten's medium containing 10IU/$m\ell$ PMSG, 10 IU/$m\ell$ HCG supplemented with or without myo-inositol. Subsequently, they were cultured for additional 22 h in Witten's medium without hormone supplemented with or without myo-inositol. When the porcine oocytes were cultured in maturation medium containing myo-inositol, the proportion of metaphase II oocytes 44h after culture was higher in the myo-inositol group(P<0.05). To study effects of cumulus cell on the maturation induced by myo-inositol, we examined the maturation status of cumulus-enclosed or cumulus-denuded porcine follicular oocytes. The rates of maturation were significantly higher in the cumulus-enclosed oocytes(P<0.05). However, the maturation rates of cumulus-denuded oocytes cultured in medium containing myo-inositol were higher than those of control group(P<0.05). Our results suggest that myo-inositol may affect meiotic progression of porcine follicular oocytes and supplementation of myo-inositol in maturation medium may be useful for the in vitro maturation of porcine follicular oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.1-7
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• 1989

배양중인 생쥐 난자의 성숙에 미치는 배양액 내 calcim의 영향을 알아보기 위하여 1.71mM $Ca^{2+}$을 처리한 배양액과 $Ca^{2+}$이 존재하지 않는 배양액에서 난자를 배양하여 불꽃 원자 흡수 분광 광도계를 이용하여 배양된 난자의 $Ca^{2+}$농도를 측정하였다. 1) $Ca^{2+}$처리한 배양액에서 배양된 cumulus-cell이 제거된 (denuded oocyte)난자들은 시간이 지남에 따라 $Ca^{2+}$농도가 높게 나타났고, 2) $Ca^{2+}$처리하지 않은 배양액에서는 denuded난자는 3시간째 배양될때 부터 $Ca^{2+}$양이 줄어 들었다. Cumulus-enclosed난자는 $Ca^{2+}$존재하에서는 GVBD가 일어났다고 생각되는 4시간까지 계속 증가를 보인 반면 $Ca^{2+}-free$에서는 배양되지 않은 난자와 거의 차이가 없게 나타났다. 3) 핵막붕괴가 일어난 후부터 15시간까지 배양시컸을 때에는 CEO와 denuded oocyte에서 공히 $Ca^{2+}$의 농도는 다시 증가된 상태로 계속 되었다. 이런 결과로 미루어 보아 난자 성숙시 부터 성숙과정이 끝날때가지 exteral $Ca^{2+}$이 요구되고 있음을 증명해 주고있다. 그러나 이러한 세포질내의 $Ca^{2+}$및 bound calcium이 난자 성숙시부터 어떤 역활을 하고 있는 기작에 대해서는 좀 더 연구가 있어야겠다.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.3
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• pp.317-324
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• 2004

The present study was conducted to investigate the involvement of nitric oxide (NO) in cumulus expansion, oocyte mortality and meiotic maturation of porcine cumulus enclosed oocytes (CEOs) cultured in two different models when gonadotropins, including follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) were presented or not. And the interaction between NO and $\beta$-mercaptoethanol ($\beta$-ME), a free radical scavenger was also investigated. Two models refer to spontaneous maturation model and hypoxanthine (HX) medium model. All the 3,433 eligible CEOs were incubated at $39^{\circ}C$ and the cumulus expansion, oocyte morphology and nuclear phase were evaluated 44 h after incubation. (1) In spontaneous maturation model, NO stimulates the cumulus expansion and $\beta$-ME delayed it. NO doesn't affect the oocyte meiotic resumption but inhibits the oocytes to develop to metaphase II. (2) In HX medium model, NO or $\beta$-ME doesn't affect the expansion in the absence of gonadotropins, but in the presence of gonadotropins, NO or $\beta$-ME inhibits the expansion. In the presence of gonadotropins, NO inhibits the oocyte meiotic resumption and it especially inhibits the oocyte to develop to metaphase II, and $\beta$-ME reverses such inhibitory effects. The cooperation of gonadotropins and $\beta$-ME stimulates the meiotic resumption and especially, promotes the CEOs to develop to metaphase II in both models. Moreover, HX might contribute to the fragility of oocyte zona pellucida and gonadotropins, nitric oxide and $\beta$-ME could alleviate it separately, and cooperatively. It is concluded that NO exerts different functions in two models and $\beta$-ME affected the functions of NO in different models.