• Journal of Embryo Transfer
• /
• v.26 no.4
• /
• pp.251-256
• /
• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• Journal of Embryo Transfer
• /
• v.18 no.3
• /
• pp.263-267
• /
• 2003

This study was carried out to test the efficacy of pharmacological inhibitors of the cell cycle transition in keeping bovine oocytes at the germinal vesicle(GV) stage and the reversibility of this inhibition. Bovine oocytes were incubated for 22∼24 hrs in the presence of various inhibitors : cycloheximide (2$\mu\textrm{g}$/$m\ell$), 6-DMAP (2 mM), and roscovitine (50$\mu$M). Bovine oocytes cultured with any of the inhibitors were significantly blocked at the GV stage. Reversibility of pharmacological inhibitors was assessed by culturing oocytes an additional 22∼24 hours in inhibitor-free medium. Examination of oocytes revealed that the inhibitory effect was fully reversible and effect of resuming meiotic progression on nuclear maturation varied according to the various inhibitors. This study suggests that cycloheximide, 6-DMAP and roscovitine can be applied to control meiotic arrest and resumption in maturation culture of bovine oocytes in vitro. More investigations are needed to better understand how the cell cycle of oocyte is blocked without problems to future developmental competence.

• The Korean Journal of Zoology
• /
• v.19 no.1
• /
• pp.1-6
• /
• 1976

The glycogen content of the oocytes at the various stages of meiotic division induced during culture was determined by a microspectrophotometer. The PAS intensity decreased gradually as the meiotic resumption progressed. The amount of glycongen was also decreased in the degenerated ova. Is is concluded that the glycogen consumption in necessary for the meiotic resumption and that the glycogen loss while the germinal vesicle is intact seems to lead degeneration. These results suggest that the endogenous glycogen is important to support meiosis.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.72-72
• /
• 2002

Oocytes maturation, characterized by germinal vesicle (GV) breakdown, formation of the first meiotic spindle, expulsion of the first polar body and arrest in metaphase of second meiotic division (MII), occurs in preovulatory follicles in response to the surge of gonadotropin and leads to an ovulated oocyte in vivo. However, meiotic resumption in vitro occurs spontaneously following removal of cumulus-oocytes complexes (COCs) from the follicle. (omitted)

• Clinical and Experimental Reproductive Medicine
• /
• v.36 no.4
• /
• pp.231-236
• /
• 2009

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.7
• /
• pp.972-979
• /
• 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
• /
• v.24 no.1
• /
• pp.1-11
• /
• 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.

• Clinical and Experimental Reproductive Medicine
• /
• v.23 no.1
• /
• pp.87-94
• /
• 1996

For evaluating the suitability of human follicular fluid(HFF) as protein supplement in ART, this preliminary study was performed to examine the maturation promoting activity of HFF on mouse follicular oocytes in vitro. Mouse follicular oocytes were collected from ovaries of 21-28 day old ICR mice by puncturing the antral follicles with fine needle at 48 hours after PMSG injection. The oocytes were rinsed and cultured in modified Whittingham's $T_6$ medium containing purines or dbcAMP to maintain meiotic arrest, and different concentrations of HFF were added into the culture medium to examine the effect of HFF on releasing the oocytes from the suppressive influence of the meiotic inhibitors. As a control for HFF, the maturation promoting activity of human fetal cord serum(HFCS) was investigated and compared with the activity of HFF. While HFF was separated, by molecular weight(M.W), into high M.W. fraction(M.W>30,000) and low M.W. fraction(M.W<30,000) and the effects of the fractions on meiotic resumption were investigated in the presence of the meiotic inhibitors. Also hormone analysis was performed to compare the content of hormones in HFF with that in HFCS. Same concentrations of HFF and HFCS induced similar germinal vesicle break down(GVBD) rates of the oocytes meiotic arrested by purines(4mM hypoxanthine+0.75mM adenosine), but the extrusion rate of 1st polar body(PB) of the oocytes cultured in HFF(65.0%, P<0.05) was significantly higher than that(51.6%) in HFCS. While, in the presence of 200 M dbcAMP, the maturation promoting activity of HFF (GVBD: 70.5%, $p<10^{-6}$; 1st PB extrusion: 67.1%, $p<10^{-3}$) was significantly greater than that of HFCS(GVBD: 35.2%; 1st PB extrusion: 41.1%). The oocytes cultured in the fraction of HFF containing high M.W. components showed higher meiotic maturation rates than the oocytes cultured in the low M.W. fraction of HFF. Gonadotropins and $E_2$ were known to improve the completion of maturation changes, and the levels of these hormones were higher in HFF than in HFCS. Therefore, HFF was more effective than HFCS to use for promoting meiotic resumption of mouse oocytes in vitro.

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

• Korean Journal of Animal Reproduction
• /
• v.20 no.4
• /
• pp.427-432
• /
• 1997

These studies were performed to approach the precise pathway inducing the meiotic inhibitory action of hypoxanthine on mouse follicular oocytes and to identify the cause of detrimental effect of hypoxanthine on viability of the oocyte in vitro. In addition, a correlation between the meiotic inhibitory effect and the detrimental effect of hypoxanthine was investigated. Mouse follicular oocytes at germinal vesicle(GV) stage were collected from the ovaries of ICR mice by puncturing the antral follicles with a fine needle, at 48 hours after PMSG injection. Oocytes were cultured in Modified Whittingham's T6 media containing hypoxanthine and several materials that involved in metabolism of hypoxanthine, and the effects of the materials on the actions of hypoxanthine were investigated by observing germinal vesicle breake down (GVBD), 1st polar body (PB) extrusion and viability of the oocytes. Phophodiesterase significantly reduced the meiotic inhibitory effect of dbcAMP but did not influence on the inhibitory effect of hypoxanthine. Allopurinol and 6-MP significantly enhanced the meiotic inhibitory effect of hypoxanthine, but the materials themselves also showed the meiotic inhibitory action like hypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase significantly enhanced the meiotic inhibitory effect of hypoxanthine, on the contrary HGPRT itself promoted meiotic resumption of the oocytes. Catalase did not induce any change in the meiotic inhibitory effect of hypoxanthine, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD did not reduce the deterimental effect of hypoxanthine. In conclusion, the meiotic inhibtory effect of hypoxanthine may be caused by guanyl dervartives converted from hypoxanthine via salvage pathway, and superoxide anion may partially participate in the inhibitory effect of hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes be cused by hydrogen peroxide produced during the metabolism of hypoxanthine.