• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.169-174
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• 2007

Despite many efforts to improving canine in vitro maturation(IVM), the efficiency is still low compared to that of other mammalian species. The present study investigated the effects of gonadotropin, epidermal growth factor and cysteine supplementation on in vitro maturation of canine oocytes. Cumulus-oocyte complexes (COCs) were matured in basic medium (TCM-199 containing 10% FBS, 0.11mg/ml sodium pyruvate supplemented with or without $10{\mu}g/ml$ FSH, 10ng/ml EGF and 0.57mM cysteine) for 72 hr at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After culture, oocytes were stained with Hoechst 33342 $(10{\mu}g/ml)$ for 30 min at $4^{\circ}C$, and assessed their nuclear status (GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphasse I, MII: metaphase II, UK: unknown stage). No differences were observed in GV, MI and MII rate except GVBD rate between with and without gonadotropins addition respectively (6.7%, vs. 17.2%). Supplementation of 10ng/ml of EGF in hormones added IVM medium resulted in significantly (p<0.05) higher MII rate (4.54% vs.7.06%). Although there are no significantly difference, total of MI and MII rates were increased by adding cysteine. In conclusion, the present study indicates that supplementation of $10{\mu}g/ml$ LH and FSH, 10ng/ml EGF and 0.57mM cysteine in canine IVM medium show a positive influence on the progression of maturation to MII at 72 hr.

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

• The Korean Journal of Zoology
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• v.29 no.3
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• pp.181-189
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• 1986

The present study was undertaken to investigate whether the known adenylate cyclase activators, forskolin and cholera toxin, would affect the germinal vesicle breakdown (GVBD) and the production of cAMP in mouse oocytes in vitro. To do this, in vitro oocyte culture method and adenylate cyclase assay were employed. In response to different concentrations of forskolin (20 to 80 $\\mu$g/ml) added to a culture medium, the percentage of GVBD significantly decreased (56 to 31%) in a dose-dependent manner as compared to that of control (63%). This inhibitory phenomenon by forskolin was reversible since the rate of GVBD was returned to the control level when the oocytes were transferred to a control medium following exposure to forskolin (80 $\\mu$g/ml). Treatment of cholera toxin (10 to 1, 000 ng/ml) was, however, ineffective in suppressing GVBD. When forskolin (10 to 80 $\\mu$g/ml) was added to the mouse oocyte extracts, cAMP production significantly increased by 5 to 18 fold, whereas cholera toxin (10 to 1, 000 ng/ml) was no longer effective. In addition, treatment of guanidyl-imidodiphosphate (GppNHp, 100 $\\mu$M), which is an activator of the regulatory unit of adenylate cycleas, with forskolin did not exhibit any changes in cAMP production as compared to that induced by forskolin alone. Neither cholera toxin nor cholera toxin plus GppNHp (100 $\\mu$M) exhibited any differences in mouse oocytes. From the above results, the suppression of GVBD by forskolin may be mediated by a high level of intracellular cAMP in mouse oocytes. It appears that the changes in intracellular cAMP level may an important role in the mouse oocyte maturation.

• Journal of Animal Science and Technology
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• v.57 no.8
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• pp.27.1-27.6
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• 2015

This study was designed to investigate the effects of ${\alpha}$-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of ${\alpha}$-tocopherol (0, 5, 10, $15{\mu}g/ml$), oocytes were incubated at $39^{\circ}C$ with 5 % $CO_2$ for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that ${\alpha}$-tocopherol had no effect on GVBD and MII as compared to control group, but when ${\alpha}$-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.41-47
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• 1992

1. In vitro system, LR and FSR accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus. 2. Caffeine (2mM) in the fetilization medium was required not only for inducing zona penetrating ability of boar also for developing to the male pronucleus of the penetrat- ing spermatozoa in vitro. 3. The germinal vesicle (GV)stage was observed for the first 17.6 hr;germinal vesicle break-down (GVBD)stage between 17.6~26.4 hr ;metaphase I (M-I)from 26.4 - 30. 9hr;anaphase I(A-I)ranged from 30. 9~33.4hr;telophase I(T-I) at 33.4~34.4hr; and metaphase II(M-II) at 34.4-48hr. 4. The addition of 10%(v /v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (p<0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. 5. The presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.

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

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.375-383
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• 1998

This study was conducted to investigate the effect of $\beta$-mercaptoethanol ($\beta$-ME) and cysteamine on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of $\beta$-ME for 36h, the GVBD rates were 91.6, 92.5, 91.8, 91.9 and 92.7%, respectively, and the maturation rates of the oocytes with metaphase-II were 49.6, 41.7, 32.5, 34.1 and 35.4%, respectively. Thus, lower maturation rate was shown in $\beta$-ME treated groups of 50, 100 and 200 $\mu$M as compared to control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of $\beta$-ME, the GVBD rates of porcine oocytes were 91.8, 90.4, 92.5, 91.2 and 93.9%, respectively, and the maturation rates were 71.9, 58.8, 56.7, 62.2 and 56.5% respectively. All treated groups of $\beta$-ME showed lower maturation rates than the control group (P＜0.05) 2. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of cysteamine for 36h, the GVBD rates were 90.6, 86.3, 88.2, 87.2 and 90.0%, respectively, and the maturation rates were 53.8, 45.1, 54.4, 57.5 and 63.3% respectively. Especially, the maturation rate of 200 $\mu$M treated group was significantly higher than those of control group (P＜0.05). After 44h of culture in the same treatments of cysteamine, the GVBD rates of porcine immature oocytes were 89.5, 93.1, 85.1, 89.8 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase- II were 84.2, 77.6, 66.0, 67.8 and 78.3%, respectively. Especially, the maturation rates of 50 and 100 $\mu$M treated groups were significantly lower than those of control group (P＜0.05).

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.179-191
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• 1996

In-vitro culture has provided new inforrnation on mechanisms of oocytes rnaturation and results obtained in vitro have led to new questions. In porcine, follicular and oocyte size have the crucial importance for the oocytes maturation. The addition of hormones to the culture medium was found to accelerate and facilitate meiotic maturation. The presence of some factors in serum trigger the resumption of meiosis and support the maturation of oocytes in vitro. The maturation rate of porcine oocytes was also increased by supplementation of porcine follicular fluid to the culture medium. The growth factors can stimulate nuclear maturation and enhances cytoplasnic maturation of oocytes by interaction with gonadotropins. The maturation-promoting factor brings about GVBD and the subsequent maturational events in oocytes. However, cAMP can block the spontaneous meiotic maturation of oocytes in culture. The understanding of these influences is a prerequisite to enhancing in vitro maturation of porcine oocytes.

• The Korean Journal of Zoology
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• v.30 no.1
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• pp.1-9
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• 1987

Cyclic AMP (cAMP) was known to play a key role in the regulation of cumulus expansion and oocyte maturation of mammalian cumulus-oocyte complexes (COC's) in vivo and in vitro. The present experiments were conducted to know how intracellular level of cAMP in these cells is controlled. Intracellular cAMP level was modulated by culturing mouse CGC's with an adenylate cyclase stimulator, forskolin, a phosphodiesterase inhibitor, 3-isobutyl-1-methyixanthine (IBMX), human chorionic gonadotrophin (HCG), or follicle stimulating hormone (FSH). The rate of cumulus expansion and germinal vesicle break-down (GVBD) was checked after culture and used as a biological end point. Forskolin in the medium began to stimulate the expansion of the complexes at 1 nM and induced maximum expansion (80~90%) at 0 1~10 $\mu$M. The expansion rate was reduced to 60% when forskolin concentration was increased to 100 $\mu$M. Oocyte GVBD occurred normally (75~82%) in the presence of 10 $\mu$M of forskolin, but partial suppression was appeared at 100 pM of the drug (40%). IBMX also stimulated the expansion from the concentration of 0.01 pM and induced full expansion (81~89%) between the concentration of 1-1000 $\mu$M. Meiotic resumption was occurred normally under 10 $\mu$M of IBMX, but suppressed drastically from the concentration of 100 $\mu$M. The minimum exposing time to hormone or drugs required to trigger cumulus expansion was two minutes with HCG, 15~30 minutes with FSH and fors kolin, and two hours with IBMX. The data presented here seemed to imply that intracellular cAMP level in cumulus cells is regulated by both adenylate cyclase and phosphodiesterase and cumulus expansion is induced by a peak of cAMP while meiotic arrest is maintained by continuous presence of cAMP.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.221-225
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• 2011

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide ($H_2O_2$), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decrease with addition of $H_2O_2$, and were significantly (p<0.05) lower in medium with 0.1 mM $H_2O_2$ than control group. Also, the rate of degenerated oocytes was increased in as $H_2O_2$ concentration in eased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When $H_2O_2$ concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM $H_2O_2$ and (3) control medium with 1.0 mM $H_2O_2$ along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. $H_2O_2$ decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity, against oxidative stress caused by $H_2O_2$. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM $H_2O_2$ alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.