• Korean Journal of Animal Reproduction
• /
• v.13 no.2
• /
• pp.70-78
• /
• 1989

Mouse eggs recovered from oviducts at one hourly intervals between 10 and 20 hours after administration of hCG were fixed, stained and then investigated the rate of in vitro fertilization and nuclear maturation. In case of out- bred ICR mice, ovulations were occured between 11 and 13 hours after hCG injection. The stages of in vitro maturation of eggs recovered from female mice at various times after hCG injection were metaphase I, anaphase I, telophase I and metaphase II. However the majority was metaphase I(17.6 to 44.4%) and metaphase II(42.9 to 80.0%) stage. When the eggs were inseminated with epididymal spermatozoa, the fertilization rate was declined as the egg recovery time after hCG administration was delayed. That is, the proportion of eggs undergoing fertilization became higher(68.1 to 77.4%) in the eggs at 12 to 15hr after injection of hCG than those(17.5 to 56.4) at 16 to 20 hr after injection of hCG. Also, when nuclear maturation of the unfertilized eggs were observed at 8 hours after insemination, the majority was in metaphase I and metaphase II and no anaphase I and telophase I were observed.

• Reproductive and Developmental Biology
• /
• v.28 no.4
• /
• pp.235-240
• /
• 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
• /
• v.37 no.4
• /
• pp.193-197
• /
• 2013

In order to investigate the effects of antioxidants on the culture of mouse preantral follicles in vitro, we examined the effects of taurine, glutathione and catalase on their growth and maturation. Addition of taurine was not effective on the survival of preantral follicles. However, metaphase II rates of oocytes within preantral follicles were significantly higher in 1 mM treated group than in control and 10 mM treated group (p<0.05). Glutathione did not improved the rates of survival and metaphase II oocytes. However, metaphase II rates of oocytes progressively decreased with increasing glutathione concentration. Catalase also showed that the rates of survival and metaphase II oocytes progressively decreased with increasing concentration. Especially, all of preantral follicles cultured in medium containing 100 IU/ml catalase were degenerated. These results suggest that low concentraion of taurine, as an antioxidant, have positive effect on the culture of mouse preantral follicles in vitro.

• Korean Journal of Animal Reproduction
• /
• v.24 no.4
• /
• pp.375-383
• /
• 2000

The nuclear maturation and developmental competence of immature, oocytes collected from donors at various morphology of corpus luteum (CL) and fertilized in vitro was investigated by comparing the meiotic activity and the yields of embryos. Ovaries were divided and classified into 4 groups as the following criteria : Group 1 ; ovaries showed evidence of recent ovulation (corpus hemorragicum). Group 2 ; apex of CL was red or brown. Vasculization was limited to periphery of CL. Group 3 ; apex of CL was orange or tan. Vasculization was covered over apex of CL. Group 4 ; CL was light yellow to white and firm in texture and the vascular network on the surface of CL had disappeared. Modified TCM 199 was used for maturation in vitro of immature oocytes and development was induced by using TLP-PVA as a basic medium. When oocytes collected from each group of donors had been matured for 4, 14, and 24 hours in vitro, the proportion of oocytes reaching metaphase I and metaphase II were not different among oocytes from 4 group of ovaries. Mature metaphase II stage of oocytes in each group was first observed at 14 hours, whereas completion of maturation of. oocytes in each group was at 24 hours. Luteal morphology of ovaries had little effect on the proportion of embryos reached 2 cells and 8 cell stage. However, the proportion of embryos cleaved to morula and blastocyst stage was significantly higher in the oocytes obtained from group 1 and 3 than in the oocytes from group 2 and 4 (p＜0.05). This data suggest that reproductive status of the donor significantly influence the yield of in vitro embryos.

• Reproductive and Developmental Biology
• /
• v.31 no.2
• /
• pp.71-76
• /
• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• Reproductive and Developmental Biology
• /
• v.30 no.4
• /
• pp.301-305
• /
• 2006

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48hr of 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 (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%, 51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.11 no.6
• /
• pp.2124-2128
• /
• 2010

In this study, we determined effects of the mitogen-activated protein kinase (MAPK) inhibitor, U0126 on meiotic maturation, microtubule organization and actin filament assembly in the porcine oocyte. The phosphorylated MAPK was first detected at 12 h after the initiation of maturation cultures, fully activated at 24h, and remained until metaphase II. Treatment of germinal vesicle (GV) stage oocytes with $20{\mu}M$ U0126 completely blocked MAPK phosphorylation, but germinal vesicle breakdown (GVBD) was normally proceeded. However, the oocytes didn‘t progress to the metaphase I. The inhibition of MAPK resulted in abnormal spindles. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, MAPK activity plays an important regulatory role in GV chromatin configuration and meiotic progress in porcine oocyte maturation.

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

• Journal of Embryo Transfer
• /
• v.19 no.3
• /
• pp.283-289
• /
• 2004

This study was conducted to determine the effects of incubation time after cooling on mouse meiotic spindle and chromosome alignment and the optimal incubation time for their restoration. Oocytes at the metaphase II were obtained from superovulated mice. Control oocytes were held at 37$^{\circ}C$ during the experiment. Oocytes were rapidly cooled to $0^{\circ}C$, held for 30 minutes, warmed and incubated at 37$^{\circ}C$ for 5, 15, 30, 60 and 120 minutes, respectively. The morphological features of spindle and chromosomes in oocytes were evaluated by immunofluorescent staining. Meiotic spindle of control oocytes exhibited a normal-looking bipolar configuration(barrel-shaped) and highly fluorescent microtubles. The chromosomes were clustered in a discrete bundles at metaphase plate. Disassembly of meiotic spindle and chromosome dispersion were occurred immediately after chilling of oocyte. Fluorescence intensity index(FIS), normal chromosomes aligned and normal spindle configuration were compared according to incubation time at 37$^{\circ}C$. Restoration of a barrel-shaped spindle and normal chromosome alignment was occurring after 5 minutes incubation at 37$^{\circ}C$, improved as a incubation time increased, and decreased gradually after 120 minutes incubation(P<0.05). The optimal incubation time for restoration of meiotic spindle and chromosomes in cooled oocytes was 60 minutes.

• Reproductive and Developmental Biology
• /
• v.32 no.3
• /
• pp.147-152
• /
• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.