The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% CO2, 95% air, 38℃. The in vitro maturation rate to MⅡ stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44～72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, OC and COCs. Activity of MMP-2 in the OC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the COCs 36 hr. Expression of TIMP-3 protein in the COCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

The aim of this study is to investigate the effect of porcine epididymal fluid (pEF) on in vitro-maturation and subsequent fertilization of porcine follicular oocytes. Porcine cumulus-oocytes complexes retrieved from antral follicles were cultured in tissue culture medium (TCM)-199 supplemented with pEF of different concentrations. At 48 h after culture, development of oocytes to germinal vesicle (GV) breakdown, metaphase I, anaphase-telophase I, and metaphase II were examined. Significant (p<0.05) increase in the proportion of oocytes developed to MII stage was observed in oocytes cultured in pEF-containing TCM-199 than in oocytes cultured in pEF-free TCM-199 (46.2% vs 16.7%), which was a dose-dependent manner. Subsequently, the proportion of monospermic fertilization were significantly (p<0.05) increased in oocytes cultured in the TCM supplemented with pEF than those cultured in pEF-free TCM-199 (51.0% vs 24.1%). In the second series of experiment, the percentage of MII oocytes was significantly (p<0.05) increased after exposure of oocytes to pEF during the first 22 h period of culture than after exposure of oocytes to pEF during the next 24 h of culture, while no significant difference in the percentage of monospermy was observed. The results of this study demonstrate that pEF contains at least enhancing component(s) for nuclear maturation.

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

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'C and 97'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'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.

This study was carried out to investigate the effects of the supplementation of glutamine, glucosamine and glutathione on the porcine oocytes on IVM rates. Cocs were incubated in NCSU-23 supplemented with at glucosamine, glutamine and glutathione for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered with mineral oil and cultured in a incubator (, 5% , 95% air). The IVM rates of oocytes cultured in NCSU-23 supplemented with 0.5, 1.0, 2.0 and 4.0 mM glutamine for 48 hrs were , , and , respectively. The IVM rates of oocytes cultured in NCSU-23 supplement with 2.0, 5.0, 7.0, 10.0 mM glucosamine for 48 hrs were , , and , respectively. The IVM rates of oocytes cultured in NCSU-23 supplemented with glucosamine were no significantly increased compare to the control (). The IVM rate of oocytes cultured in NCSU-23 supplemented with 3.0, 5.0, 7.0, 10.0 mM glutathione for 48 hrs were , , , , respectively. The IVM rate of oocytes cultured in NCSU-23 supplemented with glutamine and glutathione were significantly increased co~pared to those control (). Glucosamine did not affect the IVM rates of oocytes. IVM rates of oocytes cultured in NCSU-23 medium for 48 hrs were significantly increased compared to the cultured for 40 hrs.

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) is one of the recently developed proteomic technologies which is based on capturing proteins and peptides by chemically modified surfaces and highly sensitive for the analysis of complex biological samples. In the present study, to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in porcine oocytes at the in vitro matured metaphase II (MIIl) and germinal vesicle (GV) stages. By selected CM10 chip, 16 candidates were found to be up-regulated in GV stage oocytes compared with in MII stage oocytes, their molecular weights were 8,180 (2 candidates), 10,226 (5 candidates), 15,767 (5 candidates) and 16,770 (4 candidates) Da respectively. And the expression of 29 candidates were higher in MII than in GV stage oocytes, their molecular weight were 10,832 (3 candidates), 17,743 (8 candidates), 20,122 (3 candidates), 22,131 (3 candidates), 24,857 (7 candidates) and 33,507 (5 candidates) Da, respectively. The expression of selected 13 candidates (0.2 and 1.0 % error tolerances) were analyzed using real time RT-PCR. The proteins that differentially regulated during oocyte in vitro maturation in the pigs may be potential biomarkers of oocyte maturation and quality.

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional h. Control oocytes were denuded after h of IVM. The size of the perivitelline space was larger at 40 h of IVM () than at 30 h ( p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ () and D- oocytes () than in control oocytes ( p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.