• Korean Journal of Animal Reproduction
• /
• v.19 no.3
• /
• pp.191-195
• /
• 1995

Bovine follicular oocytes were matured in vitro for 3D hr and exposed to Dulbecco's phosphate buffered saline containing 7% ethanol for 7 minutes for their activation, and incubated in TCM199 containing 10% fetal calf serum (FCS) and cytochalasin D (CD). When the oocytes were exposed to cytochalasin D for 0, 5, 10 and 15 hr, 5 hr showed highest developmental rate to blastocyst (16%), expanded blastocyst (13%) and hatched blastocyst (7%). Also when the oocytes were cultured for 7 hours in medium contai ning 0, 2.5, 5 and 7.5 ${\mu}\textrm{g}$/ml cytochalasin D, 2.5 ${\mu}\textrm{g}$/ml showed highest developmental rate to blastocyst (13%), expanded blastocyst (7%) and hatched blastocyst (4%). In conclusion, cytochalasin D played very important role for parthenogenetic development of follicular oocytes and 5 hr of exposure time to CD and 2.5 ${\mu}\textrm{g}$/ml CD in the medium was optimal for development of the follicular oocyte.

• Korean Journal of Agricultural Science
• /
• v.35 no.2
• /
• pp.167-176
• /
• 2008

Supplementation of serum and estrogen in in vitro maturation(IVM) medium was shown to improve embryo development and quality in several species. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with canine serum or estrogen on IVM of canine oocytes. As results, in experimental 1, IVM oocytes collected from follicular stage ovaries to MII stages($10.2{\pm}1.5%$) was higher (p<0.05) with 10% canine estrus stage serum than control($1.3{\pm}1.6%$), anoestrus stage serum($4.0{\pm}1.6%$), luteal stage serum($2.7{\pm}1.7%$) and 10% FBS($1.3{\pm}1.6$). In experimental 2, 10% canine estrus stage serum supplementation has highest maturation rate to MII stages($10.0{\pm}1.8%$) and there were significant differences(P<0.05) with another treatment in follicular stages group. In order to investigate the synergic effect of estrous serum and estrogen supplementation, different estrous stage groups of oocytes were cultured with 2 ug/ml estrogen plus various concentrations of different reproductive stage serum and FBS(experimental 3). As results, the rate of maturation to metaphase II(MII) stage was significantly higher(p<0.05) in oocytes from the follicular stage supplemented with estrogen and 10% canine estrus stage serum(11.5%) compared to the other groups(6.0 - 8.8%). The present study was demonstrated that canine serum and the estrus cycle of the bitch affect the meiotic competence of oocytes. Hormonal influences within the follicle may be one of the factors responsible for the greater proportion of maturation of oocyte to MII from bitches at the follicular phase.

• Clinical and Experimental Reproductive Medicine
• /
• v.48 no.4
• /
• pp.347-351
• /
• 2021

Objective: We investigated the impact of vitamin D₃ (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor (VDR) in mouse ovarian follicles at different stages. Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0, 25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcriptase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR. Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages. Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed.

• Reproductive and Developmental Biology
• /
• v.29 no.2
• /
• pp.121-125
• /
• 2005

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as $<100{\mu}m,\;<100{\mu}m\;to\;<110{\mu}m,\;<110{\mu}m,\;to\;<120{\mu}m,\;>120{\mu}m,$. Oocytes were cultured in culture medium supplemented with $10\%\;FBS,\;0.4\%\;BSA,\;10\%$ porcine follicular fluid (pFF), $10\%$ canine serum (CS), or $10\%$ canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes $(>120{\mu}m)$ was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of $>110{\mu}m$ groups was significantly higher than that in $<100{\mu}m$ group (p<0.05). The oocytes cultured in $10\%$ FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in $10\%$ FBS-, $0.4\%$ BSA-, and $10\%$ pFF-supplemented groups were significantly higher than those in $10\%$ CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

• Korean Journal of Animal Reproduction
• /
• v.26 no.2
• /
• pp.165-171
• /
• 2002

Follicular fluid (FF) contains an oocyte maturation inhibitor with unknown chemical properties. This study was carried out to chemically define the factor(s) inhibiting cumulus cell denudation (CD) and first polar body formation (PBF). Porcine FF (PFF) was extracted with methanol and the extract was serially separated using gel filtration on Superose 12 and Superdex columns. A Superdex fraction was derived with PITC and analyzed with an amino acid analysis column. The results obtained are as follows; PFF had an activity inhibiting both CD and PBF of porcine primary oocytes. Superdex fractions RV2.11 prepared from PFF exhibited an activity inhibiting CD and PBF. By amino acid analysis, the fraction RV2.11 appeared to be proline having the same activity inhibiting CD and PBF. In conclusion, PFF had oocyte maturation inhibitors, of which proline should inhibit CD and PBF.

• Proceedings of the Zoological Society Korea Conference
• /
• 2000.10a
• /
• pp.190.1-190
• /
• 2000

No Abstract, See Full Text

• Korean Journal of Animal Reproduction
• /
• v.15 no.3
• /
• pp.207-220
• /
• 1991

This study was carried out to investigate the ultrastructural changes of ooplasm and follicular membrane of oocytes, obtained from 150 of 3-year-old female rainbow trout(Oncorhynchus mykiss). All data were collected from March in 1989 to February in 1990, and from August to October in 1991. The size of the nucleoli and number of the yolk granules increased as the oocyte growed. Yolk granules were deposited in the oocyte as crystalline granules. Due to the presence of large early and late maturing oocytes, their ovaires were enlarged, transparent and granular. The lattice was broken down at hydration, leaving the egg transparent. As thepercentages of fish in LPO and EMO stage increased from September to October, Mean GSI values increased. Follicle cells such as granulosa cell and thecal cell change a squamous into cuboid shape in LPO and EMO stage. Seasonal changes in the microscopic appearance of the ovaries were well correlated with those in bothgonadosomatic index and macroscopic apearance. Under the natural conditions,t he ovarian follicle influences the histological development and periodical secretion of the hormones, sufficient for a oogenesis and gonadal steroid production. The electrophoretic pattern of major band in mature stage was much thicker(70∼110k dalton) than that in previtellogenic phase.

• Korean Journal of Animal Reproduction
• /
• v.22 no.3
• /
• pp.253-264
• /
• 1998

Porcine follicular oocyte, collected from antral follicles (2~5 mm in diameter) of gilt ovaries were matured in vitro porcine follicular fluid (pFF) with PMSG (pFF-PMSG) buffer with at 37$^{\circ}C$ under 5% CO2 in air their ability of maturation promoting factor (MPF), of GV and GVBD formation was examined followed during time after in vitro culture. Formation of second metaphase was observed in 57.6% and 71.2% of matured in with pFF-PMSG buffer to 45 and 50 hours after invitro. Porcine oocytes cultured in pFF-PMSG for various periods of up to 30 hours were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in pFF-PMSG buffer in vitro. In oocytes matured in pFF-PMSG, H1K activity was at the 30 hours after culture and increased about 15 fold than at the germinal vesicle stage with before at the cultured in vitro. This pattern is similar to those reported in non-mammalian species and su, pp.rts the concepts that H1K is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals. These results suggest that the maturation pFF-PMSG buffer used influences the fluctuation pattern of H1K activity and biological characteristics of porcine oocytes cultured in vitro.

• Clinical and Experimental Reproductive Medicine
• /
• v.8 no.1
• /
• pp.1-12
• /
• 1981

It has been already suggested that specific macromolecules in follicular fluid produced by granulosa cells may play a role in suppressing further meiotic maturation of the oocytes. In general, the search for specific macromolecules in follicular fluid using immunological methods has not been rewarding. These studies were designed, by applying more effective immunological methods than conventionally employed, (l) to identify whether some unknown macromolecules are present in the porcine follicular fluid or not, and (2) to clarify the relationship between the oocytes and the specific macromolecules in the follicular fluid. The results obtained were as follows; (1) porcine follicular fluid contained two specific proteins, which were not present in pig plasma and serum. (2) each of two proteins showed electrophoretically fast alpha-globulin and beta-globulin mobilities. (3) these proteins seemed to have inhibitory effect on the maturation of mouse oocytes in vitro. From these results, it can be assumed that pig follicular fluid contains specific proteins which seem to be intra-follicular inhibitor(s) of oocyte maturation.

• Journal of Embryo Transfer
• /
• v.12 no.2
• /
• pp.181-188
• /
• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.