• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.111-116
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• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.27-33
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• 1999

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.11
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• pp.1557-1563
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• 2005

Ovarian follicular fluid contains both stimulatory and inhibitory agents that influence the growth and maturation of oocyte. In the present study, an attempt was made to isolate and study the biological properties of ovarian follicular fluid peptide(s) in buffaloes. Bubaline ovarian follicular was made steroid- and cell-free. A protein fraction was obtained by saturation (30-35% level) of the follicular fluid with ammonium sulfate. The protein fraction was purified with Sephadex-G 50 gel filtration chromatography and a single peak was obtained in the eluant volume, which was lyophilized. SDS-PAGE of the lyophilized fraction revealed a single band and the molecular weight of the peptide was 26.6 kDa. The peptide stimulated the cumulus cell expansion and in vitro maturation rate of oocytes in buffaloes in a dose dependent manner when it was incorporated at different dose levels (0, 10, 25, 50, 100 and 1,000 ng $ml^{-1}$ of maturation medium). The basic culture medium consisted of TCM 199 with Bovine serum albumin (0.3%). The in vitro maturation rates were comparable to those obtained with a positive control medium (TCM 199+20 ng EGF $ml^{-1}$+steer serum (20%)). Further purification and biological assays may throw more light on the nature and functions of this peptide.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.171-178
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• 1989

These experiments were carried out to investigate the effect of a co-culture with granulosa cells on in vitro maturation, fertilization and development of bovine follicular oocytes. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of diameter 2-6mm. Bovine oocytes were matured in vitro for 24-26 hr and then fertilized in vitro using epididymal spermatozoa capacitated by preincubation for 2-3hr in BO solution containing BSA(5mg/ml) and caffein(25mM). Eight hours after insemination, the oocytes were cultured in a co-culture system with granulosa cells. The rates of maturation of the follicular oocytes cultured in a co-culture system with granulosa cells were 83.1%, the rate of fertilization of the follicular oocytes culture in a co-culture in a co-culture system with granulosa cells were 76.9%, respectively. No significant difference are observed between control and treatment in maturation and fertilization rates. The rates of embryos developed to 2-, 4-, 8-, 16-cell and monula stages after co-cultured with granulosa cells were 65.8, 57.9, 39.5, 34.2 and 34.2%, respectively. The value for 16-and morula stages were significantly higher (P<0.05) than those of the embryos cultured in the basic medium.

• Animal Bioscience
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• v.34 no.11
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• pp.1766-1775
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• 2021

Objective: The oxidative stress status and changes of chicken ovary tissue after shading were studied, to determine the mechanism of the effect of shading on follicular development. Methods: Twenty healthy laying hens (40 weeks old) with uniform body weight and the same laying rate were randomly divided into two groups (the shading group and normal light group). In the shading group, the cage was covered to reduce the light intensity inside the cage to 0 without affecting ventilation or food intake. The normal lighting group received no additional treatment. After 7 days of shading, oxidative stress related indicators and gene expression were detected. Results: Analysis of paraffin and ultrathin sections showed that apoptosis of ovarian granulosa cells (GCs) increased significantly after light shading. Enzyme linked immunosorbent assay results revealed that the levels of total antioxidant capacity, malondialdehyde, superoxide dismutase (SOD), glutathione, catalase (CAT), and other substances in the sera, livers, ovaries, and follicular GCs of laying hens increased significantly after shading for 7 days; and reactive oxygen species (ROS) levels in the livers of laying hens also increased significantly. ROS in the serum, ovarian and GCs also increased. After shading for 7 days, the levels of 8-hydroxy-2 deoxyguanosine in the sera and ovarian tissues of laying hens increased significantly. Cell counting kit-8 detection showed that the proliferation activity of GCs in layer follicles decreased after shading for 7 days; the expression level of the anti-apoptotic gene B-cell lymphoma-2 in ovarian tissue and follicular GCs was significantly reduced, and the expression levels of pro-apoptotic caspase 3 (casp3), and SOD, glutathione peroxidase 2 (GPX2), and CAT were all significantly increased. Conclusion: Oxidative stress induced by shading light has a serious inhibitory effect on follicular development during reproduction in laying hens.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1113-1116
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• 2009

The objective of this study was to compare the follicular population during spontaneous and $PGF_2{\alpha}$ induced oestrous cycles in Nili-Ravi buffaloes. In Exp.1, (n = 13 oestrous cycles) follicular population was monitored using ultrasonography on alternate days. Buffaloes were monitored for ovarian follicles from day 0 (first oestrus) until next oestrus. These animals were observed for oestrus twice daily using a teaser bull. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves during the oestrous cycle. The mean number of small, medium and large follicles among various days of the oestrous cycle between two and three waves of follicular development were not significantly different (p>0.05). In Exp. 2, follicular population 3 days before oestrus was compared in buffaloes undergoing spontaneous (n = 12 oestrous cycles) and $PGF_2{\alpha}$ induced (n = 6) luteolysis. The mean number of small and large follicles increased (p<0.05) and the number of medium follicles decreased (p<0.05) during the 3 days before oestrus in buffaloes undergoing induced luteolysis as compared to those with spontaneous luteolysis. These results showed that the mean number of small, medium and large follicles among various days of the oestrous cycle were similar between the two and three waves of follicular development, and three days before oestrous the number of small, medium and large follicles altered due to induced luteolysis on day 9, compared to those with spontaneous luteolysis.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.217-221
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• 2010

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1~2 mm, 2~6 mm and 6~10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2~6 mm follicle and 5 spots from follicle fluid of 6~10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2~6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6~10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.2
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• pp.65-69
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• 1985

1) Rabbit follicular oocytes from preovulatory follicles were cultured for 12 hr in vitro and fertilized in vivo by transferring the oocytes to the first foster-mother. 2) Two youngs were bron from transferred embryos from the first foster-mother to the second foster-mother. This demonstrates that in vitro cultured follicular oocytes are normal and they can develop into normal young born when transferred to the foster-mother. 3) A simple chemically defined culture medium, salt sol. with glutamine (2mM), which was developed by Bae and Foote(1975) proves fully good enough for rabbit follicular oocyte culture. We call this B-F medium. 4) Twelve hours culture in vitro of the rabbit follicular oocyte may be a proper culture time for further development.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.145-150
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• 2009

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) ${\sim}4$ 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 $6{\sim}8$ 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.