• Asian-Australasian Journal of Animal Sciences
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• 제29권8호
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• pp.1065-1074
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• 2016

Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-${\beta}$) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review.

• 한국가축번식학회지
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• 제14권2호
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• pp.101-106
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• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.19-29
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• 1993

Plasminogen activator (PA)-plasmin system in follicular fluid is involved in the process leading to follicular rupture at ovulation. It is well known that PA is closely associated with cellular differentiation and tissue remodeling on evidences from the study of normal and malignant tissues. This study was designed to ascertain a potential role of PA in the ovarian folliculogenesis. Immature Sprague-Dawley rats were injected with pregnant mare serum gonadotropin, followed by injection of serine protease inhibitor (SPI; mixture of 1 mol/L benzamidine and 1 mol/L amino-caproic acid) into the unilateral ovarian bursa. In the control study, mechanical effect of bursal injection and contralateral ovarian effect SPI were ruled out. Total antral follicular areas relative to total ovarian cross-sectional areas was siginificantly lower in SPI-injected ovary than in saline-injected ovary. SPI injection decreased the relative antral follicular area by 33 % respectively. Electron microscopic finding of granulosa cell in the atretic follicle showed the presence of pyknotic nucleus, blurring of neucleolemma, degeneration of mitochondria and dilation of endoplasmic reticulum. After induction of ovulation with hCG, the number of oocytes released was significantly decreased in SPI-injected oviduct than in saline-injected oviduct. From above results, author discussed that PA may play a role not only in ovulation but also in some processes of folliculogenesis.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1997년도 춘계 학술대회
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• pp.16-18
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• 1997

• 한국가축번식학회지
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• 제19권3호
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• pp.191-195
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• 1995

본 연구는 체외성숙한 소의 난포란을 ethanol로 활성화시켰을 때 cytochalasin에 노출한 시간과 농도가 난포란의 배반포단계로의 발달에 미치는 영향을 검토하였다. 30시간 체외성숙시킨 소 난포란을 7% ethanol이 든 Dulbecco's phosphate buffered saline에 7분간 노출시켜 활성화를 시킨 후, cytochalasin D가 첨가된 TCM＋199＋10% fetal calf serum에서 일정시간 배양하여 세포주기를 억제시켰다. 난포란을 활성화시킨 후, 0, 5, 10 그리고 15시간 cytochalasin D(5$\mu\textrm{g}$/ml)에 노출시켰을 때, 5시간에서 가장 높은 배반포(16%), 팽윤배반포(13%), 부화배반포(7%)로의 발달률을 보였다. 또한 cytochalasin D 0, 2.5, 5 그리고 7.5$\mu\textrm{g}$/ml에서 7시간 노출시켰을 때, 2.5$\mu\textrm{g}$/ml에서 가장 높은 배반포(13%), 팽윤배반포(7%), 부화배반포(4%)로의 발달률을 보였다. 결론적으로 cytochalasin D는 난폰란의 단위발생에 중요한 역할을 하며, 성숙한 난포란은 cytochalasin D 2.5$\mu\textrm{g}$/ml에서 5시간 노출하였을 때 가장 높은 배반포로의 발달률을 보여주었다.

• 한국가축번식학회지
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• 제13권2호
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• pp.105-112
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• 1989

These experiments were carried out to obtain the basic information for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2-6mm of diameter. Bovine oocytes were matured in vitro for 24-26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM199 supplemented with hormones, pyruvate, FBS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2-3 hours in BO solution containing bovine serum albumin(5mg/ml) and caffein(2.5mM). Insemination was made by introducing about 10-15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after inseminatin the eggs were transferred to TCM 199 supplemented with FBS(10%) for in vitro development. The results obtained in these experiments were summarized as follows : 1. The maturation rate of oocytes following incubation for 24-26 hours was 78.4%(228/291). 2. Of total 250 oocytes, 172 embryos extruded 2nd polar body following in vitro culture with spermatozoa for 20 hours, and the rates of embryos developed to 2-, 4-, 8-, 16-cells and morula or early blastocyst were 64.0, 39.2, 22.0, 15.2 and 11.2%, respectively. 3. The time needed for development to 2-, 4-. 8-, 16-cell stage and morula was 42.5$\pm$5.4, 58.0$\pm$9.2, 74.4$\pm$11.5, 96.1$\pm$13.4 and 119.0$\pm$18.2 hours, respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1239-1246
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• 2016

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development.

• Clinical and Experimental Reproductive Medicine
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• 제23권3호
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• pp.277-282
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• 1996

Serum inhibin concentrations, determimed by radioimmunoassay, were measured in women undergoing pituitary suppression with Decapeptyl and subsequently ovarian stimulation with Highly Purified-Metrodin(HP-FSH) to appraise follicular development. Early follicular basal serum inhibin level correlated with the number of oocytes retrieved(r=0.89, n=8, p<0.05). The number of oocytes retrieved showed a significant correlation with serum inhibin level on the day of hCG administration(r=0.73, n=8, p<0.05). The number of mature oocytes showed a significant correlation with serum inhibin level on the day of hCG administration(r=0.73, n=8, p<0.05). These data suggest that: (1) In the early follicular phase, basal serum inhibin may be a valid index to predict ensuing follicular growth : (2) In the preovulatory phase, maximum serum inhibin may be one of the indexes of follicular development during hyperstimulation cycles.

• 한국가축번식학회지
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• 제14권1호
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• pp.1-8
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• 1990

These experiments were carried out to investigate the effects of cumulus cells for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6 mm of diameter. Bovine oocytes were matured in vitro for 24~26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hours in BO solution containing BSA(5mg/ml) and caffeine(2.5mM). Insemination was made by introducing about 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and HEPES(25mM), cultured for 7~8 days with 10~15 eggs/well in 4-well multidishes(Nunc Co.) forming cumulus cell monolayer. The results obtained in these experiments were summarized as follows ; 1. The majority of the follicular oocytes with compacted cumulus cells existed in GV stage while those with dispersed or denuded cumulus cells existed GVBD and M II stage. 2. After 24~26 hours maturation, the maturation rates of the follicular oocytes cultured in TCM-199 containing hormones were slightly higher than those of oocytes cultured in medium without hormones, and the frequency of cumulus compacted or denuded oocytes reaching M II stage cultured in medium containing hormones was 75.7% or 51.7%, respectively(P<0.05). 3. After 20 hours in vitro insemination, percentages of ova fertilized were 61.4% or 51.4%, respectively, for cumulus oophorus intacted or removed, and increased frequency of ova with both male and female pronuclei was found when cumuli were present(P<0.05). 4. The rates of embryos developed to 2-, 4-, 8-, 16-cell and morula or blastocyst stage after cocultured with cumulus cells were 65.0%, 45.3%, 34.7%, 28.0% and 22.7%, respectively. The results for momla or blastocyst stage were significantly higher than those of the embryos cultured in the basic medium(P<0.05).

• 한국발생생물학회지:발생과생식
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• 제20권4호
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• pp.315-329
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• 2016

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-${\gamma}$) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-${\gamma}$ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-${\gamma}$ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-${\gamma}$ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-${\gamma}$ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (${\geq}1,000U/mL$) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-${\gamma}$ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-${\gamma}$ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.