• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.7
• /
• pp.972-979
• /
• 2008

It has been recently shown that expression of the LH receptor (LHR) in cumulus cells is related with FSH-induced meiotic resumption of mouse cumulus enclosed oocytes (CEOs). However, to date, it is still unclear whether LHR expression in cumulus cells plays a key role during FSH-induced oocyte maturation. The purpose of this study was to characterize the functional role of LHRs in cumulus cells. CEOs were isolated from eCG-primed preovulatory follicles and cultured in hypoxanthine (HX) arrested medium. LHR protein expression in cumulus cells was time-dependent increasing during the process of FSH-induced oocyte maturation. While the sense oligodeoxynucleotide (ODN) had no effect, antisense ODN inhibited FSH-induced LHR expression and meiotic resumption. Moreover, this antisense ODN against LHR could inhibit FSH-induced mitogen-activated protein kinase (MAPK) phosphorylation. This study suggested that LHR expression in cumulus cells is involved in FSH-induced oocyte meiotic resumption, which process is possibly regulated by MAPK cascade.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.75-75
• /
• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.2
• /
• pp.207-216
• /
• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.73-80
• /
• 1998

This study was undertaken to evaluate the interaction between cumulus cells and TGF $\beta$1 on in vitro maturation in porcine oocytes. No differ ences were found in maturation rates when follicular oocytes were cultured in medium with various concentrations of TGF $\beta$. At 24 h after maturation, the oocytes matured to metaphase-II were found in medium with TGF $\beta$ regardless of cumulus cells. On the other hand, the maturation rates were significantly(P < 0.01 higher cumulus-enclosed(70 and 52%) than cumulus-denuded oocytes(35 and 26%) in medium with or without TGF $\beta$ at 48 h after culture. In a another experiment, the same maturation rates (54-71%) were observed when cumulus-enclosed oocytes were cultured with various addition time of TGF $\beta$. However, the maturation rates in cumulus-denuded oocytes were significantly (P < 0.05) higher in medium added at 0~24 h (59%) or 24-48 h(57%) after culture than in medium with(27%) and without(38%) TGF $\beta$ for 48 h. These results indicated that cumulus cells is essential for in vitro maturation in porcine oocytes but TGF $\beta$ can promote oocytes maturation in cumulus-free oocytes.

• Korean Journal of Animal Reproduction
• /
• v.14 no.1
• /
• pp.1-8
• /
• 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).

• Korean Journal of Animal Reproduction
• /
• v.19 no.2
• /
• pp.135-140
• /
• 1995

The study was conducted to investigate on the survival rate of bisected porcine embryos co-cultured in 10% FCS(v/v)＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 72 hrs after bisection. In vitro survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos cultrued in 10% FCS＋TCM-199 media contaning PMSG, hCG, PMSG＋hCG, hCG＋$\beta$-estradiol 0 to 20 hrs and 20 to 40 hrs were 37.6% and 37.5%, 28.6% and 28.6%, 35.7% and 28.8%, 30.8% and 23.1%, 38.5% and 30.7%, respectively. The survival rate of bisected embryos co-cultured in TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells was significantly higher than that of non co-culture. 2. The survival rate of bisected porcine embryos co-cultured 10% FCS＋TCM-199 media containing oviductal epithelial cells 4 to 5 hrs and 20 to 24 hrs were 42.9% and 38.5%, respectively. 3. The survival rate of bisected porcine embryos co-cultured in 10% FCS＋TCM-199 media containing cumulus cells 4 to 5 hrs and 20 to 24 hrs were 40.0% and 35.7%, respectively.

• Korean Journal of Animal Reproduction
• /
• v.14 no.4
• /
• pp.253-261
• /
• 1990

This study was carried out to investigate the factors affecting development in vitro of follicular oocytes fertilized in vitro in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follciles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM0-199 containing 10% FCS and hormones (0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$). The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution containing caffeine(5mM) and heparin(10$\mu\textrm{g}$/ml). Twenty-four hours after insemination, the oocytes were cultured in vitro and then the effects of cumulus cell layer, co-culture with cumulus cells, bovine oviduct epithelial cells from ampulla or isthmus on development of ova, were studied. The results obtained are summarized as follows : 1. The in vitro development degree of oocytes attached with compact and dense layered cumulus cells was higher than that with 3~4 layered cumulus cells to be 9~16cells(P<0.01). 2. When the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells or cumulus cells, the development rate to be morula was 20.2% and 12.7%, respectively and the rates were higher than that of control, 2.1%(P<0.05). 3. The development rate to be morula was 15.8% and 23.8%, respectively when the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells from ampulla or isthmus, and the rates were higher than that of control, 0%(P<0.05%).

• Korean Journal of Animal Reproduction
• /
• v.17 no.4
• /
• pp.271-278
• /
• 1994

The aim of this study was to compare the two culture systems 1) co-culture with cumulus cells and 2) chemically defined medium supplemented with amino acids (CR1aa) and fetal calf serum (FCS) of in vitro produced bovine embryos from follicular oocytes in vitro. Bovine follicular oocytes were collected from ovaries of slaughtered cows and matured in TCM199 supplemented with 10% FCS and hormones (1$\mu\textrm{g}$/ml FSH-P and 1$\mu\textrm{g}$/ml oestradiol-17$\beta$)24 hours at 39$^{\circ}C$ under 5% CO2 in air. The capacitation of spermatozoa from ejaculated or frozen bull semen was induced by centrifugation through Percoll density gradient (45%, 90%). Then capacitated spermatozoa (1$\times$106/ml) were inseminated into 50${mu}ell$ droplet containing matured follicular oocytes and incubated for 40~42 hours. Cleaved embryos of 2~4cell stage were transferred to the co-culture with cumulus cells and/or CR1aa medium supplemented with FCS. In semen source, the developmental rates to the blastocyst and the hatched blastocyst stages were higher in ejaculated semen(27.6% and 14.9%) than those of frozen-thawed semen(18.3% and 11.8%), respectively. In two culture systems, the proportions of embryonic development upto the blastocysts and the hatched blastocysts were higher of CR1aa medium (22.1% and 12.1%) than those of cumulus cell co-culture (16.8% and 5.1%), respectively. The number of cells in exapnded blastocysts was slightly higher in cumulus cells co-culture (122.6$\pm$8.5) than that in CR1aa medium (117.9$\pm$5.9). The present results indicated that the early development of in vitro produced bovine embryos can be maintained efficiently in CR1aa medium as well as in co-culture with cumulus cells.

• Korean Journal of Animal Reproduction
• /
• v.20 no.3
• /
• pp.353-360
• /
• 1996

The study was conducted to determine the optimal cumulus cells, glucose and superoxide dimutase(SOD) levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for morulae and blastocyst development. Oocytes were cultured for 0~8 days in TCM-199 medium supplemented with 20% FCS, cumulus cells and with different glucose and SOD levels. The results are summarized as follows; 1. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing cumulus cells and 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 21.1, 25.0, 23.3, 17.9 and 26.3, 25.7, 23.1, 19.4% respectively and there was significant differences on the development to the molurae and blastocysts stage among the cumulus cells and glucose levels. 2. The in vitro developmental rates of bovine oocytes cultured in TCM-199 medium containing 0.1, 0.5, 1.0, 5.0mM glucose levels 0~3 and 0~8 days after insemination were 11.3~24.5% and 17.3~25.0%, respectively. 3. The in vitro developmental rates bovine oocytes cultured in TCM-199 medium containing 100, 200, 300, 500 $\mu\textrm{g}$/ml SOD levels 0~3 and 0~8 days after insemination were 12.5~22.9% and 12.9~22.2%, respectively. Hight levels of SOD(500$\mu\textrm{g}$/ml) significantly reduced the rates ofmolurae and blastocysts stage(P<0.05).

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.11-11
• /
• 2001

In this study, we examined the developmental potential of reconstructed bovine embryos and the fate of donor mitochondria during their preimplantation development after nuclear transfer. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69%(56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed among blastomeres and could be identified up to the 8- to 15-cell stages. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer These results suggest that donor mitochondria disappear from recipient cytoplasm before 16-cell stage following nuclear transfer in reconstructed bovine embryos.