Developmental Capacity of Mouse Oocytes within Preantral Follicles Cultured in Medium Supplemented with Gonadotrophins

Kim, D.H.,Kang, H.G.,Kim, M.K.,Han, S.W.,Chi, H.J.,Lee, H.J.,Lee, H.T.,Chung, K.S. 韓國家畜繁殖學會 2000 Reproductive & developmental biology Vol.24 No.4

본 연구는 다양한 농도의 FSH 와 LH 에서 배양된 생쥐 preantral follicles내 난자의 발생능력을 조사하고, 이러한 조건에서 배양된 난자-난구세포 복합체에서 황체화의 지표인 cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc)와 퇴행화의 지표인 cytochrome P450 17α-hydroxylase (P450_(17α)) mRNA의 발현정도를 조사하고, 또한 progesterone과 testosterone의 분비농도를 살펴보기 위하여 실시하였다. 체외성장된 난자의 배반포까지의 발달능력은 100 mIU/ml FSH 단독첨가군 (30.2%)과 100 mIU/ml FSH+10mIU/ml LH 첨가군 (28.0%)이 100mIU/ml FSH+100mIU/ml LH 첨가군 (22.0%)보다 높은 결과를 나타냈다. 그리고 배반포의 평균 세포수에 있어서도 FSH 단독첨가군 (50.9±26.l)과 100mIU/ml FSH+10 mIU/ml LH 첨가군 (51.0±26.1)이 100mIU/ml FSH+100mIU/ml LH 첨가군 (45.2±15.1)보다 많은 것으로 조사되었다. 난자-난구세포 복합체에서 P450scc와 P450_(17α)의 발현은 LH의 첨가농도가 증가함에 따라서 증가하였으며, 그리고 progesterone과 testosterone의 분비도 증가를 하였다. 특히, P450scc와 P450_(17α)의 발현 그리고 progesterone과 testosterone의 분비는 100mIU/ml FSH+100mIU/ml LH 첨가군 에서 다른 첨가군들에 비하여 유의하게 증가하였다. 따라서, 이러한 결과들은 성선자극호르몬이 preantral follicles의 체외배양을 위해서는 필수적이지만, LH 첨가농도의 증가는 난자의 발생능력을 감소시킨다는 것을 보여주었다. 그리고 이러한 결과에 대한 원인의 하나는 황체화의 지표인 P450scc와 퇴행화의 지표인 P450_(17α) 발현의 증가에 의한 progesterone과 testosterone의 분비증가에 기인한 것으로 추정된다. 결론적으로, 본 연구는 배양액내에 100mIU/ml FSH 혹은 100mIU/ml FSH+10 mIU/ml H의 첨가가 생쥐 preantral follicles의 체외배양을 위한 적정조건임을 제시하고 있다. The present study was conducted to examine the developmental capacity of mouse oocytes within preantral follicles cultured various concentrations of FSH and LH and the expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and cytochrome P450 17 a-hydroxylase (P450_(17α)) mRNA, as luteinization and atretic marker, in these culture conditions. In addition, we investigated the concentrations of progesterone and testosterone in culture medium. The developmental potential upto blastocyst of the oocytes grown jn vitro was higher in the FSH alone (30.2%) and 10 mIU/ml LH and 100 mlU/ml FSH treated (28.0%) groups than in the 100 mlU/ml LH and 100 mIU/ml FSH treated group (22.0%). And the mean numbers of cell per blastocyst was higher in the FSH alone (50.9±26.1) and 10 mIU/ml LH and 100 mIU/ml FSH treated (51.0±21.1) groups when compared to the 100 mIU/ml LH and 100 mlU/ml FSH treated group (45.2±15.1), The expressions of P450scc and P450_(17α) mRNA in the oocyte-cumulus complexes were increased with increasing of LH concentration, and also the secretions of progesterone and testosterone were increased. Especially, in the 100 mIU/ml LH and 100 mlU/ml FSH treated group, the expression of P450scc and P45017a were significantly increased, and the secretion of progesterone and testosterone were significantly increased. Therefore, these data show that gonadotrophins are essential for the in vitro culture of pre antral follicles, but that increasing of LH concentration is reduced the developmental capacity of oocytes. The cause of these findings may be due to increasing of progesterone and testosterone secretion by the enhance of P450scc and P45017" mRNA expressions, as markers of luteinization and atresia. Conclusively, this study suggest that supplementation of 100 mlU/ml FSH or 10 mlU/ml LH and 100 mIU/ml FSH may be optimal condition for the culture of mouse pre antral follicles.

Transmission and Death Rates in Transgenic Mice Containing Growth Hormone Receptor Gene

Jin, D. I.,Kim, H. J. 한국가축번식학회 2001 Reproductive & developmental biology Vol.25 No.1

To study the signaling effect of growth hormone (GH) in vivo on animal physiology, transgenic mice containing GH Receptor (GHR) gene fused to metallothionein promoter were produced by DNA microinjection into one-cell stage embryos. Three founder mice were produced with transgenic mice with approximately 4∼6 copies of GHR genes and transgene was transmitted into the progeny. The founder mice were mated with normal mice to produce F_(1) mice, and intergation and transmission of transgene were checked by polymerase chain reaction and Southern blot methods. Transmission rate of GHR transgenic mice were 20∼50% in F_(1) generation and 50% in F_(2) generation which means that some founder mice were mosaic and transgene in F_(1) mice was transmitted to F_(2) progeny with Mendelian ratio. Death rate of GHR transgenic mice after birth was about 10∼30% in F_(1) and F_(2) progenies indicating that GHR gene may affect death of transgnenic progeny.

동결속도와 평형시간이 소 미성숙 난자의 동결 융해후 생존율에 미치는 영향

양병철,양보석,성환후,임기순,최선호,장원경,진동일,임경순 한국가축번식학회 2001 Reproductive & developmental biology Vol.25 No.1

The present study was undertaken to investigate the effects of cooling rate and equilibration time on the survival, in vitro maturation and development to embryos of frozen-thawed bovine immature oocytes(Germinal Vesicle Stage). The cryoprotectants are used 10% ethylene glycol(EG) as permeating cryoprotectant and 0.05M sucrose(S) or trehalose(T) as low molecular weight nonpermeating cryoprotectants and 5% ficoll(F) or polyvinylpyrrolidone(PVP) as high molecular weight nonpermeating cryoprotectants. Four freezing solution were uysed in this experiment(EFT: 10% EG + 5% F + 0.05M T, EFS: 10% EG + 5% F + 0.05M S, EPT: 10% EG + 5% P + 0.05M T, EPS: 10% EG + 5% P + 0.05M S). The best equilibration time and freezing solution was 15 min in EPT(83% survival rate of frozen-thawed bovine immature oocytes). When frozen-thawed bovine oocytes were cultured following IVM and IVF, there was no significant difference in cleavage and development rates among the EFT, EFS, EPT and EPS solutions. When 9 blastocysts derived from frozen bovine oocytes were transferred to 6 recipients, two recipients were pregnant. And one was aborted at 45 days of pregnancy and the other had a stillbirth.

Effects of Slow Freezing on Development of Blastomeres Separated from Mouse Preimplantation Embryos

진동일 한국가축번식학회 2000 Reproductive & developmental biology Vol.24 No.3

The development of single blastomeres isolated from 2-, 4- and 8-cell mouse embryos and the ability of such blastomeres to survive slow freezing were studied. Of 223, 60 and 188 single blastomeres isolated from 2-, 4- and 8-cell mouse embryos, respectively, 111 blastomeres (49.8%) from 2-cell embryos, 12 blastomeres (20.0%) from 4-cell embryos and blastomeres (16.5) from 8-cell embryos developed into blastocysts after culture for 96 hrs. The recovery rate was 54.2% (65/120), 46.4% (13/28) and 24.3% (17/70) of blastomeres derived from 2-, 4- and 8-cell embryos following freezing and thawing and the survival of frozen-thawed blastomeres was 27.1% (16/59), 36.4% (4/11) and 17.6% (3/17), and respectively. The apparently six normal fetuses were obtained from frozen-thawed blastomere from 2-cell embryos after transferring into the recipients. These results indicate that mouse blastomeres isolated from preimplatation stage embryos can survive storage in liquid nitrogen following slow freezing.

생쥐 Preantral Follicles의 체외성장 및 성숙에 있어서 Gonadotrophins의 역할

김동훈,지희준,강희규,한성원,이훈택,정길생,이호준 韓國家畜繁殖學會 1999 Reproductive & developmental biology Vol.23 No.1

The present study was designed to investigate the effects of gonadotrophins on in vitro growth and maturation in mouse preantral follicles. Ovaries were removed from 12-day-old ICR mice. Follicles were dissociated enzymetically in Leibovitz L-15 medium containing 1 mg/ml collagenase and 0.2 mg/ml DNase I. The follicles were cultured on Transwell-COL membrane inserts in six well cluster dishes for 10 days. The culture medium was aMEM medium supplemented with 5% fetal bovine serum and FSH or HMG. After 10 days of growth in vitro, follicles were allowed to mature for 18-20 hr in medium supplemented with 1.5 IU/ml hCG. The oocytes were then denuded of their cumulus cells and assessed maturation status. Concentrations of oestradiol and progesterone were measured with a radioimmunoassay. Oocyte diameter was determined with an ocular micrometer. The survival and Metaphase Ⅱ rates of oocytes were significantly higher in FSH treatment groups than in control group (p<0.001), but there were no differences among the groups of treated FSH concentration. The survival and Metaphase Ⅱ rates of oocytes in HMG treatment group (60.9 and 40.6%) were higher than in FSH treament group (76.6 and 48.2%) and control group (49.2 and 7.1%). The survival and Metaphase Ⅱ rates of oocytes on both FSH and LH treatment groups were no differences among the ratios of FSH and LH. Diameter of oocyte was no differences among the treatment groups, but smaller than compared to in vivo grown oocyte. Through the entire culture period, secretions of oestradiol and progesterone were significantly less in control group than in HMG and FSH treatment groups. These results suggest that gonadotrophins playa key role in in vitro culture of mouse preantral follicles. Especially, addition of FSH and LH should be more effective than FSH alone.

토끼 수정란 체외 배양액의 개발에 관한 연구

임경순,진동일,김대경,김성우,정소용,최화식 한국가축번식학회 1998 Reproductive & developmental biology Vol.22 No.1

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19∼20hr after superovulation induction and incubated at 39℃ in 5% CO_(2) for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA appeared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of mediums. BSA and taurine together in RDH medium showed the additive effects on embryo development and cell number.

Establishment of Embryonic Stem Cells Derived from Rabbit Embryos

강회성,임경순,최화석,신영수,진동일 한국가축번식학회 2001 Reproductive & developmental biology Vol.25 No.3

To establish rabbit Embryonic Stem (ES) cells, rabbit one-cell embryos were collected and cultured in vitro to blastocysts. Blastocysts were co-cultured with mouse embryonic fibroblasts (MEF), rabbit embryonic fibroblasts (REF) or STO cells expressing LIF (SNL). Although rabbit ES cells were isolated with low efficiencies, total 8 ES cell lines were kept in vitro with normal colony shape. The MEF was the best feeder for rabbit ES cell isolation in regard to growth rate and undifferentiated morphology. The doubling time of rabbit ES cells in MEF was about 84 hours and the undifferentiated morphology was maintained following passing and freezing processes. These rabbit ES cells were differentiated into embryoid body following the culture in the uncoated dishes, indicating that they were undifferentiated stem cells.