• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.100-100
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• 2002

As an preliminary experiment for making transgenic animals producing human follicle stimulating hormone (hFSH), we tried to express recombinant hFSH gene in vitro. hFSH is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. Genes encoding the common gonadotrophin alpha subunit and FSH-specific beta subunit were inserted into retroviral vectors under the control of the rat beta actin promoter. Gene transfer to the Chinese hamster ovary (CHO) cells was done by infection of the retroviruses harvested from PT67 packaging cells transfected with recombinant retrovirus vector DNA. After selection with G4l8, PCR and RT-PCR analyses of the G4l8-resistant CHO cells showed successful transfer and expression of both ${\alpha}$ and ${\beta}$ fragments of the FSH gene.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.220-220
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• 2004

The purpose of this study was to development of transgenic cow using the nuclear transfer. To secrete hFSH in urea, the vector was constructed with UPII promoter. The fetal fibroblast cells (KbFF) were constructed from pregnant day 45 male fetus. The hFSH genes were cotransfected with pcDNA3 (neo) vector to KbFF cells by electroporation. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• 제11권5호
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• pp.486-490
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• 1998

The present study examined the follicular populations prior to and during superovulation and investigated their relationship with superovulatory response in crossbred cattle. Eleven animals were administered i.m. 8 doses of Folltropin of 2.5 ml each (1.75 mg/ml) spread over 4 days beginning on Day 10 of oestrous cycle, and 30 and 20 mg Lutalyse was given alongwith the 5th and 6th injections of Folltropin, respectively, to induce luteolysis. The animals were artificially inseminated 48, 60 and 72 h after the first Lutalyse injection. The number of corpora lutea (CL) was recorded by palpation per rectum and embryos were recovered non-surgically on Day 6 (Day 0 day of superoestrus). The ovarian follicular population was examined by transrectal Ultrasonography 15 h prior to and 52 h after the first FSH injection, and then on the day of superoestrus and the day of flushing. The follicles were classfied on the basis of diameter as small (3-5 mm), medium (6-9 mm) and large (${\geq}10mm$). The total number of follicles increased significantly (p < 0.01) from $2.45{\pm}0.35$, 15 h prior to the first FSH injection to $8.09{\pm}1.12$, 52 h after the first FSH injection and then further to $13.27{\pm}1.89 $ on the day of superoestrus. A positive correlation was observed between the number of small follicles 15 h prior to the first FSH injection (r = 0.60, p < 0.05), the number of large follicles 52 h after the first FSH injection (r=0.59, p < 0.05) and the number of CL. The follicular population prior to and during superovulation was, however, not significantly different between high (> 6 CL) and low responders (${\leq}6CL$). The present study suggests that the follicular populations undergo dynamic changes during superovulation and that follicular populations prior to superovulation have a limited application as an indicator of the superovulatory response.

• Asian-Australasian Journal of Animal Sciences
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• 제6권3호
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• pp.411-416
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• 1993

We assessed effects of ovine luteinizing hormone (oLH) and follicle-stimulating hormone (oFSH) on the granulose and theca layers from the four largest follicles, $F_1-F_4$ of hens which had been hypophysectomized 12 h before expected ovulation. Ovine LH (0.4 mg), oFSH (0.4 mg) or oLH in combination with oFSH (0.4 mg each) was injected intravenously 6 h after hypophysectomy. Progesterone, testosterone and $estradiol-17{\beta}$ levels of the granulose and theca layers which were removed 6 h after hormone injection, were measured by radioimmunoassay. Progesterone contents of $F_1-F_3$ granulosa layer at 12 h after hypophysectomy were much lower than those of control hens. This reduced progesterone level was restored partially by the injection of oLH alone for $F_1$, while no follicles responded to oFSH treatment. In contrast, the injection of oLH in combination with oFSH resulted in high progesterone content of the granulose layer from all four follicles. Progesterone content of the theca layer was negligible in all treatments. Simultaneous injection of oLH and oFSH also elevated $estradiol-17{\beta}$ level accumulating in the theca layer from all follicles, of which much higher concentrations of $estradiol-17{\beta}$ were observed when comparison were made to each of their corresponding controls. No appreciable change in testosterone contents of two layers was observed in the present experiments. These results suggest that oFSH augments function of oLH to stimulate the production of progesterone in the granulose layer and $estradiol-17{\beta}$ in the theca layer.

• Clinical and Experimental Reproductive Medicine
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• 제16권1호
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• pp.69-79
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• 1989

Steroid hormone profiles during luteal phase of clomiphene citrate(CC)/human menopausal gonadotropin(hMG)/human chorionic gonadotropin(hCG)-stimulated in vitro fertilization (IVF) cycles and of follicle-stimulating hormone(FSH)/hMG/hCG-stimulated IVF cycles were compared. In seventy three cycles stimulated with CC/hMG/hCG regimen, follicles were aspirated during exploratory laparotomy and yielded 7 pregnancies, and in 83 cycles stimulated with FSH/hMG/hCG regimen, follicles were aspirated by laparoscope and made 13 pregnancies. Serum estradiol($E_2$) and progesterone($P_4$) levels were determined on days 2, 5, 7, and 9 after follicle aspiration. The FSH/hMG/hCG regimen was more effective than the CC/hMG/hCG regimen in folliculogenesis, ie, ovarian stimulation, follicular phase $E_2$ peak levels, oocyte maturation, and the number of retrieved oocytes. There was no significant difference between luteal serum $P_4/E_2$ ratio of the two regimens, suggesting that secretory endometrial build-up ability for implantation may not differ each other. Several significant correlations were observed between follicular phase seum $E_2$ peak levels and luteal phase serum $E_2$ and $P_4$ levels in the FSH/hMG/hCG-stimulated cycles but any correlation was not significant in the CC/hMG/hCG-stimulated cycles, suggesting that somewhat more follicles may eventually fall in atresia even after attaining dominant stage in the CC/hMG/hCG-stimulated cycles than the FSH/hMG/hCG-stimulated cycles.

• Clinical and Experimental Reproductive Medicine
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• 제17권1호
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• pp.29-44
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• 1990

In 105 patients with the past history of poor response to the previous controlled ovarian hyperstimulation(COH) due to poor follicular growth or premature LH surge, the effectiveness of pituitary suppression with gonadotropin-releasing hormone agonist(GnRH agonist) in IVF/GIFT program was evaluated in 112 cycles of COH using a combination regimen of Leuprolide acetate (Lupron TAP Pharmaceuticals, USA) and FSH/hMG or pure FSH from May to December, 1989 at SNUH. Starting on day 21 of the menstrual cycle(MCD #21, Day 1), Lupron (1.0mg/day, subcutaneous) was administered once a day till next MCD #3(suppression phase). After the confirmation of pituitary suppression, ovarian follicular growth was stimulated with FSH/hMG or pure FSH from MCD #3(Day + 1), and Lupron was continued with hMG or FSH until hCG administration (D 0) (stimulation phase). After suppression phase, serum E2 level decreased from 183.7${\pm}$95.1(Day 1) to 17.4${\pm}$12.3pg/ml (Day +1), and serum progesterone level from 19.17${\pm}$8.67 to 0.12${\pm}$0.05ng/ml. But there was no decresas in serum LH and FSH levels; LH from 12.74${\pm}$6.21 to 15.49${\pm}$4.93mIU/ml,FSH from 7.60${\pm}$3.84 to 8.58${\pm}$3.15 rnlU/ml. There was no occurrence of premature LH surge during COH. Eleven cycles(9.8%) were cancelled due to poor follicular growth during stimulation phase, and 3 cycles (3.0%) failed in the transvaginal oocytes fretrieval. Serum E2 level was 1366.8${\pm}$642.4 on D 0 and 1492.3${\pm}$906.9pg/ml on D+1. 7.00${\pm}$3.32 follicles(FD${\geq}$12mm) were observed on D 0, and 6.11${\pm}$4.15 oocytes were retrieved, with the oocyte retrieval rate per follicle of 95.0%. 3.59${\pm}$2.57 oocytes were fertilized and cleaved with the oocyte cleavage rate of 55.7%. In 83 IVF patients, 4.08${\pm}$2.39 embryos were transferred, and 16 pregnancies were obtained with the pregnancy rate per ET 2.39 mebryos were transferred, and 16 pregnancies were obtained with the pregnancy rate per ET of 19.3%. In 6 GIFT patients, 7.83${\pm}$3.31 oocytes were retrieved and transferred with maximum number of 6, but no pregnancy was obtained. When compared with the previous 108 cycles of COH using FSH/hMG or pure FSH regimen, the cancellation rate during COH was significantly decreased, and all the parameters of the outcome of COH including the pregnancy rate were increased. These data suggest that GnRH agonist therapy for pituitary suppression is an effective adjunct to the current gonadotropin regimens for COH in IVF/GIFT and can increase the probability of oocytes retrieval and pregnancy, especially in the previous poor responders.

• 한국수정란이식학회지
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• 제19권1호
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• pp.61-66
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• 2004

한국 재래산양 체내수정란 생산에 대한 발정동기화 및 과배란 유도방법과 회수된 수정란의 동결 융해 후 생존율을 조사하였다. 발정동기화를 위해 CIDR+FSH 및 CIDR+PMSG의 방법을 이용한 결과, 배란점 및 회수된 수정란의 수는 CIDR+FSH 처리구에서 16.3개 및 9.4개, CIDR+PMSG 처리구에서 16.4개 및 8.7개를 나타내어 두 처리구간에 유의적 차이는 인정되지 않았다. 회수된 수정란을 형태학적으로 평가한 결과 CIDR+FSH 처리구에서 Gade A, B, C 및 D는 75.8%, 15.2%, 4.5% 및 4.5%를 나타낸 반면 CIDR+PMSG 처리구에서는 52.5%, 16.4%, 16.4% 및 14.8%였으며, 이식 가능한 수정란 (Grade A, B) 수는 CIDR+FSH 처리구가 유의적(P<0.05)으로 높게 나타났다. 회수된 수정란의 완만 동결 융해 후 생존성은 CIDR+FSH 처리구에서 73.3%, CIDR+PMSG 처리구에서 63.3%이었으며, 두 군간의 유의적차이는 인정되지 않았다. 따라서 본 결과는 한국 재래산양 체내수정란의 생산과 회수된 수정란의 보존을 위해서 CIDR+FSH로 발정동기화 시키는 것이 효과적이었다.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.45-56
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• 2002

Objectives: Recently, recombinant FSH (rFSH) has been manufactured using a Chinese hamster ovary cell line transfected with the gene encoding human FSH. Both rFSH and urinary gonadotropin (uFSH) could be used for controlled ovarian hyperstimulation (COH). However, uFSH implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, and low purity. The purpose of this study was to evaluate the efficacy of rFSH in human IVF-ET program. Materials and Methods: A total of 508 infertile women was enrolled in this study. They are classified into rFSH group (n=177) or uFSH group (n=331), and all of them were matched by age and cause of infertility in same period. The $Puregon^{(R)}$ (Organon, Holland) was used as rFSH, and the Metrodin-$HP^{(R)}$ (Serono, Switzeland) and $Humegon^{(R)}$ (Organon, Holland) was used as uFSH. We subdivided the patients into three age groups. The outcomes of IVF-ET program were analyzed using the statistical package for social sciences (SPSS). Results: There was no significant differences in the level of estradiol on hCG injection day, the numbers of retrieved oocytes, matured oocytes, fertilized oocytes, transferred embryos, frozen embryos between the two groups. The total dose (IU) of gonadotropin for COH was significantly lower in the rFSH group compared to uFSH group ($1339{\pm}5491.1$ vs $2527.8{\pm}1075.2$ IU, p<0.001). Clinical pregnancy rate per embryo transfer in the rFSH group showed increasing tendency, compared to the uFSH group, but there was no statistical significance (35.2% vs 29.3%). Our results demonstrated that the relative efficiency of rFSH compared with uFSH is higher in older patients. Conclusions: The ovarian stimulatory effect and clinical outcome of recombinant FSH was similar to that of the urinary gonadotropin. The IVF-ET cycles with significantly lower dose of gonadotropin in rFSH group showed comparable results. Therefore, we suggest that recombinant FSH is more potent and effective than urinary gonadotropin.

• 한국가축번식학회지
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• 제24권4호
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• pp.395-406
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• 2000

본 연구는 다양한 농도의 FSH 와 LH 에서 배양된 생쥐 preantral follicles 내 난자의 발생능력을 조사하고, 이러한 조건에서 배양된 난자 -난구세포 복합체에서 황체화의 지표인 cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc)와 퇴행화의 지표인 cytochrome P450 17 $\alpha$ -hydroxylase (P450$_{17{\alpha}}$ ) mRNA의 발현정도를 조사하고, 또한 progesterone과 testosterone의 분비농도를 살펴보기 위하여 실시하였다. 체외성장된 난자의 배반포까지의 발달능력은 100 $m\ell$U/$m\ell$ FSH 단독첨가군 (30.2%)과 100 $m\ell$U/$m\ell$ FSH$\pm$l0$m\ell$U/$m\ell$ LH 첨가군 (28.0%)이 100$m\ell$U/$m\ell$ FSH＋100$m\ell$U/$m\ell$ LH 첨가군 (22.0%) 보다 높은 결과를 나타냈다. 그리고 배반포의 평균 세포수에 있어서도 FSH 단독첨가군 (50.9$\pm$26.1)과 100$m\ell$U/$m\ell$ FSH＋10 $m\ell$U/$m\ell$LH 첨가군 (51.0$\pm$26.1)이 100$m\ell$U/$m\ell$ FSH＋100$m\ell$U/$m\ell$ LH 첨가군 (45.2$\pm$15.1) 보다 많은 것으로 조사되었다. 난자 -난구세포 복합체에서 P450scc 와 P450$_{17{\alpha}}$의 발현은 LH의 첨가농도가 증가함에 따라서 증가하였으며, 그리고 progesterone과 testosterone의 분비도 증가를 하였다. 특히, P450scc 와 P450$_{17{\alpha}}$ 의 발현 그리고 progesterone과 testosterone의 분비는 100$m\ell$U/$m\ell$ FSH＋100$m\ell$U/$m\ell$ LH 첨가군에서 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$_{17{\alpha}}$ 발현의 증가에 의한 progesterone과 testosterone의 분비증가에 기인한 것으로 추정된다. 결론적으로, 본 연구는 배양액내에 100$m\ell$U/$m\ell$ FSH 혹은 100$m\ell$U/$m\ell$ FSH+10 $m\ell$U/$m\ell$ LH 의 첨가가 생쥐 preantral follicles의 체외배양을 위한 적정조건임을 제시하고 있다.

• 한국수정란이식학회지
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• 제12권1호
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• pp.49-56
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• 1997

This study was carried out to establish an effective and practical system for commercialization of embryo production techniques by analyzing several factors influencing in vivo embryo production on condition of donors treated FSH-P and SUPER-OV in Korean native cattle. In vivo embryos were flushed 226 times from 128 donors. The results obtained for the factors influencing in vivo embryo production by conditionof donors treated FSH-P and SUPER-OV were as follows :1. Age and parity of donors did not affect the transferable and freezable embryos among the treatments in FSH-P but the transferable and freezable embryos were decreased after 6 years old and 4th parity in SUPER-OV(P<0.01). 2. The production of embryos on the weight of donors was higher in over 400kg of body weight in FSH-P(P<0.01) and was higher in over 450kg than 400~450kg of body weight in SUPER-OV(P<0.05). For FSH-P embryo production was better responded in 350~450kg of body weight with 30~32mg doses, and showed a better result in over 450kg body weight with 32~34mg doses.(Key Words : in vivo embryo, donors, FSH-P SUPER-OV)