• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.165-172
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• 1997

the pupose of this study was to investigate the effects of gonadotropin and nitric oxide (NO) on the expression of mouse follicular bad and bax genes that are known induce apoptosis. Large and midium size follicles of immature mice were obtained at 0, 24, and 48 hours time intervals after Pregnant Mare's Serum gonadotropins(PMSG, 5 I.U.) injection. Preovulatory follicles collected at 24 hrs after PMSG injection were cultured with or without various chemicals such as gonadotropin, gonadotropin Releasing hormone(GnRH), testosterone, Sodium nitroprusside (SNP) for 24 hrs at $37^{\circ}C$. After 24 hrs culture, the culture media was used for nitrite assay and total RNA was extracted, subjected to RT-PCT for the analyses of bad and bax expression. We found that expression of bad and bax genes in follicles was markedly reduced before and after in vivo priming with hCG. When the preovulatory follicles were cultured for 24 hrs in culture media with PMSG and hCG, the expression of bad and bax genes was decreased. Moreover, SNP (NO generating agent) can significantly suppress the expression of bad and bax genes in follicles when apoptosis was induced by GnRH agonist and testosterone. At the same time, nitrite production of culture media was increased in GnRH agonist + SNP, testosterone + SNP and SNP treated groups than control group. These data demonstrated for the first time that peptide hormones and NO may play important roles in the regulation of mouse follicular differentiation and may prevent apoptosis via supressing the expression of bad and bax genes.

• Clinical and Experimental Reproductive Medicine
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• 제30권1호
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• pp.47-55
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• 2003

Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

• 한국가축번식학회지
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• 제17권4호
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• pp.279-285
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• 1994

The objective of the present study was to characterize the cell cycles of granulosa cell populations during follicular maturation in cattle by using flow cytometer. Granulosa cells were isolated from bovine preovulatory antral follicles of F1(>10mm), F2(5~20mm), F3(3~4mm) and F4(1~2mm) diameter and fixed and stained with fluorochromes that selectively bine to DNA. Flow cytometer equipped with a laser excitation system was used to analyze the intensity of fluorescence from stained cells. Forward angle light-scatter(FSC) and 90$^{\circ}$light-scatter(SSC) signals were adopted to measure the size and the granularity of granulosa cells. As a results of FSC/SSC analysis, granulosa cell populations(G1 phase of cell cycle) from each follicle were relatively regular in size and granularity, regardless of follicular size. However, their distribution in granularity was greater than that in size. Most of granulosa cell populations collected from each follicle were distributed in G0/G1, S and G2/M phases. As the follicles approached to ovulation the percentage of cells in the proliferative phases of cell cycle (S and G2/M) decreased significantly, but there was a concomitant increase in the percentage of granulosa cells in G1 phase. Therefore, these data indicate the proportion of main populations to cell cycle of granulosa cells may be changed from proliferative phase to G1 phase during follicular maturation in cattle.

• Clinical and Experimental Reproductive Medicine
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• 제15권1호
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• pp.25-34
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• 1988

The intrafo1licular echoes of cumulus oophoruses within ovarian follicles were assessed with the use of ultrasound in 86 women taking part in an in vitro fertilization(IVF) or gamete intrafallopian transfer(GIFT) program, stimulated with pure follicle-stimulating hormone(FSH)/human menopausal gonadotropin(hMG)/human chorionic gonadotropin (hCG). When intrafo1licular echoes were clearly separated from the follicular wall or relatively dispersed within the follicle, they were considered to be a dissociated cumulus, and when they were only slightly prominent from the follicular wall, they were suspected to be a nondissociated cumulus. A cumulus was seen in 62.1% of the follicles larger than 10 mm diameter and 75.1% of them were dissociated. The larger the follicles in size, the more the cumuluses in number and dissociation. The number of follicles and intrafollicular echoes per woman was not different whether or not she would be pregnant, but the number of dissociated cumuluses was significantly more in pregnant women. The number of observed dissociated cumuluses correlated significantly with the number of recovered mature oocytes. When an intrafollicular echo is seen, it can be taken as evidence of a sign of maturity of that particular follicle and oocyte. Ultrasonographic monitoring of intrafollicular echoes and follicular size is very helpful to predict follicular maturation in ovulation stimulation cycles.

• Clinical and Experimental Reproductive Medicine
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• 제41권1호
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• pp.21-28
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• 2014

Objective: To investigate the association of individual follicular fluid (FF) leptin and adiponectin levels with the quality of the corresponding oocyte and embryo. Methods: We prospectively enrolled 67 women who underwent controlled ovarian hyperstimulation with 89 FF samples. FF and the corresponding oocyte was obtained from a single dominant preovulatory follicle at the time of oocyte retrieval. Concentrations of leptin and adiponectin were measured by enzyme-linked immunosorbent assay in an individual follicle. The oocyte quality, fertilization rate, and corresponding embryo development were assessed. Results: The FF level of leptin was significantly associated with body mass index (r=0.334, p<0.01). The FF adiponectin level was significantly higher in the normal fertilization group than the abnormal fertilization group (p=0.009) in the non-obese women. A lower FF leptin level was associated with a trend toward mature oocytes, normal fertilization, and good embryo quality, although these relationships were not statistically significant. The leptin:adiponectin ratio of FF did not differ significantly according to oocyte and embryo quality. The quality of the oocyte and embryo was not associated with the FF leptin level tertile. However, the normal fertilization rate was positively associated with FF adiponectin level tertile. There was a trend towards improved oocytes and normal fertilization rates with the lowest tertile of the FF leptin:adiponectin ratio, but this difference was not statistically significant. Conclusion: Our results suggest that a high FF adiponectin concentration could be a predictor of normal fertilization. However, the FF leptin concentration and leptin:adiponectin ratio is not significantly related to oocyte maturity and corresponding embryo development.

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.18-23
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• 2011

Objective: Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment. Methods: Immature rats were injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) to induce the growth of multiple preovulatory follicles and 10 IU of human chorionic gonadotropin (hCG) to induce ovulation. Immature rats were also treated with diethylstilbestrol (DES), an estrogen analogue, to induce the growth of multiple immature follicles. Northern blot analysis was performed to detect gene expression. Cell-type specific localization of Prx I mRNA were detected by in situ hybridization analysis. Results: During follicle development, ovarian Prx I gene expression was detected in 3-day-old rats and had increased in 21-day-old rats. The levels of Prx I mRNA slightly declined one to two days following treatment with DES. A gradual increase in Prx I gene expression was observed in ovaries obtained from PMSG-treated immature rats. Furthermore, hCG treatment of PMSG-primed rats resulted in a gradual stimulation of Prx I mRNA levels by 24 hours (2.1-fold increase) following treatment, which remained high until 72 hours following treatment. In situ hybridization analysis revealed the expression of the Prx I gene in the granulosa cells of PMSG-primed ovaries and in the corpora lutea of ovaries stimulated with hCG for 72 hours. Conclusion: These results demonstrate the gonadotropin and granulosa cell-specific stimulation of Prx I gene expression, suggesting its role as a local regulator of follicle development.

• Asian-Australasian Journal of Animal Sciences
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• 제20권2호
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• pp.184-193
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• 2007

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

• 한국수정란이식학회지
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• 제26권4호
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• pp.237-244
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• 2011

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by realtime-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and postovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

• Reproductive and Developmental Biology
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• 제31권3호
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• pp.181-186
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• 2007

DNA 결합 단백질 억제(Inhibitor of DNA binding protein) 또는 분화억제(Inhibitor of differentiation) 인자인 Id는 네 종류(Id1-4)가 있으며, 세포의 증식과 분화, 혈관 형성 및 세포자가사멸 등에 정 또는 부의 조절인자로서 널리 알려져 있다. 하지만 아직까지 번식생리 분야에서 이들 유전자들의 발현이나 기능에 관해 알려진 것은 거의 없다. 따라서 본 연구에서는 쥐 난소에서 난포의 발달에 따른 Id3 mRNA의 발현 양상을 살펴보고자 실시하였다. 쥐에게 PMSG주사 후, 3, 6, 12, 24, 36 및 48시간째에 난소를 회수하여 고정, 탈수 및 파라핀 포매를 실시하였다. In situ hybridization 실험을 위하여 anti-sense와 sense Id3 cRNA 탐침자를 제작한 다음 난소 절편에 반응시켰다. 반응이 끝난 난소 절편은 NTB-2 유광제에 노출시킨 후 현상액에 반응시켰다. 모든 처리가 끝난 슬라이드는 H&E 염색을 실시한 다음 현미경하에서 hybridization 감도를 1+에서 4+로 구분하여 평가하였다. 난자에서는 Id3 mRNA 감도가 원시와 제1차 난포에서 ${\geq}2+$으로 확인되었으나, 제2차, 우성 및 배란전 난포에서는 1+ 이하로 관찰되었다. 한편, 과립막세포에서는 우성과 배란 전 난포에서 Id3 mRNA가 3+ 또는 4+로 강하게 발현되는 것을 확인하였다. 이상의 결과를 종합하여 보면, Id3 mRNA는 난포의 발단 단계 또는 난포세포에 따라 특이적으로 발현하는 것으로 보아 난포의 발달과 밀접한 연관이 있을 것으로 사료된다.

• 한국수정란이식학회지
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• 제17권2호
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• pp.123-128
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• 2002

The present study aimed at determining the effective dose of Folltropin, a follicle timulating hormone (FSH), on superovulation in indigenous cows of Bangladesh. Fifteen regularly cycling 5～7 years old dry cows, weighing 200～250 kg with 2.5～3.0 body condition scores (BCS) were divided into three groups (n＝5). Individual groups were superovulated with 100, 200 or 300 mg of Folltropin per animal. The superovulation treatment was initiated at Day 10 or Day 11 of the estrous cycle (Day 0＝day of estrus). Alfaprostol (6 mg) was injected to each cow 72 h after the initiation of superovulation treatment to induce eestrus. After confirming standing estrus, the cows were inseminated 2～3 times, 12 h apart, depending on the duration of estrus. At Day 6 or Day 7, individual horns of the uterus were flushed with 150～200 $m\ell$ of phosphate buffered saline supplemented with BSA (0.2％), penicillin (100 IU／$m\ell$) and streptomycin (100 $\mu\textrm{g}$／$m\ell$) using a two-way foley catheter. The embryos were concentrated, removing the excess medium through an embryo filter, and identified under a stereomicroscope. The identified embryos were collected, washed four times, evaluated and graded as excellent, good, fair or poor. The excellent, good and fair embryos were considered as transferable quality embryos. The mean (range). numbers of embryos collected vs. transferable quality embryos far 100, 200 and 300 mg of Folltropin were 4.5 (1～10) vs. 3.5 (1～8); 2.5 (1～4) vs. 1 (0～2) and 0.0 (0～0) vs. 0.0 (0～0), respectively, Folltropin at a dose of 100 or 200 mg produced suitable ovarian stimulation for superovulation in indigenous zebu cows of Bangladesh. A dose of 300 mg or more Folltropin consistently caused preovulatory corpora lutea formation in the ovaries and resulted in zero embryo recovery.