• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.62-62
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• 2001

Recently, production of transgenic animal by nuclear transfer has been known as a useful method. The production of cloned offspring derived from nuclear transfer depends upon a variety of factors such as species, donor cells type and cell cycle, and source of recipient ova. Therefore, we attempted a different transgenic methods using follicular granulosa cells (GCs). In general, ovulated GCs undergoes lutenization and transformation in vitro which might defective effects on developmental potential. In order to avoid the GCs transformation in vitro culture system, we introduced a direct injection of retrovirus into the follicles and then collected them mechanically from ovaries of 6-8 week-old ICR mice. Retrovirus vector constructed with pLN $\beta$ EGFP was injected into the follicles. The follicles are cultured in $\alpha$ -MEM supplemented with human FSH, LH and ITS in Costar Transwell dish for 4 days. Survival rate of virus injected follicles was 52.1% (12/23) and expression rate of EGPP gene was 33.3% (4/12). In this study, we found GCs performed transgenesis in our culture system. In addition, the GCs in follicle may be developed in vivo like environment rather than in vitro environment. Thus, the use of GCs as donor cells may be useful in the nuclear transfer for cloning of genetic modification. Therefore, these results suggest that follicular GCs can be transfected by viral vector during folliculogenesis in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제16권1호
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• pp.81-91
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• 1989

It has been suggested that the prognosis for fertility of the infertile patients with healed pelvic tuberculosis is very poor. Total 60 patients(77 cycles) with previous history of pelvic tuberculosis who underwent IVF-ET from January 1988 to March 1989 at SNUH were classified into three groups according to the principal histopathological lesions : tuberculous endometritis group(N=20, 28 cycles), tuberculous salpingitis group(N=32, 37 cycles) and pelvic peritoneal tuberculosis group(N=8, 12 cycles). To evaluate the effects of previous pelvic tuberculous lesions on ovarian follicular growth and development in controlled ovarian hyperstimulation for IVF-ET and its final outcome, serum E2 levels on the day of hCG administration(Day 0) and the day after hCG administration(Day +1), the number of ovarian follicles with mean diamete ${\geqq}$ 12 mm on Day 0, the number of oocytes retrieved by transvaginal aspiration, and pregnancy rate per cycle were measured and compared with control group(N=123, 161 cycles). There were no significant differences in cancellation rate during controlled ovarian hyperstimulation, total dosage of FSH and hMG administrated, menstrual cycle date(MCD) of hCG injection, serum E2 levels, the number of ovarian follicles with mean diameter ${\geqq}$ 15 mm, and the number of oocytes retrieved between pelvic tuberculosis group and control group. But in pelvic tuberculosis group, the number of ovarian follicles with mean diameter 12-14 mm, total number of ovarian follicles(${\geqq}$ 12 mm), and pregnancy rate per cycle were significantly decreased. These data suggest that previous pelvic tuberculous lesions have no significant adverse effects on the ovarian response to gonadotropin stimulation. IVF-ET proved to be an useful treatment modality for infertile patients with previous history of pelvic tuberculosis in spite of its relatively lowered pregnancy rate.

• 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.

• Animal cells and systems
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• 제3권4호
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• pp.385-391
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• 1999

Changes in the levels of prostaglandian F$_{2a}$ (PGF$_{2a}$) and E$_2$ (PGE$_2$) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12-O-tetradecanoylphorbol-13-acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF$_{2a}$ to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE$_2$ increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF$_{2a}$ induced ovulation but PGE$_2$ suppressed FPH- or PGF$_{2a}$-induced oocyte ovulation. Basal levels of PGs Increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell-enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOS. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF$_{2a}$ plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) thecal epithelium layer is responsible for the PGs production in Rana.

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1237-1243
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• 2002

We have developed a reliable and noninvasive method for swine genotyping of single locus nuclear gene with aged single hair follicles delivered by general mail. The method is based on booster and nested PCR amplification with step-wise increase of primers and dNTPs concentrations followed by restriction endonuclease digestion. To establish this method, the ryanodine receptor (RYR 1) locus which is an economically important trait in swine industry was employed for genotyping experiment. The 3-step PCR amplication method is much less dependent on the quantity and quality of template DNA and produces enough amplification product for the detection on the ethidium bromide-stained gel such as RFLP analysis. A total of 120 pigs were subjected to the RYR 1 genotyping analysis using three-step PCR method which amplified enough quantity of PCR products from the aged single hair follicles for RFLP analysis and genotyping results were identical to the results of the corresponding ethanol-fixed skeletal muscle tissue. This approach will be a great help for porcine breeders and investigators in genotyping of swine. They can receive genotyping results later by simply plucking single hairs of their pigs at farm and sending them in general mail to the diagnostic laboratory which eliminates the inconveniences to collect ear tissue or blood cells from pigs, or the investigator's need for travel to farms in order to collect fresh hair samples.

• Asian-Australasian Journal of Animal Sciences
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• 제20권12호
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• pp.1827-1831
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• 2007

Effects of daidzein on expression of mRNAs of gonadotropin receptors (FSHR, LHR) and P450 aromatase (P450arom) were evaluated in ovarian follicles of white silky fowls. The hens were 13 months old in the post-peak period of egg laying and were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at 10 mg/kg for 7 consecutive weeks. The mRNA expression of related genes was measured by semi-quantitative RT-PCR in the granulosa layers of the preovulatory follicle (PRF: F1, F2 ...) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF) and atretic follicle (ATF). Results showed that daidzein supplementation significantly increased the number of SYF and LWF (p<0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F3 to F1, but LHR mRNA displayed opposite developmental changes. P450arom mRNA was highest in the SYF, but was very low in the granulosa layers after follicles finished selection. Treatment with daidzein resulted in increased mRNA expression of FSHR in F3 granulosa layer, LHR in granulosa layers of F3 to F1 and P450arom in LWF (p<0.05). These results indicated that dietary supplementation of daidzein up-regulated mRNA expression of gonadotropin receptors and P450arom to improve the development of preovulatory follicles in white silky fowls after the peak-laying period.

• 대한수의학회지
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• 제39권1호
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• pp.27-33
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• 1999

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.

• Molecules and Cells
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• 제38권4호
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• pp.304-311
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• 2015

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

• Clinical and Experimental Reproductive Medicine
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• 제27권2호
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• pp.145-149
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• 2000

Objective: The present study was conducted to evaluate the viability of germ cells from the adult and fetal ovarian tissues after vitrification followed by xenografting. Method: The human adult ovarian tissues were obtained from 33 years old patient, and the fetal ovarian tissues were obtained from 22 weeks and 25 weeks in gestation. Ovarian tissues were cryopreserved by vitrification with 5.5 M ethylene glycol (EG 5.5) and 1.0 M sucrose as cryoprotectants. Adult and fetal ovarian tissues were pre-equilibrated with EG 5.5 at room temperature for 10 and 5 minutes, respectively and plunged into liquid nitrogen immediately. Frozen-thawed tissues were xenografted into NOD-SCID mice to evaluate the viability and capacity for further growth of the primordial follicles. Grafts were recovered from the recipients 4 weeks after transplantation and histological analysis was accomplished. Result and Conclusion: Grafts recovered 4 weeks after transplantation contained less number of oocytes and primordial follicles compared to that of the fresh tissues. Survived follicles were mainly primordial and intermediary with larger diameter and more granulosa cells. It is confirmed that 1) the ovarian tissues were healthy and the germ cells were survived after vitrification, and 2) the survived fetal primordial follicles after vitrification resumed the growth in the xenografts.

• 한국동물학회지
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• 제39권4호
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• pp.410-418
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• 1996

Gangiloside는 포유동물세포에 편재하는 막성분으로서, 이들은 세포상호간의 접착, 분화 및 정보전달과정에 과여하는 것으로 알려지고 있다. Rat난소는 주요한 Gangiloside로서 GM3를 함유하고 있으며, 본 연구에서는 폐쇄난포에서 이들의 분포여부와 난포의 발달 과정에서의 변화여부를 조사하기 위하여, Rat 난소의 동결절편을 이용해 GM3를 포함 11종류의 Gangilo-series Gangiloside에 대해 특이한 단일항체로서 염색시켰다. 폐쇄난포들에서 GM3는 난포발달이 진행되는 동안 시간적, 공간적으로 서로 다른 양식으로 발현하였다. 그러나 GM1을 포함한 다른 종류의 Gangiloside들은 면역조직화학적으로 검출되지 않았다. 일차난포에서 관찰되는 폐쇄난포들에서 GM3는 모든 교막세포와 난자에 인접한 과립막세포의 일부에서 발현하였다. 이차난포의 시기에서 이들 폐쇄난포의 GM3는 모든 교막세포와 과립막세포들에서 발현하였다. 이어서 발달하고 있는 그라프난포의 시기에서 관찰되는 폐쇄난포의 GM3발현은 이차난포에서의 분포패턴과 유사함을 보여 주었다.