• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• 대한임상검사과학회지
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• 제38권1호
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• pp.72-81
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• 2006

Wee1 is a kinase regulator of the M-phase promoting factor (MPF; a complex of cdc2 and cyclin B1). The present study was undertaken to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. The expression of wee1 and the correlated cell-cycle components, namely cdc2, cyclin B1, and cdc25C, were evaluated by immunohistochemistry. In addition, the expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2 existed. Each component except cdc25C was found cytoplasmic in the oocytes at all stages of follicles, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian somatic cells and to a small extent in granulosa cells of the growing follicles. To further confirm the expression of cell-cycle components in the primordial follicular oocytes, day1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. From the results of the present study, we concluded wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by the inhibitory phosphorylation of cdc2. The expression of all these proteins in the granulosa cells of growing follicles may regulate their mitosis concurrently with the growth of oocytes and follicles.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.183-187
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• 2009

This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is $2{\sim}4\;mm$ and $6{\sim}10\;mm$, were collected from ovary of slaughtered pig that each follicle of diameter $1{\sim}4\;mm$ and $6{\sim}10\;mm$. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $200{\mu}l$. Immobilized pH gradient(IPG) strip used 18 cm, $3{\sim}10\;NL$. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of $6{\sim}10\;mm$ follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kDa protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development.

• 한국동물위생학회지
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• 제36권2호
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• pp.127-132
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• 2013

An 11-year-old poodle bitch was presented for investigation of multicentric mammary masses. Abdominal sonography and radiography demonstrated abnormal enlargement of uterus and ovaries. Blood analysis revealed high progesterone concentration. The ovariohysterectomy and mastectomy were performed. Histopathologically, the mammary masses revealed complex carcinoma-tubulopapillary carcinoma with papillary pattern and tubule pattern. In the uterus, cystic endometrial hyperplasia was observed. Scattered inflammatory cells were observed in the endometrial stroma and mucinous material was protruded from endometrial surface. Also, in the ovaries, bilateral ovary granulosa cell tumor was detected. The bitch made a complete recovery following the ovariohysterectomy and mastectomy. This case was a very rare multiple tumor occurrence with bilateral ovary granulosa cell tumor and mammary complex carcinoma. High progesterone concentration was characterized clinically in the bitch.

• 한국수정란이식학회지
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• 제20권1호
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• pp.71-77
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• 2005

Relaxin과 insulin이 돼지 난포 과립막세포의 스테로이드 호르몬 분비에 미치는 영향을 연구하기위하여 체외에서 황체화된 과립막세포에서 prosesterone과 $17{\beta}-estradiol$의 생산을 조사하였다. 돼지난포 과립막세포를 혈청 존재하에 배양접시에 부착 후 48시간 동안 체외배양하고 무혈청 배지에서 24시간 배양하였다. Relaxin과 insulin의 용량의존성을 확인하기 위하여 다양한 농도 (10, 100, 1,000 ng/ml)를 각각 무혈청 배지에 첨가하였다. 병합 효과를 알아보기 위하여 100 ng/ml relaxin과 100 ng/ml insulin을 단독 혹은 병합하여 처리하였는데 황체호르몬 (100 ng/ml)을 처리한 경우와 처리하지 않은 경우 모두를 조사, 분석하였다. 최종 배양이 끝난 배양액을 수집하여 RIA법으로 proses-terone과 $17{\beta}-estradiol$의 농도를 조사하였다. Relaxin과 insulin은 용량이 증가될수록 progesterone의 생산을 증가시켰으나 $17{\beta}-estradiol$의 생산에는 아무런 영향이 없었다. 병합실험에서는 relaxin과 insulin 단독 또는 병합시 황체호르몬 존재 하에서 progesterone의 생산을 증가시켰으나 $17{\beta}-estradiol$의 생산에는 아무런 영향이 없었다. 결론적으로 relaxin과 insulin은 돼지 황체화 과립막세포의 progesterone 분비를 증가시키지만 $17{\beta}-estradiol$의 생산에는 효과가 없었으며 병합에 의한 상승효과는 없었다. Progesterone 생산에 미치는 relaxin과 insulin의 효과는 황체호르몬의 존재에 의해 증대되었다.

• Clinical and Experimental Reproductive Medicine
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• 제32권3호
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• pp.269-277
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• 2005

Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.

• 대한의생명과학회지
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• 제12권2호
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• pp.105-112
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• 2006

This study was undertaken to investigate the morphological changes between normal and atretic follicle after gamma irradiation and treatment of follicle stimulating hormone (FSH). The ovaries of each group of treated immature mice were prepared the paraffin sections after 0, 6, 12, and 24 hours (hrs) of those treatment. Hematoxylin-eosin (HE) stain, reticulin stain, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) immunohistochemical stain were performed on the each paraffin sections. As the results of HE staining, the condensed nuclei of oocytes were observed in the atretic primordial follicles, on the other hand the condensations of granulosa cell nuclei were prominent in the atretic primary, preantral, and antral follicles. Only the granulosa cells of atretic follicle were stained specifically with TUNEL staining but not stained in the theca cells, which suggested granulosa cells degenerated through apoptosis. In the reticulin staining, the basement membranes of atretic follicle which was stained weakly showed irregular structure and detachment from the follicles. The ratio of normal to atretic follicle in control and FSH treated group was about 33% but this ratio increased rapidly over 90% in the 6, 12, and 24 hrs group after the irradiation. It could be suggested that the gamma irradiation is the useful tool far the induction of follicle atresia and immunohistochemical staining methods are essential in the study of follicle atresia.

• Clinical and Experimental Reproductive Medicine
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• 제13권1호
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• pp.29-38
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• 1986

In order to study the mechanism of follicular atresia, follicles were classified into the normal groups and the atretic ones, according to the criteria with or without corpus luteum, size of follicles, vascularization, status of granulose cells and the hypertrophy of theca layers in the porcine ovary. To estimate the binding capacity of human chorionic gonadotropin (hCG) receptor on the granulosa cells during atresia, hCG were iodinated by the chloramine-T method and then purified through the column chromatography. The concentration" of hCG receptor in each group were measured by the hCG receptor binding assay. Binding capacity in large normal follicles were 1.16%, but atretic ones were 0.45%. But in medium and small follicles (below 6mm in diameter), the binding capacity in normal follicles were 0.09%, but atretic ones were 0.05%, which was lower than those of large follicles. The present ( ) that the concentrations of hCG receptors on granulosa cells is decreased when the follicles become atretic and be used as a sort of creteria for the identification of follicles atresia.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.6-11
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• 2006

Our previous study demonstrates a rapid activation of atypical PKC${\zeta}$ by the ovulatory dose of LH/hCG. The present study was therefore designed to identify PKC${\zeta}$ regulated-genes in rat ovarian preovulatory granulosa cells. Preovulatory granulosa cells cultured in the presence of myristoylated PKC${\zeta}$ pseudosubstrate peptide were subjected to identify differentially expressed genes by using anneling control primer RT-PCR. As a result, among sixteen genes identified, six genes (testin, glypican-4, retrovirus SC1, connective growth factor, aminolevulinic acid synthase1 and serum- inducible kinase) were rapidly stimulated by hCG. Northern blot analysis demonstrated that all these genes were rapidly stimulated by hCG and declined thereafter. In situ hybridization analysis revealed the expression of these genes in granulosa cells of preovulatory follicles. The present study demonstrates time- and cell-specific expression of PKC${\zeta}$-regulated genes, and may imply that these genes play a specific role(s) during LH-induced ovulation.