Expression of peroxiredoxin I regulated by gonadotropins in the rat ovary / 대한생식의학회지

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.

Mechanisms of Granulosa Cell Transition from Proliferation to Differentiation During the Ovulatory Process in Rodents: Role of Interferon-alpha System / 대한불임학회지

No abstract available.

In silico Analysis of Downstream Target Genes of Transcription Factors / 대한불임학회지

OBJECTIVE: In the previous study, we compiled the differentially expressed genes during early folliculogenesis.1 Objective of the present study was to identify downstream target genes of transcription factors (TFs) using bioinformatics for selecting the target TFs among the gene lists for further functional analysis. MATERIALS AND METHODS: By using bioinformatics tools, constituent domains were identified from database searches using Gene Ontology, MGI, and Entrez Gene. Downstream target proteins/genes of each TF were identified from database searches using TF database (TRANSFAC(R) 6.0) and eukaryotic promoter database (EPD). RESULTS: DNA binding and trans-activation domains of all TFs listed previously were identified, and the list of downstream target proteins/genes was obtained from searche of TF database and promoter database. Based on the known function of identified downstream genes and the domains, 3 (HNF4, PPARg, and TBX2) out of 26 TFs were selected for further functional analysis. The genes of wee1-like protein kinase and p21WAF1 (cdk inhibitor) were identified as potential downstream target genes of HNF4 and TBX2, respectively. PPARg, through protein-protein interaction with other protein partners, acts as a transcription regulator of genes of EGFR, p21WAF1, cycD1, p53, and VEGF. Among the selected 3 TFs, further study is in progress for HNF4 and TBX2, since wee1-like protein kinase and cdk inhibitor may involved in regulating maturation promoting factor (MPF) activity during early folliculogenesis. CONCLUSIONS: Approach used in the present study, in silico analysis of downstream target genes, was useful for analyzing list of TFs obtained from high-throughput cDNA microarray study. To verify its binding and functions of the selected TFs in early folliculogenesis, EMSA and further relevant characterizations are under investigation.

Control Mechanisms of Ovarian Follicle Development by Follicle Stimulating Hormone and Pituitary Adenylate Cyclase-activating Polypeptide / 대한불임학회지

OBJECTIVE: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. METHODS: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 (10-6 M). RESULTS: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. CONCLUSION: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.

Control Mechanisms of Ovulation by Pituitary Adenylate Cyclase-Activating Polypeptide / 대한불임학회지

No abstract available.

Expression of Apoptosis Gene Bok in the Rat Ovary / 대한산부인과학회잡지

OBJECTIVE: 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 rat ovary during the follicle development. METHODS: We used the female rats of SD strain. Bok mRNA levels in the ovary were determined by Northern blot analysis. In situ hybridization were performed to determine the specific ovarian cell type expressing Bok mRNA. RESULTS: Northern blot analysis of ovaries obtained from immature rats revealed increasing levels of Bok mRNA during postnatal development. The major cell types expressing Bok mRNA were the granulosa cells of preantral and atretic follicles. Treatment of immature rats with diethylstilbestrol (DES) for 24-48 h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in rats removed DES for 24- 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 rats with pregnant mare's serum gonadotropin (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 PMSG-primed rats with human chorionic gonadotropin (hCG) stimulated strongly ovarian Bok mRNA expression at 6-9 h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. In adult estrus cyclic ovaries, Bok gene expression was higher on proestrus and estrus than metaestrus and diestrus. Moreover, the highly increased expression of Bok mRNA was found in rat ovaries at 48 h after hypophysectomy. 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.

Expression of Proapoptotic Bcl-2 Family Member in the Mouse Ovary (i) / 대한불임학회지

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 21~23 days of age. Ovaries were collected 1~3 days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for 1~2 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 24~48 h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for 24~48 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 6~9 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.

Expression of Apoptotic Genes in Mouse Preimplantation Embryo Development / 대한불임학회지

OBJECTIVES: The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. MATERiALS AND METHODS: Two-cell embryos were retrieved from iCR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. RESULTS: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. in contrast, the expression of Mcl-1 and Bok was not significantly different. CONCLUSION: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.

Regulation of Steroid Thyroid Hormone Receptor 3 (TR3) mRNA Expression by Luteinizing Hormone in Human Early Luteinized Granulosa Cells / 대한산부인과학회잡지

OBJECTIVE: The present study examined the gonadotropin regulation of TR3 gene expression by luteinizing hormone (LH) in cultured human luteinized granulosa cells. METHODS: TR3 mRNA levels were detected by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method in cultured human luteinized granulosa cells collected from patients undergoing in vitro fertilization. RESULTS: TR3 transcript was transiently induced by LH, reaching maximum levels 1 hr after stimulation, in a dose-dependent manner. LH-stimulated TR3 expression was abolished by actinomycin D, but was superinduced by cycloheximide. Treatment of luteinized granulosa cells with Rp-cAMP, an inhibitor of protein kinase A, as well as, chelerythrin, an inhibitor of protein kinase C, suppressed LH-stimulated TR3 mRNA levels. In addition, forskolin and TPA mimicked the LH action on the induction of TR3 gene, implying the role of protein kinase A and C activation. CONCLUSION: Taken together, the present study demonstrates that TR3 gene was rapidly and transiently induced by LH in human luteinized granulosa cells. The results imply that TR3 may play a role in ovulation by initiating a cascade of ovulation-specific gene expression in response to LH.

Stimulation by EGF, bFGF and GnRH of Ovarian Pituitary Adenylate Cyclase-Activating Polypeptide Gene Expression in Cultured Rat Preovulatory Follicles / 대한불임학회지

No abstract available.