• The Journal of Korean Obstetrics and Gynecology
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• v.24 no.1
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• pp.27-41
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• 2011

Objectives: This study was designed to investigate the effect of Ojeok-san(OJS) on the estradiol valerate(EV)-induced polycystic ovaries(PCO) in rats. Methods: PCO was induced by single intramuscular injection with estradiol valerate(EV)(4mg) in female rats. Normal group(n=8) were injected with sesame oil and orally administrated distilled water for sixty days. PCO control group (n=8) were injected with EV and orally administrated distilled water for sixty days. OJS treated group(n=8) were injected with EV and orally administrated OJS for sixty days. Then, we measured weights of body, ovaries, adrenal glands and uterus of rats. The histomorphometrical changes of ovaries were also evaluated. And we observed the NGF and CRF expression by immunohistochemistry. Results: The results were as follows - The weights(mg) of ovaries in OJS treated group($57.4{\pm}9.4$) were significantly increased(p < 0.05) compared with PCO control group($42.3{\pm}8.5$). - The numbers of mature follicles in OJS treated group($9.3{\pm}2.5$) were significantly increased(p < 0.05) compared with PCO control group($6.1{\pm}2.1$). - The expressions of NGF-immunoreactive cells in the ovarian granulosa cells in OJS treated group were weaker than PCO control group. Conclusion: From the these results, we concluded that Ojeok-san(OJS) has inhibitory effect on the development of EV-induced polycystic ovary. And it's effect maybe related with decreased NGF activities in the ovary.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.15-23
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• 2006

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.

• The Korean Journal of Zoology
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• v.33 no.4
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• pp.435-445
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• 1990

Gonadotropin releasing horrnone (GnRH) is known to be extrahypothalamically localized with a broad range including gonad. It remains, however, unknown whether GnRH is locally synthesized in the gonad. The present srudy aims to identity expression and cellular localization of GnRH-Iike mRNA and immunoreactive GnRH in the rat gonad. GnRH radioimmunoassay and chromatographic extracts on G-50 sephadex column showed that rat gonadal extracts contained a substantial amount of immunoreactive GnRH similar to the hypothalamic and synthetic GnRH. Although a wide distribution of immunostainable GnRH-like molecule with different cell types in the rat ovary was observed, the major cell population hybridized with GnRH probe appears to be granulosa. theca cells and corpus luteum. Immunoreactive GnRH-Iike peptides were distributed m various regions of testis, including spermatogenic cells, Sertoli cells and Leydig cells. In situ hybridization revealed that positive signals of GnRH-Iike mRNA were predominandy present in Sertoli cells within some seminiferous tubules, but absent in the outside of seminiferous tubules in the testis. This study clearly demonstrated that GnRH-Iike molecule present in the rat gonad may be resulted from the local synthetic machinery of GnRH supporting the notion that this peptide may act as autocrine and/or paracrine role in intra-gonadal communication.

• Development and Reproduction
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• v.21 no.2
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• Development and Reproduction
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• v.2 no.2
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• pp.173-178
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• 1998

Leptin, the product of the obese gene, is produced by adipose tissue and is known to be a hormone concerned with regulation of appetite and metabolism. Recent reports have shown that leptin is associated not only with obesity but also with female reproduction, but it has not yet been ascertained whether leptin acts directly on the ovaries or indirectly via the hypothalamus or pituitary pathway. The object of this study is to determine the expression of leptin and its receptor in the ovaries of 3 and 8 weeks old rats by immunohistochemistry and RT-PCR. In the ovaries of 3 and 8 weeks old rats, leptin was stained in the theca cells and portions of granulosa cells of atretic follicles, whereas leptin receptors was stained in interstitial cells and ova of preantral follicles. The RT-PCR results showed that leptin receptor mRNA was expressed in the ovaries of both immature and adult rats, while leptin mRNA was not. In conclusion, leptin mRNA was not expressed in the ovaries, however, leptin was detected by immunohistochemistry. Compared to leptin itself, leptin receptors in the ovaries were ascertained by both RT-PCR and immunohistochemistry. These results suggest that leptin is related to the regulation of the physiological functions of the ovaries.

• Development and Reproduction
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• v.6 no.2
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• pp.123-129
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• 2002

Leptin, a product of the obese gene, is associated not only with obesity but also with female reproductive function, but it has not yet been ascertained whether leptin acts directly on the ovary or indirectly via the hypothalamus-pituitary pathway. Therefore, the object of this study was to investigate the expession of leptin and its receptor in the rat ovary by immunohistochemistry and RT-PCR during the estrous cycle. Immunohistochemistry results showed that leptin was stained in the theca cells and in part of granulosa cells in atretic follicles, whereas leptin receptor was localized in the interstitial cells and ova in preantral follicies. In particular, leptin and its receptor in atretic follicles displayed more intensive staining compared to those in normal follicles. During the estrous cycle, the mRNA expression of leptin and its receptor in the ovary was detected by RT-PCR and estradiol, progesterone, and leptin levels in the serum was measured by ELISA. The leptin level in the serum on metestrous phase was significantly higher than that on estrous phase. Similar to leptin level, progesterone level increased on metestrous phase. Leptin mRNA was not detected throughout the estrous cycle, whereas leptin receptor mRNA was expressed on all phases of estrous cycle excepting the diestrous phase. These results suggest that leptin might be directly involved in the regulation of ovarian function in rat.

• The Journal of Korean Obstetrics and Gynecology
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• v.24 no.3
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• pp.48-72
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• 2011

Objectives: This study was designed to investigate the effects of Yongdamsagantang on the polycystic ovary(PCO) induced by estradiol valerate(EV) in rats. Methods: After administrating Yongdamsagan-tang to PCO induced rats, we measured the weight of body, ovaries, adrenal glands, and uterus of rats. The observation through naked eye and histopathological observation of ovaries were evaluated. Also, the number of follicle and corpora lutea and content of androstenedione(ADD) and total estrogen were evaluated. The expressions of nerve growth factor(NGF) and corticotropin releasing factor(CRF) were analyzed by immunohistochemistry. The breeding rate and number of implantation with normal male rats were evaluated. Results: - The weight(mg) of ovaries in YST treated group($73.8{\pm}7.6$) was significantly increased(p<0.001) compared with control group($54.3{\pm}4.5$). - The number of mature follicles in YST treated group($7.3{\pm}2.4$) was significantly increased(p<0.01) compared with control group($3.5{\pm}1.2$). - The number of atretic follicles in YST treated group($9.0{\pm}1.5$) was significantly decreased(p<0.01) compared with control group($13.4{\pm}3.8$). - The number of cystic follicles in YST treated group($3.1{\pm}1.1$) was significantly decreased(p<0.01) compared with control group($6.0{\pm}2.0$). - The number of corpora lutea in YST treated group($3.8{\pm}2.1$) was significantly increased(p<0.001) compared with control group($0.3{\pm}0.7$). - The expression of NGF-immunoreactive cells in the ovarian granulosa cells in YST treated group was lesser observed than control group. - The expression of NGF-immunoreactive cells in the adrenal cortex in YST treated group was lesser observed than control group. - The breeding rate in YST treated group(100 %) was significantly increased (p<0.05) compared with control group(50 %). - The number of implantation in YST treated group($6.4{\pm}4.7$) was significantly increased(p<0.05) compared with control group($1.4{\pm}2.6$). Conclusions: We concluded that Yongdamsagan-tang activates the maturation of follicles, normal ovulation, breeding rate and number of implantation. Therefore, this may be effective for the treatment of anovulation, amenorrhea and sterility of PCOS patients.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.