• Development and Reproduction
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• v.5 no.2
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• pp.123-129
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• 2001

When mammalian oocytes undergo maturation, cumulus cells surrounding the oocyte exhibit remodeling of their structure known as cumulus expansion. Many molecules including hyaluronic acid participate in this remodeling. The present study aimed to investigate a possible existence of matrix metalloproteinases(MMPs) in the extracellular matrix(ECM) of human oocyte-cumulus complex. ECM was extracted from the human oocyte-cumulus complex. Gelatin gel zymogram of ECM exhibited 7 gelatinases having molecular weight of 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, and 84kDa. This gelatinase profile was very different from that of ovarian mural granulosa cell extract or white blood cell extract, indicating that the oocyte-cumulus complex donating ECM was free from other than cumulus cells. When ethylenediaminetetraacetic acid or 1', 10'-phenanthroline was added to the reaction buffer during zymographic development, almost gelatinase activities were abolished, suggesting that they were MMPs. Following incubation of ECM in the presence of aminophenylmercuric acetate, an activator of proMMPs, 4 gelatinases of 240kDa, 180kDa, 97kDa, and 84kDa disappeared with the concomitant appearance of 80kDa, 65kDa, and 60kDa gelatinases. Based upon these observation, it is suggested that ECM of the human oocyte-cumulus complex consists of gelatinases, presumed to be MMP-2 and MMP-9 isoforms.

• Development and Reproduction
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• v.7 no.2
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• pp.127-136
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• 2003

The present study was conducted to investigate gene expression profile of mouse ovaries during the primordial-primary follicle transition. We isolated total RNA from mouse ovaries at day1(contains only primordial follicles) and day5(contains both primordial and primary follicles) and synthesized cDNA using annealing control primers(ACP, Seegene, Inc., Seoul, Korea). Using 80 different ACPs for PCR, we cloned, sequenced, and analyzed identities of 41 differentially expressed genes(DEGs). According to BLAST analysis, sequences of 33 clones significantly matched database entries, 4 clones were novel, and 4 clones were ESTs. We selected 8 DEGs with interesting functions, Anx11 and Pepp2-Pending highly expressed in day1 ovary, while Apg3/Autlp-like, BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3, Suil-rsl, and TIAP/m-survivin highly expressed in days ovary, and confirmed their different expression between day1 ovaries and days ovaries using semi-quantitative RT-PCR. There was no false positive result. Using in situ hybridization, we found that almost all of genes studied were expressed in the oocyte from primordial follicle stage but expression decreased from primary follicle stage. Meanwhile their expression was increased in cuboidal granulosa cells. Different expression of BPOZ and TIAP/m-survivin between primordial and primary follicles was confirmed by using laser capture microdissection followed by real-time PCR BPOZ and TIAP/m-survivin expressed 4.5 and 3.4 fold higher in primary than primordial follicles, respectively. List of genes obtained from the present study will provide insights for the study of mechanism regulating primordial-primary follicle transition.

• 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.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.131-138
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• 1995

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 $\mu$g /mL of FSH and LH and 1 $\mu$g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.167-173
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• 2008

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-$\beta$, EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-$\beta$ gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.181-186
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• 2007

Inhibitor of DNA binding protein or inhibitor of differentiation(Id) is largely considered as positive and/or negative regulators of proliferation, differentiation, angiogeneisis, and apoptosis. The four Id genes(Id1, Id2, Id3, and Id4) were known in mammals. However, little is known about the expression and function of these genes in reproductive physiology. Among them, this study was conducted to analyze the expression pattern of Id3 mRNA on folliculogenesis in rat ovary. After PMSG administration, the ovaries were obtained at 3, 6, 12, 24, 36, and 48hrs, fixed, dehydrated, and paraffin embedded. For in situ hybridization, anti-sense and sense Id3 cRNA probes were prepared and applied to the ovarian section. The ovarian sections were coated with NTB-2 emulsion. After that, the slides were developed and counterstained with hematoxylin and eosin staining. The hybridization signal was estimated on a scale of 1+ to 4+. In oocyte, the intensity of Id3 mRNA in primordial and primary follicles was scored at ${\geq}2+$, but the intensity was less than 1+ in secondary, dominant, and preovulatory follicles. In granulosa cells, the Id3 mRNA was strongly expressed(3+ or 4+) in dominant and preovulatory follicles. Taken together, Id3 mRNA was expressed specifically at follicle stages and follicular tissue and might be closely related with follicle development.

• Applied Microscopy
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• v.33 no.2
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• pp.117-130
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• 2003

This research investigates the fine structural as well as the morphological changes of the mouse ovarian tissue after irradiation of various dose rates of 6 MeV LINAC radiation. The normal structure of the ovarian tissue is consisted of various stages of follicles including primordial and growing follicles, and ovarian stromal connectives. When we observed the ovarian tissues irradiated with a dose rate of 200 cGy/min using light and electron microscopes, granular cells in growing follicles are in irregular shape unlike normal follicles. Small segments of cells scattered in follicular antrum among granular cells. We could observe neutrophils and macrophages around the segments, which means the cells already got in the process of decease owing to the effects radiation. With coincident to the increase of the dose rate of x-ray irradiation as 400 or 600 cGy/min, the mature follicles appeared as an irregular form and the granular cells surrounding oocyte also deformed comparing to their normal counterparts. The granulosa cells within mature follicle are already occurred necrotic change and apoptosis. The nuclei in some cells got so fragmented that the segments formed the shape of a horseshoe or scattered in small and condensed pieces. All the cells at a granular layer irradiated with a dose rate of 600 cGy/min show typical characteristics of apoptosis. The neutrophils involved in inflammatory reaction appear evidently in follicular antrum of growing follicles, and macrophage scattered with residual and apoptotic bodies.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• The Journal of Korean Obstetrics and Gynecology
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• v.20 no.1
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• pp.99-113
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• 2007

Purpose : This study was designed to investigate the effects of Jeongkyeong-Tang(JKT) on the polycystic ovary(PCO) induced by estradiol valerate(EV) in rats. Methods : PCO was induced by single intramuscular injection with 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. JKT treated group(n=8) were injected with EV and orally administrated JKT for sixty days. Then we measured weights of body, ovaries and adrenal glands, and measured content of serum estrogen. The histomorphometrical changes of ovaries were also evaluated. The expressions of nerve growth factor(NGF) were analyzed in the central nervous system, adrenal glands and ovaries by immunohistochemistry. Results : - The weights(mg) of ovaries in JKT treated group (69.7${\pm}$6.7) were significantly increased( p<0.001) compared with PCO control group(46.7${\pm}$12.2). - The numbers of secondary follicles in JKT treated group(4.00${\pm}$l.31) were significantly increased(p <0.05) compared with PCO control group(2.25${\pm}$1.39). - The numbers of mature follicles in JKT treated group(5.50${\pm}$1.51) were significantly increased(p<0.01) compared with PCO control group(2.88${\pm}$1.13). - The numbers of atretic follicles in JKT treated group(2.75${\pm}$l.16) were significantly decreased(p<0.001) compared with PCO control group(6.88${\pm}$2.03). - The numbers of corpora lutea in JKT treated group(4.13${\pm}$1.46) were significantly increased(p<0.01) compared with PCO control group(2.13${\pm}$1.46). - The contents(pg/ml) of serum estrogen in JKT treated group(115.18${\pm}$18.29) were significantly decreased(P<0.01) compared with PCO control group(153.06${\pm}$29.47). - The expressions of NGF-immunoreactive cells in the ovarian granulosa cells in JKT treated group were lesser observed than PCO control group. Conclusion : From the above results, we concluded that Jeongkyeong-Tang has inhibitory effect on the development of EV-induced polycystic ovary. And it's effect may be related with decreased NGF activities in the ovary.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.135-141
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• 2011

Objective: Ovarian angiogenesis plays an important role in folliculogenesis. However, little is known about the expression of angiogenic factors during follicular development according to female age. Stromal cell derived factor-$1{\alpha}$ (SDF-$1{\alpha}$) plays a role in granulosa cell survival and embryo quality as an angiogenic chemokine. Leptin is also involved in folliculogenesis and angiogenesis. This study examined expression of SDF-$1{\alpha}$ and leptin, and their effects on the expression of angiogenic factors in the ovary during follicular development according to female age. Methods: Ovaries were collected from C57BL mice of two age groups (6-9 weeks and 24-26 weeks) at 6, 12, 24, and 48 hours after 5 IU pregnant mare's serum gonadotropin (PMSG) injection. The expression of ovarian SDF-$1{\alpha}$ and leptin mRNA was evaluated by RT-PCR. In the organ culture experiment, the ovaries were cultured in transwell permeable supports with Waymouth's medium treated with various doses of SDF-$1{\alpha}$(50-200 ng/mL) or leptin (0.01-1 ${\mu}g$/mL) for 7 days. Then, mRNA expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and visfatin were examined in the cultured ovaries. Results: Expression of SDF-$1{\alpha}$ and leptin in the ovary was significantly lower in the aged mouse group compared to the young mouse group ($p$ <0.05). Expression of these two factors increased with follicular development after PMSG administration. SDF-$1{\alpha}$ treatment stimulated visfatin expression in a dose-dependent manner, while leptin treatment significantly increased eNOS expression. Conclusion: These results suggest that decrease of ovarian SDF-$1{\alpha}$ and leptin expression may be associated with aging-related reduction of ovarian function. SDF-$1{\alpha}$ and leptin may play a role in follicular development by regulating the expression of angiogenic factors in mouse ovaries.