Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence..

The proliferation of embryonic cells or adult stem cells in tissue is critically regulated during development and repair. How limited the proliferation of cells, so far, is not much explored. Cell-cell contact proliferation inhibition is known as a crucial mechanism regulating cell proliferation in vitro and in vivo. In this study we examined the characters of mouse subcutaneous adipose derived stem cells (msADSC) whether they lost or get contact inhibition during in vitro culture. The characters of msADSC growth after confluence were analyzed using confocal microscope and the expression profiles of contact inhibition related genes were analyzed according to the morphological changes using real-time PCR method. msADSC showed overlapping growth between them but not after passage 14. The cell shapes were also changed after passage 14. The expression profiles of genes which are involved in contact inhibition were modified in the msADSC after passage 14. The differentiation ability of msADSCs to adipocyte, chondrocyte and osteocyte was not changed by such changes of gene expression profiles. Based on these results, it is revealed that smADSC were characterized by getting of strong cell-cell contact inhibition after passage 14 but the proliferation and developmental ability were not blocked by the change of cell-cell contact proliferation inhibition. These finding will help to understand the growth of adipose tissue, although further studies are needed to evaluate the physiological meaning of the cell-cell contact proliferation inhibition during in vitro culture of msADSC.

Manganese () is a trace element that is essential for normal physiology, and is predominantly obtained from food. Several lines of evidence, however, demonstrated that overexposure to exerts serious neurotoxicity, immunotoxicity and developmental toxicity, particularly in male. The present study aimed to evaluate the effect of 0, 1.0, 3.3, and 10 mg/kg/day doses of on the reproductive organs in the immature female rats. Rats (PND 22; S.D. strain) were exposed to () dissolved in drinking water for 2 weeks. The animals were sacrificed on PND 35, then the tissues were immediately removed and weighed. Histological studies were performed using the uteri tissue samples. Serum LH and FSH levels were measured with the specific ELISA kits. Body weights of the experimental group animals were not significantly different from those of control group animals. However, ovarian tissue weights in 1 mg and 3.3 mg dose groups were significantly lower than those of control animals (p<0.05 and p<0.01, respectively). Uterine tissue weights of 3.3 mg dose groups were significantly lower than those of control animals (p<0.01), while the 1 mg dose and 10 mg dose failed to induce any change in uterine weight. Similarly, only 3.3 mg dose could induce the significant decrease in the oviduct weight compared to the control group (p<0.05). Non-reproductive tissues such as adrenal and kidney failed to respond to all doses of exposure. The uterine histology revealed that the exposure could affect the myometrial cell proliferation particularly in 3.3 mg dose and 10mg dose group. Serum FSH levels were significantly decreased in 1mg dose and 10 mg groups (p<0.05 and p<0.01, respectively). In contrast, treatment with 1 mg dose induced a significant increment of serum LH level (p<0.05). The present study demonstrated that exposure is capable of inducing abnormal development of reproductive tissues, at least to some extent, and altered gonadotropin secretions in immature female rats. Combined with the well-defined actions of this metal on GnRH and prolactin secretion, one can suggest the might be a potential environmental mediator which is involved in the female pubertal process.

It is suggested that carbohydrate metabolites may involve in the development of morula to blastocyst but many of the mechanisms are not unmasked. Two-cell stage embryos were collected and examined the effects of lactate on the development of blastocyst in vitro. The expression profiles of lactate dehydrognase (Ldh) genes and aquaporin (Aqp) genes were analyzed with RT-PCR. The successful development from morula to blastocyst was dependent on lactate concentrations. The expression profiles of Ldh genes were changed by the lactate concentration. Ldha was expressed in morula stage at 10 mM lactate, and in blastocyst stage at lactate free condition. Ldhb was expressed in morula stage at 10 mM and 20 mM lactate, and in blastocyst stage at 10 mM lactate. Aqp genes were also showed different expression patterns by the lactate concentrations. Aqp3 was expressed in hatching embryo at 120 hr post hCG administration (hph) which was cultured in BWW medium and lactate free condition. Aqp7 was expressed in hatching embryos at 120 hph which was cultured at 10 mM lactate condition. Also Aqp8 was expressed in hatching embryo at BWW and 20 mM lactate condition. Aqp9 was expressed in morula at BWW and 10 mM lactate condition, and in blastocyst at BWW. Based on these results, it is suggested that concentration of lactate in the medium and the level of lactate synthesis in embryo is critical factor for blastocoels formation. In addition it is suggested that LDH may involve the AQPs expression in embryos

Hatching occurred in the time dependent manners and strictly controlled. Although, the hatching processes are under the control of muti-embryotrophic factors and the expressed G proteins of cell generate integrated activation, the knowledge which GPCRs are expressed during hatching stage embryos are very limited. In the present study, which G proteins are involved was examined during blastocyst development to the hatching stage. The early-, expanded-, and lobe-stage blastocysts were treated with various activators and H series inhibitors, and examined developmental patterns. Pertusis toxin (PTX) improved the hatching rate of the early-stage blastocyst and lobe-formed embryos. Cholera toxin (CTX) suppressed the hatching of the early-stage blastocyst and expanded embryos. The effects of toxins on hatching and embryo development were changed by the H7 and H8. These results mean that PTX mediated GPCRs activation is signaling generator in the nick or pore formation in the ZP. In addition, PTX mediated GPCR activation induces the locomotion of trophectoderm for the escaping. CTX mediate GPCRs activation is the cause of suppression of hatching processes. Based on these data, it is suggested that various GPCRs are expressed in the periimplantation stage embryos and the integration of the multiple signals decoding of various signals in a spatial and temporal manner regulate the hatching process.

Sex steroid hormones are key molecules to prepare the decidual response and their levels are important in this process. Imbalances of the levels of steroid hormones are cause of implantation failure and other diseases including physical weakness. Androgen replacement therapy or selective androgen receptor modulator are used to overcome various diseases but long-term use may cause of side effects. In previous report, it is suggested that the steroid hormonal complexes derived from pig enhance the proliferation of satellite cell. Therefore, to evaluate the possible usage of steroid hormonal complex derived from pig testis (tS-C), the effects of tS-C on uterine response were studied using the model of artificial decidua. tS-C did not disturb the rhythmical estrus cycle. Artificial-induced decidual response was normally induced in tS-C administered mice. The histological characters of the decidua of tS-C administered mice were not different from the vehicle. The expression patterns of molecular markers of decidua were not different between vehicle and tS-C group. Collectively these results suggested that tS-C does not disturb the uterine responsibility to the embryo. In addition, our results suggested that tS-C can be applied to overcome the various problems such as loss of muscle mass and anemia.

Lipid metabolites involved in cellular regulation as signaling mediators. Prostaglandins (PGs), metabolites of lipid are involved to pregnancy at the time of implantation but the functional roles of PGs on embryo development are still controversy and largely unknown. In previous report, the levels of and at embryos of morula stage and blastocyst stage were explored (Cheon et al., 1998). In this study, the previous suggestion was confirmed and the possible downstream mediator of prostaglandin and prostaglandin on the expansion and hatching of mouse embryo was examined. As expected, developmental rate of the blastocyst to expanded stage was a concentration-response curve that showed the highest expansion rate at 10 , but at 100 , the rate was decreased. In contrast to the , stimulated expansion without toxicity at highest concentration. Cotreatment of PGs with indomethacin overcame the inhibitory effects of indomethacin in expansion. Exogenous PGs also improved the development of expanded embryos to the hatching stage. Besides, PGs receptors' transcripts detected at blastocyst. was caused of calcium fluctuation in the blastocyst but did not. The changes of intracellular calcium concentration were different between indomethacin pretreated embryos and non-treated embryos. Based on these results it is suggested that PGs work as paracrine and/or autocrine factors through calcium and the others which were not identified in this study.

The tremendous changes of uterine endometrium are observed during early pregnancy and protease and their inhibitors are involved in regulation of cell proliferation and remodeling of the tissues through remodeling the extracellular matrix (ECM). Some of the proteases and protease inhibitors have been suspected to a factor in endometrial changes but many parts of their expression profiles and the physiological roles are not uncovered. To evaluate the functional roles of them, in this study the expression profiles of proteases and protease inhibitors were analyzed using real-time quantitative PCR analysis. Mmp9 (matrix metalloproteinase 9) mRNA levels peaked on day 4 at the time of implantation. On the other hand, Ela2 (neutrophil elastase, NE) mRNA levels were peaked on day 2 of pregnancy. Its expression were decreased until day 4 of pregnancy but increased rapidly until day 7 of pregnancy and decreased again. NE inhibitor Slpi (secretory leukocyte protease inhibitor, SLPI) mRNA levels were related with the implantation stage and with the levels of Ela2. At the time of implantation the expression levels of Slpi mRNA were about 5 times higher than the Ela2 mRNA in the uterus. In the implantation stage embryos, Mmp9 specific mRNA was only detected at the blastocyst. On the other hand, the expression level of SLPI was higher than that of the Ela2 mRNA at blastocyst and 4.5 day p.c. embryos. Based on these results it is suggested that MMP9, SLPI, and NE have important physiological role in embryo implantation both in uterus and embryos.

A fertilized oocyte can get the competence for implantation through cleavage and stage-specific gene expression. These are under the control of autonomous and exogenous regulators including physiological culture condition. Endogenous and exogenous growth factors are considered as critical regulators of cleaving embryos during travel the oviduct and uterus. In this study, an effort was made to evaluate comprehensively the quality of embryos for implantation, grown in media enriched with EGF and PAF. The study evaluated developmental rates on given time, blastulation and hatching rates, and adhesion rates. Developmental rates of blastocyst to the hatching stage were significantly high in PAF treated group compared to the control in a dose-dependent manner but not in EGF group. Implantation rates were significantly high both PAF and EGF in a dose-dependent manner. H7, a PKC inhibitor, blocked the process of hatching of the blastocysts but combined treatment of EGF and PAF enhanced the hatching and implantation of blastocsyts. Based on these results it is suggested that EGF and PAF support acquirement of implantation competence at blastocyst stage through a PKC pathway.

Pivotal roles of steroid hormones in uterine endometrial function are well established from the mouse models carrying the null mutation of their receptors. Literally androgen belongs to male but interestingly it also detected in female. The fluctuations of androgen levels are observed during reproductive cycle and pregnancy, and the functional androgen receptor is expressed in reproductive organs including uterus. Using high throughput methodology, the downstream genes of androgen have been isolated and revealed correlations between other steroid hormones. In androgen-deficient mice, uterine responses to exogenous gonadotropins are impaired and the number of pups per litter is reduced dramatically. As expected androgen has important role in decidual differentiation through AR. It regulates specific gene network during those cellular responses. Recently we examined the effects of steroid hormonal complex containing high level of androgen. Interestingly, on the contrary to the androgen-alone administration, the hormonal complex did not disturb the decidual reaction and the pubs did not show any morphological abnormality. It is suspected that the complexity of communication between other steroid hormone and their receptors are the reasons. In summary, androgen exists in female blood and it suggests the importance of androgen in female reproduction. However, the complex interactions with other hormones are not fully understood compared with estrogen and progesterone. The further studies to evaluate the possible role of androgen are needed and important to provide the in vivo rational for the prevention of associated pregnancy complications and help human's health.