During implantation period uterus undergoes functional and histological changes including cell proliferation, differentiation, apoptosis. This delicate condition is regulated by various factors such as steroid hormones, cytokines, and growth factors. From previous our studies, we showed the spatio-temporal expression of Müllerian inhibiting substance (MIS) and MIS receptor Ⅱ (MISR II) in mouse uteri during pregnancy. In present study investigated the role of MISRⅡ in proliferation and differentiation in the uterus. The decidualization markers were dramatically decreased during treated rhMIS and MISRⅡ viral particles in in vitro decidualization model. The intensity of PAS staining and Oil-Red-O staining were dramatically decreased by treatment of rhMIS and overexpression of MISRⅡ. MIS/MISRⅡ also suppressed stromal proliferation regulate the differentiation of stroma cell during decidualization. MIS suppress the stromal proliferation and decidual differentiation along with its receptor. Put together it is suggested that MIS works as paracrine and autocrine factors for cell proliferation regulator and differentiation regulator during implantation.

Early growth response 1 (Egr1) is an inducible zinc finger transcription factor. Egr1 binds specific GC-rich region. Egr1 mRNA is rapidly and transiently expressed in pre-ovulatory follicles by LH and expressed in decidual cell by estrogen. Progesterone receptor (PR) is a nuclear transcription factor that is induced in granulosa cells of pre-ovulatory follicles following the LH surge. PR regulates ADAMTS1, which downstream gene of PR. In previous study, we observed ADAMTS1 mRNA expression pattern changed in Egr1 KO mice. Therefore, we expected that progesterone receptor gene expression is directly regulated by early growth response 1 in mouse ovarian granulosa cell. We could found the ER binding domain, Egr1 binding domain and CAAT box in PR promoter using the web tool AliBaba 2.1. We construct the PR promoter vectors truncated ER binding domain, Egr1 binding domain, CAAT box, respectively. We also construct the Egr1 expression vector using pcDNA 3.1 (+) vector. Granulosa cells are isolated from female ICR mice after 12h PMSG injection. To confirm the Egr1 overexpression, we performed western blot. For reporter assay, we used Dual-Luciferase reporter assay system. In conclusion, Egr1 may regulate PR expression in granulosa cell.

Environmental conditions during early mammalian embryo development are critical and some adaptational phenomena are observed. However, the mechanisms underlying them remain largely masked. Previously, we reported that AQP5 expression is modified by the environmental condition without losing the developmental potency. In this study, AQP11 was examined instead. To compare expression pattern between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP11 by whole mount immunofluorescence. When the fertilized embryos were developed in the maternal tracts, the level of Aqp11 transcripts was decreased dramatically until 2-cell stage. Its level increased after 2-cell stage and peaked at 4-cell stage, but decreased again dramatically until morula stage. Its transcript level increased again at blastocyst stage. In contrast, the levels of Aqp11 transcript in embryos cultured in vitro were as follows. The patterns of expression were similar but the overall levels were low compared with those of embryos grown in the maternal tracts. AQP11 proteins were localized in submembrane cytoplasm of embryos collected from maternal reproductive tracts. The immune-reactive signals were detected in both trophectoderm and inner cell mass. However, its localization was altered in in vitro culture condition. It was localized mainly in the plasma membrane of the blastocysts contacting with external environment. The present study suggests that early stage embryo can develop successfully by themselves adapting to their environmental condition through modulation of the expression level and localization of specific genes like AQP11.

Adipogenesis is critical in development and homeostasis of energy metabolism. However, in these days, the obesity has become prevalent and became a cause of medial complication. Various applications have been suggested to prevent or decrease accumulaiton of energy in fat cells. However, those have little usefulness and have various side effects. Diphlorethohydroxy-carmalol (DPHC) is a phlorotannin compound, with various biological activities in vitro and in vivo. In here, we studied that DPHC could modify the accumulation of fat on integument. The size of adipocytes and thickness of the subcutanous fat tissue was analyzed after treatment of cosmetics contained 0, 0.01, 0.1, 1, or 10 % DPHC using NIS Element D 4.10.00 software (Nikon). The viability and proliferation of cell was analyzed after 0, 0.4, 2, 10, or 50μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay (R&D system, Cat # 48090-025-k) and measurment of doubling time. Accumulation of lipids in differentiating preadipocytes was analyzed with spectrophotometer after Oil Red-O staining. The size of adipocyte and thickness in skin was decreased in DPHC treated mice. The metabolic activity and doubling of 3T3-L1 were suppressed by DPHC in concentration dependent manner. DPHC also inhibit accumulation of lipids in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC has antiobesity effects in integument through suppress accumulation of lipids and suppress the proliferation and differentiation both of adipose stem cells and precursor cells.

Developing preimplantation embryos require appropriate energy source and express stage specific gene expression for proper development. During early stage embryo development, major energy sources were pyruvate and lactate, after then glucos is used as a main source. Aquaporins (AQPs), also, is suggested as key molecules for blastocoels formation, and energy and meytabolic homeostasis. In this study, we analyzed the expression profiles of AQPs and lactate dehydrogenase (LDH) which convet lacte to pyruvate and back. During development in vivo condition, the expression patterns can be classificed six clusters. AQP2,-3, -5, -8, -9, and -11 were detected at various stages but others were not. Cluster 1 is for an only express at blastocyst stage. Cluster 2 is for an incrase continuosuly from 4-cell stage. Cluster 3 is for a peak at both 4-cell and blastocyst stages. Cluster 4 is for a sharp decrease at morula stage. Cluster 5 is for a sharp decrase at 2-cell and morula stages. Cluster 6 is for continuous decrease after 4-cell stage. Cluster 7 is for no expression AQPs. LDHs expression has three patterns. First is for sharp decrase at both 2-cell stage and morula a stage. Second is for a continuous decrease from 4-cell stage. Third is for an existings until fertilized oocyte, 1-cell stage. Interestingly the expression profiles of AQPs and LDHs were totally changed by in vitro culture. All of the AQPs and LDHs were detected except AQP8. The leves of LDHA and LDHB were significantly decreased in vitro but those of LDHC and LDHD were increased. These results suggest that early stage embryos themselves adaptate to their conditon through modulation of the specific gene expression such as AQPs and LDHs.

The biological activity of tissue specific stem cell is under the control of their specific microenvironment and the exogenous chemicals derived from digestive tract can be one of the constructing factors of that. It is suggested that the extract of brown algae Ishige okamurae has antioxidant-, apoptosis induction-, and antiinflammatory- effects. On the other hand, a few studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied the effect of the extract of I. okamura on the cellular activity and differentiation of 3T3-L1 preadipocyte to adipose cell. The viability of cell was analyzed using 3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Adipogenesis of 3T3-L1 cell was analyzed after induction in the induction medium containing the I. okamurae extract. The cellular activity was high compared with the vehicle and 0.05 mM caffeine in all groups of I. okamurae extract treated cells. The extract of I. okamura inhibited accumulation of lipids in 10 and 50 μg/ml. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. These results suggest that the extract of I. okamurae increases the cellular viability of adipose precursor cells. On the other hand, it suppresses the differentiation of preadipocyte to adipocyte and accumulation of lipids in concentration-dependent manners. It may be possible that the major component of the extract can be applied in the control of adipose tissuegenesis.

Although, one of the etiologies of localized lipodystrophy of the subcutaneous connective tissue (cellulite) is the histological alternation of adipose tissue, the characteristics of expression of the components of extracellular matrix (ECM) components during adipogenesis are not uncovered. In this study, the effects of caffeine and Ishige okamurae originated diphlorethohydroxycarmalol (DPHC) on the expression of extracellualr fibers was analyzed with quantitative RT-PCR during differentiation induction of mouse subcutaneous adipose derived stem cells (msADSC) into adipocyte. The expression levels of Col1a, Col3a1, and Col61a were decreased by the adipogenci induction in a time-dependent manners. However, Col2a mRNA and Col4a1 mRNA expressions were oposit to them. Caffeine and DPHC stimulated the changes of the expression of these collagens. Eln mRNA expression was increased by induction. DPHC stimulated the expression of it. Mfap5 mRNA expression was deceased in both adipogenic cell and matured adipocytes. Caffeine suppressed the expression of Mfap5 but the effect of DPHC was different by the concentration. The expression of bioglycan, decorin, and lumican were also modified by caffeine and DPHC in a concentration-dependent manner. Based on this study, we revealed firstly the effects of caffeine and DPHC on the expression of collagens, elastin, and glycoproteins during adipogenesis of msADSCs. Those results suggest that DPHC may have antiadipogenic effect and has more positive effets on normal adipose tissue generation and work as suppressor the abnormality of ECM structure. Such results indicate that DPHC can be applied in keeping the stability of the ECM of adipogenic tissues.

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12－14 day old mice and these were cultured individually in α-minimal essential medium (α-MEM) supplemented with 5% fetal bovine serum (FBS), 100 mIU/㎖ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, 100 μg/ml penicillin and 50 ㎍/㎖ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

Brown algae is variety of biological compounds, including xanthophyll, pigments, fucoidans, phycocolloids, and phlorotannins. Several studies concerning these types of compounds have pointed out the variety of biological benefits associated with the algae, including antioxidant, anticoagulant, antihypertension, antibacterial, and antitumor activities. Diphlorethohydroxy- carmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. Numerous studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied that effect of DPHC isolated from Ishige okamurae modified the accumulation of fat on preadipocyte, 3T3-L1 cells in vitro. First, the viability of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Second, proliferation of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment through measure doubling time. 3T3-L1 cell differentiation into adipocyte was analyzed after induction in the induction medium containing DPHC. The metabolic activity was suppressed by DPHC in concentration dependent manner. Doubling of 3T3-L1 was delayed by the treatment of DPHC in concentration dependent manner. DPHC also inhibit accumulation of triglyceride in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC suppress proliferation of adipose precursor cell and differentiation into adipocytes.

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are widely distributed in the human diet through crops, beans, fruits, vegetables and red wine. The specific health effects that anthocyanins might have in vivo are not known, although there are several possibilities related to obesity, cardiovascular disease, and cancer. In this study we used human subcutaneous adipose mesenchymal stem cells (hADSC) and mouse subcutaneous adipose mesenchymal stem cells (mADSC) to evaluate the effects of anthocyanins. And we examined the effect of cell activity and adipocyte differentiation by Cyanidin-3-O-glucoside (C3G), Delphinidin-3-ß-D-glucoside (D3G) that are among the anthocyanin family and black soybean extract. Using MTT assay method, we tested cellular metabolic activity. In mADSC, cell activity is significantly decreased by C3G and D3G (50 uM, 100 uM, and 200 uM), and black soybean extract (100 ug/ml and 200 ug/ml). In hADSC, cell activity is significantly decreased only by C3G (50 uM, 100 uM and 200 uM) unlike in mADSC. Cell activity is significantly increased of 100 uM D3G and black soybean extract (50 ug/ml, 100 ug/ml and 200 ug/ml). In mADSC, 50 uM C3G promoted differentiation into adipocyte but no effect in other concentration. D3G suppressed the differentiation of mADSC at 100 uM and 200 uM. 50 ug/ml black soybean extract promoted differentiation of mADSC, but 200 ug/ml black soybean extract suppressed differentiation. In hADSC, 50 uM, 100 uM and 200 uM C3G suppressed differentiation. 100 uM D3G promoted differentiation into adipocyte, but 200uM D3G suppressed it. Black soybean extract suppressed the differentiation into adipocyte at 50 ug/ml, 100 ug/ml and 200 ug/ml. These data showed that the responsibility to the C3G and D3G were different between hADSC and mADSC. Interestingly the responsibility to the black soybean extract was similar between hADSC and mADSC. Based on them, it is suggested that there are species-specificity to the cellular responsibility to the anthocyanins in subcutaneous ADSC.

During implantation, endometrial cells undergo functional and structural changes, and support the successful embryo development. This reaction is known as decidualization and is critical to placental formation and to prevent the uterine functions. This progress is achieved by complex communication of regulators such as hormones, cytokines and growth factors. Some of the TGF-b superfamily members such as inhibin, activin, TGF-β, and bone morphogenetic proteins (BMP) involve in uterine modulation during pregnancy. Müllerian inhibiting factor (MIF) is a member of TGF-β superfamily and regulate folliculogenesis, but its expression and roles in uterus are not clear. In this study, we investigated the expression of MIF and its receptor Ⅱ in decidualizing endometrium. Interestingly its expression was detected in the fully decidualized cells. Its receptor II was detected in undifferentiated stromal cells. MIF expression was increased by decidual maturation and MIF receptor II was decreased by decidual reaction. MIF expression was induced by estrogen and its receptor II was increased by only progesterone in the stroma cells primed with estrogen. In the uterus of delayed implantation model mice, MIF expression was peak after 6 hr of estrogen administration. MIF receptor II expression was not induced. It means that MIF and MIF receptor II are expressed in the stroma cells with the specificity on physiological status. Based on them, it is suggested that MIF may work as paracrine factors in uterus during decidualization.

Keeping the intact germinal vesicle (GV) is essential for maintaining the capacity of mammals including human. It is maintained by very complex procedures along with folliculogenesis and is a critical step for getting competent oocyte. So far, a few mechanisms involved in folliculogenesis are known but GV arrest mechanisms are largely unrevealed. Cyclic AMP, a adenosine derived substance, have been used as inhibitor of germinal vesicle breakdown as a putative oocyte maturation inhibitor. In this study, we examined the potency of adenosine as GV maintainer and a possible signaling mediator for that. A1, A2b, and A3 were detected in cumulus cells of cumulus enclosed-oocyte (CEO). Intact of germinal vesicle was not kept like in follicle but the spontaneous maturation was inhibited by exogenous adenosine. It is inhibited with concentration dependent manners. Intracellular calcium level of cumulus was extensively increased after adenosine treatment. Based on these results it is suggested that one of the pathway for GV arrest by adenosine and its receptors is calcium mediated signaling pathway in CEO.

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.

Sulfotransferase 1E1 (SULT1E1, EST) is responsible for the sulfation and inactivation of betaestradiol at physiological concentrations. SULT1E1 null mice have reduced fertility by the disfunction of placenta and ovulation. Based our previous data it was revealed that the ovulation ability of 6-month old tiEsr1KO female mice is similar with the SULT1E1 female mice. In this study the possible relations in ovulation between estrogen and SULT1E1 was examined using real-time PCR and RIA methods in tiEsr1KO model mice. During the induction of superovualtion SULT1E1 gene expression peaked just before ovulation (6 hr posthCG injection) in control mice. As expected the expression patterns were similar between control and 4-weelk old tiEsr1KO female mice. Serum levels of E2 were increased in both tiEsr1KO mice and wild type mice but its level was higher 3 times in tiEsrKO mice than the wild type. Its levels became same between them after hCG administration. On the other hand, 6-month old mice shows the the dramatical increasing the SULT 1E1 expression during folliculogenesis, their expression was increased more than 100 times after hCG 6hr compared with PMSG 12hr control. In contrast with the 4-week old tiEsr1KO female mice, the expression levels of SULT1E1 gene expression were higher levels more than 20 times at 12 hr post PMSG injection. On the other hand, the expression levels of the other SULT family genes, SULT1A1 and SULT2A1 were very low compared with the SULT1E1 and did not show fluctuation during that period. Based on these results, it is suggested that the functional roles of estrogen during folliculogenesis may be regulated by SULT1E1through the metabolism of 17-beta estradiol and that the expression of SULT1E1 gene may be under the control of the levels of estrogen.

Apoptosis of the cell is one of the key steps in tissue remodeling and functional differentiation. At the time of implantation there are dramatically remodeling and functional differentiations of uterine endometrium. Decidualization is the process by which the uterine stromal cells proliferate and differentiation into morphologically and functionally distinct decidual cells. Bax is one of the key molecules in apoptotic process during that time. It is known that Bax expression have characters of time specificity and tissue specificity but the role of implantation is not largely unknown. The aim of this study was to examine the embryo implantation and decidualization reaction in order to understand the role of pro-apoptotic Bax gene as a major regulator of apoptosis of decidual cells. Some of Bax knockout mice have coat pigmentation defects involving white color. The number of blue bands after embryo transfer (ET) was not different between the control and Bax-knockout (Bax-KO) black coat (BC) and white coat (WC). Decidua were normally differentiated in both control and Bax-KO BC, but deciuda were not detected in Bax-KO WC at 96 hr post ET. Moreover, embryos of Bax-KO WC stayed in dormant stage on the embryonic day 7.5 and day 9.5. These results suggest that Bax and its related unknown genes may be a key molecule in implantation and uterine decidual reaction.

Hematopoietic stem cells (HSCs) can self-renew and can differentiate to a variety of specialized blood cells). The proliferation and homing of HSCs are strictly regulated both in the system level and local level. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, potent species-specific stimulator of granulocyte-macrophage, eosinophil, megakaryocyte and erythroid progenitors. In clinical purpose GM-CSF has been used as hematopoietic growth factor. It can promote the mobilization of HSC from bone marrow to peripheral blood. The number of HSCs mobilized into blood can be modulated by the kinds of cytolines. However, the information for the cytokine which promote mobilization is limited. Basic fibroblast growth factor (bFGF or FGF-2) induces the change of niche and affects the maintenance and differentiation of HSCs. FGF-2 positively regulates hematopoiesis, by acting on stroma cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. In this study, we investigated the effect of FGF-2 on HSCs mobilization and proliferation compare to GM-CSF. GM-CSF and FGF-2 were injected for 2 or 5 days into peritoneum of CD-1 mice (6～8 wks old) and sampling the bone marrow and peripheral blood. The bone marrow cells and peripheral blood were analyzed using FACS. In GM-CSF group, the number of HSCs was significantly increased by 2 days of injection but was significantly decreased in 5 days of injection. On the other hand the number of HSCs was significantly increased by the administration of FGF2 both in 2 days and 5 days. GM-CSF and FGF-2 are all increased the number of HSC both in bone marrow and peripheral blood. From these results, it is revealed that chronic administration of GM-CSF does not cause of the increase the number of HSCs. On the other hand, FGF2 can stimulate the proliferation of HSC without inhibition by the treatment period. It is suggested that GM-CSF and FGF2 may use different mechanisms to stimulate the HSCs proliferation. IP: 220.149.***.

Estrogen related receptor β(Esrrb)는 오르판 수용체 중 하나로 전분화능 관련유전자인 Oct4와 Nanog의 발현을 조절함으로써 줄기세포의 미분화를 유지시키고, 지속적인 자기 복제를 가능케 하는 유전자로 알려져 있다. 또한 Feng 등 (2009)은 체세포에 Oct4, Sox2와 함께 Esrrb 유전자를 함께 도입하면, 유전자가 변형된 체세포가 배아 줄기세포와 유사한 유도만능줄기세포로 리프로그래밍(reprograming)되어 진다는 결과를 보고한 바 있다. 본 연구에서는 인간 ESRRB 단백질을 양수유래줄기세포 내로 직접도입하는 방법을 개발하고, 이를 통해 전분화능 관련유전자의 기능 조절을 확인하고자 하였다. 클로닝 된 인간 short-form ESRRB를 세포투과 펩타이드(cell-penetrating peptide, CPP)의 일종인 R7(아르기닌 7개)에 접합(Fusion)하였고, 합성단백질 (R7-ESRRB-His6)의 형태로 배양중인 인간 양수 유래 줄기세포에 처리하여 세포내로 도입하였다. R7-ESRRB-His6 단백질은 5시간 내에 세포막을 통과하였고, 24시간 내에 핵 내로 이동하였다. 또한 핵 내로 이동한 ESRRB 단백질은 OCT4와 NANOG 유전자의 발현을 증가시켰을 뿐만 아니라, 또 다른 전분화능 관련유전자인 SOX2의 발현도 함께 증가시킨다는 것을 확인하였다. 이상의 결과는 세포투과 펩타이드와 유전자의 접합을 통해 생산된 R7-ESRRB-His6 합성단백질이 양수유래줄기세포내로 원활하게 도입되는 것을 확인하였고, 유전자의 변형 없이 전분화능 관련유전자의 기능을 조절할 수 있는 방법임을 확인하였다.

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.

Insulin like3 (INSL3, Relaxin like factor) is a critical regulator in testis translocation through Leucin rich G-protein coupled receptor 8 (Rxfp2,LGR8) during embryogenesis. In female, INSL3 and their receptor expressed in growing follicle and revealed their function in oogenesis. However, its role is not much evaluated. 6 weeks old C57BL/6 female mice used for measure the expression of INSL3 mRNA and their receptor expression by follicular stage. Follicle cell specific RNA were got from the theca cell and granulose cells which were isolated using LCM. To know the role of INSL 3 in theca cell, Rxpf2 were overexpressed during primary culture of theca cells, which were isolated from tertiary follicles after 12 hr PMSG injection and transduced the Rxfp2 using lenti virus. After 10 days of culture, the proliferation of theca cells was analyzed using EdU. Using Alzet osmotic pumps INSL3 was administered for 3 days into the ovary during superovulation induction. The INSL3 mRNA levels were significantly high in the theca cells of preovulator follicles after hCG injection. But granulose cells showed decreasing expression by growth of follicle. INSL3 stimulated the proliferation of theca cells in vitro which overexpressed the Rxpf2. By the administration of INSL3 into ovary caused the dramatical decrease the number of ovulated oocyte. Based on these results, we know that INSL3 stimulates the theca cell proliferation in as follicle stage specific manners, and estrogen is a modulator of this INSL3 mRNA expression. It suggests that the disturbance of the expression regulation of INSL3 and its receptor cause the unregulated theca cell proliferation and failing the rupture of grown follicle.