Post-ejaculation of sperms into the female reproductive tract, acquisition of sperm capacitation is an essential step in the fertilization process. Accordingly, during in-vitro fertilization, the successful fertilization requires necessarily induction of capacitation in the retrieved sperms. To date, many candidate substances have been considered as capacitation inducers. However, there were no reports about the comparison of efficiency inducing sperm capacitation among diverse capacitation inducers. Therefore, we tried to determine an inducer showing the best capacitation performance in mouse sperms by comparing the preimplantation development of in-vitro-fertilized embryos using sperms experiencing capacitation by a variety of capacitation inducers. For these, calcium, progesterone, bovine serum albumin (BSA), heparin, lysophosphaticylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentration of each inducer were determined by accessing ratio of sperms experiencing acrosome reaction using coomassie G-250 blue staining. Subsequently, in vitro fertilization was performed using sperms incubated in each optimized concentration inducer. The ratio of fertilized oocytes was observed. As the results, Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 μM progesterone, 50 mM heparin, and 100 μM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization. From these results, we could identify that, among diverse sperm capacitation inducers, 2.7mM calcium and 0.3% (w/v) BSA were the most effective inducers for in vitro fertilization.

Heparin-bindin epidermal growth factor (HB-EGF) is one of the EGF family to be expressed at the time of implantation in the mouse uterus. Although HB-EGF has been shown to stimulate the development of embryo and uterus in the mouse, its correlation between cell adhesion molecules remains undefined. Integrin , one of the cell adhesion molecules, is an important mediator of cell-substratum and cell-cell adhesion in implantation. In the present studies, we investigated the effects of HB-EGF on the embryonic development, initiation of implantation and expression of integrin in in vitro culture, blocking of HB-EGF, RT-PCR and immunofluores cence analysis. The results showed that HB-EGF significantly improved the developmental rate of hatched embryos (24.1%, p<0.01) and outgrowth embryos (42.5%, p<0.01). On the other hand, this growth factor showed no offset before the hatching embryonic stage. Analysis of RT-PCR showed that HB-EGF upregulated the expression level of integrina subunit genes on the preimplantation embryo and outgrowth of blastocyst (120hr and 144hr after hCG injection). Immunofluorescence analysis showed that the integrin subunits localized at the pericellular borders and cell-cell contact areas. Increase in fluorescence intensity was observed in the HB-EGF treated embryos. Intrauterine injection of an anti-HB-EGF antiserum at day 3 significantly decreased the number of implantation sites (14.4, p<0.01) and significantly increased the number of recovered embryos(6.4, p<0.05) at day 5. From these results, it imply that HB-EGF improve the embryo development and accelerated the expression of integrin in the preimplantation mouse embryos.

In vitro developmental ability of early preimplantation monse embryos was shown to be depend on the embryonic stages, media and snpplements and their interaction(Experiment 1). The development of I-cell embryos were more promoted in the complex medinm(Ham's Fl0) than in the simple one(m-KRB), but that of 2-cell embryos showed the reverse effect. The bovine serum albumin(BSA) as a medium snpplement more promoted the development of I- and 2-cell embryos, compared with human fetal cord serum(HCS). On the other hand, the harmful effect of HCS was especially shown on the early cleavage in the embryonic development of the two stages. The effect of serum, in the respect of interaction between media and snpplements. was also more significantly appeared in m-KRB than Ham's Fl0. In the experiment 2, when the harmful effect of HCS was compared with that of fetal bovine serum(FBS), the former more promoted the development of l - and 2-cell embryos than the latter. The effect of HCS was more significantly shown in the development of I-cell than that of 2-cell embryos. Conclusively, as I- and 2-cell embryos were different in the requirements for the in vitro development. the optimal medium and supplement have to be selected for each embryonic stage. It is also respected to the better result if it take into consideration into the kinds of sera when serum is used for culture of early preimplantation embryos.

Amnionless (AMN) is a plasma membrane protein that binds to cubilin and megalin in various epithelia and mediates endocytosis of extracellular ligands. This function has been studied in the kidney where it plays a key role in vitamin B12 and vitamin D homeostasis. Present study aimed to elucidate developmental pattern of expression of AMN during the peri-implantation period in mouse embryos. In an effort to understand functional role of AMN in the histiotropic nutrition in blastocyst, endocytotic function of AMN for apoplipoprotein was examined in blastocyst. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. To obtain embryos, females were mated with males. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG by flushing oviducts and uterus, and we also obtained gestation day 6.5, 7.5 and 8.5 embryos in uterus. All samples were subjected to quantitative RT-PCR, whole-mount immunofluorescence and immunohistochemistry analysis. To analyze endocytotic function of AMN, we examined uptake experiment of FICT labeled apolipoprotein A-I (ApoA-I-FITC) following functional blocking of AMN in blastocysts. AMN and cubilin mRNA was expressed in all developmental stages of mouse embryos. Megalin was the first detected at morula stage. AMN protein was expressed in trophectoderm (TE) and inner cell mass (ICM). AMN and cubilin were expressed in visceral endoderm of GD 6.5 and 7.5 embryos and visceral yolk sec of GD 8.5 embryos. In normal IgG treated embryos, ApoA-I-FITC was detected in intracellular vesicles of TE and ICM. However, in the presence of anti-AMN antibody, ApoA-I-FITC was weakly detected in apical surface of plasma membrane of TE. To date, AMN has been believed to be expressed in visceral endoderm of post implantation embryos. Our results demonstrated that AMN is the important molecular partner of cubilin and megalin in the preimplantation embryos and that AMN mediates endocytosis of apoplipoprotein, which may play a crucial role in embryonic development and normal growth via supporting histiotropic nutrition during peri-implantation period.

Coxsackievirus and adenovirus receptor (CAR) is a member of the Ig-type superfamily of cell adhesion molecules. In polarized epithelial cells CAR is expressed at the tight junction. The mouse CAR gene is composed of at least eight exons, and CAR splice variants that differ at the end of the cytoplasmic tail have been identified in a number of tissues. The present study aimed to examine the expression of (CAR), a TJ protein sealing the muticellular contact point during preimplantation embryos and role of CAR in the formation and integrity of the blastocoel. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG in oviducts and uterus, and we also obtained gestation day 5 and 6.5 embryos in uterus. All samples were subjected to RT-PCR, immunofluorescence and immunohistochemistry analysis. To analyze epithelial permeability of CAR, we examined permeability of FITC-labeled dextran (MW 40 kDa) following functional blocking of CAR in blastocysts. Long isoform of CAR mRNA was expressed from throughout the preimplantation development and markedly increased at morulae stage onward. Small amount of short isoform CAR mRNA was expressed at blastocyst stage. On Western blot, 64 kDa protein was detected together with 43 kDa protein corresponding to short and long forms CAR, respectively in blastocysts. CAR immunoreactivity was found in cell contacts between blastomeres from 4-cell stage onward. Under Ca2+ switching condition blocking antibodies for CAR increased the permeability of blastocysts to FITC-dextran, a permeability tracer. At 5 dpc, trophoblasts of the implanting embryos were immunoreactive with anti-CAR IgG. At 6.5 dpc, the egg cylinder stage in mouse, the visceral and parietal endoderm were immunoreactive with anti-CAR IgG. Our results suggest that alternative splicing of CAR transcript is highly dependent on the development of expanding blastocyst. CAR may play a crucial role as a barrier to adenovirus infection and adhesion molecule for epithelial permeability during peri-implantation period.