Search

Duringin vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, FshrmRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4,and Ptgs2expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg,and Tace/Adam17expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 μl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.

This paper analyzes the performance of the Reconstruction Finance Bank (RFB) in order to shed light on the role of development banks in fostering economic growth. The RFB played a large role in Japan's transition from war-time command economy to a market economy in the early post-war period. We use individual firm level data on sales, profits, and loans from the RFB, and find that, initially, the RFB was making loans to firms with below-average performance. We then find that this was partly a result of political interventions into the loan policy of the RFB. In fact, we also find evidence of improvements in the performance of the RFB after its loan policy became more independent. Implications for developing economies are also discussed. J. Japan. Int. Econ., Dec. 1995, 9(4), pp. 486-504. Faculty of Economics, The University of Tokyo, Bunkyo-ku, Tokyo 13, Japan.Copyright 1995, 1999 Academic Press

Our microarray analyses revealed that pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) family and related molecules are expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and exhibit functions similar to those in macrophages. Specifically, we showed that when COCs were cultured with the TLR4 ligand bacterial lipopolysaccharide (LPS), the expression of Il-6, Tnfa and Ptgs2 were induced. However, the endogenous ligand for TLR family and its physiological role in ovulated COCs was not analyzed. Recently, it was reported that hyaluronan fragments, but not the high molecular weight hyaluronan polymers, activate TLR2 and TLR4. Since cumulus cells produce a hyaluronan rich matrix during the ovulation process, and because the matrix is broken-down by sperm-secreted hyaluronidase during fertilization, we hypothesized that hyaluronan fragments generated during matrix degradation might act as an endogenous ligand for TLR2 and/or TLR4 on cumulus cells and be required for successful fertilization. To examine this, mouse ovulated COCs were cultured for 2 hr with either LPS or Pam3Cys, ligands for TLR4 or TLR2, respectively. Each ligand increased mRNAs encoding specific cytokines/chemokines: Il6, Mip2, Ccl5 ∼7-fold, ∼10-fold, ∼15-fold, respectively. When ovulated COCs were cultured with hyaluronan fragments or treated with hyaluronidase, the expression of Il6, Mip2, Ccl5 mRNAs also increased with peak levels observed using hyaluronan fragments at 100 μg/ml or hyaluronidase at 1 IU/ml for 2 hr. Anti-TLR2 and anti-TLR4 monoclonal antibodies significantly suppressed hyaluronan fragment- and hyaluronidase- induced expression of these genes. Thus, TLR2/TLR4 on cumulus cells of ovulated COCs are functional and activated by hyaluronan fragments. To determine the role of activated TLR2/TLR4 on cumulus cells in fertilization process, ovulated COCs were cultured with 1x105sperm in the presence or absence of anti-TLR2 and anti-TLR4 antibodies. Using a Bioplex Protein Array system we identified 17 cytokine and chemokine factors that were secreted by COCs. Secretion of 10 factors, CCL-5, G-CSF, IL-1a, IL-6, IL-9, IL-12 IL-17, MCP-1, MIP-1a, and TNFa was significantly up-regultaed by co-culture with sperm. The addition of anti-TLR2 and anti-TLR4 antibodies significantly suppressed the secretion of CCL-5, G-CSF, MCP-1, MIP-1a, and IL-6, concomitantly with the reduction of fertilization rate to 32 % as compared with that in oocytes without antibodies (75 %). Additionally, Ccr1, Ccr2, Ccr3 and Ccr5 mRNAs that encode CCL5, MCP-1, and MIP-1a receptors were expressed in spermatozoa. Sperm motility was enhanced by specific ligands suggesting that the chemokines secreted from cumulus cells of ovulated COCs induces sperm capacitation and subsequent oocyte penetration by mechanisms dependent on TLR2/4 activation. Supported in part by JSPS- 18688016 (MS), HD-16229, HD07459 (JSR) (platform)

It has been known that cumulus cells support oocyte meiotic and cytoplasmic maturation. Our previous study showed that the EGF like factors (amphiregulin, epiregulin and betacellulin) are expressed in granulosa cells and cumulus cells and act on cumulus cells to induce cumulus cell-oocyte complex (COC) expansion and oocyte maturation in mice and in pigs. Additionally, when ovulated mouse COCs were cultured with amphiregulin, this factor activated/phosphorylated the EGF receptor (ErbB1) and maintained cumulus expansion and the expression of specific genes when expression of endogenous amphiregulin is decreased markedly. Moreover, we have shown recently that the maintenance of the COC matrix and the secretion of specific chemokines from cumulus cells enhance successful fertilization via supporting sperm motility and induction of sperm capacitation. These data suggested that additional ligand members for the ErbB family are required to act to maintain cumulus cell function after ovulation. However, there is little information about what mechanisms regulate cumulus cells in oviductal COCs. Therefore, this study was designed to determine the kinetic expression patterns of ErbB family and the ligand in cumulus cells in vivo and in vitro, and then activate the ErbB signaling pathway to stimulate oocyte maturation in COCs in culture. For this, intact follicles and cumulus cells were collected at early, antral and ovulatory stages of follicle, or from COCs in the oviduct at specific time intervals following eCG and hCG treatment of gilts. FSH/eCG increased levels of EGF receptor and ErbB3 protein in cumulus cells, whereas ErbB2 remained constant and ErbB4 was down regulated. Immuno-histochemistry localized ErbB3 to cumulus cells after eCG and hCG stimulation. Because we found high expression of neuregulin 1 (Nrg1) mRNA in cumulus cells from our rat COC micro-array database, and because RT-PCR analyses showed that Nrg1 expression in cumulus cells of porcine COCs was increased after hCG stimulation and reached maximum levels after ovulation, we sought to determine if NGR1 might regulate cumulus cell functions via ErbB3. When porcine COCs were cultured in vitro with FSH alone, the expression of Nrg1 in cumulus cells did not increase. Therefore, to determine if NRG1 might enhance the actions of FSH, NRG1 was added to FSH-containing medium at 24 hr of culture and COC expansion and oocyte maturation were analyzed after an additional 20-hr culture (total 44 hr culture period). The addition of NRG1 strongly induced the phosphorylation of ErbB3 but not ErbB1, which resulted in the activation of both ERK1/2 and PI 3-kinase-PKB pathways in cumulus cells. The COCs exhibited improved cumulus cell survival (no apoptosis) and sustained expansion. The developmental competence of fertilized oocytes to the blastocyst stage was also enhanced by NGR1. From these results, we show for the first time that NRG1 is a novel COC secreted EGF-like factor that acts via autocrine mechanisms to activate ErbB3 expressed on cumulus cells within COCs after ovulation, and thereby helps maintain cumulus cell function to improve oocyte developmental competence, maybe due to suppress oocyte aging. Supported in part by JSPS-18688016 (MS).(poster)

In mice, LH surge induces the expression of EGF-like growth factors, ampiregulin (AREG) and epiregulin (EREG) in both granulosa cells and cumulus cells, which act on cumulus cells via paracrine and autocrine pathway (Park et al., 2004; Shimada et al., 2006). Because AREG and EREG are synthesized as transmembrane precursors, the cleavage enzymes such as TACE/ADAM17, are required for releasing the EGF peptide, whereas the expression level is universal during ovulation process in mice model. To clear the role of EGF like factors in pig, we collected granulosa cells and cumulus cells from the eCG and/or hCG-priming ovaries. The results in pig model revealed that the expression pattern of EGF like factors and the protease distinctly differs from mouse model. Areg and Ereg were increased by eCG in granulosa cells and cumulus cells of follicles prior to the LH/hCG surge. hCG priming did not affect the Areg and Ereg expression whereas the level of Tace/Adam17 mRNA was significantly increased by hCG, concomitantly with the induction of Ptgs2 expression and progesterone accumulation within follicular fluid. Therefore, we estimated that in pig, the activation of EGF like factors is dependent on the induction of Tace/Adam17 expression by LH surge, and the interaction among the TACE/ADAM17-induced EGFR activation, PGE2 and progesterone plays an important role in cumulus cell function during ovulation process. To clear this, we used in vitro culture system of porcine COCs. When COCs were cultured with FSH /LH, Areg, Ereg, Tace/adam17, Ptgs2, Cyp11a1 and Pgr mRNA expressions were increased. The addition of PTGS2 inhibitor, NS398 to FSH/LH-containing medium suppressed Tace/Adam17 mRNA expression and ERK1/2 phosphorylation in cumulus cells, but did not affect the expressions of Areg and Ereg mRNA. When COCs were cultured with FSH/LH and PGR antagonist, RU486, Areg and Ereg expression was not affected whereas Tace/Adam17 expression was significantly down-regulated by the drug. Moreover, EGF receptor tyrosine kinase inhibitor, AG1478 decreased the levels of Ptgs2 and Cyp11a1 expression, suggesting that EGFR pathway regulated both PGE2 and progesterone production. From these results, we concluded that in pig, after LH surge Tace/Adam17 is expressed via PGE2 and progesterone dependent manner, inducing the release of EGF domain and then activation of EGFR, which results in PGEs and progesterone production. This positive feedback pathway impacts cumulus expansion of porcine oocytes in vivo and in vitro. Supported in part by JSPS-19880020 (YY) and JSPS-18688016 (MS)

Several in vitro systems using cumulus oocyte complexes (COCs) recovered from early antral follicles have been designed to permit meiotic maturation of oocytes in the pig and other species. To date, these have been partially but not completely successful, however the in vitro matured oocytes exhibit limited developmental competence as compared with that in oocytes matured in vivo. Therefore, this study was undertaken to determine the precise temporal patterns of steroid hormone production and the expression of specific genes in follicular cell types in vivo and then apply these patterns to stimulate oocyte maturation in COCs in culture. For this, follicular fluid, granulosa cells and cumulus cells were collected from early stage, antral and ovulatory follicles at specific time intervals following eCG and hCG treatment of gilts. The results showed several similarities but also distinct differences from the well-characterized patterns in mouse and rat models. As in the rodent, FSH/eCG stimulated expression of Cyp19 mRNA and increased production of estrogen coordinately with up-regulated proliferation of granulosa cells and cumulus cells. However, unlike the rodent, eCG induced the expression of Lhcgr and Pgr in porcine cumulus cells as well as granulosa cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge in the pig model. Moreover, progesterone and PGR were critical for FSH-induced expression of Lhcgr mRNA in cumulus cells of cultured COCs. The expression of Lhcgr mRNA in cumulus cells was associated with the appearance of functional LH receptors and the ability of LH to induce, in cumulus cells as well as granulosa cells, prostaglandin production, release of EGF like factors and ADAMTS-1 expression, promoting COCs expansion and oocyte maturation. Based on the unique expression and regulation of Pgr and Lhcgr in cumulus cells, we designed a novel porcine COC culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from early stage follicles were pre-cultured with FSH and estradiol for 10h at which time progesterone was added for another 10h. After 20 hr, COCs were moved to fresh medium containing LH, EGF and progesterone. The COCs exhibited improved cumulus cell proliferation (and no apoptosis), greater expansion and more highly regulated changes in oocyte maturation. Moreover, the oocytes matured in this revised COC culture system exhibited greater developmental competence to the blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species, such as mouse, pig and human need to be characterized and used to increase the effectiveness of hormone stimulation regimens in each species. Supported in part by JSPS-18688016 (MS)

We have developed a compact atomic beam flux monitor by means of surface ionization. The properties were studied by measuring various rare-earth elements (Ce, Nd, Sm, Dy, Yb). The proportional region between the beam flux and the surface ionization ion current was observed for every element, which indicates that with this monitor, the atomic beam flux can be determined from the observed ion current. For Nd, Sm, Dy, and Yb, the ion current was proportional to the atomic beam flux up to 100-200 Å/s. On the other hand, the ion current for Ce saturated around the beam flux of 30 Å/s. This can be explained by the fact that since the vapor pressure of Ce atoms is low they do not desorp fast enough from the filament, and the filament is covered with Ce atoms. The coverage of the filament limits the measurable range of the monitor.

We have measured the secondary electron emission yield from a uranium surface bombarded by uranium ions from a laser ion source at impact energies of 300-3000 eV. The uranium surface was prepared by the deposition of uranium atoms, and the uranium ion beam was generated from a laser ion source. Secondary electrons were not emitted under a threshold energy of about 1000 eV. Above this threshold energy, the secondary electron emission yield increased linearly with the impact energy, and became 0.12 at the impact energy of 3000 eV.