Nihon Sanka Fujinka Gakkai zasshi最新文献

Fertilization is the process including many events such as maturation of egg and sperm, attachment, binding, acrosomal reaction, penetration, fusion, cortical reaction, zona reaction and nuclear fusion of both gamete, whereby individual gametes from the female and male unite to create offspring. Although the reason for mechanism of fertilization is still not clearly understood, this process may accelerate the rate adaptation in evolution. In this special lecture, I would like to present our experimental and clinical results especially concerning with morphological, physiological, biochemical and molecular approach on the mechanism of fertilization. 1. Development and maturation of follicles and oocytes. It is well known that pituitary FSH, LH control the ovarian function. Follicular development and ovum maturation are also controlled by both pituitary gonadotropins and local factors such as autocrine and paracrine agents. When hMG is injected during 1-6 day of menstrual cycle, several dominant follicles are developed. If hMG is injected after selection of dominant follicles, only one dominant follicle develop in the ovary. When PMS-treated immature rats were injected with immature or mature follicle fluids, rats injected with mature follicular fluid showed strongly suppress in the ovarian weights and numbers of ovulated follicles. Also mature follicle suppress aromatization from and androstenedione to estradiol. These findings mean that mature follicular fluid contains inhibitory factors. Apoptosis of granulosa cells and follicular steroids are related to fertilization. 2. Intracellular calcium of oocyte. Intracellular calcium concentration is known to start to increase in a periodic manner after fertilization in oocytes of mammalians. In 65% of tested mouse oocytes, fertilization occurred during 4 hours observation after sperm insemination in vitro. An initial long lasting intracellular calcium concentration was observed and followed by periodic manner. This calcium oscillation is inhibited by calcium blockers such as verpamil and nifedipine, but increased by high concentration of extracellular calcium concentration in the medium. Role of increase of intracellular calcium are understood to prevent polysperm and activate metabolism of oocytes. 3. Glucose metabolism of oocytes. Mouse embryo utilizes pyruvate as an essential nutrient until the 8-cell stage, and glucose thereafter. We have devised non-radiometrie and enzymatic microassay method to measure glucose, deoxyglucose, deoxyglucose 6-phosphate incorporated into individual mouse oocytes and preimplantation embryo. In parallel, the activities of several enzymes of glycolytic pathway were also determined. In this study, glucose metabolism is necessary to develop in fertilized ova with changing activity of enzymes. 4. Molecular bases of ovarian fluid. The zona pellucida ZP is involved in a number of events in fertilization, all these fertilization events occur in the oviduct. Oviductal gl

Ovarian cancer is one of the significant and deadly disease. Since 1980 when cisplatin was introduced in the chemotherapy, about 30% of the patients with advanced disease have achieved 5-year survival. However, remaining patients have had progressive disease or recurrence after achieving NED. Forty-seven% of recurrent disease was discovered as distant metastasis, while at initial therapy. In the recurrent disease, distantly metastatic lesions were encountered more frequently than those in primary disease. In the recurrent tumor, expression of immunohistochemical markers of malignancy, such as p53 protein and CD44v6 antigen were increased. These clinical data suggest that recurrent ovarian cancer which are exposed to anticancer agents attain increased metastatic potential. In order to assure that anticancer agent contribute to this increment, an experimental system using two human ovarian cancer cell lines (HRA, KF) and nude mice in which cancer cells were exposed to cisplatin in vivo was introduced. Cancer cells exposed to cisplatin in vivo (treated cells) made spontaneously more metastatic nodules in the mouse lung than those exposed to PBS (untreated cells). This result suggest that cisplatin induce the increase of metastatic potential of cancer cells in vivo. Treated cells showed higher invasiveness compared with untreated cells when inoculated in the footpad. Three major factors which were generally proposed to be necessary for cancer cell to give rise to invasion, such as attachment to extracellular matrix, production of proteolytic enzyme, and cellular mobility. For all of these factors, treated cells were superior to untreated cells. These results obtained suggests that cisplatin could increase the metastatic potential of cancer by enhancing potential of invasion. To investigate the mechanism of this phenomenon from the standpoint of genetic mutation, clonal analysis of experimental cancer in vivo was performed using southern blot method. Cancer cells before inoculation to the mice consisted of multiple clones. In 5 week after inoculation, tumor was wholely occupied by only one clone which showed one band on the lane. At this point cisplatin were administered. In 6 week, new single clone appeared with different band pattern from that of the clone at the administration of cisplatin. Furthermore, the cisplatin-induced new clone metastasized to the lung, while no metastasis was observed in the mouse with PBS-treated tumor during the same period. These data suggest that increased metastatic potential after cisplatin treatment is due not to selection but to creation of highly metastatic clone caused by potential of genetic mutation of cisplatin. In conclusion, chemotherapeutic agent has a potential to create highly malignant cancer cells as well as a potential to kill cancer cells.

Implantation is a complex process accomplished by synchronization and interactions between embryo and endometrium by local exchange of signals including a number of cytokines and growth factors and direct cell-cell and cell-matrix contact. However, the research in early events of human implantation is still in its infancy. This presentation comprises the results of our attempts to investigate the mechanisms of human implantation process in its early stage by cell-biological method, including establishment of experimental implantation model in vitro. 1. Human trophoblast of early stage of gestation showed active cell locomotion, active endocytosis, and invasion of endometrial cell monolayer in mixed cultures. Trophoblast invasion was later arrested by transformed endometrial cells similar to decidual cells in vivo. These results appeared to indicate the interactions between trophoblast and endometrial cells in implantation. 2. Coculture system of rabbit preimplantation blastocyst and endometrial epithelium reformed from isolated endometrial epithelial cells on basement membrane matrix (Matrigel) simulated the in vivo rabbit implantation processes. This coculture system may provide a useful experimental implantation model. 3. A human trophoblast cell line was established from chorionic tissues of normal early pregnancy. These cells were cytotrophoblast-like morphology and endocrine functions. They formed the villous structures similar to those in vivo in culture on Matrigel and invasion of Matrigel was observed. These indicated the extracellular matrix may affect the morphology and function of invading trophoblast in implantation site. 4. Human endometrial epithelial single cells were cultured on Matrigel. Reconstruction of gland followed by epithelium formation quite similar to in vivo structures by migration and proliferation of isolated cells was demonstrated. Height of gland was promoted by estrogen and initiation of epithelization was upregulated by platelet-derived growth factors. This system revealed the extracellular matrix regulated morphogenesis of endometrial epithelium in vivo and is an essential substrate in experimental implantation model of endometrial epithelium. 5. Parallel cultures of endometrial epithelial cells on Matrigel were carried out with the IVF. ET patients to evaluate the endometrial morphology at time of ET. Endometrial cultures were initiated in previous cycles on Matrigel and the sera of patients were added to her own cultures from 1st day of IVF treatment cycle. Evaluation of reformed epithelium revealed the apparently unsuitable morphology for implantation in group of patients who eventually failed in pregnancy. This system may provide a useful measures in evaluation of endometrial receptivity and modality of treatment for endometrial aberrations. 6. Cyclic changes of extracellular matrix components in endometrium were investigated. Collagen I, III, IV, V were immunohistochemically estimated. Relative levels

Tumor cells produce urokinase-type plasminogen activator (uPA) in an enzymatically inactive proenzyme form (pro-uPA). Secreted pro-uPA can immediately bind to the specific uPA receptors (uPAR) on tumor cell surface with high affinity. The uPAR specifically recognizes enzymatically inactive pro-uPA and active high molecular weight-uPA (HMW-uPA) by their growth factor-like terminal domain. uPAR is a glycoprotein of approximately 55 kDa; the affinity for uPA is high (0.2 nM) and the rate of dissociation is low. Receptor-bound uPA catalizes the formation of plasmin on the cell surface to generate the proteolytic cascade that contributes to the breakdown of basement membrane and extracellular matrix. The plasma membrane uPAR has attracted considerable attention because of its role in migration and tissue invasion by mononuclear phagocytes and malignant cells. In some cell types uPAR localizes uPA to cell-cell and cell-substratum contact sites, providing the possibility of a directional proteolysis that may be involved in cell migration and invasion. Recently it has been reported that competitive displacement of uPA from uPAR resulted in decreased proteolysis, suggesting that the cell surface is the preferred site for uPA-mediated protein degradation. Various very different approaches to interfere with the expression or reactivity of uPA or uPAR at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, inhibitors and recombinant or synthetic uPA and uPAR analogues. Recently we have reported that a highly purified urinary trypsin inhibitor (UTI) efficiently inhibits soluble and tumor cell-surface receptor-bound plasmin. UTI inhibits not only tumor cell invasion in an in vitro assay but also production of experimental and spontaneous lung metastasis in an in vivo mouse model. The anti-invasive effect is dependent on the anti-plasmin activity of UTI. UTI peptide, which inhibits plasmin activity, synthesized by an automated peptide synthesizer showed mouse 3LL cell invasion inhibitory activity. UTI and the effective peptide inhibited tumor cell invasion through Matrigel. UTI did not inhibit tumor cell proliferation or the binding of the cells to Matrigel. Also, UTI did not inhibit chemotactic migration of tumor cells to fibronectin. It is likely that UTI acts as a protease inhibitor. We attempted to synthesize conjugates between ATF and UTI. Thus, conjugating a physiological plasmin inhibitor to ATF might target it to reduce cell-associated proteolytic activity to the close environment of the uPAR-expressing tumor cell surface and subsequently may effectively inhibit tumor cell invasion and metastasis, because the cell surface uPAR might be a critical component of the metastatic machinery. A method of conjugation of the UTI domain II (HI-8), to the receptor-binding amino-terminal fragment (ATF) of uPA has been developed utilizing the heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyldit

The development and growth of gynecological cancers are related to steroid hormone actions. Alternatively, this prompts us to study biological contribution of sex steroids for invasion and metastasis in gynecological cancers. The first step of metastasis is the detachment of tumor cells. The adherens junction forms a main cell-to-cell junctional complex, mainly consisting of E-cadherin, alpha- and beta-catenins, etc. Estrogen suppressed the expression of their mRNAs, and the adhesive function of cells via adherens junction in endometrial cancer cells. Progestin and danazol reversed the estrogen-induced suppression. Estrogen enhanced invasiveness of endometrial cancer cells though the reconstituted basement membrane and interstitium using the Boyden chamber. Progestin reduced the estrogen-induced invasiveness. The final step of metastasis is tumor-derived neovascularization for growth of metastatic cancer cells. Progestin inhibited basic fibroblast growth factor (FGF) activity, which mainly contribute to tumor-derived neovascularization, regardless of growth-inhibition in some endometrial cancers. Progestin inhibits basic FGF in well-differentiated (WD) endometrial cancer cells, but not in poorly differentiated (PD) endometrial cancer cells. TNP470, a inhibitor of vessel endothelial proliferation, inhibited directly basic FGF in the PD. Therefore, the adequate combination therapy of progestin and TNP470 could efficiently inhibit angiogenic potential of heterologous endometrial cancers. The ratio of estrogen receptor exon 5 splicing variant (ER delta E5) to wild type-ER mRNA expression increased in some metastatic lesions of cancers. The dominant expression of ER delta E5 mRNA might be related to metastatic potential of gynecological cancers. Progesterone receptor from A (PR-A), initiated from in-frame AUG present in the PR from B (PR-B) mRNA, lacks the N-terminal 164 amino acids of PR-B, and acts as a progestin-dependent, trans-dominant repressor of PR-B function and other steroid receptor function. The expression of PR-B mRNA was dominantly expressed in all metastatic gynecological cancers given. This might be related to metastatic potential of gynecological cancers. To know tumorigenic potential of sex steroid receptors, ER, PR-A and PR-B genes were transfected to NIH3T3 cells. Transfected cells with PR-A gene alone formed a few colonies in double soft agar. On the other hand, the cells with PR-B and ER genes under the presence of estradiol formed plenty of colonies. Therefore, overexpression of PR-B under the absence of PR-A might be related to tumorigenic potential. In conclusion, estrogen could enhance some steps of metastasis in endometrial cancers, and progestin could inhibit the estrogen-induced events, regardless of growth-inhibition. Relative over-expression of ER exon 5 splicing variant, and PR-B might contribute to metastatic potential in gynecological cancers.