[Elucidation of the mechanism of fertilization and clinical application of assisted reproductive technology].

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 glycoprotein 200-240 KD has been identified from oviductal zona pellucida. Monoclonal antibody of oviductal glycoprotein reacted with ZP of oviductal egg but not with the ovarian egg. Anti-ZPO antibody inhibit to bind sperm to ZP. Sequences in mouse and hamster oviduct specific glycoprotein are estimated, this glycoprotein mRNA was observed in only oviduct by northern blotting method. These molecular gene expression was observed by in situ hybridization in the oviduct of estrous cycle of hamster. 5. Microinsemination of sperm. Microinsemination of sperm into oocyte is widely used in clinical medicine. Sperm penetration assay (hamster test) is useful method to estimate fertilization capacity of sperm. But immotile sperm cannot estimate it. So modified micro sperm penetration assay was established to estimate fertilization capacity of sperm by using micro-manipulator. Subzonal sperm injection (SUZI) and intracytoplasmic sperm injection (ICSI) promotes fertilization and cleavage rate in immotile