Akira Tsuiki

Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction

OBJECTIVES The mechanism of human sperm-oolemma adhesion and penetration as well as localization of fibronectin on the sperm head and its relation to fertilization were investigated. DESIGN Sperm-oolemma interaction was examined with an in vitro assay of the human sperm-zona-free hamster egg interaction. Localization of fibronectin on the surface of human spermatozoa was observed by the back scattered electron imaging mode of the scanning electron microscopy (SEM). RESULTS It was confirmed by observations under SEM that the anterior tip of the sperm head is the first to come into contact with the egg plasma membrane but that the equatorial segment of the sperm head is the first to be trapped by microvilli of the plasma membrane and that the postacrosomal region is first incorporated into ooplasm. Localization of fibronectin on the equatorial segment of the human sperm head was detected by SEM. Antifibronectin antibodies inhibited human sperm-oolemma adhesion significantly. CONCLUSIONS Important involvement of fibronectin in the gamete interaction was made clear by the fact that fibronectin is localized in the region where a spermatozoon is fused first with the egg plasma membrane during fertilization and that the sperm adhering to the egg is inhibited by antifibronectin antibodies.

Sperm-Egg Interactions Observed by Scanning Electron Microscopy

Successful in vitro fertilization requires mature oocytes in which the first polar body has been extruded and capacitated sperm capable of penetrating the zona pellucida. In this study we made a time sequential observations on human sperm-egg interactions by SEM in two experimental systems. Human sperm-human zona pellucida interaction: Cytoplasmic processes of corona cell extend around sperm head. Spermatozoa took different angles in attaching or penetrating to the zona pellucida. The head of some spermatozoa bound to the zona were vesiculated, suggesting the progression of the acrosomal reaction. Initially, the anterior part of the sperm head penetrates from the pore of the zona pellucida. Human sperm-zona-free hamster egg interaction: Most spermatozoa lie flat on the vitellus surface covered with numerous microvilli, but a few are oriented perpendicular to the vitellus surface. Most bound sperm had lost their acrosomal caps, because a ridge exists at the leading edge of the equatorial segment. Initially most microvilli appeared to grasp and immobilize the anterior tip of the sperm head. But as gamete interaction proceeded, microvilli were overlying the postacrosomal region and were observed adjacent to the plasma membrane of the postacrosomal region. The postacrosomal region is first incorporated into the ooplasma, the anterior tip of sperm head being the last portion to be incorporated. The microvilli of the oolemmal surface where sperm penetrated did not show major changes in size or in appearance, and the so-called incorporation cone was not observed.

Fibronectin in reproduction

The fibronectin (FN) levels in human follicular fluids have been shown to correlate well with follicular size and oocyte maturity, suggesting a role of FN in oocyte maturation. When added to the culture medium, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS), which specifically inhibits the cell-binding of FN, has been shown to inhibit both spontaneous resumption of meiosis and gonadotropin-releasing hormone-induced meiosis of the oocytes. In another set of experiments, GRGDS has been found to inhibit the in vitro cleavage of mouse embryos by a still unknown mechanism.

Measurement of urinary estrogen level with the urinary estrogen micrometry method in in vitro fertilization and embryo transfer therapy

We have evaluated a new micrometering kit using hemagglutination inhibition reaction for determination of total estrogens in urine of normal fertile women and women undergoing in vitro fertilization and embryo transfer (IVF-ET). The micrometering kit was sensitive enough to monitor follicular maturation, and its technique was simple and rapid. Results were obtained in 2.5 hours. By the analysis of the individual estrogen levels in urine collected every 4 hours from 20 IVF-ET patients, the following results were found, with a few exceptions: once the level had exceeded 60 ng/ml, the onset of the luteinizing hormone surge was induced within the next 72 hours; when the level had been under 60 ng/ml, the onset was not induced within the next 24 hours

In vitro fertilization and embryo transfer at Tohoku University, Sendai, Japan

100 mg/day of clomiphene-citrate is administered for 5 days from the fifth day of the menstrual cycle. Ultrasonography and highly sensitive immunoassay of urinary estrogen (Mochida Co., Japan) are used to decide the beginning of urinary LH measurement every 4 hr. We do laparoscopic egg collection 2628 hr after the onset of LH surge or 36-37 hr after HCG injection, depending on the maximum follicular diameter and urinary LH level measured with Hi-Gonavis (Mochida Co.). Incubation is done with Wittinghams T 6 medium including 10% (IM) or 15% (GM) inactivated patients serum under conditions of mixed gas (5% CO2, 5% 02, 90% N2). Six hours after preincubation of the ovum, 3-5 • 10~/ mt of washed sperm is added and incubated for 16 hr of fertilization time. Then the embryo is moved to growing medium and cultured for 36 hr. Fifty-six hours after egg recovery, the embryo is replaced into the uterus with Monash ET Catheter Type 2. Table I. Clinical Results of IVF and ET (February-September 1983, Tohoku University, Sendai)