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We obtained a unique CD46 cDNA, STc/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CY). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum. This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42,000, approximately 20,000 smaller than conventional CD46. These properties of the STc/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STc/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types. Since the STc/CY4 isoform may behave like sperm CD46 in cellular localization and post-translational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STc/CY4. Rosette formation was seen most effectively between hamster eggs and STc/CY4-expressing CHO cells. These results infer that O-glycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule. The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.

Abstract:A new medium which contains amino acids, electrolytes, glucose, lactate and pyruvate at concentrations adjusted to those in human follicular fluid was prepared and named human follicular fluid (HFF) medium. We compared the developmental ability of mouse embryos cultured in HFF and in three commercially available media, i.e., human tubal fluid (HTF), α-modified minimal essential medium (αMEM) and Ham's F-10. When mouse maturing oocytes obtained from ovarian follicles were cultured in HFF or αMEM/10% fetal calf serum (FCS), extrusion of the first polar body was observed in 71 and 72% of the oocytes, respectively. The oocytes matured in HFF/10% FCS showed good developmental competence to the blastocyst stage compared with those matured in HTF or Ham's F-10/10% FCS after in vitro fertilization. The supplementation of amino acids in follicular fluid to HTF medium (HTF(FF)) did not improve the development to the blastocyst stage after IVF of maturing oocytes. The HFF medium was applied to in vitro culture of blastocysts using mouse embryos obtained from oviducts. After 80 hours of incubation in each medium containing 0.5% bovine serum albumin (BSA), the percentages of expanded blastocysts in HFF and HTF(FF)/0.5% BSA were significantly higher than those in HTF and αMEM (89% and 78% vs. 39% and 34%, respectively). Blastocysts and morulae were transferred to the medium CMRL 1066/20% FCS, and their developmental competence was assessed. Embryos derived from blastocysts and morulae cultured in HFF and HTF/0.5% BSA appeared to have good developmental competence, with the formation of an inner cell mass and outgrowing trophoblast, but those cultured in HTF(FF) and αMEM/0.5% BSA did not. Amino acids at high concentrations supplemented in αMEM produced a large amount of ammonium which is harmful to embryo development in extended cultures. These results indicate that the HFF medium was beneficial for both in vitro oocyte maturation and embryo development in the extended culture.

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