Cytochemical study on the substances(ZP1-3, sp56, acrosin)working at mammalian fertilization.

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1. Distribution of ZP3 receptor on the mouse spermatozoon : ZP3 is one of the three glycoproteins in the zona pellucida, and significant for the sperm recognition, binding and induction of acrosome reaction. The localization of ZP3 receptor on the spermatozoa was determined by colloidal gold-conjugated ZP3 and surface replica technique. The gold particles were detected on the outer surface of the plasma membrane covering the dorsal edge of the sperm head.2. Localization(1), molecular image (2), and formation (3) of sp56 :(1) sp56 has specific affinity for ZP3. By immuno- cytochemical and surface replica techniques, we demonstrated that sp56 localized at the same area, where the ZP3 receptor was demonstrated in 1. It suggests that sp56 satisfies conditions as a ZP3 receptor.(2) Molecular image of sp56 was observed by low angle rotary shadowing method. The octamer of sp56, which is the form in vivo, appeared as a four-leafed clover, having a maximum diameter of approximately 50nm. Each leaf is thought to be composed of a sp56 dimer.(3) During spermiogenesis, sp56 was observed in the acrosome. A question, when sp56 appear on the outer surface of the plasma membrane, remained to be answered.3. Formation and localization of aquaporin 7 (AQP7), a water channel protein, during spermiogenesis in the rat testis : Small size of spermatozoa is indispensable for penetration through the zona pellucida. Present immunohistochemical study demonstrated that AQP7 first appears in round spermatid. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity in the elongated spermatid. The result suggests that AQP 7 works as a water channel, since it is localized on the membrane covering the condensing and diminishing cytoplasm.