Purification and characterization of sperm maturation antigens and their stabilizing factors

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Mammalian spermatozoa functionally mature during epididymal passage. This maturation is called sperm maturation in the epididymis. A monoclonal antibody (MAB) T21 (class IgM) from a hybridoma cell line that i established was employed to systematically analyze the phenomenon. Following results were obtained during the grant period.1. MAB T21 recognized a sperm maturation antigen, a sialoglycoprotein of about 54,000 dalton (sgp54).2, Due to masking of the epitope (cryptodeterminant) by sialic acid, it was unsuccessful to purify the sgp54 utilizing antigen-antibody affinity chromatography. Better purification was obtained by combination of WGA- or lentil-lection affinity and ion-exchange chromatography.3, Sialidase treatment demonstrated that the epididymal epididymal epithelial cells (clear cells) secreted sgp54 which was carried by small vesicles and membranous structures into the epididymal lumen, and then it attached to the immature spermatozoa. Binding of sgp54 to spermatozoa was inf … More luenced by a factor, possibly an antigen stabilizing factor (ASF). The molecular weight of ASF was estimated 50,000-60,000. The amount of the factor in the epididymal fluid seemed to be poor. Purification protocol of the ASF was improved using combination of column-chromatography technique. Assay protocol was improved, but the immunoreactivity of the sperm after indirect immunofluorescence technique must be judged by human eyes. This judging step was a time-comsuming work, and one might criticize the judging way was not objective. I think that more objective method is to be developed to get confirmative data.4, In vitro co-culture of immature sperm with sgp54 suggested that the sgp54-receptor existed on the immature sperm plasma membrane. The receptor was supposed to be synthesized during spermatogenesis. Interestingly, the rat and hamster mature spermatozoa also had the sgp54 receptor, even though these animals did not have sgp54 molecules. Such a kind of receptor will be present in mammalian sperm surface to attach to their own maturation molecule. Less