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  • Title: Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse.
    Author: O'Brien DA.
    Journal: Biol Reprod; 1987 Aug; 37(1):147-57. PubMed ID: 3651542.
    Abstract:
    Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine [( 35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-10(6) cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constituents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.
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