SPERMATID METABOLISM SYMBIOSIS WITH SERTOLI CELL

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It is proposed to extend earlier studies on spermatid metabolism to relate the functions of this cell to the so-called "nursing" function of Sertoli cells. A system for culturing Sertoli cells on collagen has been developed to permit the Sertoli cell to assume its true columnar shape in vitro so that two compartments (basal and adluminal) can develop and the cell can differentiate beyond the flattened form seen on plastic surfaces. The cells will be perfused in a leucite chamber designed for the purpose. The classical properties of the cell (morphology, protein synthesis - including androgen-binding protein, synthesis of nucleic acid, cyclic AMP and lactate, together with responses to hormones) will be examined to compare with Sertoli cells in the conventional culture system. We will investigate the mechanisms involved in production of the ionic gradients characteristic of the seminiferous tubule in vivo (high K+; low Na+ in adluminal compartment), and the effect of hormones on the gradient. The nature and number of proteins secreted into the adluminal compartment will be determined. Finally, attempts will be made to co-culture Sertoli cells with germ cells (spermatogonia; spermatocytes) introduced into the basal compartment. Light and electron microscopy (including time-lapse cinematography), will be used to detect differentiation of germ cells (spermatocytes - spermatids) and the occurrence of residual bodies following co-culture. Germ cells will be labeled with (3H) thymidine in vivo of by genetic markers to distinguish cells from donor rats added to recipient Sertoli cells from adherent contaminating germ cells from the recipient rat.