REGULATION OF LEYDIG CELL MITOSIS AND DIFFERENTIATION

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The capacity for testosterone production in the adult male results from increases in Leydig cell numbers during puberty. Leydig cells increase in number through a gradual sequence of events in which Leydig cell precursors initially proliferate, than lower their rate of mitosis as they begin to express proteins that are characteristic of adult Leydig cells, most notably, steroidogenic enzymes. Studies of purified Leydig cells conducted by the PI and others, have defined discrete stages within the continuum of progressive Leydig cell differentiation: spindle shaped, highly proliferative progenitor Leydig cells; immature Leydig cells, which, though less proliferative, remain in the cell cycle, contain steroidogenic organelles, synthesize steroids, but secrete 5alpha-reduced androgens rather than testosterone; adult Leydig cells, which do not divide and which secrete primarily testosterone. Luteinizing hormone (LH) controls Leydig cell function through LH receptors that are present in abundant numbers that the adult stage. However, Leydig cell progenitors have negligible numbers of LH receptors and are comparatively unresponsive to this hormone. These considerations led us to hypothesize that the beginning of Leydig cell development is regulated by other factors such as steroidogenic factor-1 (SF-1). LH is reported to increase Leydig cell numbers and is known to increase levels of testosterone production. This application focuses on two processes in which LH is unlikely to be the controlling stimulus: the decrease in mitotic rate during the transition from progenitor to immature Leydig cell; and stimulation of the androgen-metabolizing enzymes 5alpha- reductase and 3alpha-hydroxysteroid dehydrogenase, which lower testosterone secretion by progenitor and immature Leydig cells. Mullerian inhibiting substance (MIS) will be investigated because targeted deletion of the MIS gene causes Leydig cell hyperplasia, indicating that this factor is normally a growth inhibitor. In experiments planned for Specific Aim 1, we will determine if the reduction in progenitor Leydig cell mitosis, that is observed between days 14 and 21 postpartum in the rat, caused by MIS; establish whether testosterone-induced increases in mitosis of immature Leydig cells are required for terminal differentiation; investigate the involvement of estrogen in growth inhibition of the Leydig cells, because MIS is observed to regulate aromatase, the enzyme that catalyzes estradiol synthesis. The experiments planned for Specific Aim II will individually evaluate the regulation of testosterone biosynthetic and androgen- metabolizing enzyme activities by LH, testosterone, SF-1, and MIS; determine the extent of control of these enzymes by co-factor availability; and identify instances where steroidogenic enzyme activities are catalyzed by novel Leydig cell isoforms.