PARENT SESSION
STEROIDOGENESIS AND HORMONE ACTION IN THE TESTIS
Kent
7:30 AM-10:00 AM

(108) EFFECTS OF MITOCHONDRIAL DISRUPTORS ON LEYDIG CELL STEROIDOGENESIS AND THE STEROIDOGENIC ACUTE REGULATORY (StAR) PROTEIN .

Allen, John¹, Diemer, Thorsten², Janus, Paul¹, Thordardottir, Steinunn¹, Hales, Dale¹, ¹ Department of Physiology & Biophysics, Chicago, IL² Department of Urology, Giessen, DE

ABSTRACT- Mitochondrial disruption may be a mechanism which inhibits Leydig cell steroidogenesis. Previous studies have shown that properties of the mitochondria including the electrochemical gradient (_m) and ATP synthesis are necessary for Leydig cell steroidogenesis and function of the steroidogenic acute regulatory (StAR) protein. As of yet, the dependent factors necessary for steroid synthesis are unclear. The objective of the present study was to examine how different mitochondrial disruptors influence Leydig cell steroidogenesis and levels of StAR. Mouse tumor Leydig cells (MA-10) were treated with 8-Br-cAMP for 3 h ± agents that target and disrupt mitochondria. These agents included the uncoupler; CCCP; the pH altering K⁺/H⁺ exchanger; nigericin; the ATP synthase inhibitor, oligomycin; and the oxidants; Na⁺ arsenate and H₂O₂. Post treatment, media were collected and subjected to progesterone radioimmunoassay (RIA) and levels of StAR protein and mRNA were determined by Western and Northern blot analyses. RIA data indicated progesterone production was significantly decreased after treatment with all the mitochondrial disruptors tested: 10 M oligomycin, 5 M CCCP, 100 M Na⁺ arsenate, and 10 M nigericin all potently inhibited progesterone synthesis by more than 90%. Western blot analysis of StAR protein showed that nigericin significantly reduced StAR levels, and oligomycin completely inhibited StAR expression. Treatment with CCCP, Na⁺ arsenate and H₂O₂ significantly reduced levels of the 30kDa mature form of StAR protein, but surprisingly caused the unprocessed 37 kDa pre-cursor form to accumulate. Accumulation of the unprocessed form of StAR indicates that translocation and normal processing of StAR in the mitochondria was prevented. Northern blot results showed that StAR mRNA levels were not affected by any of the agents, indicating the observed changes in StAR occurred post-transcriptionally. These results demonstrate that perturbing mitochondria by uncoupling , altering pH or blocking ATP synthesis will reduce expression and normal processing of StAR protein and prevent steroidogenesis. It appears that intrinsic properties of mitochondria are essential for Leydig cell steroidogenesis, and that altering mitochondrial homeostasis will prevent steroid synthesis.

KEY WORDS: Leydig cell, StAR, steroidogenesis, reactive oxygen