OR06-6 Impaired 17,20-lyase activity in mice lacking cytochrome b5 in testicular Leydig cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR06-Molecular Mechanisms of Estrogen Action and Androgen Synthesis
Basic
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 12:45 PM
Room 256 (Moscone Center)
Varun Sondhi*1, Jiayan Liu2, Susan Matthew2, David J Mangelsdorf3 and Richard Joseph Auchus4
1UT Southwestern Medical Center, 2University of Michigan, 3U.T. Southwestern Medical Center, Dallas, TX, 4University of Michigan, Ann Arbor, MI
Cytochrome P450c17 (CYP17A1, steroid 17α-hydroxylase/17,20-lyase) is the sole enzyme capable of converting 21-carbon progestins to 19-carbon androgens. The 17,20-lyase activity is more vulnerable to mutations than the 17-hydroxylase activity and requires cytochrome b5 (CYB5A) for maximal activity. The physiologic importance of CYB5A in human physiology has been validated by the description of two kindred with 46,XY DSD due to CYB5A deficiency, in which 17,20-lyase activity is selectively impaired, leading to testosterone but not cortisol deficiency. To study the biochemical consequences of CYB5A deficiency in an intact animal and in steroidogenic tissue, we generated mice lacking Cyb5a in the Leydig cell (LCb5KO), crossing Cyb5a(lox/lox) and Cre(+/Sf1) animals. The LCb5KO animals were born in a normal Mendelian ratio. We examined the male mice and observed normal fertility with no overt phenotype. Testicular histology revealed no differences between LCb5KO and WT animals. Homogenates from LCb5KO testes had normal progesterone (P)-to-17α-hydroxyprogesterone (17OHP) conversion but low 17OHP-to-androstenedione (A) and testosterone (T) metabolism. The ratio of the hydroxylase to lyase activity was observed to be 1.7 in the WT and 4.5 (3-fold higher) in the LCb5KO testes due to deficient lyase activity in the knockout animals. Addition of recombinant CYB5A to in vitro assays had no effect on the WT lyase activity or the hydroxylase/lyase ratio but restored the LCb5KO lyase activity and ratio to the level of WT animals. We examined the effects of Cyb5a knockout on steroid production using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Exogenous hCG administration gave a large increase in serum steroids for both the LCb5KO and WT animals. In the LCb5KO animals, this rise was accompanied by the accumulation of 17OHP in serum, which led to a 17OHP/(A+T) ratio of 8.3%, compared to 0.19% in the WT animals, a 44-fold increase. These data demonstrate the physiological significance of Cyb5a in the Cyp17a1 lyase reaction. We demonstrate that the decrease in Cyp17a1 lyase activity in LCB5KO animals can be rescued by the addition of exogenous CYB5A. The residual Cyp17a1 lyase activity in LCB5KO mice, unlike human beings, is sufficient to maintain androgens at a level that leads to normal reproductive phenotype. Importantly, we found that 17OHP is undetectable by LC-MS/MS in WT mice but accumulates in the LCb5KOs upon hCG stimulation.

Nothing to Disclose: VS, JL, SM, DJM, RJA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH Grant R01GM086596
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