The studies proposed in this MERIT extension application address the molecular mechanisms by which age-induced changes in the microenvironment of Leydig cells, the cells responsible for testicular androgen biosynthesis, lead to reduced testosterone formation. We propose the following: (i) Changes in the balance between reactive oxygen accumulation and the antioxidant defense system result in increased oxidative stress which, in turn, results in damage to the signal transduction cascade at the plasma membrane and thus in reduced responsiveness of aged Leydig cells to luteinizing hormone (LH). (ii) As a consequence, aged cells produce less cAMP than young cells, resulting in reduced intracellular cholesterol transport into the mitochondria and therefore reduced substrate availability for testosterone.
The first aim i s to determine the mechanism by which increased oxidative stress elicit reductions in cAMP and testosterone production in aged Leydig cells. We will test the hypothesis that in response to increased oxidative stress, there is defective coupling of LH receptors (LHR) to Gs proteins, resulting in reduced cAMP in response to LH stimulation.
The second aim will test the hypothesis that as a consequence of the blunted ability of LH to stimulate cAMP production in aged Leydig cells, there are reduced levels of translocator protein (TSPO) and steroidogenic acute regualtory protein (STAR)-mobilized cholesterol, and thus less efficient transfer of cholesterol to the inner mitochondrial membrane. We further hypothesize that the reduced expression of TSPO in aged cells results from that reduced expression of Natural Antisense Transcripts (NATs).
The third aim i s to determine how the age-related changes in Brown Norway rat Leydig cells compare to those in aging human testes. With this aim, we will translate our findings in the rat to the human. To this end, we will measure testosterone and ROS levels in intratesticular fluid from young and aged men, capture Leydig cells from testicular biopsies to analyze steroidogenic enzyme expression and the expression of proteins involved in reactive oxygen production and in the antioxidant defense system, and examine proteins and lipids damaged by changes in the pro/antioxidant.balance using fixed tissue.

Public Health Relevance

Serum testosterone levels decline as men age. This decline has relevance to quality of life issues in men, including osteoporosis, cognition and libido. Studying how testosterone decreases will provide new insights into how Leydig cells cope with stressors that are present (or increase) with aging, shed light on the underlying molecular basis for age-related functional changes in the Leydig celts, and might provide approaches for their prevention or reversal.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AG021092-12
Application #
8536718
Study Section
Special Emphasis Panel (NSS)
Program Officer
Fuldner, Rebecca A
Project Start
2002-09-01
Project End
2016-05-31
Budget Start
2013-09-01
Budget End
2014-05-31
Support Year
12
Fiscal Year
2013
Total Cost
$368,764
Indirect Cost
$121,113
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Zhou, Liang; Beattie, Matthew C; Lin, Chieh-Yin et al. (2013) Oxidative stress and phthalate-induced down-regulation of steroidogenesis in MA-10 Leydig cells. Reprod Toxicol 42:95-101
Beattie, Matthew C; Chen, Haolin; Fan, Jinjiang et al. (2013) Aging and luteinizing hormone effects on reactive oxygen species production and DNA damage in rat Leydig cells. Biol Reprod 88:100
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Midzak, Andrew S; Chen, Haolin; Aon, Miguel A et al. (2011) ATP synthesis, mitochondrial function, and steroid biosynthesis in rodent primary and tumor Leydig cells. Biol Reprod 84:976-85
Chen, Haolin; Zhou, Liang; Lin, Chieh-Yin et al. (2010) Effect of glutathione redox state on Leydig cell susceptibility to acute oxidative stress. Mol Cell Endocrinol 323:147-54
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Fan, Jinjiang; Traore, Kassim; Li, Wenping et al. (2010) Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate on hormone-stimulated steroidogenesis in MA-10 mouse tumor Leydig cells. Endocrinology 151:3348-62
Chen, Haolin; Pechenino, Angela S; Liu, June et al. (2008) Effect of glutathione depletion on Leydig cell steroidogenesis in young and old brown Norway rats. Endocrinology 149:2612-9

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