1. CELLULAR CHARACTERIZATION OF ANDROGEN RECEPTORS AND STEROID RECEPTOR COACTIVATOR-1 IN THE LEVATOR ANI AND EXTENSOR DIGITORUM LONGUS SKELETAL MUSCLES OF ADULT MALE MICE Jamie A. Simon, S. Marc Breedlove, and Cynthia L. Jordan Neuroscience Program and Department of Psychology, Michigan State University, East Lansing MI 48824
INTRODUCTION
The SNB, or spinal nucleus of the bulbocavernosus, is a sexually dimorphic group of motoneurons that innervate the bulbocavernosus (BC) and levator ani (LA) skeletal muscles.
Androgen acts on androgen receptors (AR) in these muscles to promote survival and growth of muscle fibers and also to retrogradely promote survival of SNB motoneurons during development.
Furthermore, the size of LA muscle fibers is reduced by castration and restored with testosterone treatment in adulthood.
The extensor digitorum longus (EDL) is not similarly responsive to androgens, as this muscle is not sexually dimorphic and does not change size after androgen manipulation.
We are interested in which cell types in the muscles confer androgen responsiveness, and mediate androgen action on the SNB system.
We are also interested in steroid receptor coactivator 1 (SRC-1) and its role in mediating androgen responsiveness.
METHODS
Muscles were dissected from C57/BJ6 male mice, placed in OCT filled cryomolds and frozen in liquid nitrogen.
10um Cryostat sections were generated and quadruple labeled for AR (black), Basal lamina (red), Acetylcholine Receptors (ACHR, green) and DAPI (blue).
A second series was quadruple labeled for SRC-1 (black), Basal lamina (red), ACHR (green), and DAPI (blue).
Using combined transmitted and fluorescent light the number of AR positive/negative fibroblasts and myonuclei were counted at the neuromuscular junction and in non synaptic regions. The same was done for SRC-1.
Basal lamina and ACHR staining show the NMJ A B
A) Basal Lamina (red) thickening of muscle fiber in EDL. B) ACHR (green) staining of neuromuscular junction in EDL.
Basal lamina staining marks boundary of fibers B A
RESULTS
We found that the percent of AR positive myonuclei in synaptic and nonsynaptic muscle fibers is increased in the LA compared to the EDL.
In the LA, the percent of SRC-1 positive myonuclei is increased with respect to nonsynaptic myonuclei.
A) Basal lamina (red) and SRC-1 (black) staining in EDL. Black arrow shows a myonuclei positive for SRC-1. B) DAPI staining shows a negative myonuclei (white arrow).
Nonsynaptic
Synaptic 10 20 30 40 50 60 70 80 90
100
Percent
EDL LA
Percent AR Positive Myonuclei a b 5 10 15 20 25 30 35 40
Percent
EDL LA
Nonsynaptic
Synaptic
Percent AR Positive Fibroblasts
Nonsynaptic
myonucleus
Synaptic myonucleus
NMJ
Terminal Schwann cell
Fibroblast cell
Muscle fiber
CONCLUSIONS
AR in muscle fibers, and not in fibroblasts may be involved in mediating androgen responsiveness in the LA.
SRC-1in muscle fibers may also help mediate androgen responsiveness in the LA. 5 10 15 20 25 30 35 40
Percent
EDL LA
Nonsynaptic
Synaptic
Percent SRC-1 Positive Fibroblasts 50 60 70 80 90
100
Percent SRC-1 Positive Myonuclei b
Myonuclei = inside the basal lamina
Fibroblasts = outside the basal lamina, between fibers.
Schwann cells = between fibers, with own basal lamina
ACKNOWLEDGEMENTS
Grant info
a=Significantly different from respective LA
b=Significantly different from respective Synaptic group