Estrogens regulate humans and rabbit epididymal contractility through the RhoA/Rho-kinase pathway.
BACKGROUND
We have previously demonstrated that oxytocin (OT) and endothelin-1 (ET-1) peripherally regulate epididymal motility in an estrogen-dependent way. Because RhoA/Rho-kinase (ROCK) pathway is a contractile effector downstream to both OT and ET-1 receptors, we hypothesized an estrogenic modulation of OT- and ET-1-induced contraction through the up-regulation of RhoA/ROCK signaling.


OBJECTIVE
To evaluate the effect of changing endocrine milieu on RhoA/ROCK pathway in the epididymis.


METHODS
We induced a pharmacological hypogonadotropic hypogonadism in rabbits and replaced hypogonadal animals with different sex steroids (testosterone, T, or estradiol valerate, [E(2v)]). Effects of estrogen deprivation were also evaluated in rabbits chronically treated with the P450-aromatase inhibitor letrozole. An "in vitro" model of human epididymal smooth muscle cells was established and stimulated with sex hormones (72 hours). Protein and mRNA expression and functional activity of RhoA/ROCK signaling were studied by quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, western blot analysis, cell migration and by "in vitro" contractility studies using the ROCK inhibitor Y-27632.


METHODS
Effects of sex steroids on expression and functional activation of RhoA/ROCK signaling in rabbit epididymis and human epididymal smooth muscle cells.


RESULTS
The relaxant effect of Y-27632 on ET-1-pre-contracted epididymal strips was significantly reduced in hypogonadal rabbits, as well as in letrozole-treated animals. T supplementation normalized T plasma levels, but not Y-27632 epididymal strip sensitivity. E(2)v not only completely restored Y-27632 responsiveness but even amplified it, indicating an estrogenic up-regulation of RhoA/ROCK pathway. Accordingly, ROCK1 protein and gene expressions were strongly induced by E(2)v but not by T. The estrogen-induced up-regulation of RhoA/ROCK signaling was confirmed in human epididymal smooth muscle cells.


CONCLUSIONS
Our results suggest that estrogens regulate epididymal motility by increasing RhoA/ROCK signaling, and therefore calcium sensitivity, which tunes up responsiveness to contractile factors.
Fibbi
Benedetta
B
Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Filippi
Sandra
S
Morelli
Annamaria
A
Vignozzi
Linda
L
Silvestrini
Enrico
E
Chavalmane
Aravinda
A
De Vita
Giulia
G
Marini
Mirca
M
Gacci
Mauro
M
Manieri
Chiara
C
Vannelli
Gabriella Barbara
GB
Maggi
Mario
M
D000818:Animals; D004703:Endocrine System; D019332:Endothelin-1; D004822:Epididymis / Q000187:drug effects*; D047629:Estrogen Receptor beta / Q000378:metabolism; D004967:Estrogens; D005832:Genital Diseases, Male; D006801:Humans; D007006:Hypogonadism; D000077289:Letrozole; D008297:Male; D009570:Nitriles; D011817:Rabbits; D015398:Signal Transduction; D013739:Testosterone / Q000097:blood; D014230:Triazoles; D054460:rho-Associated Kinases / Q000096:biosynthesis*; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis*
D019332:Endothelin-1; D047629:Estrogen Receptor beta; D004967:Estrogens; D009570:Nitriles; D014230:Triazoles; D013739:Testosterone; D000077289:Letrozole; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein