Characterization of HSP20 expression in rat myometrium during pregnancy

The underlying mechanisms regulating uterine contractions during labour are still poorly understood. Heat shock protein 20 (HSP20) is a stress protein present at high levels in vascular smooth muscle and is implicated in cyclic nucleotide dependent smooth muscle relaxation, yet before this study was undertaken HSP20 expression and regulation in uterine smooth muscle, or myometrium, were completely unknown. Since HSP20 has been implicated in smooth muscle relaxation, I hypothesized that HSP20 would be highly expressed in the rat myometrium during early and mid pregnancy, but its expression would be downregulated during the late stages of pregnancy, as the muscle becomes activated and the onset of labour approaches. Northern blot analysis demonstrated that HSP20 mRNA detection was significantly decreased from day (d) 22 - d23 of gestation compared to nonpregnant (NP) samples and from d22 - one day post-partum (PP) compared with d6 (p < 0.05). Immunoblot analysis showed that detection of HSP20 was significantly decreased at d23 compared to dl2 and dl5 (p < 0.05). HSP20 detection also significantly decreased at PP compared to dl5 (p < 0.05). Immunofluorescence analysis demonstrated that after dl5, plasma membrane-associated localization of HSP20 decreased markedly in both circular and longitudinal muscle layers. My results demonstrate that HSP20 mRNA and protein are highly expressed during early and mid- pregnancy and then expression markedly decreases during late pregnancy and labour. -- During pregnancy, the myometrium exhibits a very noticeable change in size and phenotype which is believed to be modulated by both mechanical and hormonal influences originating, in part, from within the fetal genome. Since decreased HSP20 expression near term correlates with decreased levels of circulating progesterone, I also studied the effects of progesterone delayed labour and early progesterone withdrawal, using the receptor antagonist RU486, on HSP20 mRNA and protein detection. HSP20 was detectable near cell membranes at much higher levels in the longitudinal muscle layer of progesterone-treated pregnant rats (delayed labour) at all gestational timepoints examined compared with controls. Early progesterone withdrawal had no effect on HSP20 mRNA or protein detection in the myometrium. -- I also examined the role of uterine stretch in regulation of HSP20 expression during late pregnancy and labour. Using unilaterally pregnant rats, I investigated the changes in HSP20 mRNA and protein detection in the myometrium. Gravidity did not affect the detection of HSP20 mRNA or protein at dl9, but did cause a significant decrease in HSP20 mRNA (p < 0.10) and protein (p < 0.05) detection in the gravid horn at labour as compared to the non-gravid horn. Immunofluorescence analysis did not demonstrate any differences in HSP20 detection in situ at dl 9, but verified the decrease in HSP20 detection in the gravid horn at labour, with a decrease in staining near myocyte membranes. These findings suggest that expression of HSP20 in rat myometrium is not likely dependent on circulating progesterone levels, but may be negatively affected by stretch of the myometrium at term labour