Development of Prepubertal Mouse Interpubic Joint Sensitivity to Estrogen and Relaxin.

Abstract

Relaxin (RLX) induces uterine growth and causes the cartilaginous caps of the interpubic (IP) joint to resorb and form a flexible ligament, facilitating parturition in humans, guinea pigs and mice. In non-pregnant mice, estrogen priming is required for increasing sensitivity to RLX resulting in uterine growth and IP ligament lengthening. This RLX-induced growth of the IP ligament is the basis for a quantitative, in vivo bioassay. In 1955, Crelin and Levin reported that estrogen priming of prepubertal mice increased IP joint sensitivity to a crude extract of RLX in an age-dependent manner with sensitivity developing only after postnatal week (PNW) 3. However, the molecular basis for estrogen-dependent sensitivity of the IP joint during prepubertal development is unknown. Thus, objectives were to: 1) confirm when mouse IP joint sensitivity to RLX develops and 2) determine the effects of estrogens and RLX on estrogen receptor-alpha (ESR1) expression in the IP joint and uterus during prepubertal development. Since estrogens bind ESR1 in the reproductive tract, ESR1 was a marker of interest with respect to uterine and IP ligament growth. Mice were studied: 1) before (PNW 2 at d 14; n = 10/group) and 2) after (PNW 5 at d 35; n = 12/group) reported development of IP joint sensitivity to hormones. Mice were primed with estradiol cyclopentylpropionate (E) or peanut oil vehicle (C) and six days later treated with purified porcine RLX or benzopurpurine-4B vehicle (to facilitate slow release of RLX) 24 h before sacrifice. IP ligament lengths were measured using standard bioassay methods for RLX: a transilluminator equipped with a micrometer. Uterine and IP joint proteins were examined by immunoblotting and quantified by densitometry. At PNW 2 and 5, uterine weight increased (p < 0.05) in response to E and E+RLX when compared to controls. Crelin and Levin defined significant ligament lengthening as an IP separation of 0.5 mm or greater. In this study, eight out of nine E+RLX treated mice at PNW 2 met that criterion. Relaxin increased IP ligament length in E-primed mice when compared to controls (p < 0.05) at both PNW 2 and 5. At PNW 5, RLX alone reduced ESR1 protein in the uterus (p < 0.05) and IP ligament (p < 0.001). These studies suggest that the E-primed mouse uterus and IP joint are sensitive to purified porcine RLX as early as PNW 2. Detection of tissue sensitivity to E+RLX in mice after two weeks of life differs from the original Crelin and Levin observations and may be due to differences in methods of IP ligament measurement, type of estrogen used for priming and/or the purity of the RLX used in these two studies. This sensitivity of immature mouse tissues to E and RLX is consistent with hormone responsiveness reported in neonatal tissues of pigs and rats and may be important for understanding how neonatal exposure to such growth-promoting hormones affects long-term development. The significance of a decline in uterine and IP ligament ESR1 at PNW 5 in response to RLX is unclear. That E priming was effective in preventing the RLX-induced decline in ESR1 in both tissues points to the importance of estrogen in maintaining its own receptor. Estrogens increase uterine RLX receptor expression in rats and pigs and may play a similar role in mouse tissues. Thus, further studies are needed to evaluate expression of the RLX receptor during development of the mouse uterus and IP ligament. (Support: Aresty and GH Cook Undergraduate Research Programs) (poster)