Identification of relaxin gene expression and protein localization in the uterine endometrium during early pregnancy in the pig

The corpus luteum is the primary source of circulating relaxin (RLX) in female pigs. However, a preliminary experiment in our laboratory identified RLX in the uterine endometrium of a dav 16 nreanant ailt that had been ovariectomized on day 8 of pregnancy, Hnd the pregnancy maintained via progesterone replacement therapy. Therefore, our objective was to examine the uterus as a potential extraovarian source of RLX during the estrous cycle and early pregnancy in pigs. Reproductive tissues were collected from pregnant (n 2 3/day) and nonpregnant (n 2 S/day) gilts on days 10 (n = 6), 12 (n = 6), 14 (n = 6), 16 (n = 8), 18 (n = 6), and 20 (n = 6) of the estrous cycle and pregnancy and on day 42 (n = 2) of gestation. To verify that the RLX identified in uterine tissues was not of ovarian origin, three additional pregnant gilts were ovariectomized on day 8, and the pregnancy was maintained by progesterone replacement therapy until day 16, when the reproductive tissues were collected for immunohistochemistry. The uterine tissues were scored for specific RLX immunostaining and analyzed for the effects day and pregnancy by analysis of variance. Within the uterine endometrium, the luminal epithelium of pregnant pigs contained more RLX than that of nonpregnant pigs (P 5 0.001). However, there was no difference in either the glandular epithelium or stromal tissues (P 2 0.10). Increased RLX in the luminal epithelium of pregnant pigs was detected on both days 18 (P 5 0.05) and 20 (P 5 0.001) of gestation compared to that in nonpregnant pigs on similar days of the estrous cycle. An effect of day was also observed for RLX immunostaining in the luminal epithelium (P 5 0.005), but not in glandular or stromal tissues (P 2 0.10). In both pregnant and nonpregnant pigs, luminal epithelial RLX immunostaining was faint on days 10-12. Thereafter, RLX immunostaining increased on day 14 to reach peak levels on day 16. In nonpregnant pigs, RLX immunostaining dropped to low levels on day 18 and became faint to absent by day 20 of the estrous cycle. In pregnant pigs, RLX immunostaining remained elevated, but appeared to decline slightly by day 20 and became undetectable by day 42 of gestation. RLX immunolocalization in the glandular epithelium was detected on all days in pregnant and nonpregnant pigs, whereas stromal-specific immunostaining was not observed. In addition, RLX was immunolocalized in both the luminal and glandular epithelium of uteri from pregnant gilts 8 days after ovariectomy. Northern blot analysis identified the approximately l-kilobase RLX messenger RNA in RNA isolated from luteal and endometrial tissues. Reverse transcription of endometrial messenger RNA and subsequent polymerase chain amplification with porcine RLX gene-specific primers identified RLX gene expression in both pregnant and nonpregnant endometrial samples between days 10 and 20. We conclude that the porcine uterine endometrium is an additional source of RLX production during early pregnancy and the estrous cycle. Local uterine production may allow RLX to exert its uterotropic actions during the period of pregnancy establishment. (Endocrinology 135: 2517-2525, 1994)