| Repulsive guidance molecule b (RGMb) is the first member of the RGM family shown to enhance bone morphogenetic protein (BMP) signaling by acting as a BMP co-receptor. In this study, the expression patterns of RGMb in rat uterus and ovary were analyzed by IHC, real-time PCR and western blotting. Meanwhile, the expression levels in tissues of Hu sheep was also measured by RT-PCR. These results provide an experimental basis to study the regulatory mechanisms of RGMb in the reproduction. The main results are as follows:1 Expression patterns of RGMb in rat uterus and ovary during the estrous cycleTo investigate the localization and expression of RGMb in rat uterus and ovary during the estrous cycle, this study detected estrous cycle phases of rats using vaginal smears. RGMb mRNA levels in rat uterus measured by real-time PCR were significantly higher during metesterus and diestrus than during proestrus and estrus(P< 0.05). Western blotting indicated that RGMb protein was significantly lower during estrus compared with the other three stages (P< 0.05). Immunohistochemistry revealed that RGMb protein was mainly localized to the uterine luminal and glandular epithelial cells of the endometrium. RGMb mRNA and protein in the ovary remained unchanged during the estrous cycle. RGMb protein was expressed in the oocytes of all follicles. Weak staining for RGMb protein was also found in corpora lutea. RGMb was not detected in granulosa cells and stromal cells. Taken together, RGMb expression in the uterus and ovary across the estrus cycle demonstrate that RGMb may be involved in the regulation of uterine function, follicular development as well as luteal activity.2 The effects of exogenous hormones on RGMb in rat uterusTo demonstrate the effects of exogenous hormones on RGMb expression,30 immature rats were divided into 6 groups, and injected daily with sesame oil, E2, P4, E2+P4, E2+ ICI 182780 and P4+ RU486 for 3 days. The results showed that steroid hormones increased uterine weight. There is a trend that the RGMb mRNA level of E2 group was lower than the vehicle, and P4 group was higher than the vehicle. The expression of RGMb mRNA in the uterus significantly decreased by the coordination of E2 and P4 compared to the vehicle(P < 0.05), and E2 group was significantly lower than P4 (P< 0.05). Uterine expression of RGMb mRNA was significantly decreased by ICI 182780 (an ER antagonist) and RU486 (a PR antagonist) compared to E2 and P4, respectively (P< 0.05). These results suggest E2 decreased RGMb expression in rat uterus under the the coordination of progesterone, and P4 stimulated RGMb expression.3 Expression patterns of RGMb in Hu sheepPrevious studies have demonstrated that RGMb was widely expressed in rodents. However, the expression patterns in domestic animals including Hu sheep have not yet been reported. The present study is to clone part of RGMb cDNA sequence from total RNA by using RT-PCR method in male Hu sheep, and to analyze the expression pattern of RGMb mRNA in heart, liver, kidney, testis, and epididymis (caput, corpus and cauda). Sequencing analysis indicated that the 985bp cDNA fragment was 84% identical to the reported mouse RGMb mRNA,84% to the rat,86% to the human,88% to the pig,95% to cattle and 99% to the of RGMb mRNA. The level for RGMb mRNA was lowest in liver, and was significantly higher in reproductive tissues (epididymis) than that in heart, liver and kidney (P< 0.05). These results provided a theoretical basis for further exploring the function of RGMb gene in reproduction and utilization of Hu sheep. |
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