Hand2os1 Regulates the Secretion of Progesterone in Mice Corpus Luteum

Abstract

The corpus luteum plays a key role in pregnancy maintenance and estrous cycle regulation by secreting progesterone. Hand2os1 is an lncRNA located upstream of Hand2, with which a bidirectional promoter is shared and is involved in the regulation of cardiac development and embryo implantation in mice. The aim of this study was to investigate the expression and regulation of Hand2os1 in the ovaries. Here, we used RNAscope to detect differential expression of Hand2os1 in the ovaries of cycling and pregnant mice. Hand2os1 was specifically detected in luteal cells during the proestrus and estrus phases, showing its highest expression in the corpus luteum at estrus. Additionally, Hand2os1 was strongly expressed in the corpus luteum on day 4 of pregnancy, but the positive signal progressively disappeared after day 8, was detected again on day 18, and gradually decreased after delivery. Hand2os1 significantly promoted the synthesis of progesterone and the expression of StAR and Cyp11a1. The decreased progesterone levels caused by Hand2os1 interference were rescued by the overexpression of StAR. Our findings suggest that Hand2os1 may regulate the secretion of progesterone in the mouse corpus luteum by affecting the key rate-limiting enzyme StAR, which may have an impact on the maintenance of pregnancy.

Keywords: Hand2os1; corpus luteum; lncRNA; mouse; progesterone.

Figure 1

RNAscope of Hand2os1 expression in the ovary on days 1–18 of pregnancy (D1–D18) and days 1–5 of postpartum (PD1–PD5). Positive expression results in a brown color. (A) Hand2os1 expression and localization in ovaries during the estrus cycle. (B) Hand2os1 expression in ovaries during pregnancy. CL, corpus luteum; F, follicle. Scale bar = 50 µm.

Figure 2

Hand2os1 expression in the hCG-induced CL formation and regression model for different hours (24 h, 48 h, 72 h, 96 h) was detected by RNAscope. Positive expression results in a brown colour. CL, corpus luteum. Scale bar = 50 µm.

Figure 3

Effects of Hand2os1 on proliferation and apoptosis in mouse LCs collected on day 4 of pregnancy. (A) The efficiency of Hand2os1 silencing and overexpression were determined using qRT-PCR 24 h after transfection with si-Hand2os1 and pcDNA3.1-Hand2os1, respectively. GAPDH was used as the reference gene for normalization. (B) The proliferation of LCs was determined by CCK-8 assays after silencing or overexpression of Hand2os1 for 24 h. (C) The apoptosis of LCs was analyzed by FCM after transfection with the Hand2os1 overexpression vector, followed by treatment with 1 µM PGF_2α for 24 h. The data represent the mean ± SEM from three independent experiments. ** p < 0.01 compared with si-NC group, and different number of asterisks on bars indicate significant differences (p < 0.05).

Figure 4

Expression analyses of StAR, Cyp11a1, and Hand2os1 during luteinization of granulosa cells. The mRNA levels of (A) StAR, (B) Cyp11a1, and (C) Hand2os1 were detected by qRT-PCR after primary granulosa cells were treated with 100 ng/mL LH for 0, 12, and 24 h. GAPDH was used as the reference gene for normalization. The data represent the mean ± SEM from three independent experiments. b (p < 0.05), c (p < 0.01) compared with 0h group (a), the same letter indicates no significant difference and different letters means significant difference (p < 0.05).

Figure 5

Effects of Hand2os1 on the expression of steroidogenic enzymes and progesterone production in LH-induced luteinized granulosa cells. The mRNA levels of (A) StAR and (B) Cyp11a1 were detected by qRT-PCR in 1 IU/mL LH-treated granulosa cells after transfection with pcDNA3.1-Hand2os1 for 0, 12, and 24 h. (C) Meanwhile, the concentration of progesterone in the culture supernatants were determined by ELISA. GAPDH was used as the reference gene for normalization. The data represent the mean ± SEM from three independent experiments.

Figure 6

Effects of Hand2os1 on progesterone production in LCs. LCs were isolated from the CL on D4 of pregnancy following transfection with si-Hand2os1 or pcDNA3.1-Hand2os1 for 24 h. Then, (A) the concentration of progesterone in the culture supernatants was determined by ELISA, and the expression levels of the steroidogenic enzyme (B) StAR and (C) Cyp11a1 genes were analyzed by qRT-PCR. GAPDH was used as the reference gene for normalization. The data represent the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01 compared with si-NC group, and different number of asterisks on bars indicate statistically significant differences (p < 0.05).

Figure 7

Effects of StAR overexpression on the progesterone production inhibited by si-Hand2os1 in LCs. qRT−PCR analyses of (A) StAR and (B) Hand2os1 mRNA levels 24 h after transfection with si-Hand2os1 and pcDNA3.1-StAR. (C) The concentration of progesterone in the culture supernatants were determined by ELISA. GAPDH was used as the reference gene for normalization. The data represent the mean ± SEM from three independent experiments. ** p < 0.01 compared with si−NC group, and different number of asterisks on bars indicate statistically significant differences (p < 0.05).