Luteal dysfunction in pregnant women is associated with early pregnancy loss, making the study of structure and function of the corpus luteum (CL) critical. Luteinizing hormone (LH) plays a crucial role in the mammalian female reproduction majorly by regulating luteal development. In rats, the luteotropic roles of LH have been widely investigated but its role in the process of luteolysis has received little attention. In this study, we explored the luteolytic actions of LH during different stages of pregnancy in rats. Repeated administration of LH during the late and mid-stages of pregnancy led to functional luteolysis during both stages, while structural luteolysis was observed only during the late-stage. We analyzed the involvement of cAMP/PKA/CREB pathway, MAP kinases and β-arrestins to elucidate the molecular mechanism of LH-mediated luteolysis. The results indicate that the repeated administration of LH causes LH/CGR desensitization along with an increase in β-arrestin 1 expression, while luteal expression of MAP kinases remained unaffected. Further, siRNA-mediated depletion of β-arrestin 1 in primary luteal-cell cultures prevents initiation of the luteolysis process to some extent during both the stages of pregnancy, underscoring its role in LH mediated-luteolysis. In conclusion, the luteolytic actions of LH appear to involve more than one signaling pathway and cAMP/PKA/CREB pathway appears to be the key regulator. This is the first report to show a positive correlation between β-arrestin 1 and 20α-hsd expression. These findings have implications for our understanding of the molecular pathways that regulate luteolysis.
Gestational diabetes mellitus (GDM), a condition in which the state of pregnancy induces the development of diabetes, is characterized by heightened maternal insulin resistance. The levels of sex steroid hormones generally increase during pregnancy. It is thought that imbalance in the levels of steroids like estradiol (E2) and progesterone (P4) with respect to each other, may increase susceptibility towards GDM. To understand the metabolic effects of these steroids, ovariectomized (OVX) rats were treated with E2 or P4 at dosages mimicking the true hormonal status as in pregnancy. E2 significantly reduced the body weight gain (145.4±1.4% to 108.3±0.8%, p<0.001, n≥12) as well as the cumulative food intake (391.3±14.6 g to 312.5±9.0 g, p<0.001) over the course of the 23 day-treatment period. It also decreased the quantity of accumulated gonadal white adipose tissue (GWAT) in the body (3.3±0.2 g to 1.1±0.1 g, p<0.001) and repressed expression of lpl (1.3±0.2 fold, p<0.05) and other lipogenesis markers. P4, on the other hand, enhanced lpl expression (3.7±0.2 fold, p<0.001), but did not affect the total quantity of GWAT. Further, E2 treatment brought about an increase in the expression of insulin sensitivity markers like insr in the GWAT (4.5±0.6 fold, p<0.001) and soleus skeletal muscle (6.2±0.3 fold, p<0.001), as well as an increase in the protein levels of GLUT4. GDM susceptibility in pregnant women is most commonly associated with SNPs in the tcf7l2 gene, the product of which is an effector of the canonical Wnt signaling pathway. It has also been reported that certain actions of steroid hormones are mediated by Wnt signaling. Moreover, we found that tcf7l2 and other components of this pathway (β-catenin protein, lrp5) were up-regulated following treatments with E2 (3.8±0.2 fold, p<0.001 in GWAT; 5.3±0.2 fold, p<0.001 in soleus) and P4 (2.1±0.2 fold, p<0.05 in GWAT; 2.9±0.3 fold, p<0.001 in soleus). We therefore hypothesized that the metabolic actions of these steroids may be mediated by Wnt signaling. To test this hypothesis, we conducted experiments in which OVX rats treated with steroids as described above, were additionally treated with niclosamide (NIC), a Wnt pathway inhibitor. NIC in conjunction with E2 increased GWAT accumulation and lipogenesis, thereby reversing the action of E2. NIC treatment in OVX rats did not change these parameters, indicating that this effect is specific to the inhibition of Wnt signaling modulated by E2. Additionally, NIC inhibited the E2-modulated increase in insulin sensitivity in GWAT and soleus. Taken together, the results suggest that the actions of E2 on insulin sensitivity and lipogenesis are mediated by the Wnt signaling pathway. No such observation was made with respect to the effect of P4 on lipogenesis. Understanding the mechanistic actions of these steroids may play an important role in devising methods to prevent conditions like GDM before its onset.
The shift in maternal energy metabolism characteristic of pregnancy is thought to be driven by various hormonal changes, especially of ovarian and placental steroids. Imbalances in circulating estradiol (E2) and progesterone (P4) levels during this period are often associated with metabolic disturbances leading to development of gestational diabetes mellitus (GDM). Since abnormalities in the Wnt pathway effector TCF7L2 are commonly associated with occurrence of GDM, we hypothesized that the canonical or β-catenin-dependent Wnt signaling pathway mediates the metabolic actions of E2 and P4. In the present study, we observed that E2 enhanced insulin sensitivity and inhibited lipogenesis while P4 increased lipogenic gene expression – in 3T3-L1 adipocytes, and in adipose tissue and skeletal muscle of ovariectomized (OVX) rats when the dosage of E2 and P4 mimicked that of pregnancy. Both E2 and P4 were also found to up-regulate Wnt signaling. We used niclosamide (Nic) to inhibit the steroid-mediated increase in Wnt signaling in adipocytes and OVX rats, to test our hypothesis. The insulin-sensitizing and anti-lipogenic actions of E2 were found to be mediated by the canonical Wnt pathway, but the effects of P4 on lipogenesis appeared to be independent of it. Additionally, it was observed that inhibition of Wnt signaling by Nic hastened adipogenic differentiation, and the inhibitory effect of E2 on differentiation was prevented by Nic. The findings presented in this study highlight the role of steroids and Wnt pathway in glucose and lipid metabolism and are relevant in understanding the pathophysiology of metabolic disorders arising from hormonal disturbances.
Luteal dysfunctions lead to fertility disorders and pregnancy complications. Normal luteal function is regulated by many factors, including luteinizing hormone (LH). The luteotropic roles of LH have been widely investigated but its role in the process of luteolysis has received little attention. LH has been shown to have luteolytic effects during pregnancy in rats and the role of intraluteal prostaglandins (PGs) in LH‐mediated luteolysis has been demonstrated by others. However, the status of PG signaling in the uterus during LH‐mediated luteolysis remains unexplored. In this study, we utilized the repeated LH administration (4×LH) model for luteolysis induction. We have examined the effect of LH‐mediated luteolysis on the expression of genes involved in luteal/uterine PG synthesis, luteal PGF2α signaling, and uterine activation during different stages (mid and late) of pregnancy. Further, we analyzed the effect of overall PG synthesis machinery blockage on LH‐mediated luteolysis during late pregnancy. Unlike the midstage of pregnancy, the expression of genes involved in PG synthesis, PGF2α signaling, and uterine activation in late‐stage pregnant rats' luteal and uterine tissue increase 4×LH. Since the cAMP/PKA pathway mediates LH‐mediated luteolysis, we analyzed the effect of inhibition of endogenous PG synthesis on the cAMP/PKA/CREB pathway, followed by the analysis of the expression of markers of luteolysis. Inhibition of endogenous PG synthesis did not affect the cAMP/PKA/CREB pathway. However, in the absence of endogenous PGs, luteolysis could not be activated to the full extent. Our results suggest that endogenous PGs may contribute to LH‐mediated luteolysis, but this dependency on endogenous PGs is pregnancy‐stage dependent. These findings advance our understanding of the molecular pathways that regulate luteolysis.
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