|dc.description.abstract||The Corpus luteum (CL) is a dynamic endocrine structure that develops in the ovary from the remnants of the ovulated follicle and plays a critical role in the control of estrous/menstrual cycle as well as in establishment and maintenance of pregnancy. Its main function is to secrete a steroid hormone progesterone (P4). During non-fertile reproductive cycles, CL undergoes regression but its life span gets extended during fertile cycles including pseudopregnant condition. The growth and function of CL are regulated by luteotropic and luteolytic factors. The luteotropic factors mainly include prolactin, luteinizing hormone (LH), P4 and growth hormone, whereas the luteolytic factors include oxytocin and prostaglandin F2α (PGF2α). LH plays a critical role in the growth and maintenance of CL function in pregnant rats. However, it appears that the effects of LH on CL vary with the stage of pregnancy. Accordingly, it has been observed that LH functions as a luteotropic factor during the first half of pregnancy, but during the second half of pregnancy its luteolytic effects may predominate. While the luteotropic role of LH in several species has been widely accepted and extensively investigated, its luteolytic role has received scant attention. In this work, studies have been carried out to examine the luteolytic actions of LH with a view to elucidate the molecular mechanism/s involved in the initiation of LH-mediated luteolysis in pregnant rats. It has been reported by others that administration of repeated injections of LH leads to initiation of luteolytic events during late pregnancy. In the current studies, the same method has been standardized to initiate luteolysis and to examine the mechanism/s by which repeated administration of LH causes luteolytic effects. Two stages of pregnancy were chosen for the studies; late stage (gestation day 19 to 20) and mid stage (gestation day 8 to 10) of pregnancy.
Stocco and co-workers have reported initiation of functional luteolysis upon repeated LH administration during the late stage of pregnancy. The results from the current study suggest that repeated LH administration exerts luteolytic effects both during late and mid stages of pregnancy. However, it was observed the luteolytic effects of repeated LH administration were more pronounced during late stage of pregnancy since both functional and structural luteolysis were seen. On the other hand, expression of markers of only functional luteolysis increased during the mid stage of pregnancy. Studies were carried out to delineate the mechanism by which repeated LH administration mediates its luteolytic effects. Based on previous studies and the data obtained from high throughput microarray analysis, the involvement of cAMP/PKA/CREB pathway, MAP kinase family of serine/threonine kinases, β-arrestins and prostaglandin signaling in the initiation of LH-mediated luteolysis were chosen for further analysis. It was observed that the repeated administration of LH desensitized the cAMP/PKA/CREB pathway. Activation of the cAMP/PKA/CREB pathway through forskolin treatment in desensitized primary culture of luteal tissue prevented the initiation of luteolytic events during both the stages of pregnancy. Of the MAP kinases examined, protein levels of pp38 kinase decreased significantly in rats receiving multiple injections of LH during the late stage of pregnancy. The physiological relevance of the down regulation of pp38 MAP kinase requires further investigation. To further analyse the role of β-arrestins during the process of luteolysis, two approaches were employed. In the first approach, a β-arrestin biased agonist, dg-CG was utilised to increase the endogenous levels of β-arrestin 1 whereas in the second approach, endogenous β-arrestin 1 levels were depleted through siRNA approach. It was observed that the increased expression of β-arrestin 1 had no effect on markers of luteolysis upon dg-CG administration during late stage of pregnancy suggesting that increased expression of β-arrestin 1 alone is not enough to initiate the process of luteolysis. On the other hand, depletion of β-arrestin 1 by siRNA approach resulted in inhibition of the luteolytic events during both stages of pregnancy. Remarkably, the inhibitory effect of β-arrestin 1 depletion was more pronounced during the mid pregnancy stage. Further, the results indicated that late pregnancy stage was more responsive to PGF2α signaling. To confirm the participation of endogenous PGF2α during luteolysis induced by repeated LH administration, production of prostaglandins was inhibited by employing diclofenac (DIC) a prostaglandin biosynthesis inhibitor. After confirming inhibition of the genes associated with PGF2α synthesis by DIC treatment, multiple LH injections were administered to DIC treated rats to examine luteolytic effects of LH in the absence of endogenous PGF2α signaling. It was observed the repeated administration of LH caused only functional luteolysis in the absence of endogenous PGF2α signaling. Overall the results from the present studies suggest that luteolysis is a complex process and relies on different pathways during different stages of pregnancy examined. Desensitization of the cAMP/PKA/CREB appears to initiate the process of luteolysis during both the stages of pregnancy. However, during the late stage of pregnancy PGF2α though important, plays a limited role. A positive correlation between expression of β-arrestin 1 and markers of luteolysis was observed during both the stages of pregnancy||en_US