| dc.description.abstract | The human placenta develops by an association of maternal and fetal tissues and performs many important functions during pregnancy. It facilitates the transport of nutrients from the mother to the fetus, the exchange of gases between the two, and the removal of waste products from the fetus. It also serves to protect the fetus from maternal immunological attack. Besides these functions, the placenta also synthesizes a large variety of proteins, peptides, and steroids. While some of the proteins synthesized by the placenta have been characterized as hormones or enzymes, a majority of them have been only physicochemically characterized and remain poorly understood in terms of their function.
While it is true that the lack of information on the function of these proteins is partly due to the non-availability of a suitable animal model, it is intriguing that relatively very little is known about the factors that may be involved in the regulation of synthesis and secretion of placental proteins. Two factors which may possibly regulate the synthesis of proteins by the placenta are growth factors and steroid hormones. An important fact that can be considered in favor of steroids as possible factors involved in regulating protein synthesis in the placenta is that the level of the steroids estradiol 17- and progesterone increases steadily with the growth of the placenta. While the availability of specific inhibitors for steroid hormones permits us to undertake studies on their function by selectively inhibiting their synthesis or action, the absence of specific inhibitors limits the possibilities of a study of growth factors.
Among the steroid hormones, estradiol 17- was chosen for this study as it is one of the earliest hormones to be synthesized by the conceptus. Estrogen biosynthesis has been detected in the preimplantation blastocyst, and estrogen has been implicated to have a role in its development. The presence of estrogen receptors has been demonstrated in preimplantation embryos from the two-cell stage of development onwards, suggesting that estrogen could be one of the important and earliest regulators of protein synthesis in the blastocyst. Studies using antiestrogens have established that estrogen is obligatory for implantation and the maintenance of early pregnancy in primates. Although the need for estrogen during pregnancy is clear, knowledge of its role in cellular functions which mediate these physiological responses during pregnancy is still rudimentary.
Earlier studies in our laboratory have demonstrated a role for estradiol 17- in the synthesis of proteins in the human placenta and also in the synthesis and secretion of human chorionic gonadotropin. During these studies, several proteins were found to be regulated by estradiol 17-, including a protein of apparent molecular weight 45,000, and we chose this protein for further characterization due to the consistency of its stimulation by estradiol 17- in several batches of placenta and also its relative abundance.
The objective of the present study is to establish the involvement of estradiol 17- in the regulation of the 45 kDa protein and to purify and characterize it to enable its use as a marker for estradiol 17- action in the human placenta.
In Chapter 1, a brief account of the structure and function of the placenta with special emphasis on its ability to produce a variety of proteins, peptides, and steroid hormones is presented. In addition, a detailed account of the steroid hormones produced by the placenta, their biosynthesis and action, the rationale in considering the placenta as a target tissue for estradiol 17- action, and the specific objectives in undertaking this study are discussed.
Results of experiments which established the involvement of estradiol 17- in the regulation of the 45 kDa protein are described in Chapter 2. This chapter also describes the efficacy of CGS 16949A as an aromatase inhibitor in the human placenta. This compound brought about a significant reduction in the level of estradiol 17- in the first-trimester human placenta as monitored by a radioimmunoassay for estradiol 17-. Results of some preliminary studies which demonstrate the presence of mRNA for estrogen receptor in the human placenta are also presented in this chapter.
Placenta from the first trimester was chosen for these studies as it is metabolically more active than term placenta. Placental villi were incubated in the presence or absence of the modulator whose effect was examined. SDS-PAGE analysis of equal quantity of tissue proteins from control and treated samples was carried out. Addition of estradiol 17- resulted in a significant increase in the intensity of a protein of apparent molecular weight 45,000 upon treatment with estradiol 17-, as quantitated by laser densitometric scanning. The extent of stimulation was, however, relatively less than in the case of estrogen-stimulated proteins from other target tissues. The reason for this could be that unlike many other organs (such as the uterus and the liver), the placenta is not only the site of action of estradiol 17- but also synthesizes large quantities of estradiol 17-. The important functional units of the placenta, viz., the cytotrophoblast and the syncytiotrophoblast, are therefore already exposed to large concentrations of estradiol 17-, and it is difficult to observe stimulation over the “basal” state. To overcome this problem, we have employed specific inhibitors of estradiol 17- synthesis and action. These inhibitors are reported to act as pure antiestrogens.
Following incubation of first-trimester human placental minces in vitro with CGS 16949A (an aromatase inhibitor), there was a marked reduction in the intensity of the band corresponding to 45 kDa. This was found to be true even after incubation with ICI 182780 (an estrogen receptor antagonist), thus providing additional evidence for a role for estradiol 17- in the regulation of this protein.
As there are very few reports on the stimulation of specific proteins by estradiol 17- in human placenta, it was considered worthwhile to purify and characterize this protein so that it can be used as a marker for the action of estradiol 17- in human placenta. The 45 kDa protein was purified to homogeneity using conventional methods. Attempts to raise antibodies in rabbits as well as mice resulted in the production of low-titer antibodies, indicating that it may be a conserved protein. The procedure followed for purification of and production of antibodies against the 45 kDa protein and also the attempts at standardization of an immunoprecipitation assay (using antiserum raised in this study) are described in Chapter 3.
Simultaneously, in an attempt to establish the identity of the protein, N-terminal sequencing of the protein was undertaken. Failure to obtain an N-terminal sequence indicated that it might have a blocked N-terminus. The N-terminal sequences of the internal peptides generated by proteolytic digestion pointed to the identity of this protein with actin. On the basis of identity of partial sequence, molecular weight, and isoelectric point, the presence of a blocked N-terminus (actin has an acetylated N-terminus), and the results of Western blotting studies with the commercially available actin antiserum, it was concluded that the estradiol 17--stimulated 45 kDa protein from the human placenta is actin. The results of N-terminal sequencing of the internal peptides and the identification of the 45 kDa protein as actin, and attempts at developing an immunoprecipitation assay for actin with commercially procured actin antiserum, are presented in Chapter 4.
Although the results of studies with inhibitors of estrogen biosynthesis and action have clearly established a role for estradiol 17- in the regulation of synthesis of the 45 kDa protein, only marginal stimulation was observed following addition of estradiol 17-. In view of this, attempts were made towards obtaining additional evidence for the stimulation of actin by estradiol 17-. Our efforts at standardizing an immunoprecipitation protocol for actin were not successful in spite of several modifications of protocol. This could have been due to an inherent property of actin, such as its tendency to undergo spontaneous polymerization/depolymerization under in vitro conditions. Therefore, an alternative approach was undertaken to demonstrate the stimulation of actin by estradiol 17-. The DNase I inhibition assay is a method described in literature for the quantitation of actin which can be carried out under denaturing conditions, thus circumventing the problems encountered due to the existence of actin in multiple states of polymerization. This method has been successfully employed to quantitate actin levels in tissue homogenates. The demonstration of an increase in the levels of actin in first-trimester human placental minces upon incubation with estradiol 17- is also described in Chapter 4.
The possible significance of the stimulation of actin by estradiol 17- in human placental function and also the possible mechanisms involved are discussed in Chapter 5. | |
