Overview

Gene transcription: The primate trophoblast exhibits cell-specific and developmentally regulated gene transcription. We are studying the transcriptional control of the placental growth hormone variant, which is unique to the primate placenta. We will identify transcription factors and mechanisms which direct cAMP-stimulated transcription to gain insight into how hormone gene expression is coupled to the differentiation of proliferating cytotrophoblast to terminally differentiated syncytiotrophoblast. We are also using embryonic stem cells which spontaneously differentiate into trophoblasts in culture, and rhesus preimplantation embryos to gain insight into the transcriptional control of the specification of the trophoblast lineage.

Immunology: The placenta and the fetus represent an immunologically foreign tissue which escapes rejection by the maternal immune system. Nonpolymorphic MHC class I molecules on trophoblast cells may protect the fetus from immune rejection by the mother. We have cloned rhesus placental class I molecules and will examine their interactions with NK cells and lymphocytes of the maternal immune system.

Placental growth hormone physiology: The physiology of the placental growth hormone variant is poorly understood. Since its secretion rises during the second trimester, while pituitary growth hormone declines, the placental growth hormone becomes the primary circulating somatotropin in pregnant women during the second half of gestation. This places it in a position to be the primary regulator of maternal metabolism, with particular reference to adaptations to the metabolic demands of the fetus. In addition, the presence of GH receptors in cells in the uterus as well as the placenta suggests an additional potential role as a local paracrine regulator. The importance of of appropriate regulation of maternal metabolism for fetal growth and well-being is an additional area we are planning to develop with the primate model. This will interface with new efforts in the cellular and molecular biology of the placenta in the setting of diabetes, in collaboration with clinicians in the Division of Maternal-Fetal Medicine.

Women's Health Studies: A developing interest in angiotensin II receptors and receptor signaling, namely angiotensin II control of vascular flow mediated at the level of the endothelium is being undertaken. It is well known that there are surprising adaptations to pregnancy which occurred in the uterine artery, but a molecular approach had previously been lacking in this field. The tools necessary for such studies, namely a well-characterized cell model and the isolation of the cDNA sequences of interest in the animal model, namely the sheep have been developed in our department. In so doing we were able to extended this work to include development of mRNA quantification techniques for the ovine AT1-R as well as the target end- proteins the receptor regulates, namely endothelial nitric oxide synthase (eNOS) and cyclooxygenase 1 (COX-1). To date we have succeeded in showing pregnancy increases the expression of many genes, and established a well characterized cell model which will allow dissection of the cellular mechanisms underlying pregnancy-induced increases in uterine artery blood flow necessary for fetal survival and optimal growth/development. In addition we have isolated the sheep AT1-R full length cDNA and shown it is not subject to the alternate tissue-specific splicing proposed by some to occur in the human. We are now isolating the AT1-R gene from a genomic library. The molecular methods developed have also been use to monitor the elevation of eNOS and COX-1 mRNA expression in vivo during pregnancy and more recently, throughout the ovarian cycle as well as in animals infused with different steroids. We have thus shown that ovarian steroids play a role in increasing the expression of these gene products but also the relative role of estrogen and progesterone differs in each case. A future is to extend this work through isolation of eNOS and COX-1 genes, in addition to the AT1-R gene, and then examine the differential regulation of each gene's expression using reporter constructs transfected into the cell culture model.

Another exciting area developing at this time from the studies of two graduate students is the finding that different agonists (growth factors vs heterotrimeric G-protein coupled receptors) can differentially activate the eNOS Vs COX-1 pathways so each agonist can theoretically have a unique action on vascular function. In addition, in collaboration with Lubo Zhang at Loma Linda University, CA, we have shown ATP but not AII, activates an increase in cellular Ca2+ in endothelial. Additional graduate student studies show that AII instead activates alternate signaling pathways more commonly associated with growth factors (Mitogen Activated Protein Kinase - MAPK). Furthermore, the time course of activation of vasodilator production in response to AII is like that of growth factors, i.e., slow and sustained, while that in response to ATP is more rapid and transient. We have also recently reported that cytosolic phospholipase A2 is present in the UA endothelium, and is elevated in pregnancy (Di et al (Submitted). This is relevant because cPLA2 is a known convergence point in cell signalling, being regulated by both Ca2+ and MAPK pathways. Thus exciting new areas are opening up which, with the newly developed cellular and molecular tools, will allow a dissection of the mechanisms underlying differential control of vascular endothelial cell function by AII.

Fetal Studies: The use of Betamethasone as an agent to accelerate maturation of lung function in preterm infants has revolutionized the care of preterm infant care. This drug, however, suppresses adrenal function and it is a matter of concern that the consequences of this suppression are still poorly understood. We are actively investigating the molecular basis for the adverse effect of betamethasone on rabbit fetal adrenal function and attempting to develop a more complete profile of its action of bioactive steroid levels. Adrenocortical studies have also been undertaken with a number of outside laboratories in order to study both zonal expression and zonal function of AT1-R. Studies with Dr Alan Conley (Assistant Professor at Dept Population Health and Reproduction, School of Veterinary Medicine, UC-Davis, Davis, CA), and Dr. Catherine Coulter (Assistant Professor, Dept. Physiology, Univ. Adelaide, Adelaide, South Australia) of immunohistochemical localization of AT1-R and 17(-hydroxylase (P450c17) expression in adult and fetal sheep have shown directly an inverse relationship exists in vivo which extends previous work, and we have now revealed a previously unknown role for this receptor in suppressing adrenal cortisol biosynthesis mid-term. In the adrenocortical area again, recent have also resulted in a collaboration to study mechanisms underlying altered regulation of adrenal function in subordinate (non-reproductive) female primates.