Frank Talamantes - US grants

The mouse conceptus produces two lactogenic hormones, placental lactogen (PL) and midpregnancy lactogen. The objectives of this proposal are (1) to study the regulation of secretion of PL and (2) to examine the biological activities of PL and midpregnancy lactogen and two other lactogenic hormones, prolactin (PRL) and growth hormone (GH), in several target tissues. Regulation of PL secretion by the pituitary, ovaries, fetus and decidua and by growth factors will be studied. In addition, the influence of genetic factors on mPL secretion will be examined. A system for primary culture of placental trophoblast giant cells in serum-free medium will be established and the effects of various hormones and growth factors on mPL secretion and on cell growth will be examined. PL, midpregnancy lactogen, PRL and GH are all present during pregnancy in the mouse. The effects of each of these hormones on target tissues will be studied to assess the relative potencies of the hormones in each tissue and to determine whether tissues at different stages of pregnancy respond preferentially to some of the hormones. The effects of these hormones on mammary gland casein, Alpha-lactalbumin and fatty acid synthesis will be determined. The ability of these hormones to stimulate mammary gland epithelial cell growth and to bind to mammary gland receptors will be evaluated. The effects of these hormones on somatomedin produced by maternal and fetal liver will be studied. The ability of these hormones to stimulate fetal growth will be assessed. The studies proposed in this application will be carried out with hormones purified from the mouse to study the endocrinology or pregnancy in the mouse. Thus the results of these studies will not be subject to problems of interpreting data collected from heterologous systems. PL is present in humans and serum PL levels have been used as one index of fetal well-being. The studies in this proposal will provide information about the physiology of this hormone and the interaction of this hormone with other hormones with similar activities during pregnancy in an animal model. The information obtained from studies in this animal model may prove relevant to the human.

It has been estimated that up to 15,000 children born each year in the United States are affected by one or more symptoms of fetal alcohol syndrome (FAS). FAS includes central nervous system disturbances, characteristic facial malformations, other skeletal and soft tissue malformations, and prenatal and postnatal growth retardation. Growth retadation is one of the most prevalent features of FAS. A large number of tissues and processes that influence prenatal and postnatal growth are adversely affected by alcohol consumption during pregnancy, including endocrine systems that regulate growth. The overall goal of this project is to assess consequences of prenatal alcohol exposure on endocrine systems that regulate prenatal and postnatal growth using the mouse as an animal mode. Mice will be treated with alcohol beginning on day 5 of pregnancy and continuing until parturition. Alcohol will be administered in a liquid diet. Changes in food consumption due to alcohol will be controlled for by including both pair-fed controls and controls fed ad libitum in all experiments. To determine whether prenatal alcohol exposure affects circulating concentrations of hormones that regulate growth, the concentrations of placental lactogen, growth hormone, somatomedins and insulin in the maternal and fetal serum and in amniotic fluid will be determined in alcohol-treated and control mice at several times during pregnancy. In addition, concentrations of growth hormone, somatomedins and insulin will be measured in the offspring of alcohol-treated animals at different ages. To determine whether prenatal alcohol exposure affects the sensitivity of target tissues to growth regulating hormones, basal and hormone-stimulated hepatic glycogen synthesis, ornithine decarboxylase activity, somatomedin production, skeletal muscle amino acid uptake, and cartilage metabolism will be examined in alcohol-exposed and control fetal and postnatal mice.

The mouse placenta produces two placental lactogens: mouse placental lactogen-I (mPL-I) and mPL-II. The long-term objectives of the project are to understand what functions these hormones perform in various target tissues and to understand what regulates their production and concentrations in the fetal and maternal blood. Although relatively little is known at present about the biological activities and regulation of secretion of PLs in various species, it is clear from what is known, that PLs participate in several important processes during pregnancy, such as stimulating mammary gland differentiation, regulating maternal and fetal intermediary metabolism, and stimulating fetal growth. In the long term, investigating the physiology of PLs will contribute to a better understanding of the processes that influence fetal health and survival during pregnancy. The studies outlined in this renewal application are a continuation of previous work in this laboratory on the biological activity and regulation of secretion of mPL-I and mPL-II. A primary culture system will be developed for examining the regulation of mPL-I secretion. It will be used to screen mPL-I secretagogues. The biological activity of mPL-I will be examined with respect to its ability to inhibit prolactin surges in midpregnant mice, using recombinant mPL-I. The regulation of secretion of mPL-II will be examined in several series of experiments. The mechanism by which the pituitary gland suppresses the maternal serum mPL-II concentration will be investigated. A mPL-II-releasing factor present in the decidua will be purified and its physiology will be examined. Effects of gonadotropin-releasing hormone and growth hormone-releasing hormone on the synthesis and release of mPL-II and total placental proteins will be examined and the presence of these peptides in the mouse placenta will be investigated. The biological activity of mPL-II in the maternal mammary gland will be examined with respect to the stimulation of alpha- lactalbumin production. experiments will also be performed to identify proteins whose synthesis is stimulated by mPL-II in fetal mouse hepatocytes.

DESCRIPTION (Adapted from the applicant's abstract): The gene encoding the growth hormone receptor is also the source for a GH-binding protein. In human and rabbit, GHBP is generated by proteolysis of GHR. In rodents, GHR and GHBP mRNAs are alternatively spliced from the GHR/GHBP gene. Both proteins are expressed in numerous tissues, but expression of GHBP is characterized by complexity not observed for GHR. Whereas GHR is an integral membrane protein, GHBP is present in soluble intracellular, membrane-associated, and secreted forms. The functions of GHBP in cells where it is expressed as a soluble or membrane- associated protein are unknown. The long-term goal of the studies of GHR and GHBP is to understand how their expression from a common gene is regulated and how GHR and the different forms of GHBP are involved in mediating GH action. The specific interest is in the up-regulation of GHR/GHBP gene expression during pregnancy in the mouse. This represents the most dramatic change in expression of this gene observed to date in any species, making it a good model for studies of GHR/GHBP gene expression. Considering the diverse metabolic and growth-promoting effects of GH, regulation of this gene is probably an important component of the complex physiological changes necessary for a successful pregnancy. The specific aims of this revised continuation application are to: 1. Examine mechanisms regulating GHR/GHBP gene expression in the mouse. The multiple 5' noncoding exons of the GHR/GHBP gene expressed in liver and placenta will be characterized. The function of the proximal promoter regions associated with these exons will be analyzed. The contribution of transcriptional regulation to up-regulation of GHR/GHBP gene expression during pregnancy will be assessed. 2. Examine regulation of GHR/GHBP gene expression by GH and fetal/placental factors. Direct effects of GH and fetal/placental factors implicated in regulating expression in vivo will be examined in vitro. If a direct effect of fetal/placental factors is observed, the factor(s) will be characterized. 3. Characterize the GH-binding proteins present in several tissues. Studies of GHR measuring the binding of GH to cells or membrane preparations may be measuring binding to GHR and to membrane-associated GHBP. A GHR-specific RIA will be developed and used with an existing GHBP-specific RIA to determine the distribution of the proteins in membrane and soluble fractions of several tissues as a function of pregnancy.

9407939 Talamantes Several peptides originally identified in the hypothalamus are also present in the placenta. One of these peptides is growth hormone-releasing hormone. The factors that regulate expression of the growth hormone-releasing hormone gene in the placenta and its function in the placenta are not understood. This project will address two basic questions: (1) what are some of the structural features in the mouse growth hormone-releasing hormone gene that regulate its expression in the placenta? and (2) does placental growth hormone-releasing hormone regulate the production of a group of hormones in the placenta which are known to play important roles in maintaining a successful pregnancy? The latter hormones include the placental lactogens. These questions are a logical extension of the very limited research that has been carried out in this field so far, and the information gained from this project will provide a good basis for future work in an area that could prove to be very important to understanding placental physiology. ***

The mouse placenta produces two placental lactogens (PLs); mPL-I and mPL- II. The long term objectives of the project are to understand the functions of these hormones in various target tissues and to understand what regulates their production and concentrations in the fetal and maternal blood. Although relatively little is known about the biological activities and regulation of secretion of PLs in various species, it is clear that PLs participate in several important processes during pregnancy. Investigating the physiology of PLs will contribute to a better understanding of the processes that influence fetal health and survival; during pregnancy. These studies are a continuation of previous work in this laboratory on the biological and regulation of secretion of mPL-I and mPL-II. Several experiments include examining the regulation of secretion of proliferin (PLF) and PLF-related protein (PRP), which are proteins that are structurally similar to the PLs and are also produced by the mouse placenta. The specific aims of this project are: (1) The primary structure of calcyclin, a stimulator of mPL-II secretion will be determined. The protein will be localized in the conceptus by in situ hybridization, and the presence of binding sites for decidual calcyclin in the placenta will be assessed. Effects of decidual calcyclin on PL-I, PLF and PRP secretion will be examined in vivo. (2) Effects of epidermal growth factor, transforming growth factors alpha and beta, tumor necrosis factor-alpha, interleukin (IL)-beta, and IL-6 alone and in combinations, will be determined on giant cell differentiation and on mPL-I, mPL-II, PLF and PRP secretion by placental cells from various days of pregnancy. The sites of production of these agents in the conceptus will be determined by in situ hybridization. (3) The structure of the genes for mPL-I and mPL-II will be determined, and the promoter region will be examined for the presence of known response elements. Putative regulators of mPL-I and mPL-II expression identified in this way will be examined in vitro and in vivo for effects on mPL-I and mPL-II secretion. (4) A putative stimulator of mPL-I secretion produced by the pituitary gland will be characterized and if it appears to be novel, it will be purified and characterized. (5) An inhibitor of prolactin secretion produced by the placenta will be purified. Its interaction with mPL-I and mPL-II in regulating prolactin secretion will be examined in vitro. (6) A novel 29K insulin-like growth factor binding protein (IGBP) whose production is induced by mPLs will be purified and its primary structure will be determined. The liver will be examined for the presence of 29K IGFBP. The gestational profile of 29K IGFBP in liver and mammary gland will be determined. Regulation of 29K IGFBP by mPL-I and mPL-II in liver and mammary gland will be examined. (7) Proteins regulated by mPL-I and mPL-II in maternal liver will be identified.

DESCRIPTION: (adapted from the investigator's abstract) Pregnancy provides significant protection against breast cancer development in women that give birth before the age of 20 years. Similarly, parous rats are refractory to chemically induced mammary tumorigenesis. The mechanisms by which parity affects mammary cancer incidents will be investigated by the investigator and coworkers in this proposal using the rat as a model. Pregnancy is characterized by elevated concentrations in serum of hormones that regulate the development of the mammary gland and affect mammary carcinogenesis. This proposal examines the hypothesis that these hormonal changes, along with changes in the levels of hormone and growth factor receptors in the mammary gland, played a decisive role in reducing mammary gland susceptibility to carcinogenesis. In specific aim I, mammary tissue and isolated mammary epithelial cells from virgin rats previously treated with carcinogen, which are highly susceptible carcinogenesis, will be transplanted into gland-free pads of parous hosts, which are refractory to carcinogenesis, and vise versa to determine whether refractory mammary tissue becomes susceptible and if susceptible mammary tissue become refractory to carcinogenesis in an appropriate hormonal environment. In addition, isolated mammary tumor cells will be transplanted into gland-free fat pads of refractory and susceptible rats to determine whether the hormonal environment of both these groups of rats is capable of sustaining the growth of already established mammary tumors. In specific aim II, the role of growth hormone (GH), prolactin (PRL) and ovarian steroids in mediating the parity-associated refractoriness to chemically induced carcinogenesis will be examined. Somatostatin analog and bromocriptine will be used to reduce the circulating concentrations of GH and PRL, respectively, in virgin rats to the concentrations in parous rats. GH and PRL will be administered in osmotic pumps to parous rats to obtain the concentrations in serum of virgin animals. The GH and PRL manipulations will be carried out in intact rats and ovariectomized animals, untreated or treated with constant dosage of 17beta-estradiol and/or progesterone. All these experimental groups and untreated control groups will be injected with carcinogen and tumor incidence compared.

The mouse placenta produces two placental lactogens: mouse placental lactogen-I (mPL-I) and mPL-II. The long-term objectives of the project are to understand what functions these hormones perform in various target tissues and to understand what regulates their production and concentrations in the fetal and maternal blood. Although relatively little is known at present about the biological activities and regulation of secretion of PLs in various species, it is clear from what is known, that PLs participate in several important processes during pregnancy, such as stimulating mammary gland differentiation, regulating maternal and fetal intermediary metabolism, and stimulating fetal growth. In the long term, investigating the physiology of PLs will contribute to a better understanding of the processes that influence fetal health and survival during pregnancy. The studies outlined in this renewal application are a continuation of previous work in this laboratory on the biological activity and regulation of secretion of mPL-I and mPL-II. A primary culture system will be developed for examining the regulation of mPL-I secretion. It will be used to screen mPL-I secretagogues. The biological activity of mPL-I will be examined with respect to its ability to inhibit prolactin surges in midpregnant mice, using recombinant mPL-I. The regulation of secretion of mPL-II will be examined in several series of experiments. The mechanism by which the pituitary gland suppresses the maternal serum mPL-II concentration will be investigated. A mPL-II-releasing factor present in the decidua will be purified and its physiology will be examined. Effects of gonadotropin-releasing hormone and growth hormone-releasing hormone on the synthesis and release of mPL-II and total placental proteins will be examined and the presence of these peptides in the mouse placenta will be investigated. The biological activity of mPL-II in the maternal mammary gland will be examined with respect to the stimulation of alpha- lactalbumin production. experiments will also be performed to identify proteins whose synthesis is stimulated by mPL-II in fetal mouse hepatocytes.