Peter J Hansen - US grants

Long-term objectives: A better understanding of the role of uterine secretions could lead to methods to 1) reduce post-fertilization pregnancy failure and 2) control the rate of fetal growth. Objectives are to understand the functional and biochemical characteristics of the uterine milk proteins (UTMP), the major proteins secreted by the endometrium of the sheep uterus during pregnancy. Preliminary findings that UTMP inhibit T\lymphocyte function in vitro and bind to IgA suggest they may protect the fetus from immunological attack by the mother. Specific aims and methods: (1) Characterize the binding of IgA to UTMP. Binding of 125-IgA to UTMP conjugated to Sepharose will be determined in several experiments. Binding data will be analyzed by Scatchard Analysis to determine the affinity constant. Binding of 125I-IgA to UTMP-Seph in the presence of IgG, IgM and other proteins will be evaluated to determine specificity of the binding reaction. Proteolytic fragments of 125I-IgA will be tested for binding to UTMP-Seph to determine if UTMP binds to the Fc or F(ab)2 portion of IgA. To verify that binding occurs in utero, uterine fluid will be fractionated by gel filtration and analyzed by immunological procedures for the presence of IgA-UTMP complexes. (2) Test whether binding of UTMP to IgA inhibits biological functions of IgA. UTMP will be tested for their ability to inhibit binding of 125I-IgA to Fc receptors on bacterial and lymphocyte surfaces. If UTMP are found to bind to the F(ab)2 portion of IgA, the ability of UTMP to inhibit binding of specific IgA antibodies to antigen will be evaluated. (3) Determine whether UTMP are similar proteins to secretory component by evaluation of immunological cross-reactivity using immunodiffusion techniques. (4) Test whether UTMP have immunosuppressive activity in utero by determining whether infusion of UTMP in utero prevents rejection of intrauterine skin grafts. (5) Determine the types of oligosaccharide chains on the two forms of UTMP by gel filtration, glycopeptide mapping, lectin binding and gas chromatography. (6) Determine if the formation of the two UTMP from a common, lower molecular weight precursor is due to glycosylation of the precursor. The precursor and the two UTMP will be immunoprecipitated from endometrial tissue treated with a glycosylation inhibitor. The precipitates will be analyzed by SDS electrophoresis to determine if the formation of the two UTMP from the precursor occurs in the absence of glycosylation.

The purpose of the 20th Annual Meeting of the American Society for Reproductive Immunology (ASRI) is to l) share research concerning important new developments in reproductive immunology, 2) promote intellectual exchange between basic scientists and clinicians in the various disciplines of reproductive immunology and 3) provide mentorship for new scientists interested in reproductive immunology. The meeting, which is open to all interested scientists, will be organized around six plenary sessions. Plenary Session I, entitled "New Concepts in Reproductive Immunology", will highlight recent discoveries that have changed the understanding of mechanisms allowing the fetal allograft to elude the maternal immune system.. Plenary Session II will focus on cellular immune function in the reproductive tract, Plenary Session III will be devoted to reproductive microbiology and virology and Plenary Session IV will focus on regulatory molecules involved in the dialogue between the immune and reproductive systems. Plenary Session V is entitled "Nutritional Immunology and Reproduction" and will consist of a series of cutting-edge presentations on an emerging idea that nutritional regulation of immune function can have an impact on reproductive biology. Plenary session VI, entitled "Clinical Management of Patients with High-Risk Pregnancies Due to Immunological Problems", addresses the ultimate goal of research in reproductive immunology to apply results to he clinical setting. Invited speakers for session VI are in the forefront of clinical science related to management of women patients with infertility associated with immunological causes. Funds from NIH are being requested for 1) financial support for 20 graduate students, postdoctoral scientists, residents and junior faculty who wish to attend the meeting and 2) expenses for 8 invited speakers. It is critical that trainees attend the 20th Annual Meeting of the ASRI to learn about the most recent work in reproductive immunology, meet people who are doing cutting-edge research in reproductive immunology, and to get critical appraisal and feedback regarding their scientific efforts from reading reproductive immunologists. Travel support for speakers is requested since, as a small society, the ASRI is dependent upon sponsoring agencies to provide support for invited speakers at the annual meeting.

DESCRIPTION (provided by applicant): The developmental program of the preimplantation embryo is readily modified by alterations in the microenvironment. The resultant change in trajectory of development can have long-acting effects that extend into post-natal life. Male and female embryos can respond differently to environmental modification during the preimplantation period. Recent data from our laboratory with bovine embryos indicate that sexual dimorphism in response to changes in the microenvironment could be mediated by gender-specific responses to maternal regulatory signals. Treatment of embryos with colony-stimulating factor 2 (CSF2) from Day 5-7 of development caused gender-specific alterations in characteristics of embryos after transfer to females and recovery at Day 15 of gestation. If this phenomenon of sexual dimorphism is a general one, gender-specific analysis will be required to understand basic mechanisms controlling development, environmental effects on embryonic development, and therapeutic approaches to improving fertility in women. The current proposal seeks to determine the extent to which regulation of embryonic development depends on gender. There are two immediate goals: to develop a model for understanding the molecular basis for how CSF2 exerts gender-specific effects on embryonic development and to evaluate whether the phenomenon of sexual dimorphism in regulation of preimplantation development is widespread (i.e., exists for more than one regulatory molecule). To meet these objectives, we will take advantage of a large amount of preliminary data from our laboratory that describes how CSF2 and two other uterine regulatory factors, insulin-like growth factor 1 (IGF1) and dickkopf-related protein 1 (DKK1), modify development of the bovine blastocyst. In particular, we will test whether actions of CSF2, IGF1 and DKK1 on embryos seen previously occur in a gender-specific manner when embryos are produced using X- or Y-sorted spermatozoa. For Aim 1, gender differences in response of bovine morulae and blastocysts to CSF2 will be determined. Four experiments will be conducted to evaluate sexual dimorphism in regulation of the embryo by CSF2 with respect to gene expression in morulae and blastocysts, inhibition of apoptosis, and increase in numbers of cells in the inner cell mass of the blastocyst. Aim 2 will determine whether sexual dimorphism exists for other regulatory factors by testing whether IGF1 and DKK1 exert actions on bovine embryos in a gender-specific manner. Experiments will be performed to determine differences between male and female embryos with respect to IGF1 actions on blastocyst development, gene expression and apoptosis and with respect to DKK1 actions on allocation of cells in the blastocyst to cells of the epiblast, primitive endoderm, and trophectoderm lineages. These experiments are important to develop research models for delineating mechanisms by which sexual dimorphism is established (Aim 1) and to determine the importance of sexual dimorphism as an important determinant of regulation of embryonic development (Aim 2).

PROJECT SUMMARY The developmental program of the preimplantation embryo is modified by alterations in its microenvironment. The resultant change in development can have long-acting effects that extend into postnatal life. Inappropriate signaling between mother and embryo can lead to increased pregnancy loss and long-term changes in health. Research on developmental programming could lead to elimination of adverse outcomes associated with IVF and result in new approaches to improve human health and animal production generally. One characteristic of developmental programming during the preimplantation period is sexual dimorphism in the modification in adult phenotype. Sex differences in embryonic responses to changes in the microenvironment are likely mediated in part by sex-specific responses of the embryo to maternal regulatory signals. One molecule involved in this phenomenon is colony stimulating factor 2 (CSF2). In cattle, treatment of morula and blastocyst stage embryos with CSF2 changed embryonic characteristics at a later point in pregnancy (Day 15) in a sex-specific manner and, at least in female offspring, resulted in calves with increased growth rates in the first year of life. The long-term goal is to understand how the microenvironment of the embryo interacts with sex to shape the developmental program. The overall objectives of the current proposal are to 1) elucidate mechanisms by which CSF2 causes differential programming actions on male and female embryos and 2) characterize the consequences of actions of CSF2 on the preimplantation embryo in vitro and in vivo for subsequent embryonic and postnatal phenotype. It is hypothesized that sex-dependent responses to CSF2 depend upon differences between male and female embryos in remodeling of DNA methylation by CSF2 and that these changes in DNA methylation result in long-acting effects on the developmental program that have consequences for postnatal phenotype. There are three specific aims. For Aim 1, it will be tested whether differences between male and female preimplantation embryos in programming actions of CSF2 on the Day 15 embryo are dependent upon DNA methylation. For Aim 2, the importance of CSF2 for embryonic development in vivo will be evaluated by testing consequences of knocking out the CSF2 receptor for embryo elongation, gene expression and DNA methylation in male and female embryos. Aim 3 will focus on ascertaining whether exposure of the preimplantation female embryo to CSF2 programs development to alter postnatal phenotype in a manner that improves dairy cow productivity. Through these aims, we will elucidate importance of remodeling of the methylome for sex-dependent actions of CSF2 (Aims 1) and ascertain whether actions of CSF2 occur not only in vitro but also for embryos developing in utero (Aim 2). Finally, the experiment for Aim 3 represents the first attempt to enhance productivity of a food animal through use of a developmental programming molecule to manipulate development in the preimplantation period. .