The Long-Term Goal of this project is to study the role of luteal cell interactions during growth, differentiation and regression of corpora lutea (CL). The Specific Objectives of the proposed research are to study: 1) the ontogeny of contact-dependent intercellular communication between luteal cell types in vitro by evaluating three stages of the estrous cycle (corresponding to rapid growth, differentiation and regression; Exp. 1), 2) the in vitro effects of luteinizing hormone (LH) and prostaglandin F/2alpha (PGF) on contact-dependent intercellular communication and second messenger systems of luteal cells (Exp. 1 and 2), 3) the effects of second messenger systems on contact-dependent intercellular communication between luteal cells in vitro (Exp. 3), 4) the relationship of gap junctional intercellular communication with progesterone production by luteal cells (Exp. 1 and 3), 5) the presence of gap junction proteins in luteal cells and tissues (Exp. 1 and 4), and 6) the in vivo effects of LH and PGF on intercellular communication of luteal cells (Exp. 4), during luteal growth, differentiation and regression. The CL exhibits dynamic structural and functional changes during the course of the estrous cycle. Corpora lutea contain several cell types that interact to maintain normal tissue function. These cells may interact by paracrine or endocrine pathways or through gap junctional channels. Structural and functional gap junctions have been demonstrated in luteal tissues of several species, and probably play a critical role in proliferation, differentiation and regression of luteal cells. In addition, growth, differentiation and regression of the CL are regulated by systemic and local factors, including luteinizing hormone and prostaglandin F/2alpha. These systemic and local regulators affect luteal cell second messengers (i.e., cAMP, protein kinase C and/or calcium), which are known to modulate gap junctions. However, the role of contact- dependent (gap junctional) interactions in coordination of luteal growth, differentiation and regression has not been well defined. To study contact-dependent interactions, we will utilize ovine CL, whose morphology and function have been studied extensively. In the proposed experiments, interactive laser cytometry will be used to examine contact- dependent intercellular communication between luteal cell types in the presence or absence of regulators of luteal function. Interactive laser cytometry also will be used to measure intracellular concentrations of cAMP, protein kinase C or calcium in individual live cells in the presence or absence of regulators of luteal function. Moreover, presence of gap junction proteins will be evaluated in luteal cell cultures and in luteal tissues. Progesterone concentrations in media will be used as an index of luteal cell differentiated function, and also as an estimate of the relationship between gap junctional communication and progesterone secretion. DNA and protein contents of cultured luteal cells as well as immunohistochemical markers of steroidogenic capacity will be used to evaluate cell population dynamics and to determine cell numbers at the end of culture. Elucidation of contact-dependent mechanisms that regulate cellular interactions will provide insight into the coordination of cellular processes during luteal growth, differentiation and regression.
