Infertility problems have accelerated over the past decade. Fertility is regulated by pulses of gonadotropin releasing hormone (GnRH) from the hypothalamus that differentially regulate gonadotropin release. It is not known how changing frequencies of GnRH pulses regulate differential expression of LH and follicle stimulating hormone (FSH) genes. Cytochemists over the past 30 years have speculated that one site for differential regulation might be the monohormonal gonadotrope population. Studies during the past decade that detected mRNAs for the gonadotropin beta subunits showed that monohormonal LH gonadotropes were more abundant just before the LH surge, suggesting they may be selectively increased, perhaps by the rapid pulses of GnRH. This study is proposed to test the hypothesis that rapid pulses of GnRH will selectively stimulate a subset of monohormonal LH gonadotropes that also express a higher density of GnRH receptors. Slower pulses of GnRH may favor monohormonal FSH cells. The proposed studies will use emerging new dual in situ hybridization technology to learn mechanisms behind the regulation of monohormonal or bihormonal expression of LHb and FSHb genes. Aim 1 studies will determine if monohormonal gonadotropes are selectively stimulated to increase, in vivo during proestrous or estrous. They will compare expression of cells after brief culture (24h) with expression of gonadotropins in tissue sections from cycling female rats. The sections will be collected in a novel Tissue Microarray, which allows cytochemical labeling and analysis of hundreds of sections/slide. The image analysis will provide information about the percentages of gonadotrope subsets and the density and area of their label for each gene product. These studies will identify times during the cycle when monohormonal gonadotropes are abundant, and use this information to collect and purify separated fractions of these subsets by combined elutriation and bio-magnet techniques. The cells from these fractions will be used in Aim 3 studies of secretion, and samples of their mRNA saved for future gene profiling. Single monohormonal and bihormonal gonadotropes will be collected by Laser Capture Microdissection and stored in homogeneous cellular groups, or, singly, for future gene profiling. Aim 2 studies will learn if the monohormonal LH gonadotropes express more GnRH receptors at midcycle. Triple labeling techniques that detect 3 mRNAs or heteronuclear RNAs (hnRNA) and an mRNA will be developed to optimize the detection of CmRH receptors in monohormonal or bihormonal gonadotropes. Aim 3 studies will use perifusion systems to learn if monohormonal LH cells increase in response to rapid pulses of GnRH. These studies will also test if slower pulses cause an increase in monohormonal FSH cells. Aim 3 studies will also compare responses of gonadotropes in mixed cultures with those in the purified populations collected in Aim 1 studies to learn if the magnitude or pattern of the responses depends on factors from neighboring pituitary cells. The overall objective of these studies will be to use a combination of established and emerging new technologies learn more about the plasticity of the gonadotrope population, as we develop two methods whereby we can collect purified, normal gonadotropes for future gene profiling. The fact that nearly half of LH or FSH gonadotropes are monohormonal, with respect to mRNA expression, suggests a great potential that has not been recognized. An understanding of mechanisms that selectively regulate gene expression in these cells will provide important information about how fertility is regulated.

R21HD047467

2004-07-01

2007-06-30