Combined immunohistochemistry for gonadotropin-releasing hormone (GnRH) and pro-GnRH, and in situ hybridization for GnRH messenger ribonucleic acid in rat brain

Experiments were performed to explore the distribution of neurons containing pro-GnRH and GnRH in the rat brain and to determine the correspondence of immunoreactive peptides and pro-GnRH mRNA. Using avidin-biotin immunohistochemistry on free floating vibratome sections it was found that pro-GnRH- and GnRH-containing cells exhibited a similar distribution within the preoptic area-basal hypothalamus region. Within individual neurons pro-GnRH was primarily detected in the cell soma and proximal fibers, whereas the decapeptide was present in cells, fibers, and nerve terminals. Combined avidin-biotin immunohistochemistry for GnRH or pro-GnRH peptides and in situ hybridization for pro-GnRH mRNA using a cRNA probe revealed that the peptides and mRNA could be detected in the same cells. In both male and female rats pro-GnRH mRNA was localized primarily in GnRH-containing cells; however, not all immunoreactive GnRH neurons contained detectable levels of pro-GnRH mRNA, and not all neurons containing pro-GnRH mRNA contained GnRH peptides. In proestrous females a close correlation existed between the total number of neurons containing GnRH and those containing pro-GnRH mRNA (r = 0.84-0.9), while in intact male rats the correlation was not as high (r = 0.56). These results document the distribution of pro-GnRH and GnRH in the rat preoptic area-basal hypothalamus and describe the extent of colocalization of GnRH peptide and pro-GnRH mRNA in proestrous females and intact male rats. Further work will determine how each of the molecular components is regulated during different reproductive states.