Studies of immunoreactive gonadrotropin releasing hormone (GnRH) in the rat anterior pituitary.

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Abstract
The unlabeled antibody enzyme staining technique was used to localize endogenous gonadotropin releasing hormone (GnRH) in the male rat pituitary gland. In ultrathin sections specific staining was localized to the large granules of gonadotropes. No staining was observed on plasma membranes even if GnRH was applied exogenously. Absorption of the anti-GnRH with synthetic GnRH significantly reduced staining intensity; absorption with luteinizing hormone (LH) or FSH had no effect. Studies of the effect of different fixatives showed 4% p-formaldehyde to be optimal for the preservation of both immunoreactivity and cell ultrastructure. No immunoreactive GnRH was detected in tissue fixed in OsO4 or combinations of OsO4 and aldehydes. To compare the distribution of the granule-associated immunoreactive GnRH and endogenous gonadotropin, consecutive sections were stained with either anti-GnRH or anti-LH.beta.. Specific staining for both hormones was demonstrated in many of the same cells and in many identical secretory granules. To correlate the intracellular immunoreactive GnRH with gonadotropin secretion, pituitary cells in 4-day monolayer cultures were treated with exogenous GnRH. One group of cultures was used to quantitate FSH and LH secretion while a 2nd group was processed for EM immunocytochemical localization of GnRH. While the pituitary cells secreted FSH and LH in a dose-dependent manner in response to exogenous GnRH, there was no difference in the density of granule-associated immunoreactive GnRH in any culture. While immunoreactive GnRH may be found within intracellular granules of pituitary cells in vivo and in vitro, the distribution and amount of reaction product, as evaluated densitometrically, correlates with neither the gonadotropin secretion nor the concentration of exogenous GnRH. The physiological significance of intracellular immunoreactive GnRH therefore remains unclear.