Luteinizing Hormone-releasing Hormone mRNA Research Articles

Medial Preoptic Microimplants of the Antiestrogen, Keoxifene, Affect Luteinizing Hormone‐Releasing Hormone mRNA Levels, Median Eminence Luteinizing Hormone‐Releasing Hormone Concentrations and Luteinizing Hormone Release in Ovariectomized, Estrogen‐Treated Rats

Abstract We examined the temporal changes in plasma luteinizing hormone (LH) levels, median eminence luteinizing hormone-releasing hormone (LHRH) concentrations and LHRH mRNA levels in estrogen-treated, ovariectomized rats with empty or antiestrogen- containing microcannulae stereotaxically implanted into the medial preoptic area. Neither treatment disrupted the negative feedback effects of estrogen on LH secretion, but antiestrogen (Keoxifene) blocked the afternoon LH surges. In rats exhibiting LH surges, median eminence LHRH concentrations were similar at 0800, 1200 and 1600 h, but they were significantly elevated by 2000 h. In contrast, no alterations in LHRH concentrations occurred in the Keoxifene-treated group. LHRH mRNA levels in control rats were significantly elevated at 1200, 1600 and 2000 h compared with 0800 h, but LHRH mRNA levels in Keoxifene-treated rats did not change significantly over the time period examined. When we compared treatment effects over time we saw that serum LH levels were significantly higher in control than Keoxifene-treated rats only at 1600 and 2000 h. Median eminence LHRH concentrations did not differ between treatment groups until 2000 h when control animals had significantly higher levels than those of Keoxifene-treated animals. LHRH mRNA levels in Keoxifene-treated rats were significantly higher than those of controls at 0800 hand significantly lower at 1600 h. No differences in LHRH mRNA levels were detected between groups at either 1200 h or 2000 h. In summary, although it was not clear on which neuronal system estrogen acted, depriving medial preoptic neurons of this steroid in systemically estrogenized rats certainly disrupted the neural mechanisms involved in surge, but not basal LH release. In addition, neither LHRH mRNA levels nor median eminence LHRH concentrations showed variations within the period studied when the estrogen-sensitive mechanisms involved in LH release were disrupted. Therefore, the changes in LHRH mRNA levels and LHRH concentrations in the median eminence seen in surging animals probably resulted from the same neural events which triggered LH release.

Testosterone stimulates LH-RH-like mRNA level in the rat hypothalamus

To elucidate whether testosterone (T) replacement to castrated male rats may promote luteinizing hormone-releasing hormone (LH-RH) biosynthesis in the hypothalamus, an RNA-blot hybridization assay was employed. Poly(A) mRNA fractions from hypothalami (40–50) of intact, castrated plus vehicle, and castrated plus T groups were isolated, blotted on nitrocellulose paper and hybridized with 32P-end labelled LH-RH oligonucleotides (29 mer) which is complementary to the rat LH-RH mRNA. LH-RH-like mRNA level markedly attenuated 2 weeks following castration and T replacement significantly increased LH-RH-like mRNA level, comparable to about 80% of that observed in intact male rats. In addition, LH-RH contents and release from hypothalami were studied, to examine the correlation with LH-RH gene expression and secretory activity of LH-RH. Castration significantly reduced LH-RH contents and LH-RH release in vitro. T replacement restored LH-RH contents and release comparable to those shown in the intact group. This study demonstrates for the first time that T replacement increases LH-RH-like mRNA level in the hypothalami of castrated rats suggesting that T may act at the pretranslational level. This change in LH-RH gene expression is parallel with alteration of LH-RH content and release.

Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA.

A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormone-releasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffinity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.

Attempts to immunoprecipitate the LHRH precursor synthesized in cell free systems

In order to determine the molecular weight of the Luteinizing Hormone Releasing Hormone (LHRH) precursor, poly(A)-RNA from rat hypothalami and human placenta were translated in two mRNA dependent cell free translation systems. Total translation products were immunoprecipitated with two antisera that recognized LHRH high molecular weight forms. After SDS-polyacrilamide slab gel electrophoretic analysis of the immunoprecipitated material and fluorography, we detected in both tissues a protein of 50,000 daltons with the No. 1076 antiserum. This peptide was not immunoprecipitated by the No. 743 anti-LHRH antiserum or by non-immune rabbit serum. However, this protein was not displaced by excess LHRH added during the immunoprecipitation and seemed to be present in species where LHRH has not been reported. These data demonstrated that the LHRH mRNA is present in very low amounts in hypothalamus or placenta and that the sensitivity of the assay is not high enough to recognize it.