Effects of intracerebroventricular infusion of genistein on gonadotrophin subunit mRNA and immunoreactivity of gonadotrophins and oestrogen receptor-α in the pituitary cells of the anoestrous ewe

Abstract

The present study was designed to demonstrate whether genistein, a synthetic phytoestrogen, infused into the third ventricle of the brain could affect the gonadotrophic cells regarding the presence of oestrogen receptor-α immunoreactivity and gonadotrophin subunit mRNA hybridising reaction in the ewe. Ewes ( n = 7), aged 2 years, in early anoestrous season were infused with Ringer–Locke solution (control, n = 3) or 10 μg/100 μl/h of genistein ( n = 4) into the third ventricle over a 5 h period and slaughtered the following morning. Immunoreactivity of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestrogen receptor-α (ERα) was determined in the adenohypophysis by immunohistochemistry using antibodies raised against LHβ, FSHβ, and ERα. Messenger RNA analyses were performed by non-isotope in situ hybridisation using sense and antisense riboprobes produced from β subunits of LH and FSH cDNA clones. Computer image analysis was used to determine the percent of cells exhibiting immunohistochemical and/or hybridising reaction. It was found that in ewes infused with genistein, the percentage of LH-positive cells and the density of immunoreactive-LHβ material decreased significantly ( P ≤ 0.001), but the percentage of mRNA LHβ-expressing cells and the intensity of the hybridisation signal increased significantly ( P ≤ 0.001), compared to the vehicle-infused animals. The genistein infusions had no effect on the immunoreactivity of FSH cells or on the expression of mRNA for FSHβ. The percentage of ERα-positive cells increased significantly after genistein infusions ( P ≤ 0.001) and this increase was significant in the LH but not in FSH cells ( P ≤< 0.001). In conclusion, we suggest that genistein can stimulate the expression of immunoreactive ERα in the pituitary LH-cells but not in FSH-cells and change the endocrine activity of LH-producing cells of anoestral ewes.