Localization of Immunoreactive Gonadotropin-releasing Hormone and Relative Expression of Its mRNA in the Oviduct During Pregnancy in Rats

Abstract

S U M M A R Y This study was designed to determine the cellular and ultrastructural distribution of the gonadotropin-releasing hormone (GnRH) and the relative expression of its mRNA in the oviduct of rats during different time points (days 7, 9, 16, and 20) of pregnancy. Immunofluorescent localization and confocal microscopic techniques were used to determine the cellular distribution of GnRH in the oviduct. Immunogold electron microscopy indicated its localization at the ultrastructural level, and real-time PCR was used to study the expression pattern of GnRH mRNA in the oviduct during pregnancy. In general, GnRH was localized within the epithelial cells lining the oviductal lumen at each selected time point. A strong correlation between the fluorescence intensity of GnRH-immunoreactive cells and the relative expression of GnRH mRNA was noted on days 7 and 16, followed by a plateau by day 20. At the ultrastructural level, uniform labeling of colloidal gold particles was observed in secretory vesicles and lamella of the luminal epithelium as well as the lumen of the oviduct.Collectively, these results demonstrate for the first time that the oviductal epithelium synthesizes and secretes the decapeptide GnRH during pregnancy in rats, which may have a possible role in postimplantation embryonic development and the maintenance of pregnancy. (J Histochem Cytochem 55:525-534, 2007)

Expression and Localization of Gonadotropin-releasing Hormone Receptor in the Rat Oviduct During Pregnancy

“…A significant decrease in the signal intensity of GnRHR protein during day 16 of pregnancy, when compared to the other selected times points (days 7 and 20), is indicative of a decrease in the expression of the receptor protein by mid-pregnancy (day 16). As the expression of the ligand (GnRH) in the rat oviduct during day 16 is highest (Sengupta et al 2007), low expression of its classical receptor GnRHR at this particular time point of pregnancy is indicative of the downregulation of the receptor expression by high-ligand concentration. Desensitization of cultured pituitary gonadotrophs as a consequence of downregulation of a number of pituitary GnRHR by highligand (GnRH) concentrations has been previously documented in the literature (Zilberstein et al 1983;Naik et al 1984;Garcia-Palencia et al 2007).…”

“…28S and 18S rRNA bands of representative samples loaded on an Agilent 2100 Bioanalyzer using RNA 6000 nano assay kit (Agilent Technologies; Santa Clara, CA) helped in assessing the integrity of the extracted RNA. Two mg of RNA samples from each tissue type (oviduct and pituitary) were reverse transcribed to cDNA following manufacturer's instructions provided in the Taqman Reverse Transcription Reagent kit (Applied Biosystems; Foster City, CA) with reaction conditions described previously by us (Sengupta et al 2007). cDNA samples thus obtained were stored at 220C for real-time PCR studies.…”

“…To avoid amplification of contaminating genomic DNA, primers specific for GnRHR and 18S rRNA (housekeeping gene) were designed across the exon/intron boundaries. Testing of the primer efficiency, quan- Sengupta et al (2007). A comparative CT method (Qiagen; Valencia, CA) was used to compare the relative expression of GnRHR mRNA in the rat oviduct during different days of pregnancy, using day 7 as the calibrator.…”

“…Rat pituitary was used as a positive control for validation of the experiment. The detailed calculation method has been described elsewhere (Sengupta et al 2007). …”

“…As an initial approach towards investigation of the existence of a receptor-mediated autocrine/paracrine role of oviductal GnRH during pregnancy in rats, we designed the present study that aims at elucidation of the expression pattern of immunoreactive GnRHR and its mRNA during the considered time points (days 7, 9, 16, and 20) of pregnancy. The selected time points during pregnancy correspond to changes in the serum profiles of progesterone, critical for induction and maintenance of pregnancy in rats related to changes in the circulating levels of luteinizing hormone and prolactin (Morishige et al 1973;Sengupta et al 2007). …”