Gonadotropin‐Releasing Hormone mRNA and Gonadotropin β‐Subunit mRNA Expression in the Adult Female Rat Exposed to Ethanol In Utero
Authors: Wilson Melinda E. , Marshall Margaret T. , Bollnow Melanie R. , McGivern Robert F. , Handa Robert J. 
Published: April 2006

Previous studies have demonstrated that exposure of female rats to ethanol in utero results in long‐term deficits in reproductive function, including a delayed onset of puberty and an early onset of acyclicity. In the present studies, we determined if changes in reproduction are correlated with changes in gonadotropin‐releasing hormone (GnRH) mRNA expression in the brain or gonadotropin subunit mRNA expression in the anterior pituitary gland. We used in situ hybridization histochemical techniques to examine the density of GnRH mRNA and the distribution of GnRH mRNA‐containing cells in the basal forebrain, and reverse transcription‐polymerase chain reaction (RT‐PCR) to quantitate the β‐subunit mRNA of luteinizing hormone (LHβ) and follicle‐stimulating hormone (FSHβ) in the anterior pituitary gland of adult (3 months of age) fetal alcohol‐exposed (FAE) female rats. For GnRH mRNA measurements, animals were gonadectomized 4 days before use. Three groups of animals were examined. FAE females were derived from pregnant dams fed a liquid diet containing 35% ethanol‐derived calories from gestational day 14 until parturition. Dams of control animals were either pair‐fed (PF) an isocaloric diet with sucrose substituted for ethanol or maintained on normal laboratory rat chow [chow‐fed (CF)]. Serial blood samples taken by indwelling right atrial cannulae demonstrated significantly smaller pulses of LH (p < 0.05) and FSH (p < 0.05) in ovariectomized FAE females at 3 months of age, compared with PF and CF controls. Distribution of GnRH mRNA‐containing cells was mapped throughout the forebrain, and the number of autoradiographic silver grains/ cell was determined. GnRH mRNA‐containing soma were greatest in the diagonal band of Broca and in the anterior parts of the medial preoptic area. There was no difference in the distribution of GnRH mRNA‐containing cells or grain density/cell between experimental groups. To examine pituitary gonadotropin β‐subunit gene expression in ovariectomized and in estrogen‐treated FAE females, rats were ovariectomized and 4 days later given an injection of estradiol benzoate (10 μ/kg body weight, sub‐Q in oil) or vehicle. Animals were killed on the following day in the morning or the evening. Ovariectomized FAE oil‐treated females had significantly lower levels of FSHβ mRNA (p < 0.05), but not LHβ mRNA versus control‐fed rats. In response to estrogen, a surge of LH occurred in the evening in all animals that was significantly reduced in FAE females (p < 0.02). Pituitaries from these same animals were harvested for mRNA analysis of LHβ and FSHβ mRNA by semiquantitative RT‐PCR. Coincident with the reduced afternoon surge of LH, LHβ mRNA was lowered in estrogen‐treated FAE females (p<03). FSHβ mRNA was decreased by estrogen in all groups (p <05). These data demonstrate that the deficits in gonadotropin secretion seen in ovariectomized adult FAE females are not correlated with changes in the distribution of GnRH cells or the steady‐state levels of GnRH mRNA and gonadotropin β‐subunit mRNAs. After estrogen treatment, gonadotropin β‐subunit mRNA expression and gonadotropin secretion are altered in adult FAE females.