Reproductive Axis Disruption of Mice Lacking Estrogen Receptor β in GnRH Neurons

Presentation Number: OR30-2
Date of Presentation: June 23rd, 2014

Jones B Graceli*1, Silvia Capellino2, Andrew Schoeffield3, Horacio Novaira2, Fredric Edward Wondisford4, Andrew Wolfe2 and Sally Radovick5
1Federal University of Espirito Santo, Vitoria, Brazil, 2Johns Hopkins University School of Medicine, Baltimore, MD, 3Loyola University Maryland, Baltimore, MD, 4Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, 5Johns Hopkins School of Medicine, Baltimore, MD

Abstract

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ER alpha (ERα) and beta (ERβ). Lack of functional ERα and ERβ genes in vivo significantly affects reproductive function, however, the target tissues and signaling pathways in the hypothalamus are unclear. Here, we describe the generation and reproductive characterization of a GnRH neuron-specific ERβ knockout mouse model (GERβKO), as well the development of a specific antisera for mouse ERβ (mERβ). GERβKO mice displayed a delay in vaginal opening (WT: 27.0±0.3 vs GERβKO: 29.6±0.4 d; p≤0.05, n=4-8) and first estrus (WT: 28.4±0.3 vs GERβKO: 32.5±0.8 d; p≤0.05, n=4-8). They also had differences in cycle length, with GERβKO mice having a longer proestrus phase (WT: 1.5±0.1 vs GERβKO: 2.0±0.1d, p≤0.05, n=4-8). Female GERβKO mice had lower serum LH (WT: 0.6±0.03 vs GERβKO: 0.37±0.05 ng/ml; p≤0.01, n=4-10) and FSH (WT: 4.9±0.9 vs GERβKO: 1.6±0.8 ng/ml; p≤0.01, n=4-10) levels.  Further, impaired development of ovarian follicles was noted with reduced number of antral follicles and corpora lutea (p≤0.05, n=3) in the GERβKO mice. In males, there were no differences in the timing of the onset of puberty or the LH and FSH levels between WT and GERβKO mice. In order to confirm deletion of ERβ in GnRH neurons, we developed a novel antisera for mERβ since discrepancies in immunoreactivity have been reported for ERβ antibodies. The antisera was developed using 18 specific amino acids (CSTEDSKSKEGSQNLQSQ) in the C-terminus of the mERβ conjugated to keyhole limpet hemocyanin protein. Wistar rats were immunized and antisera characterized by western blot analysis in tissue panels from WT and mice genetically modified to lack ERβ (CERβO). The size of the detected ERβ protein isoforms was consistent with the predicted molecular size in protein extracts obtained from the hypothalamus, uterus, ovary and mammary gland of WT mice but was absent in CERβO female mice. Male WT mice expressed ERβ in the hypothalamus, testes and epididymus, which was not present in CERβO mice. The pattern of ERα expression was similar in CERβO and WT mice. This novel ERβ antisera confirmed ERβ deletion in GnRH neurons of GERβKO mice. Thus, deletion of ERβ of GnRH neurons in vivo results in a delay of pubertal development and impaired gonadotropin secretion disrupting periodic reproductive cycling. This work supports the hypothesis that the ERβ in GnRH neurons has an important function in the modulation of the reproduction axis.

 

Nothing to Disclose: JBG, SC, AS, HN, FEW, AW, SR