Activation of estrogen receptor-α induces gonadotroph progesterone receptor expression and action differently in young and middle-aged ovariectomized rats

Ana Gordon 1 Rafaela Aguilar 1 Jose C. Garrido-Gracia 1 Silvia Guil-Luna 0 Raquel Sa nchez-Cespedes 0 Yolanda Milla n 0 Juana Martn de las Mulas 0 Jose E. Sa nchez-Criado 1 0 Departments of Comparative Pathology, University of C ordoba , C ordoba, Spain 1 Departments of Cell Biology, Physiology and Immunology, University of C ordoba , C ordoba, Spain background: We attempted to define the effect of estrogen receptor (ER)a activation on gonadotroph progesterone receptor (PR) expression (mRNA and protein) and action (GnRH-stimulated and GnRH self-priming) in short- and long-term ovariectomized (OVX) rats. methods: Two weeks or 1 year after OVX, rats were injected over 3 days with 125 mg/kg of estradiol benzoate (EB), 7.5 mg/kg of the selective ERa agonist propylpyrazole triol (PPT), or 15 mg/kg of the selective ER modulator tamoxifen (TX). Controls were given 0.2 ml oil. The last day of ER analog treatment, half of the rats in each group received 25 mg/kg of progesterone (P). The next day, anterior pituitaries were removed and analyzed for PR-AB mRNA and protein. Gonadotrophin secretion in incubated pituitaries was also measured. results: (i) PR mRNA expression was higher in young than in middle-aged OVX rats although PR protein was absent in pituitaries from both groups of OVX rats; (ii) activation of ERa reduced gonadotroph hypertrophy and increased PR mRNA and protein expression (EB . PPT . TX) more efficiently in young than in middle-aged rats, (iii) ER agonists elicited GnRH-stimulated LH and FSH secretion in young but only FSH secretion in middle-aged OVX rats, (iv) evaluated by peak LH concentrations, GnRH self-priming was observed in both groups of OVX rats and (v) P down-regulated PR protein expression in young, and to a lesser extent, in middle-aged OVX rats, in close association with PR-dependent GnRH self-priming. conclusions: Middle-aged OVX rats exhibited clear-cut LH, but not FSH, secretory defects in pituitary sensitivity to estrogen and P. Introduction Ovarian cyclicity in mammals depends on the endocrine interaction of the components of the hypothalamus pituitary ovary uterus axis (Feder, 1981). Estral/menstrual cyclicity depends on negative and positive feedback mechanisms. In terms of the ovarian positive feedback mechanism, estradiol (E2) is the main component acting through estrogen receptor (ER)a and b isoforms on the hypothalamus pituitary system in both rats (Fink, 1988), and women (Messinis, 2006). At the pituitary level, E2, sensitizes the pituitary to GnRH (Fink, 2000) and induces progesterone receptor (PR)-dependent (Collins and Hodgen, 1986; Batista et al., 1992; Waring and Turgeon, 1992) GnRH self-priming (Fink, 1995). All this results in the pro-estrous afternoon (Smith et al., 1975) or midcycle (Hoff et al., 1983; Knobil and Hotchkiss, 1988) pre-ovulatory gonadotrophin surges (Fink, 1988, Messinis, 2006). LH surge-dependent ovarian progesterone (P) secretion enhances the positive E2 feedback on LH surge. Activation of E2-dependent & The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: (Szabo et al., 2000; Turgeon and Waring, 2006) gonadotroph PR during pro-estrous afternoon or midcycle increases the magnitude of the LH surge in rats (Rao and Mahesh, 1986; Sanchez-Criado et al., 1990; Turgeon et al., 1999; Szabo et al., 2000) and women (Chang and Jaffe, 1978; Hoff et al., 1983; Collins and Hodgen, 1986; Batista et al., 1992; Brensing et al., 1993). In addition, ovarian P secreted around the time of the LH surge once P activates its own receptor, down-regulates PR in rats (Turgeon et al., 1999; Szabo et al., 2000; Turgeon and Waring, 2000) and women (Messinis and Templeton, 1990; Gill et al., 2002) through degradation by the 26S proteasome (Lange et al., 2000). This results in a reduction in PR protein expression and extinction of PR-mediated LH secretion, GnRH-stimulated LH secretion and GnRH self-priming (Chappell et al., 1999). Therefore, it seems clear that ovarian P is involved in both the LH surge magnitude and termination in both rats (Turgeon and Waring, 2000) and women (Dafopoulos et al., 2006). Beginning around 10 months of age in female rat, or during midlife in woman, the diminished ovarian follicular reserve is followed by a period of transition characterized by irregular ovarian cycles. In the rat, this period is characterized by extended phases of persistent estrus before entering persistent diestrus, acyclicity and anovulation (Mandl, 1961; Scarbrough and Wise, 1990; Pellicer et al., 1995). In women, menstrual irregularity (perimenopause) is followed by menopause in which the low circulating E2 concentrations render the positive feedback mechanism inactive. The complete process of ovarian senescence until the anovulation stage is not precisely defined and the magnitude of the E2-exposed pituitary response to GnRH has not been quantified. This disruption appears to be caused by changes in the pituitary ovarian axis (Cooper et al., 1980), and an altered sensitivity of the pituitary to ovarian steroids seems possible (Nass et al., 1984). Young and middle-aged ovariectomized (OVX) rats, besides being a valid model for the study of the relationship between ovarian steroids and pituitary function, may be considered surgical menopause and postmenopause models, respectively (Alonso et al., 2006). Accordingly, the aim of the present study was to determine the effect of ERa activation on gonadotrope PR expression (mRNA and protein) and action (GnRH-stimulated gonadotrophins secretion and GnRH self-priming) in 2 weeks (young) and 1-year-old (middle-aged) OVX rats. Materials and Methods Animals, surgery and general conditions Adult female Wistar rats weighing 170 + 15 g were housed under a 14 h light:10 h darkness cycle (light on at 0500 h) and 22 + 28C room temperature, with ad libitum access to rat chow and tap water. Rats were included in the experiments after showing at least three consecutive 4-day regular estrous cycles. Bilateral ovariectomy (OVX) was performed under light ether anaesthesia at a random stage of the estrous cycle. All experimental protocols were approved by the Ethical Commitee of the University of C ordoba, and experiments performed in accordance with rules on laboratory animal care and international law on animal experimentation. Experimental groups and treatments Two weeks and 1 year after OVX, rats were sc injected over three days with: (a) 0.2 ml oil; (b) 125 mg/kg estradiol benzoate (EB, Sigma Chemical Co. St. Louis, MO, USA); (c) 7.5 mg/kg of the selective ERa agonist, propylpyrazole triol (PPT, Tocris Cookson Ltd, Avonmouth, UK) (Stauffer et al., 2000); and (d) 15 mg/kg of the selective estrogen receptor modulator (SERM), tamoxifen (TX, Sigma) (Bellido et al., 2003). On the last day of each analog treatment half of the OVX rats in each group were given 25 mg/kg proges (...truncated)