On the day of proestrus in the rat, rising plasma levels of estradiol (E) act in concert with progesterone (P) to trigger a preovulatory release of gonadotropins. Cellular levels of galanin mRNA in GnRH neurons are increased in association with the proestrous surge of gonadotropin secretion; however, the relative contribution made by E and P to the induction of galanin mRNA expression in GnRH neurons is unknown. We investigated the role of E and P in the induction of galanin gene expression in GnRH neurons by examining the effects of different combinations of E (estradiol benzoate; 50 μg and P (5 mg)) on the LH surge and the concomitant induction of galanin mRNA in GnRH neurons. We sacrificed ovariectomized adult rats after 1 of 4 treatments: Group 1: vehicle control (n=6); Group 2: P alone (n=7) Group 3: E alone (n=7); Group 4: combined E/P (n=6); the animals were killed at 18.00 h at the time of the LH surge. The brains from these animals were processed by double‐labelin situhybridization to allow measurement of galanin mRNA levels in GnRH neurons. GnRH neurons were identified with a digoxigenin‐labeled cRNA probe for GnRH mRNA, and galanin mRNA was detected and measured simultaneously with an35S‐labeled cRNA probe coupled with computerized grain counting. Estimation of cellular levels of GnRH mRNA was accomplished with single‐labelin situhybridization, an35S‐labeled GnRH cRNA probe and computerized grain counting. We observed a 3‐fold induction of galanin mRNA in the GnRH neurons of animals treated with E alone compared with those treated with the vehicle alone (vehicle: 13±2vsE: 42±4 grains/cell (g/c); P<0.01); LH levels in the E‐treated animals were elevated, albeit moderately, with respect to the vehicle controls. Compared with vehicle‐treated animals, those treated with the combination of E and P showed a 5‐fold induction of galanin mRNA in GnRH neurons (68±9 g/c), which was significantly (P<0.01) greater than that observed in the animals treated with E alone; in addition, the magnitude of the LH surge was much greater (P<0.05) in the E/P‐treated group compared with the E alone group. In contrast, compared to the vehicle controls, animals treated with P alone (15±2 g/c) showed no discernable effect on galanin mRNA levels; moreover, no LH surge occurred in the P alone group. Neither the number of identified GnRH cells nor their content of GnRH mRNA differed significantly among the experimental groups (GnRH mRNA signal: vehicle controls: 153±6vsE: 159±6vsE/P: 153±3vsP: 148±8 g/c). We conclude that while E is the primary ovarian signal inducing galanin mRNA expression in GnRH neurons and the LH surge itself, P plays a facilitato
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