The Journal of Neuroscience, March 1995, 75(3): 2272-2279 Progesterone Enhances an Estradiol-Induced Increase in Fos lmmunoreactivity in Localized Regions of Female Rat Forebrain Anthony P. Auger and Jeffrey D. Blaustein Neuroscience and Behavior Program and Psychology Department, University of Massachusetts, Amherst, Massachusetts 01003 In female rats, the onset of reproductive behavior depends ceptivity is abolishedby ovariectomy and reinstatedby injection on the sequential presence of estradiol followed by pro- of steroid hormones(Boling and Blandau, 1939). The sequential gesterone. Although treatment with high doses of estradiol treatment with estradiol and progesteroneis important for the has been shown to increase immunostaining for the Fos induction of sexual receptivity (Boling and Blandau, 1939; Pow- protein, an immediate early gene product that is expressed ers, 1970). In ovariectomized rats, estradiol primes the animal upon cellular activation, another report conflicts with this for a subsequentinjection of progesteroneabout 48 hr later and finding. However, the previous reports agree that subse- progesterone-facilitated receptivity occurs within a few hours quent treatment with progesterone has no apparent effect (Boling and Blandau, 1939). on Fos expression. In order to resolve this discrepancy and One mechanismby which steroid hormones act in some tis- investigate possible effects of progesterone, we used Fos sues is by binding to intracellular receptors causing a confor- immunocytochemistry combined with computer-aided im- mational change that allows the receptors to bind to DNA (Jen- age analysis. In experiment one, we found that treatment sen et al., 1968; Walters, 1985). Once the steroid-receptor com- with 5 @g of estradiol increased Fos immunoreactivity plex binds to DNA, it can regulate gene transcription, therefore (Fos-IR) within a section of the medial preoptic area and protein synthesis, and ultimately neuronal function (Walters, the dorsal medial hypothalamus. Subsequent treatment 1985). Some outcomes of estradiol and/or progesteronetreat- with 500 pg of progesterone 1 hr before perfusion in- ment are changesin second messengersystems (Etgen and Pe- creased the intensity of the immunostaining within the me- titti, 1986), peptide expression(Treiser and Wardlaw, 1992), and dial preoptic area and the dorsal medial hypothalamus, al- neurotransmitterrelease (Etgen et al., 1992). though it had no significant effect on Fos-IR cell number. With the development of Fos immunocytochemistry, it is now In experiment 2, a lower concentration of Fos antiserum possible to determine the expression of immediate early gene was used in order to diminish the immunostaining sensi- products that suggest neuronal activity (Morgan et al., 1987). tivity to a level in which no increase of Fos-IR cell number Fos, as other immediate early gene products (e.g., jun), is a was observed after treatment with estradiol. Under these transcription factor that is expressedrapidly in responseto cel- immunocytochemical conditions, subsequent treatment lular stimulation and may then regulate the expressionof other with progesterone increased the number of Fos-IR cells in proteins (Sheng and Greenberg, 1990; Curran, 1992). Fos im- the medial preoptic area, the dorsal medial hypothalamus munocytochemistry has been used to identify neuronswhich re- and the steroid receptor-rich area lateral to the ventrome- spond to physiologically relevant stimuli such as vaginal-cer- dial hypothalamus. Thus, treatment with behaviorally effec- vical (Tetel et al., 1993) and photic stimulation (Chambille et tive doses of both estradiol and progesterone induces Fos al., 1993). expression in localized regions of female rat brain. Previous studieshave shown that treatment with estradiol in- [Key words: estradiol, progesterone, steroid hormones, ducesFos expressionin the rat uterus, chick oviduct, and MCF-7 proto-oncogenes, c-fos, hypothalamus, immunocytochem- cells in vitro (for review, see Schuchard et al., 1993; Weisz and istry, image analysis] Bresciani, 1993). Progesteronehas been suggestedto either de- creaseFos expression(Kirkland et al., 1992) or have no effect During the estrous cycle, reproductive behavior in female rats in the rat uterus (Gibbs et al., 1990). Although estradiol has been dependson an increasein estradiol levels followed by a surge shown to induce Fos expressionin restricted areasof the female of progesteronea few hours before the onset of sexual receptiv- rat brain (Cattaneo and Maggi, 1990; Insel, 1990; Jenneset al., ity (Boling and Blandau, 1939; Smith et al., 1975). Sexual re- 1992), another report found no induction of Fos by estradiol (Gibbs et al., 1990). However, previous reports are in agreement Received May 16, 1994; revised Sept. 19, 1994; accepted Sept. 23, 1994. in showing that subsequenttreatment with progesteroneis with- This research was supported by Grants NS 19327 from the National Institutes out apparent effect on Fos expressionin the rat brain (Gibbs et of Health and RSDA MH 00885 from the National Institute of Mental Health. al., 1990; Insel, 1990). We gratefully acknowledge Marc Tetel, John Meredith, and Joanne Turcotte for technical assistance. We also thank Brad Powers and George Wade for In order to determine if estradiol and/or progesteroneinduce comments on the manuscript. Fos expression in female rat brain under conditions similar to Correspondence should be addressed to Jeffrey D. Blaustein, Psychology thoseused to induce sexual behavior, we immunocytochemically Department, Tobin Hall-Box 37710, University of Massachusetts, Amherst, MA 01003-7710. stained Fos protein after giving behaviorally effective dosesof Copyright 0 1995 Society for Neuroscience 0270-6474/95/152272-08$05.00/O steroid hormones. In addition, we used computer-aidedimage The Journal of Neuroscience, March 1995, 15(3) 2273 analyses, as this enables the detection of cells with subtle in- the section were at the maximum gray level of 255. Two additional sections were analyzed to confirm that the gray scale criteria had been creases in Fos expression. Furthermore, with the aid of image met. This allowed the gray level range of Fos-IR within each cell to be analysis, changes in the intensity of Fos immunoreactivity (Fos- from 0 to 255 (0 beine white. 255 being black). Once the camera gain IR) in individual cells among the treatment groups can be dis- and black levels were adjusted, they remained calibrated for all sectTons tinguished. to be analyzed through a given area. Average background pixel density for each section was determined by averaging the maximum pixel den- Materials and Methods sity of five 225 square pixel areas in Fos-IR poor areas of that section. The density threshold option was then adjusted so that the minimum to Animals. Female Sprague-Dawley rats (200-250 gm) obtained from be analyzed was set to the average background pixel density. One care- Charles River Breeding Laboratories, Inc, (Wilmington, MA) were fully matched section per animal through each area was then analyzed group housed for 1 week in a 14:lO 1ight:dark cycle. All rats were then bilaterally making adjustments for background variations only, using ovariectomized under methohexital sodium anesthesia (52 mg/kg body the above procedure. Fos-IR cell number and maximum pixel density weight, Brevital, Eli Lilly and Co., Indianapolis, IN) prior to experi- (i.e., optical density) per cell was recorded in each section bilaterally. ment. Neuroanatomical areas analyzed. Representative sections of areas Hormone treatment. One week following surgery, experimental rats associated with reproduction that contain steroid receptors, such as the were injected with 5 pg of estradiol benzoate (dissolved in 0.1 ml of medial amygdala, the medial preoptic area, the ventromedial hypothal- sesame oil) followed by either 500 )*g of progesterone (dissolved in 0.1 amus, and the dorsomedial hypothalamus, were matched as described ml of sesame oil containing 5% benzyl alcohol and 15% benzyl ben- in the rat brain atlas of Paxinos and Watson (1986; Fig. lA-C). In zoate, n = 4) or vehicle (0.1 ml sesame oil, n = 5) approximately 48 addition, the area lateral to the ventromedial hypothalamus described in hr later. In addition, control rats (n = 5) were injected with vehicle at Figure 1A was examined, as this is a site containing an abundance of both times. One hour after the final injection, the rats were perfused. progestin (J. D. Blaustein, unpublished observations) and estrogen-re- Perfusion. Animals were anesthetized with sodium pentobarbital and ceptor immunoreactive cells (Blaustein, 1993). chloral hydrate. The heart was then exposed and the left ventricle was Statistics. Bilateral measurements of each area were pooled and an- iniected with 5000 U of sodium heoarin dissolved in 1 ml of saline alyzed using SIGMASTAT Statistical Analysis System 1 .Ol (Jandel Sci- (6.15 M). The right atrium and the left ventricle were incised prior to entific, Corta Madera, CA). Results from the one-way analyses of vari- insertion of a cannula, through the incision of the left ventricle, into the ance were considered statistically significant at a probability level of aorta. Seventy-five milliliters of saline preceded the flow of 250 ml of less than 0.05. fixative (2% acrolein in 0.1 M sodium phosphate buffer; pH 7.2) through the cannula. Perfusion pressure was maintained at 100 mm Hg with a Results flow rate of 25 ml/min for 10 min. After the brains were removed and blocked, they were placed into 0.1