Estradiol Induces Hypothalamic Progesterone

Behavioral Neuroscience 2002, Vol. 116, No. 2, 198 –205

Copyright 2002 by the American Psychological Association, Inc. 0735-7044/02/$5.00 DOI: 10.1037//0735-7044.116.2.198

Estradiol Induces Hypothalamic Progesterone Receptors but Does Not Activate Mating Behavior in Male Hamsters (Mesocricetus auratus) Before Puberty Russell D. Romeo

Christine K. Wagner

Michigan State University

University of Massachusetts

Heiko T. Jansen

Stefani L. Diedrich and Cheryl L. Sisk

Washington State University

Michigan State University

This study investigated pubertal changes in neural and behavioral responses to estradiol. Gonadectomized pre- and postpubertal male hamsters (Mesocricetus auratus) were treated with 0.00, 0.05, 0.10, or 0.25 mg estradiol and tested 1 week later for sexual behavior with a receptive female. Estradiol activated behavior in postpubertal, but not prepubertal, males. In contrast, estrogen receptor ␣ (ER␣) and progesterone receptor (PR) immunoreactivity in forebrain nuclei that mediate mating behavior was similar in pre- and postpubertal males. Thus, absence of a behavioral response before puberty is not associated with reduced levels of steroid receptors. Because estradiol induced PR in prepubertal males, these data also suggest that ER␣ is functional before puberty. Therefore, gonadal steroids facilitate male reproductive behavior only after as-yet-unidentified developmental processes occur during puberty.

Previous research has examined the neural mechanisms underlying the pubertal maturation of behavioral responsiveness in the male Syrian hamster, in which the steroid-sensitive forebrain neural circuit mediating reproductive behavior is well characterized (Lehman, Powers, & Winans, 1983; Lehman, Winans, & Powers, 1980; Newman, 1999; Wood, 1996, 1998; Wood & Newman, 1995a). Studies conducted thus far suggest that the inability of testosterone to activate sexual behavior in prepubertal males is due, at least in part, to unresponsiveness of the prepubertal brain to the androgenic metabolite dihydrotestosterone (DHT; Romeo, Cook-Wiens, Richardson, & Sisk, 2001). The lack of a behavioral response to testosterone or DHT before puberty is not associated with an absence of androgen receptor in the mating circuit (Meek et al., 1997; Romeo et al., 2001). Furthermore, these receptors appear to be functional before puberty, as a testosterone-induced increase in hypothalamic aromatase activity, which is an androgen receptordependent response (Roselli, Horton, & Resko, 1987), is equivalent in prepubertal and postpubertal males (Romeo, Wade, Venier, & Sisk, 1999). Male mating behavior is dependent on estrogen as well as testosterone and DHT (Meisel & Sachs, 1994). Testosterone is converted in the brain to estradiol by the intracellular aromatase enzyme. Treatment of castrated postpubertal males with estradiol reinstates some components of mating behavior (e.g., mounting; DeBold & Clemens, 1978), whereas inhibition of the aromatase enzyme in gonad-intact males decreases reproductive behavior (Floody & Petropoulos, 1987; but see Cooper, Clancy, Karom, Moore, & Albers, 2000). The behavioral response to centrally produced estrogen is mediated through the estrogen receptor (ER; Meisel & Sachs, 1994). There are two known forms of the ER, ER␣ and ER␤. The former appears to be the predominant ER responsible for activation of mating behavior, as male mice that

One hallmark of pubertal development in males is the increased production of testosterone by the testes. The pubertal rise in testosterone levels is accompanied by the appearance of reproductive behavior, suggesting that the maturation of sexual behavior is dependent on elevated levels of the steroid. However, low prepubertal levels of testosterone are not the sole explanation for the lack of mating behavior before puberty. For instance, when juvenile males are treated with a dose of testosterone that fully activates sexual behavior in postpubertal males, the juveniles engage in little, if any, mating behavior (Baum, 1972; Larsson, 1967; Meek, Romeo, Novak, & Sisk, 1997; Sisk, Berglund, Tang, & Venier, 1992; So¨dersten, Damassa, & Smith, 1977). Therefore, not only do steroid hormone levels increase during pubertal development, but responsiveness of the neural circuit mediating steroid activation of reproductive behavior increases as well. The pubertal processes that mediate this dramatic developmental change in steroid responsiveness remain largely unknown.

Russell D. Romeo, Stefani L. Diedrich, and Cheryl L. Sisk, Department of Psychology and Neuroscience Program, Michigan State University; Christine K. Wagner, Department of Psychology, University of Massachusetts; Heiko T. Jansen, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Program in Neuroscience and The Center for Reproductive Biology, Washington State University. Christine K. Wagner is now at the Department of Psychology, State University of New York at Albany. A preliminary report of these data was presented at the annual meeting of the Society for Neuroscience, Miami, Florida, October 1999. This research was supported by National Science Foundation Grant IBN 96 – 02169 and IBN 99 – 85876. We thank Jane Venier, Chris Cutter, and Jennifer Pfau for their valuable assistance. Correspondence concerning this article should be addressed to Cheryl L. Sisk, Department of Psychology and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824. E-mail: [email protected] 198

PUBERTY, MATING, AND ER␣- AND PR-IMMUNOREACTIVITY

lack the ER␣ gene but have the ER␤ gene intact engage in relatively little reproductive behavior (Wersinger et al., 1997). The lack of a behavioral response to testosterone in prepubertal males could thus arise from a relative lack of estrogenic action, androgenic action, or both. Previous research has established that prepubertal male hamsters are behaviorally unresponsive to DHT (Romeo et al., 2001), indicating that cellular responses to activation of the androgen receptor are different in the prepubertal and postpubertal hamster brain. Whether the estrogenic metabolite of testosterone elicits similar cellular and behavioral effects in juvenile and postpubertal males is presently unknown. Castrated prepubertal and postpubertal males have equivalent numbers of ER␣containing cells in the neural circuit that mediates reproductive behavior (Romeo, Diedrich, & Sisk, 1999), but it is not known whether estrogen is capable of facilitating mating behavior in juvenile males. Likewise, it has not been established whether the expression of ER␣ is altered by estradiol treatment in prepubertal males or whether ER␣ is a functional transcription factor at this developmental stage. Thus, the present study had three objectives. The first was to determine whether puberty is associated with a change in behavioral responsiveness to estrogen. The second was to compare ER␣ levels in the behavioral neural circuit of prepubertal and postpubertal males in the presence of estrogen. Finally, because estrogen positively regulates the expression of progesterone receptor (PR) primarily by means of an ER␣-dependent mechanism (Moffatt, Rissman, Shupnik, & Blaustein, 1998; Roy, MacLusky, & McEwen, 1979), the third objective was to assess the functionality of the ER in prepubertal males by quantifying estrogen-induced hypothalamic PR in prepubertal and postpubertal males.

Method

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or 0.25 mg EB. All brain sections from Experiment 1 were used for ER␣ immunocytochemistry and other studies. Thus, to generate additional tissue for Experiment 2, hamsters were treated in an manner identical to that of Experiment 1 (n ⫽ 6 per age and treatment), except that no behavioral test was given. That is, 1 week after castration and implantation (i.e., at either 28 or 49 days of age), all hamsters were weighed, killed by overdose, and perfused as described below. This tissue was processed for PR immunocytochemistry as described below.

Tests for Male Reproductive Behavior All hamsters in Experiment 1 were tested between 1400 and 1700 (2–5 hr after lights-off). The male was placed in a 10-gallon aquarium (51.0 cm long ⫻ 26.0 cm high ⫻ 31.5 cm wide) and allowed to acclimate for 5 min before the introduction of the receptive female. Stimulus females were ovariectomized under methoxyflurane anesthesia at least 2 weeks before behavioral testing commenced, and they were made sexually receptive by sequential injections of EB (10.0 ␮g sc in 0.1 ml sesame oil, 48 hr before testing) and progesterone (0.1 mg sc in 0.1 ml sesame oil, 4 hr before testing). The behavioral tests were videotaped under red-light illumination with a Panasonic Color Video Camera (Model WV 3250). Videotapes were later scored to determine the amount of time spent in anogenital investigation (AGI) of the female and the number of mounts, intromissions, and ejaculations achieved by the male. Criteria for these behaviors have been established previously (Meek et al., 1997). Briefly, AGI was scored when the male investigated the perianal region of the female. A mount was recorded if a male mounted the female with an orientation that would allow an intromission to occur. An intromission was recorded if the male achieved vaginal penetration. An ejaculation was recorded if, after a series of intromissions separated by brief intervals, the male was temporarily disinterested in the female (20 – 60 s) and engaged in extensive autogrooming. All videotapes were scored by a single experimenter who was unaware of the hamster’s hormonal condition.

Subjects

Blood and Tissue Collection

Male Syrian hamsters (Mesocricetus auratus) were bred at Michigan State University (East Lansing, MI). All subjects were weaned at 21 days of age and singly housed in clear polycarbonate cages (37.5 cm long ⫻ 33.0 cm high ⫻ 17.0 cm wide) with ad-lib access to food (Teklad Rodent Diet No. 8640, Harlan, Madison, WI) and water. The vivaria were on a 14:10-hr light– dark schedule (lights off at 1200), and the temperature was maintained at 21⫾ 2 °C. All subjects were treated in accordance with the National Institutes of Health guidelines (National Institutes of Health, 1986), and the protocols were approved by the Michigan State University All-University Committee for Animal Use and Care.

On the day of perfusion (i.e., at either 28 or 49 days of age), hamsters from Experiments 1 and 2 were weighed and administered an overdose of sodium pentobarbital (130 mg/kg ip). Before perfusion, blood samples were obtained by cardiac puncture, and at the same time, the presence of the steroid pellet was confirmed to verify that it had remained in the hamster during the course of the experiment. The hamsters were intracardially perfused with 100 ml buffered saline rinse followed by 150 ml of 4% (wt/vol) paraformaldehyde. The brains were removed and postfixed for 1 hr in 4% paraformaldehyde, then transferred to a phosphate-buffered saline (PBS) solution containing 20% (wt/vol) sucrose. Approximately 48 hr after the brains had sunk in the sucrose solution, 40-␮m coronal sections were generated on a cryostat and stored in cryoprotectant (Watson, Weigand, Clough, & Hoffman, 1986) at –20 °C until immunocytochemistry was performed. In Experiment 1, behavioral and anatomical data from 2 prepubertal hamsters (1 treated with a 0.05-mg pellet and 1 treated with a 0.25-mg pellet) were excluded from all analyses because of poor tissue quality resulting from inadequate fixation during perfusion.

Experimental Design Experiment 1. All hamsters were castrated under methoxyflurane anesthesia at either 21 or 42 days of age and implanted with a 3-week timed-release pellet (Innovative Research of America, Sarasota, FL) containing either 0.00 (blank pellet), 0.05, 0.10, or 0.25 mg of ␤-estradiol-3benzoate (EB; n ⫽ 6 per age and treatment group). One week after castration and implantation (i.e., at either 28 or 49 days of age), all hamsters were given a 15-min mating behavior test with a hormone-primed estrous female (see below). All hamsters were sexually naive before the behavioral test. Immediately after the behavioral test, the hamsters were weighed, killed by overdose, and perfused as described below. Tissue was then processed for ER␣ immunocytochemistry as described below. Experiment 2. This experiment was conducted to investigate whether the ER␣ present in the forebrain mating circuit is functional in males before pubertal development. This was assessed by quantifying the amount of PR in prepubertal and postpubertal males treated with 0.00, 0.05, 0.10,

ER␣ and PR Immunocytochemistry In Experiment 1, brains from postpubertal and prepubertal gonadectomized males treated with a blank or 0.05-mg EB pellet were used for ER␣ immunocytochemistry. These treatment groups were chosen for study because they demonstrated the most robust behavioral differences (see Results section). In Experiment 2, brain tissue from all groups was used for PR immunocytochemistry (i.e., prepubertal and postpubertal males treated with either 0.00, 0.05, 0.10, or 0.25 mg EB). In both experiments, every

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ROMEO, WAGNER, JANSEN, DIEDRICH, AND SISK

fourth brain section from all hamsters was processed in a single immunocytochemical run. ER␣ immunocytochemistry. Free-floating sections were rinsed four times in PBS with 0.1% Triton X-100 (PBS–TX) for 5 min each. Tissue was then incubated in 1% H2O2 for 10 min, followed by four rinses in PBS–TX. Subsequently, sections were incubated in 0.1% bovine serum albumin (BSA) for 1 hr and for 72 hr at 4 °C in DAKO (1D5) mouse anti-ER␣ (1:500, DAKO, Glostrup, Denmark) in PBS–TX and 0.1% BSA. Sections were then brought to room temperature in the primary antibody for 2 hr. After the 2-hr equilibration, sections were incubated for 1 hr in biotinylated donkey anti-mouse immunoglobulin G (IgG; 1:500, Jackson ImmunoResearch Laboratories, West Grove, PA; preadsorbed against hamster and other species serum proteins). Sections were then rinsed four times in PBS–TX and incubated in avidin– biotin horseradish peroxidase complex for 1 hr (1:100, Vectastain ABC Elite Kit, Vector, Burlingame, CA). Sections were again rinsed four times in PBS–TX, followed by a 5-min rinse in a 0.05-M Tris buffer with 1.5% NaCl (1.5T buffer). For the chromagen reaction, sections were incubated in 0.05% 3,3⬘-diaminobenzadine (DAB) with the 1.5T buffer and 0.01% H2O2. The DAB reaction was terminated by four rinses in PBS. Sections were mounted on gelatincoated slides, dried, dehydrated, cleared in xylenes, and coverslipped with DPX Mountant (Fluka Chemical Corp., Milwaukee, WI). PR immunocytochemistry. Immunocytochemistry was performed on free-floating sections by using a rabbit polyclonal antibody (DAKO) directed against the DNA binding domain of the human PR. This antibody detects both the A and B isoforms of PR (Traish & Wotiz, 1990). All incubations were performed at room temperature unless otherwise indicated. Free-floating sections were rinsed in 0.5 M Tris-buffered saline (TBS) three times to remove the cryoprotectant. Sections were then incubated in 1% sodium borohydride in TBS for 10 min, rinsed in TBS four times for 5 min each, and incubated in TBS containing 20% normal goat serum (NGS), 1% H2O2, and 1% BSA for 20 min. PR antiserum was diluted 1:1,000 in TBS containing 2% NGS, 0.3% Triton X-100, and 0.02% sodium azide (TBS–TX) for 65 hr at 4 °C. After three rinses in TBS–TX, the sections were incubated for 90 min in biotinylated goat anti-rabbit IgG (Vector Laboratories) at a concentration of 5␮g/ml in TBS–TX. After two rinses in TBS–TX and two rinses in TBS, the sections were incubated in avidin– biotin horseradish peroxidase complex for 1 hr (1:100; Vectastain ABC Elite Kit). After three rinses in TBS, the sections were incubated in phosphate buffer containing 0.012% DAB, 0.018% NiCl2, and 0.075% H2O2 for 10 min. The reaction was terminated by three rinses in TBS. Sections were mounted on gelatin-coated slides, dried, dehydrated, cleared in xylenes and coverslipped as before.

Microscopic Analysis of Estrogen and PR Immunoreactivity Number of ER␣- and PR-immunoreactive cells. In Experiment 1, the number of immunoreactive cell profiles per unit area, referred to as number of ER␣-immunoreactive (ER␣-ir) cells, was determined for the medial preoptic nucleus (MPN), the magnocellular region of the medial preoptic nucleus (MPNmag), the posteromedial subdivision of the bed nucleus of the stria terminalis (BNSTpm), and the anterior and posterior subdivision of the medial amygdala (MeA and MeP, respectively). These areas were chosen for examination because they are components of the neural circuit that mediates reproductive behavior in this species (Wood, 1996, 1998; Wood & Newman, 1995a). In Experiment 2, the number of PRimmunoreactive (PR-ir) cell profiles per unit area, referred to as the number of PR-ir cells, was analyzed only in the MPN because PR-ir staining intensity in the other nuclei was relatively low, even in EB-treated males, and did not permit reliable cell counts. Bilateral counts were made in two sections for each nucleus, except for the BNSTpm and MPNmag, in which bilateral counts were made in one section. When two sections were analyzed, they were separated by 160 ␮m and anatomically matched across hamsters. With bright-field microscopy, nuclei were centered under a ⫻10 objective, and the magnification was

then raised to ⫻40. Cells that fell within the area of an ocular grid (62,500 ␮m2) were counted. Data are expressed as the mean number of ER␣-ir or PR-ir cells per 62,500 ␮m2. The experimenter was unaware of the age and hormonal treatment of the hamsters during all of the microscopic analyses. Optical density of PR immunoreactivity. In Experiment 2, in addition to the number of PR-ir cells per unit area, the relative amount of PR immunoreactivity per cell was determined by measuring the optical density of immunoreactivity in individual cell nuclei. One section through the MPN was chosen for each hamster, anatomically matched across hamsters, and the side of the MPN with the higher number of PR-ir cells was chosen for analysis. Microscopic images of the PR-ir cells in the MPN were captured on the computer screen by means of a ⫻100 oil immersion objective. NIH Image software (Rasband, 1996) was used to analyze these captured images. The optical density of individual cell nuclei of all immunoreactive cells within two 110 ␮m ⫻ 75 ␮m sampling fields within the MPN was measured. In addition, the optical density of five cell-sized areas of neuropil per sampling field was determined for each hamster and averaged as a measurement of background optical density. The optical density for each PR-ir cell was then corrected by subtracting the average background value. The mean corrected optical density of all cells within the sampling field was calculated for each hamster and is referred to as relative PR-ir per cell.

Estradiol and Luteinizing Hormone (LH) Radioimmunoassays Estradiol and LH concentrations were measured only in hamsters from Experiment 1. Plasma concentrations of estradiol were measured with the Coat-A-Count Estradiol-6 Kit (Diagnostic Products, Los Angeles, CA). To validate this assay for estradiol measurements in the Syrian hamster, samples from intact and castrated male plasma pools were assayed at two different dilutions, and proestrous and estrous female pools were assayed at five different dilutions. These results were compared to a standard curve generated from human serum-based calibrators included in the kit with estradiol concentrations ranging from 10 to 2,000 pg/ml. Estradiol values obtained from the diluted pools were parallel to the standard curve and maintained linearity throughout its range. The lower limit of detectability of the assay was 14.59 pg/ml. The intra-assay coefficient of variation was 13.8%. Plasma LH concentrations were measured with reagents in the rat LH kit obtained from A. F. Parlow and the National Hormone and Pituitary Program at the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK). To validate this assay for LH measurements in the Syrian hamster, samples from intact and castrated male plasma pools were assayed at five different dilutions. These results were compared to a standard curve generated with the LH reference preparation ranging from 0.8 to 30 ng/ml. LH values obtained from the diluted pools were parallel to the standard curve and maintained linearity throughout its range. Values are reported as nanogram equivalents of NIDDK-rLH-RP-3. The lower limit of detectability was 0.66 ng/ml, and the intra-assay coefficient of variation was 8.6%.

Statistical Analysis In Experiments 1 and 2, the effects of age and EB treatment on all dependent measures were analyzed by two-way analyses of variance (Age ⫻ Dose). Significant main effects were further analyzed with Fisher’s protected least significant difference tests, and significant interactions were examined with Tukey’s honestly significant difference tests. Differences were considered significant at p ⬍ .05. Data are presented as means (⫾ SEM). In Experiment 1, 1 postpubertal male in the 0.00-mg EB group was an outlier on the intromissions variable as determined by the Dixon’s outlier test (Sokal & Rohlf, 1981, p. 413; p ⬍ .05), and was 4 standard deviations above the mean for his group on mounts. Therefore, data from this subject were excluded from all statistical analyses.


Results Experiment 1 Peripheral and hormonal measures. On the day of castration, postpubertal males had significantly heavier paired testis weights compared with prepubertal males, F(1, 37) ⫽ 463.14, p ⬍ .05 (see Table 1). The low testicular weights of the prepubertal males are characteristic of testes not producing appreciable levels of testosterone or mature sperm (Romeo, Wade, et al., 1999), indicating that these males were indeed prepubertal and had not undergone any significant testicular maturation. Increasing doses of EB resulted in increasing circulating plasma estradiol concentrations: main effect of treatment, F(3, 37) ⫽ 18.31, p ⬍ .05 (see Table 1). Castrates treated with the blank pellet or the 0.05-mg pellet of EB had significantly lower circulating levels of estradiol compared with the castrates treated with either the 0.10- or 0.25-mg dose of EB. Furthermore, castrated males treated with the 0.25-mg pellet of EB had significantly greater amounts of circulating estradiol than the castrates treated with the 0.10-mg pellet of EB. There was no significant interaction between age and EB treatment on plasma estradiol concentrations. That is, a given dose of EB produced equivalent levels of circulating estradiol in prepubertal and postpubertal males. A significant interaction between age and EB treatment on circulating plasma levels of LH was found, F(3, 37) ⫽ 16.31, p ⬍ .05 (see Table 1). All doses of EB reduced plasma LH to undetectable levels in prepubertal males, whereas in postpubertal males, only the 0.10- and 0.25-mg pellets of EB resulted in undetectable LH. Behavior. There were significant main effects of both age, F(1, 37) ⫽ 12.44, p ⬍ .05, and estrogen, F(3, 37) ⫽ 7.82, p ⬍ .05, on AGI, such that postpubertal males engaged in significantly more AGI than prepubertal males, and estradiol increased AGI significantly above that of castrated, blank-treated controls (see Figure 1A). A significant interaction was found between age and EB treatment on mounts, F(3, 37) ⫽ 4.88, p ⬍ .05 (see Figure 1B). Post hoc tests revealed that treatment of postpubertal males with either the 0.05- or 0.10-mg pellet of EB resulted in a significant increase in mounts compared with the postpubertal males receiving the blank control pellet. In contrast, EB treatment had no effect on mounting behavior in prepubertal males.

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There was a significant interaction between age and EB treatment on intromissions, F(3, 37) ⫽ 3.93, p ⬍ .05 (see Figure 1C). The 0.05-mg dose of EB significantly increased the number of intromissions in postpubertal males, but not in juveniles. None of the doses of EB significantly increased intromissions in prepubertal males. No ejaculations were observed in any postpubertal or prepubertal group. ER␣ immunoreactivity. For the four groups of hamsters in which ER␣ immunoreactivity was assessed (e.g., prepubertal and postpubertal males treated with either 0.00 or 0.05 mg of EB), there was a significant interaction between age and estrogen treatment on the number of ER␣-ir cells in the MPN, F(1, 18) ⫽ 4.93, p ⬍ .05 (see Figure 2). Although the number of ER␣-ir cells was similar in EB-treated and control postpubertal males, prepubertal males treated with the blank pellet had significantly more ER␣containing cells than EB-treated juveniles. No other main effects or significant interactions on the number of ER␣-ir cells were found in any of the other nuclei analyzed.

Experiment 2: PR Immunoreactivity There were significant main effects of EB treatment on both the number of PR-ir cells, F(3, 39) ⫽ 29.19, and the intensity of PR immunoreactivity per cell, F(3, 39) ⫽ 19.18, in the MPN ( ps ⬍ .05, see Figures 3A and 3B). Specifically, both prepubertal and postpubertal males treated with EB had significantly more PR-ir cells and more PR immunoreactivity per cell than prepubertal and postpubertal males treated with the blank pellet. Thus, estrogen treatment did not differentially regulate PR immunoreactivity in the juvenile and postpubertal males. Figure 4 shows the similar upregulation of PR immunoreactivity in the MPN of EB-treated prepubertal and postpubertal males.

Discussion These data demonstrate that puberty is associated with a change in behavioral responsiveness to estrogen. Estradiol activates mounting and intromissions in postpubertal, but not prepubertal, males. This reduced behavioral response to EB in prepubertal males is not associated with a lack of ER␣, as ER␣ immunoreactivity is similar in EB-treated juvenile and postpubertal males

Table 1 Mean (⫾ SEM) Plasma Estradiol and Luteinizing Hormone Concentrations and Body and Paired Testis Weights From Hamsters in Experiment 1 Age group and EB dose (mg) Prepubertal 0.00 0.05 0.10 0.25 Postpubertal 0.00 0.05 0.10 0.25

Plasma estradiol (pg/ml)

Plasma luteinizing hormone (ng/ml)

Body weight (g)

51.40 ⫾ 3.12 95.51 ⫾ 10.54 340.90 ⫾ 65.98 847.84 ⫾ 282.12

3.70 ⫾ 1.09 0.66 ⫾ 0.00 0.66 ⫾ 0.00 0.66 ⫾ 0.00

55.17 ⫾ 3.07 58.80 ⫾ 1.74 60.80 ⫾ 2.29 59.67 ⫾ 3.38

0.165 ⫾ 0.017 0.200 ⫾ 0.014 0.215 ⫾ 0.012 0.180 ⫾ 0.025

73.56 ⫾ 2.99 134.44 ⫾ 15.00 318.15 ⫾ 24.97 548.11 ⫾ 60.39

12.36 ⫾ 1.64 5.37 ⫾ 0.94 0.66 ⫾ 0.00 0.66 ⫾ 0.00

112.00 ⫾ 4.39 104.00 ⫾ 4.22 108.67 ⫾ 3.18 108.17 ⫾ 2.43

2.110 ⫾ 0.258 1.922 ⫾ 0.208 2.175 ⫾ 0.148 2.300 ⫾ 0.150

Note. EB ⫽ estradiol benzoate. Testes were weighed on the day of castration (i.e., at either 21 or 42 days of age).

a

Paired testis weight (g)a

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prepubertal nervous system is behaviorally unresponsive to both the estrogenic and androgenic components of the facilitation of sexual behavior by testosterone. In neither case is this explained by a lack of nuclear receptor function.

Pubertal Change in Behavioral Response to Estradiol The fact that EB activated mounts and intromissions, but not ejaculations, in postpubertal males is in agreement with previous reports (DeBold & Clemens, 1978; Floody & Petropoulos, 1987). In stark contrast to the postpubertal males, prepubertal males treated with EB did not display either of these behaviors, even though their levels of estradiol were similar to those of the EBtreated postpubertal males. Thus, these behavioral data indicate that postpubertal males are more responsive than juveniles to the activating effects of estradiol on male mating behavior. Contrary to its effect on mounting and intromissive behavior, EB was able to activate AGI in the juveniles, albeit to a lesser degree than in the postpubertal males. Pheromonal stimulation received by the male during AGI of the female is essential for the display of subsequent mating behavior in this species (Wood, 1998; Wood & Newman, 1995b). Thus, these results indicate that pheromonal stimulation received by an EB-treated male may be

Figure 1. Number of seconds engaged in anogenital investigation (AGI; A) and number of mounts (B) and intromissions (C) in prepubertal and postpubertal male hamsters given a 15-min behavioral test with a receptive female. Hamsters were castrated 1 week before the behavioral test and were implanted with a pellet containing either 0.00 (blank), 0.05, 0.10, or 0.25 mg estradiol benzoate (EB). Postpubertal males engaged in significantly more AGI than prepubertal males, and EB increased AGI significantly above that of castrated, blank-treated controls. All values are means (⫾ SEM). In Panels B and C, asterisks indicate a significant difference between prepubertal and postpubertal males, and daggers indicate a significant difference between EB-treated males and blank-treated control males within an age group ( p ⬍ .05).

within cell groups in the forebrain neural circuit that mediates reproductive behavior. Our findings in Experiment 2 show that estradiol significantly induces PR in the MPN of both postpubertal and prepubertal males. Because estradiol induction of PR is mediated primarily by ER␣ (MacLusky & McEwen, 1978; Moffatt et al., 1998; Roy et al., 1979), the present data suggest that ER␣ in the MPN of male hamsters is functional before pubertal development. Thus, taken together with our previous experiment with DHT-treated males (Romeo et al., 2001), we can conclude that the

Figure 2. Mean (⫾ SEM) number of estrogen receptor ␣ immunoreactive (ER␣-ir) cells in a 62,500-␮m2 sampling field in the medial preoptic nucleus (MPN), medial preoptic nucleus, magnocellular nuclei (MPNmag), posteromedial bed nucleus of the stria terminalis (BNSTpm), anterior portion of the medial amygdala (MeA), and posterior portion of the medial amygdala (MeP) of prepubertal and postpubertal male hamsters that were castrated 1 week before perfusion and implanted with a pellet containing either 0.00 or 0.05 mg estradiol benzoate (EB). Asterisk indicates a significant difference between EB doses ( p ⬍ .05).


Figure 3. The number of progesterone receptor immunoreactive (PR-ir) cells in a 62,500-␮m2 sampling field (A) and the relative amount of PR-ir per cell (mean optical density, B) in the medial preoptic nucleus of prepubertal and postpubertal males that were castrated and implanted with a pellet containing either 0.00 (blank), 0.05, 0.10, or 0.25 mg estradiol benzoate (EB). All values are means (⫾ SEM). Asterisks indicate significant differences between EB-treated males and blank-treated males ( p ⬍ .05).

necessary, but not sufficient, to activate the full suite of mating behaviors. Prepubertal and postpubertal males show a similar increase in neuronal activation in response to female pheromones, as indexed by Fos-immunostaining, in the chemosensory components of the mating circuit (Romeo, Parfitt, Richardson, & Sisk, 1998). However, only postpubertal males show a pheromone-

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induced increase in testosterone secretion (Romeo et al., 1998). Therefore, chemosensory information is detected by prepubertal males, but it elicits different neuroendocrine responses before and after puberty. Thus, the absence of mating behavior in the EBtreated prepubertal males may be the result of inappropriate integration of chemosensory cues with steroidal information in the juvenile brain, which in turn does not permit the proper coordination of further reproductive behaviors. The estrogen-dependent increase in anogenital investigation in both prepubertal and postpubertal males was unexpected, because in hamsters, interest in female pheromones has been reported to be under androgenic regulation (Gregory, Engel, & Pfaff, 1975; Powers & Bergondy, 1983). However, a recent study showed that male ER␣-knockout mice engage in less AGI than do wild-type mice, indicating that ER␣ may play a role in mediating this behavior (Wersinger & Rissman, 2000). The present data indicate that estrogen has a positive impact on the amount of time a male will spend in AGI of an estrous female, and that EB can activate this behavior before puberty. It is interesting that the lowest dose of EB used in this study was the most effective in facilitating mating behavior in postpubertal males. We reported a similar phenomenon with testosterone, in that a higher dose was less effective at facilitating adult mating behavior than a lower dose (Meek et al., 1997). This low dose produced circulating testosterone levels equivalent to those found in intact postpubertal males. The subjects treated with the highest doses of steroid in this and our previous study did not appear ill or agitated. Thus, we do not suspect that high steroid levels reduce behavior indirectly by compromising normal physical capabilities or redirecting behavior. Instead, high levels of steroid may directly inhibit the neural processes that modulate reproductive behavior. For example, LHRH (luteinizing hormone-releasing hormone) facilitates the display of mating behavior in both males and females (Beyer, Gonzalez-Flores, & Gonzalez-Mariscal, 1997; FernandezFewell & Meredith, 1995; Meredith & Howard, 1992; Pfaff, 1973). Because high doses of steroid inhibit the secretion of

Figure 4. Progesterone receptor immunoreactivity in the medial preoptic nucleus of prepubertal and postpubertal males treated with pellets containing either 0.00 or 0.05 mg estradiol benzoate (EB). Scale bar ⫽ 50 ␮m.

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LHRH, they may result in a reduction in behavior through inhibition of LHRH or other neuropeptides required for expression of behavior. Further experiments will be needed to explore this possibility.

ER␣ and Pubertal Change in Behavior The number of ER␣-ir cells in the MPNmag, BNSTpm, MeA, and MeP was not significantly affected by EB treatment or age, indicating that the reduced behavioral responsiveness to EB in prepubertal males is not associated with reduced levels of ER in these brain regions. Treatment with EB did reduce the number of ER␣-ir cells in the MPN of castrated prepubertal males. Although this finding could be related to the inability of estrogen to facilitate behavior in prepubertal males, we think it unlikely because the EB-treated postpubertal males, which did engage in mating behavior, had numbers of ER␣-positive cells in the MPN equivalent to those of EB-treated prepubertal males. Taken together, the ER␣-ir data demonstrate that the differential behavioral responsiveness to EB shown by juvenile and postpubertal males is not mediated by a difference in the number of ER␣-containing cells in the mating circuit.

necessary neural event for expression of male mating behavior in adult mice (Phelps, Lydon, O’Malley, & Crews, 1998), rats (Witt, Young, & Crews, 1994, 1995), hamsters (Romeo & Sisk, 2001), and lizards (Crews et al., 1996). Thus, the absence of mating behavior in prepubertal males may be related to a lack of PR activation at this developmental stage.

Conclusions In summary, we have established that prepubertal males are less responsive than postpubertal males to the activational effects of EB on reproductive behavior and have similar levels of ER␣ in the neural circuit that mediates male mating behavior. Estradiol increases PR in the MPN of both prepubertal and postpubertal males. We conclude that the behavioral unresponsiveness to estrogen exhibited by prepubertal males is not due to an absence of functional ERs in the steroid-sensitive forebrain mating circuit. These data suggest that steroids facilitate a behavioral response only when other conditions are met, such as the appropriate interpretation and integration of sensory stimuli or social context. Thus, behavioral potentials are recast during puberty as a result of maturation of neural circuits responsible for the integration of responses to external and internal stimuli.

Estradiol Induction of PR and Functionality of the ER Treatment with EB significantly induced PR in the MPN of both postpubertal and prepubertal males. Because ER␣ primarily mediates EB-induced PR in the hypothalamus (MacLusky & McEwen, 1978; Moffatt et al., 1998; Roy et al., 1979), these data suggest that ER␣ in the MPN of males is capable of activating transcription before pubertal maturation. Furthermore, the fact that all doses of EB suppressed LH secretion in the pubertal males provides further support that the ERs are functional before pubertal development. The distributions of ER␣- and PR-ir cells within the MPN overlapped significantly, suggesting that the increase in PR immunoreactivity in response to EB exposure occurs in the same cells that contain ER␣. Indeed, when a double-labeled study of ER and PR was performed in guinea pigs, estrogen-induced PRs were found only in ER-positive cells (Blaustein & Turcotte, 1989). We infer from these data that the increase in PR-ir cells in the MPN of EB-treated prepubertal and postpubertal males occurs in ER␣expressing cells.

PR and Pubertal Change in Behavior ER is a transcription factor that regulates the expression of estrogen-responsive genes. Therefore, the absence of mating behavior before puberty may be due, in part, to the absence of certain steroid receptor cofactors that allow the ER to induce the appropriate expression of behaviorally related genes in the prepubertal nervous system. The gene (or genes) ultimately responsible for the lack of mating behavior in juveniles was not determined in the present study. The present data indicate that the absence of an estradiol-induced increase in PR expression is not responsible for the lack of sexual behavior before pubertal development. However, these data do not rule out the possibility that other ERregulated genes are not transcribed correctly before puberty, which in turn leads to the lack of copulation observed in prepubertal males. It is interesting to note that activation of the PR is a

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Received June 1, 2001 Revision received October 2, 2001 Accepted October 17, 2001 䡲