Q ~ 4...? E. 1.23 . . .c «w. in: . a: 2. 1,. 2%.?“ 1. s2»: .7... VM 2.? r133... .. h» «25.3.. .. .9... .1 $an 3.. . {3.5 a . .. . . .1 r! .1 «a? .. .3“... V «W52 . -9 w ' ) 1 93'. S. 2.3:. a... a: 1 Wk»? FREE.- )u» t. f. - wvh. N: . 1%.”va #2 no... : 71 ion?“ a? . ‘6‘. f .33 .7» 23. .. .. ._ «3 3w... iii. 1,“ . 55.1. 5,. r. at... .1 . x... .. 1.5:}: Q... 3.87 .‘ F“ 2.0....” a: .5 H can?» 1 , 5 maul ‘73, I ., v -'v \.‘ “11 z, u at Th! " ‘ ’1 full This is to certify that the dissertation entitled THE PUBERTAL MATURATION OF MALE SEXUAL BEHAVIOR: THE ROLE OF STEROID HORMONES, THEIR RECEPTORS: AND PHEROMONES presented by Russell D. Romeo has been accepted towards fulfillment of the requirements for Ph.D. degree in Psychology/Neuroscience Majtgprofessor Date 2/‘zl/ol MS U is an Affirmative Action/Equai Opportunin Institution 0- 12771 LEIRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 C'JClRC/DateDue.p65~p. 15 THE PUBERTAL MATURATION OF MALE SEXUAL BEHAVIOR: THE ROLE OF STEROID HORMONES, THEIR RECEPTORS, AND PHEROMONES BY Russell D. Romeo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology and Neuroscience Program 2001 ABSTRACT THE PUBERTAL MATURATION OF MALE SEXUAL BEHAVIOR: THE ROLE OF STEROID HORMONES, THEIR RECEPTORS, AND PHEROMONES BY Russell D. Romeo Prepubertal animals differ vastly from adults in their social behaviors. These behavioral changes during puberty must be mediated by changes in the structure and/or function of the central nervous system. In the present dissertation, the male Syrian hamster (Mesocricetus auratus) was used to investigate the neural mechanisms responsible for the pubertal change in reproductive behavior. The full suite of adult mating behaviors in this species is dependent on both steroidal hormones (internal information) and chemosensory cues (external information), thus this model allows us to investigate how puberty affects the ability of an organism to process both endogenous and exogenous information. A hallmark of pubertal development is the increased production and secretion of the steroid hormones testosterone and progesterone. Since mating behavior emerges after the pubertal rise of these steroids, it was hypothesized that the absence of sexual behavior prior to puberty was due to the lack of stimulation by these hormones. I have found that exogenously adminstered testosterone, and its androgenic and estrogenic metabolites, are capable of activating sexual behavior only in adult males, indicating that not only do these steroids increase during pubertal development, but the neural responsiveness to these hormones increases as well. Using immunocytochemistry, it was demonstrated that this reduced responsiveness to steroids prior to puberty is not mediated by a lack of androgen or estrogen receptors within the neural circuit that mediates sexual behavior. It was also shown that activation of the progesterone receptor in prepubertal males does not facilitate their ability to engage in copulation. I found that after pheromonal stimulation, prepubertal and adult males respond with an equivalent level of activity in brain regions that are imperative for chemosensory processing and male sexual behavior. However, adults are dissimilar to juvenile males in that adults experience a rise in testosterone after pheromonal exposure, indicating that adult males are integrating and processing the chemosensory cues differently before and after pubertal development. Thus, I have found that the lack of mating behavior prior to puberty is not mediated by the availability of steroids, the absence of their receptors, or basic chemosensory processing. However, the lack of a neuroendocrine reflex prior to puberty, suggests that the pubertal maturation of mating behavior is dependent upon the refinement of sensory processing and integration of stimuli, both internal and external. To my family, Thank you for your continuous love and support. iv ACKNOWLEDGMENTS I would like to thank my graduate advisor, mentor, and friend Dr. Cheryl Sisk without whose knowledge, guidance, and advice this dissertation would not be possible. I greatly appreciate the enormous amount of time you have devoted to me and my work. Your tireless effort has given me the tools and confidence to succeed. Thank you. Thanks to Drs. Antonio Nunez, Keith Lookingland, and Juli Wade for serving on my dissertation committee. I thank you for your insightful comments that have made this a stronger dissertation. I would also like to thank you for the advice you have given me at all stages of my education. I would also like to thank the technical maven Jane Venier and the greatest group of graduate students and postdocs that I have been lucky enough to work with, namely Drs. Leslie Meek, Yu Ping Tang, and David Parfitt, and Heather Richardson, Kalynn Schulz, and Lyle Burgoon. You have made this lab not only a intellectually stimulating environment, but also a great place to work. I would like to thank the host of undergraduates that have helped me with my research projects including, Jon Arbogast, Jennifer Campbell, Kristina Davis, Stefani Diedrich, Alric Hawkins, Chung Lee, Casey McGovern, and Aaron Nelson. Their effort and time are greatly appreciated. TABLE OF CONTENTS LIST OF TABLES ............................................. iX LIST OF FIGURES ............................................. x INTRODUCTION ................................................ 1 Male Sexual Behavior ................................... 3 Hormonal Regulation of Male Sexual Behavior ............ 4 Pheromonal Regulation of Male Sexual Behavior .......... 5 The Forebrain Neural Circuit that Mediates Male Sexual Behavior .................................. 8 Pubertal Maturation of Male Sexual Behavior ........... 16 Section I. Contribution of the androgenic metabolite dihydrotestosterone and the androgen receptor in the pubertal maturation of male sexual behavior ................ 21 Rationale ............................................ 21 Methods .............................................. 22 Results .............................................. 26 Discussion ........................................... 33 Section II. Contribution of the aromatase enzyme, estrogen, and estrogen receptor in the pubertal maturation of male sexual behavior ......................... 37 Experiment I. Androgenic regulation of hypothalamic aromatase activity in prepubertal and adult males ..... 37 Rationale ....................................... 37 Methods ......................................... 38 Results ......................................... 43 Discussion ...................................... 51 Experiment II. The role of estrogen and the estrogen receptor in the sexual behavior of prepubertal and adult males ........................................... 54 Rationale ........................................ 54 vi Methods .......................................... 56 Results .......................................... 59 Discussion ....................................... 66 Summary ............................................... 69 Section III. Contribution of progesterone and progesterone receptors in the pubertal maturation of male sexual behavior ............................................ 7O Experiment I. Estrogenic regulation of the progesterone receptor in prepubertal and adult males ................................................. 7O Rationale ........................................ 70 Methods .......................................... 71 Results .......................................... 76 Discussion ....................................... 81 Experiment II. The effect of progesterone receptor antagonists on adult male sexual behavior ............. 83 Rationale ........................................ 83 Methods .......................................... 84 Results .......................................... 86 Discussion ....................................... 86 Experiment III. The sexual behavior of prepubertal males treated with estrogen and progesterone .......... 91 Rationale ....................................... 91 Methods ......................................... 92 Results ......................................... 94 Discussion ...................................... 96 Summary .............................................. 98 Section IV. Contribution of chemosensory cues in the pubertal maturation of male sexual behavior ............... lOO Rationale ........................................... 100 Methods ............................................. 102 vfi Results ............................................. 105 Discussion .......................................... 108 Section V. Integration and Conclusions ................... 116 REFERENCES ............................................... l 2 6 Wfi LIST OF TABLES Table 1. Mean (1 SEM) seminal vesicle weight (mg / 100 g body weight) of prepubertal and adult males treated with seven daily injections of either 0, 500, 1000 pg DHT. Table 2. Mean (i SEM) plasma testosterone concentrations and paired testis weight. Table 3. Mean (i SEM) plasma estradiol and luteinizing hormone concentrations and paired testis weight. Table 4. Mean (i SEM) duration of AGI and the frequency of mounts. Table 5. Mean (i SEM) seminal vesicle and paired testis weight. n LIST OF FIGURES Figure 1. Cartoon of the neural circuit that mediates male mating behavior in the male Syrian hamster (horizontal plane). Abbreviations; AOB, assessory olfactory bulb; BNSTpm, posteromedial subdivision of the bed nucleus of the stria terminalis; MeA, anterior subdivision of the medial amygdala; MeP, posterior subdivision of the medial amygdala; MOB, main olfactory bulb; MPN, medial preoptic nucleus; MPNmag, magnocellular subdivision of the medial preoptic nucleus. Figure 2. Number of seconds engaged in anogenital investigation (AGI; A), and frequencies of mounts (B), intromissions (C), and ejaculations (D) in a 15 min behavioral test in prepubertal and adult males given daily injections of either 0, 500, or 1000 pg of DHT for seven days. Asterisk indicates a significant difference from prepubertal males. "a" indicates that adults that received 1000 pg of DHT are significantly different from the adults that received either the 0 or 500 pg dose of DHT. All values are means 1 SEM. Figure 3. AR—ir cells/62,500 pm2 in the MPN, MPNmag, BNSTpm, MeA, and MeP of prepubertal and adult males treated daily for seven days with either 0, 500, or 1000 pg of DHT. Asterisks indicate a significant difference between prepubertal and adult males within a dose of DHT. All values are means : SEM. Figure 4. AR-ir in the MPN of a prepubertal (A) and adult (B) male treated with 1000 pg of DHT. Bar, 50pm. Figure 5. 3H-Estradiol production (fmol / mg protein / min) after incubation of hypothalamic homogenates with 250 nM 3H— testosterone for 15, 30, 60, or 120 min. Estradiol production increased at a lower rate between the 30 and 120 min time points. Figure 6. 3H—Estradiol production (fmol / mg protein / min) in hypothalamic homogenates from intact prepubertal and adult males. Asterisk indicates significant difference. All values are means i SEM. Figure 7. 3H-Estradiol production (fmol / mg protein / min) in hypothalamic homogenates from castrated adult males treated with either 0 or 2.5 mg of testosterone (A), castrated prepubertal males treated with either 0 or 2.5 mg of testosterone (B), and castrated adult and prepubertal males treated with 2.5 mg of testosterone (C). Asterisks indicate significant differences. All values are means : SEM. Figure 8. Number of seconds engaged in AGI (A) and number of mounts (B) and intromissions (C) in prepubertal and adult males implanted with a pellet containing either 0, 0.05, 0.10, or 0.25 mg of EB. Asterisks indicate adults are significantly different from their prepubertal counterparts. "a" indicates significantly different from the blank—treated controls within an age. All values are means i SEM. Figure 9. The number of ERa-ir cells/62,500 pm2 in the MPN, MPNmag, BNSTpm, MeA, and MeP of prepubertal and adult males implanted with a pellet containing either 0 or 0.05 mg of EB. Asterisk indicates that castrated prepubertal males have a significantly greater number of ERa-ir cells compared to castrated adults. All values are means i SEM. Figure 10. ERa-ir in the MPN of a prepubertal (A) and adult (B) male treated with 0.05 mg of EB. Bar, 50pm. Figure 11. The number of PR—ir cells / 62,500 pm2 (A) and relative amount of PR-ir per cell (mean o.d.; B) in sections from the MPN exposed to different dilutions of primary antibody. Tissue was from adult castrates treated with a 0.05 mg pellet of EB for one week. Figure 12. Plasma progesterone concentrations in prepubertal and adult males castrated and implanted with a pellet containing either 0, 0.05, 0.10, or 0.25 mg of estradiol. Asterisks indicate that adults have significantly higher circulating levels of progesterone than prepubertal males. All values are means 1 SEM. Figure 13. The number of PR-ir cells / 62,500 pm2 (A) and the relative amount of PR-ir per cell (mean o.d.; B) in the 1:1000 primary antibody condition in the MPN of prepubertal and adult males that were castrated and implanted with a pellet containing either 0, 0.05, 0.10, or 0.25 mg of estradiol. Males treated with the 0 mg pellet had significantly fewer PR—ir cells and less PR—ir per cell than males treated with either the 0.05, 0.10, or 0.25 mg pellet of estradiol. All values are means 1 SEM. Figure 14. The number of PR—ir cells / 62,500 pm2 (A) and the relative amount of PR-ir per cell (mean o.d.; B) in the 1:5000 primary antibody condition in the MPN of prepubertal and adult males that were castrated and implanted with a pellet containing either 0, 0.05, 0.10, or 0.25 mg of estradiol. Males treated with the 0 mg pellet had xi significantly fewer PR—ir cells and less PR—ir per cell than males treated with either the 0.05, 0.10, or 0.25 mg pellet of estradiol. All values are means i SEM. Figure 15. PR-ir in the MPN of a prepubertal (A) and adult (B) male treated with a blank pellet and a prepubertal (C) and adult (D) male treated with a pellet containing 0.05 mg of EB. Sections were from the 1:1000 primary antibody condition. Bar, 50pm. Figure 16. The frequencies and latencies (sec) of mounts, intromissions, and ejaculations in adult males treated with RU486 (2 mg/kg) or the oil vehicle. Asterisk indicates a significant difference. All values are means i SEM. Figure 17. The frequencies and latencies (sec) of mounts, intromissions, and ejaculations in adult males treated with ZK98299 (6 mg/kg) or the oil vehicle. Asterisk indicates a significant difference. All values are means 1 SEM. Figure 18. Plasma progesterone (ng/ml) in castrated prepubertal males treated with either a 0, 0.25, 0.50, 1.5, 2.5, 5, 7.5, 10, 15, 25, or 35 mg pellet of progesterone for one week. Bars that share a letter are not significantly different from each other. Numbers in the parentheses are the number of animals that comprise that mean. The gray bar represents the average plasma progesterone level in a sexually behaving castrated adult male implanted with a 0.05 mg pellet of estradiol (E). All values are means : SEM. Figure 19. Plasma testosterone concentrations in prepubertal and adult males exposed to VS or a clean cotton swab. Asterisk indicates adult males exposed to VS had significantly higher circulating levels of testosterone than the adults exposed to a clean cotton swab (t tests). All values are means i SEM. Figure 20. Fos—ir cells / 62,500 lunz in the MPN, MPNmag, BNSTpm, MeP, MeA, and LSept of prepubertal and adult males exposed to VS or a clean cotton swab. Asterisks indicate that animals exposed to VS had significantly greater numbers of Fos-ir cells than animals exposed to a clean cotton swab. All values are means + SEM. Figure 21. Photomicrographs of Fos-ir cells in the BNSTpm of a prepubertal (A) and adult (B) male exposed to a clean cotton swab and a prepubertal (C) and adult (D) male exposed to VS. Arrowheads are outlining the approximate area of the nucleus that was analyzed. f, fornix; Bar, 100pm. xfi LIST OF ABBREVIATIONS Anatomical Abbreviations AOB BNST BNSTpm MeA MeAMY MeP MOB MPN MPNmag assessory olfactory bulb bed nucleus of the stria terminalis posteromedial subdivision of the bed nucleus of the stria terminalis anterior subdivision of the medial amygdala medial amygdala posterior subdivision of the medial amygdala main olfactory bulb medial preoptic nucleus magnocellular subdivision of the medial preoptic nucleus General Abbreviations AGI ANOVA AR BSA CV DAB DHT TX E ER ir NGS PBS PR TBS TLC VS anogenital investigation analysis of variance androgen receptor bovine serum albumin coefficient of variation diaminobenzidine dihydrotestosterone triton X—lOO estradiol estrogen receptor immunoreactive normal goat serum phosphate buffered saline progesterone receptor testosterone tris buffered saline thin layer chromatography vaginal secretion xfii INTRODUCTION The internal and external demands placed on an animal change dramatically throughout its lifespan. To meet these challenges, animals undergo equally drastic changes in their physiology and behavior. One stage of development that typifies a lifespan change is puberty. Prepubertal animals differ vastly from adults in their social behaviors, responses to sensory stimuli, and responses to environmental stressors (Primus and Kellogg, 1990; Primus and Kellogg, 1989; Slob, Huizer, and Van Der Werff Ten Bosch, 1986). These impressive behavioral changes during puberty must be mediated by changes in the structure and/or function of the central nervous system. Therefore, the study of pubertal development of the nervous system addresses the extremely important and interesting question of how the brain mediates profound behavioral changes, and equally as important, how these behavioral changes feed back and impact the brain. The study of pubertal development in animals is also important for its obvious implications in understanding human adolescence. For example, the morbidity and susceptibility to psychological disorders (e.g., schizophrenia and depression, suicide, and violent behavior/aggression) increase during adolescence (Conger and Petersen, 1984; Hammen and Rudolph, 1996; Masten, 1987). These problems may be mediated by malfunctions in the normal neural and endocrine changes associated with pubertal development (Buchanan, Eccles, and Becker, 1992; Gooding and Iacono, 1995; Lerner, 1985). Thus, studying puberty through a neurobehavioral perspective may provide important insights into how perturbations of normal pubertal processes may result in inappropriate responses to environmental stimuli in adulthood. We use the male Syrian hamster (Mesocricetus auratus) to investigate the neural mechanisms that are responsible for the pubertal change in reproductive behavior. We have chosen to use this animal model for three reasons. First, the full suite of mating behaviors in the adult male hamster is dependent on both steroidal hormones (internal information) and chemosensory cues (external information; Wood, 1998; Wood and Coolen, 1997; Wood and Newman, 1995c), thus allowing us to investigate how puberty affects the ability of an organism to process both endogenous and exogenous cues. Second, the neural circuit that mediates the mating behavior of adults in this species has been well described and functionally characterized (Coolen and Wood, 1998; Lehman, Powers, and Winans, 1983; Lehman, Winans, and Powers, 1980; Newman, 1999; Wood, 1996b; Wood, 1998; Wood and Newman, 1995b) as well as the endocrinological profile of the male as he progresses through puberty (Miller, Whitsett, Vandenbergh, and Colby, 1977; Vomachka and Greenwald, 1979). Finally, we have demonstrated that behavioral responses to testosterone change as a function of pubertal development in this species (Meek, Romeo, Novak, and Sisk, 1997). Therefore, we are provided with a large body of information describing the neuroanatomy and reproductive physiology of the adult, and, more importantly, an animal model that processes steroidal information differently before and after pubertal development. Whether Syrian hamsters process chemosensory cues differently before and after puberty is unknown. Therefore, the study of the pubertal maturation of male mating behavior in this species will lead to a deeper understanding of how internal and external cues may interact to affect an individual progressing through pubertal development. Male Sexual Behavior Male sexual behavior in most rodent species follows a highly stereotyped pattern of behaviors, at least when observed in a laboratory setting (Meisel and Sachs, 1994). First, the male chemoinvestigates the anogenital region of the female to assess her reproductive status. If the male determines the female is in estrus, as evidenced by particular pheromones and/or behavioral postures emitted by her, the male will attempt to mount the female's hindquarters. At first, mounts are typically not accompanied by thrusting, so the penis does not make contact with the vagina during a mount. When a male thrusts during mounting, and the penis penetrates the vagina, this behavior is termed an intromission, and is the third basic behavior to emerge during copulation. After a series of intromissions (e.g., 10—15), the male will ejaculate. Following a brief refractory period (i.e., 30 sec to a few minutes depending on the species), the male will return to mounting and intromitting with the female until another ejaculation occurs. This sequence of behaviors will continue until the male reaches sexual satiety. The latency to engage in these behaviors depends upon the experience of the male, and to some degree the female’s experience (Bradley and Meisel, 2000). However, the sequence in which these behaviors are displayed remains fixed. Interestingly, the pubertal emergence of reproductive behavior follows the same sequential order as described above. That is, chemoinvestigatory behavior and mounting are the first behaviors to emerge during pubertal development followed by intromissions and ejaculations (Miller et al., 1977). Hormonal Regulation of Male Sexual Behavior Physiological and behavioral aspects of male reproduction are typically temporally associated. That is, gametogenesis, steroidogenesis, and sexual behavior are linked, often waxing and waning with each other over the lifespan (Crews, 1984; for interesting exceptions see, Crews, 1984; Mendonca, Chernetsky, Nester, and Gardner, 1996). Therefore, mating behavior typically increases as testosterone (T), the major androgen secreted by the testes, increases. Indeed, when circulating levels of T are manipulated experimentally (i.e., castration, hormonal injections, or implants), T levels and behavior show a positive correlation (reviewed in, Luttge, 1979; Meisel and Sachs, 1994). However, T is not the only steroid hormone regulating male sexual behavior. It has been shown in a variety of species that its metabolites, such as estradiol (Beyer, Morali, Naftolin, Larsson, and Pérez—Palacios, 1976; Carroll, Weaver, and Baum, 1988; Christensen and Clemens, 1975; Davidson, 1969; DeBold and Clemens, 1978; Floody and Petropoulos, 1987; Luttge, 1979; deersten, 1973; Steel and Hutchison, 1988) and dihydrotestosterone (DHT; Butera and Czaja, 1989a; DeBold and Clemens, 1978; Payne and Bennett, 1976; Whalen and DeBold, 1974), are important mediators of male reproductive behavior. In males, these estrogenic and androgenic metabolites of T are formed locally in the brain by the intracellular aromatase or reductase enzymes, respectively (Meisel and Sachs, 1994). Recently, even progesterone has been implicated in the control of male sexual behavior (Crews, Godwin, Hartman, Grammer, Prediger, and Sheppherd, 1996; Lindzey and Crews, 1988; Lindzey and Crews, 1992; Phelps, Lydon, O'Malley, and Crews, 1998; Witt, Young, and Crews, 1994; Witt, Young, and Crews, 1995; Young, Greenberg, and Crews, 1991). The origin of progesterone in males is most likely from both the testes and adrenals, since neither gonadectomy nor adrenalectomy alone significantly influences progesterone titers (Kalra and Kalra, 1977). Steroid hormones affect behavior directly through the central nervous system. Specific intracellular receptors for these signals are found in various limbic areas (e.g., hypothalamus and amygdala) in nuclei that form the neural substrate for male sexual behavior (Cottingham and Pfaff, 1986; Pfaff, 1968; Wood, Brabec, Swann, and Newman, 1992; Wood and Newman, 1995a). Steroids bind to their intracellular receptors, which in turn, bind to hormone response elements within promoter regions of DNA to alter gene transcription, protein synthesis, and ultimately cellular function (Tsai and O'Malley, 1994). In a variety of species, intracerebral application of these gonadal steroids into the hypothalamus (Butera and Czaja, 1989a; Butera and Czaja, 1989b; Christensen and Clemens, 1974; Crews et al., 1996; Davidson, 1966; Johnston and Davidson, 1972; Lisk, 1967; Lisk and Bezier, 1980; Morali, Hernandez, and Beyer, 1986; Rozendaal and Crews, 1989; Wood and Newman, 1995d) and/or amygdala (Baum, Tobet, Starr, and Bradshaw, 1982; Wood, 1996a; Wood and Newman, 1995d) causes an increase in mating behavior in castrated adult males. Taken together, these data indicate that T and progesterone secreted from the testes (and adrenals) by themselves, and in conjunction with metabolites of T, affect steroid-sensitive brain regions in limbic areas, that in turn, activate and facilitate copulation. It should be noted that the efficacy of a particular steroid to activate mating behavior depends on the brain area it is acting on. This issue will be expanded on below when discussing the steroid-sensitive neural circuit that mediates male mating behavior. Pheromonal Regulation of Male Sexual Behavior Mammalian reproductive physiology and behavior are largely affected by olfaction (Powers and Winans, 1975). For example, just the odor of an estrous female causes erection and seminal emission in adult male rats (Sachs, 1997; Sachs, Akasofu, Citron, Daniels, and Natoli, 1994). The male hamster exemplifies the importance of olfactory cues in reproduction since adults will not engage in copulation unless olfactory cues from the female are present (Wood, 1998; Wood and Newman, 1995c). In hamsters, the major olfactory cue from the female is a pheromone found in her vaginal secretions around the time of estrus. Males begin to show an interest in these secretions after the onset of pubertal development (e.g., 40 days of age; Johnston and Coplin, 1979), and the interest in these secretions has been reported to be under androgenic regulation (Gregory, Engel, and Pfaff, 1975; Powers and Bergondy, 1983). Generally, reproductive behavior has two main facets: arousal and performance (reviewed in, Everitt, 1990). The necessity of pheromonal cues for the initiation of male sexual behavior in hamsters illustrates the significance of arousal. That is, if this sensory information is not received and processed in the proper context, then copulation will not ensue. Thus, developmental changes in the animal's ability to interpret these sensory cues as arousing will influence the likelihood that it will engage in the performance aspects of mating behavior. The olfactory bulbs are the first central brain structures that process olfactory information. The bulbs are composed of the main and assessory olfactory bulbs (MOB and AOB, respectively). The MOB processes volatile odorants, while the AOB processes pheromonal information received from the vomeronasal organ. Interfering with olfactory bulb function (i.e., ablation or inactivating with zinc sulfate) causes a decrease in reproductive behavior (Cain and Paxinos, 1974; Murphy and Schneider, 1970; Powers and Winans, 1975; Rowe and Edwards, 1972). Interestingly, the magnitude of the decrement in behavior one observes after interfering with the bulbs depends on the species of animal being studied. For example, bulbectomy of the rat leads to a partial failure in mating behavior (Rowe and Edwards, 1972), while in the hamster this procedure completely abolishes mating behavior (Murphy and Schneider, 1970). Taken together, these studies indicate that olfactory input to the brain is vital for normal mating to take place, but that the dependence on olfactory information demonstrates some species specificity. The Forebrain Neural Circuit that Mediates Male Sexual Behavior In the male hamster, steroidal and pheromonal information is integrated in a forebrain circuit that is composed of nuclei in the hypothalamus and amygdala (Wood, 1996b; Wood, 1997; Wood, 1998), which ultimately project to the spinal cord via the midbrain tegmentum to affect the motor aspects of mating behavior (Meisel and Sachs, 1994). The correct integration of this internal (steroidal) and external (pheromonal) information is necessary for reproductive behavior to occur. A cartoon of this mating circuit is provided in Figure 1. The function of these various nuclei are discussed below. Amygdala: The first relay for the olfactory information is the amygdala where both the MOB and AOB send their axons. The MOB projects to the more lateral aspects of the cortical nucleus of the amygdala, while the AOB projects to the more medial aspects (Lehman and Winans, 1982). The medial amygdala (MeAMY) can be subdivided into the anterior medial amygdala (MeA) and posterior medial amygdala (MeP). It has been proposed that these two subdivisions process different types of information (reviewed in, Wood and Newman, 1995b). For instance, selective lesions of the MeA completely abolish chemoinvestigatory behavior, but this component of mating behavior is only modestly affected by lesions of the MeP (Newman, Parfitt, and Kollack—Walker, 1997). These data suggest that the MeA primarily transduces chemosensory information. Conversely, the MeP has a greater number of steroid receptor-containing neurons than the MeA (Wood et al., 1992; Wood and Newman, 1995b), suggesting that the MeP relays mostly steroidal information. Thus, in experiments described later in this dissertation the MeAMY is typically subdivided into the MeA and MeP. However, this is not to say Rostral Olfactory Bulbs “ AOB MOB MPNmag MPN ‘ BNSTpm O O \‘ /’ Caudal Figure 1. Cartoon of the neural circuit that mediates mating behavior in the male Syrian hamster (horizontal plane) . Abbreviations: AOB, assessory olfactory bulb; BNSTpm, posteromedial subdivision of the bed nucleus of the stria terminalis; MeA, anterior subdivision of the medial amygdala; MeP, posterior subdivision of the medial amygdala; MOB, main olfactory bulb; MPN, medial preoptic nucleus; MPNmag, magnocellular subdivision of the medial preoptic nucleus . lO that the MeA and MeP do not share some overlap in their function. For instance, both nuclei receive chemosensory information, and both are replete with steroid hormone receptors (Meek et al., 1997; Romeo, Diedrich, and Sisk, 1999; Wood et al., 1992; Wood and Newman, 1995a; Wood and Newman, 1995b). Paradoxically, the MeP shows a greater expression of Fos, a marker of neuronal activity (Morgan and Curran, 1991), than the MeA after the male has been exposed to female pheromones (Fiber, Adames, and Swann, 1993; Fiber and Swann, 1996; Kollack—Walker and Newman, 1997; Swann and Fiber, 1997). There are reciprocal connections between the MeA and MeP (Coolen and Wood, 1998). Thus, whether the more robust increase in activity of the MeP compared to the MeA is due to direct olfactory information from the bulbs or indirect olfactory information from the MeA is unclear. Taken together, these studies indicate that the MeAMY processes both steroidal and chemosensory information, but with some degree of specificity depending on whether the anterior or posterior portion of the nucleus is involved. Ablation of the MeAMY disrupts the male’s ability to engage in copulation (Giantonio, Lund, and Gerall, 1970; Harris and Sachs, 1975; Kostarczyk, 1986; Lehman et al., 1980), but this deficit may be secondary to the inability of the male to sense the pheromonal cues provided by the female (Lehman et al., 1980). Moreover, local steroidal implants in the MeAMY facilitate mating behavior in males (Baum et al., 1982; Wood, 1996a; Wood and Newman, 1995d). However, this 11 effect appears to be due to the aromatization of T to estrogen, since local application of estradiol, a potent form of estrogen, is more effective than DHT in facilitating male sexual behavior (Wood, 1996a), and intracerebral injections of an androgen receptor blocker aimed at the MeAMY are unable to significantly reduce male mating behavior (McGinnis, Williams, and Lumia, 1996). The effects of an intracerebral implant of progesterone in the MeAMY on male sexual behavior has not been investigated. Thus, the role of progesterone in this area is unknown. It should be noted that these lesion and implant studies look at the MeAMY as a whole since the limited anatomical resolution inherent in these techniques would not allow a finer distinction. Taken together, these data show that the MeAMY is the first brain region in this circuit where both the external (chemosensory cues) and the internal (hormonal stimulation) information start to converge to allow for the full display of male reproductive behavior (Wood, 1998; Wood and Coolen, 1997; Wood and Newman, 1995b; Wood and Newman, 1995c). Bed NUcleus of the Stria Terminalis: The bed nucleus of the stria terminalis (BNST) receives projections from both the anterior and posterior portions of the MeAMY via the stria terminalis (Wood, 1998), and a sparse projection directly from the AOB (Newman et al., 1997). Similar to the MeAMY, this brain region represents another level where chemosensory cues and hormonal stimulation interact. The 12 BNST is composed of several subdivisions, one of which is the posteromedial subdivision (BNSTpm). This subnucleus of the BNST shows increases in Fos—immunoreactivity after exposure to female pheromones (Fiber et al., 1993; Fiber and Swann, 1996; Kollack—Walker and Newman, 1997; Swann and Fiber, 1997), and has a high concentration of steroid hormone receptors (Meek et al., 1997; Wood et al., 1992; Wood and Newman, 1995a; Wood and Newman, 1995b), indicating that the BNSTpm is responsive to both steroidal and chemosensory information. It has been shown in adult male rats and hamsters that when the BNST is ablated, males engage in less sexual behavior than their sham-lesioned counterparts (Claro, Segovia, Guilamon, and del Abril, 1995; Emery and Sachs, 1976; Liu, Salamone, and Sachs, 1997; Powers, Newman, and Bergondy, 1987; Valcourt and Sachs, 1979). Similar to that which is found in a male hamster with lesions of the MeAMY, this deficit in mating behavior may be secondary to the inability of the male to sense the pheromonal cues provided by the female (Powers et al., 1987). It has also been shown that local application of T to the BNST is capable of initiating male sexual behavior (Wood and Newman, 1995c; Wood and Newman, 1995d). The effects of locally administered DHT, estrogen, or progesterone in the BNST on male mating behavior have not been tested. Therefore, whether T is acting directly or indirectly through one if its metabolites, or if progesterone is capable of activating mating behavior when 13 implanted into the BNST is presently unknown. As noted before, these lesion and implant studies look at the BNST as a whole because of the limited anatomical resolution of these techniques. In summary, these data indicate that the BNST, and in particular the BNSTpm, is similar to the MeAMY in that it is responsive to chemosensory cues and is an important component of the steroid—sensitive neural circuit that mediates male mating behavior. Hypothalamus: In hamsters, there are two separate subnuclei of the hypothalamic preoptic area that have been implicated in the control of male sexual behavior, the magnocellular portion of the medial preoptic nucleus (MPNmag), and the medial preoptic nucleus (MPN). These nuclei receive projections from the MeAMY via the stria terminalis and ventral amygdalofugal pathway (Wood, 1998). These subregions of the hamster hypothalamus express Fos after exposure to female pheromones (Fiber et al., 1993; Fiber and Swann, 1996; Kollack—Walker and Newman, 1997; Swann and Fiber, 1997), and these areas have a high concentration of steroid hormone receptors (Meek et al., 1997; Romeo et al., 1999; Wood et al., 1992; Wood and Newman, 1995a; Wood and Newman, 1995b). Thus, similar to the MeAMY and BNSTpm, these nuclei integrate chemosensory cues and hormonal stimulation. It has been shown in numerous species that when the medial preoptic area is removed males engage in less sexual l4 behavior (Cherry and Baum, 1990; De Jonge, Louwerse, Ooms, Evers, Endert, and Van De Poll, 1989; Floody, 1989; Giantonio et al., 1970; Ginton and Merari, 1977; Hansen, Kohler, Goldstein, and Steinbusch, 1982; Heimer and Larsson, 1966/1967; Larsson and Heimer, 1964; Ryan and Frankel, 1978; Yahr and Gregory, 1993). Moreover, local application of either T, estrogen, or progesterone in the anterior hypothalamus is capable of initiating male sexual behavior (Butera and Czaja, 1989a; Butera and Czaja, 1989b; Christensen and Clemens, 1974; Crews et al., 1996; Davidson, 1966; Johnston and Davidson, 1972; Lisk, 1967; Lisk and Bezier, 1980; Morali et al., 1986; Rozendaal and Crews, 1989; Tang and Sisk, 1991; Wood and Newman, 1995d). Furthermore, unlike in the MeAMY, the androgenic metabolite DHT is effective in activating male sexual behavior when implanted into the preoptic/anterior hypothalamic area (Butera and Czaja, 1989a; Butera and Czaja, 1989b; Johnston and Davidson, 1972; Rozendaal and Crews, 1989; but see, Lisk and Bezier, 1980). These data indicate that the MPNmag and MPN are responsive to chemosensory cues in the hamster, and are two more important nuclei in the steroid—sensitive neural circuit that mediates male reproductive behavior. Summary: The network of nuclei discussed above represents the forebrain neural circuit that mediates male reproductive behavior. The steroid-sensitive feature of each component of this circuit demonstrates redundancy in that 15 hormonal stimulation in any one of these areas is sufficient to facilitate mating behavior in a male with an intact olfactory system (Wood and Newman, 1995d). However, the various nuclei in this circuit do show some differences with respect to which hormone causes the greatest behavioral response and what specific aspect of mating behavior (e.g , mounting, intromission) is affected (Wood, 1996b; Wood, 1997). The connections between these steroid— and pheromone— sensitive nuclei are reciprocal (Coolen and Wood, 1998). Thus, it is possible that as olfactory information proceeds from the amygdala to the bed nucleus and the hypothalamus these areas can feed back on the amygdala to alter its functioning. Furthermore, depending on the hormonal milieu and the developmental stage of the animal, these areas may be more or less responsive to steroidal and pheromonal information. Hence, the cross-talk between these nuclei may change so that a proper behavioral response may be initiated in the context of a particular set of stimuli, both internal and external. Pubertal Maturation of Male Sexual Behavior One benchmark of pubertal development in the male is the marked increase in T secretion by the testes. For instance, in the Syrian hamster serum T levels begin to increase slowly between 21 and 28 days of age and reach adult—like levels at approximately 50 days of age (Meek et al., 1997; Miller et 16 al., 1977; Sisk and Turek, 1983; Vomachka and Greenwald, 1979), while in the rat, the time course is slightly longer (T titers do not start to increase until around 45 days of age; Ketelslegers, Hetzel, Sherins, and Catt, 1978; sodersten, Damassa, and Smith, 1977). In both hamsters and rats, the pubertal maturation of mating behavior appears to be correlated with these endocrine changes since increases in androgen secretion precede the display of sexual behavior (Miller et al., 1977; sodersten et al., 1977). Similar to an experiment by Miller et al. (1977), we have shown that 28 day old male hamsters engage in little, if any, reproductive behavior, while 49 day old males can be observed engaging in all aspects of reproductive behavior (Meek et al., 1997). One obvious mediator of this increase in mating behavior is the increase in T secretion by the testes. We conducted a study (Meek et al., 1997) to address this question by castrating and clamping the T levels of prepubertal (21 days of age) and adult (42 days of age) male hamsters by immediately implanting them with a pellet containing either 0, 2.5, or 5 mg of T after castration. It should be noted that the 2.5 mg pellet of T provides back adult—like levels of circulating T. One week after castration and implantation (i.e., at either 28 or 49 days of age), all animals were given a 10—min behavioral test with a receptive female to assess their ability to engage in sexual behavior. It was found that males treated with T, regardless of age, engaged in significantly greater amounts of anogenital l7 investigation. In contrast, only adults treated with T showed significant increases in the number of mounts, intromissions, and ejaculations. T did not activate these behaviors in juvenile males. Similar behavioral results were obtained with rats and ferrets in that when prepubertal males are treated with a dose of T that fully activates sexual behavior in adults, prepubertal males still engage in little or no reproductive behavior (Baum, 1972; Larsson, 1967; Sisk, Berglund, Tang, and Venier, 1992; deersten et al., 1977). Therefore, the lower levels of T experienced by prepubertal males are not solely responsible for the lack of mating behavior exhibited prior to puberty. These data indicate that the nervous system of adults is more responsive than prepubertal males to the activating effects of T on reproductive behavior. The changes that take place in the nervous system during pubertal development that mediate this alteration in responsiveness remain largely unknown, and is the focus of the present dissertation. Since androgen receptors are the mediators of the intracellular actions of T, we hypothesized that the differential responsiveness to T before and after puberty is mediated by differential expression of the androgen receptor (AR) in prepubertal and adult males. Specifically, this hypothesis predicts that the greater behavioral responsiveness to T exhibited by the adults compared to juveniles is mediated by a greater number of ARs in the adult. Indeed, in support of this hypothesis it has been 18 demonstrated that pubertal maturation is accompanied by an increase in AR expression in the hypothalamus and amygdala in gonadally intact males (Kashon and Sisk, 1994; Meek et al., 1997). T generally upregulates AR—immunoreactivity in adults (Kashon, Arbogast, and Sisk, 1996; Menard and Harlan, 1993; Prins and Birch, 1993). Therefore, we processed the brain sections from the T—treated adult and prepubertal males to investigate whether T increased AR—immunoreactive (AR—ir) cell number in the mating circuit to a greater extent in adult compared to prepubertal males. T increased AR to the same degree in prepubertal and adult males in the MPN and MeAMY. Surprisingly, we found that T increased the number of AR—ir cells to a greater degree in prepubertal males compared to adults in the BNSTpm and MPNmag. These data clearly indicate that the lack of behavioral responsiveness to T in prepubertal males is not due to an inability of T to increase AR in the neural circuitry that underlies sexual behavior in the adult male hamster. Furthermore, these data show that the presence of AR in the mating circuit may be necessary, but not sufficient, to mediate the pubertal increase in male reproductive behavior. The few studies mentioned above are the sum total of our knowledge on the pubertal maturation of male reproductive behavior. Thus, the enormous amount of research that has been done on the steroidal and neural control of adult sexual 19 behavior stands in stark contrast to the dearth of studies investigating the pubertal development of this behavior in males. The purpose of the present dissertation is to begin to fill this gap in our understanding by elucidating the role that steroid hormones, their receptors, and pheromones play in the pubertal maturation of male mating behavior. Specifically, the lack of a behavioral response to T prior to puberty could be due to a lack of either the androgenic or estrogenic action, or both, to T's overall action. Moreover, the effects of progesterone on the pubertal maturation of male sexual behavior has not been established. Thus, this dissertation will investigate whether the metabolites of T, namely DHT and estrogen, or progesterone are capable of activating male mating behavior prior to puberty. Furthermore, receptors for these steroids will be assessed in the mating circuit before and after pubertal development to determine whether pubertal changes in responsiveness to these sex steroids is mediated by changes in the number of cells expressing these receptors. Finally, this dissertation will examine whether the mating circuit processes chemosensory cues from the female differently before and after pubertal development. 20 Section I. Contribution of the androgenic metabolite dihydrotestosterone and the androgen receptor in the pubertal maturation of male sexual behavior. Rationale T can be intracellularly reduced to the androgenic metabolite DHT, which facilitates sexual behavior in adult male hamsters (DeBold and Clemens, 1978; Payne and Bennett, 1976; Whalen and DeBold, 1974). Thus, the lack of a behavioral response to T in prepubertal males could arise from a relative lack of androgenic action by DHT. We have previously shown that prepubertal males are behaviorally unresponsive to T (Meek et al., 1997), indicating that cellular responses to activation of the AR by T are different in the prepubertal and adult brain. Whether the androgenic metabolite DHT elicits similar cellular and behavioral effects in pre— and post—pubertal males is not known, and this question was the focus of the present study. The steroid-sensitive cell groups that comprise the limbic components of this neural circuit express relatively high levels of AR, to which DHT binds (Wood et al., 1992; Wood and Newman, 1995a). DHT increases AR-immunoreactivity in this circuit in adult males (Wood and Newman, 1995a; Wood and Newman, 1999). In the present experiment we compared mating behavior and AR—immunoreactivity in prepubertal and adult castrates treated with DHT to determine whether puberty 21 is associated with changes in neural and behavioral responses to an androgenic metabolite of T. Methods Subjects and Treatment Male Syrian hamsters used in the present study were bred at Michigan State University (East Lansing, MI). All animals were weaned from their mothers at 21 days of age and singly housed in clear polycarbonate cages (37.5 X 33 X 17 cm) with ad libitum access to food (Teklad Rodent Diet No. 8640, Harlan, Madison, WI) and tap water. The animals were maintained on a 14 hr light / 10 hr dark light—dark schedule (lights off at 1200 hr EST) and the temperature was kept at 21:2W3. All animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Michigan State University All— University Committee for Animal Use and Care. All animals were castrated under methoxyflurane anesthesia at either 21 (prepubertal) or 42 (adult) days of age. Males received a daily injection (at 1000 hr) of either 500 or 1000 pg of DHT, or the sesame oil vehicle (n=5—6). Hormone treatment began on the day of castration. On the seventh day of treatment, all animals were given a 15—min mating behavior test with a hormone-primed estrous female. Immediately following the behavioral test, animals were weighed, euthanized, and perfused as described below. 22 Tests for Male Reproductive Behavior All animals were sexually naive prior to the 15—min behavioral test and were tested 2—5 hr after lights out (4—7 hr after the seventh injection of DHT). The male was placed in a 10—gal glass aquarium (51 X 26 X 31.5 cm) and allowed to acclimate for 5 min before the introduction of the receptive female. Stimulus females were bilaterally ovariectomized under methoxyflurane anesthesia and subsequently made behaviorally receptive by sequential injections of estradiol benzoate (10 pg in 0.05 ml sesame oil, so, 48 hr prior to testing) and progesterone (250 pg in 0.05 ml sesame oil, sc, 4 hr prior to testing). The behavioral tests were videotaped under dim red light illumination with a Panasonic Color Video Camera (WV 3250). Videotapes were scored to assess the amount of time spent in anogenital investigation (AGI) of the female, and the number of mounts, intromissions, and ejaculations achieved by the male. Video tapes were scored by a single experimenter who was blind to the hormonal condition of the animals. Tissue Collection Immediately after the 15 min behavioral test, animals were weighed, administered an overdose of sodium pentobarbital (130 mg/kg, ip), and perfused. Prior to perfusion, seminal vesicles were removed, seminal fluid was expressed, and the wet weight was recorded. Animals were 23 intracardially perfused with 100 ml of buffered saline rinse followed by 150 m1 of 4% paraformaldehyde. Brains were then removed and postfixed for 1 hr in 4% paraformaldehyde and then transferred to a phosphate buffered saline (PBS) solution containing 20% sucrose. Approximately 48 hr after the brains had been immersed in the sucrose solution, 40pm coronal sections were made on a cryostat and stored in cryoprotectant (Watson, Weigand, Clough, and Hoffman, 1986) at —2CPC until the AR immunocytochemistry was performed. Androgen Receptor Immunocytochemistry Every fourth brain section from each animal was processed in a single immunocytochemical run. Sections were rinsed 5 times in 0.1 M PBS to remove the cryoprotectant. Sections were then incubated sequentially in 0.1 M glycine in 0.1 M PBS (30 min), 0.3% H202 in PBS (10 min), 4% normal goat serum (NGS; Vectastain ABC Kit, Burlingame, CA) in 0.3% Triton X-100 in PBS (PBS—TX; 1 hr), 0.25 pg/ml rabbit anti-AR in PBS-TX (PG—21-18a, obtained from G. S. Prins, Michael Reese Hospital, Chicago, IL; 48 hr), secondary antibody (goat-anti—rabbit, Vectastain Elite Kit, 1:200 in PBS—TX; 24 hr), and avidin—biotin—horseradish peroxidase complex (Vectastain ABC Elite Kit, 1:50 in PBS—TX; 2 hr). For the chromogen reaction, sections were incubated for 5 min in 0.05% diaminobenzidine (DAB), 2% 500 nM NiC12 and 0.075% 24 H202. Sections were rinsed 3 times in PBS between incubations in each reagent. All incubations were at room temperature except for the primary antibody, which was at 4%: Sections were mounted on gelatin-coated slides, dried, dehydrated, cleared in xylenes, and coverslipped. To test for nonspecific binding, sections were processed as described above, but in the absence of either primary or secondary antibody. Absence of either antibody eliminated all detectable immunoreactivity. Analysis of Androgen Receptor Areal Density The areal density (cells per unit area) of AR-ir cells, referred to as the number of AR—ir cells, was determined for the MPN, MPNmag, BNSTpm, MeA, and MeP. These areas were chosen for analysis because they are the steroid—containing forebrain nuclei of the neural circuit that mediates mating behavior (Kollack—Walker and Newman, 1997; Newman, 1999; Newman et al., 1997; Wood, 1996b; Wood, 1998; Wood et al., 1992; Wood and Newman, 1995b; Wood and Newman, 1999). These nuclei were located within the respective sections by their relative position to the 3rd ventricle (e.g., MPN), fiber tracts (e.g., BNSTpm, MeA, and MeP), or in relation to each other (e.g., MPNmag is ventral to the BNSTpm). For the MPN, MeA, and MeP, bilateral counts were made in two anatomically matched sections separated by 160pm. For the BNSTpm and MPNmag, a bilateral count was made in a single section 25 anatomically matched across animals. Using bright—field microscopy, nuclei were centered in the field of View under a 10X objective, and then the magnification was increased using a 40X objective. AR—ir cell profiles that fell within the . 2 area of a square ocular grid (62,500pm ) were counted. Data . 2 are expressed as the mean number of AR—ir cells/62,500pm . Slides were coded so that the experimenter was blind to the age and treatment of the animal during microscopic analysis. Statistical Analysis All peripheral, behavioral, and central variables were analyzed by two—way ANOVAs (age X dose). Fisher’s PLSD tests and Tukey HSD tests were used to probe significant main effects and interactions, respectively. Differences were considered significant when p < 0.05. Data are presented as means : SEM. Results Peripheral and Behavioral Measures There were significant main effects of both age and DHT treatment on the weight of the androgen-sensitive seminal vesicles (Table 1). Posthoc tests indicated that seminal vesicle weight was heavier in adults compared to prepubertal males (p < 0.05), and that males treated with either the 500 or 1000 pg dose of DHT had significantly heavier seminal 26 Table 1“. IMean (i_SEM) seminal vesicle weight (mg / 100 g body weight) of prepubertal and adult males treated with seven daily injections of either 0, 500, or 1000 pg DHT. DHT (pg) Prepubertal Adult 0 15-99i1-47 62.70ill.37 500 145.28i18.52* 269.48:32.42* 1000 138.69i17.18* 246.75:14.22* *Significantly different from the 0 pg-treated controls. 27 vesicles than males of the same age treated with the oil vehicle (p < 0.05). Main effects of both age and DHT treatment were found on the length of time males spent in anogenital investigation of the receptive female (Figure 2A; both p < 0.05). Across the hormone treatment groups, adult males engaged in a greater amount of AGI than prepubertal males (p < 0.05). Across both age groups, males receiving either the 500 or 1000 pg dose of DHT engaged in more AGI than animals that received the oil vehicle (p < 0.05). Adult males engaged in a greater number of mounts compared to prepubertal males, regardless of DHT treatment (Figure 2B; p < 0.05). Figure 2C depicts the significant interaction between age and DHT treatment on the number of intromissions during the behavioral test (p < 0.05). Adult males treated with the 1000 pg dose of DHT engaged in a significantly greater number of intromissions than prepubertal males treated with the same dose of DHT (p < 0.05). The posthoc tests also revealed that the adult males treated with the 1000 pg dose of DHT engaged in a significantly greater number of intromissions than the adults treated with either the 0 or 500 pg dose of DHT (p < 0.05). There was a significant main effect of age on the number of ejaculations such that adult animals engaged in a significantly greater number of ejaculations than the 28 -o—Prepubertal . A100 +Adult m 10‘. 0 ' a 8‘ 3 8°? c 6‘ :60‘ 3 4: g 40: E 2 20 O- c :r o o d 560 1600 d 560 1600 m 24. “a c m1.2j D C 20‘ c 2 - o . , m 16: z; 1 2 12‘ £0-6 E 1 3 o 3. g b 4‘ ii? = .. 0. v fl 3 "' 04 I: - a d 560 1600 d 560 1600 Dose of DHT (pg) Dose of DHT (pg) Figure 2 . Number of seconds engaged in anogenital investigation (AGI; A) and frequencies of mounts (B) , intromissions (C) , and ejaculations (D) in a 15 min behavioral test in prepubertal and adult males given daily injections of either 0, 500, or 1000 pg of DHT for seven days . Asterisk indicates a significant diff— erence from prepubertal males . "a" indicates that adults that received 1000 pg of DHT are significantly different from the adults that received either the O or 500 pg dose of DHT. All values are means 1 SEM. 29 prepubertal animals regardless of the dose of DHT received (Figure 2D; p < 0.05). None of the prepubertal animals treated with DHT were observed to exhibit any mounting, intromissive, or ejaculatory behavior. AR-Immunoreactivity Figure 3 shows the number of AR—ir cells within the brain regions examined. Two—way ANOVAs revealed a significant main effect of DHT treatment on the number of AR— ir cells in the MPN, BNSTpm, and MeP (p < 0 05). Fisher’s PLSD posthoc tests showed that both age groups treated with either the 500 or 1000 pg dose of DHT had a significantly greater number of AR—ir cells than vehicle—treated animals (p < 0.05). ANOVAs revealed significant interactions between age and DHT treatment in both the MPNmag and MeA (both p < 0.05). The number of AR—ir cells in the MPNmag was significantly greater in prepubertal males treated with the 500 pg dose of DHT than their adult counterparts (p < 0.05). In the MeA, the number of AR—ir cells was greater in adult males treated with 1000 pg DHT compared to their prepubertal counterparts (p < 0.05). Photomicrographs in Figure 4 show AR—ir in the MPN of a prepubertal and adult male treated with 1000 pg of DHT. 30 16"]M"P"'N DPrepubertaI' ZGGJMeA IAdult . * 120‘ 160. .—. 120- 80. . 801 40- 40 NE 0 0 500 1000 160 .. 1 lMPNmag 2°" MeP O l * c . .—. 160 “'1 120 ‘ N 120 no \ so- - E 80- 8 40' 40 L 'T 0- - o _ I: o 500 1000 o 500 1000 < 1601— BNSTpm 120- 80- 4o- 0 o 500 1000 Dose of DHT (pg) Figure 3. AR-ir cells/62, 500 pm2 in the MPN, MPNmag, BNSTpm, MeA, and MeP of prepubertal and adult males treated daily for seven days with either 0, 500, or 1000 pg of DHT. Asterisks indicate a significant diff— erence between prepubertal and adult males within a dose of DHT. All values are means i SEM. 31 Figure 4. AR—ir in the MPN of a prepubertal (A) and adult (B) male treated with 1000 pg of DHT. Bar, 50pm. 32 Discussion These data demonstrate that prepubertal males are less responsive than adults to the activational effects of DHT on male reproductive behavior. These results indicate, therefore, that the lack of a behavioral response to T in prepubertal males includes a specific unresponsiveness to the androgenic component of T’s overall action. Furthermore, this experiment shows that the inability of DHT to activate mating behavior in juvenile males is not associated with a relative lack of DHT-induced ARs in most of the nuclei that comprise the neural circuit mediating male mating behavior. We previously observed a similar dissociation between sexual behavior and brain AR in which juvenile and adult males were treated with T (Meek et al., 1997). Specifically, T increased the expression of AR in the steroid-sensitive mating circuit in prepubertal males, but did not activate their sexual behavior. Thus, the present data are in agreement with this earlier study, and indicate that the presence of ARs in this neural circuit may be necessary, but not sufficient, to facilitate the display of male mating behavior. Adult males treated with 1000 pg of DHT did have a significantly higher number of AR—ir cells in the MeA compared to the prepubertal males treated with the same amount of DHT. Since 1000 pg of DHT was capable of activating intromissive behavior in the adult but not 33 juvenile males, the greater number of AR—containing cells in the adult MeA is correlated with the adult’s greater responsiveness to DHT on this behavioral measure. However, intracerebral implantation of estradiol, but not DHT, in the medial amygdala of castrated adult males activates their mating behavior, suggesting that androgenic stimulation of this area alone is not sufficient to a elicit a behavioral response (Wood, 1996a). Furthermore, T treatment induces equivalent amounts of AR—ir cells in the MeA (and elsewhere) in gonadectomized prepubertal and adult males, yet prepubertal males still do not engage in mating behavior (Meek et al., 1997). Therefore, the greater number of AR— containing cells in the MeA of adult males treated with 1000 pg of DHT is probably not responsible for their greater behavioral responsiveness to DHT. However, the greater number of ARs in the MeA of adult males could mediate neural responsiveness to other motivated behaviors that are influenced by androgens, such as aggression (Payne, 1974). It should be noted that the prepubertal males treated with the 500 pg dose of DHT had a greater number of AR— containing cells in the MPNmag compared to the adults that were treated with 500pg of DHT. The functional significance of the higher levels of AR expression in the MPNmag of androgen—treated prepubertal males is unknown. However, this finding further demonstrates the dissociation between AR levels and sexual behavior since these relatively high levels 34 of AR in the prepubertal MPNmag at this dose did not contribute to any behavioral activation in response to DHT. Unlike mounting, intromissions, and ejaculations, AGI was activated by DHT in prepubertal males, although to a lesser degree than in adults. The pheromonal stimulation received by the male during chemoinvestigatory behavior is necessary for the subsequent display of mating behavior in this species (Wood, 1998; Wood and Newman, 1995c). These results suggest that the pheromonal cues received by a DHT— treated male may be, like the presence of AR, necessary, but not sufficient, to activate the full suite of reproductive behaviors in juveniles. DHT was effective in increasing the amount of brain AR— ir and the wet weight of the androgen-responsive seminal vesicles at both ages, indicating that the prepubertal male is responsive to DHT at some level. Importantly, the similar increase in brain AR—ir in most of brain regions examined in the prepubertal and adult males suggests that animals exposed to the same dose of DHT received similar central androgenic stimulation, regardless of age. Thus, the inability of peripherally administered DHT to activate mating behavior prior to puberty cannot be fully explained by unequal exposure of the brain to DHT at the two ages. In summary, these results demonstrate that prepubertal males are less responsive than adults to the behavior— activating effects of DHT. Furthermore, the inability of DHT to activate mating behavior in juvenile males is not 35 associated with a relative lack of DHT-induced ARs in most of the nuclei that comprise the neural circuit mediating male mating behavior. Thus, the lack of mating behavior observed in androgen—treated juveniles compared to adults must be mediated, at least in part, by differences in cellular processes downstream of AR induction and activation. This conclusion does not rule out the possibility that prepubertal males are also unresponsive to the estrogenic component of T’s actions. This issue is investigated in the next set of experiments. 36 Section II. Contribution of the aromatase enzyme, estrogen, and estrogen receptors in the pubertal maturation of male sexual behavior. Experiment I. Androgenic regulation of hypothalamic aromatase activity in prepubertal and adult males. Rationale T is converted intracellularly to estradiol in peripheral and central tissues by aromatase. In the brain, estrogenic metabolites of T play a major role in the expression of male sexual behavior (reviewed in, Luttge, 1979; Meisel and Sachs, 1994). For example, systemic injections of estradiol benzoate to castrated male hamsters induce mounting behavior (DeBold and Clemens, 1978), while aromatase inhibitors decrease copulatory behaviors (Floody and Petropoulos, 1987; except see, Cooper, Clancy, Karom, Moore, and Albers, 2000). The aromatase enzyme is present in brain regions that mediate male sexual behavior, such as the amygdala and hypothalamus (Callard, Mak, and Solomon, 1986; Hutchison, Hutchison, Steimer, Steel, Powers, Walker, Herbert, and Hastings, 1991; Roselli, Horton, and Resko, 1985), and appears to be positively regulated by androgens in some regions of the hypothalamus (Abdelgadir, Resko, Ojeda, Lephart, McPhaul, and Roselli, 1994; Hutchison et al., 1991; Negri—Cesi, Celotti, and Martini, 1989; Roselli et al., 1985; Wagner and Morrell, 1996; but see, Callard et al., 1986). 37 The capacity to engage in steroid—dependent reproductive behavior increases during pubertal maturation. As mentioned in the Introduction, not only is there an increase in circulating levels of T during this time, but responsiveness of the neural circuit to the behavioral actions of T increases as well. We hypothesize that this increased behavioral responsiveness to T in adults is mediated, at least in part, by the efficacy with which T is aromatized to estradiol (E) in the hypothalamus. This hypothesis leads to two related predictions. First, in intact males, aromatase activity within the behavioral neural circuit should be greater in adults than in juveniles. Second, T treatment of castrated males should increase aromatase activity to a greater extent in adults than in juveniles, which would result in higher local concentrations of E to activate male reproductive behavior in adults. As a test of this hypothesis, aromatase activity, as measured by the conversion of 3H—T to 3H-estradiol (3H—E) , was assessed in hypothalamic homogenates obtained from intact and from castrated and T— treated adult and prepubertal male hamsters. Methods Subjects and Treatment Male hamsters were bred at Michigan State University (E. Lansing, MI) from stock obtained from Charles River (Kingston, NY). Animals were housed and cared for as described previously (Subjects and Treatment, Section I). 38 Four experiments were conducted because the number of samples that can be run in a single assay is limited. Experiment 1 characterized the amount of hypothalamic aromatase activity in untreated, intact prepubertal and adult male hamsters. In this experiment, 63- (adult, n=8) or 28— (prepubertal, n=8) day old male hamsters were weighed and rapidly decapitated. Whole hypothalami, blood samples, and testes were collected as described below. Experiments 2—4 investigated the effects of T on hypothalamic aromatase activity before and after puberty in male hamsters. Experiment 2 assessed the effects of T on aromatase activity in adult males. Adult males (60 days of age) were castrated under methoxyflurane anesthesia and implanted with a 3—week time—released pellet (Innovative Research of America, Sarasota, FL) containing either 0 mg (n=7) or 2.5 mg of T (n27). One week after castration and implantation, hamsters were weighed, rapidly decapitated, and hypothalami and blood samples were collected as described below. Previous work has shown that aromatase activity is maximally increased within a week of T treatment (Roselli, Horton, and Resko, 1987). Furthermore, the difference in T—stimulated sexual behavior observed in prepubertal and adult male hamsters is observed one week after T treatment (Meek et al., 1997). Thus, animals received one week of T exposure in Experiments 2—4. Experiment 3 assessed the effects of T on aromatase activity in juvenile males. In Experiment 3, prepubertal males (21 days of age) were castrated and implanted with either a 0 mg 39 (n=7) or 2.5 mg (n=6) pellet of T. One week after treatment, tissues were collected as in Experiment 2. Experiment 4 directly compared the effect of T on aromatase activity in juvenile and adult males. Prepubertal (21 days of age, n=6) and adult (60 days of age, n=8) males were castrated and implanted with a 2.5 mg pellet of T. One week after treatment, tissues were collected as in Experiment 2. Tissue Collection Animals were rapidly decapitated by a guillotine. Trunk blood samples were collected and centrifuged. Plasma was removed and stored at -2UT3until radioimmunoassays were performed (see below). Brains were quickly removed and the hypothalamus was dissected on a stainless steel surface on wet ice with a razor blade. Coronal cuts were made directly anterior to the optic chiasm and at the posterior end of the hypothalamus, just anterior to the mammillary bodies. Then a horizontal cut was made just ventral to the anterior commissure as it crossed the midline. Finally, the brain was placed on the dorsal surface and the optic chiasm and tissue lateral to the hypothalamus was removed. The dissected hypothalamus was then snap frozen in dry ice and stored at -JHPC until the aromatase assays were performed (see below). Assay for Steroid.Métabolizing Enzymes Individual hypothalami were homogenized in 600 pl of 250 rmM sucrose/50 mM potassium phosphate buffer. Assays were 40 conducted with minor modifications from those used in lizard brain tissue (Wade, 1997). Initially, validation assays that varied the incubation time and substrate concentration were run on adult male hamster hypothalamic homogenates to determine the appropriate assay conditions (details presented with Results). Once the assay was validated, experiments were conducted using duplicate 200 pl aliquots of hypothalamic homogenates incubated for 25 min with 250 nM substrate. The tissue homogenates were added to test tubes in which 3H—T (New England Nuclear, Boston, MA) had been dried. In all cases, substrate was repurified by thin layer chromatography (TLC) before use. Samples were incubated at IYFC with a NADH/NADPH generating system, and the reaction was terminated by freezing the tubes in a methanol/dry ice bath. Steroids were extracted from homogenates 3 times with diethyl ether. Androgens were then separated from estrogens twice by phenolic partition, and estrogens extracted 3 times with ethyl acetate. Androgenic and estrogenic products were applied to TLC plates following the addition of radioinert carrier steroids (Steraloids, Wilton, N. H.). TLC plates containing estrogens were run twice in ether:hexane (3:1), and the products visualized by exposure to iodine vapors. Plates containing androgens were run twice in chloroformzethyl acetate (4:1), and the products were Visualized under ultraviolet irradiation following a primulin spray. Regions containing the steroids of interest were 41 scraped from the plates, and after the addition of 400 pl H41 steroids were eluted from the silica—gel in 2 ml methanol. A fraction of the eluate was mixed with Bio—safe cocktail II (Research Products International, Santa Cruz, CA) and counted in a Beckman liquid scintillation counter (LS 6500). Each sample was corrected for counter efficiency, volume, and background counts in tubes incubated with buffer and cofactors but no tissue. Results were also corrected for recovery efficiency, which was determined by the addition of a known quantity of 3H—E or 3H—T (approximately 150,000 dpm) to tubes processed in parallel. Protein content in each assay tube was determined with the method of Bradford (Bradford, 1976; Bio—Rad kit, Hercules, CA) using bovine serum albumin (BSA) as the protein standard. To confirm their authenticity, samples of all steroid products were recrystallized with radioinert steroids (Steraloids, Wilton, N. H.) to constant specific activity using ethanol and water (details presented with Results). Testosterone Radioimmunoassay Plasma concentrations of T were measured in two different assays using the Coat—A—Count Total Testosterone Kit (Diagnostic Products, Los Angles, CA). This assay has been validated in our laboratory for the measurement of plasma T concentration in the Syrian hamster. The lower limit of detectability of both assays was 0.1 ng/ml. The 42 intraassay coefficients of variation (CV) were 5.8% and 4.1%, and the interassay CV was 10%. Statistical Analysis The data for each experiment were analyzed using two— tailed t tests. Differences were considered significant when p < 0 05. All data are reported as mean : SEM. Values for the Km and Vfia were generated from a Lineweaver-Burk plot X using a regression line in Statview 4.1 (Abacus Concepts, Inc., Berkeley, Ca). Results validation of aromatase assay Samples of E were twice verified by recrystallization to constant specific activity in ethanol and water (93 6/85 8, 77.0/77 7; crystals. E 10. U c 50 o .g-a 3 0 0 20 E 500 U) .5 15. l g 400 g E 300- -- 10- o g :200- I h 5- g E a, 100- iii 0 _I 0 3 2 900 m g l C 3 1 .g 2- I _g 500- I E '-'-' - a 2 - IE 1‘ a. 300- I.” c ‘ 2 . G 3 6 Treatment Treatment Figure 16. The frequencies and latencies (sec) of mounts, intromissions, and ejaculations in adult males treated with RU486 (2 mg/kg) or the oil vehicle. Asterisk indicates a significant difference. All values are means i SEM. 87 20 16J I .‘L’ c 12. 3 § 8‘ 4. 0 30 m c l g 20- / I .2 y E g 10- H E / 0 - fl ‘" l C .2 2- H 2 3 .‘E. 1‘ LIJ 0 Treatment Latency to Mount Latency to Eiaculate Latency to Intromit 250 200: 1 50- 1 00' 50' Cl Oil a ZK98299 §\81 400 300' 200* 100- 600 400- 200- .\\\ .\A_ a . a Treatment Figure 17. The frequencies and latencies (sec mounts, intromissions, and ejaculations in adult males treated with ZK98299 (6 mg/kg) or the oil vehicle. Asterisk indicates a significant difference. All values are means 1 SEM. 88 v of significantly affected by RU486 or ZK98299, all other behavioral measures showed trends such that RU486- or ZK98299—treated males performed more poorly than their oil— treated controls. These results are in agreement with previous studies that have shown that the PR plays an important role in the reproductive behavior of adult male rats, mice, and lizards (Crews et al., 1996; Lindzey and Crews, 1988; Lindzey and Crews, 1992; Phelps et al., 1998; Witt et al., 1994; Witt et al., 1995; Young et al., 1991). However, the behavioral effects of blocking PR in the hamster are not as robust as those seen in the other species mentioned above. It is possible that higher doses of RU486 or ZK98299 or a different time course would cause a greater deficit in male mating behavior, as seen in other species. In hamsters, however, doubling the dose of RU486 does not decrease their behavior below that observed in the present experiment (Romeo, unpublished observation). Unfortunately, due to limited availability of ZK98299, only one dose of the antagonist could be tested in the present study. We can not rule out the possibility that ZK98299, although not acting through the hormone binding domain, may still not completely block PR activation in the hamster. However, ZK98299 has been shown to inhibit progesterone— stimulated fibronectin secretion from chicken granulosa cells (Asem and Conkright, 1995; Conkright and Asem, 1995; but see, Moudgil, Nath, Bhakta, and Nakao, 1991). Since chickens, like hamsters, share the single amino acid switch in the 89 hormone binding domain (Benhamou et al., 1992), these studies together with the present data suggest that this PR antagonist is capable of at least partially blocking PR activation in animals that have the cysteine substitution in the hormone binding domain. Paradoxically, RU486 was able to significantly block mounting behavior in adult male hamsters even though RU486 has been reported to be a poor PR antagonist in hamsters (Gray and Leavitt, 1987; Okulicz, 1987). These studies used cytosolic uterine PR in female hamsters as the substrate for RU486, and the possibility remains that male and female and/or brain and uterine PR are subtly different. It should be noted that RU486 administered to hormonally—primed estrous female hamsters does not block lordosis (Romeo, unpublished observation), a female receptive posture mediated by PR activation (Mani, Blaustein, Allen, Law, O'Malley, and Clark, 1994b; Moguilewsky and Raynaud, 1979; Ogawa, Olazabal, Parhar, and Pfaff, 1994; Parsons, MacLusky, Krey, Pfaff, and McEwen, 1980). RU486 does bind to and antagonize the glucocorticoid receptor (GR; Moguilewsky and Philibert, 1985), albeit with half of the relative binding affinity RU-486 has for the PR. In contrast, ZK98299 binds the GR with orders of magnitude less affinity than RU486 (Koper, Molijn, van Uffelen, Stigter, and Lamberts, 1997). Thus, it is possible that the significant reduction in mounting behavior of RU486-treated males may be through RU486’s interactions with the GR, while ZK98299’s effects may be more specifically through the PR. Taken together, 90 although RU486 was effective in reducing mounting behavior in male hamsters, it is not presently understood through what mechanism this effect is mediated. In summary, these data suggest that PR activation contributes to the display of male mating behavior in adult hamsters. Thus, it is possible that the lack of reproductive behavior exhibited by prepubertal males may be, at least in part, the result of inadequate PR stimulation, as the previous experiment demonstrated significantly lower levels of circulating progesterone in juvenile compared to adult males. Experiment III. The sexual behavior of prepubertal males treated with estrogen and progesterone. Rationale We have shown PR upregulation in the MPN is similar in estrogen—treated prepubertal and adult males, suggesting that the ER is functional prior to puberty. It should be noted that T is also capable of upregulating the expression of PR in the MPN of prepubertal males (Romeo, unpublished observation), most likely through T's conversion to estradiol. It has been demonstrated that estrogen-treated prepubertal males engaged in less mating behavior and have significantly lower circulating levels of progesterone compared to estrogen—treated adults. This developmental difference in plasma progesterone levels was not affected by 91 estrogen treatment. Since PR activation appears to contribute to the facilitation of male mating behavior in adults, it is possible that the lack of reproductive behavior in prepubertal males is mediated by the relatively low levels of progesterone at this developmental stage, and hence, reduced PR activation. The present set of experiments had two objectives. First, we needed to established what dose of exogenous progesterone given to juvenile males would provide circulating adult-like levels of progesterone. The second was to tested the hypothesis that adult—like levels of progesterone will facilitate the display of mating behavior in estrogen—treated prepubertal males. Methods Subjects and Treatment Prepubertal male hamsters used in these experiments were obtained from Charles River (Kingston, NY). Animals arrived at 18 days of age with their mothers. At 21 days of age, all animals were weaned from their mothers, weighed, and treated as described below. Animals were housed and cared for as described previously (Subjects and Treatment, Section I). In Experiment 1, animals were castrated under isoflurane anesthesia and implanted with a pellet containing either 0, 0.25, 0.50, 1.5, 2.5, 5.0, 7.5, 10, 15, 25, or 35 mg of progesterone (n=4-7). One week after castration and implantation (i.e., at 28 days of age), animals were weighed 92 and trunk blood samples were collected. Plasma samples were stored at —20%2Luujl.the progesterone assay was performed (see below). Plasma samples from sexually behaving adult males that had been castrated and treated with a pellet containing 0.05 mg of EB were run in the same progesterone assay as the samples obtained from the progesterone—treated prepubertal males. These samples were included in the assay to establish what dose of progesterone provides a prepubertal male with progesterone levels similar to adult males, which have equivalent levels of PR in the MPN as estrogen—treated juvenile males, but engage in greater amounts of mating behavior than estrogen—treated juvenile males. In Experiment 2, animals were castrated under isoflurane anesthesia and implanted with a pellet containing 0.05 mg of EB. This dose of EB was chosen since it was the most effective dose at activating reproductive behavior in adult males (Section II, Experiment 2) and has been shown to induce similar levels of PR—ir in the MPN of prepubertal and adult males (Section III, Experiment 1). In addition to the EB pellet, half the animals received a pellet containing either 15 mg of progesterone or a blank pellet (n=6). This dose of progesterone was chosen based on the results obtained in Experiment 1 (see Results below). One week after castration and implantation (i e., at 28 days of age) all animals were given a 15—min mating behavior test two to four hr into their dark cycle with a hormone primed estrous female as described in Section I (Tests for Male Reproductive Behavior). 93 Progesterone Radioimmunoassay Plasma concentrations of progesterone were measured using the Coat—A-Count Progesterone Kit (Diagnostic Products). The lower limit of detectability of the assay was 0.06 ng/ml. The intraassay CV was 10%. Statistical Analysis In Experiment 1, plasma levels of progesterone were analyzed by a one-way ANOVA and significant differences were probed with Fisher’s PLSD. In Experiment 2, all behavioral measures were analyzed by t tests. Differences were considered significant when p < 0.05. Results EXperiment 1: Plasma progesterone levels in progesterone— treated prepubertal males The one—way ANOVA revealed a significant effect of progesterone treatment on plasma progesterone levels (Figure 18). Posthoc testes revealed that plasma progesterone levels were not significantly elevated above that of blank—treated controls until animals received either the 10, 15, 25, or 35 mg pellet of progesterone. More importantly, these data show that the 15 mg pellet results in plasma progesterone concentrations that most closely resemble the circulating levels of progesterone in sexually behaving adult castrates treated with 0.05 mg of EB (Figure 18). 94 40 r: DPrepubertal d E ‘ ISexually Behaving Adult 2» 30- l v c (5) '3' c = l 2 2°" (4) a.) b,c (5) :5, (5) g 10. a a a aib 3%” 3;” a; P.-. '6) (I) (I) (”J“) ‘4’ H G 0 025050125 2:5 510 715 1'0 1'5 2'5 3'5 0.05 Dose of Progesterone (mg) E (mg) Figure 18. Plasma progesterone (ng/ml) in castrated prepubertal males treated with either a 0 , 0 . 25, 0 . 50, 1.5, 2.5, 5.0, 7.5, 10, 15, 25, or 35 mg pellet of pro- gesterone for one week. Bars that share a letter are not significantly different from each other. Numbers in the parentheses are the number of animals that comprise that mean. The gray bar represents the average plasma progesterone level in a sexually behaving castrated adult male implanted with a 0 . 05 mg pellet of estradiol (E) . All values are means i SEM. 95 Experiment 2: Mating behavior of estrogen— and progesterone— treated prepubertal males There were no significant differences in any aspect of mating behavior measured. That is, prepubertal males treated with EB and progesterone engaged in equivalent levels of AGI and mounting as prepubertal males treated with EB only (Table 4). Neither group of males engaged in any intromissive or ejaculatory behavior. There was also no significant difference between either group of males in their latency to mount (data not shown). Discussion Taken together, these data demonstrated that mating behavior is not activated in prepubertal males treated with EB and supplemented with progesterone. Thus, even when PR and plasma progesterone levels are equated between prepubertal and adult males, prepubertal males still engage in relatively little reproductive behavior. It appears, therefore, that activation of the PR by progesterone is not the rate limiting factor in the pubertal maturation of male reproductive behavior. It is interesting to note that progesterone is not the only ligand to activate the PR. Dopamine and LHRH have been implicated in the activation of the PR (Mani, Allen, Clark, Blaustein, and O’Malley, 1994a, and Beyer et al., 1997, respectively) and facilitation of sexual behavior in adults (Bitran and Hull, 1987; Mani et al., 1994a; Melis and 96 TABLE 4. Mean (i SEM) duration of AGI and the frequency of mounts. Age (days)— AGI (sec) Mounts treatment 28—EB+Blank , 44.5:7.2 0.3:0.2 28-EB+Progesterone 41-3i4 9 0-7i0 4 97 Argiolas, 1995, and Beyer et al., 1997; Fernandez-Fewell and Meredith, 1995, respectively). Thus, it is possible that the ligand—independent activation of PR, either by dopamine or LHRH, may contribute to the pubertal maturation of male sexual behavior. Experiments to address these questions are currently in progress. Summary Experiment I of this section showed that prepubertal males treated with EB have an equivalent increase in PR expression in the MPN as EB-treated adults. Since one action of an activated ER is to increase transcription of the PR gene, these data suggest that the ER is functional prior to puberty. Also in Experiment I, it was shown that adult males have higher circulating levels of progesterone compared to prepubertal males, regardless of EB treatment. As demonstrated in Experiment II, activation of the PR contributes to the display of sexual behavior in the adult male Syrian hamster. Thus, it was reasoned that although estrogen treatment of prepubertal males upregulates their PR expression in the MPN to a similar level as adults, these PRs in the juvenile’s hypothalamus may not be activated because of the relatively low levels of circulating progesterone at this age. However, as was shown in Experiment III, even when PR and plasma progesterone levels are equated between prepubertal and adult males, prepubertal males still do not engage in mating behavior. In conclusion, it appears that 98 activation of the PR by progesterone is not the rate limiting factor in the pubertal maturation of male reproductive behavior. 99 Section IV. Contribution of chemosensory cues in the pubertal maturation of male sexual behavior. In Experiments in Sections I, II, and III, we have demonstrated that prepubertal males are unresponsive to the behaviorally activating effects of both the androgenic and estrogenic components of T, and progesterone. Furthermore, the unresponsiveness to these steroid hormones is not due to a lack of their respective receptors within the neural circuit that mediates sexual behavior. Finally, the ARs and ERas in the mating circuit appear to be functional. Taken together, it appears that the lack of mating behavior exhibited by prepubertal males must be mediated by processes other than the availability of these steroids, the absence of their receptors, or the receptors’ functionality. Thus, this last research chapter will focus on the role that pheromones may play in mediating the pubertal maturation of male mating behavior. Rationale As mentioned in the Introduction, full expression of male reproductive behavior in the Syrian hamster is dependent on both pheromonal cues from the female and the presence of gonadal steroids (Meisel and Sachs, 1994; Wood and Newman, 1995b). The pheromones are contained within female hamster vaginal secretions (VS) and stimulate the male vomeronasal System (Fernandez—Fewell and Meredith, 1994), leading to 100 increased AGI and mounting by the male (Darby, Devor, and Chorover, 1975). The male’s interest in VS is increased by the presence of circulating androgens (Powers and Bergondy, 1983). We have demonstrated that chemoinvestigation of the female is stimulated in castrated adult and prepubertal males treated with T, DHT, or E. However, the steroid-treated adults display more mounts and intromissions than the prepubertal males. These results suggest that, despite similar interest in the female, steroid—treated adult and juvenile males may process the chemosensory information from the female differently. Adult male ferrets (Wersinger and Baum, 1997), hamsters (Fernandez-Fewell and Meredith, 1994; Fiber et al., 1993; Fiber and Swann, 1996; Kollack—Walker and Newman, 1997; Swann and Fiber, 1997), and rats (Bakker, Baum, and Slob, 1996; Bressler and Baum, 1996) respond to chemosensory cues from an estrous female with increased expression of the immediate— early gene product Fos within various forebrain nuclei, which is indicative of increased neuronal activity in these areas (Morgan and Curran, 1991). When adult male hamsters are exposed to VS, cells within subdivisions of the MeAMY, BNST, and MPN express Fos (Fernandez—Fewell and Meredith, 1994; Fiber et al., 1993; Fiber and Swann, 1996; Kollack—Walker and Newman, 1997; Swann and Fiber, 1997). These data suggest that VS causes an increase in neuronal activity in brain regions that mediate chemosensory processing and male sexual behavior in the adult male hamster. In the present 101 experiment, we investigated the influence of sexual maturity on Fos production in response to VS in the male hamster. We tested the hypothesis that the different amount of reproductive behavior observed in prepubertal and adult males is the result of a pubertal change in the processing of chemosensory information, which leads to a greater degree of VS—induced neuronal activation within the behavioral circuit in adults compared to juveniles. This hypothesis predicted that the Fos response to VS will be greater in adults than in juvenile males. Methods Subjects and Treatment Twelve weanling (21 days of age) and twelve adult (80 days of age) male hamsters were obtained from Charles River (Kingston, NY). All animals were sexually naive, and housed and cared for as described previously (Subjects and Treatment, Section I), except that the Vivarium light—dark cycle was 14 hr light/10 hr dark (lights on at 0600 hr EST). After a one week acclimation period, half of the animals in each age group were given either a clean cotton swab or a cotton swab containing VS. Thus, there were four treatment groups (n=6): (i) 28—day—old prepubertal males exposed to a clean swab, (ii) 28—day-old prepubertal males exposed to a VS swab, (iii) 87—day—old adult males exposed to a clean swab, and (iv) 87—day—old adult males exposed to a VS swab. The VS was collected onto the swab immediately before the test from 102 naturally cycling females on the day of estrus. The swabs were given to all animals in their home cage in the light phase (between 1300 and 1700 hr EST) of their light-dark schedule. All animals (control and VS) were observed to place the cotton swab in their cheek pouch. Therefore, the only difference between the control and VS-exposed animals is that the animals receiving the swab with VS presumably were able to deliver VS to their vomeronasal organ. Tissue Collection One hour after the introduction of the swab into the home cage, animals were perfused and brains were collected and sectioned as described in Section I (Tissue Collection). Blood samples were obtained Via cardiac puncture prior to the perfusion. Fos Immunocytochemistry Every fourth section from each brain was processed simultaneously during a single immunocytochemical procedure. Free—floating sections were washed 3 times for 5 min in TBS, aumd incubated in rabbit anti c-fos (diluted 1:1000 in TBS vwith 0.25% TX; a polyclonal antibody raised in rabbit against antino acids 3—16 of c-fos p62 of human origin; Santa Cruz, 1C”: #8245, Santa Cruz, CA) for 48 hr at 4%:. Subsequently, Secrtions were incubated in biotinylated goat anti—rabbit IgG kflijluted in 1:1000 in TBS with 0.25% TX; Vector) and avidin— biCHZin horseradish peroxidase complex (Vectastain ABC Kit). 103 each for 1 hr at room temperature. Horseradish peroxidase was visualized with a 0.0125% DAB solution containing 0.06% hydrogen peroxide with 0.015% nickel chloride in TBS for 5 min. Sections were mounted on gelatin—coated slides, dried, dehydrated, cleared in xylenes and coverslipped. To test for nonspecific staining, brain sections were processed as described above following omission of primary antiserum and/or secondary antiserum. Omission of the primary and/or secondary antiserum eliminated all detectable Fos— immunoreactivity. Analysis of Fos Areal Density The number of immunoreactive cell profiles per unit area (areal density), referred to as number of Fos—immunoreactive (Fos-ir) cells, was determined in an identical manner and for the same brain regions described in Section I (Analysis of Androgen Receptor Areal Density). However, in addition to the mating circuit, the lateral septum (LSept) was analyzed as a control nucleus to demonstrate brain region specificity for the Fos response to VS, since this nucleus does not respond to pheromones with an increase in Fos expression (Kollack-Walker and Newman, 1997). Testosterone Radioimmunoassay Plasma T concentrations were measured using the Coat-A— Count Total Testosterone Kit (Diagnostic Products). The lower 104 limit of detectability of the assay was 0.08 ng/ml. The intraassay CV was 7.7%. Statistical Analysis Two—way ANOVAs (age X treatment) were used to analyze all data. Significant main effects were probed using Fisher’s PLSD tests. Differences were considered significant when p < 0.05. All data are presented as mean i SEM. Results Peripheral.Measures Regardless of treatment condition, adult males had significantly heavier seminal vesicles and testes (both p < 0.05, Table 5), and significantly higher plasma T concentrations (p < 0.05, Figure 19) compared to the prepubertal animals. An interaction between age and treatment on plasma T concentrations approached significance (p = 0.06), such that the adult males exposed to VS tended to show an elevation in T secretion while prepubertal animals exposed to VS did not. Indeed, when t tests were conducted within the two ages, VS-exposed adults had significantly higher plasma T concentrations than the adults exposed to a clean cotton swab (p < 0.05), while exposure to VS did not significantly alter plasma T concentrations of prepubertal animals (Figure 19). 105 TABLE 5. DMfifll (i SEM) seminal vesicle and paired testis weight. ‘ F 9. Age (days)- Seminal vesicles (g) Paired Testes (g) i condition i 28—Control 0.078 : 0.004 1.035 1 0.066 r 28—VS 0.058 i 0.007 0.869 : 0.061 87—Control 0.239 i 0.015 2.105 i 0.214 87-VS 0.284 i 0.025 2.293 i 0.141 106 3.5 c , Control E 3.0- I VS 3», . 5 2.5- * ,5. . c 2.01 9 o 1.5- 4- . to O 1.0‘ 4-0 3 0 5‘ I: ' - 0 Prepubertal Adult Figure 19 . Plasma testosterone concentrations in prepubertal and adult males exposed to VS or a clean cotton swab. Asterisk indicates adult males exposed to VS had significantly higher circulating levels of testosterone than the adults exposed to a clean cotton swab (t tests) . All values are means : SEM. 107 Fos—Immunoreactivity There was a significant main effect of treatment on the number of Fos—ir cells in the BNSTpm, MPNmag, MPN, and MeP (all p < 0.05, Figure 20), but no effect of age and no interaction in these areas. Specifically, there was a greater number of Fos—ir cells in the BNSTpm, MPNmag, MPN, and MeP in animals that were exposed to VS compared with those that were exposed to a clean cotton swab. The greatest increase in Fos—ir cells was in the BNSTpm where VS exposure led to approximately a three—fold increase in the density of Fos-ir cells (Figure 21A—D). In the MPNmag, MPN, and MeP, VS exposure led to approximately a 1.5- to 2—fold increase in Fos-ir cells. There was no effect of age or treatment on the number of Fos-ir cells in either the MeA or the LSept of prepubertal and adult males (Figure 20). Discussion Compared to animals exposed to a clean cotton swab, both prepubertal and adult males exposed to VS have a greater amount of Fos-immunoreactivity in the BNSTpm, MPNmag, MPN, and MeP, all of which are essential for chemosensory processing and male sexual behavior (Wood, 1998; Wood and Newman, 1995b; Wood and Newman, 1995c). Furthermore, the Fos response to VS is equivalent in the two age groups, which suggests that VS results in comparable neuronal activation in juvenile and adult males. Importantly, not all nuclei examined showed an increase in Fos—immunoreactivity in 108 2 NW 50 40: 30- 20- Fos-ir cells / 62,500 pm 50- 40 30 20' Figure 20 . means i SEM. 10(- W Control VS MPNmag 10: l I 0. ,, BNSTpm 50 Me 50 40: 30: 20' 10- 50 40: 30 20 1 0 0 Control LSept VS Fos—ir cells / 62,500 pm2 in the MPN, MPNmag, BNSTpm, MeA, MeP, and LSept of prepubertal and adult males exposed to VS or a clean cotton swab. Asterisks indicate that animals exposed to VS had significantly greater numbers of Fos-ir cells than animals exposed to a clean cotton swab. All values are 109 Figure 21. Photomicrographs of Fos—ir cells in the BNSTpm of a prepubertal (A) and adult (B) male exposed to a clean cotton swab and a prepubertal (C) and adult (D) male exposed to VS. Arrowheads are outlining the approximate area of the nucleus that was analyzed. f, fornix; Bar, 100pm. 110 response to VS exposure (e.g., LSept & MeA), indicating that the stimulation provided by VS is not merely general activation of the brain in response to a novel stimulus. The induction of Fos—ir by exposure to VS in the BNSTpm, MPNmag, and MeP of adult male hamsters in the present experiment is in general agreement with much of the previously published literature (Fernandez-Fewell and Meredith, 1994; Fiber et al., 1993; Fiber and Swann, 1996; Kollack-Walker and Newman, 1997; Swann and Fiber, 1997). The observed increase in Fos—immunoreactivity in MPN of VS— exposed animals has not been consistently found in the other studies mentioned above, but in each case there was a trend toward greater Fos expression in the MPN after exposure to VS (except in Swann and Fiber (1997) in which the MPN was not analyzed). It is possible that different experimental conditions, such as previous sexual experience or time of day of testing, could account for cross—experiment differences in Fos expression in the MPN. It should be noted that other investigations of the Fos response to chemosensory cues from an estrous female found a lack of a response in castrated males (Bressler and Baum, 1996; Fiber and Swann, 1996; Paredes, Lopez, and Baum, 1998). Thus, the relatively low, but detectable, levels of circulating T observed in the prepubertal male hamster must be sufficient to allow a Fos response to occur. Although it has been shown that nestling (7-14 days of age) and prepubertal (28-40 days of age) male hamsters are 111 attracted to VS (Johnston and Coplin, 1979), this is the first report to our knowledge that shows prepubertal males exposed to VS respond with equivalent levels of Fos expression in the same brain regions as adult male hamsters. However, this does not exclude the possibility that the pheromonal cues provided by an estrous female are interpreted differently by adult and juvenile males. For instance, Johnston and Coplin (1979) suggest that VS might act as a stimulus to facilitate nest location by pups. However, once sexual maturation is achieved these cues may become exclusively a sexual stimulus. It would be interesting to investigate the pattern of neuronal activation in response to VS in the brain of male pups and/or weanlings to see if they are similar to the patterns observed in prepubertal and adult males in the present study. Nonetheless, even if these pheromonal cues are being interpreted differently, the brain regions examined in the present study appear to be as responsive in the prepubertal as in the adult male to the pheromonal stimulation provided by VS. Intact adult male hamsters spend almost twice as much time investigating the anogenital region of a receptive female than prepubertal males (Meek et al., 1997). Therefore, the vomeronasal system of adults may be provided with greater amounts of pheromonal stimulation in a naturally occurring behavioral encounter, resulting in the pubertal increase in mating behavior observed in the adult male. However, Meek et al. (1997) also reported that when T 112 implants equated the circulating levels of hormone in castrated juvenile and adult males, AGI was activated to the same degree at both ages, presumably providing equal amounts of pheromonal stimulation in both juvenile and adult animals. Furthermore, we have shown that DHT and E can stimulate AGI in prepubertal males above that of age—matched controls. Yet, mounting and intromissions were activated by steroid in the adult group only. The present data suggest that this inability of steroid—treated prepubertal males to engage in the appetitive aspects of mating behavior is not due to an insensitivity of the juvenile brain to chemosensory cues. Therefore, this would suggest that some cellular process downstream of these events is functional in the adult but immature in the prepubertal animal, leading to the differential amounts of reproductive behavior exhibited by the animals at these two developmental stages. For instance, the chemosensory information may not be integrated properly in the prepubertal brain, or other neural events that are required contemporaneously with neuronal activation by pheromones may not be completely developed in the prepubertal animal. It is interesting to note that T-treated female rats (Bressler and Baum, 1996; Paredes et al., 1998) and hamsters (Fiber and Swann, 1996) have a Fos response to estrous female odor cues similar to that observed in the T—treated males. However, the full suite of male sexual behaviors are not exhibited by these females. Hence, the prepubertal male and adult female brain may have similar neural characteristics 113 that do not permit chemosensory facilitation of a behavioral response. The significant increases in circulating levels of T in adult animals exposed to VS in the present experiment are also in agreement with the existing literature (Macrides, Bartke, Fernandez, and D'Angelo, 1974; Pfeiffer and Johnston, 1992), and a recent study in our laboratory that described the time—course of the endocrine response to VS in prepubertal and adult males (Parfitt, Thompson, Richardson, Romeo, and Sisk, 1999). Our data suggest (Parfitt et al., 1999 and present study) that adults exposed to VS respond with a greater amount of T secretion compared to the VS- exposed juveniles. The ability to initiate a transient increase in T secretion may facilitate the greater amount of sexual behavior observed in adult male hamsters compared to juvenile males. In female rats, hormonal treatments that result in episodic increases in estrogen prior to a behavioral test lead to a greater amount of lordosis compared with treatments that provide constant levels of estrogen (Kow and Pfaff, 1975; SOdersten, 1985). Therefore, the transient increase in T secretion in response to pheromonal stimulation, which in the present experiment was more pronounced in adults, may facilitate sexual behavior in animals experiencing this acute change in steroid hormone secretion. In summary, we have found that in response to VS stimulation, prepubertal and adult males respond with 114 equivalent levels of Fos—immunoreactivity in brain regions that are imperative for chemosensory processing and male sexual behavior. Therefore, it appears that the inability of the prepubertal male to perform the full repertoire of male reproductive behaviors is not due to a lack of a neuronal activity in response to the pheromonal cues present in VS. 115 Section V. Integration and Conclusions I have demonstrated that prepubertal males are unresponsive to the behaviorally activating effects of both the androgenic and estrogenic components of T. Furthermore, the absence of a behavioral response to these steroids is not due to a lack of ARs or ERas within the neural circuit that mediates sexual behavior. These receptors appear to be functional prior to puberty, since T and estrogen treatment causes upregulation of hypothalamic aromatase activity and PR expression, respectively, in prepubertal and adult males. I have also shown that blocking PR reduced mating behavior in adult males, but activation of the PR in EB-treated prepubertal males with adult—like levels of progesterone did not facilitate their ability to engage in sexual behavior. Taken together, we have demonstrated that the lack of mating behavior exhibited by prepubertal males must be mediated by processes other than the availability of these steroids, the absence of their receptors, or the receptors’ functionality. It is possible that the greater behavioral response to steroids in adult compared to prepubertal males is related to differences in their hormonal histories. That is, in all the studies we conducted, prepubertal males experienced extremely low levels of gonadal steroids prior to treatment, whereas adults had experienced 2—3 weeks of increasing testosterone prior to treatment. Male reproductive behaviors are more readily evoked in gonadectomized adults when steroid 116 treatment is begun sooner, rather than later, after castration (Hamburger-Bar and Rigter, 1977; Meisel and Sachs, 1994; Olsen and Whalen, 1984). This difference in responsiveness to hormones is presumably because the proteins, neural connections, and other conditions necessary for mating behavior need only be maintained when steroids are replaced at the time of gonadectomy, whereas they need to be restored when steroids are replaced long after gonadectomy. However, “restoration” versus “maintenance” is unlikely to explain the absence of reproductive behaviors in steroid— treated prepubertal males compared to adults, because even adult males that have not experienced gonadal hormones for several weeks still show some behaviors after 7—14 days of steroid replacement (Hamburger—Bar and Rigter, 1977; McGinnis, Mirth, Zebrowski, and Dreifuss, 1989; Olsen and Whalen, 1984; Valenstein and Young, 1955). In addition to the studies investigating steroid hormones and their receptors, we have shown thatjuveniles are able to detect pheromones in an estrous female’s VS, and that the mating circuit is similarly activated in prepubertal and adult males in response to these chemosensory cues. Thus, it appears from the data summarized above that the striking differences in mating behavior exhibited by prepubertal and adult males are not reflected by equally impressive differences in AR, ERa, or estrogen-induced PR levels, or basic chemosensory processing. 117 If these parameters are not responsible for the lack of mating behavior observed prior to puberty, the question still stands as to what mediates the pubertal maturation of male sexual behavior. The distinct possibility remains that other areas of the central and peripheral nervous systems integral for successful reproduction to occur, such as the midbrain tegmentum (Brackett and Edwards, 1984; Eibergen and Caggiula, 1973), nuclei in the lumbar spinal cord (Breedlove, 1984), pelvic ganglion (Keast and Saunders, 1998), and penile musculature (Sachs, 1982), have not yet fully developed in the prepubertal male to allow the proper motor responses to be coordinated and displayed. Interestingly, pubertal development has been implicated in the morphological and phenotypical alteration of neurons in the spinal nucleus of the bulbocavernosus (Goldstein, Kurz, and Sengelaub, 1990)and pelvic ganglion (Keast and Saunders, 1998). Although these areas do not integrate the steroidal and pheromonal information necessary for mating behavior to emerge, the contribution of these structures to the pubertal maturation of male reproductive behavior warrants continued investigation. In addition to steroids, various neurotransmitters and neuropeptides are capable of activating and modulating mating behavior in adult males (reviewed in, Bitran and Hull, 1987; Meisel and Sachs, 1994). For example, dopamine and LHRH have been implicated in the facilitation of male sexual behavior in adults (Bitran and Hull, 1987; Mani et al., 1994a; Melis 118 and Argiolas, 1995, and Beyer et al., 1997; Fernandez—Fewell and Meredith, 1995, respectively). Thus, it is possible that factors such as these are not functioning in an effective manner prior to puberty, which in turn may underlie the lack of mating behavior in prepubertal males. Studies investigating these possibilities are currently being pursued in our laboratory. The assumption that the absence of reproductive behavior in prepubertal males is mediated by the lack of a particular factor(s) may be incorrect. Instead, the possibility exists that too much of some factor(s) is inhibiting the juvenile male from engaging in copulation. For example, serotonin is generally thought to inhibit male sexual behavior (Ahlenius, Larsson, and Svensson, 1980; Baum and Starr, 1980). Hence, the absence of mating behavior observed in juvenile males may be due to a greater serotonergic input in prepubertal compared to adult males in key areas of the brain that mediate reproduction. It would be interesting to test whether a serotonergic antagonist could facilitate reproductive behavior prior to puberty. In addition to the possibilities mentioned above, the role of chemosensory processing in pubertal males must be considered. Even though the prepubertal and adult mating circuits show similar activation in response to female pheromones, we have found that adults are dissimilar to prepubertal males in that adults experience a rise in T after pheromonal exposure, which is not observed in juvenile males. 119 These findings have been replicated and extended in a recent experiment that characterized the levels of T and LH at various time points after prepubertal and adult males were exposed to VS (Parfitt et al., 1999). This study demonstrated that 60—min after exposure to VS, adult males had significantly elevated T levels and an increase in LH secretion compared to VS-exposed prepubertal males. Furthermore, this study showed that LH levels do not increase ‘ -1. Dir-A1 appreciably in juvenile males in response to VS at any time point tested. Taken together, it appears that activation of the hypothalamic—pituitary-gonadal (HPG) axis by pheromones is greater in adult compared to juvenile males. The lack of HPG activation in response to pheromones prior to puberty indicates that prepubertal males are integrating chemosensory cues differently than adult males. Since proper integration of chemosensory cues are an integral neural event for the full suite of mating behaviors to be displayed, the absence of a neuroendocrine reflex in the juvenile may be an important mediator of the lack of reproductive behavior observed at this age. As alluded to in the Introduction, we hope to obtain a deeper understanding of how internal and external cues interact to affect the physiology and behavior of an individual progressing through puberty. The different endocrine response to chemosensory cues exhibited by prepubertal and adult males provides an interesting possibility of how the interaction of these cues may change 120 during pubertal development. Specifically, adult male rats and hamsters respond to the scent of an estrous female with a increase in dopamine secretion in the MPN (Hull, Du, Lorrain, and Matuszewich, 1995; Schulz, unpublished observation). It is not known if prepubertal males respond to an estrous female with a central release of dopamine. It is possible that, similar to the lack of HPG activation, prepubertal males may not respond to pheromones with an elevation in dopamine secretion in the MPN. If this were the case, then a potential scenario would be that increased steroid production during pubertal development primes the MPN with an increase in PR expression. Pheromonal stimulation received by males would lead to a significant increase in dopamine secretion in the MPN of adults only. Since dopamine can activate PRs through a ligand-independent mechanism (Mani et al., 1994a), these activated PRs could then facilitate copulatory behaviors necessary for successful reproduction. This possibility illustrates how dopamine secreted from the MPN in response to an estrous female may be an event where the internal (steroids) and external (chemosensory) cues converge and interact to facilitate reproductive behavior during pubertal maturation. One of our goals in studying the pubertal maturation of mating behavior is to gain a better understanding of how puberty impacts the development of the central nervous system in general. Perinatal development is often viewed as the major window of time for the organization of neural circuits 121 by steroids (MacLusky and Naftolin, 1981). These early organizational effects of steroids on the nervous system supposedly determine the behavioral potentials of an organism in adulthood (Becu—Villalobos, Iglesias, Diaz—Torga, Hockl, and Libertum, 1997; Phoenix, Goy, Gerall, and Young, 1959). However, the perinatal period is probably not the only period of development during which the sensitivity of the central nervous system to steroid hormones can be determined or influenced. Arnold and Breedlove (1985) suggest that endocrine changes that occur well after neonatal development may have profound effects on the organization of neural circuits. Since a hallmark of pubertal development is the increased production and secretion of gonadal steroid hormones, puberty may be another, perhaps critical, period of steroid-dependent neural development. Good evidence exists for organizational effects of steroid hormones during puberty on social interaction (SI) in a novel environment and open—field ambulation, two behaviors used as inverse indices of anxiety. Adult male rats show less SI and open—field ambulation than prepubertal males (Primus and Kellogg, 1989; Slob et al., 1986, respectively). If animals are gonadectomized before puberty and tested in adulthood, they display high levels of SI and open—field ambulation, similar to those displayed by juveniles (Brand and Slob, 1988; Primus and Kellogg, 1990). Testosterone replacement at the time of gonadectomy permits the normal pubertal change in these behaviors. In contrast, animals 122 gonadectomized after puberty engage in the same amount of SI and open-field ambulation as intact adults (Brand and Slob, 1988; Primus and Kellogg, 1990). Thus, the presence of gonadal steroids during puberty results in a behavioral change that does not require the continued presence of steroid, an effect that fits the traditional definition of organizational effects of steroid hormones. Estrogen formed from the aromatization of testosterone mediates the puberty- related decrease in SI (Kellogg and Lundin, 1999). Taken together, these studies demonstrate that behavioral potentials can be recast during puberty by androgenic and estrogenic steroids, and that the critical period for the organization of neural circuits by steroids may be extended into pubertal development. Direct empirical evidence exists that demonstrates remodeling of the nervous system during pubertal development. For instance, we have demonstrated that the amygdala exhibits morphological plasticity during puberty, such that the MeP is larger in adult compared to prepubertal males, while the MeA is smaller in adult males compared to juveniles (Romeo and Sisk, 2001). These changes may reflect underlying cellular processes (e.g., altered cellular excitability, changes in protein synthesis) related to the pubertal change in motivated and social behaviors (e g., mating, aggression, affiliation). We have also found that adult males that have been castrated before puberty have a greater number of AR-ir cells in the MPN than adult males that have been castrated 123 after puberty (Romeo, Diedrich, and Sisk, 2000), indicating that the presence of gonadal steroids during pubertal development decreases the number of AR—containing cells in the MPN. These data suggest that a hormone—dependent change in androgen sensitivity or responsiveness is one outcome of normal pubertal development, and provides further support for the concept that puberty is a developmental stage during which hormones shape steroid—sensitive brain regions. we Expression of adult reproductive behaviors may depend on steroid—independent maturational events that must occur in conjunction with the pubertal rise in testosterone. Thus, I prepubertal males may not engage in mating behaviors even ' when treated with steroids because a steroid-independent neural process or a change in metabolic signals that is permissive for behavior has not yet occurred. However, rats castrated prior to puberty and then treated with hormones in adulthood respond behaviorally to steroids as if they were prepubertal (Larsson, 1967), suggesting steroid—independent maturational events are not the only determinant of the pubertal increase in mating behavior. Structural changes in the brains of humans during pubertal development have been reported. For instance, a magnetic resonance imaging (MRI) study has shown that the volume of the amygdala increases in boys during adolescence, while hippocampal volume increases during puberty in girls (Giedd, Vaituzis, Hamburger, Lange, Rajapakse, Kaysen, Vauss, and Rapoport, 1996). Furthermore, recent studies have 124 demonstrated that the ratio of gray to white matter changes in the frontal and parietal lobes as a function of puberty (Giedd, Blumenthal, Jeffries, Castellanos, Liu, Zijdenbos, Paus, Evans, and Rapoport, 1999; Paus, Zijdenbos, Worsley, Collins, Blumenthal, Giedd, Rapoport, and Evans, 1999; Sowell, Thompson, Colin, Jernigan, and Toga, 1999), such that white matter volume increases during adolescence. 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