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Embryonic Temperature and Gonadal Sex Organize Male-Typical Sexual and Aggressive Behavior in a Lizard with Temperature-Dependent Sex Determination* TURK RHEN AND DAVID CREWS Section of Integrative Biology, School of Biological Sciences, University of Texas, Austin, Texas 78712 ABSTRACT Temperature during embryonic development determines gonadal sex in the leopard gecko, Eublepharis macularius. Moreover, both embryonic temperature and gonadal sex influence adult behavior. Yet it remains unclear whether the effects of embryonic temperature and gonadal sex on behavior are irreversibly organized during develop- ment. To address this question, we gonadectomized adult females and males generated from a temperature that produces mostly females (30 C) and a temperature that produces mostly males (32.5 C). Females and males from both temperatures were then treated with equivalent levels of various sex steroids. We found that both embryonic temper- ature and gonadal sex had persistent effects on the expression of male-typical sexual and aggressive behaviors. For example, adult females do not scent mark and display very little courtship and mounting behavior even when treated with levels of hormones (pri- marily androgens) that activate these behaviors in males. In contrast, species-typical aggressive displays were less sex specific and were activated by both dihydrotestosterone and testosterone (T) in males and by T in females. Nevertheless, the average duration of aggressive displays was significantly shorter in T-treated females than that in T-treated males. With regard to submissive behavior, androgens de- creased flight behavior in males, but had no effect in females. Em- bryonic temperature had enduring effects on certain behaviors in males. For instance, males from a male-biased embryonic tempera- ture scent-marked more than males from a female-biased embryonic temperature when treated with dihydrotestosterone or T. Conversely, and across hormone treatments, males from a female-biased embry- onic temperature mounted more than males from a male-biased em- bryonic temperature. Finally, treatment with 17b-estradiol decreased submissive behavior in males from a male-biased embryonic temper- ature compared with that in males from a female-biased embryonic temperature. Courtship and aggressive behavior were not influenced by temperature. These results strongly suggest that male-typical behaviors in the adult leopard gecko are permanently organized by both embryonic temperature and gonadal sex during development. (Endocrinology 140: 4501–4508, 1999) WHEREAS SEX chromosomes determine gonadal sexin mammals and birds (1), embryonic temperature determines sex in some lizards, many turtles, and all croc- odilians (2–4). Despite this dramatic difference in the signal that initiates testicular or ovarian development, it appears that much of the molecular machinery for gonadogenesis is evolutionarily conserved. Indeed, genes clearly involved in mammalian sex determination [e.g. anti-Mu¨llerian hormone, steroidogenic factor-1, Wilms’ Tumor (WT-1) gene, and Sry- like, High Mobility Group Box-like (SOX)9 gene] (5–7) have also been identified and implicated in avian sex determina- tion (5, 8–10) and temperature-dependent sex determination (TSD) in reptiles (5, 11–13). Moreover, the gonadal anlagen is initially bipotential and consists of a cortical region that gives rise to the ovary and a medullary region that gives rise to the testis in all amniotic vertebrates (14). Considering such similarities in gonadal differentiation, a fundamental ques- tion is whether other aspects of sexual differentiation are also alike in mammals, birds, and reptiles with TSD. In this respect, the sexual differentiation of reproductive and aggressive behavior is very well studied in mammals and birds and depends upon the sexually dimorphic pro- duction of steroids by the differentiated gonads. Our under- standing of sex differences in these behaviors is based on the organization-activation paradigm formulated by Pheonix et al. (15). Classically, behavioral activation is the process by which circulating sex steroids affect specific neural sub- strates to induce sexual or aggressive behavior in adults that are exposed to the appropriate external stimuli (i.e. individ- uals of the opposite or same sex, respectively). For example, sex differences in the display of male-typical sex behavior occur because of sex differences in plasma testosterone (T) levels in intact rats. In fact, exogenous T can activate male- typical mounting behavior in gonadectomized male and fe- male rats (reviewed in Refs. 16–19). In contrast, castrated male rats do not display female-typical sex behavior when treated with a sex steroid regimen [i.e. 17b-estradiol (E2) followed by progesterone] that activates lordosis in ovari- ectomized female rats. A perinatal T surge in male, but not female, rats causes this dimorphic response to hormonal activation of female sex behavior in adulthood (reviewed in Refs. 16 and 19). Such permanent developmental effects of sex steroids on subsequent behavior are called organiza- tional effects. Although details vary, the basic paradigm of organiza- tional vs. activational effects of sex steroids has been sup- ported in a variety of mammals and birds. For instance, female quail are demasculinized (i.e. organized) by circu- lating estrogens during the perinatal period and lose the ability to display male-typical mounting behavior when Received March 22, 1999. Address all correspondence and requests for reprints to: Dr. Turk Rhen, Section of Integrative Biology, School of Biological Sciences, Uni- versity of Texas, Austin, Texas 78712. E-mail: turkrhen.uta@mail. utexas.edu. * This work was supported by Individual National Research Service Award MH-11369 from the NIMH (to T.R.), NSF Dissertation Improve- ment Grant IBN-9623546 (to T.R.), and NIMH Grant MH-57874 (to D.C.). 0013-7227/99/$03.00/0 Vol. 140, No. 10 Endocrinology Printed in U.S.A. Copyright © 1999 by The Endocrine Society 4501 treated with T as adults (20, 21). Gonadectomized male and female quail, when treated with E2 as adults, can display female-typical receptive behavior in response to male sexual overtures. Copulatory behavior in the zebra finch also fits this general pattern, even though its song system is paradoxical in that exogenous E2 organizes the male phenotype (22). Thus, the sexual differentiation of reproductive behaviors in birds can be classified as acti- vational and/or organizational in nature. In contrast, very little is known about the sexual differentiation of behavior in reptiles with TSD. In the leopard gecko, Eublepharis macularius, an embryonic temperature of 30 C produces a female-biased sex ratio (ap- proximately one male to three females), whereas 32.5 C pro- duces a male-biased sex ratio (approximately three males to one female) (23). Furthermore, both embryonic temperature and gonadal sex influence reproductive and aggressive be- havior in intact adult leopard geckos (24, 25; reviewed in Ref. 26). However, it is unclear whether these effects are organi- zational or activational, because embryonic temperature and gonadal sex also influence adult sex steroid physiology. For example, female leopard geckos have lower circulating levels of T and 5a-dihydrotestosterone (DHT) than males and nor- mally do not exhibit male-typical sex behaviors (24, 25, 27, 28), yet females treated with male-typical levels of T can display male-typical courtship behavior (27). However, in this latter study, there was no quantitative comparison be- tween levels of courtship behavior in males and females given identical hormone treatments and tested in exactly the same manner. Consequently, it is not clear whether sex dif- ferences in courtship behavior are purely activational in nature. Similarly, males from the male-biased incubation tem- perature (i.e. 32.5 C) are more aggressive but less sexually active toward females than are males from the female- biased incubation temperature (i.e. 30 C) (25). Males from the male-biased incubation temperature also have lower E2 levels than males from the female-biased incubation temperature, whereas their T levels are similar (28, 29). Overall, the combined data clearly show that sexual dif- ferentiation of behavior in the leopard gecko depends upon both gonadal sex and embryonic temperature (re- viewed in Ref. 26). Nevertheless, it is uncertain whether such effects are activated or organized because there has been no systematic examination of temperature and sex effects on reproductive and aggressive behavior while controlling for circulating hormone levels. A definitive answer to this question would provide fun- damental information concerning sexual differentiation of behavior in a reptile with TSD. The following study of male-typical behaviors was designed to determine whether the sexes are behaviorally organized in the way that the mammalian and avian sexes are organized. An- other goal was to determine whether embryonic temper- ature has permanent (i.e. organizational) effects on behav- ior within each sex. Overall, this experiment illuminates how embryonic temperature and gonadal sex during de- velopment and sex steroids in adulthood act and interact to influence sexual and aggressive behaviors in the leop- ard gecko. Materials and Methods Animals Animals were treated according to a research protocol approved by the university’s institutional animal care and use committee. Leopard gecko eggs from our captive breeding colony at the University of Texas were collected within 24 h of oviposition and candled for fertility. Fertile eggs were placed in individual cups filled with moist vermiculite (1 part water/1 part vermiculite) and split between two constant incubation temperatures (30 and 32.5 6 0.1 C). An incubation temperature of 30 C produces a female-biased sex ratio, whereas 32.5 C produces a male- biased sex ratio (23). Geckos hatched from these eggs were raised in isolation for 49–52 weeks as previously described (25). Leopard geckos reach sexual maturity at roughly 45 weeks of age (28). Surgical and hormonal manipulation Approximately equal numbers of adult males and females from each incubation temperature were gonadectomized under cold anesthesia. At the same time these animals were implanted sc with SILASTIC brand tubing (Dow Corning Corp., Midland, MI) containing cholesterol (C), E2, DHT, or T for a fully factorial experimental design, with embryonic temperature, gonadal sex (before gonad removal), and adult hormone treatment as main effects. Although sample sizes ranged from 8–15 for each group, all except 1 group had 10 or more individuals (see Table 1). Implant length was 10 mm for C, E2, and DHT and 20 mm for T. Otherwise, implants were identical in dimension (id, 1.47 mm; od, 1.95 mm), were packed in the same manner, and were all soaked in reptilian Ringers solution for 24 h before surgery. Animals were allowed 4 weeks to recover after surgery/implantation, and then behavior was tested. One day after behavior testing was completed, a blood sample was taken via cardiac puncture for RIA to confirm hormone delivery. Animals were then killed, dissected, and examined for residual gonadal tissue. Gon- adectomies were complete in all cases. Behavior testing We used a behavior testing procedure similar to that described by Flores et al. (25). Briefly, animals were tested three times for 5 min each time in a neutral cage with one of two types of stimulus animals to assess levels of male-typical and female-typical sexual and aggressive behavior (six tests total per animal). In this paper we report the results of behavior tests in which experimental animals were exposed to intact vitellogenic females (i.e. sexually receptive females) on 3 consecutive days; each experimental animal interacted with a given female only once. This set of tests allowed us to examine the factors controlling the display of male-typical behaviors toward female stimulus animals. Experimental animals were first placed in a neutral cage (43 3 22 3 20 cm) with a clean paper towel as a liner. Stimulus females were then placed, facing the experimental subject, in the same cage. Subject animal and stimulus female behavior was recorded using a keypad timer (Witt/Timer Pro- gram courtesy of Diane Witt, NIH, Bethesda, MD). Tests ended after 5 min or if an attack or attempted copulation occurred. In contrast to the testing protocol used previously (25), experimental animals in the cur- rent study were tested on 6 consecutive days (vs. over a 5-week period) and were first tested with female stimulus animals for 3 days and then with male stimulus animals for 3 days (vs. a randomized sequence). The latter change was made because aggressive behavior of stimulus males toward experimental animals could alter subsequent behavior and thus would have confounded our measures of male-typical and female- typical sexual behaviors. We measured scent marking, courtship (i.e. tail vibrations), and mounting (i.e. body grips) as male-typical sex behaviors in our exper- imental animals. We also recorded aggressive (i.e. high posture display and attacks) and submissive (i.e. flight) behaviors. In a sexual encounter, a male slowly approaches a female, first licking the substrate or the air with his tongue and then licking the female. An attractivity pheromone in the skin of females (30) elicits a male-typical tail vibration that creates an audible buzz and a tactile vibration of the substrate. During these encounters males may also drag their preanal pores on the substrate, presumably to deposit pheromones in a scent-marking behavior. Males then body grip the female’s skin with their jaws during courtship and mounting. Body grips are a major component of mounting behavior, as 4502 SEXUAL DIFFERENTIATION OF BEHAVIOR IN A LIZARD Endo • 1999 Vol 140 • No 10 they position the male for copulation and nearly always accompany intromission. We measured the cumulative duration (in seconds) of scent marking, tail vibration, and mounting (i.e. body grip) behaviors. Overall, these behaviors are a fairly complete index of male-typical sex behavior. We also measured high posture duration (an aggressive dis- play) and the frequency of tests in which an attack occurred as an index of aggressive behavior. Conversely, submissive behavior was recorded as the cumulative duration (in seconds) of flight from the stimulus female. RIA On the day after the last behavior test (with a male), a blood sample was drawn from each experimental animal by cardiocentesis using a heparinized 1-cc syringe with a 25-gauge needle. Blood was centrifuged at 3000 rpm for 10 min at 4 C, and plasma was stored in plastic microfuge tubes at 280 C until assayed for levels of DHT, E2, and T. The antibodies used for RIA were DT3–351 for DHT, E26–47 for E2, and T3–125 for T (Endocrine Sciences, Inc., Calabasas Hills, CA). Column chromatogra- phy and RIAs were performed as previously described (28). Recoveries averaged 57%, 56%, and 70% for DHT, E2, and T, respectively. Assay sensitivity was 71 pg DHT/ml plasma, 92 pg E2/ml plasma, and 86 pg T/ml plasma. For a pooled plasma sample, intraassay coefficients of variation were 16%, 18%, and 17% for DHT, E2, and T, respectively. Interassay coefficients of variation for the same sample were 18%, 17%, and 13% for DHT, E2, and T, respectively. We also ran quality control standards of known concentration in the low, medium, and high ranges of the standard curve for each steroid. For DHT, intraassay coefficients of variation were 12%, 6%, and 6% in the low, medium, and high parts of the curve, respectively. Interassay coefficients of variation for DHT were 18%, 9%, and 11% in the low, medium, and high parts of the curve, respectively. For E2, intraassay coefficients of variation were 11%, 4%, and 6% in the low, medium, and high parts of the curve, respectively. Interassay coefficients of variation for E2 were 10%, 8%, and 9% in the low, medium, and high parts of the curve, respectively. For T, intraassay coefficients of variation were 9%, 4%, and 5% in the low, medium, and high parts of the curve, respectively. Interassay coefficients of variation for T were 14%, 9%, and 10% in the low, medium, and high parts of the curve, respectively. Statistical analyses All data were analyzed using embryonic temperature, gonadal sex (before gonadectomy), adult hormone treatment, and day of testing as main effects in a four-way repeated measures design. All dependent variables, scent marking, tail vibration, body grip, high posture, and flight durations, were analyzed with univariate ANOVA. Independent variables were considered nonsignificant when P . 0.05. Dependent variables are presented as least squares mean 6 one se. Post-hoc com- parisons were made using the Dunn-Sida´k method to provide a signif- icance level of a9 5 1 2 (1 2 0.05)1/k, where k is the number of individual comparisons for an experimentwise a 5 0.05 (31). Hormone concentra- tions were first log transformed and then compared using Tukey’s honestly significant difference test. All statistics were performed using version 3.1 of JMP (32) for Macintosh (Apple Computer, Inc., Cupertino, CA). Results Hormone levels As expected, treatment with SILASTIC capsules contain- ing E2, DHT, and T elevated plasma levels of these hormones above the levels observed in geckos treated with C (see Table 1). Importantly, treatment with a given steroid resulted in equivalent levels of hormones in gonadectomized female and male leopard geckos from each embryonic temperature. Consequently, our experimental manipulations achieved the desired goal, which was to separate the normally confound- ing effects of embryonic temperature and gonadal sex on sex steroid physiology and behavior. The steroid levels pro- duced by these implants are in the normal physiological ranges for intact males and/or females of this species (24–28). Scent marking behavior Scent marking behavior was organized by embryonic tem- perature [F(1,458) 5 12.1; P 5 0.0005], gonadal sex [F(1,458) 5 24.0; P , 0.0001], and a significant interaction between em- bryonic temperature and gonadal sex during development [F(1,458) 5 12.1; P 5 0.0005]. Specifically, females never scent marked regardless of their embryonic temperature (results not shown), whereas, overall, males from the male-biased temperature marked significantly more than did males from the female-biased temperature (see Fig. 1). Scent marking was activated by adult hormone treatment [F(3,458) 5 5.8; TABLE 1. Circulating concentrations of DHT, E2, and T (nanograms per ml plasma) in female and male leopard geckos from two incubation temperatures (30 or 32.5 C) after receiving SILASTIC implants filled with C, DHT, E2, or T Treatment groups Circulating steroid levels Sample size Sex Temperature (C) Hormone E2 DHT T Female 30 C 1.4 6 0.3a 0.3 6 0.1a 0.3 6 0.05a 13 E2 7.2 6 2.4 b 0.3 6 0.1a 0.4 6 0.1a 10 DHT 2.9 6 0.4a,c 42.9 6 5.3b 1.6 6 0.2b 14 T 1.7 6 0.3a 17.4 6 1.6b 172 6 15c 15 32.5 C 0.8 6 0.1a 0.2 6 0.05a 0.3 6 0.07a 11 E2 6.7 6 1.3 b,c 0.2 6 0.04a 0.2 6 0.05a 10 DHT 1.2 6 0.2a 39.6 6 8.3b 1.4 6 0.3b 10 T 1.6 6 0.2a 16.7 6 3.8b 190 6 23c 10 Male 30 C 1.0 6 0.5a 0.4 6 0.3a 0.3 6 0.05a 10 E2 8.1 6 1.7 b 0.3 6 0.2a 0.4 6 0.1a 10 DHT 1.1 6 0.3a 35.0 6 4.3b 1.3 6 0.2b 10 T 2.4 6 0.3a,c 17.9 6 4.4b 227 6 15c 10 32.5 C 1.4 6 0.3a 0.3 6 0.2a 0.4 6 0.1a 8 E2 7.8 6 1.1 b 0.4 6 0.2a 0.3 6 0.1a 10 DHT 2.0 6 0.4a,c 44.2 6 6.3b 1.5 6 0.3b 10 T 2.0 6 0.2a 14.7 6 2.6b 187 6 25c 10 Mean hormone levels are shown in nanograms per ml plasma 6 1 SE. Numbers of samples assayed are shown. For each hormone, groups with different superscripted letters are significantly different from each other using Tukey’s post-hoc comparisons. SEXUAL DIFFERENTIATION OF BEHAVIOR IN A LIZARD 4503 P 5 0.0006], but there was also a signif