Could neonatal testosterone replacement prevent alterations induced by prenatal stress in male rats?

Life Sciences 78 (2006) 2767 – 2771 www.elsevier.com/locate/lifescie

Could neonatal testosterone replacement prevent alterations induced by prenatal stress in male rats? Oduvaldo Caˆmara Marques Pereira a,*, Maria Martha Bernardi b, Daniela Cristina Ceccatto Gerardin a,b b

a Department of Pharmacology, Institute of Biosciences, UNESP, Botucatu, Brazil Department of Pharmacology, Institute of Biomedical Sciences, USP, Sao Paulo, Sao Paulo, Brazil

Received 16 August 2005; accepted 31 October 2005

Abstract The present study was designed to examine whether testosterone replacement is able to prevent some effects of maternal restraint stress — during the period of brain sexual differentiation — on endocrine system and sexual behavior in male rat descendants. Pregnant rats were exposed to restraint stress for 1 h/day from gestational days 18 to 22. At birth, some male pups from these stressed rats received testosterone propionate. The neonatal testosterone replacement was able to prevent the reduction in anogenital distance at 22 days of age observed in pups from stressed pregnant rats as well as prevents the decrease in testosterone levels during the adulthood of these animals. Testosterone replacement in these males also presented an improvement in sexual performance. In this way, testosterone replacement probably through increasing neonatal level of this hormone was able to prevent the later alterations caused by the prenatal stress during the period of brain sexual differentiation. D 2005 Published by Elsevier Inc. Keywords: Prenatal stress; Brain sexual differentiation; Testosterone; Sexual behavior; Male rat

Introduction Sexual differentiation of the hypothalamus of male and female rats involves complex phenomena and an important participation of estrogen, as well as androgens (Dohler, 1991). In male rats, testosterone surges markedly on days 18 – 19 of gestation (Weisz and Ward, 1980) and again during the first few hours following parturition (Baum et al., 1988; Corbier et al., 1978; Lalau et al., 1990; Slob et al., 1980). During this period of brain sexual differentiation, testosterone or its metabolites are fundamental for masculinization and defeminization of sexual behavior, for the establishment of gonadotropin secretion patterns, and also for various morphological indices. In the absence of testosterone or its metabolites, sexually dimorphic structures and functions are feminized (Rhees et al., 1997).

* Corresponding author. Fax: +55 14 3815 3744. E-mail address: [email protected] (O.C.M. Pereira). 0024-3205/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.lfs.2005.10.035

The stress response induced by physical or emotional challenges has been recognized as a profoundly disruptive factor in reproductive function in both males and females (Velazquez-Moctezuma et al., 1993; Wang et al., 1995; RetanaMarques et al., 1998; Rhees et al., 1999; Ward et al., 2002). The prenatal stress during the critical stage of hypothalamic differentiation is related to reduced fertility and fecundity (Anderson et al., 1986) and leads to altered sexual behavior (Ward, 1984) in male offspring through an increased corticosterone level. During stressful situations the activation of the hypothalamus –pituitary – adrenal axis was greater in prenatally stressed animals compared to non-stressed controls. Thus, prenatal stress partly affects adult behavior by altering the regulation of the hypothalamus– pituitary– adrenal axis (Szuran et al., 2000). Prenatal stress can alter masculine function even more directly by reducing plasma testosterone levels in adult males (Anderson et al., 1985; Pollard and Dyer, 1985). It has been hypothesized that prenatal stress disrupts the normal maternal hormonal milieu and suppresses the fetal testosterone peak on gestational days (GD) 18 and 19, a necessary peak for the later expression and maintenance of male sexual behavior

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(Ward and Weisz, 1984). High plasma levels on corticosterone are also associated with depression of the circulating androgen level (Kime et al., 1980; Moore and Miller, 1984; Nowel, 1980; Tokarz, 1987). In humans, it is well established that adverse prenatal or early childhood events have a profound influence on neurodevelopment, altering central nervous system function later in life (Fride and Weinstock, 1988) and potentially contributing disorder and cognitive and psychiatric disorders (Fumagalli et al., 2005). At gestation (22 – 23 days) the gross appearance and histological detail in rat brain is like that of a human fetus of about 14 –17 weeks (Bayer et al., 1993). Thus, some neuronal systems that are present at birth in the human continue to develop in the rat for several days or weeks after parturition (Weinstock, 2001). The data from the present study confirm also the results obtained in previous study realized in male rats offspring exposed to prenatal stress (Gerardin et al., 2005) by showing a reduction in anogenital distance at 22 days old as well as a reduction in plasmatic testosterone level and a delay in the latencies of first mount and intromission and a decrease in number of ejaculations in adulthood. How to prevent this alteration becomes fundamental importance. So, we propose to evaluate whether neonatal testosterone replacement could restore some alterations caused by prenatal stress during the hypothalamus sexual differentiation. Thus, the aim of present study was to replicate this previous study in order to verify whether neonatal testosterone replacement may be able to prevent endocrine and sexual alterations caused by prenatal stress.

– Stressed group + TP: some male pups (n = 17) were obtained from different dams that were restrained according to the same procedure in stressed group. At birth these male received testosterone propionate dissolved in corn oil, 10 Ag/animal (s.c.). The pups from the different groups were immediately fostered to recipient dams (8 pups/recipient dams) that had not been manipulated during the gestation and delivered on the same day. The pups were culled to six males and two females to ensure the presence of both sexes in the litters. They were left with each dam until weaning (23 days of age). For each set of experiments, a maximum of two male siblings was taken from each litter in order to avoid ‘‘litter effects’’. The animals used in this study were maintained in accordance with Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and approved by the Bioscience Institute/UNESP Ethical Committee for Animal Research (Protocol number: 065/03). Body weight and anogenital distance during the pre-weaning period of male pups At birth and on postnatal day 22 (PND 22), the average offspring’s body weight of five litters was done. At birth and on postnatal day 22 (PND 22), ten male pups per group were utilized to obtain the anogenital distance through a verniercaliper. Plasmatic testosterone quantification on postnatal day 75 (PND 75)

Materials and methods Animals and experimental groups Wistar rats were used as the parent generation. They were kept in a controlled environment with temperature at 25 T 1 -C; humidity of 55 T 5%; 12 h light/dark cycle (lights on at 6:00 a.m.) and had free access to regular lab chow and tap water. Virgin female rats (200 T 10 g) were mated overnight. The onset of pregnancy was confirmed by the presence of spermatozoa in vaginal smears on the following morning and was considered day 1 of gestation. On GD22, all dams were weighed, anaesthetized with sodium pentobarbital (40 mg/kg, ip), and laparotomized to obtain male pups, which were divided according to treatment, as described below. – Control group: some male pups (n = 18) were obtained from different dams that were not manipulated during gestation. At birth these male received only vehicle (s.c.). – Stressed group: some male pups (n = 18) were obtained from different dams that were restrained in a Plexiglas cylinder (with variable diameter and 16 cm length) for 1 h from gestational day (GD) 18 to 22. The removable restraining shield was readjusted to the tightest setting the expanding body size of the pregnant animals would allow. At birth these male received only vehicle (s.c.).

Blood samples were collected through the abdominal aorta into heparin-coated syringes (always at 09:00 a.m.) from seven male rats per group. Blood was centrifuged (2500 rpm for 20 min at 2 -C) and the plasma testosterone concentration determined by competitive immunoassay using the IMMULITE\ Total Testosterone Test (Diagnostic Products Corporation, USA). The antibody used was highly specific for testosterone and the test had an analytical sensitivity of 0.10 ng/ ml. The intra-assay coefficient of variation was 4.8%. Sexual behavior evaluation on PND 75 At least ten sexually inexperienced male pups per group were observed under red-light illumination during the dark phase of their cycle. For the test, female rats in their estrus phase (induced by estradiol benzoate 20 Ag/kg, i.p., 24 h before test) were used (Arteche et al., 1997). Each male was placed into a Plexiglass cage, and after 5 min, the female was introduced. For 30 min, the following parameters were recorded: mount (the male normally mounts from the rear, sometimes posing his forelegs over the female’s back, and makes rapid anteroposterior pelvic thrusts), intromission (vaginal penetration, this behavior starts with a mount, but suddenly the male makes a deep thrust forward and stops pelvic thrusting, then vigorously withdraws and always licks

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As demonstrated in Table 1, the average offspring’s body weight of litters was not altered either at birth or on PND22 by prenatal restraint stress nor was it affected by neonatal testosterone replacement. Anogenital distance was not altered at birth, but it was decreased on PND22 in male pups stressed prenatally when compared with the others two groups. When the male pups were stressed prenatally and received neonatal testosterone, the anogenital distance on PDN22 was the same as that of the control group. Adult rats stressed prenatally presented reduced plasmatic testosterone level in relation to control group. Recuperation in plasmatic testosterone level was observed in the adulthood of prenatally stressed males that received testosterone replacement at birth (Fig. 1). Data from male sexual behavior evaluation are shown in Table 2. Prenatal stress induced a delay in latencies to the first mount and to the first intromission, as well as a decrease in the number of ejaculations. The other parameters were not significantly altered. However, when prenatally stressed males were treated with neonatal testosterone, they presented an improvement in the latencies to the first mount and to the first

Table 1 Average of the litters of offspring’s body weight and anogenital distance (at birth and PND22) of male pups from control, stressed, and stressed plus testosterone groups Weight (g) At birth

Stress + TP

A 5 Control

4

Stress

B

3

Stress+TP

2 1

Fig. 1. Plasma testosterone concentration from adult male rats of control, prenatally stressed, and prenatally stressed plus neonatal testosterone groups. Data are means T SEM of 7 animals per group. Different capital letters indicate significant difference ( p < 0.05, test of Tukey – Kramer).

intromission, observed by the reduction in these parameters when compared to stressed group.

Results

Stress

A

Groups

The results were analyzed by descriptive statistics for determination of normal distributions of data. Then, the Tukey –Kramer and Bonferroni tests were employed, with the results considered significant if p < 0.05.

Control

6

0

Statistical analysis

Groups

7 Testosterone levels (ng/ml)

his genitals), and ejaculation (starts with an intromission, but after vaginal penetration the male remains on the female for 1 – 3 s) latencies; number of mounts and intromission until the first ejaculation; mount and intromission latencies after the first ejaculation; and number of postejaculatory mounts and intromissions. If a male did not mount or intromit within 10 min, the evaluation was ended and the male was considered sexually inactive (Agmo, 1997).

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Anogenital distance (mm) PND22

A

5.84 T 0.35 (5) 5.77 T 0.19A (5) 5.73 T 0.14A (5)

At birth A

39.38 T 1.44 (5) 37.87 T 1.03A (5) 43.37 T 2.01A (5)

PND22 A

2.84 T 0.11 (10) 3.23 T 0.14 A (10) 2.81 T 0.14A (10)

14.04 T 0.52A (10) 11.72 T 0.43B (10) 13.62 T 0.47A (10)

Data are means T S.E.M. Numbers in parentheses represent the number of litters (weight) and animals (anogenital distance) per group. Different capital letters indicate vertical significant difference ( p < 0.05, test of Tukey – Kramer).

Discussion In animal models, social stress leads to a variety of behavioral changes, particularly involving emotionality-linked behaviors such as anxiety, defensiveness, and substance selfadministration, as well as in social and sexual behaviors. It also produces many changes in the brain, affecting neuronal structure and survival as well as neurochemical transmission (Blanchard et al., 2001). Plasma testosterone in the stress group was lower than control levels on day 19 of gestation and Table 2 Effects of prenatal stress and prenatal stress plus neonatal testosterone on sexual behavior of male descendants at PND 75 Parameters

Groups Control

Stress

Stress + TP

Latency to first mount (s) Number of mounts without intromission Latency to first intromission (s) Number of intromission Latency to first ejaculation (s) Postejaculatory mount latency (s) Postejaculatory intromission latency (s) Number of postejaculatory intromission Number of ejaculation

81.50 T 21.57A (10 / 11) 2.40 T 0.52A (10 / 11)

203.30 T 43.31B (10 / 11) 2.20 T 0.51A (10 / 11)

88.50 T 28.76 (10 / 10) 5.20 T 1.26A (10 / 10)

84.40 T 20.84A (10 / 11) 20.70 T 2.23A (10 / 11) 874.70 T 115.01A (10 / 11) 1172.20 T 117.94A (10 / 11) 1172.20 T 117.94A (10 / 11)

232.10 T 41.53B (10 / 11) 27.50 T 6.01A (10 / 11) 1119.00 T 186.70A (8 / 11) 1370.00 T 174.81A (7 / 11) 1370.00 T 174.81A (7 / 11)

97.30 T 27.96A (10 / 10) 23.70 T 3.74A (10 / 10) 1198.60 T 125.08A (8 / 10) 1433.00 T 90.08A (7 / 10) 1433.00 T 90.08A (7 / 10)

12.70 T 1.06A (10 / 11)

12.85 T 3.73A (7 / 11)

9.57 T 1.51A (7 / 10)

2.5 T 0.22A (10 / 11)

1.37 T 0.18B (8 / 11)

1.50 T 0.18B (8 / 10)

A,B

Data are means T SEM. Numbers in parentheses represent the number of animals that presented the behavior/total number of animals. Different capital letters indicate horizontal significant difference ( p < 0.05, test of Bonferroni).

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marginally lower on day 17. The diminished fetal surge in plasma testosterone characteristic of prenatally stressed males seems to be the result of a depression in gonadal steroidogenesis (Ward et al., 2003). The anogenital distance at birth has been shown to predict the volume of the sexual dimorphic nucleus in the preoptic area (SDN-POA) of the hypothalamus in adulthood (Faber and Hughes, 1992) and its reduced size confirms change in this nucleus described in the male after prenatal stress. At 22 days of age, the prenatally stressed males presented a reduction in anogenital distance. The reduction in anogenital distance is related to an inadequate action or release of testosterone and could be an early indication of impaired sexual activity in adulthood (Keshet and Weinstock, 1995). When these stressed males rats were treated with testosterone at birth, there was recuperation in anogenital distance, showing that the reduction in anogenital distance was probably connected to an inadequate action or release of testosterone during this period. Data with different forms of gestational stress, including immobilization, noise or overcrowding (Dahlo¨f et al., 1978; Keshet and Weinstock, 1995; Williams et al., 1998) also showed that the anogenital distance in male neonates was reduced in this condition. On the other hand, certain pharmacological substances (e.g., alcohol) experienced during pregnancy in combination with certain environmental conditions (e.g., stress) can lead to relatively limited reductions in the plasma testosterone of the male fetuses and neonates. These changes are sufficient to prevent full sexual behavior masculinization, but leave adequate amounts of testosterone to ensure normal virilization of gross anatomical structures such as the penis, testicular size, and epididymis (Ward et al., 1994). Plasma testosterone has been reported to be unaffected in the adult offspring by prenatal stress (Ward, 1984; Ward et al., 1996). However, in the present study, male rats exposed to stress in later prenatal life exhibited a decrease in adulthood testosterone level. Prenatal stress during late gestation may also have caused an incomplete masculinization of the central nervous system leading to a lack of tonic gonadotropin secretion, which is responsible for the testosterone levels in adult males (Arena and Pereira, 2002). Therefore, when the prenatally stressed male rats received a neonatal replacement of testosterone, they presented recuperation in testosterone levels in adulthood. So, male sexual behavior in adult mammals is modulated by testosterone (Robbins, 1996); and it requires normal functioning of the hypothalamic – pituitary – testicular axis (Agmo, 1997). The above consideration confirmed the importance of adequate levels of testosterone at the right time for normal adult sexual activity (Weinstock, 2001), despite the importance of many other relevant neuroendocrine factors, like dopamine and serotonin. In humans, sexual differentiation of the SDN – POA probably occurs after birth but could depend on processes that were programmed much earlier during gestation (Swaab and Hoffman, 1995). Regarding sexual behavior, the prenatal stress during late gestation disrupted it, as demonstrated by delayed latencies to the first mount and to the first intromission, as well as

decreased number of ejaculations (Gerardin et al., 2005, and present study). These data agree with a study showing that males exposed to stress experienced a reduction in testosterone during fetal development, which led to a moderately attenuated potential for male copulatory behavior (Ward et al., 2003). The neonatal testosterone replacement also improved sexual behavior by decreasing the latencies to the first mount and intromission in stressed male rats, although the replacement was not able to restore the number of ejaculations. This reduced number of ejaculation suggest that the testosterone level in perinatal period was not sufficient or others factors may be involved in this process. Moreover, it seems that the sexual behavior of these animals is somehow in between that of the control and the stressed groups. So, it is demonstrated that the neonatal testosterone replacement could, in same way, have improved the male sexual behavior observed in the present study. Practically all the control rats are sexually active (10 / 11) while at least 1 / 3 of the stressed male are not completely active since male-typical behavior was altered in some aspects. This ratio in the stressed plus testosterone is between that of control and stressed animals. It was also demonstrated that the administration of testosterone to stressed pregnant rats (immobilization and illumination stress) during the last days of gestation (days 15 –22 of gestation), and to their pups after parturition, prevented the demasculinization and feminization of the brain (Do¨rner et al., 1983). The data from the present study demonstrated that a single dose of testosterone at birth was able to prevent the alterations induced by prenatal stress in male rats. In summary, the present results confirms that prenatal stress through a decreased testosterone level during the critical period of male brain sexual differentiation decreased both anogenital distance at 22 days of age and testosterone level in adulthood as well as impaired the sexual behavior. So, a single testosterone replacement at birth was able to prevent at least some alterations observed in rats exposed to prenatal stress. Acknowledgments We are grateful to Eunice Oba, Ph.D. for help in testosterone determination and to FAPESP for financial support. (Proc: 01/ 03458-0)This work constituted part of the Doctoral Thesis presented to the Universidade de Sa˜o Paulo-USP, in 2005, by Daniela C. C. Gerardin. References Agmo, A., 1997. Male rat sexual behavior. Brain Research Protocols 1, 203 – 209. Anderson, D.K., Rhees, R.W., Fleming, D.E., 1985. Effects of prenatal stress on differentiation of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of rat brain. Brain Research 332, 113 – 118. Anderson, R.H., Fleming, D.E., Rhees, R.W., Kinghorm, E., 1986. Relationships between sexual activity, plasma testosterone, and volume of the sexually dimorphic nucleus of the preoptica area in prenatally stressed and non-stressed rats. Brain Research 370, 1 – 10. Arena, A.C., Pereira, O.C.M., 2002. Neonatal inhalatory anesthetic exposure: reproductive changes in male rats. Comparative Biochemistry and Physiology 133C, 633 – 640.

O.C.M. Pereira et al. / Life Sciences 78 (2006) 2767 – 2771 Arteche, E., Strippoli, G., Loirand, G., Pacaud, P., Candenas, L., Molto´, J., Souto, L., Fernandez, J., Norte, M., Martı´n, J.D., Savineau, J., 1997. An analysis of the mechanisms involved in the okadaic acid induced contraction of the estrogen-primed rat uterus. The Journal of Pharmacology and Experimental Therapeutics 282, 201 – 207. Baum, M.J., Ooms, M., Vreeburg, J.T.M., Slob, A.K., 1988. Immediate postnatal rise in whole body androgen content in male rats: correlation with increased testicular content and reduced body clearance of testosterone. Biology of Reproduction 38, 980 – 986. Bayer, S.A., Altman, J., Russo, R.J., Zhang, X., 1993. Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat. Neurotoxicology 14, 83 – 144. Blanchard, R.J., McKittrick, C.R., Blanchard, D.C., 2001. Animals models of social stress: effects on behavior and brain neurochemical systems. Physiology & Behavior 73, 261 – 271. Corbier, P., Kerdelhue, B., Picon, R., Roffi, J., 1978. Changes in testicular weight and serum gonadotropin and testosterone levels before, during, and after birth in the perinatal rat. Endocrinology 103, 1985 – 1991. Dahlo¨f, L.G., Hard, E., Larsson, K., 1978. Influence of maternal stress on the development of the fetal genital system. Physiology & Behavior 20, 193 – 195. Dohler, K.D., 1991. The pre and postnatal influence of hormones and neurotransmitters on sexual differentiation of the mammalian hypothalamus. International Review of Cytology 131, 1 – 57. Do¨rner, G., Do¨tz, F., Do¨cke, W.D., 1983. Prevention of demasculinization and feminization of the brain in prenatally-stressed rats by perinatal androgen treatment. Experimental and Clinical Endocrinology 81, 88 – 90. Faber, K.A., Hughes Jr., C.L., 1992. Anogenital distance at birth as a predictor of volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus and pituitary responsiveness in castrated adult rats. Biology of Reproduction 46, 101 – 104. Fumagalli, F., Bedogni, F., Slotkin, T.A., Racagni, G., Riva, A., 2005. Prenatal stress elicits regionally changes in basal FGF-2 gene expression in adulthood and alters the adult response to acute or chronic stress, Neurobiology of Disease — in press, available on line. Fride, E., Weinstock, M., 1988. Prenatal stress increases anxiety related behavior and alters cerebral lateralization of dopamine activity. Life Sciences 42, 1059 – 1065. Gerardin, D.C.C., Pereira, O.C.M., Kempinas, W.G., Florio, J.C., Moreria, E.G., Bernardi, M.M., 2005. Sexual behavior, neuroendocrine, and neurochemical aspects in male rats exposed penatally to stress. Physiology & Behavior 84, 97 – 104. Keshet, G.I., Weinstock, M., 1995. Maternal naltrexone prevents morphological and behavioral alterations induced in rats by prenatal stress. Pharmacology Biochemistry and Behavior 50, 413 – 419. Kime, K.E., Vinson, G.P., Malor, P.W., Kilpatrick, R., 1980. Adrenal – gonadal relationships. In: Chester-Jones, I., Henderson, I.W.General Comparative and Clinical Endocrinology of the Adrenal Cortex vol. 3. Academic Press, New York, pp. 183 – 264. Lalau, J.-D., Aubert, M.L., Carmignac, D.F., Gre´goire, I., Dupouy, J.-P., 1990. Reduction in testicular function in rats: II. Reduction by dexamethasone in fetal and neonatal rats. Neuroendocrinology 51, 289 – 293. Moore, F.L., Miller, L.J., 1984. Stress-induced inhibition of sexual behavior: corticosterona inhibits courtship behavior of a male amphibian (Taricha granulosa). Hormones and Behavior 18, 400 – 410. Nowel, N.W., 1980. Adrenocortical function in relation mammalian population densities and hierarchies. In: Chester-Jones, I., Henderson, I.W. (Eds.). General Comparative and Clinical Endocrinology of the Adrenal Cortex vol. 3. Academic Press, New York, pp. 349 – 393. Pollard, I., Dyer, S.l., 1985. Effects of stress administered during pregnancy on the development of fetal testes and their subsequent function in the adult rat. Journal of Endocrinology 107, 241 – 245.

2771

Retana-Marques, S., Bonilla-Jaime, H., Velazquez-Moctezuma, J., 1998. Lack of effect of corticosterone administration on male sexual behavior of rats. Physiology & Behavior 63 (3), 367 – 370. Rhees, R.W., Kirk, B.A., Sephton, S., Lephart, E.D., 1997. Effects of prenatal testosterone on sexual behavior, reproductive morphology and LH secretion in female rat. Developmental Neuroscience 19, 430 – 437. Rhees, R.W., Al-Saleh, H.N., Kinghorn, E.W., Fleming, D.E., Lephart, E.D., 1999. Relationship between sexual behavior and sexually dimorphic structures in the anterior hypothalamus in control and prenatally stressed male rats. Brain Research Bulletin 50 (3), 193 – 199. Robbins, A., 1996. Androgens and male sexual behavior, from mice to men. Trends in Endocrinology and Metabolism 7, 345 – 350. Slob, A.K., Ooms, M.P., Vreeburg, J.T.M., 1980. Prenatal and early post-natal sex differences in plasma and gonadal testosterone and plasma luteinizing hormone in female and male rats. Journal of Endocrinology 87, 81 – 87. Szuran, T.F., Pliska, V., Pokorny, J., Welzl, H., 2000. Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance. Physiology & Behavior 71, 353 – 362. Swaab, D.F., Hoffman, M.A., 1995. Sexual differentiation of the human hypothalamus in relation to gender and sexual orientation. Trends Neuroscience 18, 264 – 270. Tokarz, R.R., 1987. Effects of corticosterona treatment on male aggressive behavior in a lizard (Anolis sagrei). Hormones and Behavior 21, 358 – 370. Velazquez-Moctezuma, J., Salazar, E.D., Rueda, M.L.C., 1993. The effect of prenatal stress on adult sexual behavior in rats depends on the nature of the stressor. Physiology & Behavior 53, 443 – 448. Wang, C.T., Huang, R.L., Tai, M.Y., Tsai, Y.F., Peng, M.T., 1995. Dopamine release in the nucleus accumbens during sexual behavior in prenatally stressed adult male rats. Neuroscience Letters 200, 29 – 32. Ward, I.L., 1984. The prenatal stress syndrome: current status. Psychoneuroendocrinology 9, 3 – 11. Ward, I.L., Ward, O.B., Winn, R.J., Bielawski, D., 1994. Male and female sexual behavior potential of male rats prenatally exposed to the influence of alcohol, stress, or both factors. Behavioral Neuroscience 108, 1188 – 1195. Ward, I.L., Ward, O.B., Mehan, D., Winn, R.J., French, J.A., Hendricks, S.E., 1996. Prenatal alcohol and stress interact to attenuate ejaculatory behavior but not serum testosterone or LH in adult male rats. Behavioral Neuroscience 110, 1469 – 1477. Ward, I.L., Ward, O.B., Affuso, J.D., Long III, W.D., French, J.A., Hendricks, S.E., 2003. Fetal testosterone surge: specific modulations induced in male rats by maternal stress and/or alcohol consumption. Hormones and Behavior 43, 531 – 539. Ward, I.L., Weisz, J., 1984. Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers. Endocrinology 114, 1635 – 1644. Ward, O.B., Ward, I.L., Denning, J.H., French, J.A., Hendricks, S.E., 2002. Postparturitional testosterone surge in male offspring of rats stressed and/or fed ethanol during late pregnancy. Hormones and Behavior 41, 229 – 235. Weinstock, M., 2001. Alterations induced by gestational stress in brain morphology and behaviour of the offspring. Progress in Neurobiology 65, 427 – 451. Weisz, J., Ward, I.L., 1980. Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106, 306 – 316. Williams, M.T., Hennessy, M.B., Davis, H.N., 1998. Stress during pregnancy alters rat offspring morphology and ultrasonic vocalizations. Physiology & Behavior 63, 337 – 343.