Prenatal stress feminizes and demasculinizes the behavior of males.

Male rats were exposed to prenatal or postnatal stress, or both. The prenatally stressed males showed low levels of male copulatory behavior and high rates of female lordotic responding. Postnatal stress had no effect. The modifications are attributed to stress-mediated alterations in the ratio of adrenal to gonadal androgens during critical stages of sexual differentiation. Specifically, it appears that stress causes an increase in the weak adrenal androgen, androstenedione, from the maternal or fetal adrenal cortices, or from both, and a concurrent decrease in the potent gonadal androgen, testosterone.

Maternal stress alters plasma testosterone in fetal males.

Titers of testosterone in plasma were determined by radioimmunoassay in male rat fetuses of stressed and control mothers on days 17, 18, 19, 21, and 23 (the day of birth) after conception. In fetuses of stressed mothers, testosterone concentrations were highest on day 17, declined on days 18 and 19, and then remained unchanged. In the control fetuses, testosterone increased from relatively low concentrations on day 17 to the highest amounts on days 18 and 19, and then declined. Thus, the persistence of feminine and impaired masculine sexual behavior in male offspring of stressed mothers could be due to the absence of a surge of circulating testosterone during days 18 and 19 after conception, a period postulated to be critical in the development of the central nervous system in the rat.

Fetal female rats are masculinized by male littermates located caudally in the uterus.

Female rats are masculinized in utero by male littermates sharing the same uterine horn. Increased anogenital distances in neonatal females and mounting behavior in adult females are related to the presence of males on the caudal side of the females in the uterine horn. Contrary to current beliefs, interamniotic diffusion may not be responsible for the exchange of masculinizing agents among fetuses. Since uterine blood flow in the rat is from the direction of the cervix toward the ovary, masculinizing hormones secreted by fetal males may be carried via the uterine vasculature to female littermates located further downstream.

Activation of lordotic responding in female rats by suppression of serotonergic activity.

After systemic administration of several serotonergic antagonists, female rats that had been ovariectomized, adrenalectomized, and estrogen-primed showed lordotic responding. Lordosis could also be elicited after direct placement of serotonergic receptor blockers into hypothalamic sites known to contain serotonergic terminals. None of the treatments activated the soliciting component of the estrous behavior pattern of the female rat. It is postulated that the hypothalamus contains serotonergic terminals which suppress lordotic responding.

Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers.

Testosterone, progesterone, and corticosterone titers were measured by RIA in plasma of stressed and control pregnant rats and their male and female fetuses on days 17, 18, 19, and 21 of gestation and on the day of birth. The regimen of stress used (three 45-min periods of restraint under intense illumination daily from days 14-21 of pregnancy) causes failure of masculinization and defeminization of behavioral potentials in male offspring. In fetuses of both sexes, corticosterone titers increased sharply between days 17 and 18 postconception (pc) to a peak that was maintained through day 19 and then declined. This pattern resembled that obtained for testosterone in control male fetuses in which the levels of testosterone also rose sharply between days 17 and 18 pc. Corticosterone titers were elevated in samples obtained during the middle of the stress session from both the mothers (serum) and their male and female fetuses (plasma). Increased corticosterone levels were no longer evident in samples obtained from fetuses 75-165 min after the end of a stress session. Testosterone titers were altered by stress only in male fetuses. Their testosterone levels were elevated on day 17 pc, and the surge on days 18 and 19 pc, characteristic of control males, was absent in samples obtained 75-165 min after termination of stress. Progesterone titers were not affected by stress in either mothers or their fetuses. In both stressed and control groups, progesterone concentrations were identical in male and female fetuses, were higher in mothers than in fetuses, and declined in both fetuses and mothers between days 19 and 21 pc. Thus, a persistent effect of stress was observed only on testosterone and only in males.

Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring.

Testosterone and progesterone titers were determined by RIA in the plasma of pregnant rats and their male and female fetuses from day 17 of gestation through the day of birth and in male and female neonates on days 3 and 5 post partum. Males had significantly higher mean testosterone levels than females from day 18 of gestation through day 5 post partum. Sex differences in plasma testosterone concentrations were greatest in the fetuses on days 18 and 19 of gestation when testosterone levels peaked in the males. Instances in which female fetuses had testosterone titers equal to or greater than their male littermates were found on every day of gestation except day 18. Mean testosterone concentrations in plasma of female fetuses were high throughout gestation (greater than 1000 pcg/ml). Testosterone concentrations decreased in both sexes after birth. Differences between the sexes remained significant, and although there was an overlap in the values for males and females, testosterone concentrations in females exceeded those of their male littermates in only one out of nine pairs of samples on day 5 and in none of seven pairs on day 3 post partum. There were no significant differences in progesterone levels in plasma of males and females, either pre- or postnatally. Progesterone titers changed as a function of days post conception in both the fetuses and their mothers. In the fetuses, progesterone levels declined progressively from day 18 post conception through the day of birth, while in the mother they rose from days 18 to 19 then declined between days 20 and 21 of pregnancy. Fetuses had lower progesterone titers than their mothers. From these data, we conclude that day 18 and possibly day 19 post conception represent a critical period during which the central nervous system of the male is primed by high levels of testosterone. Thereafter, the process of masculinization is completed by exposure to testosterone levels that are relatively low and need not be consistently higher than those of female littermates.

Naltrexone normalizes the suppression but not the surge of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of stressed rat fetuses.

Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time.

The prenatal stress syndrome: current status.

Exposure of female rats to stressors during the last week of pregnancy results in a selective feminization and demasculinization of adult sexual behaviors in the male offspring. No behavioral abnormalities are detectable in the female offspring, and reproductive morphological structures appear normal in both sexes. Existing data suggest that the mechanism mediating the so called Prenatal Stress Syndrome in male rats is an alteration in fetal testicular enzyme activity. This, in turn, leads to abnormal levels of testosterone, the hormone believed to masculinize sexual behavior potentials at critical stages of perinatal development. Specifically, the activity of the steroidogenic enzyme delta 5-3 beta-hydroxysteroid dehydrogenase in fetal Leydig cells and plasma titers of testosterone are low in prenatally stressed males on days 18 and 19 of gestation, a time when both of these substances reach maximal levels in control males. The implications of this model for sexual behavior differentiation in higher organisms is explored.

Prenatal and neonatal androgen exposure interact to affect sexual differentiation in female rats.

Rats were injected with oil on Days 17.5 and 18.5 of pregnancy or with 2 mg of testosterone propionate (TP) on Days 15.5 and 16.5, or Days 17.5 and 18.5, or Days 19.5 and 20.5. The female offspring were given oil or 5 micrograms of TP on Day 25 postconception. Among females exposed to TP only during prenatal ontogeny, a lower proportion of those treated on Days 17.5-18.5 of gestation displayed lordotic behavior than did the control group. Postnatal TP alone did not affect lordosis. However, all groups receiving combined pre- and postnatal TP showed impaired estrous patterns. The development of several components of morphology also was differentially affected by the timing of the androgen exposure. The data suggest that the differentiation of sexual behavior and reproductive morphology in the rat are influenced by an interaction of androgen dependent processes operating at different stages of perinatal ontogeny. Further, there may be an optimal fetal period during which androgenization sensitizes animals to low levels of testosterone circulating during neonatal development.

Prenatal stress and prepuberal social rearing conditions interact to determine sexual behavior in male rats.

The two major categories of factors known to influence adult sexual behavior potentials are the relative amounts of androgen present during specific stages of perinatal ontogeny and adequate social stimulation during prepuberal development. The possible interaction between these two was evaluated by characterizing the ejaculatory and lordotic behavior potentials of prenatally stressed and control male rats that had been weaned at 16 days of age and raised either in total social isolation or with a same-age female, a control male, or a prenatally stressed male. The decrement in male sexual behavior produced by prenatal stress was attenuated by raising the male with either a female or a control male. Social isolation alone or in combination with stress resulted in severely deficient male behavior. Peripheral skin shock promoted ejaculatory behavior in many previously noncopulating prenatally stressed males raised with other stressed males, but it was ineffective in most isolated animals. The high lordosis potential characteristic of prenatally stressed male rats was slightly lower in the group with a female cagemate and was markedly decreased by social isolation. These results support and extend the finding by Dunlap, Zadina, and Gougis (1978) that prenatal hormonal events and prepuberal rearing conditions can interact to attenuate or accentuate the effects that either treatment alone has on the development of adult sexual behavior potentials.

Prenatal beta-endorphin can modulate some aspects of sexual differentiation in rats.

Sexually dimorphic traits were studied in offspring of rats injected with 33 micrograms rat beta-endorphin (beta-END) three times daily from Day 14 to Day 21 of pregnancy. beta-END males had shorter neonatal anogenital distances than did controls and were more likely to show the female lordosis pattern as adults, but they did not differ in male copulatory behavior. When given a choice between spending time with an estrous female or a male, beta-END males showed a lower preference for the female than did control males. The number and somal size of neurons in the bulbocavernosus and dorsolateral nucleus of the lumbar spinal cord were unaffected by drug exposure. Elevated beta-END during fetal ontogeny apparently alters the differentiation of some, but not all, sexually dimorphic traits. The data suggest that endogenous opioids may contribute to the etiology of the prenatal stress syndrome.

Male and female sexual behavior potential of male rats prenatally exposed to the influence of alcohol, stress, or both factors.

Adult sexual behaviors were characterized in male rats prenatally exposed to ethanol, stress, or ethanol combined with stress; 60% to 75% of each group exhibited female-typical lordosis. A substantial proportion of males subjected to alcohol (44%) or to alcohol with stress (54%) failed to ejaculate. The adult genitalia and testicular size appeared normal in all groups. Either alcohol or stress can suppress fetal plasma testosterone. Thus, exposing pregnant dams to alcohol, particularly in association with stress, may alter the hormonal milieu of their male fetuses sufficiently to block full masculinization and defeminization of sexually dimorphic copulatory behavior potentials, but not anatomy. It appears that certain pharmacological and stressful factors can interact during fetal ontogeny to influence the process of sexual behavior differentiation.

Prenatal alcohol and stress interact to attenuate ejaculatory behavior, but not serum testosterone or LH in adult male rats.

Restraint stress reduced blood alcohol levels in pregnant rats given a liquid alcohol diet. The male offspring prenatally exposed to both stress and alcohol failed to ejaculate spontaneously, although they copulated normally following exogenous testosterone (T) administration. Males prenatally exposed only to alcohol or only to stress showed no behavioral deficits. Adult serum T and luteinizing hormone levels were normal in both of the fetal alcohol exposed male groups. It appears that the androgen threshold for ejaculatory behavior is elevated in males prenatally exposed to alcohol plus stress and cannot be realized with normal testosterone titers, but it can be attained with exogenous hormone administration. Presumably the alcohol and stress combination interfered with ontogenetic patterns of T needed to fully masculinize the fetal nervous system.

SDN-MPOA volume in male rats is decreased by prenatal stress, but is not related to ejaculatory behavior.

A computer-assisted image analysis technique was used to measure the adult volume of the sexually dimorphic nucleus of the medial preoptic area (SDN-MPOA) in prenatally stressed male rats and in groups of non-stressed males and females. The SDN-MPOA of male offspring from dams stressed daily (i.e. three 45-min exposures to physical restraint and bright light) during the last week of pregnancy was significantly smaller than in males not exposed to stress, but was larger than in females. Maternal stress has been shown to attenuate the surge in fetal plasma testosterone (T) which normally occurs on days 18 and 19 of gestation in male rats. The present results suggest that suppression of T during prenatal development leads to an incomplete masculinization of the SDN-MPOA in male rats. There was no difference in SDN-MPOA volume between males that exhibited the ejaculatory pattern when tested with estrous females and males that failed to ejaculate in either the control or prenatal stress group. SDN-MPOA volume does not appear to be predictive of masculine ejaculatory performance.

Prenatal stress alters sexually dimorphic nuclei in the spinal cord of male rats.

The spinal nucleus bulbocavernosus (SNB), the dorsolateral nucleus of the spinal cord (DLN), and the bulbocavernosus/levator ani (BC/LA) muscle complex were examined in prenatally stressed and control adult male rats, which had been screened for male copulatory behavior. There was a small but significant decrease in the number of DLN (5%) and SNB (3%) neurons in prenatally stressed males compared to controls. Prenatal stress had no effect on the somal or nuclear area of individual neurons within either nucleus, nor did it affect the weight of the BC/LA muscle complex. There were no differences in any of these measures between males that ejaculated and those did not in either the stressed or the control group. These data suggest that exposure of pregnant rats to transient environmental stressors may result in permanent alterations in androgen-sensitive CNS structures in their male offspring.

Sexually dimorphic areas in the rat medial amygdala: resistance to the demasculinizing effect of prenatal stress.

Exposure to prenatal stress blocks full masculinization of several sexually dimorphic nuclei in the brain and spinal cord of male rats. We now compare the adult volume of the medical amygdala (MA) and two of its component cell groups, posterodorsal (MePD) and posteroventral (MePV), in prenatally stressed male rats and nonstressed males and females. Previous reports of sex differences (male > female) in the overall size of the MA and the MePD component were confirmed, and we identified a previously unreported sex difference (male > female) in MePV. Prenatal stress had no effect on the size of the total MA, or of the MePD or MePV in males. Maternal stress attenuates the surge in plasma testosterone (T) which normally occurs on days 18 and 19 of gestation in male rats. This brief suppression of T during prenatal development leads to incomplete masculinization of some sexually dimorphic features of the CNS (i.e. the SDN-MPOA of the hypothalamus, and SNB and DLN of the spinal cord) but not others (i.e. the MA, MePD, and MePV). The selective effects of prenatal stress on neural differentiation may be due to differences in the onset and duration of the periods when each of these structures in most sensitive to T and/or its metabolites.

Prenatal flutamide alters sexually dimorphic nuclei in the spinal cord of male rats.

The effects of prenatal exposure to the antiandrogen flutamide on two sexually dimorphic nuclei of the lumbar spinal cord, the dorsolateral nucleus (DLN) and the spinal nucleus of the bulbocavernosus (SNB), were investigated. Rat dams were given daily injections of 5 mg flutamide or vehicle alone from day 11 through 21 of pregnancy. The spinal cords and perineal morphology of their male and female offspring were examined in adulthood. Flutamide reduced the number of SNB and DLN neurons, reduced the somal and nuclear area of SNB neurons, and reduced the weight of the perineal muscles in males. Flutamide produced no effect in females. No sexual dimorphism was found in the mean somal area of DLN neurons, but a sexual dimorphism was found in the distribution of somal areas in our samples; females had proportionately more large neurons than males. Flutamide-treated males also had proportionately more large neurons than control males but fewer than females. A sexual dimorphism was found in the nuclear areas of DLN neurons but flutamide did not influence this trait.

Prenatal stress feminizes juvenile play patterns in male rats.

Sexually dimorphic rough-and-tumble play patterns were compared in male and female rats derived from control mothers and mothers stressed from days 14-21 of pregnancy. Animals were weaned into groups of 8 consisting of 2 males and 2 females from each treatment. Play in the home cage was recorded at 25, 28, 31, 34, 37 and 45 days of age and was most intense on day 31. The overall level of play was significantly higher in control males than in females or stressed males. Control males showed higher levels of the pinning component of rough-and-tumble play than females or stressed males. No play partner preferences were detected in any group. In adulthood, a higher percentage of stressed than control males displayed the female lordotic pattern. No deficits in ejaculatory behavior occurred in the stressed males. Since maternal stress alters patterns of plasma testosterone in male fetuses, the data suggest that the sexual differentiation of social play begins during prenatal ontogeny in the rat. The present results show that sexually dimorphic behaviors displayed before puberty are incompletely masculinized in prenatally stressed males, a finding similar to that reported for a number of adult behaviors.

Prepuberal social rearing conditions and sexual behavior in control and neonatally castrated male rats.

Male and female sexual behaviors were assessed in control and neonatally castrated male rats that had been housed with a female, a control male, or a neonatally castrated male from day 16 until adulthood. Prepuberal housing conditions had no differential effect on lordosis or ejaculatory potentials of neonatally castrated males tested as adults. A smaller percentage of control males raised with a neonatal castrate ejaculated than did animals housed with a female or a control male. However, a greater proportion of control males caged with a female showed lordosis than did those living with another control male or with a neonatal castrate. The data demonstrate the modulating effects which specific types of social stimulation experienced during early life have on sexual behaviors displayed in adulthood.