Developmental programming: postnatal steroids complete prenatal steroid actions to differentially organize the GnRH surge mechanism and reproductive behavior in female sheep.

In female sheep, estradiol (E2) stimulates the preovulatory GnRH/LH surge and receptive behavior, whereas progesterone blocks these effects. Prenatal exposure to testosterone disrupts both the positive feedback action of E2 and sexual behavior although the mechanisms remain unknown. The current study tested the hypothesis that both prenatal and postnatal steroids are required to organize the surge and sex differences in reproductive behavior. Our approach was to characterize the LH surge and mating behavior in prenatally untreated (Control) and testosterone-treated (T) female sheep subsequently exposed to one of three postnatal steroid manipulations: endogenous E2, excess E2 from a chronic implant, or no E2 due to neonatal ovariectomy (OVX). All females were then perfused at the time of the expected surge and brains processed for estrogen receptor and Fos immunoreactivity. None of the T females exposed postnatally to E2 exhibited an E2-induced LH surge, but a surge was produced in five of six T/OVX and all Control females. No surges were produced when progesterone was administered concomitantly with E2. All Control females were mounted by males, but significantly fewer T females were mounted by a male, including the T/OVX females that exhibited LH surges. The percentage of estrogen receptor neurons containing Fos was significantly influenced in a brain region-, developmental stage-, and steroid-specific fashion by testosterone and E2 treatments. These findings support the hypothesis that the feedback controls of the GnRH surge are sensitive to programming by prenatal and postnatal steroids in a precocial species.

Chemical communication and reproduction in the gray short-tailed opossum (Monodelphis domestica).

The gray short-tailed opossum is one of the most widely studied of all marsupials and an important model for study of olfactory communication, particularly as it relates to pheromonal activation of reproduction. Males respond to differentially to female skin gland secretions and urine from anestrous females, while females respond only skin gland secretions, particularly that of the suprasternal gland. Divergent responses by male and female opossums to odors from these different body sources are most likely related to sex-specific production and deposition of chemical signals in this species. Female opossums do not have an estrous cycle but are stimulated to estrus by male pheromone. Females nuzzle scent marks from male suprasternal gland secretions, and thereby facilitate delivery of a nonvolatile estrus-inducing pheromone to the chemosensory epithelium of vomeronasal organ. Neuroendocrine correlates of pheromonal induction of estrus include elevated plasma estradiol and upregulation of progesterone receptors in hypothalamic regions that control reproductive behavior.

Organizational actions of postnatal estradiol in female sheep treated prenatally with testosterone: programming of prepubertal neuroendocrine function and the onset of puberty.

Prenatal testosterone (T) exposure defeminizes reproductive neuroendocrine function in female sheep, although the LH surge dysfunctions are initially less severe in gonadally intact females than in females subject to neonatal ovariectomy and estradiol (E) replacement. Because prepubertal ovarian production of E differs quantitatively and qualitatively from chronic E replacement, we tested the hypothesis that postnatal E exacerbates the consequences of prenatal T on the positive, but not the negative, steroid feedback controls of GnRH secretion. Our approach was to characterize prepubertal sensitivity to E negative feedback, the onset and maintenance of progestagenic cycles, and the LH surge response in ovary intact, prenatally untreated (control), and T-treated (T) sheep that were exposed postnatally to only endogenous E, or exposed to excess E by s.c. implant. Sensitivity to E negative feedback was reduced in T females, but excess postnatal E did not further increase LH pulse frequency. Excess E prevented ovarian cycles in several control females, and increased cycle irregularity in T females. However, the LH surge mechanism was functional in all control females (regardless of postnatal E exposure) and in some T females without excess E, but nonfunctional in T females with excess E. These findings suggest that postnatal E does not program increased resistance to E negative feedback, but excess postnatal E does disrupt other mechanisms required for ovarian cyclicity. We conclude that in this precocial species, prenatal steroids are sufficient to program controls of tonic LH secretion, but the LH surge mechanism is susceptible to further programming by postnatal E.

Behavioral and reproductive responses of female opossums to volatile and nonvolatile components of male suprasternal gland secretion.

Female gray short-tailed opossums (Monodelphis domestica) lack an estrous cycle and are induced into estrus by exposure to a pheromone in male scent marks. Behavioral and physiological responses of females to the volatile and nonvolatile components of scent marks were examined in two experiments. Young females (n=9) were tested prior to and during their first estrus for behavioral responses to scent marks, collected on a 7-ml glass vial rubbed over the suprasternal gland of a mature male. The response to volatile components of the scent mark, recorded when marked and unmarked vials were covered with a perforated shield, was compared to the response to these vials when unshielded. Estrous females nuzzled the shields over marked vials (55.8+/-8.5 nuzzles/10 min) more than the shielded clean vial (10.9+/-2.4) (P<0.05); a similar response was observed in anestrous females. Nuzzling of unshielded, scent-marked vials was higher (P<0.05) during anestrus than in the same females when in estrus. The role of nonvolatile pheromones in reproductive activation was tested in adult females (n=11) exposed for up to 14 days to a shielded, marked vial or to an unshielded, marked vial in a crossover design. All females exposed to unshielded vials expressed estrus, and 10 copulated. Only 2 females expressed estrus (significantly fewer, P<0.05), when exposed to shielded marked vials, and neither copulated. These results demonstrate that females detect and respond behaviorally to both volatile and nonvolatile components of male suprasternal gland secretion, but the estrus-inducing pheromone in these secretions is nonvolatile.

Sexual differentiation of the external genitalia and the timing of puberty in the presence of an antiandrogen in sheep.

Testicular steroids during midgestation sexually differentiate the steroid feedback mechanisms controlling GnRH secretion in sheep. To date, the actions of the estrogenic metabolites in programming neuroendocrine function have been difficult to study because exogenous estrogens disrupt maternal uterine function. We developed an approach to study the prenatal actions of estrogens by coadministering testosterone (T) and the androgen receptor antagonist flutamide, and tested the hypothesis that prenatal androgens program estradiol inhibitory feedback control of GnRH secretion to defeminize (advance) the timing of the pubertal increase in LH. Pregnant sheep were either untreated or treated with T, dihydrotestosterone (DHT) (a nonaromatizable androgen), or T plus flutamide from d 30-90 of gestation. To study the postnatal response to steroid negative feedback, lambs were gonadectomized and estradiol-replaced, and concentrations of LH were monitored in twice-weekly blood samples. Although T and DHT produced penile and scrotal development in females, the external genitalia of T plus flutamide offspring remained phenotypically female, regardless of genetic sex. Untreated females and females and males treated with T plus flutamide exhibited a pubertal increase in circulating LH at 26.4+/-0.5, 26.0+/-0.7, and 22.4+/-1.6 wk of age, respectively. In females exposed to prenatal androgens, the LH increase was advanced (T: 12.0+/-2.6 wk; DHT: 15.0+/-2.6 wk). These results demonstrate the usefulness of combining T and antiandrogen treatments as an approach to increasing prenatal exposure to estradiol. Importantly, the findings support our hypothesis that prenatal androgens program sensitivity to the negative feedback actions of estradiol and the timing of neuroendocrine puberty.

Long-term exposure of female sheep to physiologic concentrations of estradiol: effects on the onset and maintenance of reproductive function, pregnancy, and social development in female offspring.

As steroids and steroid-like compounds accumulate in the environment, it has become important to understand how low-dose exposure affects reproductive function. Ovary-intact sheep were used in a multigenerational study, to determine whether chronic exposure to low levels of estrogen disrupts reproductive function and behavior. We assessed parameters of reproductive performance in control and postnatally estradiol-treated females (Generation 1, G1), and their offspring (Generation 2, G2). In the G1 animals, 17beta-estradiol (E) was administered continuously from 4 wk of age at two doses via subcutaneous implants (ultralow E [<1 pg/ml in circulation, n = 8] or low E [1-3 pg/ml, n = 8]). Both doses delayed puberty; low E also produced pronounced prepubertal and seasonal anestrus hypogonadotropism, and delayed the onset of the second breeding season. All G1 animals conceived and produced offspring (G2), the treatment of which resulted from continuous maternal exposure during pregnancy and lactation. Behavioral observations of G2 females revealed that low prenatal E modestly masculinized play behavior and increased the frequency of attempts to displace competitors relative to ultralow E and control animals. The timing and magnitude of the LH surge also differed in prepubertal low prenatal E females relative to the controls, although these differences were not evident when retested at one year of age. These findings support the hypothesis that chronic exposure to physiologic amounts of exogenous estrogens has multigenerational effects on behavior and neuroendocrine function. Despite these disruptive steroid actions, ovarian cyclicity and fertility are not invariably compromised, pointing to an impressive resiliency of the reproductive axis to insult by exogenous estrogenic compounds.

Programming of GnRH feedback controls timing puberty and adult reproductive activity.

The timing of puberty generally differs between sexes, and this may be due to sex differences in the organization of steroid feedback systems. We propose that the reproductive neuroendocrine default sex is female. If the individual is male, the feedback control of GnRH secretion is programmed early in development, and the pubertal GnRH rise is either advanced or delayed depending upon species. This developmental programming is by androgens. Early programming also reorganizes adult reproductive neuroendocrine function to change a pattern of cyclic gamete release (periodic ovulations) requiring multiple feedback systems to that of a continuous one (spermatogenesis) requiring only the negative feedback control. The multiple feedback systems underlying the complex ovulatory cycle are innate, and in the male the unnecessary feedbacks are abolished or rendered less sensitive during development by the estrogenic, as well as the androgenic metabolites of testosterone.

Male pheromone stimulates ovarian follicular development and body growth in juvenile female opossums (Monodelphis domestica).

Female opossums are induced into estrus by direct exposure to a non-volatile pheromone in male scentmarks. Juvenile females develop this responsiveness by 150 days of age (days), and earlier (130 days) if exposed to male pheromone beginning at 90 days. The goal of this study was to examine the effect of male pheromone on body growth and ovarian follicular development in young opossums. Females (n = 28) were weaned at 56 days and caged individually with ad libitum food and water from 84 days. Body mass was recorded 2-3 times weekly and the onset of estrus was monitored by urogenital sinus cytology. Exposed females were given continuous access to adult male scentmarks from 90 days and randomly selected for necropsy at 105 and 130 days. Unexposed females were isolated from direct contact with males and their scentmarks and randomly selected for necropsy at 90, 105, 130, and 150 days. Exposed females were larger (63.5 +/- 1.1 g) than unexposed females (56.6 +/- 1.1 g) at 130 days, and 4 of 5 had expressed estrus or proestrus. Uterine mass at 130 days was higher (P < 0.05) in exposed (129.8 +/- 28.8 mg) than in unexposed (25.4 +/- 6.7 mg) females, none of which expressed estrus by 150 days. The mean number of developing, antral follicles per female increased from 1.5 +/- 1.5 at 90 days to 17.8 +/- 5.2 at 130 days. Mean diameter of developing antral follicles at 130 days was larger in exposed (534 +/- 54 microm) than in unexposed females (393 +/- 4 microm). The results of this study demonstrate that pheromonal induction of first estrus in juvenile opossums is associated with an increased rate of body growth and follicular development.