Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents.

Sex steroids exert profound influence on neural development and function through activation of intranuclear receptors. However, during sexual differentiation and at onset of puberty, intracerebral estrogen (E) availability is subsequent to these effects. The potent mitogen epidermal growth factor (EGF) activates estrogen receptor (ER)-dependent transcription in cultured cells in the absence of exogenous E. Since reproductive behavior in female rodents is the result of E-dependent transcriptional activity and protein synthesis, lordosis serves as a well established in vivo model for probing cellular and molecular mechanisms of steroid receptor-dependent behavior. Here we demonstrate that EGF can signal through the classical E receptor (ERalpha) to alter in vivo function in rodent central nervous system. EGF and EGF receptor ligands induced lordosis in a dose- and time-dependent manner in the absence of steroid treatment in ovariectomized rats and mice. Using antisense oligonucleotides, pharmacological and antibody blockade, and mutant mice, we also report that this behavioral responsiveness is mediated through ERalpha by specific stimulation of membrane-bound EGF receptors and EGF receptor-specific tyrosine kinase rather than by direct ligand activation of the ERalpha. Of biological significance, delayed onset of puberty and the absence of synchronization between reproductive behavior and ovulation was detected in intact mutant Wa-2 mice that express a naturally occurring point mutation in the EGF receptor. To our surprise, EGF-mediated behavior was independent of progesterone (P) and progesterone receptor (PR) since antiprogestins, PR antisense oligonucleotides, and targeted disruption of PR in ovariectomized transgenic mice failed to impede the display of lordosis after EGF. Finally, we also found that another growth factor, insulin-like growth factor-1, which provokes ER-dependent transcription in vitro, activates mating behavior in a similar E-independent manner. Thus, growth factor mediation of ER-targeted function may be a universal feature in the rodent central nervous system, raising critical questions about the role of growth factors in mediating ER-dependent processes in development and reproduction.

Distribution of D(5) dopamine receptor mRNA in rat ventromedial hypothalamic nucleus.

Estrogen induces lordosis through, in part, estrogen receptor (ER)-mediated synthesis of progesterone receptors (PR) in the ventromedial nucleus (VMN). In vitro, PR is activated by the neurotransmitter dopamine through D1-like receptors (1). In vivo, lordosis is induced by dopamine, an effect mediated in part by PR and D(5) dopamine receptors. The purpose of the present study was to determine mRNA distribution of D1-like receptors in the female rat brain using RT-PCR combined with punchout microdissection techniques. Employing specific primers to D(5) and D(1) dopamine receptors, we found detectable expression levels of D(5) dopamine receptor mRNA in VMN as well as the arcuate nucleus/median eminence (ArcN/ME). In contrast, D(1) dopamine receptor mRNA was detected only in VMN. By using this highly sensitive and specific RT-PCR methodology, we have confirmed the presence of D(5) dopamine receptor mRNA in an area of the brain that regulates reproductive behavior through PR. The data support the previous observation that D(5) dopamine receptors in VMN contribute to facilitation of female reproductive behavior by D1-like agonists.

In vivo regulation of central nervous system progesterone receptors: cocaine induces steroid-dependent behavior through dopamine transporter modulation of D5 receptors in rats.

To characterize the membrane pathway by which the cocaine-sensitive dopamine transporter (DAT) modulates progesterone receptor activation, steroid-dependent behavior lordosis was used in estrogen-primed ovariectomized Sprague-Dawley rats with stereotaxic implanted third ventricle cannulas. Lordosis in response to solicitous males was observed in females after intercerebral ventricular administration of DAT antagonists WIN35,428 (80 ng) and cocaine (0.016-1.6 micrograms). Significantly, antisense oligonucleotides (AS) to DAT mRNA also induced reproductive behavior. In contrast, the D1-D2 receptor membrane-repopulation inhibitor N-ethoxycarbonyl-2 ethoxy-1,2-dihydroquinoline and the D1-like antagonist SCH23390 blocked cocaine-inducible behavior. Further, facilitation of behavior by AS to the DAT was suppressed by N-ethoxycarbonyl-2 ethoxy-1,2-dihydroquinoline. Behavior was not dependent on D2 receptors, since animals pretreated with the D2 antagonist sulpride displayed lordosis after cocaine challenge. Antisense oligonucleotides to D5 but not D1 dopamine receptor mRNA suppressed reproductive behavior associated with cocaine. Microinjections of cocaine to the ventromedial nucleus (VMN) but not arcuate nucleus or preoptic area potentiated lordosis, suggesting the functional presence of DAT in the VMN. Finally, cocaine facilitation of behavior was blocked by both antiprogestin RU486 and progesterone receptor AS microinjected into either the third ventricle or the VMN. Collectively, the data provide strong evidence for cocaine modulation of reproductive behavior through presynaptic cocaine-sensitive dopamine transporters and postsynaptic D5 dopamine receptor mediation of progesterone receptor-dependent behavior in rat central nervous system.

Dopaminergic regulation of progesterone receptors: brain D5 dopamine receptors mediate induction of lordosis by D1-like agonists in rats.

To characterize the signaling pathway by which the neurotransmitter dopamine modulates progesterone receptor (PR) activation, the steroid-dependent behavior lordosis was used in estrogen-primed ovariectomized Sprague-Dawley rats with stereotaxic implanted third ventricle cannulas. Lordosis was observed in response to solicitous males in females after central administration of the D1-like agonist SKF38393 and three of its analogs (SKF77434, SKF75640, and SKF85174). In contrast, D1-like antagonist SCH23390 and D1-like/D2 repopulation inhibitor EEDQ blocked behavior inducible by the D1-like agonists. Further, antisense oligonucleotides to D5, but not D1, dopamine receptor mRNA suppressed reproductive behavior associated with D1-like stimulation. This finding provides strong evidence that dopaminergic modulation of lordosis is mediated by the novel D5 dopamine receptor. Although D1, but not D5, dopamine receptor mRNAs were detected in the ventromedial nucleus (VMN) by in situ hybridization, agonists microinjected into the VMN, but not into the arcuate nucleus or preoptic area, induced lordosis, suggesting the functional presence of D5 dopamine receptors in the VMN. Also in support, D5 receptor mRNA antisense microinjected into the VMN blocked the subsequent induction of lordosis by D1-like agonists. Finally, facilitation of sex behavior by D1-like agonists was blocked by the antiprogestin RU38486 and PR antisense oligonucleotide. Collectively, the data provide strong evidence for dopaminergic modulation of reproductive behavior through D5 dopamine receptor-mediated modulation of PR-dependent behavior in rat CNS.

Hormonal regulation of hypothalamic gene expression: identification of multiple novel estrogen induced genes.

Estrogen (E) has been shown to play a major role in hypothalamic function and is a prerequisite for progesterone (P) induced sexual behavior in female rats. In the course of studies in search of steroid induced hypothalamic genes, we discovered a surprisingly large number of E-induced genes (21 mRNAs in total). This is the largest number of E-induced genes ever identified in a single organ. Many of these mRNAs exhibit considerable magnitudes of induction and their levels were maintained typically during subsequent P treatment. Among the induced genes, several encode metabolic enzymes and may account for some of the morphological changes observed in hypothalamic neurons in response to E. Since E appears to play a major role in defining the pattern of hypothalamic gene expression in conjunction with its capacity for behavioral modulation, these newly identified cDNAs may serve as genetic markers for correlative studies of E-induced central nervous system behavior.

Cortisol feedback regulation of pulsatile ACTH secretion in fetal sheep during late gestation.

In fetal sheep, plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations increase during late gestation to surge within 72 h of birth (approximately 146 days gestation). To determine the feedback role of cortisol in control of pulsatile ACTH secretion, six chronically catheterized fetuses were treated with cortisol (1 microgram/h i.v.) for 96 h at 133 days gestation. Before (133 days), during (134 and 137 days), and after (142 days) cortisol treatment (5-min sampling for 2 h), ACTH pulses were evident in each fetus. At 134 days, ACTH pulse peak, nadir, and estimated secretory rate were significantly increased while frequency, amplitude, mean concentrations, and cortisol binding capacity (CBC) were unchanged. At 137 days, most characteristics of pulsatile ACTH secretion remained enhanced compared with pretreatment controls. At 142 days (96 h postinfusion), ACTH secretion parameters returned to pretreatment levels, but cortisol concentrations remained elevated. Cortisol infusion was then reinitiated at 142 days and, 22-24 h later, parameters of ACTH secretion increased except for amplitude, secretory rate, and CBC activity. The data indicate an absence of cortisol negative feedback regulation of pulsatile ACTH secretion. Rather, the ACTH rise that accompanied cortisol infusion suggests that cortisol exerts a positive feedforward influence on ACTH secretion in the ovine fetus near term.

Time of day of birth and absence of endocrine and uterine contractile activity rhythms in sheep.

To determine whether 24-h rhythms characterize hormone secretion and uterine activity in the pregnant sheep, blood samples were drawn every 1-4 h for 48 h from ewes and fetuses from day 120 of gestation to term. Repetitive 24-h rhythms were absent for cortisol, progesterone, and prolactin in maternal and fetal circulation and for hourly mean uterine contraction rate and amplitude. To test whether photoperiod or pineal melatonin contributes to the absence of rhythms, pineal-intact and pinealectomized ewes and their fetuses were studied in reverse photoperiod. Again, there was little evidence to suggest 24-h endocrine rhythms except for prolactin in two fetuses by cosinor analyses. Prolactin concentrations were increased in pinealectomized ewes and their fetuses. In the apparent absence of rhythms, 20 of 21 pineal-intact ewes gave birth at night; however, 6 out of 7 pinealectomized ewes gave birth during the day. Thus photoperiod and the maternal pineal gland profoundly influenced the time of day of birth in the absence of circadian endocrine or uterine activity rhythms.

Dissociation of pulsatile cortisol and adrenocorticotropin secretion in fetal sheep during late gestation.

In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 +/- 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean +/- SE) decreased between 133 and 146 days from 54 +/- 11 min to 23 +/- 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 +/- 2 ng/ml at 133 days to 44 +/- 13 ng/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 +/- 0.6 pulses/2 h) and amplitude (21 +/- 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term.

Regulation of basal adrenocorticotropin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation.

This study tested the hypothesis that arginine vasopressin (AVP) is involved in the regulation of basal ACTH secretion in the ovine fetus near term. In five fetuses challenged with AVP (1 microgram/ml, iv bolus) plasma ACTH concentrations increased to an 8-fold peak within 10 min of the preceding baseline (55 +/- 6 to 403 +/- 241 pg/ml). Cortisol in fetal circulation subsequently increased 2-fold (11 +/- 1 to 28 +/- 5 ng/ml) within 15 min of the AVP injection. The AVP-induced rise in plasma ACTH and cortisol concentrations was blocked when the fetus was pretreated with the AVP V1 receptor antagonist d(Ch2)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 micrograms/kg estimated BW, iv bolus) was administered to three fetuses, aged 137-147 days gestation, followed 40 min later by the exogenous AVP challenge, as described above. After AVP antagonist treatment, basal ACTH and cortisol concentrations were not significantly different from the preinjection baseline levels (P greater than 0.05, by analysis of variance). Moreover, plasma ACTH and cortisol remained unchanged after the AVP challenge. To further define the role of endogenous AVP in basal ACTH and cortisol secretion, the AVP antagonist was administered (five studies in two fetuses) at 30-min intervals for a total of three injections per fetus. This extended AVP antagonist regimen also failed to alter fetal circulating concentrations of ACTH or cortisol (P greater than 0.05). Cortisol in the maternal circulation was not affected by any of the fetal AVP or AVP antagonist treatments. Lambs were born at 146 +/- 2 days gestation (n = 5), within the range for the normal duration of pregnancy. These data do not support the hypotheses that AVP is involved in the regulation of basal ACTH secretion in the fetal sheep during the 10 days preceding parturition. Rather, the ability of AVP antagonist to block the AVP-induced rise in plasma ACTH and cortisol in the fetus suggests that basal and stimulated ACTH secretion are under separate regulatory mechanisms.