Estrogen (E) and progesterone exert profound influence on development and reproduction. In vitro, steroid receptor coactivators (SRCs) are nuclear proteins that interact with DNA-bound steroid receptors to potentiate their transcriptional efficiency. We examined the effects of antisense oligonucleotides to SRC-1, SRC-2, and SRC-3 on female sexual behavior and steroid receptor-mediated transcription. Rat (r) SRC-1, rSRC-2, and rSRC-3 genes were cloned. Our results reveal a significant inhibitory effect by antisense (AS) to SRC-1 and SRC-2, but not SRC-3, on hormone-induced reproductive behavior. Importantly, sexual behavior was attenuated through estrogen receptor alpha (ERalpha)-dependent, rather than progesterone receptor (PR)-dependent, transcription, as E failed to induce the synthesis of PR content in the medial basal hypothalamus, and immunoreactive PR in the ventromedial nucleus were depleted in tissue from rSRC-1-AS- and rSRC-2-AS-treated, but not rSRC-3-AS-treated, rats primed with E. Consistent with interruption of ERalpha-induced transcription, high dose of E and epidermal growth factor alone failed to induce sexual behavior in females treated with either rSRC-1-AS or SRC-2-AS. Immunoreactive SRC-1 and SRC-2, but not SRC-3, proteins were abundant in the ventromedial nucleus, thus demonstrating that the biological activities of hypothalamic steroid receptors are selectively regulated by regional distribution of specific SRCs. As SRC-1 knockout mice have only a slight loss in reproductive function, the possibility that genetic adaptation occurs during development was tested. Mouse (m) SRC-1-AS suppressed lordosis in wild-type, but not SRC-1, knockout mice, whereas mSRC-2-AS suppressed behavior in both genotypes. mSRC-3-AS had no effect in either genotype, and SRC-3 knockout mice exhibited full receptivity. Collectively, the findings clearly implicate dual regulation of ERalpha-dependent function by SRC-1 and SRC-2 in the intact female brain. In the genetic, but not acute, absence of SRC-1, up-regulation of SRC-2 serves as a critical adaptive mechanism during female development.

Adaptation to stress in vertebrates occurs via activation of hormonal and neuronal signaling cascades in which corticotropinreleasing hormone (CRH) plays a central role. Expression of brain CRH is subject to strong, brain-region specific regulation by glucocorticoid hormones and neurogenic intracellular signals. We hypothesized that Steroid Receptor Coactivator 1 (SRC-1), a transcriptional coregulator of the glucocorticoid receptor, is involved in the sensitivity of CRH regulation by stress-related factors. In the brains of SRC-1 knockout mice we found basal CRH mRNA levels to be lower in the central nucleus of the amygdala. Hypothalamic CRH up-regulation after chronic (but not acute) stress, as well as region-dependent up-and down-regulation induced by synthetic glucocorticoids, were significantly attenuated compared with wild type. The impaired induction of the crh gene by neurogenic signals was corroborated in AtT-20 cells, where siRNA and overexpression experiments showed that SRC-1 is necessary for full induction of a CRH promoter reporter gene by forskolin, suggestive of involvement of transcription factor CREB. In conclusion, SRC-1 is involved in positive and negative regulation of the crh gene, and an important factor for the adaptive capacity of stress.adaptation ͉ amygdala ͉ HPA axis ͉ neuroendocrinology ͉ transcription B rain corticotropin-releasing hormone (CRH) plays a pivotal role in the mammalian response to stress. CRH synthesized by neurons in the central nucleus of the amygdala (CeA) mediates the effect of stress on emotional states, including fear and anxiety (1, 2). From the paraventricular nucleus of the hypothalamus (PVN) CRH coordinates autonomic outflow and the activity of the hypothalamus-pituitary-adrenal (HPA) axis. The latter leads to secretion of the adrenal glucocorticoid hormones, which in turn orchestrate the adaptation to stress of virtually all tissues in the body (3-5).As part of adaptation, CRH expression itself is strongly regulated in response to stress-induced elevations of glucocorticoids and neurogenic signals. In the core of the HPA-axis, activation of the glucocorticoid receptor (GR) can repress transcription from the crh gene as part of negative feedback, whereas stress-related noradrenergic and glutamatergic excitatory signals can activate the gene, in part via activation of the transcription factor CREB. In the CeA, both GR activation and excitatory signals can lead to an increased CRH expression (6, 7). These modulations are considered crucial for stress adaptation, and a considerable amount of research has been devoted to understand how transcriptional signals are integrated at the level of the CRH promoter, both in vitro and in vivo (8, 9). Nevertheless, the regulation of the crh gene in vivo is far from understood.Transcriptional coregulators constitute an expanding class of molecules that act as mediators and integrators of signals carried by nuclear receptors, such as GR (10). The p160 coregulator Steroid Receptor Coactivator (SRC-1) is strongly expressed in br...

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. Key words: steroid receptors; D5 dopamine receptors; lordosis; antisense oligonucleotides; progesterone; estrogenIn the female rat, the ovarian steroid hormone estrogen modulates the reproductive behavior lordosis (Pfaff and SchwartzGiblin, 1988). Estrogen effect on lordosis is mediated in part by steroid receptors in the estrogen-concentrating neurons located in the ventrolateral region of the hypothalamic ventromedial nucleus (VMN). The major effect of estrogen on lordosis is thought to be mediated by progesterone receptors (PR), because (1) estrogen induces PR in the VMN, and (2) RU38486 and PR antisense oligonucleotide block the induction of lordosis by progesterone (P) (Pfaff and Schwartz-Giblin, 1988;McCarthy et al., 1993). Thus, the dependence of lordosis on estrogen and progestins can be used in vivo to probe cellular and molecular mechanisms for mammalian behavior.As ligand-dependent nuclear transcription factors, steroid receptors alter the expression of specific genes or gene networks (Evans, 1988). However, the PR can be activated in vitro in a ligand-independent manner by the neurotransmitter dopamine (Tsai and O'Malley, 1994). Dopamine is thought to act selectively through D1-like and D2 receptors (Grierson et al., 1988). Centrally administered dopamine (Mani et al., 1994) and the D1-like agonist SKF38393 (Foreman and Moss, 1979) induce lordosis in rats primed with low doses (5-10 g) of estroge...

Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition.

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.