Charles V. Mobbs

Sexual stimulation activates c-fos within estrogen-concentrating regions of the female rat forebrain.

Regions of the brain that concentrate estrogen and progesterone are thought to regulate female sexual behavior by altering gene expression and neural sensitivity to afferent stimulation. We used immunocytochemistry and in situ hybridization to examine c-fos gene expression within estrogen-concentrating regions of the forebrain following various types of sexual stimulation with or without hormone treatment. Ovariectomized rats received injections of estradiol benzoate 48 h and progesterone 4 h before testing. Control rats that had been ovariectomized at least 5 months before testing did not receive hormone treatment. Rats were then either placed into bilevel testing chambers with sexually vigorous males, received manual stimulation of the flanks, received vaginocervical stimulation with a glass rod, or were left in their home cages. Copulation with intromission and ejaculation in hormone-treated rats, or stimulation of the vaginal cervix in both hormone-treated and control rats, produced a dramatic induction of c-fos mRNA and Fos-like immunoreactivity in estrogen-concentrating regions, such as the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, ventromedial hypothalamus, lateral habenula, and medial amygdala, in addition to regions that do not readily concentrate estrogen, such as the neocortex, thalamus, and striatum. Mechanical stimulation of the flanks produced a smaller induction of Fos in these rats, whereas hormone treatment alone had no effect. These data demonstrate that afferent sensory stimulation, but not estrogen or progesterone, regulates c-fos gene expression within different estrogen-concentrating and non-concentrating regions of the female rat forebrain.

Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone

Estrogen has been shown to increase proenkephalin (PE) mRNA levels in neurons of the ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM). In this series of experiments, we examined the temporal qualities of this induction by determining both the latency of the estrogen-induced elevation in PE mRNA levels and the rate at which the message levels decline following removal of estrogen. In addition we have examined the effects of progesterone on PE gene expression in the VL-VM of estrogen-primed rats. The latency of the estrogen-induced elevation in PE mRNA levels was found to be relatively short: PE mRNA levels were increased 2-fold within 1 h of estrogen replacement. Following estrogen removal the levels of PE mRNA declined rapidly. Progesterone treatment attenuated this decline, prolonging the estrogen-induced elevation of PE mRNA levels. These results suggest that estrogen rapidly increases PE mRNA levels through a mechanism that probably involves alterations in both the rate of appearance and the rate of degradation of the message. Together, the short latency of the estrogen-induced elevation and the rapid rate of decay following estrogen removal indicate that PE gene expression is highly sensitive to fluctuating estrogen levels. The effect of progesterone suggests that this enkephalinergic system may be regulated by both estrogen and progesterone during the estrous cycle.

Infusion of antisense oligodeoxynucleotides to the oxytocin receptor in the ventromedial hypothalamus reduces estrogen-induced sexual receptivity and oxytocin receptor binding in the female rat

Exogenous administration of the neuropeptide oxytocin reliably facilitates sexual behavior in the female rat and exposure to estrogen increases oxytocin receptor (OTR) binding in the ventromedial nucleus (VMN) of the hypothalamus. We have used a novel approach to investigate the role of hypothalamic OTR in controlling behavior by infusing antisense oligodeoxynucleotides (oligo) to the 5-region of the human OTR mRNA into the VMN of hormonally primed rats. Control infusions consisted of a scrambled-sequence oligo that had little or no homology to known mRNAs. OTR antisense oligo infusion significantly reduced lordosis frequency and intensity in females primed with estrogen. There was also a significantly greater number of rejection behaviors exhibited by antisense-oligo-infused estrogen-treated females versus controls and no evidence of decreased locomotion by either treatment. In contrast to the effects in estrogen-primed-females, when females were primed to be sexually receptive with estrogen plus progesterone, OTR antisense-oligo infusion had no effect on sexual behavior. The lack of effectiveness of OTR antisense oligo in females primed with progesterone may be the result of the action of this steroid on other neurotransmitter systems that also facilitate lordosis and thereby override a deficit in oxytocin binding. Alternatively, via previously described mechanisms, progesterone may enhance the effectiveness of oxytocin binding at its receptor. In vitro receptor autoradiography in estrogen-primed females indicated a 31% reduction in VMN OTR binding in the vicinity of the cannula tip in antisense-oligo-infused females compared to controls. There was no significant difference in the level of OTR binding in the central nucleus of the amygdala.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat

Hormone effects on proenkephalin (PE) mRNA allow an opportunity to compare a brain region-specific molecular change with a quantifiable behavior. Slot blots were used to measure PE mRNA levels in the ventromedial hypothalamus (VMN) and preoptic area (POA) as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PE mRNA level in VMN, while only a small effect was observed in POA at the higher estradiol doses. Lordosis behavior, assessed manually and in mating tests, also increased monotonically with estradiol dose. The data indicate that an apparent threshold level of PE mRNA in VMN coincided wit display of behavior, and suggest further that high levels of PE mRNA, alone, are not sufficient for lordosis. While the exact relationship of the eventual product, Met-enkephalin, to female reproductive behavior remains to be determined, the parallel changes in PE mRNA level and behavior encourage further analysis.

Roles of second-messenger systems and neuronal activity in the regulation of lordosis by neurotransmitters, neuropeptides, and estrogen: A review

Many neurotransmitters and neuropeptides can affect the rodent feminine sexual behavior, lordosis, when administered in the ventromedial hypothalamus (VMH), midbrain central gray (MCG), or other brain regions. A survey of the electrophysiological and biochemical actions of these neural agents revealed that there is a very consistent association between lordosis facilitation with both the activation of the phosphoinositide (PI) pathway and the excitation of VMH and MCG neurons. In contrast, lordosis inhibition is associated, less consistently, with alterations of the adenylate cyclase (AC) system and the inhibition of neuronal activity. The findings that lordosis could be facilitated by going beyond membrane receptors and directly activating the PI pathway, suggest that this second-messenger pathway is a common mediator for the lordosis-facilitating agents. Furthermore, as in the case of stimulating membrane receptors, direct activation of this common mediator also requires estrogen priming for lordosis facilitation. Therefore, it is likely that the PI pathway is modulated by estrogen in the permissive action of estrogen priming. Indeed, a literature review shows that estrogen can affect selective isozymes of key enzyme families of the PI pathway at various levels. Such selective modulations, at several levels, could easily alter the course of a PI cascade; thence, the eventual functional outcome. These findings prompt us to propose that estrogen enables lordosis to be facilitated by a selective modulation of the PI pathway.

Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in situ Hybridization Study

We have used in situ hybridization to investigate estradiol regulation of estrogen receptor (ER) mRNA in regions of the mediobasal hypothalamus which contain ER and are related to specific neuroendocrine functions. Ovariectomized rats were treated with oil or 10 μg estradiol benzoate for 2, 4, 18 or 24 h. Brains were sectioned and hybridized with a [3 H]single‐stranded DNA probe prepared from the pORF cDNA of the human ER gene and exposed to autoradiographic emulsion for 4 months. Specificity of labeling was determined by counting the number of grains over cells in hypothalamic regions known to bind estradiol, compared to cells in the thalamus and cortex, and by comparing with sections pretreated with ribonuclease or hybridized with a [3 H]single‐stranded message‐sense (control) probe. Labeling for ER mRNA was distributed differently than glucocorticoid and thyroid hormone receptor mRNAs, and was regulated by estrogen differently than progestin receptor mRNA. These differences indicated specific hybridization for ER mRNA. ER‐expressing cells constituted 11.5% of the cells in the dorsomedial nucleus, 30% of the cells in the arcuate nucleus and 43% in the ventromedial nucleus, in close accordance with previous studies of ER autoradiography and binding. Quantitative analysis showed that the highest level of ER mRNA was present in the ovariectomized controls. ER mRNA levels fell 42% (ventromedial), 64% (arcuate), or remained unchanged (dorsomedial) 18 h following estradiol benzoate treatment. The pattern of decrease was similar for cells in the ventromedial nucleus and arcuate nucleus. These data show that estrogen regulation of ER mRNA in brain parallels that reported for MCF‐7 cells and rat uterus. These results also demonstrate that in situ hybridization can be used to detect and measure the relative level of a low abundance mRNA in a heterogeneous tissue in which only 12% to 40% of the cells in limited regions express the message.

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.

PLC-α: A common mediator of the action of estrogen and other hormones?

Abstract The phosphatidyl inositol (PI) second messenger pathway may mediate diverse effects of estrogen, including its potentiation of the effects of other hormones. Both estradiol (E2) and luteinizing hormone-releasing hormone (LHRH) induce a putative isoform of PI-specific phospholipase C-α (PLC-α). PLC-α catalyzes PI hydrolysis, which in turn can increase protein kinase C (PKC) activation, Ca2+ mobilization, and arachidonic acid metabolism. Estrogen activates the PI pathway, and components of the PI pathway can mimic or enhance some effects of estrogen. Furthermore, estrogen potentiates effects of several hormones (e.g., LHRH, prolactin, and insulin) which can also act through the PI system. PLC-α may therefore provide a common second messenger pathway mediating the potentiation by E2 of the effects of other hormones; in addition it may also mediate some or all of the many actions of E2, since components of the PI pathway can have secretory, trophic, toxic, and neuromodulatory effects.

Changes in preproenkephalin messenger RNA level in the rat ventromedial hypothalamus during the estrous cycle

To gain a better understanding of the relationship between the female rat reproductive system and preproenkephalin (PPE) expressing neurons under physiological conditions, we examined changes in PPE mRNA levels in the mediobasal hypothalamus during the rat estrous cycle by means of northern blotting and in situ hybridization histochemistry (ISHH). In the Northern blot studies, we found that PPE mRNA levels in the mediobasal hypothalamus were significantly increased by noon of proestrus compared to those in the morning and stayed high until diestrus day 1, and returned toward low levels on diestrous day 2. In contrast, measured as controls, glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were significantly higher on proestrus regardless of time of day compared to diestrus day 2, and levels of calcineurin mRNA on proestrous and estrous were significantly lower than diestrous day 1 and day 2. ISHH studies revealed that these changes in PPE mRNA levels were specific in the ventromedial hypothalamic nucleus pars ventrolateralis (VMHVL), since we could not see any significant changes in signal in other parts including ventromedial hypothalamic nucleus pars dorsomedialis and arcuate hypothalamic nucleus. In the VMHVL, PPE mRNA levels in the afternoon of proestrous were significantly higher than those in the afternoon of diestrous day 2 whereas no significant change in PPE mRNA was observed in the caudate-putamen. The present study provides additional information relevant to possible implications of PPE gene expression in female reproductive systems, since changes in PPE mRNA levels may be associated with estrogen as well as progesterone or other hormonal concentrations during the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in the regulation of heat shock protein 70 kDa and 90 kDa in the rat ventromedial hypothalamus by estrogen

The regulation of heat shock proteins 70 kDa (hsp 70) and 90 kDa (hsp90) by estradiol (EB) was examined in the ventromedial hypothalamus (VMH) of gonadectomized adult female and male rats with and without estradiol replacement. Estradiol significantly elevated hsp70 and hsp90 protein concentrations in EB-treated VMH of females, but not males, sacrificed at 12 h following steroid hormone compared with oil-vehicle controls. These findings confirm our previous observations that EB induces hsp70 and hsp90 in the female VMH16,17. The sex differences for the two proteins may occur by different mechanisms, since basal hsp70 levels were higher in males than females, whereas hsp90 levels were similar for the two sexes.