Joanne C. Turcotte

Estradiol-induced progestin receptor immunoreactivity is found only in estrogen receptor-immunoreactive cells in guinea pig brain.

A fluorescent immunocytochemical technique was developed to determine if cells in the guinea pig hypothalamus and preoptic area that contain estradiol-induced progestin receptors also contain estrogen receptors. With this technique little or no progestin receptor-immunoreactivity (PR-IR) was observed in the absence of estrogen treatment in ovariectomized guinea pigs. As has been reported previously, priming with estradiol caused a large increase in the concentration of PR-IR cells in discrete regions of the hypothalamus and preoptic area, primarily in the arcuate nucleus, ventrolateral area of the hypothalamus, periventricular preoptic area, medial preoptic nucleus, medial preoptic area, anterior hypothalamic nucleus and anterior hypothalamus. A range of lightly to intensely labeled estrogen receptor-immunoreactive (ER-IR) cells were observed in high concentration in each of these areas, as well as in some areas in which no PR-IR cells have been identified, such as the amygdala. PR-IR was only observed in cells that also had ER-IR. In some areas such as the ventrolateral hypothalamic area and arcuate nucleus, nearly all medium to highly-fluorescent ER-IR cells also contained estradiol-induced PR-IR, while in the amygdala no PR-IR was observed despite a high concentration of ER-IR cells. These results confirm the hypothesis that progestin receptors are produced in estrogen receptor-containing cells in the brain, and they suggest that these cells are the sites where estradiol and progesterone act to influence behavior and physiology.

Immunocytochemical localization of estrogen-induced progestin receptors in guinea pig brain

By using a combination of monoclonal antibodies to progestin receptors and a double-bridge peroxidase-antiperoxidase technique, a sensitive immunocytochemical method was developed for visualizing progestin receptor immunoreactivity (PR-IR) in brains of estrogen-primed guinea pigs. PR-IR neurons were observed throughout the hypothalamus and preoptic area. They were seen in largest numbers in the arcuate nucleus, periventricular preoptic regions, medial preoptic nucleus, medial preoptic area and in the ventrolateral area of the hypothalamus. Lower numbers of PR-IR positive cells were detected in the bed nucleus of stria terminalis, paraventricular nucleus and lateral hypothalamus with scattered cells seen throughout the hypothalamus and preoptic area. The PR-IR was mostly intranuclear with lighter staining in neuronal processes. Electron microscopy confirmed the predominantly intranuclear localization and further demonstrated that the reaction product was dispersed throughout the nucleus, but not associated with the nucleolus. Few PR-IR cells were observed in the absence of estradiol priming, and the reaction product was much lighter than in the presence of estradiol. Progesterone injection was without apparent effect on intensity of the PR-IR.

Estrogen receptor-immunostaining of neuronal cytoplasmic processes as well as cell nuclei in guinea pig brain

We have recently developed an immunocytochemical technique for staining estrogen receptors in cell nuclei of some cells in the guinea pig brain. With optimization of this technique, we have now been successful in staining estrogen receptor-immunoreactivity in processes of many neurons in the guinea pig brain that also contain estrogen receptor-immunoreactive cell nuclei. While reaction product is visible in cell nuclei under a wide variety of conditions, neuronal processes are darkly immunostained only with modifications of the procedure optimized for maximum sensitivity, for example, the multiple-bridge immunocytochemical procedure. Omission of Triton X-100 and/or dimethylsulfoxide, usually used to increase penetration of the antibodies, had no effect on immunostaining in processes or cell nuclei, and immunostaining was apparent in both vibratome-cut and freezing microtome-cut sections. Injection of estradiol, but not progesterone, caused the total loss of cytoplasmic estrogen receptor-immunostaining, consistent with the idea that the cytoplasmic immunostaining, like the cell nuclear immunostaining is due to the presence of estrogen receptors. In all neuroanatomical regions containing estrogen receptor-immunoreactive cell nuclei, associated processes of some, but not all cells were also immunostained. However, in certain areas, such as the midbrain central gray and the preoptic area, cytoplasmic staining was particularly dark. The cellular characteristics that result in immunostaining in some estrogen receptor-immunoreactive cells, and not in others is under investigation.

A Subgroup of LHRH Neurons in Guinea Pigs with Progestin Receptors Is Centrally Positioned within the Total Population of LHRH Neurons

Although the role of gonadal steroids in inducing the LH surge is undisputed, the mechanism(s) whereby steroids induce the release of the hypothalamic luteinizing hormone-releasing hormone (LHRH) remain(s) enigmatic. In this study we examined the issue of the presence of steroid receptors in LHRH neurons using a mammalian species that has a true luteal phase, namely, guinea pigs. Progestin receptors (PR) were localized in LHRH neurons of ovariectomized guinea pigs administered estradiol (10-20 micrograms estradiol benzoate) for 3-4 days, using several different immunocytochemical protocols. The subgroup of LHRH neurons containing PR, although small, was strategically positioned within the core of the total population of LHRH neurons. This central position was visualized in simultaneous views of three-dimensional computer reconstructions of the populations of LHRH/PR neurons and LHRH neurons. The subgroup of LHRH/PR neurons formed a thread permeating the population of LHRH neurons. We propose that in guinea pigs, LHRH neurons containing progestin receptors, are foci of activity, capable of activating a larger component of the LHRH population of cells in certain endocrine conditions, such as prior to the LH surge.

Estrogenic effects of zearalenone on the expression of progestin receptors and sexual behavior in female rats

Zearalenone is a resorcylic acid lactone compound that is produced by fungal infection of edible grains and is believed to influence reproduction by binding to estrogen receptors. In order to study the potential estrogenic effects of this compound in the brain, we examined the effects of zearalenone on the expression of neuronal progestin receptors and feminine sexual behavior in female rats. Ovariectomized rats were treated with zearalenone (0.2, 1.0, or 2.0 mg), estradiol benzoate, or vehicle daily for 3 days. They were then either perfused, and progestin receptors visualized by immunocytochemistry, or injected with progesterone and tested for sexual receptivity with male rats. Progestin receptor-containing cells were counted in the medial preoptic area and ventromedial hypothalamus. The two highest doses of zearalenone increased the concentration of neuronal progestin receptors, as did 10 microg of estradiol. The highest dose of zearalenone (2 mg) also induced progestin receptor staining density comparable to that of 10 microg of estradiol benzoate. In behavioral tests, ovariectomized animals treated with 2 mg of zearalenone followed by progesterone showed levels of sexual receptivity comparable to females treated daily with estradiol benzoate (2 microg) followed by progesterone. These studies suggest that, although structurally distinct and less potent than estradiol, zearalenone can act as an estrogen agonist in the rat brain.

Hypothalamic ovarian steroid hormone-sensitive neurons involved in female sexual behavior.

Estradiol and progesterone regulate sexual behaviors in guinea pigs and rats, at least in part, through interaction with intracellular steroid hormone receptors. In the present review of work from the laboratory of the authors, we summarize recent work which has focused on one of the sites of hormone action in female guinea pigs--the ventrolateral hypothalamus. We summarize results of earlier experiments in which the regulation of steroid hormone receptors in this area was assessed after hormonal treatments with predictable effects on female sexual behavior. We then review the results of recent tract-tracing experiments in which we have examined the projections from the steroid receptor-immunoreactive neurons in this region, as well as the afferent projections from other neuroanatomical areas, including neurons which themselves contain estrogen receptors. We also present studies on the afferent input into these neurons by noradrenergic neurons and discuss the possibility that noradrenergic input influences steroid hormone sensitivity in these neurons. Finally, we discuss the results of experiments in which Fos-immunocytochemistry was used in rats to identify the neurons responding to a particular tactile stimulus associated with female reproduction, i.e., vaginal-cervical stimulation. These experiments further define a complex neuroanatomical system, in which many of the elements are estradiol or progesterone-responsive, that is involved in the hormonal regulation of sexual behavior in guinea pigs and rats.

A Small Population of Tyrosine Hydroxylase‐lmmunoreactive Neurons in the Guinea‐Pig Arcuate Nucleus Contains Progestin Receptor‐lmmunoreactivity

We studied the co‐localization of progestin receptor‐immunoreactive (PR‐IR) cell nuclei and tyrosine hydroxylase‐immunoreactive (TH‐IR) cell bodies in guinea‐pig brain with a double antibody, immunocytochemical technique. Sections were first immunostained for estradiol‐induced PR‐IR using a peroxidase‐antiperoxidase technique with diaminobenzidine as the chromogen followed by α‐naphthol as the chromogen for TH‐IR. We examined the periventricular‐preoptic area and the arcuate nucleus, because these two sites are dense in both PR‐IR cells and TH‐IR cells (cell groups A14 and A12, respectively), and the dorsal hypothalamic Area A13, because this area contains a high density of TH‐IR cells, but few PR‐IR cells. No co‐localization was seen in the periventricular‐preoptic area or Area A13. However, a small proportion (5% to 13%) of TH‐IR cells in the arcuate nucleus was observed to have PR‐IR cell nuclei with the rostral arcuate showing the greatest concentration of co‐localized cells. In order to determine if the estradiol pretreatment required to induce PR‐IR influenced TH‐IR, TH‐IR in estradiol‐primed guinea‐pigs was compared with that of vehicle‐injected controls. This treatment did not noticeably influence the amount of TH‐IR in the arcuate nucleus. Therefore, the results of these experiments suggest that, although some of the TH‐IR neurons in the arcuate nucleus contain PR‐IR, this relationship is seen in less than 15% of the TH‐IR cells. In many cases, PR‐IR neurons were found to have TH‐IR varicosities closely associated with their cell bodies.

Further evidence of noradrenergic regulation of rat hypothalamic estrogen receptor concentration: possible non-functional increase and functional decrease.

The regulation of estrogen receptors by the alpha 2-noradrenergic system was studied. A single injection of the alpha 2-noradrenergic antagonist, yohimbine, caused a biphasic effect on the concentration of cytosol estrogen receptors in the mediobasal hypothalamus and anterior pituitary gland. A short-latency increase was seen at 1.5-3 h, followed by a longer-lasting decrease at 8-16 h. Scatchard analysis revealed that the apparent, short-latency increase is in the concentration of binding sites, not in the affinity of the receptor for [3H]estradiol. The increase in the concentration of cytosol estrogen receptors is not blocked by pretreatment with the alpha 2-noradrenergic agonist, clonidine. In addition, no increase is detected in the concentration of cell nuclear estrogen receptors accumulating in response to a saturating dose of estradiol. Therefore, the apparent increase in the concentration of cytosol estrogen receptors may not represent a functional increase in receptors. The decrease in the concentration of estrogen receptors, which occurs 8-16 h after yohimbine treatment, is also seen after injection of the alpha 2-adrenergic antagonist, idazoxan, and is not due to a change in the in vitro rate of association of the receptors with [3H]estradiol. Furthermore, the decrease seems to be a functional decrease in the concentration of receptors capable of cell nuclear accumulation in response to estradiol injection, as indicated by the results of experiments in which the concentration of cell nuclear estrogen receptors was assayed after estradiol injection. These experiments provide further support for the hypothesis that the alpha-noradrenergic system, and perhaps specifically the alpha 2-subtype, is involved in decreasing the concentration of estrogen receptors in parts of the brain and pituitary gland. This interaction provides a mechanism by which the environment could regulate the sensitivity of certain neurons to estradiol. However, the finding that the initial increase in the concentration of cytosol estrogen receptors after yohimbine treatment is not followed by the predicted increase in cell nuclear estrogen receptors after estradiol injection raises questions about the physiological relevance of the apparent increase under some conditions.

Projections of the Estrogen Receptor-Immunoreactive Ventrolateral Hypothalamus to Other EstrogenReceptor-Immunoreactive Sites in Female Guinea Pig Brain

The ventrolateral hypothalamus in female guinea pigs includes an estrogen receptor dense region adjacent to the ventromedial hypothalamus. This region is reciprocally connected with other estrogen receptor-containing areas suggesting that steroid hormone receptor-containing cells may be directly linked. Phaseolus vulgaris leucoagglutinin, an anterograde tract tracer, was specifically placed in this region with the aim of labeling some projections from estrogen receptor-containing neurons. These projections were colocalized immunocytochemically with the distribution of estrogen receptor-containing cells. Dense ventrolateral hypothalamic innervation was observed in some regions also containing a high concentration of estrogen receptor-containing cells. These regions included the medial preoptic area, the bed nucleus of the stria terminalis, the ventrolateral hypothalamus anterior and posterior to the injection site, and the midbrain central gray. A low density of ventrolateral hypothalamic fibers and terminals was observed in two regions rich in estrogen receptors, the amygdala and the arcuate nucleus. In general, ventrolateral hypothalamic fibers and terminals were present in all regions where estrogen receptors were found except the medial thalamus and habenular region. Labeled terminal boutons and perineuronal baskets were found around estrogen receptor-containing cells in most regions which contained estrogen receptor-containing cells. These close appositions were suggestive of synaptic contacts, suggesting that the ventrolateral hypothalamus may influence steroid-dependent behaviors via the modulation of estrogen receptor-containing cells. Furthermore, ventrolateral hypothalamic projections may include direct connections with estrogen receptor-containing cells, suggesting the presence of a network of interconnected estradiol-sensitive neurons involved in the regulation of estradiol-dependent functions.

Efferent Projections from the Ovarian Steroid Receptor‐Containing Area of the Ventrolateral Hypothalamus in Female Guinea Pigs

The ventrolateral hypothalamus (VLH) in female guinea pigs includes a subset of neurons which contain estrogen and progestin receptors, and which are implicated in the regulation of female sexual behavior by steroid hormones. However, little is known about where these neurons project, and consequently which other brain areas are involved in sexual behavior in female guinea pigs. The anterograde tracer Phaseolus vulgaris‐Leucoagglutinin was used to label efferents from the ovarian steroid receptor‐containing part of the VLH. To identify the correct placement of the tracer specifically within the group of neurons containing estrogen receptors, medial hypothalamic sections were also immunostained for estrogen receptors. Forebrain areas receiving dense projections from the ventrolateral hypothalamus included the bed nucleus of the stria terminalis, medial preoptic area, anterior hypothalamic area, anterior ventromedial hypothalamus, and caudal ventrolateral hypothalamus. The midbrain central gray was also heavily labeled. Moderate innervation was observed in the forebrain in the basolateral amygdala, medial preoptic nucleus, lateroanterior hypothalamic nucleus, dorsal hypothalamic areas, posterior hypothalamus, zona incerta, and in the midbrain interspersed among the central and lateral tegmental tracts. The major efferent pathways from the VLH appeared to travel rostrally through the mediobasal hypothalamus and preoptic area, and caudally via the medial thalamic nuclei and periventricular fiber system. These findings are similar to those of previous studies tracing the efferents from the ventromedial nucleus in rats and from the lateral hypothalamus in guinea pigs. Many of these areas that receive input from the steroid receptor rich area within the VLH are likely to be involved in the regulation of female sexual behavior.