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Dissemination of neoplastic cells from the primary tumor (invasion and metastasis) is a fundamentally dangerous step in multistage carcinogenesis. Recent evidence suggests that Rho GTPase-mediated signaling is linked to dissemination of cells from several different types of human tumors. The Rho family of proteins is typically associated with the regulation of cytoskeletal activity, including actin assembly, microtubule dynamics, and myosin II-dependent contractility of the actin-rich cortex. We examined the effect of overexpression of constitutively active RhoA on islands and monolayers of epithelial cells. Although newly plated cells initially formed small spread islands, there was also a significant population of cells that detached from the substrate, floated in the medium, and then could reattach to the substrate to form new colonies. Detachment of cells from transfected epithelial islands or monolayers occurred in correlation to the plane of cytokinesis after misorientation of the mitotic spindle axis. We suggest that these alterations result from Rho-induced increase of contractility of the cortex of dividing cells, which, during cytokinesis, produces a cell that has budded out of an existing layer of cells. Cell division-mediated detachment of cells from tissue structures may be an important mechanism of tumor dissemination and metastasis.

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special "leader" cells at the tops of outgrowths and "follower" cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior-posterior polarity is unknown. To dissect the roles of microtubule assembly and of Rho-dependent contractility on structural polarization of cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree of polarization by developing a highly elongate cell body with multiple narrow processes extended from the edges of the cell. Treatment of fibroblasts with nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid or polygonal cells having broad, flattened lamellae that did not form long lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not display anterior-posterior polarization. When treated with Y-27632, the cells acquired a polarized, elongate shape with narrow protrusions and wide lamellas. Nocodazole alone or in combination with Y-27632 did not change the discoid shape of epitheliocytes, however treatment with Y-27632 produced thinning of the lamellar cytoplasm. We conclude that microtubules provide the necessary framework for polarization of fibroblasts and epitheliocytes, whereas Rho-regulated contractility modulates the degree of polarization of fibroblasts and completely inhibits polarization in epitheliocytes.

The spatial organization of cell-cell adherens junctions is distinct in cultured cells from two different tissue types, specifically, epitheliocytes and fibroblasts. In epitheliocytes, contacts are localized tangentially, along contacting cell edges and in association with circumferential actin bundles. Contacts between fibroblasts are radially oriented; that is, they are perpendicular to the overlapping edges of the cells and are associated with straight bundles of actin filaments. In the present study, we establish that the spatial organization of cell-cell contacts in the epithelial cell line IAR-2 can be converted from the typical tangential pattern to the radial pattern observed in fibroblasts. This transition can be induced by treatment with two agents, phorbol 12-myristate 13-acetate and nocodazole, which have different modes of action. Inhibition of myosin contractility reverses tangential-to-radial conversion of cell-cell contacts. These data suggest that formation of radially aligned contacts depends on modulation of contractility within the actin cytoskeleton through the myosin motor protein. The results open the possibility that modulation of the spatial organization of cell-cell contacts may play important roles in regulating organization and physiological functions of epithelial tissues.

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of beta-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell-cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell-cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell-cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.

We investigated the structural and functional alterations of active lamellae during initial cell-cell collision and establishment of cell-cell contacts in wounded cultures of nontransformed rat epitheliocytes (IAR-2 line) and their ras-transformed descendants (C4 line). Typically, the leading edges of nontransformed cells formed multiple transient contacts followed by establishment of small, stable contacts that would undergo lateral expansion. Formation and expansion of the contact area was accompanied by accumulation of the cell-cell adhesion molecules E-cadherin, beta-catenin, and plakoglobin. During lateral expansion, the circumferential bundles of actin filaments, characteristic of IAR-2 cells, disassembled at the site of stable contact forming a concave arc-like actin bundle between adjacent cells at the expanding edge. Pseudopodial activity was completely inhibited in the contact zone and partially inhibited at the free lamellar edges adjacent to the zone of contact. Con A-coated beads on the plasma membrane at the zone of contact stopped undergoing centripetal transport but now moved along the cell-cell boundary. On the other hand, ras-transformed cells developed overlapping lamellae and exhibited no detectable change in activity of lamellae, localization of adhesion molecules, and organization of the actin cytoskeleton. We propose that contact-induced reorganization of cell surface adhesion molecules and the underlying cortical cytoskeleton leads to development of lateral traction that may be an essential element in inducing expansion of the contact and in inhibiting local pseudopodial activity.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.

We have recently demonstrated that (1) stimulation of alpha-adrenergic neurotransmission in the medial basal hypothalamus (MBH) of ovariectomized (OVX) estradiol-17 beta-benzoate (EB) treated female guinea pigs activates lordotic responding and that (2) inhibition of alpha-adrenergic neurotransmission in the MBH inhibits lordotic responding in EB and progesterone (EB + P) treated females (Neuroendocrinology, in press). In this study, we investigated the relative influence of selective drugs altering alpha-2-adrenergic neuronal transmission within the MBH and medial preoptic area (MPOA) on lordotic responding in EB + P primed females. In EB + P primed females the selective alpha-2-adrenergic agonist UK-14,304 increased the number of seconds EB + P treated females held lordosis at test periods starting 15 min after infusion until test periods 1.5 h after infusion into the MPOA (maximum effect at test period 30 min after infusion, 265% vehicle (VEH); P < 0.005). UK-14,304 also facilitated lordotic responding 15-30 min and 1.5 h after infusion into the MBH of EB + P primed females (maximum effect at test period 30 min after infusion, 223% VEH; P < 0.025). Furthermore, infusing the selective alpha-2-adrenergic antagonist yohimbine (YOH) into the MBH of EB + P primed females inhibited lordotic responding between 1.0 and 2.0 h after treatment (maximum 1.5 h, 42% VEH; P < 0.05). These results demonstrate that alpha-2-adrenergic neurotransmission within the regions of the MPOA and the MBH facilitates lordotic responding in the guinea pig.

Evidence has accumulated showing that the alpha 1-adrenergic antagonist prazosin decreases nuclear estrogen binding in the hypothalamus of the guinea-pig. In this study we asked if prazosin treatment alters estrogen receptor (ER) protein content as reflected by changes in ER-immunoreactivity. The monoclonal rat antibody H222 directed against ER was used to detect ER-immunoreactive (ER-ir) cells in eight specific preoptic and hypothalamic brain regions of ovariectomized Hartley strain guinea-pigs treated with estradiol benzoate and 1.0 mg/kg prazosin or vehicle. Immunocytochemical parameters which provided optimum conditions for detection of even modest changes in ER-immunoreactivity were first established. Then, using these optimum conditions, we compared 1) the mean number of ER-ir profiles, 2) the mean density of ER-ir staining, and 3) the distribution of ER-ir staining density readings, between conditions within each of the eight brain regions. No differences in any of these measures were found between prazosin- and vehicle-treated females. We also compared the percentage of ER-ir nuclear profiles across the eight cell groups investigated in estradiol benzoate- and vehicle-treated females. The medial preoptic area had by far the highest percentage (48%) of ER-ir profiles (P < 0.05) compared to all seven other brain regions (23% to 32% ER-ir cells). Our data, showing that ER-immunoreactivity is not reduced (6h) after prazosin treatment, suggests that mechanisms other than alterations in ER protein should be considered when interpreting the effects of prazosin on the retention of estradiol by nuclear or cytosolic extracts of hypothalamic lysates.

In ovariectomized (OVX) female guinea pigs sequential administration of estradiol and then progesterone (P) is usually necessary to induce the sexually receptive lordosis posture. Systemic administration of alpha-adrenergic agonists can induce lordosis in OVX guinea pigs primed with estradiol benzoate (EB) alone and facilitate lordosis in OVX females primed with EB and P. In this study, we examined the regional specificity of this regulation by infusing the alpha-adrenergic agonist clonidine (CLON) or the alpha-adrenergic antagonist phentolamine (PHEN) into discrete brain areas. CLON, infused in the medial basal hypothalamus (MBH) of EB-primed females, increased the percentage of females showing lordosis 15-45 min after infusion (p < 0.05) and also increased the number of seconds females held the lordosis posture 15 min to approximately 2.0 h after infusion (by as much as 65% at 15 min p < 0.001). CLON infused in the medial preoptic area (MPOA) of EB primed females had no effect on lordosis except at 4.0 h after infusion when it increased the number of seconds females held the lordosis posture (p < 0.05). In EB + P primed females, PHEN infused into the MBH transiently reduced the number of seconds EB + P primed females held lordosis from 30 min to approximately 2.0 h after infusion (by as much as 64% at 1.0 h; p < 0.01). Conversely, PHEN infused into the MPOA of EB + P primed females transiently increased the number of seconds females held lordosis from 15 min to 2.0 h after infusion (maximum difference 64%; p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

This experiment examined whether the imidazoline idazoxan (which binds to alpha-noradrenergic receptors and to imidazoline-preferring sites) interferes with hormone-dependent or hormone-independent lordosis responses. Ovariectomized (ovx) Strain 2 female guinea pigs which were sexually receptive after receiving estradiol benzoate (EB; 3 micrograms/d for 3 days) were injected with either idazoxan (10 mg/kg) or with vehicle at 24 hr after the last EB injection. Idazoxan significantly decreased EB-facilitated lordosis responses in these females. Castrated Strain 2 males, which show lordosis behavior without gonadal hormone administration, were injected with the same dosage of idazoxan (10 mg/kg) or with vehicle. Idazoxan did not inhibit lordosis behavior in these males.

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue.

Microinfusion of the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) into the mediobasal hypothalamus (MBH) significantly reduced lordosis in estrogen plus progesterone-treated female rats at 10 min post-infusion with recovery to pretest values by 30 min (P less than .05; Wilcoxon). Microinfusion of the specific NMDA antagonist D,L2-amino-5-phosphonopentoic acid (AP-5) into the same sites was without effect on lordosis responding of fully receptive females. There was also a significant increase in the number of females vocalizing to mounts by males after infusion of NMDA but not after infusion of AP-5 into the MBH. When NMDA was infused into the preoptic area (POA) there was no effect on lordosis responding of full receptive females, but AP-5 infusion resulted in a significant inhibition of lordosis at 10 min post-infusion. There was no difference between groups in percentage of females vocalizing after drug infusion into the POA. These results suggest that increased excitatory amino acid activity in the MBH and decreased excitatory amino acid activity in the POA inhibits lordosis behavior.

GABAergic neurotransmission has been implicated in the control of the steroid-dependent behavior, lordosis. GABA has dual effects on lordosis: it facilitates lordosis through actions in the medial hypothalamus (mHYP) and it inhibits lordosis through actions in the preoptic area (POA). In the present study, gonadally intact and ovariectomized female rats were behaviorally tested with a sexually active male. Brains were removed from sexually receptive female either 1 or 24 h after behavioral testing. There was a significant difference in endogenous GABA concentration in HYP and POA between receptive, postreceptive and ovariectomized nonreceptive females. Specifically, GABA levels in postreceptive females were higher in the HYP (20%) and lower in the POA (21%) in comparison to receptive females (p less than 0.05). There was also a significant change in binding parameters of 3H-muscimol in the HYP and POA of receptive females as compared to 24 h postreceptive and ovariectomized rats. Attempts to modulate 3H-GABA release from hypothalamic tissue slices by estrogen or progesterone in ovariectomized rats yielded no effect on this parameter.

Steroid-dependent lordosis behavior in ovariectomized (OVX) guinea pigs is attenuated by alpha 1- and/or alpha 2-noradrenergic (NE) receptor antagonists. Correlated with the decrease in lordosis after alpha 1-NE receptor blockade by prazosin is a decrease in 'cytosol' progestin receptors in the ventromedial hypothalamic nucleus (VMN). We examined whether a presumed alpha 2-NE receptor blocker (idazoxan, IDA) also affects progestin receptors. A decrease in 'cytosol' progestin receptors was found after IDA treatment of OVX, estrogen-treated guinea pigs in the VMN and the arcuate nucleus-median eminence (ARC-ME). Apparently, either prazosin or IDA can inhibit lordosis behavior and decrease 'cytosol' progestin receptors in the VMN. In contrast, idazoxan but not prazosin, decrease 'cytosol' progestin receptors in the ARC-ME.

The role of gamma-aminobutyric acid (GABA) in mediation of lordosis in the rat has been unclear. We report here that GABA plays a dual role in the mediation of lordosis and has differential effects in the medial hypothalamus (MH) and preoptic area/anterior hypothalamus continuum (POA-AH). Bilateral infusion of the GABAA antagonist bicuculline into the MH of cannulated females primed with estradiol benzoate and progesterone (EB + P) resulted in a marked and transient inhibition of ongoing lordosis. A similar pattern of inhibition was seen in females treated with EB only. In contrast, infusion of the same dose of bicuculline into the POA-AH of sexually receptive females had no effect on lordosis whereas infusion of the GABAA agonist muscimol into this site resulted in a short-term inhibition of lordosis. Furthermore, when females were treated with subthreshold doses of EB + P to induce a low level of lordosis responding, infusion of muscimol into the MH resulted in a significant enhancement of lordosis; infusion of bicuculline into the POA-AH also enhanced lordosis responding as compared to saline-infused controls. These data indicate that increased GABAergic neurotransmission in the MH facilitates lordosis whereas increased GABAergic activity in the POA-AH inhibits this behavior.

The development of the cytoplasmic progestin receptor (CPR) system in male and female guinea pigs was examined to determine if insensitivity to the lordosis promoting activity of estrogen-progestin treatment in adult males and neonatal guinea pigs is correlated with alterations in this receptor system. Gonadectomized neonatal (4-6 days old) and adult (50-65 days old) guinea pigs of both sexes were injected with estradiol benzoate (EB) and killed 40 h later. CPRs were measured in the hypothalamus (HYPO), preoptic area (POA) and cortex ( CORT ). Neonatal brain contained both high (Kd congruent to 0.1 nM) and low (Kd greater than 10 nM) affinity binders for [3H]R5020 similar to those found in adult brain. In the absence of EB-priming the concentration of CPR in HYPO, POA and CORT was lower in neonatal animals than adults. In both males and females, neither a low (1.6 micrograms) nor a high (10 micrograms) dose of EB resulted in a concentration of estrogen inducible CPR in neonatal HYPO greater than that found in adult HYPO after 1.6 micrograms EB. Even after treatment with 50 micrograms EB the concentration of CPR in neonatal female HYPO was not different from adult female HYPO after 1.6 micrograms EB. In neonatal POA, 10 micrograms EB resulted in a concentration of CPR greater than that found in adult POA after 1.6 micrograms EB. Finally, there was a sex difference in the concentration of estrogen inducible CPR in HYPO (female greater than male) but not POA of adult guinea pigs. No sex difference in CPR concentration was found in any neonatal brain area.

A cytoplasmic progestin receptor has been characterized in the brain of castrated ring doves using an in-vitro assay that measures the binding of a synthetic progestin, [3H]17 alpha,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione(promegestone; R5020). The affinity of the receptor was similar in both the hyperstriatum and the hypothalamus (Kd approximately equal to 4 X 10(-10) mol/l). Its concentration was higher in the anterior hypothalamus-preoptic area (63 +/- 4 fmol/mg (S.E.M.) protein) than in other brain regions (posterior hypothalamus, 33 +/- 5; hyperstriatum, 28 +/- 3; midbrain, 17 +/- 4 fmol/mg protein; n = 7). Progesterone and R5020 competed well for binding but oestradiol and 5 beta-dihydrotestosterone did not. Corticosterone and, to a lesser extent, testosterone and 5 alpha-dihydrotestosterone completed for binding but much higher concentrations were required than for progestins. Injections of testosterone (200 micrograms testosterone propionate daily for 7 days) significantly increased the concentration of progestin receptors in the anterior and posterior hypothalamus without having any significant effect on other brain areas. Shorter treatment, lasting for 2 days, with testosterone propionate (200 micrograms daily), 5 alpha-dihydrotestosterone (200 micrograms daily) or oestradiol benzoate (50 micrograms daily) did not always cause this increase but seven injections of oestradiol benzoate (50 micrograms daily for 7 days) were even more effective than seven injections of testosterone propionate (200 micrograms daily for 7 days). These data suggested that the sensitivity to progesterone of the brain of the bird changes as a consequence of increases in the level of testosterone in the circulation.

Estrogen induction of cytosolic progestin receptors (CPRs) in the hypothalamus-preoptic area of the female guinea pig is correlated with facilitation of female sexual behavior and gonadotropin secretion by progesterone. The present study validated a CPR microassay and determined where, within microdissected areas of the hypothalamus-preoptic area of the female guinea pig, induction of CPRs by estradiol occurs. Ovariectomized adult guinea pigs were given 20 micrograms estradiol benzoate (EB) or oil vehicle for 3 successive days. CPRs were measured using the synthetic progestin [3H]-R5020. The highest basal (no estrogen treatment) level of CPRs was seen in the arcuate-median eminence (34.1 +/- 3.7 fmol/mg). With EB treatment, the highest level of CPRs was again in the arcuate-median eminence (178.0 +/- 12.0 fmol/mg). EB-treated females also had high CPR levels in the periventricular area (88.5 +/- 10.8 fmol/mg) and the medial preoptic area (86.3 +/- 9.3 fmol/mg). Moderate levels were seen in the ventromedial nucleus of the hypothalamus (32.7 +/- 3.0 fmol/mg) and in the anterior hypothalamic nucleus (13.0 +/- 2.1 fmol/mg), but these were not significantly different from the low levels in the medial amygdala (4.5 +/- 1.2 fmol/mg) and in the dorsomedial nucleus of the hypothalamus (5.4 +/- 1.1 fmol/mg) of EB-treated females. However, EB caused a significant induction over baseline levels not only in the arcuate-median eminence, periventricular area, and medial preoptic area, but also in the ventromedial nucleus of the hypothalamus and the anterior hypothalamic nucleus. EB did not increase CPRs in the medial amygdala or the dorsomedial nucleus of the hypothalamus.

L-5-hydroxytryptophan (L-5-HTP)-induced myoclonus was used as a behavioral index of central serotonergic activity. Estradiol benzoate (EB) and progesterone (P) influenced the induction of myoclonus by L-5-HTP. When L-5-HTP was injected 46 h after EB, myoclonus was enhanced. P blocked this effect on EB when 100 or 125 mg/kg L-5-HTP (but not 80 mg/kg) was given 6 h after P in EB-primed animals. When L-5-HTP was given 3 or 11-15 h after P in EB-primed animals, there was no inhibitory effect of P on myoclonus. In fact, at the lowest dose (80 mg/kg), L-5-HTP increased myoclonus when given 3 h after P in EB-primed animals. The inhibitory effects of P in EB-primed females on myoclonus were temporally correlated with the display of lordosis, suggesting that the neural progestin receptor mechanisms that have been proposed to mediate P effects on lordosis are also involved in the inhibitory effects of P on myoclonus.

Intracranial implants of crystalline progesterone (P) were used to examine in site of action of P's facilitatory and inhibitory effects on lordosis behavior in the ovariectomized, estradiol-benzoate (EB)-primed RAT. P implanted in the medial basal hypothalamus (MBH) 1 h prior to subcutaneous (s.c.) EB injection inhibited lordosis in response to a systemic P injection 44 h after EB (concurrent inhibition). P implanted in the MBH did not facilitate lordosis when implanted 44 h after EB injection, but this same implant of P inhibited lordosis in response to P injection 68 h after EB (sequential inhibition). Cholesterol (Chol) implants in the MBH did not inhibit lordosis behavior in either the concurrent or the sequential inhibition experimental paradigms. The results indicate that the MBH is an important site of P inhibition of sexual receptivity in the rat.

Experiments were conducted to determine whether alpha 1- or alpha 2-receptors mediate noradrenergic (NA) regulation of guinea pig lordosis behavior and hypothalamic progestin receptors. When infused into a lateral cerebroventricle at a dose that inhibits lordosis and that decreases the concentration of estradiol-inducible hypothalamic progestin receptors, phenoxybenzamine decreased binding of the alpha 1-ligand [3H]WB4101 but not the alpha 2-ligand [3H]clonidine to brain membranes. Thus, under the conditions used, phenoxybenzamine appears to block alpha 1-receptors with little or no effect on alpha 2-receptors. Experiments with the selective alpha 1-antagonist prazosin also indicated alpha 1-receptor regulation of lordosis and hypothalamic progestin receptors. Prazosin inhibited lordosis induced by estradiol benzoate (EB) plus progesterone and by EB + clonidine and decreased the concentration of cytoplasmic progestin receptors in hypothalamus (but not in preoptic area or frontal cortex) of EB-primed females. The inhibition of lordosis is apparently not due to some unknown side effect of prazosin because pretreatment with a high dose of clonidine attenuated the inhibition. The possibility that a causal relationship exists between effects of alpha 1-NA transmission on hypothalamic progestin receptors and lordosis was discussed. Also, because effects of NA transmission on hypothalamic progestin receptors are dependent on prior treatment with EB, it was suggested that NA transmission might influence estradiol action in addition to progestin action in hypothalamic cells.

Because manipulations of the noradrenergic system affect both lordosis behavior and progestin receptor levels in female guinea pigs, the present study attempted to determine if the noradrenergic (NE) system affects lordosis solely because of its impact on progestin receptors. Although the progestin receptor antagonist RU486 significantly reduced progesterone-facilitated lordosis, it had no effect on lordosis induced by the alpha-NE agonist clonidine in estrogen-primed female guinea pigs. This indicates that although progesterone may facilitate lordosis in female guinea pigs via activation of progestin receptors, the alpha-noradrenergic agonist clonidine does not mediate lordosis through the same mechanism.

The distribution of muscarinic binding sites was analyzed in regions of the guinea-pig brain with semi-quantitative densitometry of [3H]N-methyl scopolamine binding, a muscarinic antagonist. In the rostral forebrain, high levels of binding were detected in the caudate putamen, nucleus accumbens and olfactory tubercle while intermediate levels of binding were observed in the medial and lateral septum, bed nucleus, and vertical and horizontal limbs of the diagonal band. The hypothalamus displayed binding that ranged from low levels in the preoptic area to intermediate levels in the mammillary nucleus. In limbic areas such as the thalamus, amygdala and hippocampus, a heterogeneous pattern of binding was evident in various subregions which tended to correspond with known innervation by cholinergic afferents. In the midbrain, binding was high in the superficial layer of the superior colliculus and the medial geniculate while intermediate binding was recorded in the lateral geniculate and the lateral aspect of the central gray. The pattern of muscarinic binding observed in the brain of the guinea-pig is similar to distributions of this binding site previously reported in the rat brain and the human brain.

[3H]Prazosin was found to bind to sites on guinea pig brain membranes with alpha 1-adrenergic receptor characteristics. Treatment of ovariectomized guinea pigs with estradiol benzoate (EB) or EB followed by progesterone (P) did not affect [3H]prazosin binding to membranes from hypothalamus, preoptic area, amygdala or cerebral cortex. When added to the incubation mixture of the assay, estradiol, P, and other steroids decreased [3H]prazosin binding but only at high concentrations. These results do not support the idea that estrogen and progestin influence reproductive physiology through effects on brain alpha 1-receptors, although limitations of the methodology employed do not completely rule out this possibility.

Nuclear progestin receptors (NPR) were measured, with an exchange assay, in neonatal (4-6 days old) and adult (50-60 days old) guinea pigs of both sexes in order to further examine deficits in the neonatal hypothalamic progestin receptor system. Both neonatal and adult hypothalamus (HYPO) and cortex ( CORT ) contained a KCl extractable high affinity (Kd congruent to 0.1 nM) binder for R5020. In neonatal guinea pigs, given 10 micrograms estradiol benzoate (EB), NPR accumulation in HYPO and CORT peaked 2-4 h after a 0.5 mg injection of progesterone (P). There was no dose response of NPR to EB in neonatal HYPO, CORT or adult CORT . In adult HYPO, 10 micrograms EB-priming resulted in a higher concentration of NPR than 1.6 micrograms EB-priming. Neonatal HYPO (1.6 or 10 micrograms EB) did not differ in concentration of NPR from adult HYPO (1.6 micrograms EB). Neonatal CORT (1.6 or 10 micrograms EB) showed a lower NPR concentration than adult CORT (1.6 micrograms EB). There was a sex difference (females greater than males) in NPR concentration in both neonatal and adult HYPO, but not CORT . These results are discussed with regard to deficits in cytoplasmic progestin receptors and behavioral insensitivity to estrogen-progestin treatment found in neonatal guinea pigs.

In this experiment, we examined the influence of estradiol on alpha 2-noradrenergic receptor binding in the female guinea pig brain with tritium-sensitive film autoradiography. Treatment of ovariectomized guinea pigs with estradiol increased alpha 2-receptor binding in regions of the preoptic area and decreased binding in the ventromedial nucleus of the hypothalamus. There was no effect of estradiol on alpha 2-receptor concentrations in other estradiol concentrating regions or in brain areas which do not concentrate estradiol.

Lordosis behavior was induced in previously unreceptive, ovariectomized estrogen-primed female guinea pigs by administration of the noradrenergic agonist clonidine. Clonidine also enhanced lordosis responding in females that were weakly receptive after estrogen priming. Unlike progesterone, the lordosis-facilitating effect of clonidine was not accompanied by a subsequent refractory period. Clonidine had a weak lordosis facilitatory effect when administered during the decline in receptivity to estrogen plus progesterone-primed animals and failed to induce lordosis when administered during the refractory period.

Cytosol androgen receptors were assayed in guinea pig brain and pituitary tissues, using [3H]R1881 as ligand. These receptors had an apparent Kd of 0.04 nM and were androgen-specific (R1881 greater than dihydrotestosterone greater than testosterone = estradiol greater than progesterone). Concentrations of cytosol androgen receptors in castrated adult male guinea pigs were 12.2, 11.6, 6.9, 2.6 and 1.3 fmol per mg protein in anterior pituitary, hypothalamus, medial preoptic area, amygdala and cortex, respectively. No significant differences in receptor levels were observed between castrated adult males and females. The concentration of androgen receptors was significantly lower in the hypothalamus, medial preoptic area and anterior pituitary of castrated neonatal males than in castrated adult male guinea pigs. The systemic injection of the alpha 1-adrenergic antagonist, prazosin, had no significant influence on androgen receptor levels in castrated males in any brain area.

The synthetic progestin, R 5020, was used to measure cytoplasmic progestin receptors in the brain and pituitary gland of ovariectomized guinea pigs. Progestin receptors with a dissociation constant of 0.1--0.3 nM were measured by gel filtration in all brain regions studied, pituitary gland and the uterus. The receptor is progestin-specific; biologically potent progestins compete well against [3H]R 5020 for binding, but androgens, glucocorticoids and estrogens do not. The concentration of the cytoplasmic progestin receptor in hypothalamus-preoptic area-septum and midbrain is decreased in vivo by behaviorally effective doses of progesterone. In the pituitary gland, hypothalamus, preoptic area-septum and midbrain, but not other brain regions, the concentration of progestin receptors increases after estradiol benzoate-priming. The increase in the concentration of cytoplasmic progestin receptors in hypothalamus-preoptic area-septum is dependent on the dose of estradiol benzoate injected. After a single injection of a dose of estradiol benzoate routinely used to facilitate the display of sexual receptivity (1.6 microgram estradiol benzoate/animal), the latency to an increase and subsequent decrease in cytoplasmic progestin receptors in the hypothalamus-preoptic area-septum correlates well with the previously reported time course for progesterone's facilitation of sexual receptivity after estradiol benzoate injection. The experiments are consistent with the notion that brain progestin receptors mediate at least some of the behavioral effects of progesterone.