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Intracerebroventricular administration of neuropeptide Y (NPY) has been shown to reduce anxiety-like effects in rodents and also profoundly alter feeding. The area postrema-lesioned (APX) rat model of food motivated behavior overconsumes palatable foods and expresses significantly higher levels of NPY in the hypothalamus than sham-lesioned control rats. For this reason, we examined APX rats in the open field test, which is a standard measure of anxiety- or fear-related behavior and also investigated NPY mRNA levels in the hippocampus, amygdala and hypothalamus. We found that APX rats display reduced anxiety-like behavior in the open field test as indicated by spending increased time in the center of the field as opposed to the perimeter of the field. NPY mRNA levels were also found to be significantly elevated in the amygdala, hippocampus and arcuate nucleus of the hypothalamus of APX rats when compared to sham-lesioned rats. These results support the action of limbic NPY to reduce anxiety-like behavior in a rodent model that appears to express higher than normal NPY levels., (Copyright 2002 Elsevier Science Inc.)

The effects of treadmill exercise training on prepro-galanin (GAL) and tyrosine hydroxylase (TH) gene expression in the locus coeruleus (LC) were examined. Male Fischer-344 rats (n=9) were assigned to 6 weeks of treadmill running. An additional group of animals comprised the sedentary home cage control group (n=9). Levels of GAL and TH messenger RNA (mRNA) in the LC were measured using in situ hybridization histochemistry with autoradiography. Levels of GAL mRNA were higher in treadmill trained animals compared to sedentary animals, but there was no effect of treadmill running on TH mRNA. These results suggest that gene expression for galanin is responsive to repeated exercise stress and may have a neuromodulatory role in LC-noradrenergic adaptation to treadmill exercise training.

Bilateral olfactory bulbectomy (OBX) in rats produces a well-characterized syndrome of behavioral, physiological, and neurochemical changes identical to those seen in depression. Previous experiments using in situ hybridization histochemistry have demonstrated that OBX increases prepro-neuropeptide-Y (NPY) and prepro-enkephalin (ENK) mRNA levels in limbic structures. The present experiments determined whether increases in peptide immunoreactivity occur in conjunction with increases in mRNA levels following OBX. In situ hybridization analyses in olfactory bulbectomized and sham-operated rats revealed increased prepro-ENK mRNA in the piriform cortex (PIR) and olfactory tubercles (OTs) of bulbectomized rats. Prepro-NPY mRNA levels were significantly increased in the PIR of bulbectomized rats as comapred to controls. Radioimmunoassays (RIAs) revealed significant elevations in ENK-like immunoreactivity in the OTs following OBX. NPY-like immunoreactivity was significantly elevated in the PIR following OBX. These data reveal that OBX-induced increases in ENK-like immunoreactivity occur concomitantly with increases in prepro-ENK mRNA, and NPY-like immunoreactivity occur concomitantly with increases in prepro-NPY mRNA.

Previous experiments have reported increased seizure susceptibility in transgenic mice lacking normal neuropeptide-Y (NPY) gene expression (i.e. NPY 'knock-out' mice). A critical issue inherent in such experiments concerns the confounding of developmental influences of NPY and its neurotransmitter functions in the mature organism. The present experiments directly addressed this issue by studying seizure susceptibility in transgenic mice possessing an inducible antisense transcript that can be experimentally manipulated to attenuate NPY synthesis. NPY-deficient and control mice were injected with kainic acid (40 mg/kg, i.p.) and several seizure-related behaviors were measured. Consistent with previously reported effects in NPY knock-out mice, significantly more NPY-deficient mice died within 24 h than control mice. In situ hybridization analyses confirmed a decrease in prepro-NPY gene expression in transgenic mice. The experiments support the hypothesis that the control of neural excitability is a prominent function of NPY.

The effects of neuropeptide-Y were examined in the forced swim model of depression in rats. Following a 15-min preswim, four groups of rats were given three intracerebroventricular (i.c.v.) injections of neuropeptide-Y (0.5, 5, or 10 microg) or saline over a 24-h period. Several behaviors were subsequently measured during a 5-min forced swim. Neuropeptide-Y treatment dose dependently increased swimming and decreased immobility. The pattern of results is consistent with that produced by serotonergic antidepressant drugs in this model.

The aim of the present studies was to determine the extent to which changes in defensive behaviors could account for some of the behavioral effects of bilateral olfactory bulbectomy (OBX) in rats. Four tests of aversively-motivated behavior were conducted in bulbectomized and sham-operated rats: activity in a dimly lit or brightly lit open field, passive avoidance, foot shock-induced freezing, and defensive withdrawal. OBX reduced the duration of immobility in the open field. Bulbectomized rats exhibited less freezing in response to foot shock than sham-operated rats. In the defensive-withdrawal test, bulbectomized rats made more transitions into and spent less time inside the covered enclosure than sham-operated rats. The experiments thus reveal two novel paradigms for assessing the behavioral effects of OBX. The results also suggest that deficits in aversively-motivated behavior, specifically defensive freezing, may comprehensively explain the putative "hyperactivity" and "passive-avoidance learning deficits" widely associated with the OBX behavioral syndrome.

The effects of bilateral olfactory bulbectomy (OBX) on prepro-enkephalin, thyrotropin-releasing hormone, and D-2 receptor mRNA levels in the ventral striatum were examined by in situ hybridization histochemistry. Pre- pro-enkephalin mRNA levels were significantly increased in the olfactory tubercle (OT), but not in the nucleus accumbens, 14 days following bilateral OBX. Levels of D-2 receptor mRNA were also increased in the OT, though to a lesser degree. Prepro-thyrotropin-releasing hormone mRNA was unaffected by OBX. A separate experiment revealed no effect of OBX on enkephalin gene expression 7 days following surgery but a comparable elevation in pre- pro-enkephalin mRNA 14 and 28 days post-surgery. The findings are consistent with previously-reported effects of dopamine lesions on striatal gene expression, suggesting that the observed effects may be mediated by deafferentation-induced alterations in dopaminergic transmission in the OT. Altered dopaminergic function in the OT may be particularly relevant to the 'anhedonia' that has been associated with the olfactory bulbectomized rat model of depression., (Copyright 1999 Harcourt Publishers Ltd.)

Effects of physical activity on brain noradrenergic response to footshock were examined. Male Fischer 344 rats were randomly assigned to shoebox cages with (AW) or without (SED) 24-hr access to an activity wheel for 4-5 weeks. Extracellular levels of norepinephrine (NE) and 3,4-dihydroxyphenyl-acetic acid (DOPAC) in the brain frontal cortex were measured in 20-min samples of microdialysate taken during a 2-hr baseline, 40 min of scrambled footshock, and a 1-hr recovery. Levels of messenger RNA (mRNA) for tyrosine hydroxylase (TH), c-fos, and prepro-galanin in the locus coeruleus were measured by in situ hybridization histochemistry with autoradiographic analysis. NE levels were the same for SED and AW rats at baseline but were elevated in SED compared with AW during and after footshock. Levels of mRNA for TH and c-fos were elevated after footshock but did not differ between SED and AW. Our findings suggest that wheel running blunts NE release in the brain frontal cortex in response to footshock but does not influence expression of the gene that encodes TH in the locus coeruleus.

Bilateral olfactory bulbectomy in the rat produces a well-characterized syndrome that is independent of anosmia. This syndrome is reversed by chronic antidepressant administration, which provides the basis for the olfactory bulbectomy model of depression. The present experiments focused on neuropeptide plasticity in central olfactory/limbic structures following olfactory bulbectomy in rats. Male Sprague-Dawley rats received bilateral surgical ablation of the olfactory bulbs, sham surgery, or no surgery and were killed either three, seven, 14 or 28 days later. Relative levels of messenger RNA encoding neuropeptide Y, somatostatin, thyrotropin-releasing hormone, and corticotropin-releasing factor precursors in the forebrain were measured by quantitative in situ hybridization histochemistry using oligonucleotide probes. Prepro-neuropeptide Y messenger RNA levels in the piriform cortex and dentate gyrus were significantly elevated in bulbectomized rats 14 and 28 days after surgery compared to sham-operated and surgically naive rats. Prepro-somatostatin messenger RNA levels in the piriform cortex were marginally increased in bulbectomized rats at these time-points. Thyrotropin-releasing hormone and corticotropin-releasing factor precursor messenger RNA levels were not altered in the brain regions studied. The results indicate that olfactory bulbectomy causes long-term increases in the expression of the neuropeptide Y gene. These findings suggest that neuropeptide Y plasticity in the olfactory/limbic system may contribute to the olfactory bulbectomy syndrome in rats, and they provide further evidence of a role for neuropeptide Y in the pathophysiology of depression.

The aim of this study was to determine whether specific growth factors, those shown to be involved using PCR and immunohistochemistry, are necessary for the in-vivo mechanisms of normal implantation in mice. The abdomen of pregnant female mice were opened surgically on day 4 to expose the uterine horns, which were microinjected with specific neutralising antibodies against PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor. At autopsy on day 12, the numbers, positions and sizes of all implantation and resorption sites were recorded. Sham-operation controls were utilised to evaluate the implantation model. Normal female mice exhibited a mean of 6.24 implantation sites per uterine horn. Sham-operated mice exhibited a 30.8% reduction in implantation compared with normals, and saline-injected mice exhibited a 45.7% reduction. Antibody-injected horns were compared with horns injected with saline and horns injected with heat deactivated antibody. All neutralising antibodies tested resulted in significant reductions in the implantation rate and the size of the implantation site. These experiments confirm, in vivo, participation of the specific growth factors tested in the mechanisms of murine implantation, as alluded to previously by evidence from PCR in vitro stimulatory and immunohistochemical work.

Enkephalins are endogenous opioid peptides that are derived from a pre-proenkephalin precursor protein. They are thought to be vital in regulating many physiological functions, including pain perception and analgesia, responses to stress, aggression and dominance. Here we have used a genetic approach to study the role of the mammalian opioid system. We disrupted the pre-proenkephalin gene using homologous recombination in embryonic stem cells to generate enkephalin-deficient mice. Mutant enk-/- animals are healthy, fertile, and care for their offspring, but display significant behavioural abnormalities. Mice with the enk-/- genotype are more anxious and males display increased offensive aggressiveness. Mutant animals show marked differences from controls in supraspinal, but not in spinal, responses to painful stimuli. Unexpectedly, enk-/- mice exhibit normal stress-induced analgesia. Our results show that enkephalins modulate responses to painful stimuli. Thus, genetic factors may contribute significantly to the experience of pain.

The effects of olfactory bulbectomy (OBX) on galanin (GAL) gene expression in the locus coeruleus (LC) were examined with quantitative in situ hybridization histochemistry. OBX increased prepro-GAL levels 3 and 14 days after surgery, as compared to sham-operated controls. Levels of mRNA encoding prepro-neuropeptide Y (NPY) were unchanged, and levels of mRNA encoding tyrosine hydroxylase (TH) were elevated in the LC only on day 3. The results indicate that GAL gene expression in the LC increases after lesioning a terminal field.

The stress-induced changes in peripheral benzodiazepine receptors (PBR) can be observed in a number of different tissues, depending upon the nature and chronicity of the aversive experience. In addition, virtually all stress procedures that cause rapid changes in PBR simultaneously increase the physical activity or metabolic rate of the subjects. The present study analyzed the contributions of rapid alterations in activity or metabolic rate with and without aversive stimulation and their subsequent impact on PBR. Mechanically induced increases in activity by forced running stress results in a significant reduction in [3H]Ro 5-4864 binding to PBR in olfactory bulb, opposite to the PBR changes in this tissue following forced cold-water swim stress. Pharmacological induction of increased locomotor activity as well as metabolic rate by d-amphetamine causes a significant increase in cardiac PBR binding, again, opposite to the response typically observed following inescapable shock stress. Finally, administration of the anxiogenic beta-carboline, FG-7142, causes increases in both hippocampus and adrenal gland PBR binding reminiscent of acute noise stress exposure. These experiments demonstrate that increased locomotor activity or metabolic rate alone is not a necessary and sufficient condition for previous stress-induced changes in PBR. Conversely, increased metabolic rate coupled with an aversive stimulus appears to be an important factor for inducing stress-like changes in PBR. This data, coupled with previous reports, suggests that rapid alterations in these sites are stressor and tissue dependent. Finally, we propose that the PBR may be involved in many aspects of the stress response including: a) a blowarning system in adrenal gland, b) participation in stress-induced hypertension via renal PBR, and c) a modulator of stress-induced immunosuppression and subsequent recovery of function or recuperation by actions on immune cells.

Galanin is a 29 amino acid neuropeptide that coexists with norepinephrine in approximately 80% of locus coeruleus (LC) neurons in the rat. The effects of chronic, naturalistic stress on preprogalanin mRNA in the LC was studied. The visible burrow system (VBS) served as the stress paradigm. Long-Evans rats (three males and two females) were housed together in the VBS for 13 days. The males established dominance hierarchies during this period. On day 14, preprogalanin mRNA in the LC was significantly elevated in subordinate rats compared to dominant and control rats, as measured by quantitative in situ hybridization. Levels of mRNA were positively correlated with the number of wounds by day 7 and day 14 and negatively correlated with body weight gain by day 14. These results suggest that the neuropeptide galanin may be overexpressed during chronic stress in rats.

Morphine dependence was experimentally induced in rats by daily injection of increasing doses of morphine for seven days. Withdrawal was precipitated in half of the morphine-dependent rats by a single injection of naloxone on day 8. Behavioral signs of withdrawal were evident in the morphine/naloxone group. Gene expression in locus coeruleus (LC) neurons was investigated using quantitative in situ hybridization analysis. Messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for precursors to galanin (GAL) and neuropeptide Y (NPY), peptides that coexist with norepinephrine in LC neurons, were not altered by chronic morphine treatment or naloxone-precipitated withdrawal. In contrast, mRNA levels for c-fos were dramatically elevated in the LC following naloxone-precipitated withdrawal. Chronic morphine treatment caused a small decrease in levels of mRNA encoding the precursor to corticotropin-releasing factor (CRF) in Barrington's nucleus. Although long-term adaptations of LC neurons have previously been implicated in the development of morphine tolerance, dependence, and withdrawal, alterations in the levels of TH, GAL, or NPY mRNA in the LC apparently do not underlie this process.

The 29 amino acid peptide galanin (GAL) coexists with norepinephrine in rat locus coeruleus (LC) neurons to a remarkably high degree. The effects of central administration of GAL were examined in three behavioral paradigms that putatively involve increases in the activity of LC neurons. GAL did not affect behavioral signs associated with naloxone-precipitated withdrawal in rats treated chronically with morphine, a condition in which the firing rate of LC neurons is dramatically increased, although the behavioral signs of withdrawal were abolished by clonidine. Foot shock induced freezing behavior was similarly unaffected by either dose of GAL but was significantly diminished by clonidine and the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF. GAL did not influence the decrease in exploratory activity in a novel open field induced by idazoxan. The behavioral activity of the peptide and route of administration were confirmed in a feeding paradigm. Doses of GAL that were inactive in the three paradigms were active in stimulating intake of a palatable food to a similar degree as clonidine-stimulated intake. These results suggest that intraventricularly administered GAL may not influence behaviors thought to be mediated by activation of neurons in the LC.

The etiology of the decrease in renal peripheral benzodiazepine receptor (PBR) binding caused by stress was studied in rats. Prior investigations suggest that the response of the renal PBR to stress occurs independently of the hypothalamo-pituitary-adrenal (HPA) axis and sympathetic nervous system. The present experiments tested the hypothesis that the renin-angiotensin system is involved in regulating the PBR. Eighty min of brief, intermittent tailshocks caused increases in plasma renin activity and decreases in renal PBR binding. The stress-induced decrease in renal PBR binding was reversed by pretreatment with captopril. Acute administration of angiotensin II (ANG II) alone caused reductions in PBR binding in kidney, heart, and cerebral cortex. These data suggest that ANG II may be an endogenous factor responsible for regulating the PBR in several tissues during stress.

Embryonic and fetal growth are generally considered to be independent of pituitary GH. However, it has been demonstrated recently that 18-day-old rat embryos and rat fetuses express GH receptors, suggesting that GH could play a role in early development. The aim of the present investigation was to determine whether preimplantation embryos also express GH receptors. Germ line competent mouse embryonic stem (ES) cells and cultured mouse preimplantation embryos were examined with Northern blot analysis, RNAse-protection solution-hybridization assays, reverse transcription/polymerase chain reaction assays and immunohistochemistry for the detection of GH receptors. Northern blot analysis of ES cells using a probe corresponding to the extracellular domain of the GH receptor demonstrated the presence of two transcripts (1.2 and 4.5 kilobases). The RNAse-protection solution-hybridization assay revealed that ES cells express approximately one sixth of the GH-receptor messenger RNA (mRNA) levels expressed in liver from pregnant mice. Treatment of cultured ES cells with retinoic acid (100 nM) for 6 days increased GH-receptor mRNA levels (P < 0.01). GH-receptor mRNA was further identified in ES cells, preimplantation embryos, muscle, liver, and placenta by a reverse transcription/polymerase chain reaction assay. In humans it has previously been shown that exon 3 of the GH-receptor is deleted in the placenta. However, none of the studied mouse tissues had a deletion of the GH-receptor mRNA corresponding to exon 3 of the human GH receptor. GH-receptor immunoreactivity was identified on the cultured ES-cells by immunohistochemistry. In conclusion, we have in the present study shown that germline competent ES cells and preimplantation mouse embryos express the GH receptor transcript and that this transcription is increased by retinoic acid in ES cells. Furthermore, the presence of GH-receptor immunoreactivity on the ES cells indicates that the GH-receptor transcript is translated.

Previous research has demonstrated that the density of peripheral benzodiazepine receptors (PBR) in rat kidney rapidly drops following exposure to 80 min of stress. The present experiments examined the contribution of the central and autonomic nervous systems in mediating this effect. Ibotenic acid lesions of the amygdaloid central nucleus (ACe), but not the lateral and basolateral amygdala, diminished the magnitude of the reduction in renal PBR binding caused by stress. Pretreating rats with methyl-scopolamine also inhibited the response of the PBR to stress. Adrenergic blockade with nadolol was ineffective. In order to test whether the PBR was under direct or indirect neural control during stress, unilateral renal denervation was performed. The stress-induced reduction in PBR binding persisted in denervated kidneys revealing that any neural control over the PBR that might exist must be indirect. Together the results suggest that the CNS may be involved in regulating the PBR during stress through the activation of intermediate, possibly hormonal, factors. The involvement of the central nervous system in the modulation of the PBR indicates the relevance of the PBR to physiological adaptations to stress.

A sexual dimorphism in stress-induced alterations in renal peripheral benzodiazepine receptors (PBR) was recently reported. The present paper includes five experiments examining the etiology of this sex difference. Surgical removal of ovaries and testes was ineffective in altering the renal PBR stress response in both male and female rats. A diurnal variation in the sexual dimorphism was observed; the difference was seen in the early part of the light cycle, while the two sexes were indistinguishable at the end of the light cycle. Finally, based on recent data indicating the importance of the renin-angiotensin system in the stress-induced decrease in renal PBR, we examined both stress-induced plasma renin activity and renal PBR reactivity to exogenous angiotensin II (AII) administration in both sexes. Female rats show both an attenuated elevation of plasma renin levels in response to inescapable shock stress and a reduced PBR response to AII administration in comparison to males. The present data indicate that the renin-angiotensin system may be a critical factor in the sexual dimorphism in the renal PBR response to stress. The possibility of this difference in the renin-angiotensin system contributing to sex differences in susceptibility to escape deficits following inescapable shock is entertained. The implications for these findings regarding the physiological function of the PBR are also discussed.

In an attempt to dissociate the relative impact of psychological vs. physiological concomitants of stress on the peripheral benzodiazepine receptor (PBR), the influence of stressor controllability and predictability was investigated in rats. In addition, the effect of a purely psychological stressor, contextually conditioned fear, was examined. The response of the PBR in rats confronted with a naturalistic threat, a cat, was also tested. Various peripheral and CNS tissues were analyzed. Specific binding of [3H]Ro 5-4864 was significantly reduced in the kidneys of subjects receiving either controllable or uncontrollable shock. Similar changes were seen in the kidneys of subjects receiving either predictable or unpredictable shock. Mean [3H]Ro 5-4864 binding in lung was reduced following both predictable and unpredictable shock, but only the reduction in the predictable shock group reached significance. Controllability appeared to protect against the stress-induced reduction in [3H]Ro 5-4864 binding in lung. Contextually conditioned fear only affected PBR in the olfactory bulb, and exposure to a cat was without effect. These data suggest that the PBR responds only to potent stressors, and psychological influences on the PBR are tissue specific.

The effects of acute exposure to angiotensin II (AII) on the renal peripheral benzodiazepine receptor were studied in rats. As little as 37.5 micrograms of AII injected s.c. over an 80 min period caused immediate reductions in [3H]Ro5-4864 binding. Scatchard analysis revealed that the reduction in [3H]Ro5-4864 binding induced by AII was due to a drop in receptor density or Bmax. The influence of AII on the peripheral benzodiazepine receptor is similar to that of stress.

This study examines the possible direct effect of interleukin-1 alpha (IL-1 alpha) upon human granulosa cells. The cells were isolated from follicles of stimulated cycles in women undergoing oocyte retrieval for in-vitro fertilization. Purified cell preparations were cultured for different time periods in the presence of IL-1 alpha and human luteinizing hormone (LH) or follicle stimulating hormone. IL-1 alpha stimulated basal as well as LH-induced progesterone accumulation. The response in terms of cyclic AMP was more complex, there was no effect of IL-1 alpha on basal cyclic AMP accumulation. However, at the highest concentration tested (50 IU/ml), IL-1 alpha enhanced cyclic AMP accumulation over that seen with LH alone. At a lower concentration, IL-1 alpha either had no effect or was slightly inhibitory to the LH-induced cyclic AMP accumulation, depending on the culture period. Our results, taken together with other findings, are compatible with the view that IL-1 alpha has a potential regulatory role in the granulosa-luteal cell transition in the human ovary.

The content of PDGF in human blastocyst culture medium (n = 8), serum (n = 12), and FF (n = 17) from natural IVF cycles was determined by an RIA specific for PDGF B-chain. The blastocysts were cultured under serum-free conditions throughout development. The findings show that PDGF B-chain is released into the culture medium of human blastocysts and that serum is positive, whereas FF is negative for PDGF.

The present review discusses the current knowledge of the molecular pharmacology and neuroanatomical and subcellular localization of both the central benzodiazepine/GABA-chloride ionophore receptor complex and the peripheral benzodiazepine receptor. It then reviews all of the literature to date on how these two receptor sites are modulated by environmental stress. The possible role of these sites in learning and memory is also discussed. Finally, a theoretical model is presented which examines the differential, and perhaps complementary, alterations of these two sites in an organism's response to stress.

Adult male and female rats were exposed to either inescapable shock or no treatment. In vitro [3H]Ro 5-4864 (4'-chlorodiazepam) binding (1 nM) to peripheral benzodiazepine receptors (PRB) in both CNS and peripheral tissues indicated no gender differences in olfactory bulb, heart, lung or adrenal gland but a significant effect was observed in renal tissue. Female rats showed an attenuated stress-induced reduction (23%) in PBR in comparison to males (55%). This difference was shown to be an alteration of Bmax and not kD by Scatchard analysis. These data are the first demonstration of a sexual dimorphism in environmentally-induced alterations in PBR.

Previous research has demonstrated that low doses of anxiogenic central benzodiazepine receptor (CBR) ligands, the beta-carbolines, improve performance in various learning and memory tests in animals if administered prior to training. The present experiments compared the effect of a beta-carboline (FG 7142) with that of a pharmacologically distinct anxiogenic compound, a peripheral benzodiazepine receptor (PBR) ligand, 4'-chlorodiazepam (Ro5-4864), in two tests of learning and memory in rats. As expected, FG 7142 significantly improved performance in a passive avoidance test. Ro5-4864 was without effect. In a shuttlebox escape test, Ro5-4864 significantly impaired performance while FG 7142 had no effect. The effect of Ro5-4864 was antagonized by the specific peripheral benzodiazepine receptor antagonist, PK 11195. These results indicate that the differential impact of CBR and PBR anxiogenic ligands on performance in aversively-motivated learning tests may be a reflection of their distinct pharmacologies.

Exposure to environmental stress causes changes in the binding of [3H]Ro 5-4864 to peripheral benzodiazepine receptors (PBRs). The influence of the central nervous system (CNS) in these stress-induced modifications is unclear. The present study examined whether pretreatment with a dose-response regimen of sodium pentobarbital would impact the stress-induced reduction in renal PBR. Administration of either a sedative/ataxic (20 mg/kg) or hypnotic (60 mg/kg) dose of pentobarbital prior to stress blocks the stress-induced decrease of [3H]Ro 5-4864 binding to renal PBR in rat. These findings suggest that higher-order, supraspinal mechanisms play a critical role in marshalling the renal PBR changes in response to stress.

In order to determine whether leukotrienes, products of the lipoxygenase pathway, are involved in ovulation, pairs of rabbit ovaries were treated with the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) while being perfused in vitro. The control ovaries from each rabbit received luteinizing hormone (LH) (1.5-2.25 micrograms ml-1) while the contralateral ovaries were treated with LH + NDGA (100 microM) or LH + CA (100 microM). The numbers of ovulations from both the LH + NDGA- and LH + CA-treated ovaries were significantly higher (P less than 0.05) than from their respective LH-stimulated controls. Treatment with NDGA alone in the perfusate did not cause any ovulation, while CA alone caused one ovulation from one of six ovaries perfused. Ovarian tissue levels of prostaglandins after 7 h of perfusion with LH + NDGA or with LH alone showed that, in five of the six ovaries perfused in this group, the tissue levels of PGE2, 6-keto-PGF1 alpha and PGF2 alpha were higher in the presence of NDGA. The mean differences were significant (P less than 0.05) for prostacyclin but not significant (P greater than 0.05) for PGE2 and PGF2 alpha. Our interpretation of the findings is that, when used for blocking the lipoxygenase pathway, NDGA and CA increase the substrate availability for the cyclo-oxygenase pathway of arachidonic acid metabolism, resulting in a net increase in prostaglandins. The increased ovarian levels of prostaglandins, especially prostacyclin, may cause the observed increase in ovulation rate. Consequently, although the leukotrienes may be involved in the mechanism of ovulation in the rabbit, their effects appear to be less pronounced than those of prostaglandins.

This work investigates the production of prostaglandin E2 (PGE2), a well known modulator for the suppression of immune cells, by pre-implantation human conceptuses from the 4-cell stage to the hatched blastocyst and by cumulus oophorus cells, these being obtained from an IVF/ET program. Cumulus cell complexes cultured for 48 h produced considerable amounts of PGE2, necessitating complete removal of their influence on the conceptus cultures. All stages of the human conceptus studied produced PGE2 during 48-h cultures, the greatest amounts from late blastocysts, and from those cultured in media containing bovine serum albumin rather than human donor serum. Indomethacin-treated control blastocysts confirmed the synthesis and release from the human conceptuses. The production of PGF2a from human blastocysts could not be demonstrated.

The present work investigates the possibility that lipoxygenase products are involved in the biochemical mechanisms of blastocyst implantation by utilizing nordihydroguaiaretic acid (NDGA) and caffeic acid (CA), inhibitors of lipoxygenase enzymes, and quinacrine (QU), an inhibitor of phospholipase-A2. It has been shown previously that inhibition of cyclooxygenase results in blockade of implantation. The inhibitors were dissolved in a standard medium and 5 microliter of the solutions were micro-injected into the uterine horns of day-4 pregnant mice. The contralateral horns acted as controls and received only vehicle. A sham-operated group provided normal controls. In 14 NDGA-treated mice, the control horns contained 40 implantations while the treated horns contained only 6 small implantations and 8 resorbing sites. These control horns were comparable to the sham controls. In 14 CA-treated mice, treated horns contained 17 small implantations plus 4 resorptions, whereas the control horns contained 26 small implantations and 4 resorptions. Twelve QU-treated mice exhibited 7 small implantations and 4 resorptions in the treated horns, plus 24 small sites and no resorptions in the control horns. Fourteen sham-operated mice had 95 implantation sites and no resorptions in their 28 horns. The results provide evidence for the involvement of the lipoxygenase enzymes and phospholipase-A2 in the initial implantation process and in the subsequent development of early pregnancy.

A previously described technique for perfusion of the isolated rabbit ovary in a recirculating system (Ahrén et al., 1975) was modified for studies of preovulatory follicular development and ovulation. Follicular ruptures were induced either by injection of human chorionic gonadotropin (hCG) to the animal 5-6 h prior to perfusion or by adding luteinizing hormone (LH) directly to the medium during perfusion. Ovulation occurred 9-14 h after hCG injection in vivo and 7-13 h after the addition of LH in vitro. Ovulations did not occur unless the ovaries were stimulated by gonadotropin in vivo or in vitro. The patterns of release of estradiol-17 beta and progesterone into the medium are consistent with the measurements reported by others on these steroids in ovarian venous blood in vivo. This experimental model is likely to be useful in further exploration of the physiology, biochemistry and anatomy of ovulation.

Ovulation was induced in rabbits by intravenous administration of human chorionic gonadotrophin (HCG), and 4-5 h later the ovaries were isolated and introduced into an in-vitro perfusion system containing synthetic medium with albumin. Rupture of follicles occurred in vitro within the physiological time range (mean 11.3 h after injection of HCG), although with a reduced frequency. Preovulatory and ruptured follicles were studied in detail by light and electron microscopy. In the granulosa layer of ruptured or preovulatory follicles cytoplasmic blebbing activity, disappearance of Call-Exner bodies and differentiation toward luteinized cells were found. Perhaps the most important sign of normal preovulatory development in vitro was that the basement membrane surrounding the granulosa layer was penetrated by projections of granulosa cells. In the absence of this penetration phenomenon the granulosa layer prolapsed out of the follicle. Immediately before rupture, follicles showed marked degeneration, restricted to the outer layers of the apical wall, which is compatible with the hypothesis that degradative enzymes are released close to the surface of preovulatory follicles. Although the majority of follicles that ovulated under in-vitro conditions showed the same kind of morphological alterations as can be seen in vivo, occasional atypical ruptures occurred without any overt signs during perfusion. Also technical manipulations of the perfusion system, e.g., nonphysiological increase of perfusion pressure, could force follicles to rupture. This illustrates the importance of careful morphological study of all ovaries perfused in vitro before conclusions are drawn.

Oestrogen administration to female mice with 'delayed' implantation induces changes in the uterus and blastocysts followed by implantation and normal gestation. The second messenger for oestrogen, adenosine 3' :5'-cyclic monophosphate (cAMP), was instilled into the uterine lumen of females with 'delayed implanting' blastocysts to determinewhether it also is able to induce implantation. Implantation of the blastocysts was induced by cAMP but the pregnancies were not maintained to tem. These results are discussed with respect to the changes in blastocysts induced by cAMP and with respect to the reported contraceptive effects of cAMP in normal mice.

Estrogen induces blastocyst implantation in diapausing female mice, increases uterine levels of adenyl cyclase and cyclic adenosine monphosphate (cylic AMP), and stimulates synthesis of ribonucleic acid and protein in both the uterus and blastocyst. Furthermore, the surface ultrastructure of trophoblast cells seen with scanning electron microscopy (SEM) changes with activation of the diapausing blastocyst by estrogen. Cyclic AMP-activated blastocysts were investigated with the use of SEM and compared to blastocysts activated by estrogen. Intraperitoneal administration of cyclic AMP was generally ineffective, whereas administration of cyclic AMP intraluminally in the uterus effectively mimicked the early estrogen activation. These findings are discussed in relation to other known activation changes in preimplantation blastocysts and with regard to similar findings after administration of various antiestrogens.

Surface cell changes at the apices of preovulatory follicles and ovulations were documented in isolated perfused ovaries from immature rats treated with pregnant mare serum gonadotropin (20 IU) and 48 h later with human chorionic gonadotropin (hCG) (10 IU). A video camera coupled to an inverted microscope and a video recorder captured the preovulatory and ovulatory events at a cellular level. At around 8 h post-hCG, the follicular apex changed from a smooth and optically homogeneous appearance into a rough surface with bleb formation and extrusions of single cells through minute perforations (early stigma formation). At approximately 10 h, a sticky material formed a basketlike structure with trapped cells (late stigma formation). At 12 to 15 h, ovulation took place at a constant speed and with no contractions of the follicular wall. This indicates that ovulation can occur with no visible circumfollicular muscular activity. Furthermore, the observations of a leakage of cells over an extended period of time indicates that the follicular wall is partly digested several hours before ovulation occurs.

Delayed-implantation mice were used to test for local implantation induction effects of prostaglandins E2 and F2 alpha. Evidence of implantation was gathered by microdissection of implantation sites and by scanning electron microscopy. Indomethacin was tested for the ability to interfere with the mechanisms of estrogen-induced, normal implantation. Both prostaglandins appear to have significant effect inducing implantation when applied locally in the uterine lumen, PGE2 being more effective than PGF2 alpha. The indomethacin clearly has a blocking effect on normal implantation. However, indomethacin appears only partially able to prevent the morphological changes indicative of the trophoblast cell transformation.

The temporal and spatial parameters of oestrogen-induced increases in RNA, protein and DNA synthesis were investigated autoradiographically in mice undergoing experimental diapause. The tritiated precursors, uridine, amino acid hydrolysate and thymide, were instilled into uterine lumen for a 15 minute pulse at various times after subcutaneous oestrogen treatment. RNA synthesis increased between 15 and 30 minutes, and protein synthesis between 30 and 45 minutes, after oestrogen administration. DNA synthesis did not increase up to 24 hours after oestorgen. No spatial correlations could be made between trophoblastic giant cell transformation and the RNA and protein synthesis although both are induced by oestrogen.

The effects of prostaglandin E2 (PGE2) on the ovulation process were studied in a recirculating perfusion model using ovaries from virgin rabbits. Ovulation frequency, time of ovulation, and progesterone release from the ovaries were examined after the addition of PGE2, either alone or with luteinizing hormone (LH) in the presence or absence of indomethacin. The stimulatory effect of LH on ovulation was totally blocked if the ovaries were exposed to indomethacin at the same time. Ovaries treated with PGE2 alone did not ovulate, and PGE2 was unable to restore indomethacin-blocked ovulation. Conversely, the frequency of ovulation was reduced in ovaries treated with PGE2 and LH compared with controls receiving only LH. There was no measurable difference in the pattern of progesterone release between ovaries simultaneously treated with LH and indomethacin and LH-treated controls. Ovaries exposed to PGE2 alone showed only a slight increase of progesterone release in the medium, while those treated with PGE2 in combination with LH in the perfusate showed a smaller progesterone release than those treated with LH alone. The results confirm the blocking effect on ovulation by indomethacin. PGE2 could not reverse this effect, but instead reduced the number of LH-induced follicular ruptures. Indomethacin had no effect on progesterone levels, while PGE2 (which alone showed a slight stimulating effect on the steroid concentration) together with LH counteracted the effect of LH on progesterone release.

Both ovaries of 31 rabbits were perfused with a chemically defined medium in vitro in a recirculation system. In one series of experiments, hCG (100 IU) was injected iv 5-6 h prior to anaesthesia and surgery. Approximately 1 h later the perfusion was started. One ovary was perfused as control while the other ovary was perfused with 5 micrograms/ml indomethacin or with indomethacin and 1 micrograms/ml PGF2 alpha. In another series of experiments the rabbits received no pretreatment prior to operation. Instead, bovine LH was added to the perfusion medium of both control and experimental ovaries. The experimental side also received either indomethacin or indomethacin and PGF2 alpha. Finally, the effect of PGF2 alpha in the absence of LH was compared to the control ovary receiving only LH. After injection of hCG in vivo, ovulations occurred in 4 of 5 control ovaries. Indomethacin completely blocked ovulation in 4 of the 5 ovaries treated, while PGF2 alpha restored ovulations in all the experimental ovaries. In the group of experiments where LH was added in vitro, ovulations were induced in all ovaries treated with varying LH doses. Furthermore, indomethacin blocked ovulation in 5 out of 7 ovaries, and PGF2 alpha restored ovulation in all ovaries. Fifty per cent of the ovaries treated only with PGF2 alpha (in the absence of LH) also ovulated. The pattern of steroid release did not differ between control ovaries, indomethacin treated ovaries, and indomethacin + PGF2 alpha treated ovaries. Ovaries treated in perfusion with PGF2 alpha alone had very low steroid levels compared to the ovaries treated with LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated ovaries from untreated, sexually mature rabbits were introduced into an in vitro perfusion system and perfused with a chemically defined medium containing albumin. The ovaries were perfused for up to 15 h (mean 11.5 h) and then processed for morphological investigation. Both at the light- and electron-microscopical levels, most of the ovaries exhibited a normal structure comparable with ovaries in situ. In two cases, however, marked accumulations of bacteria were found, although not inside the follicles. Since ovulation in the rabbit normally occurs between 9.5-13 h after mating or human chorionic gonadotrophin treatment, this model seems adequate for studies of ovulation in vitro. It is, however, important to study the ovaries microscopically after the perfusion to detect artifacts, e.g., bacterial infection, that may have influence on the process of ovulation.

LH alters ovarian steroidogenesis via adenylate cyclase (AC) activation and cAMP production. Although LH also initiates ovarian follicle rupture, evidence is lacking for involvement of cAMP in this process. This work explores the involvement of cAMP in the ovulation of in vitro perfused rabbit ovaries by comparing LH stimulation of ovaries with that of LH plus 3-isobutyl-1-methyl-xanthine (IBMX), (an inhibitor of phosphodiesterase) and of forskolin (a nonreceptor-specific activator of AC). Venous perfusates were analyzed for cAMP, progesterone, 17 beta-estradiol, and testosterone, ovaries were analyzed for cAMP, and ovulations were noted. LH, LH plus IBMX, and forskolin all increased tissue cAMP levels significantly after 0.5 h, the perfusate levels increasing rapidly thereafter reaching plateau levels, while tissue levels returned to control levels after 2.4 h. LH plus IBMX and forskolin significantly increased cAMP release over LH controls, LH plus IBMX increasing and forskolin decreasing the number of ovulations. Forskolin significantly increased progesterone release over LH controls and, although no other significant steroid differences were seen, strong tendencies existed. Although forskolin could induce ovulations and could induce significantly higher release of cAMP than LH, it resulted in a lower ovulation rate than receptor-specific LH. LH plus IBMX also induced significantly higher cAMP release than LH, as did forskolin, and resulted in a higher ovulation rate than both LH and forskolin. These findings suggest, not only that cAMP production alone is sufficient for ovulation, but also that the receptor specificity of the cAMP production is important for the number of ovulations. Since tissue levels of cAMP peak several hours before ovulation, the cAMP is probably inducing a metabolic pathway leading to ovulation.

Using a method of in vitro perfusion of the rabbit ovary with a chemically defined medium in a recirculation system, normal appearing follicular ruptures occurred following exposure of the ovaries to hCG in vivo (100 IU) or following addition of LH (0.25 microgram/ml of NIH B9) to the perfusate. The addition of oestradiol-17 beta (10 micrograms/ml) to the perfusate did not inhibit these follicular ruptures, although the follicular fluid oestradiol contents were increased more than 100-fold as compared to the control side not receiving the addition of oestradiol. These data suggest that the physiological decline of follicular oestrogen, normally observed in vivo prior to ovulation in the rabbit, is not required as part of the mechanism of ovulation and that normal appearing ovulations can occur even though follicular oestrogen levels are kept artificially elevated.

Four hatched human blastocysts obtained after in-vitro fertilization and development were placed on monolayer cell cultures of human endometrial epithelium, and subsequently examined by transmission electron microscopy. All four blastocysts became adherent to the monolayer and three implanted and exhibited outgrowth of their trophoblastic cells. During implantation the blastocysts differentiated into mural and polar trophoblastic cells, and embryonic cells including endodermal cells. The endometrial cells were displaced and stacked into a multilayer at the periphery of the implantation sites, allowing the trophoblastic cells to come in contact with the culture dish. The endometrial cells displayed local exo- or endo-cytosis where they contacted the trophoblastic cells. The trophoblastic cells were not observed to be phagocytosing endometrial cells. These observations suggest that human blastocysts portray an intrusive type of implantation during the initial stages.

Using the isolated perfused rabbit and rat ovaries as experimental models, we have studied various biochemical aspects of the ovulatory process. In rabbits, ovulations were induced by injecting hCG prior to the perfusion or by adding LH directly to the medium. In PMSG-treated rats, ovulations were induced by adding LH to the perfusion system. Steroids and other metabolites were analyzed in the perfusate and in follicular fluid. Steroid levels in follicular fluid were high early in the preovulatory development, but declined to very low levels 4 hours after LH stimulation. Levels of prostaglandins E and F rose as ovulation approached. In both perfusion models, indomethacin blocked ovulation without affecting steroid release or oocyte maturation. In the rabbit, PGF2 alpha reversed the indomethacin-induced inhibition and was able to induce follicular rupture by itself. Manipulations of the follicular fluid content of progesterone and estradiol to supraphysiological levels did not affect follicular rupture or oocyte maturation in the rabbit model. When the initial increase in LH-induced steroidogenesis was blocked by a 3 beta-ol-dehydrogenase inhibitor, ovulation was not affected. In rats, inhibition of estradiol production by an aromatase blocker did not affect the ovulatory process. When the endogenous formation of cyclic AMP is increased by pretreatment with a phosphodiesterase inhibitor, the LH-induced ovulation frequency increases in rabbits. Furthermore, forskolin, which increased the adenylate cyclase activity, stimulated steroidogenesis and induced follicular rupture. Recent experiments in the rat indicate that cyclic AMP acts on the ovulatory process via an effect on prostaglandin synthesis.

Rabbit ovaries were isolated surgically before the ovulatory gonadotrophin stimulation and perfused in vitro. Untreated, control ovaries never ovulated. Ovaries treated in vitro with ovine LH ovulated 10-14 h later and the oocytes had undergone germinal vesicle breakdown (GVB). LH induced increases in progesterone secretion from the treated ovaries. A 3 beta-hydroxysteroid dehydrogenase blocker ('Compound A') effectively reduced progesterone secretion into the perfusate and follicular fluid to very low levels but had no effect on ovulation rate or on oocyte maturation. Excessively high progesterone levels were obtained artificially in perfusates by addition of exogenous steroid; the number of ovaries ovulating was markedly reduced but there was no effect on oocyte maturation. It is concluded that the rise in progesterone that normally occurs immediately after the LH surge is not a prerequisite for ovulation in the rabbit. However, progesterone may have a modifying effect on LH-induced follicle rupture when at a pharmacologically high level.