Quantitative method for conjugated metabolites of bisphenol A and bisphenol S determination in food of animal origin by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry.

With the objectives of both generating bisphenols (BPs) conjugates occurrence data in food from animal origin but also investigating the origin of associated contamination, the present study deals with the development of an efficient analytical method aiming at monitoring both BPA and BPS conjugated metabolites in food from animal origin. The objective of such monitoring is to determine the origin of BPs contamination (FCM or animal contamination). The targeted compounds were BPA-monoglucuronide (BPA-1G), BPA-diglucuronide (BPA-2G), BPA-monosulfate (BPA-1S), BPA-disulfate (BPA-2S) and BPS-monoglucuronide (BPS-1G). The developed standard operating procedure includes a preliminary solid-liquid extraction step followed by two successive solid phase extraction (SPE) stages, using successively a non-polar phase and a strong cation exchange polymer. Quantification was achieved according to both the isotopic dilution and surrogated quantification methods, using 13C-BPA-1G and BPA-d6-1S as internal standards. Linearity was validated (R2 > 0.99) for each molecule within the concentration range [0-10] µg kg-1. Detection limits ranged from 0.02 µg kg-1 (BPA-1G in muscle, BPA-1S and BPA-2G in liver) to 0.50 µg kg-1 (BPA-2S in muscle). The strategy was then proven on liver samples collected from pregnant ewes subcutaneously exposed to BPA during 105 days, at 50 µg kg-1 per day. BPA-1G, BPA-2G and BPA-1S were detected and quantified at a concentration of 3.81 µg kg-1, 0.80 µg kg-1 and 0.09 µg kg-1, respectively. The analytical method was finally implemented on fifty unpacked food samples from animal origin in which significant free BPA concentrations were previously measured. Since no metabolites of BPA could be measured (

[An update on Candida dubliniensis]. / Actualités sur Candida dubliniensis.

Eleven years ago, Irish authors, using molecular biology, demonstrated the existence of Candida dubliniensis, a new species of Candida close to Candida albicans. Initially isolated from AIDS patients with oral candidiasis, this species was detected, even in immunocompetent patients. Recently, with new, easy to implement identification tests (latex, immunochromatography), numerous epidemiological studies were undertaken. In most studies, C. dubliniensis was most often identified in the oral cavity. In the absence of HIV infection, the proportion C. dubliniensis/C. albicans ranged from 1 to 5% but it increased to 15-20% in case of HIV infection. It should be stressed that, from an experimental point of view, the acquisition of a secondary resistance to fluconazole is more quickly obtained with C. dubliniensis that with C. albicans, this resistance remains exceptionally observed in clinical observations.

Maternal parity affects placental development, growth and metabolism of foals until 1 year and a half.

Primiparous mares are known to produce smaller foals than multiparous mares. This difference seems to be partly explained by the reduced exchange surface and volume of the placental villi in primiparous compared to multiparous placentas. The effect of maternal parity on foals' post-natal growth, metabolism and sexual maturation, however, has been given little consideration. The objectives of this work were to analyse placental biometry and structure at term, growth of foals and yearlings, their metabolism and testicular maturation at one year of age. Twenty multiparous mares (M), aged over 6 years and 12 primiparous mares (P), aged up to 5 years were artificially inseminated with the same stallion and monitored the same way until foaling. At birth, foals and placentas were measured and placentas were sampled above at the umbilical cord insertion, as well as in the pregnant and the non-pregnant horn to perform stereological analyses. Foals were weighed and measured until 540 days of age. At 120 and 360 days of age, an Intravenous Glucose Tolerance Test was performed on foals and yearlings. At 360 days of age, the males were castrated and testicular maturation analysed by RT-qPCR. At birth, P dams produced lighter and smaller foals and placentas. The foal birth weight to placental surface ratio was lower in the P compared to the M group. P Foals remained lighter than M foals until 360 days of age and smaller until at least 540 days of age. At 120 days of age, P foals had a higher glucose tolerance than M foals, and then may be less mature than M foals in terms of the control of their glucose homeostasis. At 360 days of age, the testicles of prepubertal P stallions were less mature in the P vs the M group. In conclusion, primiparous dams produce intrauterine growth restricted, less mature and smaller foals compared to multiparous dams with altered metabolism and growth until at least 540 days of age. These differences could affect the sport career of these foals, especially if it begins at an early age.

Maternal obesity increases insulin resistance, low-grade inflammation and osteochondrosis lesions in foals and yearlings until 18 months of age.

INTRODUCTION: Obesity is a growing concern in horses. The effects of maternal obesity on maternal metabolism and low-grade inflammation during pregnancy, as well as offspring growth, metabolism, low-grade inflammation, testicular maturation and osteochondrotic lesions until 18 months of age were investigated. MATERIAL AND METHODS: Twenty-four mares were used and separated into two groups at insemination according to body condition score (BCS): Normal (N, n = 10, BCS ≤4) and Obese (O, n = 14, BCS ≥4.25). BCS and plasma glucose, insulin, triglyceride, urea, non-esterified fatty acid, serum amyloid A (SAA), leptin and adiponectin concentrations were monitored throughout gestation. At 300 days of gestation, a Frequently Sampled Intravenous Glucose Tolerance Test (FSIGT) was performed. After parturition, foals' weight and size were monitored until 18 months of age with plasma SAA, leptin, adiponectin, triiodothyronine (T3), thyroxine (T4) and cortisol concentrations measured at regular intervals. At 6, 12 and 18 months of age, FSIGT and osteoarticular examinations were performed. Males were gelded at one year and expression of genes involved in testicular maturation analysed by RT-qPCR. RESULTS: Throughout the experiment, maternal BCS was higher in O versus N mares. During gestation, plasma urea and adiponectin were decreased and SAA and leptin increased in O versus N mares. O mares were also more insulin resistant than N mares with a higher glucose effectiveness. Postnatally, there was no difference in offspring growth between groups. Nevertheless, plasma SAA concentrations were increased in O versus N foals until 6 months, with O foals being consistently more insulin resistant with a higher glucose effectiveness. At 12 months of age, O foals were significantly more affected by osteochondrosis than N foals. All other parameters were not different between groups. CONCLUSION: In conclusion, maternal obesity altered metabolism and increased low-grade inflammation in both dams and foals. The risk of developing osteochondrosis at 12 months of age was also higher in foals born to obese dams.

Effect of swimming on prednisolone-induced osteoporosis in elderly rats.

We investigated the possible ameliorating and preventive effect of swimming on prednisolone-induced osteoporosis in elderly rats. A total of 48 female Sabra strain rats were randomly assigned to the following groups and treatments: (1) control (C), (2) swimming (S), (3) prednisolone-treated (CP), and (4) swimming + prednisolone (SP). An additional 8 rats were sacrificed and examined at the onset of the study. Groups C and S were sham injected; groups CP and SP were injected with prednisolone (Ultracorten), 80 mg/kg three times per week for 10 weeks. Groups S and SP swam 1 h daily, 5 days per week for 10 weeks. SP rats swam simultaneously with prednisolone administration. At the end of the swimming period, in vivo bone mineral content (BMC) measurements were performed on rat vertebrae L4-5 by single-photon absorptiometry. Later, the humerus and femur were removed for the following measurements: morphometric, bone density (BD) by Compton scattering technique, bone ion content by atomic absorption, and hydration fraction by proton magnetic resonance (PMR). We found that the humeral BD of S rats was greater by 14% for group S over C and 3% greater for group SP over CP (P less than 0.05). Vertebral BMC was higher by 15% in group S over C and 11% higher for group SP over CP (P less than 0.05). Femoral calcium (mg/g dry bone) ion content was higher by 5% in group S over C and 8% in group SP over CP group (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Median eminence dopaminergic activation is critical for the early long-day inhibition of luteinizing hormone secretion in the ewe.

In ewes, photoperiod modulates LH release. The median eminence (ME) dopaminergic activity seems to be implicated in the inhibition of LH secretion by photoperiod. This study investigated the functional importance of ME dopaminergic activity for LH secretion inhibition in three inhibitory photoperiodic treatments: after 33 long days (LD) (LD1 treatment), after 72 LD (LD2 treatment), and after 34 short days. Using reverse microdialysis on three groups of seven ewes, a solution of alpha-methyl-paratyrosine [alphaMPT, an inhibitor of tyrosine hydroxylase (TH); 10 mM in Ringer's lactate] was infused into the ME for 5 h, preceded by a 5-h control period during which only vehicle was infused, in each of the three photoperiodic treatments. AlphaMPT dramatically decreased the 3,4-dihydroxyphenylacetic acid concentration, similarly in all three photoperiodic treatments, suggesting a similar inhibition of TH activity. In the LD1 treatment, alphaMPT significantly increased LH pulse frequency (+1.22 +/- 0.46 pulse/5 h from control period, mean +/- SEM, n = 9; P < 0.05) and mean concentration (+51 +/- 28%; P < 0.001). In the other two photoperiodic treatments, alphaMPT had no significant effect on LH release. Thus, blockade of dopamine synthesis in the ME seems to stimulate LH secretion in early, but not long-term, inhibition by LD nor after the transition to short days. Therefore, dopaminergic activity of the ME seems to be critical for LH secretion inhibition in some photoperiodic inhibitory treatments but not in others.

Characterization of the short day-induced decrease in median eminence tyrosine hydroxylase activity in the ewe: temporal relationship to the changes in luteinizing hormone and prolactin secretion and short day-like effect of melatonin.

In the ewe, photoperiod modulates LH and PRL secretion as well as median eminence (ME) dopaminergic activity. The studies reported here were designed to characterize the functional significance of this photoperiodic modulation of ME dopaminergic neuron activity in relation to the regulation of LH and PRL secretion. The aim of the first experiment was to assess whether photoperiodic changes in hypothalamic dopaminergic activity were temporally linked to changes in either PRL or LH secretion. The purpose of the second experiment was to determine whether melatonin mimicked the effects of photoperiod on ME dopaminergic activity. In the first experiment, LH and PRL secretion, hypothalamic tyrosine hydroxylase (TH) activity, and catecholamine contents were determined in ovariectomized estradiol-treated ewes either during long days (LD; control group) or after 5, 25, and 76 short days (SD). SD were associated with a stimulation of LH secretion and a decrease in ME TH activity, which were both expressed only in the 76 SD group. In contrast, the SD-induced inhibition of PRL secretion was already maximal in the 25 SD group. In the second experiment, LH secretion and hypothalamic dopaminergic activity were studied in ovariectomized estradiol-treated ewes kept in LD and then treated for 0 (control), 25, or 77 days with melatonin implants producing a SD-like effect on LH secretion. Melatonin induced a decrease in PRL secretion (observed after 25 days of treatment), as well as a stimulation of LH secretion and a decrease in ME TH activity and dopamine content (observed only after 77 days of treatment). In conclusion, the decrease in ME dopaminergic activity associated with SD exposure or the SD-like effect of melatonin appears unrelated to the regulation of PRL secretion. The SD-like effect of melatonin on ME dopaminergic activity suggests that melatonin mediates the effect of SD on this activity. The regulation of ME dopaminergic activity can thus be considered a probable step in the photoperiodic regulation of LH secretion.

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone.

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:

Endotoxin disrupts the estradiol-induced luteinizing hormone surge: interference with estradiol signal reading, not surge release.

Three experiments were conducted to investigate whether the immune/inflammatory stimulus endotoxin disrupts the estradiol-induced LH surge of the ewe. Ovariectomized sheep were set up in an artificial follicular phase model in which luteolysis is simulated by progesterone withdrawal and the follicular phase estradiol rise is reproduced experimentally. In the first experiment, we tested the hypothesis that endotoxin interferes with the estradiol-induced LH surge. Ewes were either infused with endotoxin (300 ng/kg/h, i.v.) for 30 h beginning at onset of a 48-h estradiol stimulus or sham infused as a control. Endotoxin significantly delayed the time to the LH surge (P < 0.01), but did not alter surge amplitude, duration, or incidence. The second experiment tested the hypothesis that the delaying effects of endotoxin on the LH surge depend on when endotoxin is introduced relative to the onset of the estradiol signal. Previous work in the ewe has shown that a 14-h estradiol signal is adequate to generate GnRH and LH surges, which begin 6-8 h later. Thus, we again infused endotoxin for 30 h, but began it 14 h after the onset of the estradiol signal. In contrast to the first experiment, endotoxin given later had no effect on any parameter of the LH surge. In the third experiment, we tested the hypothesis that endotoxin acts during the first 14 h to disrupt the initial activating effects of estradiol. Estradiol was delivered for just 14 h, and endotoxin was infused only during this time. Under these conditions, endotoxin blocked the LH surge in five of eight ewes. In a similar follow-up study, endotoxin again blocked the LH surge in six of seven ewes. We conclude that endotoxin can disrupt the estradiol-induced LH surge by interfering with the early activating effects of the estradiol signal during the first 14 h (reading of the signal). In contrast, endotoxin does not disrupt later stages of signal processing (i.e. events during the interval between estradiol signal delivery and surge onset), nor does it prevent actual hormonal surge output. Thus, endotoxin appears to disrupt estrogen action per se rather than the release of GnRH or LH at the time of the surge.

Thyroid hormones act primarily within the brain to promote the seasonal inhibition of luteinizing hormone secretion in the ewe.

In the ewe, thyroid hormones are required for the seasonal suppression of GnRH and LH secretion, thereby maintaining an annual rhythm in reproductive activity. The primary site of action of thyroid hormones is unknown; in particular, there is no evidence to distinguish a central from a peripheral action. In this study, we test the hypothesis that thyroid hormones can act directly within the brain to promote GnRH/LH seasonal inhibition. Ovariectomized estradiol-treated ewes were thyroidectomized late in the breeding season to prevent seasonal LH inhibition. T4 was then infused for 3 months, either peripherally or centrally. Neuroendocrine reproductive state was monitored by assaying the LH concentration in biweekly blood samples. Central infusion of low dose T4, which restored a physiological concentration of the hormone in cerebrospinal fluid of these thyroidectomized ewes, promoted the neuroendocrine changes that lead to anestrus. The serum LH concentration in these animals fell at the same time as the seasonal LH decline in euthyroid controls. Neither this same T4 dose infused peripherally nor vehicle infused centrally was effective; LH remained elevated, signifying blockade of the mechanism for anestrus. Our results provide strong evidence that thyroid hormones can act directly within the brain to promote seasonal inhibition of neuroendocrine reproductive function in the ewe.

Potential for polysialylated form of neural cell adhesion molecule-mediated neuroplasticity within the gonadotropin-releasing hormone neurosecretory system of the ewe.

The GnRH neurosecretory system undergoes marked structural and functional changes throughout life. The initial goal of this study was to examine the neuroanatomical relationship between GnRH neurons and a glycoprotein implicated in neuroplasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Using dual label immunocytochemistry in conjunction with confocal microscopy, we determined that fibers, terminals, and perikarya of GnRH neurons in adult ovariectomized ewes are intimately associated with PSA-NCAM. In the preoptic area, intense PSA-NCAM immunoreactivity was evident around the periphery of GnRH cell bodies. The second goal of this study was to determine whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal changes in the activity of the GnRH neurosecretory system in ovariectomized ewes treated with constant release implants of estradiol. During the breeding season when reproductive neuroendocrine activity was enhanced, the expression of PSA-NCAM immunoreactivity associated with GnRH neurons was significantly greater than that during anestrus when GnRH secretion was reduced. This difference, which occurred despite an unchanging ovarian steroid milieu, was not observed in preoptic area structures devoid of GnRH immunoreactivity, suggesting that the seasonal change is at least partially specific to the GnRH system. The close association between PSA-NCAM and GnRH neurons and the change in this relationship in conjunction with seasonal alterations in GnRH secretion provide anatomical evidence that this molecule may contribute to seasonal remodeling of the GnRH neurosecretory system of the adult.

Endotoxin inhibits the reproductive neuroendocrine axis while stimulating adrenal steroids: a simultaneous view from hypophyseal portal and peripheral blood.

This study was designed to test the hypothesis that systemic immune challenge with endotoxin inhibits the reproductive axis centrally by suppressing GnRH pulsatile release into hypophyseal portal blood. Using alert, normally behaving, ovariectomized ewes, we sampled hypophyseal portal blood at 10-min intervals beginning 4 h before and continuing 10 h after endotoxin (400 ng/kg, iv bolus, n = 6) or saline (vehicle, iv, n = 6). Simultaneous jugular samples for measurement of LH, cortisol, and progesterone were taken, and core body temperature was monitored by telemetry. Saline had no effect on any of the parameters in control ewes. In contrast, endotoxin dramatically inhibited the reproductive neuroendocrine axis coincident with stimulating the adrenal steroids, cortisol and progesterone, and elevating body temperature. Mean GnRH collection rate and GnRH pulse amplitude were suppressed (pre- vs. 7 h postendotoxin: collection rate 0.93 +/- 0.31 vs. 0.34 +/- 0.13 pg/min; amplitude 4.13 +/- 1.33 vs. 1.30 +/- 0.41 pg/min per pulse; P < 0.05 and P = 0.01). However, endotoxin did not have a significant effect on GnRH pulse frequency. Along with inhibited GnRH secretion, endotoxin significantly suppressed mean LH concentrations (P = 0.001) and LH pulse amplitude (P < 0.05). In addition, endotoxin suppressed LH pulse frequency (P = 0.01). Coincident with reproductive inhibition, endotoxin stimulated cortisol (P < 0.001), progesterone (P < 0.01), and core body temperature (P < 0.001). We conclude that the suppressive effects of endotoxin on the reproductive axis can be mediated centrally through an inhibition of GnRH and thus LH pulsatile secretion. The coincident stimulation of cortisol, progesterone, and temperature raises the possibility that the central inhibition of the reproductive system may be a consequence of any or all of these activated parameters.

Prostaglandins mediate the endotoxin-induced suppression of pulsatile gonadotropin-releasing hormone and luteinizing hormone secretion in the ewe.

Five experiments were conducted to test the hypothesis that PGs mediate the endotoxin-induced inhibition of pulsatile GnRH and LH secretion in the ewe. Our approach was to test whether the PG synthesis inhibitor, flurbiprofen, could reverse the inhibitory effects of endotoxin on pulsatile LH and GnRH secretion in ovariectomized ewes. Exp 1-4 were cross-over experiments in which ewes received either flurbiprofen or vehicle 2 weeks apart. Jugular blood samples were taken for LH analysis throughout a 9-h experimental period. Depending on the specific purpose of the experiment, flurbiprofen or vehicle was administered after 3.5 h, followed by endotoxin, vehicle, or ovarian steroids (estradiol plus progesterone) at 4 h. In Exp 1, flurbiprofen reversed the endotoxin-induced suppression of mean serum LH concentrations and the elevation of body temperature. In Exp 2, flurbiprofen prevented the endotoxin-induced inhibition of pulsatile LH secretion and stimulation of fever, reduced the stimulation of plasma cortisol and progesterone, but did not affect the rise in circulating tumor necrosis factor-alpha. In Exp 3, flurbiprofen in the absence of endotoxin had no effect on pulsatile LH secretion. In Exp 4, flurbiprofen failed to prevent suppression of pulsatile LH secretion induced by luteal phase levels of the ovarian steroids progesterone and estradiol, which produce a nonimmune suppression of gonadotropin secretion. In Exp 5, flurbiprofen prevented the endotoxin-induced inhibition of pulsatile GnRH release into pituitary portal blood. Our finding that this PG synthesis inhibitor reverses the inhibitory effect of endotoxin leads to the conclusion that PGs mediate the suppressive effects of this immune/inflammatory challenge on pulsatile GnRH and LH secretion.

Systemic challenge with endotoxin stimulates corticotropin-releasing hormone and arginine vasopressin secretion into hypophyseal portal blood: coincidence with gonadotropin-releasing hormone suppression.

We tested the hypothesis that systemic immune/inflammatory challenge (endotoxin) activates the neuroendocrine stress axis centrally by stimulating the secretion of CRH and arginine vasopressin (AVP) into hypophyseal portal blood. In addition, we examined the temporal association between this stimulation of the stress neuropeptides and the inhibition of pulsatile GnRH and LH secretion. Using alert, normally behaving ewes, hypophyseal portal and peripheral blood were sampled simultaneously at 10-min intervals for 14 h. Temperature was monitored remotely by telemetry at the same interval. Endotoxin (400 ng/kg, i.v. bolus) or saline as a control was injected after a 4-h baseline period. Portal blood was assayed for CRH, AVP, and GnRH, and peripheral blood was assayed for cortisol, progesterone, and LH. In controls, hypophyseal portal CRH and AVP remained just above or at assay sensitivity, and cortisol showed a regular rhythmic pattern unaffected by saline and typical of basal secretion. In contrast, endotoxin potently stimulated CRH and AVP secretion into portal blood, and cortisol and progesterone into peripheral blood. Both CRH and AVP generally rose and fell simultaneously, although the peak of the AVP response was approximately 10-fold greater than that of CRH. The AVP in portal blood was not due to recirculation of hormone secreted into the peripheral circulation by the posterior pituitary gland, because the AVP increase in peripheral blood was negligible relative to the marked increase in portal blood. The stimulation of CRH and AVP coincided with significant suppression of GnRH and LH pulsatile secretion in these same ewes and with the generation of fever. We conclude that endotoxin induces central activation of the neuroendocrine stress axis, stimulating both CRH and AVP release into the hypophyseal portal blood of conscious, normally behaving ewes. This response is temporally coupled to inhibition of pulsatile GnRH and LH release as well as with stimulation of adrenal cortisol and progesterone secretion and generation of fever.

Alterations of somatotropic function in prion disease in sheep.

This study aimed at investigating the possible linkage between natural scrapie and alterations of the somatotropic axis. Scrapie-affected ewes exhibited 2-fold higher mean GH concentrations during both autumn and spring. GH pulse frequencies were higher in scrapie-affected ewes than in control animals (mean+/-S.E.M. number of pulses/24 h: 10.4+/-0.9 and 7.6+/-0.9 for scrapie-affected and control ewes respectively) suggesting the involvement of central mechanisms. GH secretion induced by administration of an alpha(2)-adrenergic agonist, which acts centrally to stimulate GH secretion, was similar between healthy and scrapie-affected ewes (ratios of the area under the curve (AUC) of GH concentration after to the GH AUC before the agonist administration were 3.6+/-1.6 and 4.9+/-1.0 for scrapie-affected and control ewes respectively). Finally, humoral markers and parameters of the metabolic status were determined to test the hypothesis that scrapie-associated alterations of GH secretion could be related to disruption of metabolic homeostasis. Glucose, insulin and urea plasma concentrations were higher in scrapie-affected than in healthy ewes. Neither leptin nor IGF-I levels were affected by scrapie. Total thyroxine (T4) was decreased in scrapie-affected ewes but free T4 and total and free triiodothyronine were not modified. In conclusion, our results showed the existence in scrapie-affected ewes of endocrine and metabolic alterations typical of acute illness proceeding, at least in part, from central mechanisms.

Photoperiodic modulation of monoamines and amino-acids involved in the control of prolactin and LH secretion in the ewe: evidence for a regulation of tyrosine hydroxylase activity.

Several neurotransmitters are implicated in the photoperiodic regulation of prolactin and luteinising hormone (LH) secretion in the ewe. This work investigated whether catecholamines, gamma-amino butyric acid (GABA), excitatory amino acids and serotonin diencephalic contents are affected by photoperiod and how such changes relate to the seasonal effects of photoperiod on LH and prolactin secretions. Moreover, to determine whether photoperiod can influence catecholamine biosynthesis, the activity of its rate limiting enzyme, tyrosine hydroxylase (TH) was also investigated. TH activity and the tissue content of the monoamines and their metabolites were measured in stalk-median eminence (SME), preoptic area (POA) and the mediobasal, mediodorsal and laterobasal aspects of the hypothalamus. Investigation of excitatory amino acids and GABA was limited to the POA and the SME. Ovariectomized ewes were initially maintained in long days (LD) for 70 days. Thereafter half the ewes remained exposed to long days and the other half were transferred onto short days (SD) for 63 to 66 days to induce a stimulation of LH secretion and an inhibition of prolactin secretion. In each photoperiodic regime, half the ewes were treated with a subcutaneous oestradiol implant (+E) and half were not (-E). As expected, short days induced a decrease in prolactin and an increase in pulsatile LH secretion. These neuroendocrine changes were associated with a decrease in the TH activity of the SME in both oestradiol treated and non treated animals (146.5 +/- 24.1, 167.6 +/- 26.5 U TH/g of tissue in LD-E and LD+E vs 83.5 +/- 12.4 and 95.0 +/- 30.2 U TH/g of tissue in SD-E and SD+E animals; P < or = 0.01). A similar and parallel short day-induced decrease was observed in the tissue content of dopamine and its metabolite, 3,4-dihydroxy-phenylacetic acid (SD level were 55% of LD levels, P < 0.05). In POA, a short day-induced decrease in dopamine (18%; P < or = 0.05) and GABA (16.4%; P < or = 0.05) content and an oestradiol-induced decrease in aspartate (15.6%; P < or = 0.05) content were found. This study provides the first report of a photoperiodic control of the synthesis activity of catecholaminergic neurones of the SME in the ewe. The photoperiod-induced changes in dopaminergic activity at the level of the SME were associated with changes in LH and prolactin secretion indicating that TH activity of dopaminergic neurones of the SME could be a critical component of the photoperiodic regulation of LH and/or prolactin secretion. In particular, this finding is in agreement with the hypothesis that photoperiod can control a dopaminergic pathway inhibitory of LH secretion and which ends in the median eminence.

Blockade of tyrosine hydroxylase activity in the median eminence partially reverses the long day-induced inhibition of pulsatile LH secretion in the ewe.

The photoperiod-induced stimulation of LH secretion is associated with a decrease in dopamine content, as well as in the activity of its rate limiting enzyme, tyrosine hydroxylase (TH), in the median eminence (ME) of the ewe. We therefore hypothesize that ME-TH activity can constitute a limiting factor of photoperiod-induced inhibition of LH pulsatile secretion. To test this hypothesis, we studied whether the inhibition of ME-TH activity can reverse the long day-induced inhibition of LH. Using microdialysis, a 3 mM solution of alpha methyl-p-tyrosine (alpha MPT; a competitive inhibitor of TH), was administered in the ME of ovariectomized ewes bearing a 0.5 cm oestradiol implant at the beginning of a LD-induced inhibition of LH secretion. The vehicle solution was infused for 4 h followed by a 3 mM alpha MPT solution infused for an additional 4 h. LH pulsatile secretory patterns within the same animal were compared between the control period and the alpha MPT period. alpha MPT infusion in the ME was associated with an increase in LH pulse frequency whereas it did not affect prolactin secretion. In conclusion, our results suggest that the inhibition of TH activity in the ME causes a stimulation of LH secretion in long-day inhibited ewes.

Blood glutathione oxidation during human exercise.

To examine the effects of increased O2 utilization on the glutathione antioxidant system in blood, eight moderately trained male volunteers were exercised to peak O2 consumption (VO2peak) and for 90 min at 65% of VO2peak on a cycle ergometer. Blood samples were taken during exercise, and for up to 4 days of recovery from submaximal exercise. During exercise to VO2peak, blood reduced glutathione (GSH) and total glutathione [GSH + oxidized glutathione (GSSG)] did not change significantly. Lactate (L), pyruvate (P), and L/P increased significantly from rest values (P less than 0.01). During prolonged submaximal exercise, GSH decreased 60% from control, and GSSG increased 100%. Total glutathione, glucose, pyruvate, and lactate concentrations and L/P did not change significantly during sustained exercise. During recovery, GSH and GSH/GSSG increased from exercise levels and significantly overshot preexercise levels, reaching maximum values after 3 days. Oxidation of GSH during submaximal exercise and its reduction in recovery suggest increased formation of active O2-. species in blood during physical exercise in moderately trained males.

Antioxidant status and indexes of oxidative stress during consecutive days of exercise.

We tested whether consecutive days of prolonged submaximal exercise would result in oxidant stress sufficient to alter blood antioxidant profiles, progressively change and exhaust blood and plasma antioxidants, and damage RNA. Eleven moderately trained males (24.3 +/- 1.1 yr) exercised 90 min at 65% peak O2 uptake on a cycle ergometer for 3 consecutive days. During day 1 exercise, blood reduced glutathione (GSH) declined 55 +/- 10% and oxidized glutathione (GSSG) increased 28 +/- 7% within 15 min. Total blood glutathione did not significantly change during exercise. GSH levels returned to baseline after 15 min of recovery. On day 3, preexercise GSH and GSSG levels were not significantly different from day 1 preexercise values; essentially similar results were obtained during exercise and recovery. During day 1 exercise, plasma total ascorbate (ascorbate + dehydroascorbate) increased from 53.8 +/- 9.3 to 59.0 +/- 11.3 microM, and percent reduced ascorbate increased from 77.6 +/- 9.3 to 87.3 +/- 9.7%. During day 3 exercise, plasma ascorbate changes were similar to those on day 1. Plasma vitamin E did not change due to exercise on either day 1 or 3. RNA adducts, urinary 8-hydroxyguanosine, did not change significantly due to exercise. Observed increases in GSH oxidation indicate the presence of oxidant stress during prolonged submaximal exercise. Similar redox changes on consecutive days of exercise, with recovery to preexercise values within 15 min, indicate no evidence of persistent or cumulative exercise effects on blood glutathione redox status.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of the circannual rhythm of reproduction by melatonin in the ewe.

Annual variations in day length are responsible for seasonal changes in reproductive activity in sheep. However, in constant photoperiodic conditions, ewes express an endogenous rhythm characterized by alternations of reproductive activity and quiescence that are not synchronized among animals. Thus, the main role of photoperiod in the natural environment appears to be the synchronization of this endogenous rhythm. Photoperiodic information is processed through a complex nervous and endocrine pathway to modulate reproductive activity. Light information perceived at the level of the retina is transformed through neural processing into an endocrine signal by the pineal gland: the nocturnal increase in melatonin release. Recent studies strongly suggest that melatonin has a hypothalamic target to modulate the reproductive neuroendocrine axis. Most LHRH perikarya are located in the preoptic area, but this region is devoid of melatonin receptors, and microimplants of melatonin placed in the preoptic area do not effect LHRH release. Thus, melatonin influences LHRH neurones indirectly and must involve interneurons. Good evidence now exists to demonstrate that a population of dopaminergic neurons with axons projecting to the median eminence is one of these interneurons.