Melatonin from slow-release implants upregulates claudin-2 in the ovine choroid plexus.

In ewes, the turnover rate of cerebrospinal fluid (CSF), mainly produced by choroid plexus (ChP), is photoperiodically modulated and is higher during short days (SDs) than long days (LDs). We demonstrated that, melatonin from continuous slow-release implants increases the expression of aquaporins, water channel-forming proteins engaged in transcellular water transport (across the plasma membrane of the cells), in the ovine ChP. This study evaluated the effect of slow-release melatonin implants on the expression of claudin-2 (CLDN2), a pore-forming protein that allows the paracellular passage (between the cells) of select inorganic cations and water, in the ovine ChP. The studies were conducted on ovariectomized, estradiol-implanted ewes during seasonal anestrus (May/June). The ewes were implanted with slow-release-melatonin implants (n = 6, Melovine 18 mg) or sham-implanted (n = 6). Blood samples were collected for melatonin and prolactin measurements. The ewes were sacrificed 40 days after the melatonin/sham implantation, and the ChPs from the brain ventricles were collected for real-time PCR and Western blot analyses. Plasma melatonin concentration reached the median value of 120.4 pg/ml (range: min/max = 29.6/447.0) or was below the detection limit 40 days after the melatonin/sham implantation, respectively. The area under the curve of the plasma prolactin concentration was significantly (P < 0.05) higher in sham-implanted ewes than in melatonin-implanted ewes. CLDN2 expression in the ChP was significantly (P < 0.05) higher in melatonin-implanted ewes than in sham-implanted ewes at both the mRNA and protein levels. This is the first evidence for the photoperiodic regulation of CLDN2 expression in the ovine ChP, since it has been shown that slow-release melatonin implants during LDs, mimicking SDs, increased the expression of CLDN2. This may partially explain the higher turnover rate of CSF observed in ewes during SDs.

The effect of melatonin from slow-release implants on basic and TLR-4-mediated gene expression of inflammatory cytokines and their receptors in the choroid plexus in ewes.

The present study concerns the effect of melatonin from slow-release implants on the expression of genes coding interleukin-1ß (Il1B), inerleukin-6 (Il6), tumour necrosis factor α (Tnf) and their receptors: IL-1 receptor type I (Il1r1) and type II (Il1r2), IL-6 receptor (Il6r) and signal transducer (Il6st), TNFα receptor type I (Tnfrsf1a) and II (Tnfrsf1b) and retinoid-related orphan receptor α (RorA) and Rev.-erbα in the ovine choroid plexus (CP) under basal and lipopolysaccharide (LPS)-challenged conditions. Studies were performed on four groups: 1) sham-implanted and placebo-treated, 2) melatonin-implanted (Melovine, 18mg) and placebo-treated, 3) sham-implanted and LPS-treated (400ng/kg of body weight) and 4) melatonin-implanted and LPS-treated. Under basal conditions, we observed weak expression of Tnf, low expression of Il1B, Il6 and Il1r2 and intermediate expression of other cytokines receptors. LPS treatment induced (P≤0.05) expression in all cytokines and their receptors, except Il6r 3h after the administration. Melatonin attenuated (P≤0.05) LPS-induced up-regulation of Il6 but had no effect on other cytokines and their receptors and up-regulated (P≤0.05) Rev.-erbα expression under basal conditions. This indicates that melatonin from slow-release implants suppresses TLR4-mediated Il6 expression in the ovine CP via a mechanism likely involving clock genes.

Interleukin-1 ß Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study.

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1ß, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1ß (75 pg/mL), or (4) NE + IL-1ß. It was found that IL-1ß abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1ß also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1ß treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1ß suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

Photoperiod affects the cerebrospinal fluid proteome: a comparison between short day- and long day-treated ewes.

Photoperiod is the main physical synchronizer of seasonal functions and a key factor in the modulation of molecule access to cerebrospinal fluid (CSF) in animals. Previous work has shown that photoperiod affects the transfer rate of steroids and protein hormones from blood to CSF and modulates choroid plexus tight junction protein content. We hypothesized that the CSF proteome would also be modified by photoperiod. We tested this hypothesis by comparing CSF obtained from the third ventricle of mature, ovariectomized, estradiol-replaced ewes exposed to long day length (LD) or short day length (SD). Variations in CSF protein expression between SD- or LD-treated ewes were studied in pools of CSF collected for 48 h. Proteins were precipitated, concentrated, and included in a polyacrylamide gel without protein fractionation. After in-gel tryptic digestion of total protein samples, we analyzed the resulting peptides by nanoliquid chromatography coupled with high-resolution tandem mass spectrometry (GeLC-MS/MS). Quantitative analysis was performed using 2 methods based on spectral counting and extracted ion chromatograms. Among 103 identified proteins, 41 were differentially expressed between LD and SD ewes (with P < 0.05 and at least a 1.5-fold difference). Of the 41 differentially expressed proteins, 22 were identified by both methods and 19 using extracted ion chromatograms only. Eighteen proteins were more abundant in LD ewes and 23 were more abundant in SD ewes. These proteins are involved in numerous functions including hormone transport, immune system activity, metabolism, and angiogenesis. To confirm proteomic results, 2 proteins, pigment epithelium-derived factor (PEDF) and gelsolin, for each individual sample of CSF collected under SD or LD were analyzed with Western blots. These results suggest an important photoperiod-dependent change in CSF proteome composition. Nevertheless, additional studies are required to assess the role of each protein in seasonal functions.

Effect of a two-week treatment with low dose of ortho-substituted polychlorinated biphenyls (PCB104 and PCB153) on VEGF-receptor system expression in the choroid plexus in adult ewes.

Ortho-substituted polychlorinated biphenyl (PCB) congeners, which constitute a large part of PCB residues found in the environment and in animal tissues, are known to exert potent vascular effects and can activate endothelial cells in the periphery and in the brain. The choroid plexus (CP) is responsible for cerebrospinal fluid (CSF) production and its epithelial cell layer is responsible for structure and functions of the blood-CSF barrier. The aims of this study were: 1) to investigate if environmentally relevant doses of PCB153 and similar doses of PCB104 caused changes in the expression of vascular endothelial growth factor (VEGF)--receptor system, which maintains CP function, and 2) to determine the level of both congeners in blood plasma after their oral administration. Studies of both congeners were performed on ovariectomized ewes treated per os with low doses (0.1 mg/kg, three times a week for two weeks) of PCB153 (n = 4) or PCB104 (n = 4) and vehicle (control, n = 4). The effects of PCB153 and PCB104 treatment on mRNA expression of two isoforms of VEGF (VEGF120 and VEGF164) and their receptors Flt-1 and KDR were determined using real-time PCR. Plasma concentration of PCBs was measured using high resolution chromatography/tandem mass spectrometry (HRGC/MS-MS). We observed that neither PCB153 nor PCB104 significantly altered the mRNA of the VEGF-receptor system in the CP. In PCB treated animals plasma concentration of PCB153 (1.425 +/- 0.16 ng/g of dry mass, DM) was about 150 times higher than PCB104 (0.009 +/- 0.007 ng/g DM). In control animals the PCB153 level was 0.14 +/- 0.031 ng/g DM, while the PCB104 level was below detection level. This indicates that increase in plasma PCB153 concentration to levels similar to those reported in humans and of PCB104 concentration to levels 100 times higher than those found in human plasma did not affect the VEGF-receptor system in the CP in adult ewes. The significantly lower increase of PCB104 than PCB153 concentration in blood after oral administration suggests different absorption of both congeners from the digestive tract.

Independent changes of thyroid hormones in blood plasma and cerebrospinal fluid after melatonin treatment in ewes.

The authors measured the effects of exogenous melatonin treatment on the concentrations of total (T) and free (f) fractions of thyroxine (T4) and triiodothyronine (T3) in cerebrospinal fluid (CSF) and blood plasma as well as the expression of their binding/transporter protein, transthyretin (TTR), in the choroid plexus of ewes from May to August. Melatonin implantation in May and July mainly prevented the decrease in plasma for fT3 and TT3 exhibited in untreated group, and induced a limited decrease in TT4 in June. By contrast, melatonin implantation prevented the decrease in CSF fT3 observed in the untreated group. No effect of melatonin was found on the expression of TTR mRNA in the choroid plexus There were a correlations between blood fT4 and CSF TT4 concentrations in both control and melatonin treated group (r(2)-0.4; P < 0.01 vs. r(2)-0.14; P < 0.05), as well as between blood fT3 and CSF TT3 concentrations but only in the melatonin-treated group (r(2)-0.26; P < 0.02). We conclude that T3, the active form of the hormone within the brain, is regulated by melatonin independently of the peripheral changes within the blood. The lack of correlation between plasma fT3 and CSF TT3 in the control group suggests that an increase in local T3 conversion could contribute as an additional source of T3 in the CSF during the period of increasing day length. These data seem to confirm a local nature for recently discovered connections between the pineal melatonin signal and thyroid-dependent seasonal biology in mammals.

Thyroid hormones in the cerebrospinal fluid of the third ventricle of adult female sheep during different periods of reproductive activity.

Thyroid hormones (THs) are obligatory for transition from breeding season to anestrus in sheep. In this process, THs act during a very limited time of the year and primarily within the brain. In ewes chronically equipped for sampling cerebrospinal fluid (CSF) from the third ventricle, we have characterized the concentrations of total and free thyroxine (T4), triiodothyronine (T3), and total reverse T3 (rT3) in the CSF during breeding season, anestrus and during a critical period required for transition to anestrus (December-March). The total T4, T3, rT3 and free T3 average concentrations (+/- SEM) in CSF were 1.5 +/- 0.07 ng/ml, 14.5 +/- 1.2 pg/ml, 43 +/- 7.4 pg/ml, and 0.6 +/- 0.05 pg/ml, respectively, and all were significantly lower (p < 0.001) than in blood plasma except free T4 (12.6 +/- 1.1 pg/ml), which was similar to that in plasma. There was a seasonal trend (p < 0.05) in the concentration of total T3 (highest in December) and free T4 (highest in November) in the CSF that does not follow that in blood plasma. During the period of transition to anestrus the CSF total T3/TT4 molar ratio and free T3/T4 ratio were significantly lower (p < 0.05 and p < 0.01, respectively) than in blood plasma, while the total rT3/T4 ratio was significantly higher (p < 0.01) at the end of this period (March). Additionally, the CSF total rT3 concentrations were also significantly correlated with the CSF total T4 levels (r = 0.57; p < 0.05). In conclusion, the CSF in sheep may serve as a considerable source of thyroid hormones for neuroendocrine events. The lack of significant changes in THs concentrations in the CSF during the period of transition to anestrus indicate that neither seasonal changes of THs circulating in the blood plasma nor THs circulating in the CSF actively drive the transition to anestrus.

Contractile effect of PGF2alpha and PGE2 on isolated branches of uterine and ovarian artery in different days of estrous cycle and early pregnancy in pigs.

The contractile effects of PGF2alpha (3 x 10(-6) to 10(-4) M) and PGE2 (10(-7) to 10(-5) M) were examined on isolated branches of ovarian artery (OA) and extramyometrial branches of uterine artery (UA) collected from pigs in the luteal (day 10-12) and follicular phase (day 17-20) of the estrous cycle, and during early pregnancy (day 10-12). Strong contraction was demonstrated in both arteries during all investigated periods in response to PGF2alpha, which was significantly higher (P < 0.01) than to PGE2, being negligible in the follicular phase. In UA, the effective dose of PGF2alpha (ED50) amounted 7.9 x 10(-6) M and 6.3 x 10(-6) M in the luteal and follicular phase, and 5.0 x 10(-6) M in early pregnancy. ED50 for PGE2 reached 5.0 x 10(-7) M in the luteal phase, and 4.1 x 10(-7) M in early pregnancy. For both prostaglandins, the contraction was much stronger (P < 0.01) in OA than in UA branches. In OA, the ED50 for PGF2alpha was 1.2 x 10(-5) M in the luteal phase and was significantly higher (P < 0.05) than in the follicular phase (3.1 x 10(-6) M) and early pregnancy (2.7 x 10(-6) M). ED50 for PGE2 amounted 7.3 x 10(-7) M in the luteal phase and 1.7 x 10(-7) M in early pregnancy. Studies showed the influence of the estrous cycle and early pregnancy on OA branches sensitivity to the contractile effect of PGF2alpha and the lack of this effect on UA branches, and the influence of the estrous cycle on UA and OA branch contraction in response to PGE2.

The effect of intramuscular injections of boar pheromone 5alpha-androstenol on the hormonal regulation of the estrous cycle in hypoosmatic gilts.

Until 1999 it was accepted that pheromones act exclusively by stimulating the dendritic receptors present in olfactory epithelium. Cycling gilts with an experimentally-disrupted neural olfactory pathway were used to test the hypothesis that boar pheromone 5alpha-androstenol may affect the secretion of hormones involved in the regulation of the estrous cycle by the humoral pathway. On day 12 of the estrous cycle the nasal cavity of gilts (n=15) was irrigated with zink sulfate solution. From day 16 to 20, the experimental group (n=10) was injected intramuscularly with 5alpha-androstenol (20 microg) twice a day. Blood samples were collected from the jugular vein at 4 h intervals on days 17-21 to estimate plasma concentration of LH, oxytocin, estradiol-17beta, testosterone and progesterone. The experimental group displayed a significantly lower mean concentration of LH than the control animals (P<0.0001). The decrease in concentration of LH was accompanied by the reduction of oxytocin (P<0.001), estradiol-17beta (P<0.001) and testosterone (P<0.01) secretion. These results demonstrated that 5alpha-androstenol influenced hormonal regulation by humoral pathway and might be considered to be the priming pheromone in gilts.

Apoptotic cell death in the porcine endometrium during the oestrous cycle.

It has been reported that apoptosis plays an essential role in controlling the physiological cell kinetics in the human and rodent endometrium but this type of death has never been studied in the porcine endometrium. The aim of this study was to investigate the apoptotic cell death in the porcine endometrium during the middle (Days 9-11) and late (Day 13) luteal phase, during the luteolysis (Day 15) and early follicular phase (Days 17-19) of the oestrous cycle. Apoptotic cells were identified by in situ DNA 3'-end labelling method. It was revealed that the greatest number of apoptotic cells in the luminal and glandular epithelium was found on Days 17-19 and on Day 15 of the oestrous cycle, respectively. In the stroma, the greatest number of these cells was found on Days 9-11. Our data have shown that in the porcine endometrium, both epithelial and stromal cells undergo apoptosis and that the number of apoptotic cells varies depending on the phase of the oestrous cycle.

Humoral pathway for local transfer of the priming pheromone androstenol from the nasal cavity to the brain and hypophysis in anaesthetized gilts.

It is generally accepted that pheromones act by stimulating of the dendritic receptors of the olfactory neurones massed in the olfactory epithelium. This study was designed to ascertain whether it is possible for the boar pheromone androstenol (5alpha-androst-16-en-3-ol) to be transported from the nasal cavity of anaesthetized gilts to the brain and hypophysis via local transfer from the blood in the perihypophyseal vascular complex. The experiment was performed on days 18-21 of the porcine oestrous cycle (crossbred gilts, n = 6). Tritiated androstenol (3H-A; total amount 10(8) d.p.m. (758 ng)) was applied for 1 min onto the respiratory part of the nasal mucosa, 4-6 cm from the opening of the nares. Arterial blood samples from the aorta and from the carotid rete were collected every 2 min during the 60 min period following administration of the steroid. Total radioactive venous effluent from the head was removed and an adequate volume of homologous blood was transfused into the heart through the carotid external vein. At the end of the experiment gilts were killed and tissue samples of the hypophysis and some brain structures were collected to measure radioactivity. In addition, corresponding control tissues were collected from three untreated gilts and from three heads of gilts 60 min after 3H-A was applied post mortem into the nasal cavity. The concentration of 3H-A was significantly higher (P < 0.0001) in the arterial blood of the carotid rete than that of aorta. The mean rate of 3H-A counter current transfer from venous to arterial blood in the perihypophyseal vascular complex, expressed as the ratio of the 3H-A concentration in arterial blood of the carotid rete to the 3H-A concentration in blood sampled simultaneously from the aorta, was 1.96 +/- 0.1. The concentration of 3H-A in plasma from the venous effluent from the head ranged from 1.3 to 1.8 pg x ml(-1). During the 60 min period of the experiment, 0.68% of the total applied dose of 3H-A was resorbed from the nasal cavity into the venous blood. Moreover, we found that 3H-A was present in the olfactory bulb (P <0.01), amygdala, septum, hypothalamus, adenohypophysis, neurohypophysis (P > 0.05) and perihypophyseal vascular complex (P < 0.01). These results demonstrate that, in anaesthetized gilts, the boar pheromone androstenol may be resorbed from the nasal mucosa, transferred in the perihypophyseal vascular complex into arterial blood supplying the brain and hypophysis, and then arrested in the hypophysis and certain brain structures. We suggest that in addition to the standard neural pathway for signalling pheromones, another pathway exists whereby androstenol, as a priming pheromone, may be resorbed from the nasal cavity into the bloodstream and then pass locally from the perihypophyseal vascular complex into the arterial blood supplying the brain and hypophysis, thus avoiding the first passage metabolism in the liver.

Humoral pathway for transfer of the boar pheromone, androstenol, from the nasal mucosa to the brain and hypophysis of gilts.

Signaling and priming pheromones play an important role in intraspecies behavioral and sexual interactions and in the control of reproduction. It is generally accepted that pheromones act by stimulating the dendritic receptors in the mucus-imbedded cilia of olfactory neurons massed in the olfactory epithelium. The boar pheromone androstenol, known to induce sexual behavior in pigs, is 1 of 2 pheromones that have been chemically defined, tritiated and thus made available for use in studies. In Experiment 1, sexually mature cyclic gilts at Days 16 to 21 of the estrous cycle were humanely killed and the heads separated from the bodies. The heads were attached to a perfusion system using heated, oxygenated, heparinized, autologous blood. A total amount of 10(8) dpm (758 ng) of 3H-5 alpha-androstenol (3HA) was either infused into the angularis oculi veins that drain the nasal cavities (n = 7) over a 5-min period or applied through intranasal catheters onto the mucose surface (n = 16) for 2 min. In both groups frequent blood samples were collected from the carotid rete and from venous effluent. Concentration of 3HA in the arterial blood of the carotid rete after direct (into angularis oculi veins) or indirect (onto the nasal mucosa) administration of 3HA into veins draining the nasal cavities was significantly higher than background radioactivity before 3HA administration (P < 0.0001 and P < 0.05, respectively). The 3HA was selectively accumulated (compared with the respective control tissue) in the neurohypophysis (P < 0.001), adenohypophysis (P < 0.01), ventromedial hypothalamus (P < 0.05), corpus mammillare (P < 0.01), and perihypophyseal vascular complex (P < 0.001). In a second in vitro experiment, active uptake of 3HA into the nasal mucosa of the proximal, respiratory segment of the nasal cavity was observed. These results demonstrate a humoral pathway for the transfer of pheromones from the nasal cavity to the hypophysis and brain. Androstenol was taken up by the respiratory part of the nasal mucosa, resorbed into blood, transported to the cavernous sinus and transferred into the arterial blood of the carotid rete (supplying the hypophysis and brain), and then selectively accumulated in the hypophysis and certain brain structures.

The inhibitory effect of hCG on counter current transfer of GnRH and the presence of LH/hCG receptors in the perihypophyseal cavernous sinus--carotid rete vascular complex of ewes.

The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle.

Local increase of ovarian steroid hormone concentration in blood supplying the oviduct and uterus during early pregnancy of sows.

Countercurrent transfer in the ovarian vascular pedicle elevates the concentration of steroid hormones in blood supplying the oviduct and periovarian part of the uterus during the estrous cycle in the pig. This study was conducted to determine whether during early pregnancy the arterial blood supply to the oviduct and uterus carries greater concentration of steroid hormone than systemic blood. The concentration of ovarian steroid hormones (progesterone, estradiol-17 beta, estrone, androstenedione and testosterone) was measured in 40 gilts on Days 12, 18, 25 or 35 of pregnancy. Silastic catheters were inserted: a) into the jugular vein, b) into the branch of uterine artery close to the ovary (proximal to the ovary) and c) into the branch of the uterine artery close to the cervix (distal to the ovary). On the day following surgery simultaneous blood samples from cannulated vessels were collected every 20 min for 3 hours. The concentration of steroid hormones was determined by radioimmunoassay. The mean concentrations of studied hormones in branches of the uterine artery proximal and distal to the ovary were significantly greater than in the jugular vein (P < 0.001) by 18 to 69% and 7 to 31%, respectively. The concentrations of hormones in proximal and distal to the ovary branch of the uterine artery were also significantly different (P < 0.001). The increase in concentrations of the measured hormones did not differ considerably between investigated days of pregnancy. It is concluded that during maternal recognition of pregnancy, formation of the corpus luteum of pregnancy, implantation of the embryo and the placenta elongation the oviduct and uterus are supplied with locally elevated concentration of steroid hormones compared to systemic blood.

Countercurrent transfer of 125I-LHRH in the perihypophyseal cavernous sinus-carotid rete vascular complex, demonstrated on isolated pig heads perfused with autologous blood.

The objective of the study was to determine whether the local permeability of luteinizing hormone-releasing hormone (LHRH) from the venous blood of the perihypophyseal cavernous sinus into the arterial blood of the carotid rete, supplying the brain and hypophysis in gilts, depends on the day of the estrous cycle, as well as to determine whether this transfer exists when LH concentration in the blood is reduced (the experimental short-loop negative feedback for LH secretion after estradiol injection in ovariectomized gilts). Experiments were conducted on isolated gilt heads with necks, on chosen days of the estrous cycle (n = 40), and on previously ovariectomized gilts treated with estradiol benzoate (EB) (n = 5) or corn oil (n = 3). After exsanguination, the gilt heads with necks were disarticulated and about 30-45 min later were supplied with autologous, oxygenated, and heated blood at a stable blood flow and pressure through the left carotid artery for 30 min. 125I-LHRH was infused into both cavernous sinuses through the cannulated angularis oculi veins for 5 min. After 125I-LHRH infusion, radiolabeled LHRH was found (P < 0.001) in arterial blood taken from the carotid rete through the open right carotid artery in all animals used in the experiment: on Days 1-2 (six gilts), on Days 12-14 (seven gilts) of the estrous cycle, and in five ovariectomized gilts during negative feedback for LH surge (40 hr after EB). No significant radioactivity of 125I-LHRH was found in the arterial blood on Days 3-5 (n = 6), 9-11 (n = 4), and 15-21 (n = 17) of the estrous cycle. A very low level of radioactivity was found in the ovariectomized control group after the injection of corn oil (n = 3). These results provide evidence for the permeability of LHRH from the venous to the arterial blood and its retrograde transport with the arterial blood to the hypophysis and brain, after the ovulation period (Days 1-2) and on Days 12-14 of the estrous cycle. This suggests that a close relationship exists between the day of the estrous cycle and LHRH permeability from the venous to the arterial blood in the perihypophyseal cavernous sinus-the carotid rete complex in gilts-and that this mechanism may be included in a short-loop feedback for LHRH secretion.

Involvement of adrenoceptors in the ovarian vascular pedicle in the regulation of counter current transfer of steroid hormones to the arterial blood supplying the oviduct and uterus of pigs.

1. On Day 10 of the oestrous cycle in pigs, after laparotomy noradrenaline (NA), methoxamine (alpha 1-adrenomimetic, M), Prazosin (alpha 1-adrenolytic, Pr) in total doses of 4 mumol, and saline were infused (10 min) into the superficial layer of mesovarium on both sides of the ovarian pedicle vasculature, close to the ovary. 2. Blood flow in the ovarian artery, heart rate and progesterone (P4) and androstenedione (A4) secretion from the ovary and their concentrations in the ovarian venous effluent, as well as the concentrations of P4 and A4 in the blood supplying the oviduct and the uterus, were determined. 3. A significant increase of P4 and A4 secretion after NA and M infusion and increased concentrations of P4 and A4 in the ovarian venous effluent were found, but these changes did not influence the counter current transfer of hormones from the venous effluent into arterial blood supplying the oviduct and the uterus. 4. Infusion of Pr caused a significant decrease of P4 and A4 secretion and their concentrations in the ovarian venous effluent and significantly increased A4 concentration in the blood supplying the oviduct and uterus. 5. The results indicate that stimulation of alpha 1-adrenoceptors in the area of ovarian vasculature did not influence, whereas block of alpha 1-adrenoceptors affected, the local concentration of steroid hormones in the blood supplying the oviduct and the part of the uterus proximal to the ovary, despite the changes in the concentrations of steroid hormones in the ovarian effluent.

Counter current transfer of beta-endorphin in the perihypophyseal cavernous sinus--carotid rete vascular complex of sheep.

This study was performed to answer the question of whether counter current retrograde transfer of beta-endorphin in the perihypophyseal cavernous sinus-carotid rete vascular complex depends on the reproductive activity of sheep and if this transfer depends on membrane Na+ K+ ATP-ase blocking by ouabain. Sheep were anaesthetised and the jugular vein and the carotid artery were cannulated on both sides. Multielectrolitical liquids (Solfin, Polfa "Kutno", Poland): 500 ml of Solfin with heparin (25,000 IU), or Solfin with heparin and ouabain (Sigma, St. Louis, USA) in concentrations of 10(-5) or 10(-4) mol 1(-1) were infused into the brain through the carotid artery. Heparinized blood was collected through the carotid artery. After exsanguination, the head with the neck was removed. The isolated head was supplied with oxygenated, heated, autologous blood diluted with Solfin (4:1) without or with ouabain in concentration of 10(-5) or 10(-4) mol 1(-1). Blood pressure and temperature were measured throughout the duration of the experiment. During the experiment 125I-beta-endorphin (7.9 x 107 dpm) dissolved in 10 ml of Solfin was infused for 5 min (5 ml) into each cavernous sinus through the angularis oculi veins. Blood samples for radioactivity measurements were collected each min from the carotid rete (through the opposite carotid artery to the artery supplying the brain with arterial blood) and from both jugular veins. In all the experiments significant 125I-beta-endorphin radioactivity was found in arterial blood supplying the brain and hypophysis in the early luteal phase in sheep. No radioactivity was found (with the exception of one animal) in sheep during seasonal anoestrus. A blockage of Na+ K+ ATP-ase by ouabain administered during exsanguination and during head perfusion with dose of 10(-4) mol 1(-1) reduced beta-endorphin counter current transfer to arterial blood, but this effect was not evident with the dose of 10(-5) mol 1(-1). Increased blood pressure was observed in all the experiments with either dose of ouabain.

Vasa vasorum of blood and lymph vessels in the broad ligament of the sheep uterus analyzed by scanning electron microscopy.

The morphology of vasa vasorum of the blood and lymph vessels in the broad ligament of the uterus was examined in 12 adult ewes. The technique of vascular corrosion casts with the usage of Mercox resin allows multiple surveys of a three-dimensional organization of vascular networks under a scanning electron microscope (SEM). The study was appended by tissue pieces dried by the critical point method for SEM and semi-thin sections stained with toluidyne blue for light microscopy. Vasa vasorum surrounded both the larger vessels and the lymph vessels emanating from the ovary and the uterus. Significant differences in location and intensity of vasa vasorum in blood vessels were observed. They were less developed in the first part of the ovarian and the uterine arteries than in their smaller branches. Significantly large, two- or even three-layer vasa vasorum surrounded the ovarian and the uterine branches of the ovarian artery as well as the ovarian veins in the area of mesovarium. In the other areas of the broad ligament of the uterus, arterial vessels of similar sizes usually had a single-layer vasa vasorum. The lymph vessels leaving the ovary and uterus were surrounded by dense, although usually single-layed, microvascular network. The presence of vasa vasorum on relatively small blood and lymph vessels of the broad ligament of uterus indicates their particular functions that are probably connected with the estrous cycle or pregnancy and changes of blood flow rate entering the ovary and uterus. However, the presence of significantly developed, multi-layer vasa vasorum networks on only some of the blood vessels indicates that it is probably one of the vessels morphological adaptations to serve the counter-current transfer of biologically-active regulators in the area of the broad ligament of the sheep's uterus.

Counter current transfer of oxytocin from the venous blood of the perihypophyseal cavernous sinus to the arterial blood of carotid rete supplying the hypophysis and brain depends on the phase of the estrous cycle in pigs.

The objective of the present study was to determine whether or not the neuropeptide, oxytocin, can move by counter current transfer from venous blood of the perihypophyseal cavernous sinus into arterial blood of the carotid rete supplying the brain and hypophysis, and also whether this exchange depends on the day of the estrous cycle. Isolated heads of gilts (n = 37), on different days of the estrous cycle, were supplied with autologous, oxygenated and heated blood at a stable blood flow and pressure through the right carotid artery for 30 min. 125I-Oxytocin (125I-OT) was infused into both cavernous sinuses through the angularis oculi veins for 5 min. After 125I-OT infusion, radiolabeled oxytocin was found in arterial blood taken from the carotid rete in all 7 gilts on Days 1-3, and in 7 of 9 gilts on Days 12-13 of the estrous cycle. In general, the level of radioactivity in arterial blood during Days 12-13 was significantly lower (p < 0.002) than during Days 1-3 of the estrous cycle. No 125I-OT was found in arterial blood from Days 4 through 11 (n = 10) or from Day 14 to the beginning of ovulation (n = 11). These results provide evidence for the counter current transfer of oxytocin from hypophyseal and brain venous effluent (cavernous sinus) to arterial blood supplying the hypophysis and brain, during the ovulation period and the late luteal phase of the estrous cycle. The meaning of this process is not as yet known and needs further study.