Janina Skipor

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.

Local increase of steroid hormone concentrations in blood supplying the uterus and oviduct in anaesthetized and conscious gilts

Sexually mature cycling gilts (n = 5) with two recorded oestrous cycles were anaesthetized on Day 10 of the oestrous cycle. On both sides of the uterus, catheters were inserted into branches of the uterine artery proximal to the ovary. The tip of each catheter was placed close to, but above anastomoses with ovarian artery branches. This allowed collection of uterine arterial blood mixed with ovarian arterial blood supplying the uterus and oviduct. A third catheter was placed in the jugular vein. Catheters were exteriorized subcutaneously to the backs of the animals for blood sample collection. Simultaneous blood samples were collected as follows: (a) in anaesthetized animals, every 5 min for 1 h immediately after catheters were inserted during surgery, and every 10 min for 2 h, when the surgery was completed; (b) from conscious animals on Day 2 after surgery, every 10 min for 2 h in the morning and every 10 min for 2 h in the afternoon. Blood plasma samples were assayed for progesterone and androstenedione by RIA method. Mean values (±SEM) for progesterone measured in samples collected from branches of the uterine arteries and from the jugular vein in anaesthetized animals were 34 ± 1.25 ng ml−1 and 26 ± 1.31 ng ml−1 of plasma, respectively. In conscious animals the measurements showed 33 ± 0.90 ng ml−1 and 24 ± 0.81 ng ml−1 of plasma, respectively. Plasma concentration of androstenedione in branches of the uterine arteries and from the jugular vein in anaesthetized and conscious animals were 107 ± 5.26 pg ml−1 and 72 ± 5.50 pg ml−1, and 78 ± 4.54 pg ml−1 and 57 ± 3.77 pg ml−1, respectively. It was concluded that counter-current transfer of steroid hormones in ovarian pedicle vasculature on Days 10–11 of the oestrous cycle significantly elevates (P < 0.0001) the local concentration of steroid hormones in arterial blood supplying the uterus and oviduct (35% for progesterone and 46% for androstenedione).

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.

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 108 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 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.

Tight junction proteins vary in the choroid plexus of ewes according to photoperiod

Sheep from temperate latitudes exhibit seasonal variations in many physiological functions such as reproduction, food intake, body weight, and pelage growth. Majority of seasonal changes are controlled by the annual photoperiodic cycle and melatonin secretion. For reproduction, the resulting key event is a modulation of the negative feedback of steroids on gonadotropin secretion. However, this seasonal effect could also depend on variable uptake of steroids by the brain. Seasonal regulation of food intake also involves numerous peripheral hormones, among which the protein hormone leptin informs the brain on the metabolic status of the animal. It has been shown previously that access of progesterone, estradiol and leptin to the cerebrospinal fluid (CSF) increases under long days. This physiological modulation of the passage of hormones to the brain could depend on regulation of the permeability of the blood-CSF barrier. This study therefore compared the tight junction proteins in the choroid plexus of ewes exposed to short days or long days. Levels of occludin, zonula occludens proteins (ZO) ZO-1 and ZO-2, afadin and cadherin were significantly higher during short days, but no statistical difference was observed for junctional adhesion molecule 1 (JAM-1), ZO-3 or claudins 1 and 5. These results are consistent with an increase in the blood-CSF barrier permeability during long days through a regulation of tight junctions and show that the permeability could depend upon physiological conditions such as photoperiodic status.

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.

On Day 10 of the oestrous cycle in pigs, after laparotomy noradrenaline (NA), methoxamine (α1‐adrenomimetic, M), Prazosin (α1‐adrenolytic, Pr) in total doses of 4 μmol, and saline were infused (10 min) into the superficial layer of mesovarium on both sides of the ovarian pedicle vasculature, close to the ovary. 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. 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. 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. The results indicate that stimulation of α1‐adrenoceptors in the area of ovarian vasculature did not influence, whereas block of α1‐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.

Cavernous sinus and carotid rete of sheep and sows as a possible place for countercurrent exchange of some neuropeptides and steroid hormones

Abstract This study was conducted in order to scrutinise the countercurrent exchange of luteinising hormone releasing hormone (LH-RH), β-endorphin and progesterone in the carotid rete and cavernous sinus of sheep and gilts. The ewes (n=30) and gilts (n=20) were anaesthetised and had catheters inserted into their jugular veins and carotid common arteries from which heparinised blood was then collected. After exsanguination, the head with the neck was immediately removed and about 30 min later supplied with oxygenated and heated autologous blood mixed with saline (1:1), through one of the carotid arteries at a stable flow rate. Blood samples were collected every minute from the carotid artery contralateral to the arterial blood supply (blood from the carotid rete) and from both jugular veins. Arterial blood pressure was continuously measured and regulated. Radiolabelled hormones 3H-progesterone (3H-P4), 125I-luteinising hormone releasing hormone (125I-LH-RH), 125I-β-endorphin (125I-En), or 51Cr-labelled red blood cells (control) were infused for 5 min into the cavernous sinuses through catheterised angularis oculi veins. Infusions were carried out on gilts and sheep which were in different phases of the oestrous cycle and on sheep during anoestrus and after oestrogen treatment. As a control, 3H-P4 was infused into the facial vein instead of into the cavernous sinus. In all experiments with 3H-P4 and 125I-En infusions, labelled hormones were found in arterial blood collected from the carotid rete. No radioactivity was present in arterial blood in the experiment with seasonally anoestrous sheep. However, 125I-LH-RH was found in the arterial blood of anoestrous oestrogen-treated sheep and of sheep in the early luteal phase. No radioactivity was ascertained in arterial blood after 3H-P4 infusion into the facial vein (control) or 51Cr-labelled red blood cells into the cavernous sinus (control). These results demonstrate that two neuropeptides (LH-RH and β-endorphin) and a steroid hormone (progesterone) can be transferred from venous to arterial blood at the cavernous sinus and carotid rete in the base of the brain in sheep and pigs. This indicates that there is a powerful exchange system for resupplying hormones to the brain and that this system is dependent on the phase of the oestrous cycle.

Profile of toll-like receptor mRNA expression in the choroid plexus in adult ewes

The blood-cerebrospinal fluid barrier (BCSFB) located in the epithelial cells of the choroid plexus (CP) forms the interface between the cerebrospinal fluid (CSF) and pathogen components circulating in the blood. The CP is also implicated in the passage of peripheral immune signals and circulation of immune cells into the central nervous system. Toll-like receptors (TLRs) are patternrecognition receptors that play a crucial role in the recognition of pathogens and triggering of the innate immune response. In sheep, ten members of the TLR family have been identified and cloned. We used real-time PCR analyses to examine the profiles of TLR mRNA expression in the CP of cerebral ventricles in healthy adult ewes. The transcripts for all ten TLRs except TLR8 were present; however, we observed a high variation in the degree of expression of the TLR5 and TLR1 genes (coefficient of variation: 61% and 46%, respectively) as well as a moderate variation in the expression of the TLR4 (34%), TLR2 (27%) and TLR6 (26%) genes. The TLR9, TLR7, TLR3 and TLR10 genes were the four receptors with relatively invariable expression levels (coefficient of variation: 7%, 8%, 16% and 17%, respectively) across the six adult ewes. The concentration of cortisol in blood collected prior to sacrificing the ewes ranged from 0.18 to 78.9 ng/ml. There was no correlation between cortisol concentration and mRNA expression of any of the examined TLRs. These data suggest that the CP has the potential to sense the presence of many bacterial and viral components and mediate responses for the elimination of invading microorganisms, thereby protecting the brain.

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.

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.