Stanisława Stefańczyk-Krzymowska

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).

The role of the endometrium in endocrine regulation of the animal oestrous cycle.

A critical analysis of the results of research in the function of the endometrium was carried out and a view point presented. The role of the endometrium in endocrine regulation of the oestrus cycle can be summarized as follows: 1. The transfer of prostaglandin F(2alpha) (PGF(2alpha)) from the uterus to an ovary, which causes luteolysis, occurs mainly via the lymphatic pathways. 2. The system of retrograde transfer of PGs enables PGF(2alpha) and PGE(2) to reach the myometrium and endometrium with arterial blood at high concentration. In the luteal phase, PGF(2alpha), together with the increasing concentration of progesterone, constricts the arterial vessels of the uterus; in the follicular phase and in early pregnancy, PGE(2) together with oestrogen and embryonic signals, relaxes the arterial vessels. In addition, this system protects the corpus luteum from premature luteolysis during the cycle and luteolysis during early pregnancy. 3. In days 10-12 of the cycle, the blood flow in the uterus decreases by 60-70% in pigs and around 90% in sheep. This causes ischaemia and local hypoxia confirmed by the presence of hypoxia inducible factor and thus remodelling of the endometrium commences. 4. The pulsatile elevations in PGF(2alpha) concentration occurring in the blood flowing out of the uterus during the period of luteolysis and the next few days, do not result from increased PGF(2alpha) synthesis as suggested in numerous studies. They are the effect of excretion of PGF(2alpha) and its metabolites together with lymph and venous blood and tissue fluids in which prostaglandin accumulates.

Retrograde transfer of ovarian steroid hormones to the ovary in the porcine periovarian vascular complex

The aim of the present study was to investigate the mechanism of the retrograde transfer of ovarian steroid hormones from the ovarian lymphatic and venous effluent to the arterial blood supplying the ovary. In the first experiment, reproductive organs were collected from gilts in the luteal (n= 10) and follicular (n= 10) phase of the oestrous cycle. The ovary with the mesovarium was isolated and perfused through the ovarian artery with warmed, oxygenated autologous blood. The concentrations of progesterone and oestradiol in ovarian arterial blood increased on passing through the ovarian artery to the ovary, in the luteal phase, from 20.3 ± 2.1 to 31.4 ± 3.9 ng ml−1 (P < 0.001) and from 6.2 ± 0.8 to 11.4 ± 1.4 pg ml−1 (P < 0.001), respectively, and in the follicular phase, from 1.2 ± 0.2 to 2.2 ± 0.4 ng ml−1 (P < 0.001) and from 8.2 ± 1.8 to 13.2 ± 2.3 pg ml−1 (P < 0.001), respectively. Approximately 17.5 ± 3.9% of the progesterone and 12.6 ± 1.7% of the oestradiol found in the ovarian venous effluent was retrogradely transferred from the ovarian venous blood to the ovary in the luteal phase. In the follicular phase, these values were 10.1 ± 2.0% and 8.6 ± 1.4%, respectively. The efficiency of retrograde transfer of oestradiol and the rate of retrograde transfer of progesterone differed between phases of the oestrous cycle (P < 0.05 and P < 0.0001, respectively). A direct relationship between the concentration of the steroids in the venous effluent and the efficiency and rate of the retrograde transfer to the ovary was not found. In the second experiment (luteal phase, n= 10; follicular phase, n= 5), the concentration of progesterone and oestradiol increased in both ovarian arterial blood (P < 0.0001) and in the venous effluent (P < 0.0001) after administration of the steroids into the lymphatic vessels of the isolated mesovarium with separated ovary. In the third experiment (follicular phase, n = 5), with the mesovarium isolated after the ovary was removed and ovarian venous blood flowing out under the force of gravity (without the blood pressure in the ovarian vein), it was demonstrated that the veno‐venous network covering the branches of the ovarian artery was supplied with the blood flowing out from the mesovarian tissue and that the filling of the veno‐venous network was dependent on the blood pressure in the ovarian artery. We conclude that the effective retrograde transfer of steroid hormones from ovarian venous and lymphatic effluent to the ovary is accomplished not only by the classical counter‐current exchange mechanism, but also as a result of complex processes that may be dependent on a specific part of the circulation of the blood and lymph in the periovarian vascular complex of the mesovarium.

Local transfer of prostaglandin E2 into the ovary and its retrograde transfer into the uterus in early pregnant sows.

This study was designed to establish (a) whether prostaglandin E2 (PGE2) can reach the ovary and oviduct by a local pathway and what is the contribution of lymphatic vessels to this transfer, and (b) whether PGE2 can permeate from venous and lymphatic vessels of the mesometrium to arterial blood and be delivered to the uterine horn during maternal recognition of pregnancy in gilts. The reproductive tract was excised from gilts (n= 10) on day 14 after mating. The uterine horn was isolated with the ovary and broad ligament and perfused with warmed and oxygenated autologous blood. A total dose of 5.5 × 107 disintegrations per min (d.p.m.) (49 ng) [3H]PGE2 was infused into the small branches of the uterine vein on the broad ligament or into the lymphatic vessels. Frequent blood samples were collected from the branch of the uterine artery and from the venous effluent. Tissue samples were collected from the uterine horn, the ovary and the broad ligament. The concentration of [3H]PGE2 was significantly higher in the ovary (P < 0.001), oviduct (P < 0.01), endometrium (P < 0.01), myometrium (P < 0.001) and mesometrium (P < 0.001) after infusion of [3H]PGE2 into lymphatic vessels than into the branches of the uterine vein. In contrast, the concentration of [3H]PGE2 was significantly higher in arterial blood supplying the uterine horn (P < 0.01) and in the venous effluent (P < 0.001) after infusion of [3H]PGE2 into the branches of the uterine vein than into lymphatic vessels. These results demonstrated local transfer of [3H]PGE2 into the ovary, oviduct and uterine horn from lymphatic and venous vessels of the mesometrium. However, the efficiency of this transfer was considerably higher after infusion into lymphatic vessels than into branches of the ovarian vein. We conclude that the lymphatic pathway is a fundamental mechanism in the local transfer of PGE2 from the uterus to the ovary and oviduct during early pregnancy in the pig.

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.

Retrograde transfer of steroid hormones to the ovary in luteal and follicular phases of porcine oestrous cycle in vivo

The efficiency of the retrograde transfer of steroid ovarian hormones from the ovarian effluent into blood supplying the ovary and the rate of its back transport to the ovary were determined for the first time in in vivo conditions. Sexually mature gilts (n= 25) were used in the physiological study. The concentration of oestradiol and progesterone in blood collected from the ovarian artery was higher in both the follicular phase (by 87.9 ± 2.9% and 150.0 ± 4.8%, respectively, P < 0.001) and the luteal phase (by 82.1 ± 3.9% and 77.7 ± 2.7%, respectively, P < 0.001) than in systemic blood reaching the initial part of the ovarian artery. The high efficiency of the retrograde transfer was not dependent on the concentration of hormones in the ovarian venous blood. However, the efficiency and rate of the retrograde transfer differed between phases of the oestrous cycle. We suggest that such effective retrograde transfer of ovarian hormones must affect the secretory function of the ovary.

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.

Antioxidant Enzyme Activity and mRNA Expression in Reproductive Tract of Adult Male European Bison (Bison bonasus, Linnaeus 1758)

Antioxidants in the male reproductive tract are the main defence factors against oxidative stress caused by reactive oxygen species production, which compromises sperm function and male fertility. This study was designed to determine the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the testicular and epididymidal tissues of adult male European bison (Bison bonasus). The reproductive tract tissues were subjected to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to quantify mRNA expression levels of five antioxidant enzymes: copper/zinc SOD (Cu/Zn SOD), secretory extracellular SOD (Ec-SOD), CAT, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and GPx5. The corpus and cauda epididymidal tissues displayed greater (p < 0.05) SOD activity compared with the testicular tissue. It was found that CAT activity was lowest (p < 0.05) in the cauda epididymidis, whereas negligible GPx activity was detected in the reproductive tract tissues. There were no detectable differences in the mRNA expression level of Cu/Zn SOD among the different reproductive tract tissues. Small amounts of Ec-SOD mRNA were found in the reproductive tract, particularly in the epididymides. The caput and cauda epididymides exhibited greater (p < 0.05) level of CAT mRNA expression, whereas PHGPx mRNA was more (p < 0.05) expressed in the testis. Furthermore, extremely large amounts of GPx5 mRNA were detected in the caput epididymidal tissue compared with other tissues of the reproductive tract. It can be suggested that the activity of the antioxidant enzymes and the relative gene expression of the enzymes confirm the presence of tissue-specific antioxidant defence systems in the bison reproductive tract, which are required for spermatogenesis, epididymal maturation and storage of spermatozoa.

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.