Endogenous carbon monoxide (CO) as a modulating factor of molecular biological clock in the hypothalamic structures involved in light transmission in pig and wild boar hybrid during long and short day season.

Mature males of a wild boar-pig crossbreed, during the long and short day season, were used for the study which demonstrates that the chemical light carrier CO regulates the expression of biological clock genes in the hypothalamus via humoral pathways. Autologous blood with experimentally elevated concentrations of endogenous CO (using lamps with white light-emitting diodes) was infused into the ophthalmic venous sinus via the right dorsal nasal vein. Molecular biology methods: qPCR and Western Blot were used to determine the expression of genes and biological clock proteins. The results showed that elevated endogenous CO levels, through blood irradiation, induces changes in genes expression involved in the functioning of the main biological clock located in suprachiasmatic nuclei. Changes in the expression of the transcription factors Bmal1, Clock and Npas2 have a similar pattern in both structures, where a very large decrease in gene expression was shown after exposure to elevated endogenous CO levels. The changes in the gene expression of PER 1-2, CRY 1-2, and REV-ERB α-ß and ROR ß are not the same for both POA and DH hypothalamic structures, indicating that both structures respond differently to the humoral signal received. The results indicate that CO is a chemical light molecule whose production in an organism depends on the amount of light. An adequate amount of light is an essential factor for the proper functioning of the main biological clock.

Changes in gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression after an increase in carbon monoxide concentration in the cavernous sinus of male wild boar and pig crossbread.

Previous studies indicate that there are at least a few regulatory systems involved in photoperiodic synchronisation of reproductive activity, which starts with the retina and ends at the gonadotropin-releasing hormone (GnRH) pulse generator. Recently we have shown indicated that the amount of carbon monoxide (CO) released from the eye into the ophthalmic venous blood depends on the intensity of sunlight. The aim of this study was to test whether changes in the concentration of carbon monoxide in the ophthalmic venous blood may modulate reproductive activity, as measured by changes in GnRH and GnRH receptor gene expression. The animal model used was mature male swine crossbred from wild boars and domestic sows (n = 48). We conducted in vivo experiments to determine the effect of increased CO concentrations in the cavernous sinus of the mammalian perihypophyseal vascular complex on gene expression of GnRH and GnRH receptors as well as serum luteinizing hormone (LH) levels. The experiments were performed during long photoperiod days near the summer solstice (second half of June) and short photoperiod days near the winter solstice (second half of December). These crossbred swine demonstrated a seasonally-dependent marked variation in GnRH and GnRH receptor gene expression and systemic LH levels in response to changes in CO concentration in ophthalmic venous blood. These results seem to confirm the hypothesis of humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and the effect of CO on GnRH and GnRH receptor gene expression.

Changes in melatonin synthesis parameters after carbon monoxide concentration increase in the cavernous sinus.

Previous studies indicate that the gaseous messenger carbon monoxide (CO) is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight. This study was designed to determine whether the increased concentration of CO in ophthalmic venous blood affects the synthesis of melatonin and therefore, whether CO released from the eye under normal lighting conditions can be a carrier of light intensity information. Thirty six mature male wild boar and pig crossbreeds (n = 36) were studied. We measured the difference in the scotophase melatonin pathway response in terms of mean concentration of increased melatonin levels after 48 hours infusion of autologous blood plasma with an experimentally induced approximately 3-fold increase in the concentration of CO into the ophthalmic venous sinus. We demonstrated in this crossbreed a marked variation in the duration and amplitude of nocturnal melatonin peak in response to increased concentration of CO in ophthalmic venous blood. During the winter this treatment limited the nocturnal melatonin rise. During the summer this same experimental treatment enhanced the nocturnal melatonin rise. Changes in melatonin levels were always associated with parallel changes in AANAT protein levels. This work demonstrates that non-physiological changes in CO concentration in ophthalmic venous blood can have an acute impact on the systemic melatonin level. These results support humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and treatment of winter seasonal affective disorder.

Countercurrent transfer of dopamine from venous blood in the cavernous sinus to the arterial blood supplying the brain - the perfused rabbit head as an experimental model.

The objective of the current study was to check whether countercurrent transfer of dopamine occurs in the cavernous sinus of the rabbit and whether the rabbit can be used as an animal model to study cavernous sinus function. After exsanguination of the animal, oxygenated and warmed (37°C) Hanseneleit-Krebs buffer with autologous or homologous blood (in a 3:1 or 1:1 ratio) was pumped through both common carotid arteries into the head (60 ml/min; 80-100 mm Hg) and radiolabeled dopamine (3(H)-DA, 10 µCi) was infused into the cavernous sinus through the angular oculi vein. Cerebral blood from the basilar artery was collected from the cannulated vertebral artery during 3(H)-DA infusion and for 10 minutes after completion of infusion. Selected brain tissue samples were collected after completion of the head perfusion. It was demonstrated that dopamine can penetrate from the rabbit's cavernous sinus to the internal carotid artery supplying the brain. Dopamine permeation was greater when the rabbit head was perfused with buffer and blood in a 3:1 ratio than with 1:1 (P<0.01). When the head was perfused with buffer and blood in a 3:1 ratio, significant radioactivity was found in samples collected from the brain basilar artery during and after 3(H)-DA infusion (P<0.001). The radioactivity was identified as 34.13 ± 2.7% unmetabolized 3(H)-DA and 65.9 ± 2.7% its metabolites. Significant radioactivity was also found in some brain tissue samples in both groups (P<0.05). The concentration of free radiolabeled dopamine particles in the dialysate of blood plasma and plasma diluted with buffer did not differ significantly. Because the structures of the cavernous sinus and cavernous fragment of the internal carotid artery of the rabbit are similar to those in humans, it suggests that rabbits can serve as a model for experimental physiological studies of cavernous sinus function and retrograde dopamine transfer in the cavernous sinus should be considered as an important link in the genesis of Attention Deficit Hyperactivity Disorder (ADHD) and Parkinson's disease.

Carbon monoxide-mediated humoral pathway for the transmission of light signal to the hypothalamus.

The gaseous messenger carbon monoxide (CO) is released from the eye into ophthalmic venous blood depending on the intensity of sunlight. Numerous neurohormones and other regulatory factors permeate from venous blood into arterial blood in the perihypophyseal vascular complex (PVC) and are transferred to the brain by the humoral pathway. This study was designed to determine whether elevated CO in ophthalmic venous blood (OphVB) affects the expression of clock genes and their transcriptional factors in the hypothalamus. Mature males of a wild boar and pig crossbreed (n=24) were used for the study. Autologous plasma with increased concentrations of CO was infused into the ophthalmic sinus (OphS) of the experimental group (n=12). The expression of clock genes (Per1, Per2, Cry1, Cry2, Rev-erb α and Rev-erb ß) and the genes of their regulators (Bmal1, Npas2, Clock, Ror ß) was estimated in two hypothalamic structures involved in the reception and transmission of light signal (the preoptic area (PA) and dorsal hypothalamus (DH)). We demonstrated that the expression of clock genes and the genes of their regulatory factors in the experimental group was altered compared with control, both in the PA and DH. The response to an increased concentration of CO differed between individual genes and the hypothalamic regions. The expression of Per1 which, according to many authors, is regulated by light, was increased in animals treated with CO both in the PA and DH, and regardless of the time of day and season. In conclusion, the current results seem to confirm the hypothesis on the function of CO in humoral transfer from the eye to structures related to the reception and transmission of light signal and the effect of CO on clock gene expression.

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.

The gaseous messenger carbon monoxide is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight.

Circadian and seasonal rhythms in daylight affect many physiological processes. In the eye, energy of intense visible light not only initiates a well-studied neural reaction in the retina that modulates the secretory function of the hypothalamus and pineal gland, but also activates the heme oxygenase (HO) to produce carbon monoxide (CO). This study was designed to determine whether the concentration of carbon monoxide (CO) in the ophthalmic venous blood changes depending on the phase of the day and differing extremely light intensity seasons: summer and winter. The concentration of CO in the venous blood flowing out from the nasal cavity, where heme oxygenase (HO) is expressed, but no photoreceptors, was used as a control. Sixteen mature males of a wild boar and pig crossbreed were used for this study. Samples of ophthalmic and nasal venous blood and systemic arterial and venous blood were collected repeatedly for two consecutive days during the longest days of the summer and the shortest days of the winter. The concentrations of CO in blood samples was measured using a standard addition method. During the longest days of the summer the concentration of CO in ophthalmic venous blood averaged 3.32 ± 0.71 and 3.43 ± 0.8 nmol/ml in the morning and afternoon, respectively, and was significantly higher than in the night averaging 0.89 ± 0.12 nmol/ml (p<0.001). During the shortest day of the winter CO concentration in ophthalmic venous blood was 1.11 ± 0.10 and 1.13 ± 0.14 nmol/ml during the light and nocturnal phase, respectively, and did not differ between phases, but was lower than in the light phase of the summer (p<0.01). The CO concentration in the control nasal venous blood did not differ between seasons and day phases and was lower than in ophthalmic venous blood during the summer (p<0.01) and winter (p<0.05). The results indicate that the gaseous messenger carbon monoxide is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight.

Relationship between contractions of the uterus and concentration of PGF2α in uterine venous blood after luteolysis in gilts.

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.

The effect of intravaginal application of estradiol and progesterone on porcine embryo development.

This study was designed to check whether intravaginal application of estradiol and progesterone on chosen days of early pregnancy affects the development and survival of porcine embryos. Crossbred gilts were assigned to control (C-1 and C-2) or experimental (T-1 and T-2) groups. Systemic blood samples were collected in groups C-1 and T-1 on days 3-18 of pregnancy. Gilts of control groups were supplied on days 2-6 and 12-17 of pregnancy with intravaginal cocoa butter suppositories. Gilts of experimental groups were intravaginally supplied on days 2-6 of pregnancy with estradiol (0.2 mg) to improve uterine blood supply and on days 12-17 of pregnancy gilts were supplied with progesterone (0.25 mg) to stimulate the secretory function of the endometrium during the periimplantation period. Systemic concentration of progesterone was elevated on days 3-6 and of estradiol on days 4, 5 and 10 of pregnancy in the experimental groups in comparison with the control gilts. The weight of the ovary and number of corpora lutea were similar in the control and experimental groups, but the number of follicles over 3 mm in diameter was higher in the experimental groups than in control (P < 0.01) on day 30 of pregnancy. The mean ovulation rate, total number of embryos and rate of embryo survival on day 30 of pregnancy were similar in the control and experimental groups. However, the weight, crown-rump length and width of embryos were greater in the experimental groups than in control (P < 0.001). The results demonstrated that the intravaginal application of estradiol and progesterone on precisely chosen days of early pregnancy stimulated embryo development. This study demonstrated that the timing of hormone administration must comply with the physiological effect of estradiol and progesterone on uterine blood supply to ensure embryo development and survival.

A local pathway for increased testosterone supply from the testis to the epididymis, vas deferens and accessory genital glands of the rat.

Radiolabeled testosterone (3H-T) was infused into the testes or left and right mesofuniculus (106 dpm) or injected into a testes (2 x 10(6) dpm). The 3H-T concentration was estimated 15 or 10 min after 3H-T infusion or injection, respectively, in the tissue samples collected from the prostate, seminal vesicles, caput and cauda epididymides, vasa deferentia and the mesofuniculi. The abdominal aorta and posterior vena cava were cannulated, and the posterior part of the body perfused with blood (at blood pressure 70-140 or 260-300 mm Hg in abdominal aorta) was used to study 3H-T transfer from the testes to venous blood and other male genital organs. The concentration of 3H-T found in the accessory genital glands, epididymes and vasa deferentia was affected by blood pressure in the abdominal aorta. The reduced blood pressure and partial blocking of blood supply to the genital organs (after ligation of both testicular arteries or the terminal part of the abdominal aorta) increased the concentration of 3H-T in accessory genital glands, vasa deferentia and epididymes. The removal of the mesofuniculi and vasa deferentia with their mesoducti reduced 3H-T concentration in the prostate, seminal vesicles and cauda epididymides. Both arterial trunks, testicular arteries and common iliac arteries, were shown to be connected by anastomoses in target organs so effectively, that supplying the male genital organs with blood by only one of them assures the transfer of testosterone from the testes to the epididymes, vasa deferentia, mesofuniculi as well as prostate and seminal vesicles. It was concluded that lymphatic vessels of the mesofunicules and of the spermatic cords as well as venous and arterial vasculature of the mesofunicules create a recently unknown pathway for the increase of testosterone supply from the testes to the epididymes, vasa deferentia and accessory genital glands of rats.