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.

Toxic effect of antidepressants on male reproductive system cells: evaluation of possible fertility reduction mechanism.

Depression is acknowledged as a major public health problem. Pharmacological treatment may cause adverse drug reactions and sexual side effects. At the same time, the knowledge of the molecular mechanisms associated with antidepressant-mediated toxicity to reproductive cells is fragmentary. The aim of this study was the multilevel evaluation of the potential toxicity of several antidepressants or antipsychotic drugs (amitriptyline, 10 µM; escitalopram, 30 µM; fluoxetine, 5 µM; imipramine, 20 µM; mirtazapine, 150 µM; olanzapine, 40 µM; reboxetine, 30 µM; venlafaxine, 250 µM) on the cells of the spermatogenesis pathway. Effects of various drugs were monitored by several methods including mitochondrial activity MTT test, fluorescent staining, real-time PCR, morphology analysis, immunofluorescence, and Western blots. Obtained results suggest the concentration- and the time-dependent cytotoxic effect. The molecular mechanism of cytotoxic effect is mediated by disturbances in the redox balance (increased production of reactive oxygen species and reactive nitrogen species), failure of enzymatic and non-enzymatic cell protection mechanisms (glutathione system, nuclear factor-κB and fibroblast growth factor 2-mediated pathways), and impairment of mitochondrial functions. In addition, we provide for the first time, to our knowledge, evidence that antidepressant treatment may contribute to spindle apparatus assembly defects and organelle distribution during cell division in vitro (alterations in the levels of small C terminal domain phosphatase-1 and -3, NuMa, and calnexin protein levels). This study sheds new light on the pathomechanisms of antidepressants action and their associated toxicity towards the reproductive system, emerging issues linked with animal or human reproductive health, and treatment of mood disorders.

Effect of neonatal exposure to endocrine-active compounds on plasma lipid and steroid concentrations, and morphology of luteal tissue in the adult pig.

To study the long-term impact of neonatal exposure to endocrine-active compounds (EACs) on plasma lipid profiles, steroid concentrations, and morphology of porcine luteal tissue, piglets were injected with testosterone propionate (TP), flutamide (FLU), 4-tert-octylphenol, ICI 182,780 (ICI), methoxychlor, or corn oil (controls) between postnatal days 1 and 10 (n = 5/group). Blood samples and corpora lutea were obtained from sexually mature gilts. The investigated compounds differentially affected plasma lipid and steroid concentrations. Moreover, we demonstrated hypertrophy of luteal cells after neonatal EAC administration. In addition, a predominant abundance of lipid droplets was found in luteal cells of TP-, FLU-, and ICI-treated animals. It seems that the pathways leading to changes in the plasma lipid profile may contribute to the development of long-term alterations that have the potential to affect luteal steroidogenic capability in pigs.

Cereal grass juice in wound healing: hormesis and cell-survival in normal fibroblasts, in contrast to toxic events in cancer cells.

Natural products and traditional medicines are of great importance. Recent studies have demonstrated, that cereal grass juice improves wound healing, however the cellular and molecular mechanisms underlying these processes have not been fully characterized. Also, the full phytochemical characteristics of freshly squeezed juices obtained from cereal grasses is still missing. Thus, in this study a multi-dimensional analysis of juice parameters like refraction value, pH, chlorophyll and flavonoids content as well as antioxidant properties was performed. The results demonstrate that the effect induced by freshly squeezed cereal juices is strictly cell type-dependent. In this study, it is shown for the first time, that in normal fibroblasts (BJ cells) low dose cereal grass juices exhibit strong adaptive response through hormetic mechanism mediated by NF-κB/HO-1 and insulin/IGF-1 anti-oxidant pathways. As consequence, the process of wound healing is significantly upregulated. In cancer cells (ES-2 cells), despite anti-oxidant defense mechanism activation, levels of ROS and RNS are elevated. This leads to enhanced O-GlcNAcylation, DNA damage and cell cycle arrest, and as a result impaired wound healing. This study provides insights into the underlying mechanisms through which cereal grass juices activate hermetic adaptation response in normal fibroblasts, and induce cytotoxic and genotoxic events in cancer cells.

Carbon monoxide modulates melatonin synthesis in porcine pineal Cells in vitro: a preliminary study.

Two main isoforms of heme oxygenase (HO-1 and HO-2), the main enzyme of heme metabolism, were identified in the pineal gland. This suggests possible interactions between the melatonin synthesis pathway and the HO system. The aim of this study was to investigate the participation of carbon monoxide (CO), an HO by-product, on the melatonin synthesis pathway. Tests were carried using primary cell cultures of porcine pineal glands. The tricarbonyldichlororuthenium (II) dimer (CORM-2) compound was used as a CO donor at concentrations of 1 and 3 µM, as low concentrations of CORM-2 affect the regulation of the melatonin synthesis pathway in pineal cells in vitro. In addition, the presence of Sn-protoporphyrin-IX, an HO inhibitor, changed the melatonin response of pineal cells. These results suggest the existence of an intermediate mechanism in the pineal gland, which is associated with HO activity, that is involved in the modulation of melatonin synthesis.

Melatonin receptors subtypes (MT1 and MT2) in the uterus masculinus of mature male european bison. Biological and seasonal reproductive role.

Regulation of seasonality in reproduction is closely related to melatonin and circadian rhythms. Melatonin affects the functions of reproductive organs through membrane melatonin receptors MT1 and MT2. The current knowledge about the presence, location and function of MT1 and MT2 receptors in the reproductive tract of an adult male European bison, seasonally breeding animal, is still missing. Frequently occurring organ in the male reproductive system of the European bison is uterus masculinus, what seems to confirm its specific role in seasonal reproduction control. Taking this into account, our study aimed to evaluate the expression of mRNA and protein synthesis for both melatonin receptors in the tissues of uterus masculinus in November and December in European bison. Protein synthesis of MT1 and MT2 receptors in uterus masculinus of mature European bisons was clearly raised in November and decreased in December. The comparable results were also found for mRNA expression of MT1 and MT2 receptors, where in November the expression level was significantly higher than in December. Therefore, we suggest that melatonin is needed in the European bison's reproductive system after a period of intensive reproductive activity in November. Probably, melatonin plays a protective function in uterus masculinus of the European bison and thus regulates the seasonal reproduction.

Male reprotoxicity associated with Sophora japonica treatment: evaluation of cellular and molecular events in vitro.

Toxicological studies are urgently needed to confirm the safety of Sophora japonica extracts for clinical use. In particular, in addition to pharmacy and pharmacology, phytotherapy, herbal medication, Sophora japonica extracts are widely used as biologically active supplements. Scientfic data suggests that some of Sophora japonica extract components have very comprehensive biological effects. In the present study, our hypothesis assumed the potential reprotoxicity of Sophora japonica extract in respect to mouse germ cells (GC-1 spg, GC-2 spd) in vitro. Specifically, we were interested in the high-performance liquid chromatography (HPLC) identification of extract components and its stress-related effects on cellular and biochemical features, such as mitochondrial metabolism, cell cycle progression, oxidative stress balance and micronuclei formation. The results indicate that Sophora japonica extract induces oxidative/nitrosative stress-mediated impairment of the mechanism for free radicals scavenging, which may provoke genotoxic events in germ cells, by cell cycle arrest and micronuclei formation. Therefore, the interplay between reactive oxygen species (ROS)/reactive nitrogen species (RNS) and antioxidant system is critical for normal testicular function maintenance in the their environment. The specific pathways and mechanisms involved in the reprotoxicity of Sophora japonica need to be further investigated.

Local regulators of seasonal reproduction processes in uterus masculinus of an adult male European bison (Bison bonasus, Linnaeus 1758).

Growth factors, hypoxia-inducible factor 1-alpha (HIF-1α) and klotho protein all have very important functions in the male reproduction; however their role in the regulation of seasonal reproductive processes in the male European bison remains unclear. Similarly, although the uterus masculinus is very frequently found in the bison, its importance and functions remain unknown. It is likely that, this organ may have secretory functions and thus be a target for various regulatory factors. Therefore, the aim of this study was to investigate expression and activity of several factors: vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF-2), transforming growth factor ß1 (TGF-ß1), nerve growth factor (NGF), insulin-like growth factor receptor (IGF-IR ß), hypoxia-inducible factor 1 alpha (HIF-1α), and klotho protein in the uterus masculinus, immediately after the season of the reproductive activity (November and December). Our study reveals that the growth factor expression levels are significantly higher in November, when compared to December, while expression of HIF-1α and klotho was higher in December. These results provide novel data on differences in the expression levels of several factors in the uterus maculinus of European bison bulls after the breeding season. The described factors may, therefore, be potent regulators of the seasonal reproduction.

Electromagnetic fields with frequencies of 5, 60 and 120 Hz affect the cell cycle and viability of human fibroblast BJ in vitro.

The impact of electromagnetic field (EMF) on humans has been described in numerous studies, but many questions are still unanswered. The aim of the experiment described in this study was to evaluate the effect of EMF on the viability of human fibroblast BJ in vitro and the percentage of cells in different phases of the cell cycle (G1/G0, S, G2/M) after 2 hours of exposure to sinusoidal continuous and pulsed EMFs with frequency of 5 Hz, 60 Hz and 120 Hz at a magnetic induction of 2,5 mT. The viability of BJ cells exposed to an EMF was estimated immediately after completion of exposure and after 24 hours. Metabolic activity of cells was assessed by MTT assay and compared to a control culture not exposed to EMFs. Cell cycle analysis was performed by BrdU incorporation. The analysis of the viability demonstrated significant differences in field efficiency, depending on its nature. Exposure of cells to pulse EMFs resulted in a decrease in their viability for each of the analyzed frequencies. Reduced viability was maintained for a further 24 hours after the end of exposure of cells to pulsed EMF. In the case of continuous field, reduced BJ cell viability was observed only at the highest applied frequency - 120Hz, and this effect maintained for the next 24 hours. Although there was no significant effect on cell viability (metabolic activity) of cells immediately after exposure to continuous EMF with a frequency of 5Hz, a significant increase was observed after 24 hours of incubation.

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.

Morphological defects of epididymal spermatozoa in male roe deer (Capreolus capreolus) during the reproductive season.

The aim of this study was to analyse the morphology of the epididymal spermatozoa of male roe deer obtained postmortem at the beginning (May), peak (July/August) and the end (September) of the reproductive season. Spermatozoal abnormalities were divided into major (associated with impaired fertility) and minor (not associated with impaired fertility) defects. The highest percentage of abnormal spermatozoa was observed in May (17.78±1.88%), with a much higher proportion of major (12.35±1.11%) than minor defects (5.43±1.59%) being observed. The percentage of abnormal spermatozoa was lowest during the peak of the reproductive season (4.97±1.13%), with the proportion of major (2.68±0.78%) and minor defects (2.28±0.45%) being comparable during this period. The percentage of abnormal spermatozoa increased again in September (11.05±1.60%), with the major defects (6.15±1.04%) slightly surpassing the minor defects (4.90±0.77%); however, total abnormalities still remained lower than those found in May. These differences were statistically significant, with the exception of the difference in minor defects between the pre-rut and post-rut periods. These results indicate that the best period to collect epididymal spermatozoa from roe deer postmortem is the peak of the reproductive season (July/August); however, they can also be recovered at the end of the reproductive season (September), as the percentage of major defects is relatively low at this time. This study provides the basis for further research to determine optimal methods for the storage and cryopreservation of epididymal spermatozoa in this species.

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.

THE INFLUENCE OF CARBON MONOXIDE ON THE SECRETION OF MELATONIN BY PINEALOCYTES MEASURED IN VITRO.

Photoperiod is considered the most important factor entraining the circannual physiological rhythms through changing circadian patterns of melatonin (MEL) secretion from the pineal gland. The pineal gland of mammals does not respond directly to light but is controlled by light via neuronal phototransduction originating in the retina. In accordance with humoral phototransduction hypothesis, the aim of this study was to determine whether an increased concentration of CO, as a carrier of a light signal in pineal cell culture, affects the synthesis of melatonin. This study demonstrates that a commonly used carbon monoxide donor (CORM-2) markedly stimulated melatonin release from pineal cells incubated in vitro in a time-dependent manner, but the mechanism whereby CO modulates MEL release needs to be further explored.

Seasonal-dependent variations in metabolic status of spermatozoa and antioxidant enzyme activity in the reproductive tract fluids of wild boar/domestic pig hybrids.

This study investigated seasonal changes in the metabolic performance of spermatozoa and activity of the antioxidant enzymes in the seminal plasma of three wild boar/domestic pigs (aged 1.5 to 2.5 years) and the activity of the antioxidant enzymes in fluids of the cauda epididymidis and vesicular glands from 16 wild boar/domestic pig hybrids (aged 1 to 3 years). Parameters of the sperm metabolic activity, such as total motility, mitochondrial functions, and measurements of oxygen uptake, ATP content and L-lactate production, were analyzed during the spring-summer and autumn-winter periods. Besides these sperm metabolic parameters, the sperm membrane integrity was also assessed. Total protein content and activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were measured in the reproductive tract fluids. There were no marked significant differences (P > 0.05) between the seasonal periods in terms of sperm motility, mitochondrial function and oxygen uptake; however, spermatozoa collected during the autumn-winter period exhibited higher (P < 0.05) ATP content and L-lactate production than those harvested during the spring-summer period. It was found that the vesicular gland fluid exhibited a higher level of SOD activity during the spring-summer period compared with the autumn-winter period. Furthermore, CAT activity in the seminal plasma and vesicular gland fluid was greater during the autumn-winter. Total protein content was significantly higher in the vesicular gland fluid, whereas the cauda epididymidal fluid exhibited greater SOD and GPx activities, irrespective of the seasonal period. The findings of this study confirmed seasonal-related differences in the metabolic performance of spermatozoa and activity of antioxidant enzymes of the reproductive tract of the boar/domestic pig hybrids.

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.

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.

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.

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 µg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

The expression of genes encoding opioid precursors and the influence of opioid receptor agonists on steroidogenesis in porcine adrenocortical cells in vitro.

Endogenous opioid peptides are involved in the regulation of the HPA-axis function and stress response mechanism. However, there is a scarcity of data on opioid involvement in the regulation of the adrenocortical endocrine function. This study was performed to: 1) establish the expression of proenkephalin, POMC and prodynorphin genes in the porcine adrenal cortex and test in vitro the influence of ACTH, angiotensin II, CRH and epinephrine on this expression, and 2) determine the effects of opioid receptor agonists on basal and ACTH- or angiotensin II-affected secretion of cortisol, aldosterone and progesterone by the cultured adrenocortical cells. Our experiment has demonstrated the presence of mRNAs for opioid precursors in cells isolated from the adrenal cortex and the significant effects of ACTH and angiotensin II, but not CRH or epinephrine, on adrenocortical transcription of the analyzed genes. Angiotensin II reduced the expression of the POMC gene but stimulated that of prodynorphin. In turn, ACTH decreased the transcription of prodynorphin. The study has also demonstrated the effects of selective opioid receptor agonists - DPLPE (delta), FK33-824 (mu) and U50,488 (kappa) - on adrenal steroidogenesis in pigs. Basal secretion of cortisol was enhanced after the activation of mu or kappa receptors, whereas ACTH-stimulated cortisol output was increased only by the mu receptor agonist. Angiotensin II-treated cells significantly decreased aldosterone secretion in the presence of the kappa receptor agonist. The present results suggest that opioid peptides are synthesized in the porcine adrenal cortex, indicating their involvement in the regulation of adrenal steroidogenesis through autocrine and/or paracrine interactions.

Antioxidant defence system of boar cauda epididymidal spermatozoa and reproductive tract fluids.

Antioxidants secreted by the reproductive tract protect spermatozoa against the toxic effects of reactive oxygen species (ROS) after ejaculation. This study aimed at characterizing the level of antioxidant protection in boar cauda epididymidal spermatozoa and fluids of the cauda epididymidis, vesicular and prostate glands. Also, this study investigated the effect of a 5-h period of dialysis on the antioxidant capacity of boar seminal plasma. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) activities were monitored in the cauda epididymidal spermatozoa or reproductive tract fluids. Also, the concentrations of total glutathione (GSH + GSSG), L-ergothioneine (ERT) and l-ascorbate and the total antioxidant status (TAS) of the fluids were measured. It was found that the cauda epididymidal spermatozoa exhibited high SOD activity and relatively low activity of PHGPx. The relative amounts of GPx, GR and GST activities in the cauda epididymidal spermatozoa were negligible, whereas CAT activity was undetectable. Greater SOD activity was found in the fluids of the cauda epididymidis and prostate gland. Furthermore, the prostate gland fluid appeared to be the main source of CAT activity in the seminal plasma, whereas the highest level of GPx activity was derived from the cauda epididymidal fluid. The reproductive tract fluids exhibited negligible amounts of GR and GST activities. It seemed that the significant amounts of GSH + GSSG, ERT and L-ascorbate in the reproductive tract fluids could have an ameliorative effect on the level of TAS in the seminal plasma. Dialysis had a marked effect on the total antioxidant capacity of the seminal plasma, which was manifested in greater activity of SOD and GPx. The findings of this study confirmed that the scavenging potential of the seminal plasma is dependent on the contributions of different antioxidants, originating in various fluids of boar reproductive tract.