Suppression of plasminogen activator inhibitor-1 (PAI-1) activity by crocin ameliorates lipopolysaccharide-induced thrombosis in rats.

The imbalance between clot formation and fibrinolysis is mainly attributed to increased levels of plasminogen activator inhibitor type 1 (PAI-1), an inhibitor of fibrinolysis closely involved in inflammatory responses such as septic shock. This increase is mediated by many factors, including reactive oxygen species (ROS). The present study was designed to evaluate the prophylactic effect of crocin, a potent natural antioxidant, on PAI-1 in the rat model of endotoxic shock. Lipopolysaccharide-infused rats (500 µg/kg) showed significant changes in thrombosis-related haematological parameters such as decrease of platelet blood counts and increase (7 fold) of PAI-1 concentration in blood plasma. No effect on t-PA activity was observed. Crocin administration in two different doses (10 mg/kg and 100 mg/kg) 30 min prior to the injection of LPS, inhibited the reduction of platelet counts and ameliorated the concentration of PAI-1 in the liver and the brain. Moreover, crocin inhibited the deposition of fibrin in the renal glomeruli. No significant changes were recorded in the healthy groups of crocin (10 mg/kg and 100 mg/kg) compared to the control group. These data demonstrate the potential of crocin to prevent LPS-induced organ injury and suggest it is worthwhile to investigate the use of antioxidants for the treatment of septicemia.

Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

The aim of this study was to determine the effect(s) of dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω-3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20-day intervals, for a total period of 120 days. The enrichment of membranes in ω-3 PUFA was manifested by the elevation of the 22:5 ω-3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω-3 PUFA-enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω-3 PUFA on sperm cells were evident within 40 days of ω-3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω-3 PUFA-rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

Chromatin integrity of ram spermatozoa. Relationships to annual fluctuations of scrotal surface temperature and temperature-humidity index.

The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SST mean) and THI (THI mean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SST mean, and THI mean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SST mean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THI mean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SST mean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa.

Histological features in the mammary glands of female dogs throughout lactation.

The objective of this study was to describe the histology of the mammary glands of female dogs throughout lactation. Twelve lactating female dogs were operated 4, 7, 10, 14, 21, 28, 35, 42, 56, 70 and 84 days post-partum; four mammary glands of each animal were excised for histological, ultrastructural and morphometric examination. During early lactation and mid-lactation, all lobes and lobules within the same gland had similar features; alveoli were well developed and distended and had a spherical to slightly ovoid structure, with muscular fibres grasping them around; inflammatory cells were seen in the inter- and intra-alveolar space; mammary lobules were separated with a scant amount of connective tissue. In late lactation, connective tissue was abundant and dense, with large numbers of inflammatory cells; alveoli appeared to be irregularly shaped and collapsing, shrunken or fully collapsed. Number of alveoli per lobule and number of epithelial cells per alveolus, as well as diameter of alveoli and height of epithelial cells decreased as lactation progressed. The third mammary glands (from caudal to cranial) had a significantly smaller number of alveoli, but not of epithelial cells per alveolus, than each of the two mammary glands caudally to that. The results suggest that progressive involution of the normal mammary gland starts around the end of the 2nd month of lactation and continues until the end of the 3rd month.

Seasonal variation of plasminogen activator activity in spermatozoa and seminal plasma of boar, buck, bull and stallion.

Plasminogen activators (PA) are proteolytic enzymes present in the spermatozoa and seminal plasma of various species. They play a role in the binding of the spermatozoon and its penetration through the layers surrounding the oocyte. Plasminogen activator activity (PAA) is modulated by hormones that have a seasonal variation, such as testosterone and melatonin. The present study investigates the seasonal variation of PA activity in sperm extracts and seminal plasma of four farm animal species: boar, buck, bull and stallion. Semen samples were collected every second week during a 12-month period and PAA was determined. With respect to sperm enzyme activity, the boar showed a peak from late January until the beginning of April, whereas the activity in the bull was at the highest levels from April until October and gradually declined during autumn and winter period. Plasminogen activator activity of stallion spermatozoa peaked during March and April, and remained low throughout the rest of the year, whereas in the buck sperm, PAA increased from late October until the end of January. No biologically significant variation was detected regarding the seminal PAA activity in any of the species studied. While seasonality of reproduction is typically studied from the female perspective, the present data provide compelling information about a factor that may affect the reproductive ability of the male.

Post-partum involution of the canine uterus - gross anatomical and histological features.

We aimed to study the normal puerperium in the bitch. Ovariohysterectomy was performed in nine bitches, each at a different day after normal whelping; their genital tract was subject to gross anatomical examination, as well as to histological examination and electron microscopy scanning. Corpora albicans were evenly distributed in the left and right ovaries and placental sites were evenly distributed among left and right uterine horns. Placental sites were initially of dark green to grey colour, later becoming dark brown; their length and height progressively decreased. Height of the myometrium and diameter of the uterine glands progressively decreased. Trophoblast-like cells were consistently observed at the placental sites and on the surface of the interplacental areas, at all time points where hysterectomy had been performed. It is suggested that involution of the canine genital tract can last up to 3 months and is slow. Continuous (up to D84 post-partum) presence of prominent placental sites should be considered a normal feature of canine uterine post-partum involution.

Effect of long-term dietary administration of oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats.

This study aimed at evaluating the protective effect of long-term dietary oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats. Twenty-four female Wistar rats were allocated to four groups of six animals each. Groups 1 (control) and 2 (CCl 4) were fed a basal diet, while groups 3 (oregano) and 4 (oregano + CCl 4) were fed the basal diet supplemented further with ground oregano at 1% level. Following six-week feeding, the rats of groups 2 and 4 were given a single intraperitoneal injection of CCl 4 at a dose of 1 mL/kg bw. Six hours after the CCl 4 injection, all animals were sacrificed, and serum, liver, kidney, and heart tissue samples were collected. Analysis results showed that the addition of oregano significantly increased the total phenolic content and the Trolox equivalent antioxidant capacity of the basal diet but had no effect on its lipid peroxidation index. Treatment with CCl 4 of rats from the CCl 4 group caused a significant increase in aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) in serum, whereas it decreased cholesterol and triglyceride content as compared to the control. It also increased the lipid peroxidation index and decreased the scavenging activities of the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) radical cation, the hydroxyl anion radical, the superoxide anion radical, and the hydrogen peroxide in all tested tissues, as compared to that of the control. Without CCl 4 treatment, diet supplementation with oregano had no effect on these biochemical parameters, excluding the hydroxyl radical scavenging activity, which was increased in all tested tissues as compared to that of the control. Feeding oregano before CCl 4 treatment resulted in a significant decline of the increase in AST, ALT, and ALP activities ( P < 0.05 vs CCl 4 group), but the recorded values could not attain those of the control group ( P < 0.05 vs control group). It significantly increased the reduced cholesterol and triglycerides ( P < 0.05 vs CCl 4 group) to values not differing from those of the control. It also resulted in a significant reduction of the increased malondialdehyde ( P < 0.05 vs CCl 4 group) to values that could not attain the levels of the control but had no significant effect ( P > 0.05) on the reduced ABTS radical cation scavenging activity. It increased significantly the reduced hydroxyl anion radical scavenging activity ( P < 0.05 vs CCl 4 group) to values that could not attain those of the control in all tested tissues except kidney. Additionally, it resulted in a significant elevation of the decreased superoxide anion radical scavenging activity in serum and liver but had no effect in kidney and heart, whereas it also resulted in a significant elevation of the decreased hydrogen peroxide scavenging activity in liver, kidney, and heart but had no effect in serum. These results suggest that dietary oregano may effectively improve the impaired antioxidant status in CCl 4-induced toxicity in rats.

Effects of lamb sucking on the bacterial flora of teat duct and mammary gland of ewes.

We aimed (i) to determine differences in bacterial flora of teat duct and mammary gland of ewes before and after suckling, (ii) to evaluate factors potentially affecting those. We collected samples of teat duct material and mammary secretion from 11 ewes immediately before and after sucking by lambs, as well as 120 min later. We processed samples bacteriologically and compared changes in infection by the Sign Test. We isolated bacteria from 3.5% duct and 1.5% secretion samples before suckling. Respective figures post-suckling were 10.6% and 2.0%, and 120 min later 6.8% and 1.5%. We recorded differences in infection of duct samples before and after suckling in 40 cases; bacteria were isolated before suckling from six samples, whereas after it from 34 (p < 0.001). Also, we recorded differences in samples collected after suckling and 120 min later in 12 cases; bacteria were isolated immediately post-suckling from eight samples, whereas 120 min later from four (p = 0.375). No significant changes were seen for secretion. We found neither difference between ewes with single or twin lambs, nor among stages of lactation. Mostly, we isolated staphylococci: 70% of isolates before suckling, 80% of isolates after it, 91% of isolates 120 min later. After suckling we also isolated two Mannheimia haemolytica strains. Suckling predisposes to entrance of bacteria into the teat; however, increased teat infections did not result in mammary infections. Isolation of M. haemolytica post-suckling indicates that lambs act as source of infection for this pathogen.

Subclinical mastitis changes the patterns of maternal-offspring behaviour in dairy sheep.

Subclinical mastitis was induced by inoculating one mammary gland in dairy ewes (n=8) with a Staphylococcus simulans isolate; control ewes (n=4) were included. The milk yield of inoculated glands decreased (P<0.001), the California Mastitis Test (CMT) score increased and the organism could be recovered from the inoculated glands. With time, there was significantly increased frequency of "hindering sucking" (P=0.016) and "head up posture" (P<0.001) in the control ewes. Infected ewes had a significantly increased frequency of "vocalisation" (P=0.013) compared to controls. There was a significant difference in the frequency of "sucking attempt" and "successful suck" (P<0.05) behaviours between lambs of the two groups. Lambs of the challenged ewes also showed significantly increased frequency and duration of these behaviours towards the uninoculated glands of their dams, rather than to the challenged glands (P<0.05); no such difference was evident for the lambs of control ewes. It was concluded that subclinical mastitis alters the sucking behaviour of both ewes and lambs.

Presence of subepithelial lymphoid nodules in the teat of ewes.

A total of 87 clinically healthy ovine teats were examined bacteriologically (by scraping the mucosa) and histologically. Teats examined were those of lactating mammary glands with no bacteria isolated (n = 23); of mammary glands after cessation of lactation with no bacteria isolated (n = 25); of lactating mammary glands with bacteria isolated (n = 22); and of mammary glands after cessation of lactation with bacteria isolated (n = 17). The salient histological feature was subepithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Subepithelial lymphoid nodules, some with germinal centres, were detected in 43 (49%) teats. The majority of lymphoid nodules was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with the presence of leucocytic activity (P < 0.001) and with the presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced subepithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion. The findings call for further investigations into the lymphoid structures of the teat; these should elucidate the role and development of mammary mucosa-associated lymphoid tissues and may lead to strategies for enhancing non-specific defence mechanisms of the mammary gland.

Effects of dietary vitamin A intake on acrosin- and plasminogen-activator activity of ram spermatozoa.

Acrosin and plasminogen activators are proteolytic enzymes of ram spermatozoa that play an essential role in the induction of the acrosome reaction, as well as the binding of spermatozoa to the oocyte and their penetration through the layers that surround the oocyte. Since vitamin A can alter gene expression in various tissues, testis included, this study was undertaken to evaluate the possible effect of vitamin A intake on acrosin- and plasminogen-activator activity. During a 20-week experiment, 15 rams of the Greek breed Karagouniki, divided to three groups, received different amounts of vitamin A per os in retinyl acetate capsules (group A, controls, 12,500 iu/animal per day; group B, 50,000 iu/animal per day; group C, 0 iu/animal per day up to the 13th week, then 150,000 iu/animal per day until the end of the experiment). Acrosin- and plasminogen-activator activity were determined by spectrophotometric methods. Vitamin A was determined in blood plasma by HPLC. No statistical differences were detected regarding the body weight of the rams or the qualitative and quantitative parameters of their ejaculate throughout the whole experiment. No statistically significant alterations of enzyme activity were detected in group B. In group C, both enzyme activities started declining in week 9. Compared with controls, maximum reduction for acrosin was 49% on week 11 and for plasminogen activators 51% in week 14. Activities returned to normal rates after vitamin A re-supplementation. To date, the main result of vitamin A deficiency was known to be arrest of spermatogenesis and testicular degeneration. A new role for vitamin A may be suggested, since it can influence factors related to male reproductive ability before spermatogenesis is affected.

Experimentally induced teat stenosis in dairy ewes: clinical, pathological and ultrasonographic features.

A strain of Staphylococcus chromogenes was introduced into the teat cistern of five ewes, teat inflammation and stenosis being the primary consequences. Initially, the inoculated teats were swollen and warm; later, a hard structure was palpated running lengthwise inside the teat, with a thick ring above the tip of the teat, which interfered with expression of milk. Mastitis, confirmed by clinical, cytological, bacteriological and histological findings, was evident 4 days after infection. Ultrasonographically, a hyperechoic line under the mucosa of the teat cistern was observed. At necropsy, the duct wall of the inoculated teats was found to be thickened. Histopathological features included leucocytic infiltration, especially under the mucosa of the teat, and extensive fibrosis in the subcutaneous tissues. S. chromogenes was recovered from scrapings from the duct and the cistern of the inoculated teats.

Involvement of the plasminogen activation system in cow endometritis.

The objectives of this study were to investigate the: (a) presence and activity of components of the "plasminogen activators/plasmin" system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30-40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1.5x10(6) IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis.

Induction of the acrosome reaction in bull spermatozoa with plasmin.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.

Dexamethasone reduces acrosin activity of ram spermatozoa.

The aim of this study was to investigate the effect of dexamethasone on acrosin activity of spermatozoa in Chios rams during autumn (breeding season for sheep in Greece), in correlation with possible changes in blood testosterone. Dexamethasone was administered in four equal consecutive intramuscular injections, one every four hours (total dose: 3 mg kg(-1)). Total acrosin activity was determined in semen samples collected 48 h before and on the 4th and 7th day and thereafter once every week until the 77th day after dexamethasone administration. Blood samples for testosterone radioimmunoassays were collected 24 h before, during dexamethasone administration and on the 4th, 7th, 14th and 21st day after administration. Total acrosin activity in spermatozoa was reduced between days 7-28 after dexamethasone administration. Dexamethasone also induced a reduction in mean value and basal level of blood testosterone and inhibited its episodic secretion between 1 and 4 days after administration. As the reduction of acrosin activity appeared relatively soon after dexamethasone administration (7th day), it is likely that the increased amount of dexamethasone did not influence the synthesis of proacrosin in the late spermatids. As glucocorticoid receptors exist in the epididymis and accessory glands in various species, dexamethasone may have a direct influence on the synthesis and/or release of acrosin inhibitors in epididymal fluid or seminal plasma. These changes in acrosin activity in ovine spermatozoa mediated by dexamethasone may be of importance regarding the role of stress in the reduction of sperm fertilizing ability.

Inhibition of human and ovine acrosomal enzymes by tannic acid in vitro.

The effect of tannic acid, a common flavonoid, on the acrosin and plasminogen activator activity and plasmin activity of human and ram spermatozoa was evaluated. Acrosin and plasminogen activator activity were determined by spectrophotometry using the chromogenic substrates N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) and H-D-valyl-L-leucyl-L-lysine-p-nitroanilide-2HCl (S-2251), respectively. In extracts from both human and ovine acrosomes, the activities of acrosin and plasminogen activators were susceptible to tannic acid inhibition. The inhibitory effect of tannic acid was observed at concentrations > 50 micromol l(-1) in a dose-dependent manner. In additional experiments, low concentrations of tannic acid significantly inhibited tissue-type plasminogen activator, urokinase-type plasminogen activator and plasmin activity in a concentration-dependent manner over the range 0.25-200 micromol l(-1). Tannic acid reduced the motility of ram spermatozoa at a concentration of 1000 micromol l(-1) after 2 and 3 h co-incubation with spermatozoa. The motility of human spermatozoa remained unchanged over the range 0.1-1000 micromol tannic acid l(-1) during 3 h co-incubation. These results indicate that tannic acid inhibited the activity of both acrosin and plasminogen activator and indicates a possible mechanism by which flavonoids exert their antifertility effects.

Dietary administration of tannic acid lowers plasminogen activator activity in the liver of C3H hepatoma bearing male mice.

BACKGROUND: This study was undertaken to investigate if low-dose dietary administration of tannic acid exerts its chemopreventive activity on hepatocarcinogenesis in male C3H mice through changes of plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) or plasmin inhibition (Pl) in the liver of C3H hepatoma bearing male mice. MATERIALS AND METHODS: Tannic acid was administered in the drinking water of hepatoma bearing C3H mice, in a concentration of 0.0075% (group B), 0.015% (group C) and 0.03% (group D) for 9 months. PAA, PAI and PI were measured by spectrophotometric methods in macroscopically healthy liver regions. RESULTS: Tannic acid induced a decrease of PAA (53%) and an increase of PAIs (50%) in the livers of group D while tannic acid received mice showed a dramatic decrease in liver carcinomas incidence (0% vs 33.3% in the control group). CONCLUSION: The decrease of PAA in the liver of C3H hepatoma bearing male mice, might be correlated with the chemopreventive activity of tannic acid in hepatoma development.

Gossypol-induced inhibition of plasminogen activator activity in human and ovine acrosomal extract.

The effect of gossypol--a polycyclic compound isolated from cotton seeds--on the plasminogen activator activity of man and ram acrosomal extracts was explored in vitro. The action of gossypol on the plasminogen activator activity was investigated by a spectrophotometric method using the chromogenic substrate S-2251. Gossypol, a known antispermatogenic agent, was found to effectively inhibit human and ovine acrosomal plasminogen activator activity. The inhibition was dose-dependent. Plasminogen activator activity from man and ram extracts was completely inhibited by 350 mumol l-1 and 300 mumol l-1 of gossypol, respectively. In additional experiments, low, non-spermicidal concentrations of gossypol (2.5-40 mumol l-1) were found to significantly inhibit plasmin activity in a dose-dependent manner. The results suggest that inhibition of both acrosomal plasminogen activator and plasmin activity is a possible mechanism by which gossypol exerts its antifertility effect, since the plasminogen activator/plasmin system plays a role in the whole process of ovum fertilization.