Melatonin administration increased plasminogen activator activity in ram spermatozoa.

The aim of the present study was to investigate the effect of melatonin on plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram spermatozoa and seminal plasma, in correlation with changes in blood testosterone. Melatonin implants (18 mg) were placed subcutaneously in sixteen Chios rams in autumn and spring. Semen samples for spectrophotometrical assays were collected 36 h before the implantation of melatonin and thereafter once a week, for 17 weeks. Blood samples for testosterone assay (RIA) were collected 8h before implantation (one sample/30 min x 7.5 h) and thereafter every 15 days for 105 days after implantation. For each ram, six parameters of testosterone were estimated: mean value, basal level, number of peaks, peak amplitude, peak duration and mean testosterone concentration during peaks. Melatonin implantation during autumn induced an increase in PAA and t-PAI in spermatozoa; melatonin implantation in spring induced an additional increase in u-PAI and PI; no change in PAA, PAI or PI was found in seminal plasma, during autumn or spring. The melatonin-induced increase of PAA, PAI and PI in spermatozoa was in positive correlation with the increase of testosterone mean value, basal level and number of peaks; the increase of testosterone parameters was greater in autumn compared to spring. Changes of PAA, PAI and PI of spermatozoa, under the influence of melatonin, might indicate changes in the fertilizing ability of spermatozoa, since plasminogen activators and their inhibitors are present on the plasma and the outer acrosomal membrane of spermatozoa and are released during the acrosome reaction.

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus.

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.

The presence of environmental pollutants in the semen of farm animals (bull, ram, goat, and boar).

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively, and may affect male reproduction function. Although several pollutants have been detected in human semen, similar studies in farm animals have not appeared. In the present study, the semen of bulls, rams, goats, and boars was assayed for the organochlorine pesticides hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, and heptachlor epoxide, for DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT), and for the PCBs congeners (PCB-52, -101, -138, -150, and -180). In all species of farm animals, the most frequently detected pollutants were p,p'-DDE (80-100% of samples), HCB (73.9-100%), and gamma-HCH (69.6-100%). Species differences in the concentrations of HCB, alpha-, beta-, and gamma-HCH, dieldrin, p,p'-DDE, p,p'-DDD, and PCBs were noted as well as differences in the concentrations of some isomers of HCH, DDT-related chemicals, and PCB congeners in the same species.

The presence of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs).

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively. The present study assessed the burden of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs). An analytical method combining a solid-phase extraction with (C(18)) for clean-up and GC-electron capture detection using a capillary column was implemented for isolation and determination of organochlorine pesticides and polychlorinated biphenyls (PCBs). Of the organochlorine pesticides, hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, heptachlor epoxide, and the DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT) were detected and of the PCBs, the congeners PCB-52, -101, -138, -153, and -180 were detected. In all species of farm animals, the most frequently detected pollutant was gamma-HCH (90-100% of samples) followed by HCB (80-100%), and p,p'-DDE (75-90.91%). Species differences in the concentrations of HCB, beta-HCH, heptachlor epoxide, and DDT-related chemicals in follicular fluid were noted as well as differences in the concentrations of some pollutants within the same species.

Effect of alpha-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa.

The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe.

The effect of melatonin implants on blood testosterone and acrosin activity in spermatozoa of the ram.

In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non-breeding season). Total acrosin activity in spermatozoa was increased between days 35-56 (autumn) and days 49-70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non-breeding season (after oestrus induction).

Circadian variations of plasminogen activator activity, tissue-type plasminogen activator antigen, plasminogen activator inhibition and plasmin inhibition in rat aorta, heart and brain are influenced by endotoxin or aspirin or endotoxin plus aspirin.

The effect of aspirin or endotoxin or aspirin plus endotoxin on the circadian variations of plasminogen activator activity (PAA), tissue-type plasminogen activator (t-PA) antigen level, plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in aorta, heart and brain of the rat was studied. In aorta the circadian variations of PAA, t-PA antigen and tissue-type PAI (t-PAI) were blunted by endotoxin, while the circadian variation of PI was reversed; t-PAI variation was blunted by aspirin plus endotoxin, while PI variation was reversed. Various disturbances of the circadian variations of PAA, t-PA antigen, PAI or PI in heart and brain were also observed. In conclusion, the circadian variations of fibrinolytic parameters in key organs are influenced by aspirin or endotoxin or aspirin plus endotoxin in a varying way or degree depending on the treatment, the parameter studied or the organ.

Sex-related differences of plasminogen activator activity and plasminogen activator inhibition in the gastric wall after chronic ethanol consumption: effect of orchidectomy or ovariectomy.

Plasminogen activator activity (PAA), tissue-type PA (t-PA) antigen level, plasminogen activator inhibition (PAI), and plasmin inhibition (PI) showed no sex-dependent differences in the stomach of the rat. Chronic ethanol consumption induced an increase of PAA and t-PA antigen and a decrease of PAI (urokinase-type PA [u-PAI]) and PI in the body and pyloric region (PI only in the pyloric region). PAA increase and u-PAI decrease in the pyloric region were greater in the male than in the female. Orchidectomy, but not ovariectomy, induced effects similar to those of ethanol. The effects of orchidectomy and chronic ethanol consumption on gastric PAA, t-PA antigen, and PAI were additive. An increased PAA and decreased PAI or PI in the stomach in cases of hypogonadism, particularly if this deficiency is combined with chronic ethanol consumption, might influence the susceptibility to pathological conditions related to or affected by the local fibrinolytic activity (PAA, PAI, or PI).

Circadian variation of plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition in aorta, heart, brain and lungs of the rat. Sex-related differences.

Plasminogen activator activity (PAA), t-PA antigen level, plasminogen activator inhibition (PAI) and plasmin inhibition (PI) showed a circadian variation in rat aorta, heart, brain, and lungs of both sexes, but in a different way depending on the organ and the fibrinolytic parameter studied and the sex as well. In general, the lowest values of PAA and t-PA antigen were found in the early morning and the highest values in late afternoon, while PAI or PI showed the opposite pattern (with the exception of lungs). In kidneys and liver the fibrinolytic parameters studied showed no circadian variation. Sex-related differences, mostly quantitative, were noted in aorta, heart, brain and kidneys, with lower PAA and t-PA antigen and higher PAI or PI in the male than in the female.

Breed and seasonal variation of plasminogen activator activity and plasminogen activator inhibition in spermatozoa and seminal plasma of the ram in correlation with testosterone in the blood.

Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI), against t-PA (t-PAI) or u-PA (u-PAI), in spermatozoa and seminal plasma as well as testosterone in the blood of Friesland, Chios, and Karagouniki rams all showed a seasonal variation with the highest values during the corresponding breeding season of the ewes (Autumn-Winter). The seasonal variation of PAA and PAI in spermatozoa or seminal plasma as well as blood testosterone was different among the three breeds studied. Increased spermatozoal PAA was observed in November and May in Friesland rams, in October and November in Chios rams, and in October in Karagouniki rams. Spermatozoal t-PAI was increased in December and June in Friesland rams, in November and December in Chios rams, and in November in Karagouniki rams. Spermatozoal u-PAI was increased in December in Friesland rams, in October and December in Chios rams, and in November and December in Karagouniki rams. Plasminogen activator activity and PAI in seminal plasma also showed similar seasonal variations. Plasminogen activator activity and PAI in spermatozoa and seminal plasma showed a positive correlation with blood testosterone. The results of the present study support our previous findings on the possible role of spermatozoal PAA and PAI in the fertilizing ability of spermatozoa.

Plasminogen activator: the identification of an additional proteinase at the outer acrosomal membrane of human and boar spermatozoa.

We have recently shown that spermatozoa of various species contain both types of plasminogen activator, the tissue-type (t-PA) and the urokinase-type (u-PA). In the present study, the localization of t-PA and u-PA in plasma membrane and outer acrosomal membrane of human and boar spermatozoa has been investigated. The identification of the type of the plasminogen activator (t-PA or u-PA) was made immunologically. In human spermatozoa, the outer acrosomal membrane and plasma membrane contained both types of plasminogen activator (t-PA and u-PA); in addition, t-PA antigen was measured. In boar spermatozoa, the outer acrosomal membrane contained only t-PA, whereas plasma membrane contained both types of plasminogen activator (t-PA and u-PA). Plasminogen activator inhibition (PAI) has also been demonstrated in plasma and outer acrosomal membranes of both species and identified as PAI-1 in membranes of human spermatozoa.

Variable response of tissue parameters of the fibrinolytic system to unilateral or bilateral adrenalectomy and unilateral or bilateral adrenal demedullation.

The effect of unilateral and bilateral adrenalectomy, unilateral and bilateral demedullation or unilateral adrenalectomy/unilateral demedullation on tissue plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) was studied in the rat. All the above treatments induced an increase of PAA in lungs. Increased PAA was also noted in brain after bilateral adrenalectomy or demedullation, as well as unilateral adrenalectomy/unilateral demedullation. In kidneys the PAA was decreased after bilateral adrenalectomy or demedullation and unilateral adrenalectomy/unilateral demedullation. In aorta only after bilateral adrenalectomy a decreased PAA was induced. In heart and liver no changes in PAA were noted. A decreased PAI expressed against tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) was found in lungs of all treated rats compared to controls. However, an increased PAI was noted in heart, liver and aorta against t-PA or u-PA after bilateral adrenalectomy and in liver after bilateral demedullation as well as after unilateral adrenalectomy/unilateral demedullation (rats with only one adrenal cortex). A dissociation between anti-t-PA and anti-u-PA response was seen in heart, liver and aorta. A variable change in PI was induced in kidneys, aorta, lungs and brain in all treatments except unilateral demedullation or unilateral adrenalectomy. Therefore, the effect of adrenalectomy or demedullation on tissue PAA, PAI and PI was variable and dependent on the extent of the treatment (unilateral or bilateral), the organ as well as the time after the treatment.

Increased plasminogen activator activity and plasminogen activator inhibition in spermatozoa and seminal plasma of the ram after serum gonadotrophin (PMSG) administration. Correlation with the increased level of testosterone in the blood.

After a single injection of serum gonadotrophin (PMSG) at the dose of 15 IU/kg, i.m. into rams testosterone in the plasma of blood showed a significant rise between 4th and 7th day post-injection. At the same time (4th-7th day) the plasminogen activator activity (PAA) in seminal plasma was found to be increased, but the plasminogen activator inhibition (PAI) expressed against t-PA (anti-t-PA) showed an increase between 32nd and 46th day. In spermatozoa a marked increase of PAA was revealed between 32nd and 46th day post-injection, while an increase of PAI (anti-t-PA) was exhibited on the 74th day. Plasmin inhibition (PI) in seminal plasma and spermatozoa showed no change compared to controls. A positive correlation has been found between increased concentrations of testosterone and PAA or PAI (anti-t-PA) in spermatozoa and seminal plasma. The induced increase of PAA in spermatozoa under the effect of testosterone might be of physiological importance, since PAA is localized to sperm membranes and might participate in the whole process of fertilization.

Sex-related differences in plasminogen activator activity and plasminogen activator inhibition of human and animal kidneys: effect of orchidectomy or ovariectomy.

Plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) have been studied with spectrophotometric methods in extracts of human, bovine, ovine and rat kidneys of both sexes. In all species studied, renal PAA (cortex or medulla) was higher in females than in males. The PAA was also higher in the medulla than in the cortex in all species and both sexes. The PAA was due to both types of plasminogen activator; tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). In the human kidney (cortex or medulla) the measurement of t-PA antigen showed that t-PA is higher in females than in males; t-PA is also higher in the medulla than in the cortex in both sexes. The PAI showed the opposite pattern in all species studied; it was lower in females than in males. It was also lower in the medulla than in the cortex. PAI-1 was identified in the human kidney. Sex-related differences in renal PAA or PAI almost disappeared after bilateral orchidectomy in rats. PI showed no sex or regional differences in the species studied. Sex-related differences in renal PAA and PAI in man and various animal species might be of physiological or pathophysiological importance.

Enhancement of plasminogen activator activity in the gastric wall after chronic ethanol consumption.

In the normal stomach of male rats marked differences in plasminogen activator activity (PAA) and plasmin inhibition (PI), but not in plasminogen activator inhibition (PAI), were noted among cardiac area, body and pyloric region. Chronic ethanol consumption (for 15 or 30 days) at the concentration of 6% or 12% in the drinking water induced an increase in PAA in the pyloric region and the body of the stomach (the higher concentration after 15 days and both concentrations after 30 days). The response was time- and dose-dependent. At the cardiac area no change of PAA was noted. Ethanol at both concentrations induced after 30 days a decreased PAI in the pyloric region and the body of the stomach, which was expressed against u-PA, but not against t-PA. A decreased PI was noted at both concentrations of ethanol after 30 days only in the pyloric region. Therefore, changes in PAA, PAI and PI after chronic ethanol consumption were dependent on the concentration, the period of the consumption and the area along the gastric wall.

Variable effect of unilateral or bilateral ovariectomy performed in young or adult animals on tissue plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition.

Unilateral or bilateral ovariectomy was performed in young and adult rats. Mainly bilateral ovariectomy induced variable changes of plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in key organs (brain, lungs, heart, aorta and kidneys). The most remarkable changes were induced after bilateral ovariectomy performed in young animals and mostly after two and three months of ovariectomy. Therefore, the effect of ovariectomy on tissue PAA, PAI or PI was variable and dependent on the extent of the ovariectomy (unilateral or bilateral), the age of the animal at ovariectomy (young or adult), the time after ovariectomy, and the organ. An additional interesting finding was the dissociation in the response of tissue anti-t-PA and anti-u-PA activities to ovariectomy in some of the organs studied.

Transplacental effect of lead compounds on tissue plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition.

The transplacental effect of lead compounds (lead acetate and tetraethyl lead) on the tissue plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) was studied in the rat. The concentration of lead in organs of the newborn showed a great variation; the distribution of lead in the organs studied depended on the dose and the stage of gestation at injection. In each organ the concentration of lead was dose-dependent. In control specimens no lead could be detected. The tissue response of PAA, PAI and PI to the lead compounds also showed a great variation; however, there was no correlation between lead concentrations and PAA, PAI or PI responses. Changes of one or more of the parameters studied (PAA, PAI or PI) were noticed in lungs, liver, heart, brain and kidneys. The PAA was due to the tissue type plasminogen activator in all organs studied; in kidneys and lungs the urokinase type of plasminogen activator was also detected. Therefore, fetal tissue PAA, PAI and PI can be affected transplacentally by lead compounds.

The effect of experimental chronic hypertension on tissue plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition.

Chronic hypertension was induced in rats after partial nephrectomy. The systolic blood pressure was significantly elevated from the first week after nephrectomy to the end of the experimentation (8th week). Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) showed a tissue- and time-dependent pattern of changes in some key organs compared to controls (sham-operated rats). Two weeks after nephrectomy (one week after the induction of hypertension) the PAA was markedly increased in lungs, heart and aorta. In aorta the PAA continued to be enhanced until the end of the experimentation (the 8th week after nephrectomy), while in heart and lungs the PAA returned to the normal eight weeks after nephrectomy. In vena cava, brain and liver no change in PAA was noticed compared to controls. Tissue PAI was mostly increased or unchanged, while tissue plasmin inhibition (PI) was unchanged. The differential response of PAA and PAI was varying not only from one organ to another or in the same organ at a given time but also in the same organ throughout experimentation. In a number of nephrectomized rats, however, hypertension was not induced. In these rats similar changes in tissue PAA and PAI were noted compared to hypertensive nephrectomized rats. Therefore, all the changes in the parameters studied should be due to the partial nephrectomy itself. In conclusion, experimentally induced chronic hypertension had not any effect on tissue PAA, PAI and PI.

Demonstration of plasminogen activator activity in the intima and media of the normal human aorta and other large arteries: immunological identification of the plasminogen activator(s).

The plasminogen activator activity (PAA) in extracts of the intima, media, and adventitia of the normal human aorta and other large arteries (carotid artery, renal artery and iliac artery) was studied with a sensitive, quantitative spectrophotometric assay using plasminogen and the chromogenic plasmin substrate S-2251. All layers of the arteries showed PAA which was highest in the adventitia, lowest in the media, while in the intima (aorta) PAA was intermediate, but much closer to that of the media. Plasminogen activator inhibition (PAI) was at the same level in all layers of the arteries studied. Plasmin inhibition (PI) was higher in adventitia than in intima (aorta), while in media the PI was intermediate. The PAA was due to the tissue-type plasminogen activator (t-PA), but not to the urokinase-type (u-PA), as judged by addition of respective antibodies. The relatively low PAA found in the intima of large arteries is therefore due to a low plasminogen activator and not a high plasminogen activator inhibitor activity or plasmin inhibitor level.