N. Kokolis

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).

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

Summary. 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 7 th 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.

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.

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

Summary. 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.

Effect of α-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.

Effect of chromosomal anomalies on the plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition in spermatozoa and seminal plasma 1. Chromosomal chimaerism XX/XY in the ram

Abstract In normal Friesland rams, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) in spermatozoa and seminal plasma, plasmin inhibition (PI) in seminal plasma and testosterone concentration in blood all showed seasonal variation. In a Friesland ram with chromosomal chimaerism XX/XY, however, although PAA, PAI and PI in seminal plasma and testosterone in the blood exhibited the same seasonal variation as in normal rams, in spermatozoa the PAA and PAI showed no seasonal variation. This is the first case in which a chromosomal anomaly has been linked with abnormal patterns in PAA and PAI.

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.

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.

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 μmol l−1 and 300 μmol l−1 of gossypol, respectively. In additional experiments, low, non‐spermicidal concentrations of gossypol (2.5–40 μmol 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.

The effect of dexamethasone on fibrinolytic parameters of ram lymphocytes

The aim of the study was to investigate the effect of dexamethasone on plasminogen activator activity (PAA), t-PA antigen level, plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram lymphocytes. The PAA of ram lymphocytes was due to t-PA (tissue-type plasminogen activator), but not to u-PA (urokinase-type plasminogen activator). Dexamethasone at the dose of 3 mg kg ˇ1 increased the PAA and t-PA antigen level of lymphocytes within 2 h after administration, while decreased the PAI 6‐14 h after administration; no change in PI was noted. When dexamethasone was administered at the dose of 0.75 mg kg ˇ1 every 4 h for 12 h, PAA and t-PA antigen level of lymphocytes showed an increase 2‐6 h after the end of the administration, while PAI and PI remained unchanged. These changes in PAA or PAI of lymphocytes, dependent on the dose or the pattern of dexamethasone administration, may be of importance regarding the role of lymphocytes and PAA or PAI in several pathophysiological conditions. # 1999 Elsevier Science B.V. All rights reserved.