Cornp. Biochem. Physiol. Vol. 81B, No. 2, pp. 519 522, 1985 0305-0491/85 $3.00+0.00 Printed in Great Britain ~C' 1985 Pergamon Press Ltd ACTIVITY OF ACROSIN INHIBITORS IN BULL SEMINAL PLASMA IN AN ANNUAL CYCLE JADWIGA TORSKA a n d JERZY STRZEZEK Chair of Animal Biochemistry, Agricultural-Technical Academy, 10-718 Olsztyn, Poland (Received 3 September 1984) Abstract--l. The spectrophotometric method was used to determine the activity of acrosin inhibitors in bull seminal plasma in particular months. 2. A highly significant increase of total inhibitor activity was noticed in spring and summer months, compared to the values recorded in autumn and winter. 3. Seasonal changes of the activity are shaped by both tow-molecule inhibitors (BUSI I, BUSI II), and high-molecule ones (BUSI 0). INTRODUCTION TWO glycopeptide inhibitors of proteinases (BUSI I and BUSI II) were isolated from bull seminal plasma, and their properties were determined (~echov~ and Fritz, 1976; Cechovdt et al., 1979a,b; Veselsk~ and ~echovfi, 1980; Zelezndt et al., 1980). These sub- stances are synthetized by epithelial cells of the male reproductive system. They form inactive bonds with acrosin and thus protect semen proteins against proteolytic effect of the enzyme liberated from dam- aged or dead spermatozoa (Fritz et al., 1975). More- over, it was suggested that the inhibitor BUSI I (which is able to inhibit activity of leucocyte pro- teinases, cathepsin G, and elastase) may play a significant role in preventing inflammation processes in the male reproductive system (Stanrikov~ et al., 1980; Veselsk~, et al., 1983). BUSI II is characterized by highly alkaline properties, and probably consti- tutes a component of the protein system stabilizing spermatozoa membranes (Cechova et al., 1979; Zel- eznh et al., 1980). Apart from the two mentioned inhibitors, bull seminal plasma contains also a trypsin acrosin in- hibitor, characterized by much higher mol. wt (~echovh and Fritz, 1976; Torska and Strze2ek, 1983). According to some authors, this inhibitor may originate from blood, and most probably its function in the seminal plasma is to aid the low-molecule inhibitors ((~echovfi and Fritz, 1976; Fritz et al., 1975). In view of significant role of these substances in regulation the acrosin activity, studies were under- taken aimed at determining the share of inhibitors from both groups (low- and high-molecule) in shap- ing the seasonal changes of inhibitor activity in the bull seminal plasma. MATERIALS AND METHODS Materials consisted of high quality bull semen (spermato- zoa mobility--above 70~, concentration--over 0.5 × 109/ cm3) collected in successive months from 20 bulls, re- productors at the Animal Insemination Station. Biochemical analyses were carried out on the seminal plasma obtained after double centrifugation of the samples at 4°C (gmigielska and Strze2ek, 1979). Total protein con- tent was determined by Biuret method (Weichselbaum, 1946), inhibitors were detected in electrophoretic plasma separation on acrylamide-agar gel (pH 8.2) (Uriel and Berges, 1968), antitrypsin activity of the inhibitors was determined by photometric method, using a VSU 2-P spectrophotometer produced by Carl Zeiss, Jena. N-a- benzoyl-L-arginine ethyl ester (Schwert and Takenaka, 1955; ~echov~ and Fritz, 1976) was used as a substrate. Inhibitor activity was measured in the plasma diluted 10-fold with a 2~ solution of acetic acid of pH2.7 and after 30min incubation of acid solutions at 37°C. The obtained data were used to calculate activity of high mol. wt inhibitor (BUSI 0), and low mol. wt ones (BUSI I and BUSI II), taking advantage of the differences in their thermostability (Torska and Strze~ek, 1983). The results are given in units per 1 cm3 of the plasma. One inhibitor unit (IU) represented an amount of the inhibitor necessary to reduce the activity of two trypsin units by 50~. RESULTS AND DISCUSSION Three bands of the inhibitor activity were discov- ered by electrophoresis in the seminal plasma of the reproductive bulls under study. Their mobility corre- sponded to inhibitors BUSI I, BUSI II and BUSI 0 (Fig. 1). The results of studies on inhibitor activity in the plasma confirmed our earlier observations that this activity was characterized by a reasonality (Strze2ek et al., 1981). As results from the data presented in Fig. 2, total activity of the acrosin inhibitors remained at a stable level in spring and summer, amounting to 32.58 _+ 13.53 and 32.36 _+ 13.06 IU/cm 3 on the aver- age. In autumn there was a significant decrease of the inhibitor activity (24.36 _+ 5.74 IU/cm3), followed by another drop in winter (19.74 + 6.15 IU/cm3). The same direction of changes was observed for both low- and high-molecular inhibitors. On the other hand, seasonal changes of total protein content in the plasma were quite different. The highest values were recorded in winter, the lowest--in spring. Analysis of the results obtained in particular months revealed that in May-June total inhibitor activity almost doubled compared to the values ob- 519