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  • Author: T. Vanha-Perttula x
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A. Jauhiainen and T. Vanha-Perttula

Summary. A synthetic substrate (p-nitrophenyl-α-D-glucopyranoside) was used to measure the acid and neutral α-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid αglucosidase. The activity of neutral α-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity.

After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid α-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid α-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid α-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid α-glucosidase was clearly different from that of the enzymes in seminal plasma.

The pH optimum of acid α-glucosidase ranged from 3·75 to 4·5 and that of the neutral enzyme from 6·5 to 7·0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.

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Saara Lampelo and T. Vanha-Perttula

Summary. Three activity peaks hydrolysing l-cystine-di-β-naphthylamide (CysNA) and two activities hydrolysing l-leucine-β-naphthylamide (LeuNA) were separated by gel filtration on Sepharose 6B from human placental tissue. The enzyme activities in the void volume and the solubilized enzyme activities with both substrates apparently are bound and free forms of the same enzymes (I) since detergent treatment caused a total disappearance of the activities in the void volume. The second distinct enzyme (II) was highly soluble and detected only with CysNA.

The particle-bound enzyme(s) had a pH optimum at 6·5 with CysNA and at about 7·5 with LeuNA. They were highly sensitive to EDTA, could be reactivated by Co2+ and Zn2+ and were more sensitive to Ni2+ and l-methionine than the soluble enzyme II. The former enzyme(s) tolerated thermal treatment better than the soluble enzyme II. The solubilized free enzyme(s) I had a molecular weight of about 309 000. The soluble enzyme II was resistant to EDTA. Its optimum was at pH 6·0 and an estimate of 76 000 for the molecular weight was obtained.

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A. Jauhiainen and T. Vanha-Perttula

Summary. The highest specific activity of β-N-acetylglucosaminidase (β-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate β-NAG activity.

The β-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8·0–4·0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high β-NAG activity in bull seminal plasma. The major secretory forms of β-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the β-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all β-NAG isoenzymes was observed at pH 5·0. They were almost totally inactivated at 60°C and about 75–80% of the activity was lost at 55°C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents.

Histochemical studies showed a strong granular (lysosomal) reaction for β-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a β-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.

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Saara Lampelo and T. Vanha-Perttula

Summary. Cystine aminopeptidase and arylamidase activities in human serum were determined by enzymic hydrolysis of l-cystine-di-β-naphthylamide (CysNA) and l-leucine-β-naphthylamide (LeuNA), respectively. The activities of both enzymes increased during pregnancy, cystine aminopeptidase 12·5-fold and arylamidase 8·3-fold.

Serum CysNA and LeuNA hydrolysing aminopeptidases were separated by gel filtration on Sepharose 6B. Serum from non-pregnant women (control) contained arylamidase (Ic), which hydrolysed LeuNA and (weakly) CysNA, and cystine aminopeptidase II, hydrolysing only CysNA. During pregnancy a new enzyme appeared in maternal serum and showed cystine aminopeptidase and arylamidase activity (Im). Maternal serum Enzyme(s) I had higher pH optima (6·5 with CysNA; 7·5 with LeuNA) and higher molecular weights (309 000) than arylamidase Ic (pH optima at 5·52–5·5 with CysNA and 7·0 with LeuNA; mol.wt ~130 000). Arylamidase Ic was more sensitive to l-methionine, but more resistant to heat than maternal serum Enzyme(s) I. Both control and maternal serum Enzyme(s) I were inhibited by EDTA, but were re-activated by Zn2+ and Co2+ with LeuNA and CysNA as substrates and by Ni2+ with CysNA. Cystine aminopeptidase Im and arylamidase Im may be a single enzyme although differences were obtained in pH optima and reactivation by Ni2+ after EDTA treatment. Since maternal serum Enzyme(s) I had biochemical characteristics similar to those of placental aminopeptidase(s) I, it is suggested that the activities are of placental origin. Cystine aminopeptidase II appeared in all sera. It differed clearly from both maternal and control serum Enzyme(s) I: it had the lowest molecular weight (76 000), a different pH optimum (6·0), and was resistant to EDTA and l-methionine. It was not as effectively inhibited by Ni2+ as was Enzyme(s) I.

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Y. Agrawal and T. Vanha-Perttula

Summary. Particles found in bovine seminal vesicle secretion were enriched by centrifugation. They varied in size and morphology and contained Mg2+,Ca2+-activated ATPase, aminopeptidase A, alanyl aminopeptidase, γ-glutamyl transpeptidase and dipeptidyl peptidase IV activities. Hyperactivation of sperm motility and the acrosome reaction were induced by these particles in epididymal spermatozoa suspended in a modified Ringer medium. The hyperactivation, analysed with a microscopic slide test, started within minutes of exposure to membrane particles and continued for 3–4 h, during which time spermatozoa underwent the acrosome reaction. Acrosome staining, phase-contrast microscopy and transmission electron microscopy revealed that the acrosome reaction started within 60 min at 37°C and affected up to 80% of spermatozoa in 4 h. These membrane particles differed from those reported previously in other species in enzyme composition, function and organ of origin.

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M. Kantola, M. Saaranen and T. Vanha-Perttula

Summary. High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7·0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.

Keywords: selenium; glutathione peroxidase; seminal plasma; human; bull

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Y. P. Agrawal, T. Peura and T. Vanha-Perttula

Summary. γ-Glutamyl transpeptidase (γ-GT), its substrate (GSH) and hydrolytic product (l-glutamic acid) were measured biochemically in mouse reproductive tissues. The epididymal caput and seminal vesicles showed the highest specific activities of γ-GT, while GSH and l-glutamic acid were widely distributed in all tissues. Histochemically, γ-GT displayed a strong apical and supranuclear reaction and a moderate basal activity in the ductuli efferentes, a weak luminal reaction in the first, a moderate apical reaction in the second and a strong apical and supranuclear reaction in the third segment of the epididymal caput. In the epididymal corpus and cauda, the γ-GT reaction was confined to the tubular lumina but an apical reaction was also present in the cauda. The daily administration of acivicin (12 mg/kg body weight), an irreversible inhibitor of γ-GT, for 14 days resulted in a 60% suppression of the enzyme activity in the epididymal caput, while the γ-GT inhibition in the kidney was >95%. The treatment caused no change in the activity of alanyl aminopeptidase. Histochemically, the basal and supranuclear γ-GT activities in the ductuli efferentes and the third epididymal segment were suppressed, but the apical reactions were maintained. The in-vivo suppression of epididymal γ-GT activity may have implications in the control of post-testicular sperm maturation.

Keywords: γ-glutamyl transpeptidase; acivicin; mouse; epididymis