Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma.

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.     

Arylsulfatases are present in seminal plasma of several domestic mammals.

Mammalian spermatozoa and seminal plasma both contain high levels of arylsulfatases (AS), enzymes that remove sulfate from sulfated glycoconjugates. In ejaculated semen of boars, 85% of AS was found in seminal plasma whereas only 13% was found in spermatozoa. A comparable distribution of AS between spermatozoa and seminal plasma was observed in other domestic mammals. The presence of AS in seminal plasma was not due to leakage from spermatozoa because sperm cells had intact acrosomes and plasma membranes after their separation from seminal plasma, and because 84% of the acrosomal marker enzyme hyaluronidase was retained in washed spermatozoa. Spermatozoa in boar semen diluted with Beltsville Thawing Solution (BTS) deteriorated faster during storage at 17 degrees C than spermatozoa stored in BTS without seminal plasma. This suggests that seminal plasma has a deleterious effect on mammalian spermatozoa. We propose that (1) sulfated glycoconjugates stabilize sperm plasma membranes; (2) AS present in seminal plasma contribute to the deterioration of spermatozoa by desulfating these glycoconjugates; and (3) AS present in seminal plasma could well play a role in sperm capacitation.     

Phospholipases A2 in the reproductive system of the bull

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.     

Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)

β-N-Acetylglucosaminidase (β-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that β-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of β-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for β-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of β-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of β-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that β-NAGase can play a significant role in the fertilization process in teleosteans.     

The role of free amino acids in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio

The present study investigated (1) the free amino acid (FAA) composition in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio, (2) enzyme systems involved in amino acid metabolism and (3) the effect of amino acids on sperm viability under in vitro storage conditions. In the seminal plasma of O. mykiss, the main FAAs were arginine, glutamic acid, isoleucine, leucine, methionine and proline, in spermatozoa cysteine, arginine and methionine. In the seminal plasma of C. carpio, the main FAAs were alanine, arginine, cysteine, glutamic acid, histidine, leucine, lysine, methionine and proline, in spermatozoa arginine, glutamic acid, histidine, leucine and lysine. When spermatozoa were incubated for 48 h together with the seminal plasma, the quantitative amino acid pattern changed in both species indicating their metabolism. In spermatozoa and seminal plasma of O. mykiss and C. carpio, the following enzymes were found to be related to amino acid metabolism: transaminases (specific for alanine, aspartate, isoleucine and leucine), decarboxylases (specific for valine and lysine), glutamate dehydrogenase and α‐keto acid dehydrogenases (substrates: 3‐methyl‐2‐oxovaleric acid and 4‐methyl‐2‐oxovalerate). These data demonstrate that amino acid catabolism by transamination, decarboxylation and oxidative deamination can occur in semen of the two species. Also activity of methionine sulphoxide reductase was detected, an enzyme which reduces methionine sulphoxide to methionine. This reaction plays an important role in antioxidant defence. To determine the effect of FAAs on the sperm viability, C. carpio and O. mykiss spermatozoa were incubated in sperm motility inhibiting saline solution containing different amino acids. Methionine had a positive effect on the sperm viability in both species. Taken together this result with the in vivo occurrence of methionine and of methionine reductase in semen, it can be assumed that this amino acid plays an important role in antioxidant defence. Also isoleucine in O. mykiss and leucine in C. carpio had a positive effect on sperm viability. As seminal plasma and spermatozoa of the two species exhibit enzyme activities to catabolize leucine and isoleucine, they might serve as additional energy resources especially during prolonged incubation and storage periods.     

Sperm biology and control of reproduction in sturgeon: (I) testicular development, sperm maturation and seminal plasma characteristics

Sturgeon (Chondrostei, Acipenseriformes) are threatened or endangered species due to overfishing and environmental degradation causing disruption of natural reproduction. Commercial sturgeon aquaculture and conservation program requires broodfish management as well as biogeographical and biological knowledge. Therefore, control of sturgeon reproduction in captivity can become as a valid tool in the field of sustainable development. The main objectives of the present review were to summarize, describe and synthesize available data about neuroendocrine control of testicular development, spermiation induction, seminal plasma characteristics and factors affecting sperm quality. In sturgeon, puberty usually occurs late in life and adult males do not spawn on an annual basis. Gonadal differentiation and spermatogonia proliferation occurs at 1?C2 and 2?C3?year-old, respectively. In spermatogenesis, environmental stimuli affect hypothalamus to release GnRH, which induce FSH release from pituitary stimulating testicular androgenesis, which is involved in spermatogonial proliferation and spermatogenesis. At spawning season, GnRH stimulates LH production from pituitary, regulating 17??,20??-dihydroxy-4-pregnen-3-one production in testis, which control sperm maturation. In captivity, hormonal treatment is essential to induce spermiation. Chemical and biochemical compounds of the seminal plasma are important to protect viability, motility and fertilizing capacity of spermatozoa. Several kinds of acrosomal enzymes have been identified in sturgeon seminal plasma; higher concentrations reported in the frozen/thawed than fresh sperm suggesting their origination from spermatozoa. Moreover, there are numerous factors that influence on sperm quality including temperature, methods for spermiation induction, stripping frequency and stress.     

Characterization of rabbit testis beta-galactosidase and arylsulfatase A: purification and localization in spermatozoa during the acrosome reaction.

Examination of the role of carbohydrates in specific recognition between spermatozoa and zona pellucida has focussed on understanding the interaction of sperm hydrolases or lectin-like molecules with zona pellucida ligands. To elucidate the role of specific spermatozoan hydrolases in gamete interaction, rabbit testis beta-galactosidase and arylsulfatase A were purified, characterized, and localized in spermatozoa. beta-Galactosidase and arylsulfatase A co-purified after affinity, size, or reverse-phase chromatography. N-Terminal amino acid analysis and enzymatic characterization suggested that neither enzyme is a testis-specific isozyme. Size chromatography indicated that both enzymes aggregated into macromolecular complexes at pH 4.0, while both dissociated at pH 8.0. beta-Galactosidase and arylsulfatase A co-localized on the sperm surface and in the acrosome and postacrosomal regions of spermatozoa. Throughout the zona-induced acrosome reaction, both enzymes remained associated with the detached acrosomal cap and postacrosomal region of acrosome-reacted spermatozoa. Because the acrosome is an acidic subcellular compartment, internal beta-galactosidase and arylsulfatase A are probably aggregated in acrosome-intact spermatozoa and dissociate as they are exposed to pH increases during the acrosome reaction.     

Morphology, chemical contents and physiology of chondrostean fish sperm: a comparative study between Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus)

The present study was carried out with two sturgeon species, Siberian sturgeon (Acipenser baerii) and sterlet (A. ruthenus) to compare spermatological parameters to better understand inter‐species differences. Significant differences between morphometric features were observed such as acrosome length, acrosome width, head length, midpiece width and flagellar length, while midpiece length did not reveal such differences. The sterlet has a shorter spermatozoon than the Siberian sturgeon. Ultrastructural parameters vary significantly in terms of length of the nucleus, diameter of the endonuclear canals (EC), size of posterolateral projections (PP) and diameter of flagellum. Mean values for density of spermatozoa in the semen, seminal plasma pH, osmolality (mOsmol kg^?1), along with Ca²⁺, Na⁺, K⁺, Cl^? ions concentrations (mm ) were determined to be 0.61 ± 0.37 × 10⁹, 8.16 ± 0.18, 77.20 ± 52.28, 0.24 ± 0.06, 31.39 ± 10.21, 3.51 ± 1.10, 14.00 ± 4.30 in A. baerii and 0.41 ± 0.32 × 10⁹, 8.13 ± 0.19, 50.74 ± 6.27, 0.16 ± 0.11, 20.11 ± 3.78, 1.26 ± 0.54, 6.11 ± 0.60 in A. ruthenus, respectively. Significant differences were observed in Na⁺, K⁺ and Cl^? concentrations in the seminal plasma as well as in sperm velocity. The percentage of motile spermatozoa did not show any significant difference between the two species. Comparing the results of this study with published literature data on sturgeon spermatozoa reveals that morphological and ultrastructural parameters of spermatozoa together with some parameters of the seminal fluid and spermatozoa velocity can be used in comparative spermatology to better understand inter‐species differences. The observed biochemical and physiological differences should be also considered for the development of methods for controlled reproduction and for sperm cryopreservation techniques.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Purification and immunological characterization of a new form of gamma-glutamyltransferase of human semen.

A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.     

Arylsulfatase of sea urchin sperm--distribution of arylsulfatase in the gonads and gametes of echinoderms.

1. Fairly high activities of arylsulfatase are found in the sperm and mature testes of all the sea urchins studied; Strongylocentrotus intermedius, Strongylocentrotus nudus, Hemicentrotus pulcherrimus and Anthocidaris crassispina, whereas the activities in the ovaries and eggs of these animals are low. 2. Neither the sand dollar, Clypeaster japonicus nor the starfishes, Asterias amurensis and Asterina pectinifera prove to have considerable activities of the enzyme in their gonads and gametes. 3. Most of the activity of arylsulfatase in the sperm of S. intermedius is found in the seminal plasma, but the significant activity is bound to the spermatozoa. 4. Part, if not all, of the spermatozoa-borne arylsulfatase is suggested to exist on the surface of spermatozoa or in the acrosome or both. 5. The ubiquitous distribution of sperm arylsulfatase in sea urchins on the contrary to its absence in starfish or sand dollar is discussed in connection with the penetration of sperm through egg investments.     

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-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 alpha-glucosidase. The activity of neutral alpha-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 alpha-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 alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-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 alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-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.     

Isolation and preliminary characteristics of β-N-acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)

The aim of this study was to characterize the enzyme β- N -acetyglucosaminidase (β-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed β-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing β-NAGase activity were found in rainbow trout milt plasma. The molecular mass of β-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the β-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4–4.8 for milt plasma and sperm extract from rainbow trout. K _m value of the β-NAGases was 0.212, 0.563, 0.779 m m for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The β-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study

Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside.     

Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.     

Meiotic Gynogenesis in the Stellate and Russian Sturgeons and Sterlet

Diploid gynogenetic progenies were obtained in the stellate sturgeon Acipenser stellatus, Russian sturgeon, A. gueldenstaedtii, and sterlet A. ruthenus by means of insemination of the eggs with UV-irradiated spermatozoa and suppression of the second meiotic division by heat shock. The gynogenetic nature of experimental fish was confirmed by RAPD-PCR analysis of DNA. Effective photoreactivation of UV-induced lesions of spermatozoa was shown in the case of illumination of the fertilized eggs with visible light. This phenomenon should be taken into account when determining the doses of irradiation that allow inactivation of the male chromosomes and incubating gynogenetic embryos. Gynogenetic stellate and Russian sturgeons are viable and can be reared in order to study the mechanism of sex determination in sturgeons.     

Isolation and purification of the IGF-I protein complex from rabbit seminal plasma: Effects on sperm motility and viability

A protein of about 150 kDa affecting sperm kinetic motility and viability was purified from rabbit seminal plasma. The incubation of rabbit sperm with this purified seminal plasma protein caused significant changes in sperm viability and motility. Moreover, the seminal protein showed a noticeable reactivating effect on immotile spermatozoa. A 10-mg amount of purified protein, added to immotile rabbit spermatozoa suspended in Tris-citrate, pH 7.4, resulted in a 48% reactivation. It is known that circulating insulin-like growth factors are bound to specific high-affinity binding proteins and form complexes with relative molecular masses of about 150 kDa. Western blotting analyses proved the existence of insulin-like growth factor in the protein purified from rabbit seminal plasma and immunofluorescence staining showed the existence of IGF-1 receptor in rabbit spermatozoa. Therefore, we suggest that the purified rabbit seminal plasma protein may represent the protein complex delivering IGF to the sperm cells thus affecting their physiological functions.     

Motility of spermatozoa from shovelnose sturgeon and paddlefish

The spermatozoa in the seminal plasma from shovelnose sturgeon Scaphirhynchus platorynchus and paddlefish Polyodon spathula were immotile with only a few spontaneously motile spermatozoa for 5-10 and 10-20 s, respectively. Spermatozoa of shovelnose sturgeon were observed to be 100% motile immediately after sperm dilution in 10 m m NaCl and 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min; and 1 to 5% of spermatozoa remain active after 360 s (P<0.01). Spermatozoa of paddlefish demonstrated the best motility 10 s after dilution in 10 m m NaCl with 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min and 1 to 5% of spermatozoa remained active after 370 s ( p <0.01). The spermatozoa of shovelnose sturgeon and paddlefish were motile in a range of osmotic pressure from 0 to 100 mosmol kg⁻¹ and 0 to 120 mosmol kg⁻¹, respectively. The best results with short-term storage of sperm from shovelnose sturgeon and paddlefish were observed in 100 m m glucose + 20 m m Tris-HCl, pH 8.5 and 150 m m glucose + 20 m m Tris-HCl, pH 8.5.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.