- Email: [email protected]
Transfer of aminopeptidase activity from prostasomes to sperm
Biochimica et Biophysica Acta 1336 Ž1997. 269–274
Transfer of aminopeptidase activity from prostasomes to sperm Giuseppe Arienti b
a,)
, Enrico Carlini a , Rosaria Verdacchi a , Carlo A. Palmerini
b
a Istituto di Biochimica e Chimica Medica, Via del Giochetto, 06126 Perugia, Italy Dipartimento di Biologia Cellulare e Molecolare, Via del Giochetto, 06126 Perugia, Italy
Received 25 November 1996; revised 18 February 1997; accepted 27 February 1997
Abstract Prostasomes are membranous vesicles Ž150–200 nm diameter. present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca2q. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. It has been demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at slightly acidic pH. In this paper, we show that an aminopeptidase activity is transferred from prostasome to sperm. This may be of particular interest since it indicates the involvement of protein in the process of fusion and because sperm may acquire new membrane-bound proteins by this procedure. q 1997 Elsevier Science B.V. Keywords: Aminopeptidase; Prostasome; Sperm; Membrane
1. Introduction Prostasomes are membranous vesicles secreted by the prostate gland w1x. Their lipid composition is peculiar; cholesterol is present in high amounts as is sphingomyelin, whereas phosphatidylcholine is less abundant w2x. Prostasomes are also rich in Ca2q, GDP, ADP and ATP w3,4x, and many proteins of their surface possess catalytic activity w3x or are involved in the immune response w5x. We would cite, among their physiological roles, the enhancement of sperm motility w6x, the liquefaction of semen w7x and immunosuppression w8–10x. A number of membrane-bound enzymes have been Abbreviations: R 18 , octadecyl rhodamine B chloride; Thesit, dodecylpolyŽethyleneglycolether. 9 ) Corresponding author. Fax: Žq39. 75-5853424. E-mail: [email protected]
described in prostasomes. Among these, Mg 2q and Ca2q-dependent ATPases w1,6x, protein kinase w11x, g-glutamyltransferase w12x, phospholipase A 2 , lactate dehydrogenase w13x and angiotensin converting enzyme w14x. Laurel et al. w15x reported an hydrolytic activity on the synthetic substrate succinylŽ alanine. 3paranitroanilide ŽSucŽAla. 3 pNA.. o-Phenantroline is an inhibitor of this enzyme but the activity can be restored following the addition of Zn2q. The reported pH optimum is 7.8, too high for the enzyme to be active in prostatic fluid. Yet, after mixing with other secretions, the pH of the seminal fluid is high enough for the full activity of the enzyme. It has been hypothesised that its physiological role could be connected to the fluidification of seminal fluid, since o-phenantroline inhibits the liquefaction of the gel w7x. It has been reported in a previous paper w16x that prostasomes ‘interact’ with sperm and we have found
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G. Arienti et al.r Biochimica et Biophysica Acta 1336 (1997) 269–274
that they can fuse to sperm, as demonstrated by the transfer of a lipophilic fluorescent dye Žoctadecyl Rhodamine B. w17x. We undertook this study with the aim of ascertaining whether the aminopeptidase activity described by Laurell et al. w15x can be transferred to sperm Ž that are devoid of this activity. . This may have two main implications: Ža. it would be a proof that not only lipid, but also protein can be transferred from prostasomes to sperm by the pH-dependent fusion mechanism and Žb. the acquisition of an enzymic activity from other membranes may give new properties to sperm and take a part in the processes that lie between the emission of semen and the fecundation of the ovum.
2. Materials and methods 2.1. Materials Hepes, Thesit ŽdodecylpolyŽethyleneglycolether. 9 . and MES were produced by Boehringer-Biochemie ŽMannheim, Germany. . Sephadex G-50 and Sephadex G-200 were obtained from Pharmacia Fine Chemicals ŽUppsala, Sweden., octadecyl rhodamine B chloride ŽR 18 . was purchased from Molecular Probes Ž Eugene, OR, USA . . N-succinyl-ala-ala-ala-p-nitroanilide ŽSucŽAla. 3 pNA., p-nitroaniline, benzamidine hydrochloride and iodoacetamide were produced by Sigma Chemical Co. ŽSt. Louis, MO, USA. . Other reagents, all of reagent grade or better, were obtained from Carlo Erba Ž Milan, Italy. , unless stated otherwise. 2.2. Semen samples and sperm preparation Fresh human semen was obtained from donors and was left 30–40 min at room temperature. We used normospermic samples w18x. Samples were centrifuged Ž800 = g for 10 min. to harvest sperm. The supernatant Ž S 1 . was used to prepare prostasomes. The pellet Ž P1 . was suspended in 30 mmolP ly1 Tris q 130 mmol P ly1 NaCl Žadjusted to pH 7.6 with HCl.. Sperm were purified by layering on 70% Percoll ŽSigma Chemical Co., St. Louis, MO, USA. and centrifuging at 600 = g for 30 min. The procedure was repeated two times and the final pellet was suspended in 30 mmolP ly1 Tris q
130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl.. This preparation was immediately used for further procedures, unless otherwise stated. 2.3. Preparation of prostasomes The supernatant ŽS 1 . was diluted Ž1 : 1, vrv. with 30 mmolP ly1 Tris q 130 mmol P ly1 NaCl Žadjusted to pH 7.6 with HCl. and was centrifuged at 1000 = g for 20 min to eliminate cell debris and residual spermatozoa. The new supernatant was then centrifuged at 105 000 = g for 120 min. The supernatant was discarded and the pellet containing prostasomes and amorphous material w19x was suspended in 30 mmol P ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. to have about 1–1.5 mg prot P mly1. Prostasomes were purified from amorphous material by chromatography on a Sephadex G-200 column Ž1.5 = 30 cm. preequilibrated with 30 mmolP ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. w3x. Prostasomes were not retained by the column and were collected with V0 . They were finally harvested by centrifugation at 105 000 = g for 120 min and suspended in the same buffer. 2.4. Characterisation of prostasomes The homogeneity of the prostasome preparation was checked by quasi-elastic light-scattering ŽQELS. using a NICOMP Model 370rVHPL submicron particle sizer with very-high-power laser Ž75-mW aircooled argon-ion 488 nm. . Particle size was about 150–200 nm. The determinations were made either at pH 5.0 or 7.5 to check that prostasomes did not aggregate at low pH. In addition, prostasomes were characterised by measuring their lipid content. The extraction of lipid from membranes was performed according to Folch et al. w20x. In some instances, chloroform extracts were used to determine the distribution of phosphorus among lipid classes. The chloroform phase was dried under a gentle stream of nitrogen and dissolved in known amounts of chloroform : methanol Ž2 : 1, vrv.. Phospholipids were separated by two dimensional thin-layer chromatography Ž6.5 = 6.5 cm, PE SIL G 250 m m, Whatman, Maidstone, UK. with: Ža. chloroform : methanol : 1.6 mol P ly1 ammonia Ž 70 : 30 : 5, vrvrv . and Ž b . chloroform : acetone : acetic acid : methanol : water
G. Arienti et al.r Biochimica et Biophysica Acta 1336 (1997) 269–274 Table 1 Lipid content of prostasomes Cholesterol a Total lipid phosphorus a Cholesterolrphospholipid ratio Phosphatidylethanolamine b Phosphatidylcholine b Sphingomyelin b Phosphatidylserine b Phosphatidylinositol b
0.8"0.1 0.4"0.1 2.00 15"4 12"3 53"12 14"3 6"3
a
Results expressed as m molPmgy1 protein"S.E.M. Results expressed as percent of total lipid phosphorus"S.E.M. The reported results are the average of three determinations.
b
Ž75 : 30 : 15 : 15 : 7.5, vrvrvrvrv.. Spots were visualised by exposure to I 2 vapours and identified with pure reference standards. After the sublimation of I 2 , spots were scraped off the plate and their phosphorus content determined w21x. The lipid composition of prostasomes is reported in Table 1. The reported data permitted to conclude that the material used by us was similar to that utilised by others w2,19x. 2.5. Insertion of R 18 into prostasomes The insertion of R 18 into prostasomes was performed as described by Hoekstra et al. w22x. The probe was dissolved in ethanol Ž 1 mg P mly1 . and 50 m l of this solution were added to 1 ml of vesicle suspension. The mixture was then kept in the dark for 1 h at room temperature. To eliminate non inserted R 18 , vesicles were chromatographed on a Sephadex G-50 column Ž0.5 = 25 cm. and eluted with 30 mmol P ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. Ž prostasomes. . The fluorescence of these preparations was stable for many hours. Surface density Žmol of probermol of lipid. was calculated comparing the fluorescence of suspensions in the presence of 0.03% Thesit to that of standard solutions of R 18 . About 50–60% of fluorophore was incorporated into vesicles and a surface density of 0.02 mol of probermol of lipid or less was obtained.
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cuvette containing 0.32 mol P ly1 sucroseq 20 mmol P ly1 MES or 2 mmolP ly1 Hepes at the required pH Ž1.85 ml. and vesicles loaded with the probe Ž 50 m l.. The fusion process was started by adding unloaded sperm Ž 100 m l. and was monitored following the increments of fluorescence at 580 nm Ž excitation 560 nm. using a Shimadzu RF5000 spectrophotofluorimeter. Slit widths were set at 5 nm for both excitation and emission. The calibration of the assay was performed taking as 100% the fluorescence produced after the addition of 1% Žf.c.. Thesit; indeed, fluorescence did not increase for further additions of the detergent. No increase of fluorescence was detected before the addition of unloaded vesicles. We investigated fluorescence self-quenching versus surface density and always used concentrations comprised in the proportionality range; for surface densities F 0.02 mol probermol lipid fluorescence quenching was linearly related to surface density. The extent of fusion Žtaking as 100% the complete intermixing of lipid phases. was calculated from the percent of fluorescence quenching relief using the following relationship w22x: F s P DŽ 1 q rrl ., were F s fusion, PD s percentage of fluorescence dequenching, r s amount of R 18-loaded lipid Žin mol. and l s amount of unloaded lipid Ž in mol.. 2.7. Determination of Suc(Ala)3 pNA hydrolysis The incubation mixture contained, unless otherwise stated, 4 mmolP ly1 SucŽAla. 3 pNA and 0.2 mol P ly1 Tris-HCl buffer ŽpH 7.5. in a final volume of 0.7 ml. The reaction was started by adding 30 m l of prostasome or sperm suspensions Ž about 0.03 mg protein. and continued for 60 min at 258C. The absorbance at 410 nm was monitored continuously with a Cary 3E spectrophotometer. Activity was usually expressed as m mol hydrolysed substrateP hy1 P mgy1 protein. 2.8. Analyses
2.6. Assay of fusion Fusion was tested by the relief of octadecylrhodamine fluorescence self-quenching w22x, that monitors lipid mixing. The assay was performed in a
Protein was determined as described w23x, cholesterol and phospholipid phosphorus assayed after digestion with 70% Žwrw. perchloric acid w21x and cholesterol as described by Rudel and Morris w24x.
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3. Results and discussion After leaving the testes, sperm have shut down plasma membrane protein and lipid synthesis; yet their plasma membrane changes dramatically as to lipid and protein composition. In a previous paper w17x, we have shown that prostasomes and sperm can fuse at slightly acidic pH and therefore the exchange of material between sperm and prostasomes may be identified as one of the mechanisms accounting for the variations in sperm plasma membrane composition. Arienti et al. w17x demonstrated the fusion between sperm and prostasomes using a lipophilic probe: octadecyl rhodamine B. These findings have shown that prostasome lipid mix with sperm lipid but did not prove any involvement of protein, that is present on prostasomes and may have a noticeable functional role because many prostasomal proteins possess catalytic activity andror regulate the immune response.
3.1. Characterisation of the aminopeptidase We tested the aminopeptidase activity of prostasomes using SucŽAla. 3 pNA as a substrate. Prostasomes were able to hydrolyse the substrate with a Vmax of 4.7 m mol P mgy1 prot P hy1 and a K M of 0.4 mM. This activity was not detectable in sperm or in the material unable to sediment at 105 000 = g. The addition of 1% Thesit to the incubation mixture was without effects either in prostasomes or in sperm, so excluding the possibility of latency.
Optimal pH was about 7.5 and the activity was stable after freezing and thawing, but was destroyed upon heating at 658C for 10 min. A number of substances Ž EDTA, up to 10 mmolP ly1, iodoacetamide 0.16–1.35 mmolP ly1, benzamidine 0.11–0.9 mmol P ly1 and DFP Ž diisopropylfluorophosphate. 0.11–0.90 mmol P ly1 . were without effect on the enzymic activity. On the other hand, the activity was inhibited by o-phenanthroline. The inhibition produced by 1 mM o-phenanthroline was reversed by the subsequent addition of 0.33 mmolP ly1 Zn2q ŽTable 2.. All together, these properties indicated that the enzyme was not a serine-protease and depended on Zn2q for activity and that this is the same activity described by Lilja and Laurel w7x. 3.2. Fusion of sperm to prostasomes The fusion of sperm to prostasomes was measured by the method of the relief of R 18 self-quenching. At pH above 7.0 no fusion was detectable by this method. Yet, it was possible to observe a relief of fluorescence self-quenching by lowering the pH of prostasome-sperm mixtures. The extent of fusion was calculated as reported previously w17x. From the extent of fusion and from the lipid composition of prostasomes, we could calculate the amount of transferred lipid as nmol P mgy1 of sperm protein ŽTable 3.. The amount of transferred lipid in 10 min depended on the ratio of donor Žprostasomes. to acceptor membranes Ž sperm. and was higher for high ratios.
Table 2 The effect of o-phenanthroline and Zn2q on aminopeptidase activity in human prostasomes o-Phenanthroline ŽmmolP ly1 .
Residual activity
1.0 0.5 0.1 0.05 0.01 0.001
0 37 87 84 91 90
a
a
Added Zn2q ŽmmolP ly1 . in the presence of 1 mmol P ly1 o-phenanthroline 1.32 0.66 0.33 0.16 0.08 0
Residual activity
0 12 76 48 8 0
Data are expressed as percent of control activity, which was 4.7 m mol hydrolysed substrateP hy1 P mgy1 protein.
a
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Table 3 Transfer of lipid and of aminopeptidase activity from prostasomes to sperm Prostasome to sperm protein ratio
a
2.5 2.0 1.6 0.8
Transferred aminopeptidase activity
b
0.80 0.69 0.67 0.36
Transferred lipid
c
375 360 346 192
a
Physiological value in human seminal fluid is 2.0 " 0.8 Ž5 determinations.. Data are expressed as the prostasomal activity Ž m mol P hy1 . found in sperm Ž1 mg protein. after fusion for 10 min at pH 5.0, at the indicated prostasome to sperm protein ratios. Non fused prostasomes were removed by washing. 1 mg protasome protein contains an activity of 4.7 m mol of hydrolysed substrateP h y 1. c Data are expressed as nmol of prostasomal lipid transferred to 1 mg of sperm protein. Calculations have been made from the values of the relief of R 18 fluorescence self-quenching. b
In this paper we measure the transfer of the aminopeptidase activity at different ratios. 3.3. Transfer of aminopeptidase Several aminopeptidases are present in the prostasomal membrane. However, the peptidase studied in this paper is absent from sperm. If sperm and prostasomes are mixed at pH 8.0 and then separated again by centrifugation, no aminopeptidase activity is transferred to sperm. However, if prostasome and sperm are mixed at pH 5.0, a substantial amount of aminopeptidase activity moves to sperm. The amount of transferred aminopeptidase activity is comparable with the percentage of fusion, as calculated by fluorescence relief of self-quenching of R 18 . The transferred activity maintained the properties of the prostasomal enzyme Že.g. sensitivity to inhibitors. . Therefore, the transfer of aminopeptidase gives further support to the idea that there is a fusion of sperm and prostasomes at acidic pH. The transfer of protein from prostasomes to other membranes ŽChinese hamster ovary cells and rat erythrocytes. has recently been described w25x; yet the observation has not been reported for sperm. Therefore, the process of fusion involves both the lipid and the protein moiety of the prostasome membrane. This also means that sperm may acquire new properties following fusion. We propose that this mechanism may be physiologically important for the maturation of sperm. Although this pH is very low Ž sperm does not survive a long time in these conditions., it was used to have an extensive fusion. On the other hand, fusion is still present at pH 6.0 w17x.
3.4. Concluding remarks In this work we demonstrate that the transfer of aminopeptidase activity from prostasome and sperm is possible at slightly acidic pH, but not at pH 8.0. This results parallels the one reported for R 18 relief of self-quenching, indicating that the fusion between prostasomes and sperm interests both the lipid and protein components of the membrane. This fact is relevant because it indicates the possibility that sperm are enriched in prostasomal protein when the pH decreases to values under 7, as it may be expected in the vaginal tract. This finding may offer a potential mechanism to explain some of the physiological roles of prostasomes, such as the enhancement of sperm motility w6x and immunosuppression w8–10x.
Acknowledgements Mr. Fernando Santi is thanked for skilful technical assistance. The Ministry for University and Scientific Research ŽMURST, Rome. and the National Research Council ŽCNR, Rome. are thanked for research grants.
References w1x G. Ronquist, Eur. J. Clin. Invest. 17 Ž1987. 241–246. w2x G. Arvidson, G. Ronquist, G. Wikander, A.C. Ojteg, Biochim. Biophys. Acta 984 Ž1989. 167–173. w3x R. Fabiani, Uppsala J. Med. Sci. 99 Ž1994. 73–112. w4x G. Ronquist, G. Frithz, Acta Eur. Fertil. 17 Ž1986. 273–276.
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w5x I.A. Rooney, J.P. Atkinson, E.S. Krul, G. Schonfeld, K. Polakoski, J.E. Saffitz, B.P. Morgan, J. Exp. Med. 177 Ž1993. 1049–1420. w6x B. Stegmayr, G. Ronquist, Scand. J. Urol. Nephrol. 16 Ž1982. 85–90. w7x H. Lilja, C.B. Laurell, Scand. J. Clin. Lab. Invest. 44 Ž1984. 447–452. w8x R.W. Kelly, P. Holland, G. Skibinski, C. Harrison, L. McMillan, T. Hargreave, K. Jam, Clin. Exp. Immunol. 86 Ž1991. 50–58. w9x G. Skibinski, R.W. Kelly, D. Harkiss, K. James, Am. J. Reprod. Immunol. 28 Ž1992. 97–103. w10x R.W. Kelly, Hum. Reprod. 10 Ž1995. 1686–1693. w11x B. Stegmayr, I. Brody, G. Ronquist, J. Ultrastruct. Res. 78 Ž1982. 206–214. w12x H. Lilja, H. Wieber, Scand. J. Clin. Lab. Invest. 43 Ž1983. 307–312. w13x I. Olsson, G. Ronquist, Arch. Androl. 24 Ž1990. 1–10. w14x F. Krassnigg, H. Niederhauser, T. Placzek, J. Frick, W.B. Schill, Adv. Exp. Med. Biol. 198 Ž1986. 477–485. w15x C.B. Laurell, H. Weiber, K. Ohlsson, G. Rasnevik, Clin. Chim. Acta 126 Ž1982. 161–170.
w16x G. Ronquist, B.O. Nilsson, S. Hjerten, ¨ Arch. Androl. 24 Ž1990. 147–157. w17x G. Arienti, E. Carlini, C.A. Palmerini, J. Membrane Biol. 155 Ž1997. 89–94. w18x World Health Organization, WHO Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interactions, 2 nd edn., Press Syndicate of the University of Cambridge, Cambridge, 1987, pp. 55–58. w19x G. Ronquist, I. Brody, Biochim. Biophys. Acta 822 Ž1985. 203–218. w20x J. Folch, M. Lees, G.H. Sloane-Stanley, J. Biol. Chem. 226 Ž1957. 497–509. w21x G.R. Bartlett, J. Biol. Chem. 234 Ž1959. 466–468. w22x D. Hoekstra, T. de Boer, K. Klappe, J. Wilschut, Biochemistry 23 Ž1984. 5675–5681. w23x M.A. Bradford, Anal. Biochem. 72 Ž1976. 248–254. w24x L.L. Rudel, M.D. Morris, J. Lipid Res. 14 Ž1973. 364–366. w25x I.A. Rooney, J.E. Heuser, J.P. Atkinson, J. Clin. Invest. 97 Ž1996. 1675–1686.
Transfer of aminopeptidase activity from prostasomes to sperm Giuseppe Arienti b
a,)
, Enrico Carlini a , Rosaria Verdacchi a , Carlo A. Palmerini
b
a Istituto di Biochimica e Chimica Medica, Via del Giochetto, 06126 Perugia, Italy Dipartimento di Biologia Cellulare e Molecolare, Via del Giochetto, 06126 Perugia, Italy
Received 25 November 1996; revised 18 February 1997; accepted 27 February 1997
Abstract Prostasomes are membranous vesicles Ž150–200 nm diameter. present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca2q. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. It has been demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at slightly acidic pH. In this paper, we show that an aminopeptidase activity is transferred from prostasome to sperm. This may be of particular interest since it indicates the involvement of protein in the process of fusion and because sperm may acquire new membrane-bound proteins by this procedure. q 1997 Elsevier Science B.V. Keywords: Aminopeptidase; Prostasome; Sperm; Membrane
1. Introduction Prostasomes are membranous vesicles secreted by the prostate gland w1x. Their lipid composition is peculiar; cholesterol is present in high amounts as is sphingomyelin, whereas phosphatidylcholine is less abundant w2x. Prostasomes are also rich in Ca2q, GDP, ADP and ATP w3,4x, and many proteins of their surface possess catalytic activity w3x or are involved in the immune response w5x. We would cite, among their physiological roles, the enhancement of sperm motility w6x, the liquefaction of semen w7x and immunosuppression w8–10x. A number of membrane-bound enzymes have been Abbreviations: R 18 , octadecyl rhodamine B chloride; Thesit, dodecylpolyŽethyleneglycolether. 9 ) Corresponding author. Fax: Žq39. 75-5853424. E-mail: [email protected]
described in prostasomes. Among these, Mg 2q and Ca2q-dependent ATPases w1,6x, protein kinase w11x, g-glutamyltransferase w12x, phospholipase A 2 , lactate dehydrogenase w13x and angiotensin converting enzyme w14x. Laurel et al. w15x reported an hydrolytic activity on the synthetic substrate succinylŽ alanine. 3paranitroanilide ŽSucŽAla. 3 pNA.. o-Phenantroline is an inhibitor of this enzyme but the activity can be restored following the addition of Zn2q. The reported pH optimum is 7.8, too high for the enzyme to be active in prostatic fluid. Yet, after mixing with other secretions, the pH of the seminal fluid is high enough for the full activity of the enzyme. It has been hypothesised that its physiological role could be connected to the fluidification of seminal fluid, since o-phenantroline inhibits the liquefaction of the gel w7x. It has been reported in a previous paper w16x that prostasomes ‘interact’ with sperm and we have found
0304-4165r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 0 3 6 - 6
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G. Arienti et al.r Biochimica et Biophysica Acta 1336 (1997) 269–274
that they can fuse to sperm, as demonstrated by the transfer of a lipophilic fluorescent dye Žoctadecyl Rhodamine B. w17x. We undertook this study with the aim of ascertaining whether the aminopeptidase activity described by Laurell et al. w15x can be transferred to sperm Ž that are devoid of this activity. . This may have two main implications: Ža. it would be a proof that not only lipid, but also protein can be transferred from prostasomes to sperm by the pH-dependent fusion mechanism and Žb. the acquisition of an enzymic activity from other membranes may give new properties to sperm and take a part in the processes that lie between the emission of semen and the fecundation of the ovum.
2. Materials and methods 2.1. Materials Hepes, Thesit ŽdodecylpolyŽethyleneglycolether. 9 . and MES were produced by Boehringer-Biochemie ŽMannheim, Germany. . Sephadex G-50 and Sephadex G-200 were obtained from Pharmacia Fine Chemicals ŽUppsala, Sweden., octadecyl rhodamine B chloride ŽR 18 . was purchased from Molecular Probes Ž Eugene, OR, USA . . N-succinyl-ala-ala-ala-p-nitroanilide ŽSucŽAla. 3 pNA., p-nitroaniline, benzamidine hydrochloride and iodoacetamide were produced by Sigma Chemical Co. ŽSt. Louis, MO, USA. . Other reagents, all of reagent grade or better, were obtained from Carlo Erba Ž Milan, Italy. , unless stated otherwise. 2.2. Semen samples and sperm preparation Fresh human semen was obtained from donors and was left 30–40 min at room temperature. We used normospermic samples w18x. Samples were centrifuged Ž800 = g for 10 min. to harvest sperm. The supernatant Ž S 1 . was used to prepare prostasomes. The pellet Ž P1 . was suspended in 30 mmolP ly1 Tris q 130 mmol P ly1 NaCl Žadjusted to pH 7.6 with HCl.. Sperm were purified by layering on 70% Percoll ŽSigma Chemical Co., St. Louis, MO, USA. and centrifuging at 600 = g for 30 min. The procedure was repeated two times and the final pellet was suspended in 30 mmolP ly1 Tris q
130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl.. This preparation was immediately used for further procedures, unless otherwise stated. 2.3. Preparation of prostasomes The supernatant ŽS 1 . was diluted Ž1 : 1, vrv. with 30 mmolP ly1 Tris q 130 mmol P ly1 NaCl Žadjusted to pH 7.6 with HCl. and was centrifuged at 1000 = g for 20 min to eliminate cell debris and residual spermatozoa. The new supernatant was then centrifuged at 105 000 = g for 120 min. The supernatant was discarded and the pellet containing prostasomes and amorphous material w19x was suspended in 30 mmol P ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. to have about 1–1.5 mg prot P mly1. Prostasomes were purified from amorphous material by chromatography on a Sephadex G-200 column Ž1.5 = 30 cm. preequilibrated with 30 mmolP ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. w3x. Prostasomes were not retained by the column and were collected with V0 . They were finally harvested by centrifugation at 105 000 = g for 120 min and suspended in the same buffer. 2.4. Characterisation of prostasomes The homogeneity of the prostasome preparation was checked by quasi-elastic light-scattering ŽQELS. using a NICOMP Model 370rVHPL submicron particle sizer with very-high-power laser Ž75-mW aircooled argon-ion 488 nm. . Particle size was about 150–200 nm. The determinations were made either at pH 5.0 or 7.5 to check that prostasomes did not aggregate at low pH. In addition, prostasomes were characterised by measuring their lipid content. The extraction of lipid from membranes was performed according to Folch et al. w20x. In some instances, chloroform extracts were used to determine the distribution of phosphorus among lipid classes. The chloroform phase was dried under a gentle stream of nitrogen and dissolved in known amounts of chloroform : methanol Ž2 : 1, vrv.. Phospholipids were separated by two dimensional thin-layer chromatography Ž6.5 = 6.5 cm, PE SIL G 250 m m, Whatman, Maidstone, UK. with: Ža. chloroform : methanol : 1.6 mol P ly1 ammonia Ž 70 : 30 : 5, vrvrv . and Ž b . chloroform : acetone : acetic acid : methanol : water
G. Arienti et al.r Biochimica et Biophysica Acta 1336 (1997) 269–274 Table 1 Lipid content of prostasomes Cholesterol a Total lipid phosphorus a Cholesterolrphospholipid ratio Phosphatidylethanolamine b Phosphatidylcholine b Sphingomyelin b Phosphatidylserine b Phosphatidylinositol b
0.8"0.1 0.4"0.1 2.00 15"4 12"3 53"12 14"3 6"3
a
Results expressed as m molPmgy1 protein"S.E.M. Results expressed as percent of total lipid phosphorus"S.E.M. The reported results are the average of three determinations.
b
Ž75 : 30 : 15 : 15 : 7.5, vrvrvrvrv.. Spots were visualised by exposure to I 2 vapours and identified with pure reference standards. After the sublimation of I 2 , spots were scraped off the plate and their phosphorus content determined w21x. The lipid composition of prostasomes is reported in Table 1. The reported data permitted to conclude that the material used by us was similar to that utilised by others w2,19x. 2.5. Insertion of R 18 into prostasomes The insertion of R 18 into prostasomes was performed as described by Hoekstra et al. w22x. The probe was dissolved in ethanol Ž 1 mg P mly1 . and 50 m l of this solution were added to 1 ml of vesicle suspension. The mixture was then kept in the dark for 1 h at room temperature. To eliminate non inserted R 18 , vesicles were chromatographed on a Sephadex G-50 column Ž0.5 = 25 cm. and eluted with 30 mmol P ly1 Tris q 130 mmolP ly1 NaCl Žadjusted to pH 7.6 with HCl. Ž prostasomes. . The fluorescence of these preparations was stable for many hours. Surface density Žmol of probermol of lipid. was calculated comparing the fluorescence of suspensions in the presence of 0.03% Thesit to that of standard solutions of R 18 . About 50–60% of fluorophore was incorporated into vesicles and a surface density of 0.02 mol of probermol of lipid or less was obtained.
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cuvette containing 0.32 mol P ly1 sucroseq 20 mmol P ly1 MES or 2 mmolP ly1 Hepes at the required pH Ž1.85 ml. and vesicles loaded with the probe Ž 50 m l.. The fusion process was started by adding unloaded sperm Ž 100 m l. and was monitored following the increments of fluorescence at 580 nm Ž excitation 560 nm. using a Shimadzu RF5000 spectrophotofluorimeter. Slit widths were set at 5 nm for both excitation and emission. The calibration of the assay was performed taking as 100% the fluorescence produced after the addition of 1% Žf.c.. Thesit; indeed, fluorescence did not increase for further additions of the detergent. No increase of fluorescence was detected before the addition of unloaded vesicles. We investigated fluorescence self-quenching versus surface density and always used concentrations comprised in the proportionality range; for surface densities F 0.02 mol probermol lipid fluorescence quenching was linearly related to surface density. The extent of fusion Žtaking as 100% the complete intermixing of lipid phases. was calculated from the percent of fluorescence quenching relief using the following relationship w22x: F s P DŽ 1 q rrl ., were F s fusion, PD s percentage of fluorescence dequenching, r s amount of R 18-loaded lipid Žin mol. and l s amount of unloaded lipid Ž in mol.. 2.7. Determination of Suc(Ala)3 pNA hydrolysis The incubation mixture contained, unless otherwise stated, 4 mmolP ly1 SucŽAla. 3 pNA and 0.2 mol P ly1 Tris-HCl buffer ŽpH 7.5. in a final volume of 0.7 ml. The reaction was started by adding 30 m l of prostasome or sperm suspensions Ž about 0.03 mg protein. and continued for 60 min at 258C. The absorbance at 410 nm was monitored continuously with a Cary 3E spectrophotometer. Activity was usually expressed as m mol hydrolysed substrateP hy1 P mgy1 protein. 2.8. Analyses
2.6. Assay of fusion Fusion was tested by the relief of octadecylrhodamine fluorescence self-quenching w22x, that monitors lipid mixing. The assay was performed in a
Protein was determined as described w23x, cholesterol and phospholipid phosphorus assayed after digestion with 70% Žwrw. perchloric acid w21x and cholesterol as described by Rudel and Morris w24x.
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3. Results and discussion After leaving the testes, sperm have shut down plasma membrane protein and lipid synthesis; yet their plasma membrane changes dramatically as to lipid and protein composition. In a previous paper w17x, we have shown that prostasomes and sperm can fuse at slightly acidic pH and therefore the exchange of material between sperm and prostasomes may be identified as one of the mechanisms accounting for the variations in sperm plasma membrane composition. Arienti et al. w17x demonstrated the fusion between sperm and prostasomes using a lipophilic probe: octadecyl rhodamine B. These findings have shown that prostasome lipid mix with sperm lipid but did not prove any involvement of protein, that is present on prostasomes and may have a noticeable functional role because many prostasomal proteins possess catalytic activity andror regulate the immune response.
3.1. Characterisation of the aminopeptidase We tested the aminopeptidase activity of prostasomes using SucŽAla. 3 pNA as a substrate. Prostasomes were able to hydrolyse the substrate with a Vmax of 4.7 m mol P mgy1 prot P hy1 and a K M of 0.4 mM. This activity was not detectable in sperm or in the material unable to sediment at 105 000 = g. The addition of 1% Thesit to the incubation mixture was without effects either in prostasomes or in sperm, so excluding the possibility of latency.
Optimal pH was about 7.5 and the activity was stable after freezing and thawing, but was destroyed upon heating at 658C for 10 min. A number of substances Ž EDTA, up to 10 mmolP ly1, iodoacetamide 0.16–1.35 mmolP ly1, benzamidine 0.11–0.9 mmol P ly1 and DFP Ž diisopropylfluorophosphate. 0.11–0.90 mmol P ly1 . were without effect on the enzymic activity. On the other hand, the activity was inhibited by o-phenanthroline. The inhibition produced by 1 mM o-phenanthroline was reversed by the subsequent addition of 0.33 mmolP ly1 Zn2q ŽTable 2.. All together, these properties indicated that the enzyme was not a serine-protease and depended on Zn2q for activity and that this is the same activity described by Lilja and Laurel w7x. 3.2. Fusion of sperm to prostasomes The fusion of sperm to prostasomes was measured by the method of the relief of R 18 self-quenching. At pH above 7.0 no fusion was detectable by this method. Yet, it was possible to observe a relief of fluorescence self-quenching by lowering the pH of prostasome-sperm mixtures. The extent of fusion was calculated as reported previously w17x. From the extent of fusion and from the lipid composition of prostasomes, we could calculate the amount of transferred lipid as nmol P mgy1 of sperm protein ŽTable 3.. The amount of transferred lipid in 10 min depended on the ratio of donor Žprostasomes. to acceptor membranes Ž sperm. and was higher for high ratios.
Table 2 The effect of o-phenanthroline and Zn2q on aminopeptidase activity in human prostasomes o-Phenanthroline ŽmmolP ly1 .
Residual activity
1.0 0.5 0.1 0.05 0.01 0.001
0 37 87 84 91 90
a
a
Added Zn2q ŽmmolP ly1 . in the presence of 1 mmol P ly1 o-phenanthroline 1.32 0.66 0.33 0.16 0.08 0
Residual activity
0 12 76 48 8 0
Data are expressed as percent of control activity, which was 4.7 m mol hydrolysed substrateP hy1 P mgy1 protein.
a
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Table 3 Transfer of lipid and of aminopeptidase activity from prostasomes to sperm Prostasome to sperm protein ratio
a
2.5 2.0 1.6 0.8
Transferred aminopeptidase activity
b
0.80 0.69 0.67 0.36
Transferred lipid
c
375 360 346 192
a
Physiological value in human seminal fluid is 2.0 " 0.8 Ž5 determinations.. Data are expressed as the prostasomal activity Ž m mol P hy1 . found in sperm Ž1 mg protein. after fusion for 10 min at pH 5.0, at the indicated prostasome to sperm protein ratios. Non fused prostasomes were removed by washing. 1 mg protasome protein contains an activity of 4.7 m mol of hydrolysed substrateP h y 1. c Data are expressed as nmol of prostasomal lipid transferred to 1 mg of sperm protein. Calculations have been made from the values of the relief of R 18 fluorescence self-quenching. b
In this paper we measure the transfer of the aminopeptidase activity at different ratios. 3.3. Transfer of aminopeptidase Several aminopeptidases are present in the prostasomal membrane. However, the peptidase studied in this paper is absent from sperm. If sperm and prostasomes are mixed at pH 8.0 and then separated again by centrifugation, no aminopeptidase activity is transferred to sperm. However, if prostasome and sperm are mixed at pH 5.0, a substantial amount of aminopeptidase activity moves to sperm. The amount of transferred aminopeptidase activity is comparable with the percentage of fusion, as calculated by fluorescence relief of self-quenching of R 18 . The transferred activity maintained the properties of the prostasomal enzyme Že.g. sensitivity to inhibitors. . Therefore, the transfer of aminopeptidase gives further support to the idea that there is a fusion of sperm and prostasomes at acidic pH. The transfer of protein from prostasomes to other membranes ŽChinese hamster ovary cells and rat erythrocytes. has recently been described w25x; yet the observation has not been reported for sperm. Therefore, the process of fusion involves both the lipid and the protein moiety of the prostasome membrane. This also means that sperm may acquire new properties following fusion. We propose that this mechanism may be physiologically important for the maturation of sperm. Although this pH is very low Ž sperm does not survive a long time in these conditions., it was used to have an extensive fusion. On the other hand, fusion is still present at pH 6.0 w17x.
3.4. Concluding remarks In this work we demonstrate that the transfer of aminopeptidase activity from prostasome and sperm is possible at slightly acidic pH, but not at pH 8.0. This results parallels the one reported for R 18 relief of self-quenching, indicating that the fusion between prostasomes and sperm interests both the lipid and protein components of the membrane. This fact is relevant because it indicates the possibility that sperm are enriched in prostasomal protein when the pH decreases to values under 7, as it may be expected in the vaginal tract. This finding may offer a potential mechanism to explain some of the physiological roles of prostasomes, such as the enhancement of sperm motility w6x and immunosuppression w8–10x.
Acknowledgements Mr. Fernando Santi is thanked for skilful technical assistance. The Ministry for University and Scientific Research ŽMURST, Rome. and the National Research Council ŽCNR, Rome. are thanked for research grants.
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