Biol. Chem. Hoppe-Seyler Vol. 373, pp. 685-689, August 1992
Isolation and Partial Characterization of the Outer Dense Fiber Proteins from Human Spermatozoa Ralf HENKEL, Thomas STALF and Werner MISKA Andrologische Forschungsabteilung, Zentrum f r Dermatologie und Andrologie der Universit t Gie en, Germany
(Received 26 August 1991 / 11 May 1992)
Summary: Flagella of human spermatozoa were separated from the sperm head by sonication at 25 kHz and subsequent density gradient centrifugation in Percoll. For isolation of the outer dense fibers (ODF), the flagellar membrane and fibrous sheath were dissolved under reducing conditions in the cationic detergent cetyltrimethylammonium bromide (CTAB) for 30, 60 and 90 min, respectively.The isolation steps were monitored by phase-contrast micro-
scopy and electron microscopy. After SDS-PAGE and silver staining two protein bands at 55 and 67 kDa could be detected. An identification of these proteins as phosphoproteins, either with molybdate/methylgreen or rhodamine B, was not possible.The obtained results indicate that the ODF proteins might have more passive elastic than active function with respect to motility of spermatozoa.
Isolierung und partielle Charakterisierung der Outer-dense-fiber-Proteine aus menschlichen Spermatozoen Zusammenfassung: Flagella menschlicher Spermatozoen wurden mittels Ultraschall bei 25 kHz und anschlie ender Dichtegradientenzentrifugation in Percoll vom Kopf getrennt. Zur Isolation der outer dense fibers (ODF) wurden Zellmembran und fibr se H lle 30, 60 bzw. 90 min in dem kationischen Detergens Cetyltrimethylammoniumbromid (CTAB) unter reduzierenden Bedingungen inkubiert. Die Kontrolle der Isolationsschritte erfolgte licht- und elek-
tronenmikroskopisch. Nach SDS-PAGE und Silberf rbung zeigten sich zwei Proteinbanden bei 55 und 67 kDa. Eine Identifikation dieser Proteine als Phosphoproteine war weder mit Molybdat/Methylgr n noch mit Rhodamin B m glich. Dieser Befund l t auf eine mehr passiv-elastische, als auf eine aktive Funktion in Bezug zur Bewegungsf higkeit von Spermatozoen schlie en.
Key terms: Outer dense fiber proteins, spermatozoa, phosphoproteins, motility.
In the flagella of spermatozoa of many species, even in different phyla, accessory structures exist in addition to the contractile axoneme and other coverings. In insects these structures are crystalloid or tubular^L2\ in cephalopods and vertebrates they are filamentous. These filaments are termed outer dense fibers (ODF).
ODF are only located in the midpiece and the principal piece of the spermatozoal flagellum^3~5^.The question about their lengths in the human is a matter of controversy. Burgos et al.[3] describe their extension over the whole length of the flagellum. On the contrary, Holstein and Roosen-Runge[6] stated that gradually tapered off ODF take up about half of the
Abbreviations: /ΛυυικνιαιιυιίΑ: Cac, Cacodylate; CTAB, cetyltrimethylammonium bromide; DTT, dithiothreitol; ODF, outer dense fibers; PBS, phosphatebuffered saline; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate;TEM, transmission electron microscopy.
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686
R. Henkel.T. Stalf and W. Miska
principal piece. Haidl and Becker^ showed by means of the negative staining technique that in normal spermatozoa the ODFtake up more than 60% of the principal piece. Agreement exists that the ODF3 and 8 through their association with the longitudinal columns of the fibrous sheath are in any case shorter than the other ones^l In preliminary examinations it was shown that absence or impaired development of the ODF is one reason of structural flagellum disturbance'7'. In most cases such spermatozoa show only local motility without any substantial progressive motility. Most examinations of ODF were performed in animals. Till now the focal points of examination, particularly in human spermatozoa, were done for the morphological characterization of spermatozoa and their flagella and the clinical relevance of flagellar disturbances18-131. Only in rats[14] and bulls [15] the biochemical characterizations were already performed partially. The aim of this study was to isolate and to characterize the proteins of human ODF. Moreover, we expect information about functional aspects of the ODF in human spermatozoa and therefore a better understanding of the mentioned flagellar disturbances.
Material and Methods Spermatozoa from normal ejaculates (WHO criteria) of the andrological outpatient clinic were washed twice with lOOniM PBS, pH 7.4, 0.2mM phenylmethylsulfonyl fluoride (PMSF). For separation of flagella from sperm heads the specimens were sonicated twice at 25 kHz for 10 s with a microtip sonicator (Bandelin, Berlin, Germany) at 4 °C and separated by subsequent density gradient centrifugation in 80% Percoll for 30 min at 4°C and 4.000 x g.The interphase between Percoll and the upper layer of 0.9% NaCl contains the flagella and their fragments. Sonication was monitored by phase contrast microscopy. After washing in PBS-PMSFthe pellet was suspended in lOmMTris/ HC1, pH 8.0, 0.5% cetyltrimethylammonium bromide (CTAB), 0.2mM PMSF, and 2% dithiothreitol (DTT) and incubated at room temperature for 30, 60 and 90 min, respectively.The anionic detergent SDS (1%), diluted in the same buffer, was used alternatively. Transmission electron microscopy (TEM) was used for control of the isolation steps.The samples were fixed in O.lM sodium cacodylate/HCl, pH 7.4 (Na-Cac/HCl), 2.5% glutaraldehyd, 1% sucrose overnight, washed twice for 2 h in O.lM Na-Cac/HCl, postfixed in 1% OsO4 and embedded in epoxi resin according to Spurr'16l Ultrathin sections were counterstained with uranylacetate/lead citrate and analysed in a Phillips TEM 201 at 60 kV. Both isolated flagella and isolated ODF were washed twice in 0.9% NaCl, dissolved in 2% SDS, 0.5% DTTand were heat-denaturated for 5 min at 98°C in a water bath. After SDS electrophoresis using a continuous gel system with 15.39% T, 2.53% C the gels were stained either with silver for general protein detection according to Blum et al. [ 17'or prepared for specific detection of phosphoproteins in SDS gels. Phosphoprotein stain was carried out either according to Cutting and Roth'18' with molybdate/methylgreen or employing the method of Debruyne' 19 '. Phosvitin, a phosphoprotetn from chicken egg white, served as positive control.The apparent molecular masses were calculated by using the following standards: phos-
Vol. 373 (1992)
phorylase b: molecular mass 94 kDa; bovine serum albumine: 67 kDa; ovalbumine: 43 kDa; carbonate dehydratase: 30 kDa: soy bean trypsin inhibitor: 20.1 kDa: a-lactalbumine: 14.4 kDa.
Results Separation of flagella from the sperm head by sonication at 25 kHz and subsequent density gradient centrifugation in Percoll resulted in a high yield of flagella in the interphase. Besides the separation of flagella and sperm heads, the flagella were also cut into fragments. As shown in Fig. IB, the pellet contains sperm heads with and without midpieces and single intact spermatozoa. In the interphase flagella and their fragments were enriched in a high degree (Fig. 1C). After 30 min of incubation in CTAB/DTTthe regular structure of the flagella (here in the region of the mid piece) with membranes, fibrous sheath, ODF and axoneme (Fig. 2A) is completely disintegrated (Fig. 2B). However, remnants of membranes and fibrous sheath are still present. After 60 min of incubation in CTAB/DTT sheath structures and membranes are clearly more disintegrated than after 30 min. But remnants of the sheath structures are partially present (not shown). Complete dissolution of the fibrous sheath and the membranes is achieved after an incubation time of 90 min. Nevertheless, a partial lysis of the ODF could be seen (Fig. 2C). However, after 30 min of incubation in SDS/DTT ODF are extensively dissolved (not shown). SDS electrophoresis of isolated flagella shows a multitude of protein bands with molecular masses between 10 and 150 kDa (Fig. 3A).Two of these bands, with apparent molecular masses of 55 and 67 kDa, could be enriched after isolation of ODF (Fig. 3B, arrowheads). Identification of these polypeptides as phosphoproteins was not possible with molybdate/ methylgreen (Fig. 4B) and rhodamine B (Fig. 4D). As positive control served phosvitin (Fig. 4A, C).
Discussion The employment of the cationic detergent for isolation of ODF from human sperm tails yielded in better results than using the anionic detergent SDS[20]. Whereas CTAB disintegrated the membranes and fibrous sheath, the ODF were largely intact. On the contrary, SDS in the given concentration of 1%^ dissolved not only membranes and fibrous sheath, but also ODF in the relatively short time of 30 min. Therefore, by using SDS as detergent it is rather difficult to obtain the proteins of ODF in pure and high yield. We suggest to use CTAB instead of SDS.The re-
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Vol. 373 (1992)
1A
Outer Dense Fiber Proteins from Human Spermatozoa
t
687
2A
200 nm
Fig. 1. A) Photomicrograph of spermatozoa (phase contrast; 250 χ). Inset: 2-fold magnification of a cut of Fig. 1 A. B) Sperm heads after sonication and separation in 80% Percoll. Flagella are separated by ultra sound either after the sperm head (arrow heads) or after the mid piece (arrows), (phase contrast; 250 x). Inset: 2.3-fold magnification of a cut of Fig. IB. C) Flagella and their fragments after sonication of spermatozoa and separation in 80% Percoll. (phase contrast; 250 x). Inset: 2-fold magnification of a cut of Fig. 1C.
Fig. 2. A) Electronmicrograph of a flagellum in the region of the mid piece. (A: Axonem; M: Mitochondria; arrow: outer dense fibers). B) Electronmicrograph of isolated flagella after 30 min incubation in CTAB/DTT. Remnants of membranes and fibrous sheath are still present (arrow heads). ODF: Outer dense fibers. C) Electronmicrograph of isolated ODF after 90 min incubation in CTAB/DTT. A partial lysis of the fiber can be seen.
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688
R. Henkel,T- Stalf and W. Miska
MM(kDa)
3A
3B
30-
20.1 — 14.4-
Fig. 3. SDS-PAGE and silver staining, A) Isolated flagella. B) Isolated ODE Two protein bands at 67 and 55 kDa are enriched, respectively (arrow heads).
Fig. 4. SDS-PAGE and phosphoprotein staining with molybdate/methylgreen (A, B) and rhodamine B (C, D). A and C) Positive control with 5 /xg phosvitin. B and D) Phosphoprotein staining of isolated ODE ODE proteins are not stained for phosphoproteins with molybdate/ methylgreen and rhodamine B, respectively. Positions of the ODE proteins are marked with arrow heads.
Vol. 373 (1992)
latively slow dissolution of the ODF by CTAB is the main reason for the better isolation and higher yields of the ODF proteins as described by Oko[20]. After SDS electrophoresis we found two protein bands with molecular masses of 55 and 67 kDa, respectively. Polypeptides of similar molecular masses (54-55 kDa; 72-75 kDa) were also detected by Baccetti et al J21^ by using only ultrasound and subsequent density gradient centrifugation in sucrose for isolation of ODF. Furthermore, these authors detected two additional polypeptides at 31 kDa and 28 kDa, respectively, and several bands in the dye front, which might be due to degradation products of proteins with higher molecular masses or an unspecific marking of the dye front. In the examined species, human, rat and bull, Baccetti et al.[21' detected in each case corresponding bands in SDS electrophoresis. In the rat, the species that has most frequently been examined, different authors^14'20"2^ found varying numbers of proteins with molecular masses between 11 and 33 kDa, and between 55 and 87 kDa, respectively, by using different methods for isolation of ODF. Differing data are reported in the literature not only for one species, the rat, but even for different species, such as bull^15'21^, rat and human^. Partly, the number of the detected proteins as well as their molecular masses are differing considerably. One possible reason for these differences might be due to the various methods for isolation of ODF. Directly comparing examinations by employing only one method were merely performed by Baccetti et alJ21^. Supposing that the ODF, at least in mammals, are phylogenetically relative old proteins, no great differences among the molecular masses should appear. However, negligible variations in the molecular masses can occur obviously. Though in closely related species these differences are petty. One reason for the greater number of proteins described in ODF of rats by most of the authors may be established in the function of these structures in the flagellum. According to Baltz et alJ24^ there is a coherence among the length of the flagella and the thickness of the ODF. Species with long sperm tails, such as hamster, rat, or guinea pig, have clearly thicker ODF than the human. At transport of spermatozoa within the epididymides, especially at ejaculation, shear forces arise, by which flagella can be disturbed. In this context the ODF should increase the tensile strength of sperm and protect them as an element of stability from these shear forces[24]. Concerning an improved stability of the very thick ODF in rat or Chinese hamster, it would be possible that the ODF contain additional proteins of lower molecular mass, which may act as "linking pieces". Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 7:19 PM
Vol. 373 (1992)
Outer Dense Fiber Proteins from Human Spermatozoa
On the other side we detected only polypeptides of lower molecular mass at deficient processing of the samples, presuming proteins as products of degradation of higher molecular mass. Furthermore, through the separation of flagella from sperm heads by means of ultrasound prior to the incubation with detergent, DNA and proteins located in the sperm head are eliminated in a physical manner. Therefore, we obtained the ODF proteins in high purity. Early investigations postulate that ODF have ATPase activity and are involved in the realization of the movement of the flagellum[25·261. Later Baccetti et al.[27] suggested a passive elastic role of the ODF. Recently, Brito et al.[15] detected phosphoserine and phosphothreonin in the bull.This indicates a more active role of the ODF in sperm motility. In the human we could not identify proteins of ODF as phosphoproteins with both molybdate/methylgreen and rhodamine B, a method, which is 3 times more sensitive than the molybdate/methylgreen method. This may be due to either a very low grade of phosphorylation, so that the detectable phosphorus is below the limit of detection, or the ODF are not phosphorylated. Taking the employed amount of ODF into consideration we suppose that human ODF contain no phosphorus. Therefore, presumably human ODF have more passive elastic functions. Summarizing all the facts, including those from the literature, it can be stated that the main question upon the function of ODF in the human still remains unanswered. The excellent technical assistance of Nadja Mertens is greatfully acknowledged.
689
References 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Baccetti, B., Bigliardi, E., Burrini A.G., Gabbiani, G., Jockusch, B.M. & Leoncini, P. (1984) J. Submicrosc. Cytol. 16, 79-84. Afzelius, B.A. (1988) /. Ultrastrct. Molec. Struct. Res. 98, 94-102. Burgos. M.H., Vitale-Calpe, R. & Aoki, A. (1970) in The Testis, vol. /., Development, Anatomy and Physiology (Johnson, A.D., Gomes, V.R. &Vandemark, N.L., eds.) Academic Press, New York, London, pp. 551-649. Irons, M.J. & Clermont, Y. (1982) Anat. Rec. 202, 463471. Serres, C., Escalier, D. & David, G. (1983) Biol. Cell. 49, 153-162. Holstein, A.F. & Roosen-Runge, E.G. (1981) Atlas of Spermatagenesis. Grosse, Berlin. Haidl, G. & Becker, A. (1991) Hautarzt 42,242-246. Fawcett, D.W. (1975) Develop. Biol. 44, 396-436. Afzelius, B.A. & Eliasson, R. (1979) J. Ultrastruct. Res. 69,43-52. McClure, R.D., Brawer, J. & Robaire, B. (1983) Fertil. Steril. 40,395-399. Escalier, D. & David, G. (1984) Biol. Cell. 50, 37-52. Escalier, D. & Serres, C. (1985) Biol. Cell. 53, 239-259. Dadoune, J.P. (1988) Human Reprod. 3, 311-318. Vera, J.C., Brito, M., Zuvic, T. & Burzio, L. (1984) /. Biol Chem. 259, 5970-5977. Brito, M., Figueroa, J., Vera, J.C., Hott, R. & Burzio, L.O. (1986) Gamete Res. 15, 327-336. Spurr, A.R. (1969) J. Ultrastruct. Res. 26, 31 -43. Blum, H., Beier, H. & Gross, H.J. (1987) Electrophoresis 8,93-99. Cutting, J.A. & Roth,T.F. (1913)Anal. Biochem. 54,386394. Debruyne, I. (1983) Anal. Biochem. 133,110-115. Oko, R. (1988) Biol. Reprod. 39,169-182. Baccetti, B., Pallini, V. & Burrini, A.G. (1976) J. Ultrastruct. Res. 57, 289-308. Price, J.M. (1973) /. Cell. Biol. 59,272a Olson, G.E. & Sammons, D.W. (1980) Biol. Reprod. 22, 319-332. Baltz, J.M.,Williams, P.O. & Cone, R.A. (1990) Biol. Reprod. 43, 485-491. Nelson, L. (1958) Biochem. Biophys. Acta 27,634-641. Gordon, M. & Barrnett, R.J. (1967) Exp. Cell. Res. 48, 395-412. Baccetti, B., Pallini,V. & Burrini, A.G. (1973) /. Submicrosc. Cytol. 5,237-256.
Ralf Henkel*, Thomas Staff and Werner Miska, Andrologische Forschungsabteilung, Zentrum für Dermatologie und Andrologie am Klinikum der Justus-Liebig-Universität, Gaffkystr. 14, W-6300 Gießen, Germany. * To whom correspondence should be addressed.
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Isolation and Partial Characterization of the Outer Dense Fiber Proteins from Human Spermatozoa Ralf HENKEL, Thomas STALF and Werner MISKA Andrologische Forschungsabteilung, Zentrum f r Dermatologie und Andrologie der Universit t Gie en, Germany
(Received 26 August 1991 / 11 May 1992)
Summary: Flagella of human spermatozoa were separated from the sperm head by sonication at 25 kHz and subsequent density gradient centrifugation in Percoll. For isolation of the outer dense fibers (ODF), the flagellar membrane and fibrous sheath were dissolved under reducing conditions in the cationic detergent cetyltrimethylammonium bromide (CTAB) for 30, 60 and 90 min, respectively.The isolation steps were monitored by phase-contrast micro-
scopy and electron microscopy. After SDS-PAGE and silver staining two protein bands at 55 and 67 kDa could be detected. An identification of these proteins as phosphoproteins, either with molybdate/methylgreen or rhodamine B, was not possible.The obtained results indicate that the ODF proteins might have more passive elastic than active function with respect to motility of spermatozoa.
Isolierung und partielle Charakterisierung der Outer-dense-fiber-Proteine aus menschlichen Spermatozoen Zusammenfassung: Flagella menschlicher Spermatozoen wurden mittels Ultraschall bei 25 kHz und anschlie ender Dichtegradientenzentrifugation in Percoll vom Kopf getrennt. Zur Isolation der outer dense fibers (ODF) wurden Zellmembran und fibr se H lle 30, 60 bzw. 90 min in dem kationischen Detergens Cetyltrimethylammoniumbromid (CTAB) unter reduzierenden Bedingungen inkubiert. Die Kontrolle der Isolationsschritte erfolgte licht- und elek-
tronenmikroskopisch. Nach SDS-PAGE und Silberf rbung zeigten sich zwei Proteinbanden bei 55 und 67 kDa. Eine Identifikation dieser Proteine als Phosphoproteine war weder mit Molybdat/Methylgr n noch mit Rhodamin B m glich. Dieser Befund l t auf eine mehr passiv-elastische, als auf eine aktive Funktion in Bezug zur Bewegungsf higkeit von Spermatozoen schlie en.
Key terms: Outer dense fiber proteins, spermatozoa, phosphoproteins, motility.
In the flagella of spermatozoa of many species, even in different phyla, accessory structures exist in addition to the contractile axoneme and other coverings. In insects these structures are crystalloid or tubular^L2\ in cephalopods and vertebrates they are filamentous. These filaments are termed outer dense fibers (ODF).
ODF are only located in the midpiece and the principal piece of the spermatozoal flagellum^3~5^.The question about their lengths in the human is a matter of controversy. Burgos et al.[3] describe their extension over the whole length of the flagellum. On the contrary, Holstein and Roosen-Runge[6] stated that gradually tapered off ODF take up about half of the
Abbreviations: /ΛυυικνιαιιυιίΑ: Cac, Cacodylate; CTAB, cetyltrimethylammonium bromide; DTT, dithiothreitol; ODF, outer dense fibers; PBS, phosphatebuffered saline; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate;TEM, transmission electron microscopy.
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686
R. Henkel.T. Stalf and W. Miska
principal piece. Haidl and Becker^ showed by means of the negative staining technique that in normal spermatozoa the ODFtake up more than 60% of the principal piece. Agreement exists that the ODF3 and 8 through their association with the longitudinal columns of the fibrous sheath are in any case shorter than the other ones^l In preliminary examinations it was shown that absence or impaired development of the ODF is one reason of structural flagellum disturbance'7'. In most cases such spermatozoa show only local motility without any substantial progressive motility. Most examinations of ODF were performed in animals. Till now the focal points of examination, particularly in human spermatozoa, were done for the morphological characterization of spermatozoa and their flagella and the clinical relevance of flagellar disturbances18-131. Only in rats[14] and bulls [15] the biochemical characterizations were already performed partially. The aim of this study was to isolate and to characterize the proteins of human ODF. Moreover, we expect information about functional aspects of the ODF in human spermatozoa and therefore a better understanding of the mentioned flagellar disturbances.
Material and Methods Spermatozoa from normal ejaculates (WHO criteria) of the andrological outpatient clinic were washed twice with lOOniM PBS, pH 7.4, 0.2mM phenylmethylsulfonyl fluoride (PMSF). For separation of flagella from sperm heads the specimens were sonicated twice at 25 kHz for 10 s with a microtip sonicator (Bandelin, Berlin, Germany) at 4 °C and separated by subsequent density gradient centrifugation in 80% Percoll for 30 min at 4°C and 4.000 x g.The interphase between Percoll and the upper layer of 0.9% NaCl contains the flagella and their fragments. Sonication was monitored by phase contrast microscopy. After washing in PBS-PMSFthe pellet was suspended in lOmMTris/ HC1, pH 8.0, 0.5% cetyltrimethylammonium bromide (CTAB), 0.2mM PMSF, and 2% dithiothreitol (DTT) and incubated at room temperature for 30, 60 and 90 min, respectively.The anionic detergent SDS (1%), diluted in the same buffer, was used alternatively. Transmission electron microscopy (TEM) was used for control of the isolation steps.The samples were fixed in O.lM sodium cacodylate/HCl, pH 7.4 (Na-Cac/HCl), 2.5% glutaraldehyd, 1% sucrose overnight, washed twice for 2 h in O.lM Na-Cac/HCl, postfixed in 1% OsO4 and embedded in epoxi resin according to Spurr'16l Ultrathin sections were counterstained with uranylacetate/lead citrate and analysed in a Phillips TEM 201 at 60 kV. Both isolated flagella and isolated ODF were washed twice in 0.9% NaCl, dissolved in 2% SDS, 0.5% DTTand were heat-denaturated for 5 min at 98°C in a water bath. After SDS electrophoresis using a continuous gel system with 15.39% T, 2.53% C the gels were stained either with silver for general protein detection according to Blum et al. [ 17'or prepared for specific detection of phosphoproteins in SDS gels. Phosphoprotein stain was carried out either according to Cutting and Roth'18' with molybdate/methylgreen or employing the method of Debruyne' 19 '. Phosvitin, a phosphoprotetn from chicken egg white, served as positive control.The apparent molecular masses were calculated by using the following standards: phos-
Vol. 373 (1992)
phorylase b: molecular mass 94 kDa; bovine serum albumine: 67 kDa; ovalbumine: 43 kDa; carbonate dehydratase: 30 kDa: soy bean trypsin inhibitor: 20.1 kDa: a-lactalbumine: 14.4 kDa.
Results Separation of flagella from the sperm head by sonication at 25 kHz and subsequent density gradient centrifugation in Percoll resulted in a high yield of flagella in the interphase. Besides the separation of flagella and sperm heads, the flagella were also cut into fragments. As shown in Fig. IB, the pellet contains sperm heads with and without midpieces and single intact spermatozoa. In the interphase flagella and their fragments were enriched in a high degree (Fig. 1C). After 30 min of incubation in CTAB/DTTthe regular structure of the flagella (here in the region of the mid piece) with membranes, fibrous sheath, ODF and axoneme (Fig. 2A) is completely disintegrated (Fig. 2B). However, remnants of membranes and fibrous sheath are still present. After 60 min of incubation in CTAB/DTT sheath structures and membranes are clearly more disintegrated than after 30 min. But remnants of the sheath structures are partially present (not shown). Complete dissolution of the fibrous sheath and the membranes is achieved after an incubation time of 90 min. Nevertheless, a partial lysis of the ODF could be seen (Fig. 2C). However, after 30 min of incubation in SDS/DTT ODF are extensively dissolved (not shown). SDS electrophoresis of isolated flagella shows a multitude of protein bands with molecular masses between 10 and 150 kDa (Fig. 3A).Two of these bands, with apparent molecular masses of 55 and 67 kDa, could be enriched after isolation of ODF (Fig. 3B, arrowheads). Identification of these polypeptides as phosphoproteins was not possible with molybdate/ methylgreen (Fig. 4B) and rhodamine B (Fig. 4D). As positive control served phosvitin (Fig. 4A, C).
Discussion The employment of the cationic detergent for isolation of ODF from human sperm tails yielded in better results than using the anionic detergent SDS[20]. Whereas CTAB disintegrated the membranes and fibrous sheath, the ODF were largely intact. On the contrary, SDS in the given concentration of 1%^ dissolved not only membranes and fibrous sheath, but also ODF in the relatively short time of 30 min. Therefore, by using SDS as detergent it is rather difficult to obtain the proteins of ODF in pure and high yield. We suggest to use CTAB instead of SDS.The re-
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Vol. 373 (1992)
1A
Outer Dense Fiber Proteins from Human Spermatozoa
t
687
2A
200 nm
Fig. 1. A) Photomicrograph of spermatozoa (phase contrast; 250 χ). Inset: 2-fold magnification of a cut of Fig. 1 A. B) Sperm heads after sonication and separation in 80% Percoll. Flagella are separated by ultra sound either after the sperm head (arrow heads) or after the mid piece (arrows), (phase contrast; 250 x). Inset: 2.3-fold magnification of a cut of Fig. IB. C) Flagella and their fragments after sonication of spermatozoa and separation in 80% Percoll. (phase contrast; 250 x). Inset: 2-fold magnification of a cut of Fig. 1C.
Fig. 2. A) Electronmicrograph of a flagellum in the region of the mid piece. (A: Axonem; M: Mitochondria; arrow: outer dense fibers). B) Electronmicrograph of isolated flagella after 30 min incubation in CTAB/DTT. Remnants of membranes and fibrous sheath are still present (arrow heads). ODF: Outer dense fibers. C) Electronmicrograph of isolated ODF after 90 min incubation in CTAB/DTT. A partial lysis of the fiber can be seen.
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688
R. Henkel,T- Stalf and W. Miska
MM(kDa)
3A
3B
30-
20.1 — 14.4-
Fig. 3. SDS-PAGE and silver staining, A) Isolated flagella. B) Isolated ODE Two protein bands at 67 and 55 kDa are enriched, respectively (arrow heads).
Fig. 4. SDS-PAGE and phosphoprotein staining with molybdate/methylgreen (A, B) and rhodamine B (C, D). A and C) Positive control with 5 /xg phosvitin. B and D) Phosphoprotein staining of isolated ODE ODE proteins are not stained for phosphoproteins with molybdate/ methylgreen and rhodamine B, respectively. Positions of the ODE proteins are marked with arrow heads.
Vol. 373 (1992)
latively slow dissolution of the ODF by CTAB is the main reason for the better isolation and higher yields of the ODF proteins as described by Oko[20]. After SDS electrophoresis we found two protein bands with molecular masses of 55 and 67 kDa, respectively. Polypeptides of similar molecular masses (54-55 kDa; 72-75 kDa) were also detected by Baccetti et al J21^ by using only ultrasound and subsequent density gradient centrifugation in sucrose for isolation of ODF. Furthermore, these authors detected two additional polypeptides at 31 kDa and 28 kDa, respectively, and several bands in the dye front, which might be due to degradation products of proteins with higher molecular masses or an unspecific marking of the dye front. In the examined species, human, rat and bull, Baccetti et al.[21' detected in each case corresponding bands in SDS electrophoresis. In the rat, the species that has most frequently been examined, different authors^14'20"2^ found varying numbers of proteins with molecular masses between 11 and 33 kDa, and between 55 and 87 kDa, respectively, by using different methods for isolation of ODF. Differing data are reported in the literature not only for one species, the rat, but even for different species, such as bull^15'21^, rat and human^. Partly, the number of the detected proteins as well as their molecular masses are differing considerably. One possible reason for these differences might be due to the various methods for isolation of ODF. Directly comparing examinations by employing only one method were merely performed by Baccetti et alJ21^. Supposing that the ODF, at least in mammals, are phylogenetically relative old proteins, no great differences among the molecular masses should appear. However, negligible variations in the molecular masses can occur obviously. Though in closely related species these differences are petty. One reason for the greater number of proteins described in ODF of rats by most of the authors may be established in the function of these structures in the flagellum. According to Baltz et alJ24^ there is a coherence among the length of the flagella and the thickness of the ODF. Species with long sperm tails, such as hamster, rat, or guinea pig, have clearly thicker ODF than the human. At transport of spermatozoa within the epididymides, especially at ejaculation, shear forces arise, by which flagella can be disturbed. In this context the ODF should increase the tensile strength of sperm and protect them as an element of stability from these shear forces[24]. Concerning an improved stability of the very thick ODF in rat or Chinese hamster, it would be possible that the ODF contain additional proteins of lower molecular mass, which may act as "linking pieces". Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 7:19 PM
Vol. 373 (1992)
Outer Dense Fiber Proteins from Human Spermatozoa
On the other side we detected only polypeptides of lower molecular mass at deficient processing of the samples, presuming proteins as products of degradation of higher molecular mass. Furthermore, through the separation of flagella from sperm heads by means of ultrasound prior to the incubation with detergent, DNA and proteins located in the sperm head are eliminated in a physical manner. Therefore, we obtained the ODF proteins in high purity. Early investigations postulate that ODF have ATPase activity and are involved in the realization of the movement of the flagellum[25·261. Later Baccetti et al.[27] suggested a passive elastic role of the ODF. Recently, Brito et al.[15] detected phosphoserine and phosphothreonin in the bull.This indicates a more active role of the ODF in sperm motility. In the human we could not identify proteins of ODF as phosphoproteins with both molybdate/methylgreen and rhodamine B, a method, which is 3 times more sensitive than the molybdate/methylgreen method. This may be due to either a very low grade of phosphorylation, so that the detectable phosphorus is below the limit of detection, or the ODF are not phosphorylated. Taking the employed amount of ODF into consideration we suppose that human ODF contain no phosphorus. Therefore, presumably human ODF have more passive elastic functions. Summarizing all the facts, including those from the literature, it can be stated that the main question upon the function of ODF in the human still remains unanswered. The excellent technical assistance of Nadja Mertens is greatfully acknowledged.
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Baccetti, B., Bigliardi, E., Burrini A.G., Gabbiani, G., Jockusch, B.M. & Leoncini, P. (1984) J. Submicrosc. Cytol. 16, 79-84. Afzelius, B.A. (1988) /. Ultrastrct. Molec. Struct. Res. 98, 94-102. Burgos. M.H., Vitale-Calpe, R. & Aoki, A. (1970) in The Testis, vol. /., Development, Anatomy and Physiology (Johnson, A.D., Gomes, V.R. &Vandemark, N.L., eds.) Academic Press, New York, London, pp. 551-649. Irons, M.J. & Clermont, Y. (1982) Anat. Rec. 202, 463471. Serres, C., Escalier, D. & David, G. (1983) Biol. Cell. 49, 153-162. Holstein, A.F. & Roosen-Runge, E.G. (1981) Atlas of Spermatagenesis. Grosse, Berlin. Haidl, G. & Becker, A. (1991) Hautarzt 42,242-246. Fawcett, D.W. (1975) Develop. Biol. 44, 396-436. Afzelius, B.A. & Eliasson, R. (1979) J. Ultrastruct. Res. 69,43-52. McClure, R.D., Brawer, J. & Robaire, B. (1983) Fertil. Steril. 40,395-399. Escalier, D. & David, G. (1984) Biol. Cell. 50, 37-52. Escalier, D. & Serres, C. (1985) Biol. Cell. 53, 239-259. Dadoune, J.P. (1988) Human Reprod. 3, 311-318. Vera, J.C., Brito, M., Zuvic, T. & Burzio, L. (1984) /. Biol Chem. 259, 5970-5977. Brito, M., Figueroa, J., Vera, J.C., Hott, R. & Burzio, L.O. (1986) Gamete Res. 15, 327-336. Spurr, A.R. (1969) J. Ultrastruct. Res. 26, 31 -43. Blum, H., Beier, H. & Gross, H.J. (1987) Electrophoresis 8,93-99. Cutting, J.A. & Roth,T.F. (1913)Anal. Biochem. 54,386394. Debruyne, I. (1983) Anal. Biochem. 133,110-115. Oko, R. (1988) Biol. Reprod. 39,169-182. Baccetti, B., Pallini, V. & Burrini, A.G. (1976) J. Ultrastruct. Res. 57, 289-308. Price, J.M. (1973) /. Cell. Biol. 59,272a Olson, G.E. & Sammons, D.W. (1980) Biol. Reprod. 22, 319-332. Baltz, J.M.,Williams, P.O. & Cone, R.A. (1990) Biol. Reprod. 43, 485-491. Nelson, L. (1958) Biochem. Biophys. Acta 27,634-641. Gordon, M. & Barrnett, R.J. (1967) Exp. Cell. Res. 48, 395-412. Baccetti, B., Pallini,V. & Burrini, A.G. (1973) /. Submicrosc. Cytol. 5,237-256.
Ralf Henkel*, Thomas Staff and Werner Miska, Andrologische Forschungsabteilung, Zentrum für Dermatologie und Andrologie am Klinikum der Justus-Liebig-Universität, Gaffkystr. 14, W-6300 Gießen, Germany. * To whom correspondence should be addressed.
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