Mol. Hum. Reprod. Advance Access originally published online on January 18, 2008
Molecular Human Reproduction 2008 14(3):151-156; doi:10.1093/molehr/gan003
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Physiological roles of semenogelin I and zinc in sperm motility and semen coagulation on ejaculation in humans
1Biomedical Engineering Center, Toin University of Yokohama, 1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan 2Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, 1024 Misaki-Koajiro, Miura, Kanagawa 238-0225, Japan 3Department of Urology, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan 4Division of Male Infertility, Center for Infertility and IVF, International University of Health and Welfare Hospital, 537-3 Iguchi, Nasushiobara, Tochigi 329-2763, Japan 5Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata 990-8560, Japan
6 Correspondence address. E-mail: yoshidak{at}cc.toin.ac.jp
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Abstract |
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At ejaculation, human sperm are considered to be mechanically trapped and become immotile in the semen coagulum by binding to semenogelins (Sgs) from the seminal vesicle and zinc ions from the prostate. However, the physiological combined roles of the protein and heavy metal on sperm motility are unknown. Here, we have first demonstrated that Sg I alone, which does not form the semen coagulum without zinc, is an inhibitor of the motility of intact human sperm at physiological concentration. On the other hand, zinc ions alone had no effect on sperm motility, but confer recovery of sperm motility that has been inhibited by Sg I at a concentration equal to or less than 1 mg/ml. These observations suggest that the roles played by Sg I and zinc on sperm motility are not mechanical but physiological. Quartz crystal microbalance analysis suggests that the sperm extract first bind to Sg I and then zinc ions which subsequently increase the protein accumulation, suggesting that Sgs inhibit sperm motility by directly binding to the sperm surface. Further accumulation of Sg I mediated by zinc ions may entrap the quiescent sperm at semen ejaculation.
Key words: semenogelin I/human sperm/motility/zinc
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Introduction |
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Semenogelin I (Sg I, a protein of molecular weight (MW) 52 kDa) and Sg II (existing as two forms of a Sg I-related protein with MWs of 71 and 76 kDa) are secreted from the seminal vesicle at ejaculation; they are the major structural components of coagulated human semen (Chaistitvanich and Boonsaeng, 1983; Lilja and Laurell, 1984, 1985). A coagulum is formed by the binding of Sgs with zinc ions from the prostate. In time, the coagulum is liquefied by hydrolysis of the Sgs into several fragments; this hydrolysis is mainly by the prostate-specific antigen (PSA) (Lilja et al., 1987).
Zinc ions are released from the prostate at ejaculation and are the other major component of mammalian seminal plasma, and their concentration in humans is 
2 mM (Arver, 1982; Arver and Sjoberg, 1982), i.e. zinc ion concentration is 100 times higher than that in blood plasma (Burtis and Ashwood, 1999). As described above, one of the major roles of zinc ions is the formation of the semen coagulum (Malm et al., 2007) when sperm is ejaculated into the female reproductive tract since these ions have a high binding affinity to Sgs (Jonsson et al., 2005). The coagulum appears to be an elastic substance, and thus, sperm are rendered immotile by the physical constraint of pressure exerted by the accumulated Sgs surrounding the sperm.
Another plausible role of zinc in human reproduction may occur after liquefaction of the coagulum, which results in the re-initiation of sperm motility. During liquefaction of the coagulum, it is known that a part of the zinc ions are freed from Sgs, and some of them bind with PSA to inhibit the hydrolysis activity when the degradation of coagulum has ceased (Malm et al., 2000). The direct effects of these free zinc ions on sperm motility have been controversial. Some reports argue that a high concentration of zinc is associated with enhanced sperm motility (Stankovic and Mikac-Devic, 1976; Caldamone et al., 1979), whereas others report that it suppresses progressive motility (Sorensen et al., 1999a).
With regard to the direct effects of Sgs on sperm motility, it has been reported that a fragment of Sgs termed the seminal plasma motility inhibitor (SPMI) (Iwamoto and Gagnon, 1988b) inhibits the motility of sperm whose plasma membrane has been removed by detergents. Further, the motility of intact sperm can be inhibited by a 1000-fold increase in the concentration of SPMI as compared to that in demembranated sperm (Iwamoto and Gagnon, 1988a). This suggests that Sgs as well as SPMI play a role in retracting injured sperm by immobilizing them. However, there is no direct evidence regarding how Sgs contribute to the motility of healthy sperm, and the fate of the zinc ions that bind to Sg fragments after liquefaction has been ignored. Nevertheless, although there are many evidences regarding the Sg and zinc interaction, the concomitant effect of Sgs and zinc on sperm motility has not been investigated.
Here, we demonstrate that Sg I at a concentration comparable to that in seminal plasma inhibits the motility of intact sperm from young healthy men. The motility is recovered upon the addition of zinc ions at Sg I concentrations equal to or less than 1 mg/ml suggesting that Sg I and zinc ions play physiologically important roles in the regulation of sperm motility during semen coagulum formation at ejaculation as well as during the liquefaction of the coagulum in the female reproductive tract.
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Preparation of sperm
Semen was collected from three human male volunteers aged between 18 and 22 years by masturbation in a sterile container after at least 48 h of abstinence. The semen samples were liquefied for 15–30 min at 37°C, following which the sperm parameters were assessed in accordance with the World Health Organization guidelines (WHO, 1999). Liquefied semen having spermatozoa that exhibited 90% motility was layered on a 99% Percoll solution containing 130 mM NaCl, 4 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2 and 14 mM fructose buffered with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 8.0) and then centrifuged at 1300xg for 30 min at 20°C. The sedimented spermatozoa were washed with Hank's solution (138 mM NaCl, 5.4 mM KCl, 0.35 mM Na2HPO4, 0.44 mM KH2PO4, 5.6 mM glucose, 0.48 mM NaHCO3, 0.52 mM MgCl2, 1.26 mM CaCl2 and 0.4 mM MgSO4) buffered with 20 mM HEPES (pH 8.0).
Informed consent was obtained from the volunteers before using their semen for research. This study was approved by the Ethical Committee/Institutional Review Board of St Marianna University School of Medicine.
Sperm motility analysis
The washed sperm were incubated at 37°C for 1 min in Hank's solution with or without Sg I in the presence or absence of 2 mM zinc chloride. Bovine serum albumin (BSA) or RNase was used as the control protein. The sperm suspension (5 µl) was introduced in a Makler counting chamber (depth = 10 µm) that was placed on a Nikon microscope (TE300; 20x, phase contrast optics) with a stage warmer (37°C) (MP-10DM; Kitazato Supply Co., Kitazato, Japan). Analyses of the motility parameters were performed using the C. IMAGING CASA system (Compix Inc., OR, USA); at least 200 cells were examined for each sample. The settings of the system were as follows: frames analysed, 15; framing rate, 30 s–1; pixel scale, 0.69 µm/pixel; cell size range, 2–40 µm; absolute maximum velocity, 800 µm/s; relative maximum velocity, 140 µm/s; moving debris filter for area, minimum = 10 µm2 and maximum = 150 µm2; moving debris filter for relative velocity, minimum = 140 diameter/s and maximum = 300 diameter/s; minimum points considered for linearity, 3; minimum points considered for amplitude of lateral head (ALH) displacement and beat cross frequency (BCF), 7; minimum velocity for ALH and BCF, 5 µm/s and maximum circulation radius, 10. The following parameters of motility were analysed: percentage of motile spermatozoa, straight-line velocity (straight-line distance), curvilinear velocity (total distance traveled divided by the total time for which the cell was tracked), linearity index (ratio of the straight-line distance to the actual tracked distance), ALH mean (deviation of the sperm head from the mean trajectory), ALH max (the maximum ALH displacement), BCF and average radius. Results were expressed as percentage of control without proteins or zinc ions. Data were analysed with a one-way ANOVA followed by post hoc Dunnet test or Tukey–kramer test if required using JMP 6.0.3 software (SAS Institute Inc., NC, USA). Statistical significance was defined as P < 0.05. Photos of the appearance of sperm suspension were prepared with an Olympus microscope and camera when the sperm motility was completely inhibited by 10 mg/ml Sg I.
Purification of Sg I
Sg I was purified from semen by using a modified method reported previously (Robert and Gagnon, 1996). Semen was treated with 8 M urea, and dithiothreitol (DTT) and iodoacetamide were sequentially added. The sample was centrifuged, and the supernatant was loaded onto a SP-Sepharose column (Amersham Biosciences, Tokyo, Japan) and eluted with a linear gradient of NaCl (0–400 mM). The Sg I content was assessed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and the fractions containing Sg I were pooled and loaded onto an HPLC column (Vydac C4; Separation Group, Hesperia, CA, USA). The fractions containing high-purity Sg I were combined, lyophilized and stored at –80°C until use. The protein concentration was measured by a bicinchoninic acid assay (Smith et al., 1985) using BSA as the standard.
Quartz crystal microbalance
The quartz crystal microbalance (QCM) is a sensitive mass-measuring device whose resonance frequency decreases linearly with the increase in mass on the QCM electrode at the nanogram scale (Okahata et al., 1999). In the present study, the device was used for detecting the molecule that binds to Sg I. The sperm extract was prepared according to a method described previously (Iwamoto and Gagnon, 1988a). Percoll-washed sperm were incubated in 10 times the volume of the demembranation solution (0.1% Triton X-100, 250 mM sucrose, 25 mM potassium glutamate and 1 mM DTT) for 5 min at 25°C and then centrifuged. An aliquot of the sperm extract supernatant was immobilized by hydrophobic interaction on the gold electrode surface of a 27-MHz QCM. The frequency of the electrode was then detected using a frequency counter equipped with a computer for recording (Affinix Q System; initium Inc., Tokyo, Japan) in the mixing chamber containing HEPES-buffered saline (HBS; 130 mM NaCl, 4 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 14 mM fructose, 10 mM HEPES, pH 8.0) at 25°C. After the frequency was stabilized, Sg I or RNase was added to the chamber, and the change in frequency in the presence or absence of 2 mM zinc chloride was recorded.
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Effects of Sg I on sperm motility
Percoll-washed spermatozoa that exhibited more than 70% motility were used for the experiments. All the parameters of sperm motility decreased with an increase in Sg I concentration except the BCF (Table I). The decreases were significant at Sg I concentrations of 10 mg/ml as compared to the untreated control. In particular, the motility, straight-line velocity, curvilinear velocity and ALH mean were significantly decreased at 5–10 mg/ml of Sg I (Table 1). This effect was not observed for the other proteins, i.e. BSA or RNase (data not shown).
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Effects of zinc and Sg I on sperm motility
No significant change was observed in all the parameters of sperm motility examined in the presence of 2 mM zinc ions alone (data not shown). However, zinc exhibited contradictory effects on the straight-line velocity and linearity depending on the concentration of Sg I, present along with zinc. Both parameters decreased in the presence of 0.5 and 1 mg/ml of Sg I as presented in Table I and in the open circles in Fig. 1B and D, although the decreases were not significant as compared to the untreated control. These values were increased with 2 mM zinc (Fig. 1B and D, closed circles). In particular, the straight-line velocity was significantly increased at 0.5–1 mg/ml of Sg I compared to the same-dose data without zinc (Fig. 1B, asterisks). No significant changes were detected with the other five parameters upon addition of 2 mM zinc as compared to the same-dose data without zinc.
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Like Sg I, RNase is a highly basic protein and is used as a control for evaluating the effects of Sg I and zinc ions on the parameters of sperm motility. In the presence of RNase and 2 mM zinc, all the parameters of sperm motility were not changed as compared to the untreated control (Fig. 1A–H, closed squares). These results suggest that the effect of zinc and Sg I on sperm motility is specific.
Agglutination of Sg I by zinc
Sperm motility was inhibited in the presence of 5–10 mg/ml of Sg I alone (Table I, Figs 1 and 2A). When 2 mM zinc ions was added to the sperm-Sg I suspension, aggregates of clot-like components containing trapped sperm appeared (Fig. 2B). These aggregates were not observed when Sg I and zinc were mixed without sperm (data not shown). These results suggest that Sg I physiologically inhibits sperm motility and that the immobilization of sperm in the aggregates is due to the physiological inhibition by Sg I as well as the physical trapping of sperm in the aggregates.
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QCM analyses demonstrated that sperm extract interacted with both the Sg I and RNase in the solution. The weight of both the proteins bound to the sperm extract increased following the addition of zinc ions (Fig. 3A and B), and the estimated molar amount of Sg I bound to the sperm extract was at least 3.2 times higher than that of RNase at the end of this experiment. Visible aggregates were observed upon the addition of over 30 µM (1.5 mg/ml) Sg I in the presence of 2 mM zinc ions (Fig. 3A). With regard to the Michaelis–Menten saturation kinetics between Sg I and sperm extracts, the Kd was 0.4 µM in the absence of zinc, and the maximum value of the amount of binding change (
mmax) was 2 µg/cm2. However, the binding did not follow Michaelis–Menten kinetics in the presence of zinc. When the amount of binding change (m) was subjected to Lineweaver–Burk plot analysis, the binding of Sg I revealed a non-linear plot (Fig. 3C). The increase in the amount of binding change was larger in the absence rather than presence of zinc at the initial injection of Sg I (Fig. 3A, inset). This suggests that Sg I binding to the sperm extract is suppressed by zinc ions when Sg I makes initial contact with the sperm extract, and directly binds to the spermatozoa. After the initial contact, the increase in binding amount became prominent in the presence of zinc, suggesting that zinc promotes reciprocal binding among Sgs. This may result in the formation of large Sg aggregates that have trapped spermatozoa that have been immobilized by the direct action of Sgs alone.
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Discussion |
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Sgs from the seminal vesicle and zinc ions from the prostate play a significant role in semen aggregation at the time of sperm ejaculation (de Lamirande, 2007; Malm et al., 2007). This aggregate is called a coagulum. The coagulum is believed to trap the sperm and immobilize them by the physical constraint of pressure (Lilja et al., 1989). Here, we show that the motility of intact human sperm is suppressed by 0.5–5 mg/ml Sg I alone, and 10 mg/ml Sg I completely inhibits sperm motility in the absence of zinc ions. The coagulum was not formed even in the presence of 10 mg/ml Sg I which is the concentration sufficient to suppress sperm motility. The coagulum was formed when both 10 mg/ml Sg I and 2 mM zinc were added to the sperm; these concentrations are comparable to those in seminal plasma (Arver, 1982). This observation appears to contradict the previous assumption that sperm immobilization at ejaculation is due to coagulum formation by the cooperative functioning of Sgs and zinc ions, and suggests the existence of the first step to immobilize sperm by Sgs alone during ejaculation. QCM analyses may provide an answer regarding this possibility. Here, we demonstrate that zinc ions reduce the interaction velocity of Sg I with the sperm extract but increase the total amount of bound Sg I, suggesting that the reaction of Sg I with sperm can be divided into two phases. The first phase obeys Michaelis–Menten saturation kinetics for the interaction between Sg I and the sperm surface, suggesting that the spermatozoa have a receptor molecule for Sg I with 1 binding site. It is possible that this phase is not interrupted by zinc ions because the present study showed that the addition of zinc ions reduced the binding velocity of Sg I with the sperm extract. The second phase involves the binding among Sg I molecules; zinc ions may participate in this binding because the addition of zinc ions increased the total amount of Sg I binding to the sperm surface. This kinetics suggests the following model that explains the effects of Sg I and zinc ions on the sperm surface: Sg I makes contact with the sperm surface and starts to immobilize them at ejaculation; subsequently, the accumulated Sg I is bridged by zinc ions and covers the sperm, resulting in coagulum formation.
We observed no significant effect of exogenous zinc ions alone on sperm motility, while intrinsic mitochondrial zinc ions were reported to play some role in human sperm motility (Sorensen et al., 1999b). On the other hand, our preliminary studies revealed that many peptides containing Sg fragments bound to zinc appeared during the liquefaction process (Fujinoki and Morisawa, unpublished work). Although the roles of these zinc-binding peptides on sperm motility are not clear, one of the Sg degradation peptides that bind to zinc could serve as a molecular shuttle so that the zinc can be transferred to the sperm nucleus. Moreover, in mammalian reproduction, sperm need to undergo a complex maturational process called capacitation. It is notable that both zinc (Huang et al., 2000) and Sg I (de Lamirande et al., 2001) function as decapacitation factors. However, their roles during fertilization in mammalian species are yet to be investigated.
Our results reveal that sperm are not immobilized merely by the physical constraint of pressure but by finely controlled molecular interactions. The molecular mechanisms underlying sperm motility and semen coagulum regulation by Sg I are still unclear, however some binding molecules of Sg I were recently demonstrated, e.g. Eppin (Wang et al., 2005) or CD52 (Flori et al., 2007). Both molecules are found to be associated with Sg-derived peptides and sperm in liquefied semen and believed to be important for human reproduction; the former may provide antimicrobial activity for sperm and the latter may provide a release of the sperm from coagulum. There is a possibility that the several complexes containing Sgs regulate the function of sperm. Sgs and zinc ions may play a key role in these complexes.
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This research was supported by the Japan Society for the Promotion of Science (JSPS) as a part of the Japan-Hungary Research Cooperative Program (2005).
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The authors are grateful to Ms Kaoru Takakura for technical assistance.
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Arver S. Zinc and zinc ligands in human seminal plasma. III. The principal low molecular weight zinc ligand in prostatic secretion and seminal plasma. Acta Physiol Scand (1982) 116:67–73.[Web of Science][Medline]
Arver S, Sjoberg HE. Calcium fractions in seminal plasma and functional properties of human spermatozoa. Acta Physiol Scand (1982) 116:159–165.[Web of Science][Medline]
Burtis CA, Ashwood ER. Tietz Textbook of Clinical Chemistry. (1999) WB Saunders Company.
Caldamone AA, Freytag MK, Cockett AT. Seminal zinc and male infertility. Urology (1979) 13:280–281.[CrossRef][Web of Science][Medline]
Chaistitvanich N, Boonsaeng V. Molecular structure of human seminal coagulum: the role of disulfide bonds. Andrologia (1983) 15:446–451.[Web of Science][Medline]
de Lamirande E. Semenogelin, the main protein of the human semen coagulum, regulates sperm function. Semin Thromb Hemost (2007) 33:60–68.[CrossRef][Web of Science][Medline]
de Lamirande E, Yoshida K, Yoshiike M, Iwamoto T, Gagnon C. Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process. J Andorol (2001) 22:672–679.
Flori F, Ermini L, La Sala GB, Nicoli A, Capone A, Focarelli R, Rosati F, Giovampaola CD. The GPI-anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen. Mol Reprod Dev (2007).
Huang YH, Chu ST, Chen YH. A seminal vesicle autoantigen of mouse is able to suppress sperm capacitation-related events stimulated by serum albumin. Biol Reprod (2000) 63:1562–1566.
Iwamoto T, Gagnon C. A human seminal plasma protein blocks the motility of human spermatozoa. J Urol (1988) a140:1045–1048.[Web of Science][Medline]
Iwamoto T, Gagnon C. Purification and characterization of a sperm motility inhibitor in human seminal plasma. J Androl (1988) b9:377–383.
Jonsson M, Linse S, Frohm B, Lundwall A, Malm J. Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen. Biochem J (2005) 387:447–453.[CrossRef][Web of Science][Medline]
Lilja H, Laurell CB. Liquefaction of coagulated human semen. Scand J Clin Lab Invest (1984) 44:447–452.[Web of Science][Medline]
Lilja H, Abrahamsson PA, Lundwall A. Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa. J Biol Chem (1989) 264:1894–1900.
Lilja H, Laurell CB. The predominant protein in human seminal coagulate. Scand J Clin Lab Invest (1985) 45:635–641.[Web of Science][Medline]
Lilja H, Oldbring J, Rannevik G, Laurell CB. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J Clin Invest (1987) 80:281–285.[Web of Science][Medline]
Malm J, Hellman J, Hogg P, Lilja H. Enzymatic action of prostate-specific antigen (PSA or hK3): substrate specificity and regulation by Zn(2+), a tight-binding inhibitor. Prostate (2000) 45:132–139.[CrossRef][Web of Science][Medline]
Malm J, Jonsson M, Frohm B, Linse S. Structural properties of semenogelin I. FEBS J (2007) 274:4503–4510.[CrossRef][Medline]
Okahata Y, Masunaga Y, Matsuno H, Furusawa H. Quantitative detection of a DNA ligase reaction on a quartz-crystal microbalance. Nucleic Acids Symp Ser (1999) 42:147–148.
Robert M, Gagnon C. Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin. Biol Reprod (1996) 55:813–821.[Abstract]
Smith P, Krohn R, Hermanson G, Mallia A, Gartnert F, Provenzano M, Fujimoto E, Goeke N, Olson B, Klenk D. Measurement of protein using bicinchoninic acid. Anal Biochem (1985) 150:76–85.[CrossRef][Web of Science][Medline]
Sorensen MB, Bergdahl IA, Hjollund NH, Bonde JP, Stoltenberg M, Ernst E. Zinc, magnesium and calcium in human seminal fluid: relations to other semen parameters and fertility. Mol Hum Reprod (1999) a5:331–337.
Sorensen MB, Stoltenberg M, Danscher G, Ernst E. Chelation of intracellular zinc ions affects human sperm cell motility. Mol Hum Reprod (1999) b5:338–341.
Stankovic H, Mikac-Devic D. Zinc and copper in human semen. Clin Chim Acta (1976) 70:123–126.[CrossRef][Web of Science][Medline]
Wang Z, Widgren EE, Sivashanmugam P, O'Rand MG, Richardson RT. Association of eppin with semenogelin on human spermatozoa. Biol Reprod (2005) 72:1064–1070.
WHO. WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction. (1999) 4th edn. Cambridge University Press.
Submitted on July 25, 2007; resubmitted on January 8, 2008; accepted on January 11, 2008.
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