1 Biología de la Reproducción, 2 Instituto de Biología y Medicina Experimental and 3 Department of Biological Chemistry, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
4 To whom correspondence should be addressed. e-mail: lucrecalvo{at}fibertel.com.ar
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: fertilization/gamete biology/sperm
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mammalian species, including humans, sperm nuclear decondensation in vivo seems to involve two distinct stages: (i) reduction of disulphide bonds in protamines; and (ii) replacement of reduced protamines by histones (Zirkin et al., 1989). The role of reduced glutathione (GSH) present in the oocyte as a disulphide bond reducer has been clearly established (Perreault et al., 1988
, 1994), but the nature of the protamine acceptor is still unknown. In fish, amphibians and Drosophila, nucleoplasmin has been shown to be the protamine acceptor in vivo and thus responsible for sperm nuclear decondensation (Ohsumi and Katagiri, 1991
; Philpott et al., 1991
; Kawasaki et al., 1994
). However, with the exception of indirect evidence of the possible involvement of nucleoplasmin in mouse sperm decondensation (Maeda et al., 1998
), these findings have not been extended to mammals.
Human sperm can be decondensed in vitro in the presence of physiological concentrations of heparin and GSH (Gaubeca-Klix et al., 1998; Reyes et al., 1989
). The mechanism of action of heparin in this process is still a matter of controversy. The presence of heparin receptors on the sperm plasma membrane has been described by several groups (Delgado et al., 1982
; Lasalle and Testart, 1992
; Carell and Liu, 2002
), and Delgado and co-workers have proposed that heparin binding to its receptors leads to the destabilization of the sperm plasma membrane, which in turn would allow the incorporation of other molecules, such as GSH, into the sperm nucleus. Alternatively, a direct effect of heparin on sperm chromatin has been suggested since heparin has a strong affinity for protamines and can combine with them to form a highly insoluble complex (Chargaff and Olson, 1938
). Direct experimental evidence is lacking and why heparin is able to decondense human sperm in vitro is not clearly understood.
Heparin has also been proposed as a sperm-decondensing agent in vivo (Lalich et al., 1989; Montag et al., 1992
). However, this seems quite unlikely, since the only cell capable of synthesizing heparin in vivo is the mast cell, and no heparin has been found in the oocytecumulus complex. On the other hand, there is ample evidence in the literature related to the presence of other glycosaminoglycans (GAGs) in the cumulusoocyte complex in different species, including human (Ball et al., 1982
; Gebauer et al., 1978
; Salustri et al., 1989
). Among these, heparan sulphate (HS) seems to be the most likely candidate to be considered as a nuclear-decondensing agent in vivo, since it is a structural analogue of heparin and, in many biological systems, behaves in the same way (Delgado et al., 1982
; Jackson et al., 1991
).
The aim of this study was to evaluate whether the human sperm-decondensing ability of heparin in vitro is related to structural characteristics of the molecule and to test the hypothesis that HS could be a human sperm-decondensing agent in vivo.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All chemicals and reagents used were obtained from Sigma Chemical Co. (St Louis, MO), unless otherwise stated.
Samples were washed twice by centrifugation at 300 g for 10 min in human tubal fluid (HTF, Irvine Scientific, Santa Ana, CA) supplemented with 0.3% bovine serum albumin (BSA). Washed sperm were swam-up in HTF containing 2.6% BSA (HTF26B) for 90 min at 37°C in an atmosphere of 5% CO2 in air. Specimen concentration after swim-up was adjusted to 35 x 106/ml and sperm were incubated in capacitating conditions in HTF26B for 18 h at 37°C in 5% CO2/95% air.
Sperm nuclear decondensation assay
Capacitated sperm were decondensed in the presence of 10 mmol/l GSH and 46 µmol/l heparin (mol. wt 13 500 Da, 170 IU/mg) in HTF26B at 37°C for 15, 30 and 60 min (Romanato et al., 2001). Controls consisted of parallel incubations with heparin or GSH alone. After each time period, a 20 µl aliquot was removed and fixed with an equal volume of 2.5% glutaraldehyde in phosphate-buffered saline (PBS). Two 5 µl aliquots were transferred onto a microscope slide, a cover-slip was placed on top and nuclear status was assessed under phase contrast in an Olympus CH2 microscope at 400x. Sperm were classified (Bedford et al., 1973
) as unchanged (U), moderately decondensed (M) or grossly decondensed (G) (Figure 1). At least 200 sperm were evaluated in each aliquot. Total decondensation achieved, %(M + G), was determined as the sum of %M and %G.
|
Sulphation characteristics of heparin and decondensing ability
To evaluate the effect of sulphation characteristics of heparin on its nuclear-decondensing ability, capacitated sperm from the same semen sample were decondensed in the presence of 10 mmol/l GSH and 46 µmol/l heparin or each of the following chemically modified structures (Syntex S.A., Buenos Aires, Argentina): partially N-desulphated (N-des), partially O-desulphated (O-des), partially N-desulphated-N-acetylated (N-des-N-Ac) and partially O/N-desulphated-N-acetylated (ON-des-N-Ac).
Total decondensation in each sample was determined as usual, following 15, 30 and 60 min of incubation in decondensing conditions.
Heparin molecular weight and decondensing ability
To determine the effect of molecular size on heparin nuclear-decondensing ability, capacitated sperm from the same semen specimen were decondensed in the presence of 10 mmol/l GSH and 46 µmol/l of one of three different molecular weight heparins: 13.5, 18.0 and 3.0 kDa (Syntex S.A., Buenos Aires, Argentina). Total decondensation in each sample was determined as usual, following 15, 30 and 60 min of incubation in decondensing conditions.
Decondensing ability of different GAGs
Capacitated sperm from the same semen specimen were decondensed in the presence of 10 mmol/l GSH and 46 µmol/l heparin or each of the following GAGs: HS, chondroitin sulphate (CS), dermatan sulphate (DS) and hyaluronic acid (HA). Total decondensation in each sample was determined as usual, following 15, 30 and 60 min of incubation in decondensing conditions.
Statistical analysis
Statistical analysis was performed using Instat Mathpad.
The effect of heparin concentration, heparin sulphation and heparin molecular weight on total decondensation was determined by repeated-measures ANOVA followed by TukeyKramers multiple comparisons test. The decondensing ability of different GAGs was evaluated by one-way ANOVA followed by TukeyKramers multiple comparisons test. Differences were considered statistically significant when P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study presents evidence that the human sperm-decondensing ability of heparin in vitro is related to structural characteristics of the molecule rather than simply due to the fact that this molecule is a polyanion that can compete efficiently with DNA for positively charged protamines. Moreover, the results presented in this study suggest that HS, a structural analogue of heparin which can be found in the oocytecumulus complex (Bellin et al., 1986; Jackson et al., 1991
; Eriksen et al., 1997
), could be involved in in-vivo human sperm decondensation.
Upon optimizing assay conditions, it was surprising to see that maximum sperm decondensation achieved was 25% despite the fact that most sperm readily decondense following ICSI. This fraction of decondensing sperm may well correspond to those cells whose plasma membrane suffered alterations during overnight capacitation. There was a significant correlation (Spearman, P = 0.005, n = 14) between the percentage decondensed and the percentage of eosin Y-stained cells after overnight capacitation (data not shown). However, this does not affect the validity of the results presented herein since the aim of the present study was to understand the mechanism whereby heparin functions as a sperm-decondensing agent in vitro, with no implications on the use of the assay as a diagnostic tool.
There is ample evidence in the literature regarding the effect of structural modifications of the heparin molecule on its biological activity (Jackson et al., 1991; Bertolesi, 1998
). As previously stated in the Results, heparin desulphation affects both the net charge of the molecules disaccharidic unit and the localization of positively and negatively charged groups. Consequently, electrostatic interactions between charged groups in the molecule will be affected. This, in turn, will induce conformational changes in the molecule which may or may not be reflected on its biological activity. In our study, ON-des-N-Ac, which possesses the lowest negative charge, showed no decondensing ability, and might lead us to believe that decondensing ability is simply related to net negative charge. However, heparin, N-des-N-Ac and O-des behaved similarly despite the fact that their net negative charges are different. On the other hand, O-des and N-des have the same net charge but did not show the same decondensing ability. Thus, the position of negatively charged groups on the disaccharidic structure appears to be more important than net negative charge per se.
Interestingly, N-des was less active than O-des, in agreement with the general idea that N-sulphations are important for the biological activity of heparin (Lindahl and Kjellén, 1991; Bertolesi, 1998
). Furthermore, Bertolesi (1998
), studying a series of biological activities of the same chemically modified heparins, also found that ON-des-N-Ac was completely inactive and that the activity of N-des-N-ac was similar to that of heparin.
The biological activity of GAGs has been shown to be influenced by molecular size. Fedarko and Conrad (1986) have proposed that this might be due to the fact that certain specific fragments of the molecule could be incorporated into the cell and eventually reach the nucleus. In this study, we tested the decondensing ability of two additional heparins, with lower and higher molecular weight than control heparin, and observed no significant differences between them, suggesting that there is no effect of molecular size (within the range 300018 000 Da) on this particular biological activity. These results are in agreement with those of Delgado et al. (1988
) who tested heparin fragments obtained by treatment of heparin with heparanases and found that heparin decasaccharides (mol. wt
3000 Da) were as active as heparin in inducing nuclear swelling. However, smaller molecular weight fragments were progressively less active, with tetrasaccharides being the smallest fragments showing any activity.
Having concluded that the decondensing ability of heparin in vitro is related to structural characteristics of the molecule, we decided to test the decondensing ability of other GAGs known to be present in the cumulusoocyte complex in an attempt to find heparins equivalent in vivo. It is important to keep in mind that heparin is not present in the cumulusoocyte complex and is, thus, not likely to play this role as previously suggested. On the other hand, HA, CS, HS, DS and keratan sulphate have been found in the female genital tract, particularly in follicular fluid, of various species (Yanagishita et al., 1979; Grimek et al., 1988
) including human (Bellin et al., 1986
).
Among the different GAGs tested in this study, only HS, a structural analogue of heparin, possessed sperm nuclear-decondensing ability in vitro, while HA, CS and DS were inactive. Moreover, the decondensing abilities of HS and heparin were similar, despite the fact that the former is significantly less sulphated than the latter, and thus possesses a smaller net negative charge. These results once again support the contention that heparins decondensing ability in vitro is related to structural characteristics of the molecule, rather than merely a consequence of its elevated negative charge.
The similarity between the biological activities of heparin and HS in vitro has already been observed in a variety of biological systems, where it has been demonstrated that HS is the active agent in vivo (Yanagishita and Hascall, 1992). Therefore, it is tempting to speculate that HS could be responsible, together with GSH, for human sperm nuclear decondensation in vivo. There is no evidence as yet indicating that GAGs are present inside the oocyte, an issue which the authors plan to address in the near future. In the meantime, the involvement of HS in sperm decondensation in vivo remains a plausible hypothesis.
Preliminary data from our laboratory show that some human follicular fluids obtained from women undergoing IVF possess sperm nuclear-decondensing ability in vitro when GSH is added to the incubation medium. Whether this activity is due to the presence of HS in these fluids remains yet to be proven and is the subject of current research in our laboratory.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bedford, J.M., Bent, M. and Calvin, H. (1973) Variations in the structural character and satibility of the nuclear chromatin in morphologically normal human spermatozoa. J. Reprod. Fertil., 33, 1929.[CrossRef][Medline]
Bellin, M., Ax, R., Laufer, N., Tarlatzis, B., DeCherney, A., Feldberg, D. and Haseltine, F. (1986) Glycosaminoglycans in follicular fluid from women undergoing in vitro fertilization and their relationship to cumulus expansion, fertilization and development. Fertil. Steril., 45, 244248.[ISI][Medline]
Bertolesi, G. (1998) Heparin and related glycosaminoglycans: effects on tumoral development. PhD Thesis. Faculty of Exact and Natural Sciences, University of Buenos Aires, Argentina.
Carrell, D. and Liu, L. (2002) Heparin binding sites are present at a higher concentration on sperm of subfertile men than donors of known fertility. Arch. Androl., 48, 147154.[CrossRef][ISI][Medline]
Chargaff, E. and Olson, K. (1938) Studies on the chemistry of blood coagulation. VI. Studies on the action of heparin and other coagulants. The influence of protamine on the anticoagulant effect in vivo. J. Biol. Chem., 122, 153167.
Delgado, N., Reyes, R., Huacuja, L., Merchant, H. and Rosado, A. (1982) Heparin binding sites in the human spermatozoa membrane. Arch. Androl., 8, 8795.[ISI][Medline]
Delgado, N.M., Reyes, R., Mora-Galindo, J. and Rosado, A. (1988) Size-uniform fragments as nuclear decondensation and acrosome reaction inducers in human spermatozoa. Life Sci., 42, 21772183.[CrossRef][ISI][Medline]
Eriksen, G., Malmström, A. and Uldbjerg, N. (1997) Human follicular fluid proteoglycans in relation to in vitro fertilization. Fertil. Steril., 68, 791798.[CrossRef][ISI][Medline]
Fedarko, N. and Conrad, E. (1986) A unique heparan sulfate in the nucleus of hepatocytes: structural changes with the growth state of the cells. J. Cell Biol., 102, 587599.[Abstract]
Gaubeca-Klix, E., Marin-Briggiler, C., Cameo, M. and Calvo, L. (1998) In vitro sperm nuclear decondensation in the presence of heparin/glutathione distinguishes two subgroups of infertile patients not identified by conventional semen analysis. In Treatment of Infertility: The New Frontiers (Abstract book). Boca Raton, FL. Abstract PO-51.
Gebauer, H., Lindner, H. and Amsterdam, A. (1978) Synthesis of heparin-like glycosaminoglycans in rat ovarian slices. Biol. Reprod., 18, 350358.[ISI][Medline]
Grimek, N., Reyes, R., Gallind, J. and Rosado, A. (1988) Characteristics of proteoglycans isolated from small and large bovine ovarian follicles. Biol. Reprod., 30, 397409.
Jackson, R., Busch, S. and Cardin, A. (1991) Glycosaminoglycans: molecular properties, protein interactions and role in physiological processes. Physiol. Rev., 71, 481539.
Kawasaki, K., Philpott, A., Avilion, A.A., Berrios, M. and Fisher, P.A. (1994) Chromatin decondensation in Drosophila embryo extracts. J. Biol. Chem., 269, 1016910176.
Lalich, R.A., Vedantham, S., McCormick, N., Wagner, C. and Prins, G.S. (1989) Relationship between heparin binding characteristics and ability of human spermatozoa to penetrate hamster ova. J. Reprod. Fertil., 86, 297302.[Abstract]
Lasalle, B. and Testart, J. (1992) Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation. J. Reprod. Fertil., 95, 313324.[Abstract]
Lee, C. and Ax, R. (1984) Concentrations and composition of glycosaminoglycans in the female bovine follicular fluid. Nature, 224, 919920.
Lindahl, U. and Kjellén, L. (1991) Heparin or heparan sulfatewhat is the difference? Thromb. Haemostasis, 66, 4448.[ISI][Medline]
Maeda, Y., Yanagimachi, H., Tateno, H., Usui, N. and Yanagimachi, R. (1998) Decondensation of the mouse sperm nucleus within the interphase nucleus. Zygote, 6, 3945.[ISI][Medline]
Montag, M., Tok, V., Liow, S., Bongson, A. and Nicolle, J.C. (1992) In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation. Mol. Reprod. Dev., 33, 338346.[ISI][Medline]
Ohsumi, K. and Katagiri, C. (1991) Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin. Dev. Biol., 148, 295305.[ISI][Medline]
Perreault, S.D., Wolff, R.A. and Zirkin, B.R. (1984) The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vitro. Dev. Biol., 101, 160167.[ISI][Medline]
Perreault, S.D., Barbu, R.R. and Slott, V.L. (1988) Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev. Biol., 125, 181186.[ISI][Medline]
Philpott, A., Leno, G.H. and Laskey, R.A. (1991) Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. Cell, 65, 569578.[ISI][Medline]
Reyes, R., Rosado, A., Hernández, O. and Delgado, N.M. (1989) Heparin and glutathione: physiological decondensing agents of human sperm nuclei. Gamete Res., 23, 3947.[ISI][Medline]
Romanato, M., Bertolesi,G., Calvo, J.C. and Calvo, L. (2001) Heparan sulfate: a putative sperm decondensing agent for human spermatozoa in vivo. J. Androl., 22(Suppl), 85.
Salustri, A., Yanagishita, M. and Hascall, V.C. (1989) Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus celloocyte complex during follicle-stimulating hormone-induced mucification. J. Biol. Chem., 264, 1384013847.
Tesarik, J. and Kopecny, V. (1989) Development of human male pronucleus: ultrastructure and timing. Gamete Res., 24, 135149.[ISI][Medline]
World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill, J.D. (eds), The Physiology of Reproduction, 2nd edn. Raven Press, New York, pp. 189317.
Yanagishita, M. and Hascall, V.C. (1992) Cell surface heparan sulfate proteoglycans. J. Biol. Chem., 267, 94519454.
Yanagishita, M., Rodbard, D. and Hascall, V. (1979) Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. J. Biol. Chem., 254, 911920.[Abstract]
Zirkin, B.R., Perreault, S.D. and Naish, S.J. (1989) Formation and function of the male pronuceus during mammalian fertilization. In Schatten, H. and Schatten, G. (eds), The Molecular Biology of Fertilization. Academic Press, San Diego, CA, pp. 91114.
Submitted on January 6, 2003; resubmitted on March 24, 2003; accepted on May 14, 2003.