ISSN 10623590, Biology Bulletin, 2015, Vol. 42, No. 6, pp. 479–485. © Pleiades Publishing, Inc., 2015. Original Russian Text © S.T. Zakhidov, V.M. Rudoy, O.V. Dement’eva, N.M. Mudzhiri, N.V. Makarova, I.A. Zelenina, L.E. Andreeva, T.L. Marshak, 2015, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2015, No. 6, pp. 565–572.
DEVELOPMENTAL BIOLOGY
Effect of Ultrasmall Gold Nanoparticles on the Murine Native Sperm Chromatin S. T. Zakhidova, V. M. Rudoyb, O. V. Dement’evab, N. M. Mudzhiria, d, N. V. Makarovac, I. A. Zeleninaa, L. E. Andreevac, and T. L. Marshakd a Biological
Faculty, Moscow State University, Moscow, 119991 Russia b Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninskii pr. 31, Moscow, 119071 Russia c Institute of Molecular Genetics, Russian Academy of Sciences, pl. Akademika Kurchatova 2, Moscow, 123182 Russia d Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia email:
[email protected] Received May 12, 2015
Abstract—The effects of ultrasmall (2–3 nm) gold nanoparticles on native epididymal sperm chromatin of CBA × C57BL/6 hybrid mice and 129/IMG mice with a mutation in the DNApolymerase iota gene were studied. It is shown that for both mouse strains after sperm incubation in a solution containing Au nanopar ticles, at 23, 37 and 60°C for 30 min followed by 1 h treatment in dithiothreitol solution, a decrease in the number of nuclei with fully decondensed chromatin was observed compared with the control. Though, the manifestation of this effect in the population of 129/IMG mice mature sperm, was weaker. Also we have dem onstrated that sperm of both strains that were incubated in a sol of Au nanoparticles at 60°C behave differently under the action of dithiothreitol. A considerable part (~80%) of sperm of CBA × C57BL/6 hybrid mice treated with Au nanoparticles showed high resistance to the action of dithiothreitol, whereas in the case of 129/IMG mice only ~30% did, and a partial or complete chromatin decondensation takes place in the remaining sperm. In general, using the method of nuclear chromatin decondensation in vitro for the native sperm, the patterns that we have identified in earlier studies on previously demembranized sperm are con firmed. DOI: 10.1134/S1062359015060138
INTRODUCTION Special studies carried out within the framework of experimental nanotoxicology must show how safe the newly created and existing nanomaterials can be. At the same time, we believe that research aimed at clari fying the impact of nanomaterials on the structural and functional integrity of the reproductive cells should play a major role, because these specialized cells of the germ line, separated from the soma in embryogenesis, provide for hereditary succession for a number of generations. Currently, gold nanoparticles are one of the most widely studied nanomaterials. They may find (and have already found) applica tions in biomedicine (Dykman and Khlebtsov, 2012). In particular, these nanoparticles are promising in terms of both diagnosis and therapy of malignant tumors (Llevot and Astruc, 2012; Vigderman and Zubarev, 2013). In the latter case, they can be used as sensitizers in photothermal therapy (Dement’eva
et al., 2012) and as means of delivery of certain drugs (Ghosh et al., 2008). In this connection, it is essential to investigate the ability of Au nanoparticles to penetrate certain cell structures, which is directly related to their size. Ultr asmall Au nanoparticles, overcoming the cell mem brane barrier, can prove not only to be in its cytoplasm, but can also then penetrate into the nucleus (Huang et al., 2012; Huo et al., 2014), where their interaction with DNA will inevitably occur. Indeed, as has been found in model experiments with cholesteric liquid crystal dispersions of DNA, ultrasmall (size 2–3 nm) Au particles can disturb its doublestranded structure (Skuridin et al., 2010; Yev dokimov et al., 2012). Apparently, this is due to their insertion into the major groove of DNA molecules in the Bform. This possibility for Au nanoparticles of a similar size was discussed previously (Pan et al., 2007). In recent years, important results were obtained on the effect of Au nanoparticles on mature male gametes of eukaryotic organisms (Wiwanitkit et al., 2009; Mor etti et al., 2012; Taylor et al., 2012; Tiedemann et al.,
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2014). Using the method of nuclear chromatin decon densation under in vitro conditions, it has been shown (Zakhidov et al., 2010) that the abovementioned ultr asmall Au nanoparticles negatively affect the organi zation of the deoxyribonucleoprotein complex (DNP) in the predemembranized epididymal spermatozoa of mice. In an experiment of this kind, when the cells are released artificially from cellular and nuclear membranes, nanoparticles offer the possibility of a more rapid and deeper penetration into the structure of chromatin and active interaction with its basic com ponents—proteins and DNA. The aim of this work is to study, using the same methodological approach, and varying the tempera ture, the efficiency of action of the ultrasmall Au nanoparticles on the DNP complex in native (i.e., not demembranized) mature sperm of CBA × C57BL/6 hybrid mice, as well as 129/IMG mice, containing a nonsense mutation in the second exon of the DNA polymerase iota gene. MATERIALS AND METHODS A gold hydrosol was synthesized by the same method (Duff et al., 1993) as in our previous studies (Zakhidov, et al., 2010, 2013). For this, 1.5 mL of 0.2 M NaOH and 1 mL of an aqueous solution of tet rakis(hydroxymethyl)phosphonium chloride with a concentration of 9.6 mg/mL were added to 45.5 mL of deionized water under stirring, and then (after 5 min) 2 mL of 1% solution of chloroauric acid were added. The reaction mixture almost instantly acquired a dark brown color, indicating the reducton of AuCl 4− ions with the formation of ultrasmall Au particles. Their average size, determined by means of a dynamic light scattering spectrometer Zetasizer Nano ZS (Malvern, United Kingdom), was equal to 2–3 nm; the particle concentration was about 1 × 1015 mL–1. The hydrosol was stored in the dark at 4°C and used no earlier than 2.5 months after the synthesis. During this time, the quite defective structure of the Au nano particles, rearranged, and they acquired pronounced metallic properties (Morozov et al., 2012). This is confirmed in particular by the appearance of the local ized surface plasmon resonance band in the absorp tion spectrum of the hydrosol. The pH value of this “mature” sol was ~5. We used mature male CBA × C57BL/6 hybrid mice (n = 4) and 129/IMG mice (n = 7) derived at the Insti tute of Molecular Genetics, Russian Academy of Sci ences, based on 129 mice who have a nonsense muta tion in the second exon of the DNA polymerase iota gene. Animal experiments were performed in accordance with the requirements of the Order of the Ministry of Health of the Russian Federation no. 267 of June 19, 2003, “On Approval of Rules of Laboratory Practice” and the ethical standards set out in the Rules of Good
Laboratory Practice (GLP), in the Helsinki Declara tion (2000). Animals were sacrificed by cervical dislocation. The epididymis was removed, the cauda epididymis was placed in saline and thoroughly crushed, and the sperm suspension was prepared. After low speed cen trifugation (1600 rpm, 15 min), the supernatant was removed, and the precipitates of spermatozoa after redispersion in saline were divided into equal portions and centrifuged again under the same conditions. After removing the supernatant, one portion of the samples was resuspended in 0.5 mL of saline (control), and the other was suspended in 0.5 mL of the Au hydrosol (experiment). Control and experimental sam ples of spermatozoa were incubated at 23, 37 or 60°С for 30 min. Thereafter, the samples were centrifuged under the same conditions, supernatants were removed, and 0.5 mL of saline was added. The samples were resuspended and centrifuged, and after removal of supernatants, 0.5 mL of 1% solution of the anionic surfactant sodium dodecylsulfate (SDS) (Sigma, United States) was added to each precipitate. The resulting suspensions were incubated at 23°С for 30 min, then 0.3 mL of 0.01 M solution of dithiothre itol (DTT) (Sigma), in TrisHClbuffer (pH 8) was added, and the incubation was continued for 1 h. After treatment of the control and experimental samples of spermatozoa in a decondensing medium, smears were prepared. Preparations were air dried, fixed in 96% ethanol for 10 min, and stained with 0.1% solution of toluidine blue (Fluka, Switzerland). The smears of the stained spermatozoa were analyzed by an Opton microscope (Germany) with a total magnifica tion of ×1000, viewing an average of 100 randomly selected fields of view. RESULTS After the treatment of epididymal sperm of CBA × C57BL/6 hybrid mice and 129/IMG mice in a solu tion containing thiol reagent DTT, both in the control and in the experiment, the nuclei differing in the degree of swelling and in the pattern of chromatin decompactization were found. As in our previous studies (Zakhidov et al., 2010, 2013), these nuclei were conditionally divided into three main types: nonde condensed (or “intact”), partially decondensed, and fully decondensed. Note that under the action of DTT in the population of CBA × C57BL/6 hybrid mice sperm treated with Au nanoparticles, there appeared many nuclei with unusual forms, many of which looked like abnormal, whereas in 129/IMG mice such nuclei were encountered both in the control and in the experiment (Figs. 1, 2). Figure 3a shows that after incubation of epididymal sperm in the saline (control) at 23, 37 and 60°С for 30 min and subsequent 30min treatment in a solution containing SDS, followed by 1 h treatment with DTT, the percentages of cells with nondecondensed, par BIOLOGY BULLETIN
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Fig. 1. Nondecondensed (ND), partially decondensed (PD), and fully decondensed (FD), as well as abnormal (ABN) nuclei in the population of epididymal sperm of CBA × C57BL/6 hybrid mice. (a, b) control (saline), (c–f) experiment (gold nanoparti cles). Stained with toluidine blue.
tially decondensed, and fully decondensed nuclei were quite close (2 : 9 : 89, 2 : 8 : 90, and 0 : 5 : 95). In con trast to the control, under the same experimental con ditions, sperm treated with Au nanoparticles demon strated relatively high resistance to the decondensing agent, as is evidenced by the percentages of nondecon densed, partially decondensed, and fully decondensed nuclei which are equal for the above—mentioned tem peratures, to 30 : 33 : 37, 30 : 40 : 30, and 78 : 22 : 0, respectively. Data on the frequency of occurrence of epididymal sperm with varying degrees of nuclear chromatin decondensation in 129/IMG mice is shown in Fig. 3b. In the control sperm after incubation in saline at 23, 37, and 60°С for 30 min followed by 30min treatment in a solution containing SDS, and then in a solution of DTT for 1 h, the percentages of nondecondensed, partially decondensed, and fully decondensed nuclei were respectively 0 : 13 : 87, 3 : 12 : 85, and 20 : 49 : 31. At the same time in the experiment under the same conditions, these types of nuclei were encountered in the following percentages: 8 : 27 : 65, 3 : 45 : 52, and 31 : 66 : 3. A comparison of the results presented in Figs. 3a and 3b shows that in the control the response of native BIOLOGY BULLETIN
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epididymal sperm of CBA × C57BL/6 hybrid and mutant 129/IMG mice to the action of DTT differs only at 60°С, while after the treatment with Au nano particles, there are differences at all incubation tem peratures. DISCUSSION As it has already been mentioned above, one of the tasks of our previous work (Zakhidov et al., 2010) was to study the influence of ultrasmall Au nanoparticles on the process of DTTinduced decondensation of the nuclear chromatin in mouse epididymal spermatozoa, which were preliminarily demembranized by SDS. In this experiment, we made the following significant changes: the native sperm were exposed to Au nano particles (and their incubation temperature in the hydrosol was varied) and only then were the cells treated with SDS and DTT. In addition, we used not only the sperm of the CBA × C57BL/6 hybrid mice, but also of the mutant 129/IMG mice. In general, our results are close to those obtained previously (Zakhidov et al., 2010). They also rather convincingly demonstrate the ability of ultrasmall Au nanoparticles to render a depressing effect on the pro
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Fig. 2. Nondecondensed (ND), partially decondensed (PD), and fully decondensed (FD) as well as abnormal (ABN) nuclei in the population of epididymal sperm of 129/IMG mice. (a–d) control (saline), (e–i) experiment (gold nanoparticles). Stained with toluidine blue.
cess of the artificial decompactization of the paternal genome in the mature murine male germ cells. Indeed, as follows from our observations, native mouse epididymal sperm of both strains after incuba tion for 30 min in a sol containing ultrasmall Au nano particles at 23, 37 or 60°C and subsequent treatment with SDS and DTT are characterized by significantly lower percentage of nuclei with fully decondensed chromatin than the control. Note, however, that for the population of gametes of the mutant 129/IMG mice, this effect was less pronounced. In other words
Au nanoparticletreated spermatozoa of the mutant 129/IMG mice showed greater sensitivity to DTT than those of the CBA × C57BL/6 hybrid mice. Thus, the effect of the utilized Au nanoparticles on the DNP complex is essentially independent on whether we are dealing with native epididymal mouse sperm or with the sperm predemembranized using SDS. Obviously, this is primarily due to the ultrasmall size of nanoparticles, allowing them to easily penetrate enough through the cell and nuclear membranes of gametes. BIOLOGY BULLETIN
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Note that in the recent years more attention has been paid to studying the ability of Au nanoparticles to penetrate into mammalian cells (including spermato zoa) and different cell structures (Murphy et al., 2008; Wiwanitkit et al., 2009; Johnston et al., 2010; Moretti et al., 2012; Taylor et al., 2012; Boyoglu et al., 2013; Dykman and Khlebtsov, 2014; Tiedemann et al., 2014). The results obtained indicate that this ability depends on the size and surface chemistry of the nano particles; moreover, it can also depend on the cell type. It is shown in particular (Moretti et al., 2012) that the cell membrane of human spermatozoa is permeable even for fairly large Au nanoparticles (with an average diameter of ~ 50 nm) modified with the biologically inert polymer polyvinylpyrrolidone. At the same time, much smaller Au nanoparticles obtained by laser abla tion (with an average diameter of 7–10 nm), stabilized electrostatically or by adsorption of oligonucleotide molecules, do not penetrate bovine spermatozoa (Tay lor et al., 2014). In our view, this result is directly due to the surface chemistry of the nanoparticles. Indeed, electrostatically stabilized nanoparticles in a medium with high ionic strength, which is usually a culture broth, can rapidly coagulate to form large aggregates, especially if the charge of the nanoparticles is not too large. Exactly this situation was described by Taylor et al. (Taylor et al., 2014). Cellular internalization of such aggregates is either impossible or very difficult (Albanese and Chan, 2011). For Au nanoparticles sta bilized by oligonucleotide, a strong specific interac tion of its molecules with the cell membrane proteins obviously took place, which also prevented the pene tration of particles into the cell. Note that our nanoparti cles, although stabilized by an electrostatic mechanism, are not substantially aggregated in saline for at least several hours. This was primarily due to their high charge (the zeta potential of the particles was approximately 40 mV). Returning to our results, it should again be empha sized that they indicate the ability of the ultrasmall Au nanoparticles to penetrate directly into the sperm nucleus. This is also confirmed by the fact of an increased resistance of the DNP complex of sperma tozoa to the action of DTT with an increased temper ature of preincubation in the gold sol. Indeed, the increase in temperature leads to a decrease in the sta bility of biological membranes (Salisbury et al., 1977), i.e., to an increase in their permeability, in particular by reducing the membrane microviscosity. At the same time, the mobility of nanoparticles (i.e., their diffusion coefficient) increases. As a result, they can penetrate into the nucleus more quickly (and perhaps more deeply). The other conditions being equal, the higher the temperature of incubation, the greater the number of Au nanoparticles in the nucleus should be. What is the possible mechanism of inhibition of chromatin decondensation under the action of gold nanoparticles penetrating into the nucleus? We recall that electrostatically stabilized nanoparticles were used in our experiments. They can quite actively inter
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Fig. 3. The frequency of occurrence of nondecondensed (1), partially decondensed (2), and fully decondensed (3) nuclei in the populations of epididymal spermatozoa of CBA × C57BL/6 hybrid mice (a) and 129/IMG mice (b).
act with free thiol groups and/or with some other functional groups in the amino acids of basic nuclear proteins, by acting as “crosslinking sites” and, conse quently, preventing chromatin decondensation. Thus, it is well known (Zirkin et al., 1985), that an obligatory condition of chromatin decondensation is the disrup tion of disulfide bridges. At the same time, the ability of thiols and dithiols to chemisorb on the gold surface may lead to the formation of S–Au–S “bridges”. In addition, we cannot exclude the interaction of Au nanoparticles with intranuclear acrosinlike pro teases, which may lead to (partial) loss of their activity. It is known (Kostomarova and Knyazeva, 1992) that normally in mammalian sperm nuclei the proteolytic activity of these unusual intranuclear enzymes is aimed mainly at degradation of a sperm specific prot aminelike proteins, slipping from the DNA molecule during decondensation of chromatin under the action of the thiol. Another possible reason for the inhibition of nuclear chromatin decondensation under our experimental conditions may be the for mation of reactive oxygen species in the presence of ultrasmall Au nanoparticles (Pan et al., 2009). These
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compounds⎯singlet oxygen, superoxide radicals, and others⎯can inhibit the enzymes belonging to the anti oxidant defense system, in particular, glutathione, which contains the sulfhydryl group, and thereby make an independent contribution to the destructive processes taking place also in the structure of the gametic DNP complex. Some studies have clearly demonstrated the ability of Au nanoparticles to reduce the locomotor activity of mature spermatozoa (Wiwanitkit et al., 2009; Moretti et al., 2012; Taylor et al., 2014). Formation in sperm of a large number of reactive oxygen species is regarded as one of the possible mechanisms of violation of loco motor function. Another possible explanation for this effect is the direct interaction of Au nanoparticles with free sulfhydryl groups located on the surface of mature male gametes (Taylor et al., 2014). 129 mouse strain occupies a special place among the variety of laboratory strains. These mice are most successfully used to obtain embryonic stem cells, have a tendency to form spontaneous teratomas in genital ridges, and are resistant to radiation. In mouse 129 and 129/IMG strains, there is a stop codon in the second exon of the DNA polymerase iota gene. Prone to errors, the DNA polymerase iota has some unusual properties: it can both cause and correct mutations in the genome; the endogenous polymerase activity manifests itself in all organs and tissues of hybrid mice embryos and is strongly inhibited at postnatal stages of development. The highest level of expression and activity of this enzyme in the organism of adult mam mals (including hybrid mice) is in the testes. However, in adult mouse 129 such activity is absent (Gening et al., 2004; Kazakov et al., 2010). As was noted above, after the contact with the ultr asmall Au nanoparticles, spermatozoa of the mutant mouse 129/IMG showed a greater sensitivity to DTT than spermatozoa of CBA × C57BL/6 hybrid mice. So ~80% of spermatozoa of the CBA × C57BL/6 hybrid mice were insensitive to the action of DTT after incu bation in the sol of ultrasmall Au nanoparticles for 30 min at 60°C. At the same time, in the spermatozoa of 129/IMG mouse strain under the same experimen tal conditions, in most cases there were nuclei with partially decondensed chromatin (66%) and only 31% of the nuclei were resistant to the decondensing agent. The observed differences are obviously caused by different packaging of the genetic material in the sperm nuclei of mice of the two strains. As a conse quence, the number of ultrasmall Au nanoparticles accumulated in the sperm nuclei and their interaction with various functional groups of the DNPcomplex (and as a result, the sustainability of chromatin to DTT action ) may be different for CBA × C57BL/6 hybrid mice and mutant 129/IMG mice. Note also that incubation of 129/IMG mice sper matozoa in saline at 60°C for 30 min resulted in a nearly threefold decrease in the percentage of nuclei with a fully decondensed chromatin compared with that in a
population of spermatozoa of CBA × C57BL/6 hybrid mice. Finally, we note yet another resut. It has been found that for both strains of mice the action of DTT in the control and in the experiment resulted in the forma tion in the population of epididymal spermatozoa of a relatively large amount of nuclei having an abnormal appearance, and to a greater extent, this effect was observed after incubation of spermatozoa in a gold sol. The fraction of such nuclei in a population of gametes in CBA × C57BL/6 hybrid mice was significantly less. There is no doubt that the emergence of such a large number of morphologically abnormal sperm nuclei in 129/IMG mice is associated with the socalled phe nomenon of immature and/or overmature of the gametic DNPcomplex formed, as well known, during spermiogenesis and sperm transport through different regions of the epididymis. In this connection we espe cially emphasize that in some seminiferous tubules of 129/IMG mice (26% versus 5% in CBA × C57BL/6 hybrid mice) spermatogenesis occurs with large devia tions from the norm (N.M. Mudzhiri et al., unpub lished data). Thus, the results of this study generally support those that we have obtained previously using the same method of nuclear chromatin decondensation in vitro. In addition, they demonstrate the ability of ultrasmall Au nanoparticles to penetrate easily through the plasma membrane of the mature male gametes and that the effect of such nanoparticles on the sperm of CBA × C57BL/6 hybrid mice and mutant 129/IMG mice is not the same for the same experimental condi tions. Obviously, this is due to the existence of some subtle genetically determined differences in the pack aging of the DNP complex in the spermatozoa of mice of these two strains. We emphasize that the actual data relating toxicity of the Au nanoparticles for the sperm are still very lim ited in number; they are ambiguous and, of course, do not allow us to fully assess the reproductive risks aris ing from the use of these nanostructures that are very promising from a practical point of view. However, the already available results indicate the possible strong impact of Au nanoparticles on the male germ cells. Thus, further studies of the spermatotoxicity of Au nanoparticles, of course, are relevant. ACKNOWLEDGMENTS This work was supported in part by the Program of the Presidium of the Russian Academy of Sciences “Wildlife: Current Status and Problems of Develop ment.” REFERENCES Albanese, A. and Chan, W.C.W., Effect of gold nanoparticle aggregation on cell uptake and toxicity, ACS Nano, 2011, vol. 5, pp. 5478–5489. BIOLOGY BULLETIN
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Translated by G. Naumova
