T. L. Marshak

Effect of Gold Nanoparticles on Mouse Spermatogenesis

The response of the mouse male germ cells exposed to gold nanoparticles (∼2.5 nm) was studied. Our investigation demonstrates that treatment with Au nanoparticles for four days does not impair the architecture of the spermatogenic epithelium. Cytogenetic evaluation using micronucleus assay showed that gold nanoparticles can affect the chromosomes of early primary spermatocytes. However, gold nanoparticles did not induce chromosome abnormalities in spermatogonial stem cells. Further, the cauda epididymal sperm was isolated on the 14th day after treatment and was incubated in SDS solution (Na dodecyl sulphate) and then in a solution containing DTT (dithiothreitol) to induce nuclear chromatin decondensation. Observations showed that after four days of treatment of spermiogenic (postmeiotic) cells with gold nanoparticles the decondensation process had no differences from the control. On the contrary, in the experiment with the same cells and period of fixation but with a single exposure to gold nanoparticles, the number of mature gametes with totally decondensed nuclei reached 100% as opposed to 44% in the controls.

Bovine sperm chromatin is not protected from the effects of ultrasmall gold nanoparticles

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of ∼3.0 nm and a concentration of 1 × 1015 particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed (“intact”), partially decondensed, and completely decondensed nuclei correlated as 40: 32: 28% and 0: 36: 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.

Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senescence-accelerated mice

Specific features of spermatogenesis were studied in senescence-accelerated mice of the strain SAMP1 after one-time injection of the chemical mutagen dipin. Quantitative and histomorphological changes in the spermatogenic epithelium proved to develop gradually. Cell loss and disorganization of spermatogenesis reached the peak as late as on days 28 and 35 after the injection. Differentiating spermatogonia manifested increased sensitivity to dipin. In prophase I of meiosis, developing spermatocytes proved to be less sensitive to the cytotoxic action of dipin at the pachytene than at the preleptotene-leptotene stages. Spermatogenesis in most seminiferous tubules was restored by day 56 after dipin treatment. At the end of the experiment (day 100), both quantitative parameters and morphological pattern of spermatogenesis did not differ significantly from those in the control. Thus, the cytotoxic action of dipin does not lead to irreversible structural disorganization of the spermatogenic epithelium in SAMP1 mice. Radioautography revealed a large proportion of highly differentiated Sertoli cells with 3H-thymidine-labeled nuclei in experimental animals. In some cases, structures resembling embryonic seminiferous tubules were revealed in the vicinity of rete testis in histological sections of testes of experimental mice. These structures contained the cells morphologically similar to gonocytes and immature Sertoli cells.

[Analysis of spermatogenesis in senescence-accelerated mice].

A comparative analysis of age-related dynamics of spermatogenesis has been performed in mutant mouse lines predisposed or resistant to accelerated senescence (SAMP1 and SAMR1 respectively). The results show that quantitative and morphohistological trends in the development of sperm cells and Sertoli cells in both lines are similar in both lines. Their comparison with data obtained in our previous studies (Zakhidov et al., 2001; Gordeeva et al., 2001) shows that sharp quantitative and qualitative changes in the structure of the spermatogenic system have occurred in senescence-accelerated mice of new generations, which confirms the fact of dynamic instability of the germinal lineage. The role of stem spermatogonial cells in restoration of spermatogenesis in animals reaching the critical age is discussed.

Gold Nanoparticles: Mutagen, Antimutagen, or Comutagen?

The effect of ultrasmall gold nanoparticles (GNPs), both direct and in combination with the alkylating mutagen dipin, on the genetic structures of male germ cells in mice were studied using the meiotic micronucleus assay. It was shown for the first time that GNPs can exhibit mutagenic, antimutagenic, and comutagenic activity depending on the experimental conditions.

Peculiarities of development of mouse male germ cells after intratesticular injection of dipin

It has been shown that a single intratesticular injection of the chemical mutagen dipin (experiment) or saline (control) into mice resulted in significant but reversible morphohistological damage of the spermatogenic epithelium. However, unlike the controls, in mutagenized testes these damages were more pronounced. Thus, the process of restoring a normal pattern of spermatogenesis was slower. In addition, on day 35 of fixation, mature gametes were almost completely absent in the cauda epididymis and a large number of sperm cells with abnormal head shape (58.5 versus 1.7% in the controls) appeared in the testes. Using spermatogonial and meiotic micronucleus assay, we found that dipin did not induce a rise in the number of gross chromosomal mutations in the spermatogonial stem cells (SSCs): on days 35, 56, and 100 postinjection, the incidence of aberrant spermatogonia and round spermatids was not significantly different from the saline control. The degree of gametic chromatin decondensation was evaluated after treatment of the cauda epididymal sperm with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). Judging by the results of the in vitro sperm chromatin decondensation on days 7, 14, 35, 56, and 100 after the injection of dipin or saline, the number of decondensed nuclei decreased sharply in the studied samples as compared with the sperm from intact animals where sperm cells with fully decondensed chromatin prevailed.

Estimation of the frequencies of induced mutations in spermatogenic cells of senescence-accelerated prone mice of the SAMP1 strain

The results obtained in this work demonstrate the dynamics of cytogenetic changes of spermatogenic cells in senescence-accelerated prone mice, strain SAMP1, after a single exposure to a chemical mutagen, dipin, at a genetically active dose of 30 mg/kg. In the time interval between days 3 and 28 the frequency of induced spermatogonial micronuclei does not significantly exceed the level of spontaneous mutagenesis. The lack of an experimental effect of micronuclei in this time interval is probably a consequence of mitotic delay and (or) of the death of a considerable part of genetically defective cells in the spermatogonial compartment. Different stages of meiosis exhibit different chemical sensibilities: the yield of round spermatids with micronuclei is maximum after treatment of early primary spermatocytes (preleptotene-leptotene stage) with dipin. The high sensibility of preleptotene and leptotene spermatocytes is confirmed by the sperm head shape abnormality assay. Chromosome damage caused by dipin in spermatogonial stem cells is irreversible, as evidenced by a sharp increase in the frequencies of spermatogonial and meiotic micronuclear aberrations within long periods after treatment. Increased genetic instability in the stem compartment does not lead to irreversible degradation of the system of development of male sex cells in senescence-accelerated SAMP1 mice.

Cytogenetic Activity of Gold Nanoparticles in Germ and Somatic Cells of 129 Mice with a Nonsense Mutation in the DNA Polymerase Iota Gene

In this study, we investigated the cytogenetic effects of single and quadruple exposure of spermatogenic cells and hepatocytes of 129 mice, which have a mutation in the gene that encodes DNA polymerase iota, to ultrasmall gold nanoparticles (GNPs). The combined effects of GNPs and chemical mutagen dipin were evaluated. In all cases, except for the experiment with the quadruple GNP injection, we observed a slight, statistically nonsignificant increase in the frequency of round spermatids with micronuclei compared to the negative control (saline). It is established that, in the intact liver of 129 mice, in all variants of the experiment, GNPs behaved as a potentially cytotoxic agent, as evidenced by the decrease in the frequency of the micronucleated hepatocytes.

Effect of ultrasmall gold nanoparticles on the murine native sperm chromatin

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 DNA-polymerase iota gene were studied. It is shown that for both mouse strains after sperm incubation in a solution containing Au nanoparticles, 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 demonstrated 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 confirmed.

A Biological Model of Accelerated Senescence. 1. The Rate of the Spontaneous Mutation Process in Spermatogenesis of Senescence-Accelerated Mice

We studied the dynamics of age-related cytogenetic changes in the developing male germ cells of mice prone to accelerated senescence (strain SAMP1) and mice resistant to accelerated senescence (strain SAMP1) by counting the spermatogonial and meiotic micronuclei and testing the defects of the spermatozoon head shape. In these animals, the accumulation of germinative mutations has a nonlinear pattern. During the entire ontogenesis of SAMP1 mice, the frequency of circular spermatids with micronuclei corresponded to the level observed upon induced mutagenesis.