Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on mammalian gametes.

Metal and alloy nanoparticles are increasingly developed for biomedical applications, while a firm understanding of their biocompatibility is still missing. Various properties have been reported to influence the toxic potential of nanoparticles. This study aimed to assess the impact of nanoparticle size, surface ligands and chemical composition of gold, silver or gold-silver alloy nanoparticles on mammalian gametes. An in vitro assay for porcine gametes was developed, since these are delicate primary cells, for which well-established culture systems exist and functional parameters are defined. During coincubation with oocytes for 46 h neither any of the tested gold nanoparticles nor the gold-silver alloy particles with a silver molar fraction of up to 50% showed any impact on oocyte maturation. Alloy nanoparticles with 80% silver molar fraction and pure silver nanoparticles inhibited cumulus-oocyte maturation. Confocal microscopy revealed a selective uptake of gold nanoparticles by oocytes, while silver and alloy particles mainly accumulated in the cumulus cell layer surrounding the oocyte. Interestingly sperm vitality parameters (motility, membrane integrity and morphology) were not affected by any of the tested nanoparticles. Only sporadic association of nanoparticles with the sperm plasma membrane was found by transmission electron microscopy. In conclusion, mammalian oocytes were sensitive to silver containing nanoparticles. Likely, the delicate process of completing meiosis in maternal gametes features high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag(+)-ions are responsible for the observed toxicity, but the compounding into an alloy seemed to alleviate the toxic effects to a certain extent.

Nonendosomal cellular uptake of ligand-free, positively charged gold nanoparticles.

Gold nanoparticles (GNPs) have interesting optical properties, such as exceptionally high quantum yields and virtually limitless photostability. Therefore, they show the potential for applications as biomarkers especially suitable for in vivo and long-term studies. The generation of GNPs using pulsed laser light rather than chemical means provides nanoparticles, which are remarkably stable in a variety of media without the need of stabilizing agents or ligands. This stabilization is achieved by partial oxidation of the gold surface resulting in positively charged GNPs. However, little is known about cellular uptake of such ligand-free nanoparticles, their intracellular fate, or cell viability after nanoparticle contact. The current work is aimed to explore the response of a bovine cell line to GNP exposure mainly using laser scanning confocal microscopy (LSCM) supported by other techniques. Cultured bovine immortalized cells (GM7373) were coincubated with GNP (average diameter 15 nm, 50 microM Au) for 2, 24, and 48 h. The detection of GNP-associated light scattering by the LSCM facilitated a clear distinction between GNP-containing cells and the negative controls. After 48 h, 75% of cells had visibly incorporated nanoparticles. No colocalization was detected with either Rab5a or Lamp1-positive structures, i.e., endosomes or lysosomes, respectivley. However, transmission electron microscope analysis of GNP-coincubated cells indicated the nanoparticles to be positioned within electron-dense structures. Coincubation at 4 degrees C did not inhibit nanoparticle uptake, suggesting diffusion as possible entrance mechanism. Although the assessment of cell morphology, membrane integrity, and apoptosis revealed no GNP-related loss of cell viability at a gold concentration of 25 microM or below, a cytotoxic effect was observed in a proliferation assay after exposing low cell numbers to 50 microM Au and above. In conclusion, this study confirmed the cellular uptake of ligand-free gold nanoparticles during coincubation apparently without using endocytic pathways.

Bioconjugated Gold Nanoparticles Penetrate Into Spermatozoa Depending on Plasma Membrane Status.

Spermatozoa are not only essential for animal reproduction they also represent important tools for the manipulation of animal genetics. For instance, the genetic labeling and analysis of spermatozoa could provide a prospective complementation of pre-fertilization diagnosis and could help to prevent the inheritance of defective alleles during artificial insemination or to select beneficial traits in livestock. Spermatozoa feature extremely specialized membrane organization and restricted transport mechanisms making the labeling of genetically interesting DNA-sequences, e.g., with gold nanoparticles, a particular challenge. Here, we present a systematic study on the size-related internalization of ligand-free, monovalent and bivalent polydisperse gold nanoparticles, depending on spermatozoa membrane status. While monovalent conjugates were coupled solely to either negatively-charged oligonucleotides or positively-charged cell-penetrating peptides, bivalent conjugates were functionalized with both molecules simultaneously. The results clearly indicate that the cell membrane of acrosome-intact, bovine spermatozoa was neither permeable to ligand-free or oligonucleotide-conjugated nanoparticles, nor responsive to the mechanisms of cell-penetrating peptides. Interestingly, after acrosome reaction, which comprises major changes in sperm membrane composition, fluidity and charge, high numbers of monovalent and bivalent nanoparticles were found in the postequatorial segment, depicting a close and complex correlation between particle internalization and membrane organization. Additionally, depending on the applied peptide and for nanoparticle sizes < 10 nm even a successive nuclear penetration was observed, making the bivalent conjugates promising for future genetic delivery and sorting issues.

Influence of gold, silver and gold-silver alloy nanoparticles on germ cell function and embryo development.

The use of engineered nanoparticles has risen exponentially over the last decade. Applications are manifold and include utilisation in industrial goods as well as medical and consumer products. Gold and silver nanoparticles play an important role in the current increase of nanoparticle usage. However, our understanding concerning possible side effects of this increased exposure to particles, which are frequently in the same size regime as medium sized biomolecules and accessorily possess highly active surfaces, is still incomplete. That particularly applies to reproductive aspects, were defects can be passed onto following generations. This review gives a brief overview of the most recent findings concerning reprotoxicological effects. The here presented data elucidate how composition, size and surface modification of nanoparticles influence viablility and functionality of reproduction relevant cells derived from various animal models. While in vitro cultured embryos displayed no toxic effects after the microinjection of gold and silver nanoparticles, sperm fertility parameters deteriorated after co-incubation with ligand free gold nanoparticles. However, the effect could be alleviated by bio-coating the nanoparticles, which even applies to silver and silver-rich alloy nanoparticles. The most sensitive test system appeared to be in vitro oocyte maturation showing a dose-dependent response towards protein (BSA) coated gold-silver alloy and silver nanoparticles leading up to complete arrest of maturation. Recent biodistribution studies confirmed that nanoparticles gain access to the ovaries and also penetrate the blood-testis and placental barrier. Thus, the design of nanoparticles with increased biosafety is highly relevant for biomedical applications.

Rational design of gold nanoparticle toxicology assays: a question of exposure scenario, dose and experimental setup.

Many studies have evaluated the toxicity of gold nanoparticles, although reliable predictions based on these results are rare. In order to overcome this problem, this article highlights strategies to improve comparability and standardization of nanotoxicological studies. To this end, it is proposed that we should adapt the nanomaterial to the addressed exposure scenario, using ligand-free nanoparticle references in order to differentiate ligand effects from size effects. Furthermore, surface-weighted particle dosing referenced to the biologically relevant parameter (e.g., cell number or organ mass) is proposed as the gold standard. In addition, it is recommended that we should shift the focus of toxicological experiments from 'live-dead' assays to the assessment of cell function, as this strategy allows observation of bioresponses at lower doses that are more relevant for in vivo scenarios.

Assessment of fetal cell chimerism in transgenic pig lines generated by Sleeping beauty transposition.

Human cells migrate between mother and fetus during pregnancy and persist in the respective host for long-term after birth. Fetal microchimerism occurs also in twins sharing a common placenta or chorion. Whether microchimerism occurs in multiparous mammals such as the domestic pig, where fetuses have separate placentas and chorions, is not well understood. Here, we assessed cell chimerism in litters of wild-type sows inseminated with semen of transposon transgenic boars. Segregation of three independent monomeric transposons ensured an excess of transgenic over non-transgenic offspring in every litter. Transgenic siblings (n = 35) showed robust ubiquitous expression of the reporter transposon encoding a fluorescent protein, and provided an unique resource to assess a potential cell trafficking to non-transgenic littermates (n = 7) or mothers (n = 4). Sensitive flow cytometry, fluorescence microscopy, and real-time PCR provided no evidence for microchimerism in porcine littermates, or piglets and their mothers in both blood and solid organs. These data indicate that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during gestation.

Current state of laser synthesis of metal and alloy nanoparticles as ligand-free reference materials for nano-toxicological assays.

Due to the abundance of nanomaterials in medical devices and everyday products, toxicological effects related to nanoparticles released from these materials, e.g., by mechanical wear, are a growing matter of concern. Unfortunately, appropriate nanoparticles required for systematic toxicological evaluation of these materials are still lacking. Here, the ubiquitous presence of surface ligands, remaining from chemical synthesis are a major drawback as these organic residues may cause cross-contaminations in toxicological studies. Nanoparticles synthesized by pulsed laser ablation in liquid are a promising alternative as this synthesis route provides totally ligand-free nanoparticles. The first part of this article reviews recent methods that allow the size control of laser-fabricated nanoparticles, focusing on laser post irradiation, delayed bioconjugation and in situ size quenching by low salinity electrolytes. Subsequent or parallel applications of these methods enable precise tuning of the particle diameters in a regime from 4-400 nm without utilization of any artificial surface ligands. The second paragraph of this article highlights the recent progress concerning the synthesis of composition controlled alloy nanoparticles by laser ablation in liquids. Here, binary and ternary alloy nanoparticles with totally homogeneous elemental distribution could be fabricated and the composition of these particles closely resembled bulk implant material. Finally, the model AuAg was used to systematically evaluate composition related toxicological effects of alloy nanoparticles. Here Ag(+) ion release is identified as the most probable mechanism of toxicity when recent toxicological studies with gametes, mammalian cells and bacteria are considered.

Gold nanoparticles interfere with sperm functionality by membrane adsorption without penetration.

Abstract To examine gold nanoparticle reprotoxicity, bovine spermatozoa were challenged with ligand-free or oligonucleotide-conjugated gold nanoparticles synthesized purely without any surfactants by laser ablation. Sperm motility declined at nanoparticle mass dose of 10 µg/ml (corresponding to ∼14 000 nanoparticles per sperm cell) regardless of surface modification. Sperm morphology and viability remained unimpaired at all concentrations. Transmission electron microscopy showed an modification dependant attachment of nanoparticles to the cell membrane of spermatozoa, but provided no evidence for nanoparticle entrance into sperm cells. A molecular examination revealed a reduction of free thiol residues on the cell membrane after nanoparticle exposure, which could explain the decrease in sperm motility. Sperm fertilising ability decreased after exposure to 10 µg/ml of ligand-free nanoparticles indicating that agglomerated ligand-free nanoparticles interfere with membrane properties necessary for fertilisation. In conclusion, nanoparticles may impair key sperm functions solely by interacting with the sperm surface membrane.

Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development.

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag(+)-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome.

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa.

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.

Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy.

Gold nanoparticles (AuNPs) have the potential to become a versatile biomarker. For further use of AuNPs labeled with functionalized molecules, their visualization in biological systems by routine laboratory tools such as light microscopy is crucial. However, the size far below the diffraction limit affords specialized parameters for microscopical detection, which stimulated the current study, aimed to determine from which size onward AuNPs, either in dispersion or cell-associated, can be reliably detected by standard confocal microscopy. First, gold colloids of size-restricted fractions are examined in dispersion. At a minimum particle size of 60 nm, detection appears to be reliable. Particle counts in dilution series confirm these results by revealing single particle detection of 60-nm colloids. Second, AuNPs are visualized and quantified in cells, which interestingly cause a phase shift in the reflection of AuNPs. Gold mass spectroscopy confirms the number of AuNPs counted microscopically inside cells. Furthermore, it demonstrates for the first time a very high diffusion rate of 15-nm particles into the cells. In conclusion, the results back the suitability of confocal microscopy for the quantitative tracking of colloidal and intracellular gold nanoparticles sized 60 nm.

Preferential loss of porcine chromosomes in reprogrammed interspecies cell hybrids.

Fusion of terminally differentiated somatic cells with pluripotent embryonic stem cells has been proposed as model for reprogramming the somatic cell genome, and may contribute to our understanding of the underlying mechanisms of this epigenetic process. We established an interspecies cell fusion model using murine embryonic stem cells (ESCs) and porcine fibroblasts. These inter-species fusion experiments yielded much lower conversion efficiency rates than murine intraspecies fusion. Nevertheless, two double-resistant mouse-pig hybrid clones could be generated. Reactivation of the porcine OCT4 gene, an essential pluripotent stem cell marker, and demethylation of the porcine OCT4 promoter in hybrid clone 1, suggested successful reprogramming of porcine chromosomes. A rapid loss of porcine chromosomes was observed during the selection phase. Spectral karyotyping (SKY) analysis showed that fusion-hybrid clone 1 carried a tetraploid mouse chromosome complement with only few pig chromosomes and/or chromosomal fragments. Hybrid clone 2 had a diploid set of murine chromosomes complements and also contained an interspecies chromosome fusion product. Interspecies cell fusion results in hybrid cells that retained the complement of mouse chromosomes and preferentially lose porcine chromosomes during colony expansion. Neither species-specific chromosomal segregation nor reprogrammed diploid porcine cells were observed. These findings indicate that generation of reprogrammed pluripotent diploid cells by cell fusion may require additional supporting provisions.

Porcine spermatozoa inhibit post-breeding cytokine induction in uterine epithelial cells in vivo.

Early endometrial cytokine responses after exposure to various inseminate components were investigated for a better understanding of the immunological reactions occurring in the porcine uterus after insemination. Baseline values were established for the mRNA concentrations of GM-CSF, IL-6, IL-10, CXCL8 (interleukin-8), Tumour Necrosis Factor alpha (TNF-alpha), TGF-beta, cyclooxygenase-2 (COX-2) and arachidonate 5-lipooxygenase (ALOX-5) in periovulatory uterine endometrial tissue using quantitative RT-PCR. Synchronized gilts were inseminated with spermatozoa diluted either in the semen extender Androhep or seminal plasma. Uterine infusions of media without spermatozoa were used as controls. Three hours after insemination sows were slaughtered and the expression of the above mentioned cytokines was measured in uterine epithelial cells. Simultaneously, the influx of polymorphonuclear neutrophilic (PMN) granulocytes into the uterus was quantified. Compared to baseline values seminal plasma (SP) and Androhep (AH) respectively, if used alone, caused a significant increase in mRNA concentrations of IL-10 (SP: 1.5-fold), TGF-beta (AH: 1.5-fold), CXCL8 (AH: 7.1-fold), TNF-alpha (AH: 1.9-fold) and COX-2 (AH: 7-fold). Surprisingly, in the presence of spermatozoa, none of the tested cytokines revealed mRNA concentrations higher than baseline values. The number of immigrated, intra-luminal PMN correlated only with mRNA concentrations of CXCL8 in presence of Androhep (r=0.51). None of the other cytokines tested seemed to be involved in the regulation of neutrophil recruitment. However, the most interesting result was the sperm-induced down-regulation in the expression of TNF-alpha, TGF-beta, IL-10, CXCL8 and COX-2 to mRNA concentration levels similar to or even below baseline values. In conclusion the results show that CXCL8 contributes significantly to uterine PMN recruitment and indicate a so far underestimated role of porcine spermatozoa in the general regulation of the uterine post-mating inflammatory response.