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.

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.

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.

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.

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.

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.

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos.

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal-epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.

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.

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.

Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa.

Using bovine embryos generated in vitro from IVF with X-sorted, Y-sorted and unsorted spermatozoa, we compared the kinetics of male and female embryo development and gene expression between male and female blastocysts. Bovine in vitro-matured oocytes (n = 8858) were fertilised with spermatozoa from each of three different bulls (X-sorted, Y-sorted or unsorted spermatozoa depending on the experiment). The cleavage rate was assessed 24, 27, 30, 33, 36, 40, 44 and 48 h post insemination (h.p.i.) and blastocyst development was recorded on Days 6-9. The relative mRNA abundance of nine genes (GSTM3, DNTM3A, PGRMC1, TP53, BAX, COX2, IGF2R, AKR1B1 and PLAC8) was analysed in male and female Day 7 blastocysts produced with sorted and unsorted spermatozoa from one bull. Cumulative cleavage rate and blastocyst yield were significantly higher in the unsorted group compared with the X- or Y-sorted group from the same bull (P < or = 0.05). Although differences existed between bulls in terms of cleavage rate, no differences were observed in cleavage rate between X- and Y-sorted spermatozoa within a bull. The blastocyst yield was significantly higher only for Bull 3 when the Y-sorted spermatozoa were used (27.1+2.8 v. 19.1+1.4 for Y- and X-sorted spermatozoa, respectively; P < 0.05). There were no differences in the mRNA abundance of the nine genes analysed between embryos of the same sex produced with sorted or unsorted spermatozoa. However, significant differences in polyA mRNA abundance were observed between male and female blastocysts for three genes (GSTM3, DNMT3A and PGRMC1; P < or = 0.05). In conclusion, the use of sorted rather than unsorted spermatozoa in IVF significantly delays the onset of first cleavage. Differences were noted between bulls, but not between X- and Y-sorted spermatozoa, and although no differences were found in terms of the mRNA abundance of the nine genes tested between sorted and unsorted spermatozoa, sex-related differences were found in the case of three genes.

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.

Sperm sex-sorting in the Asian elephant (Elephas maximus).

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro.

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa.

IVF of porcine oocytes has been carried out in many laboratories. However, polyspermic fertilization is still a major issue to be solved. It is well known that besides the nucleus, oocyte organelles and the cytoplasm have to undergo a final maturation process before they become fully competent for fertilization. Until now, it is still uncertain whether the zona pellucida (ZP) must also undergo a maturation process and what impact the maturation status may have on sperm recognition and monospermic fertilization. Our data show that the ZP undergoes biochemical changes in the final maturation phase of the oocyte prior to fertilization. During zona maturation, the induction of the acrosome reaction in spermatozoa bound to the zona pellucida shows a different time pattern. Additionally, it was shown by 2D gel electrophoresis that after maturation, ZPA moved 0.8 pI units and ZPB/ZPC 1.3 pI units in the direction of the anode, indicating increased acidity. These preliminary studies indicate that the maturation processes of the oocyte involves biochemical and functional alterations in the zona pellucida. In addition, the morphology of the porcine ZP was investigated before and after maturation at the GVI and metaphase II stage as well as 1h after onset of IVF. No significant consistent structural changes were seen between immature oocytes and those matured in vitro for 48 h. However, at 24 h, the zona structures were more similar to those in in vivo matured oocytes. This phenomenon needs to be elucidated. So far, the only way to avoid polyspermic penetration is to reduce the number of spermatozoa per oocyte used for IVF. The amount depends on the treatment of the sperm and has to be set for each individual boar.

Preselection of sex of offspring in swine for production: current status of the process and its application.

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure.

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.

Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro.

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laser-scanning microscopy, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [3H]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with alpha-amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with alpha-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitro-produced embryos.

Production of piglets using intracytoplasmic sperm injection (ICSI) with flowcytometrically sorted boar semen and artificially activated oocytes.

The purpose of the present study was to develop a protocol for the successful production of piglets employing intracytoplasmic sperm injection (ICSI) with flowcytometrically sexed spermatozoa and artificially activated porcine oocytes. In vitro matured oocytes were fertilized by ICSI using non-sorted frozen/thawed epididymal semen. Oocytes were either activated by CaCl(2), Ca(2+)-ionophore or electrical pulse. Activation and fertilization rates of sperm injected oocytes stimulated by CaCl(2)-injection were significantly higher than those without activation (70.4% versus 45.9%; 49.9% versus 33.2%, respectively; P<0.001). Activation rate of sham injected oocytes increased in parallel (11.2% versus 26.3%, P<0.05), parthenogenetic development remained low (2.8% versus 8%). Co-incubation in Ca(2+)-ionophore did not improve activation rates as compared to non-activated oocytes (44.8% versus 42.5%). Fertilization rate decreased as compared to non-treated sperm injected oocytes (36.8% versus 24.5%, P<0.05). Activation of oocytes with a single electrical pulse resulted in significantly higher activation rates in all groups of oocytes as compared to non-stimulated ones (sperm injected oocytes: 65.6% versus 43.1%, P<0.001; sham injected oocytes: 48.5% versus 5.6%, P<0.001; control oocytes: 50.7% versus 0.0%, P<0.001). Fertilization rates (32.3% versus 48.2%) and parthenogenetic development (0.7% versus 38.9%, 0.0% versus 30.9%, P<0.001) increased significantly in parallel. In addition, in four replicates of flowcytometrically sorted Y-chromosome bearing spermatozoa were injected into in vivo matured oocytes, activated with 1.2 pl of a 30 mM CaCl(2) solution. On average 85.3 fertilized oocytes were transferred surgically into four recipients. Pregnancies delivered a total of 13 male piglets. These are the first piglets born from ICSI with sorted spermatozoa.