Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.

Mechanical properties are vital for living cells, and various models have been developed to study the mechanical behavior of cells. However, there is debate regarding whether a cell behaves more similarly to a “cortical shell – liquid core” structure (membrane-like) or a homogeneous solid (cytoskeleton-like) when experiencing stress by mechanical forces. Unlike most experimental methods, which concern the small-strain deformation of a cell, we focused on the mechanical behavior of a cell undergoing small to large strain by conducting microinjection experiments on zebrafish embryo cells. The power law with order of 1.5 between the injection force and the injection distance indicates that the cell behaves as a homogenous solid at small-strain deformation. The linear relation between the rupture force and the microinjector radius suggests that the embryo behaves as membrane-like when subjected to large-strain deformation. We also discuss the possible reasons causing the debate by analyzing the mechanical properties of F-actin filaments.

Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.

Zebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.

A method was developed, which enables substances to be injected into the feeding structure (syncytium) established by Heterodera schachtii in roots of Arabidopsis thaliana. The technique was used to study the uptake of nutrients by the feeding nematode. The fluorescent dye lucifer yellow CH (LYCH) and fluorescence-labelled dextrans of different molecular weights were injected into the thin and translucent roots of A. thaliana. Such roots are a feature of this plant and they provide optimal conditions for microinjection. Injected LYCH was taken up by feeding juveniles and adults, indicated by the staining of the alimentary duct and the digestive system. Fluorescent dextrans of 3, 10 and 20 kDa but not of 40 and 70 kDa were ingested, suggesting that molecules of a maximum Stokes radius of 3·2 to 4·4 nm are taken up. It is likely that the feeding tube, forming the interface between the plant cytosol and the nematode's digestive system, is responsible for this size exclusion effect. The injected fluorescent substances were not detected in plant cells adjacent to the syncytium or in the root vascular elements. Injections into parts of roots which were infested by several nematodes revealed that feeding H. schachtii individuals may share one syncytium.

Spermatozoa obtained from hybrid male mice were treated with solubilised zonae pellucidae after a period of capacitation in vitro to induce the acrosome reaction. Single spermatozoa were selected and microinjected into the perivitelline space of mature oocytes. A high proportion of the spermatozoa acrosome-reacted affter treatment with solubilised zonae (63%). However, the fertilisation rate (37%) after subzonal microinjection of oocytes by the treated spermatozoa was not different to the fertilisation rate (40%) of oocytes microinjected with untreated spermatozoa which had a lower rate of acrosome reaction (39%). When spermatozoa were washed by high-speed centrifugation before treatment with solubilised zonae pellucidae and subzonal microinjection, the fertilisation rate (68%) was significantly higher (p < 0.001) than that (29%) for occytes microinjected with untreated spermatozoa and was found to be correlated with a high acrosome reaction rate (74%) (r = 0.8). The washing of spermatozoa by centrifugation itself did not increase the acrosome reaction rate or fertilisation rate of oocytes after microinjection. The results of this study suggests that some modifications other than the acrosome reaction are needed to enable capacitated mouse spermatozoa to fuse with the oocyte plasma membrane. These modifications were achieved by washing spermatozoa by high-speed centrifugation and the replacement of the supernatant with fresh culture medium used for capacitation. Induction of the acrosome reaction by solubilised zonae pellucidae following this treatment leads to a high fertilisation rate of oocytes by subzonal sperm microinjection.

Polyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.

Intact mouse sperm or mouse sperm tails alone, labelled with MitoTracker Green FM® fluorochrome, were injected into mouse oocytes and the cells cultured in vitro for up to 5 days. The dye stained midpiece mitochondria, the sperm tail coarse fibres and the sperm perforatorium. Intact sperm (or tails injected with separated heads) induced normal embryonic development. The mitochondria could be identified in embryos up to the 4-cell stage, remaining associated with the sperm tail. They largely disappeared by the 8-cell stage, when only a minority of embryos (6/43) could be found with small patches of mitochondria. Axonemal elements could be identified coiled up in single external blastomeres as late as day 5 blastocysts. By contrast, mitochondria as well as tail components could be identified up to 5 days after injection of sperm tails alone into non-activated oocytes and also in embryos that arrested development before the 8-cell stage. We conclude that disappearance of the labelled sperm mitochondria in normally cleaving embryos is not due to fading or inactivation of the fluorochrome marker, but is rather an event specifically tied to cell cycle activities around the second cell division.

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro.

The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (105.16 TCID50/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24–48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV–FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).

After microinjection of BVDV under the ZP, significantly more (p < 0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV–FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos.

These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.

A 5′-flanking region and partial open reading frame (ORF) of the β-actin gene (GenBank accession No. EF026001) of Nile tilapia (Oreochromis niloticus) was cloned by polymerase chain reaction (PCR) amplification. The segment included a 1643 bp regulatory sequence and a 90 bp partial ORF which encoded a 30-amino-acid peptide. The regulatory sequence comprised a 108 bp 5′ proximal promoter, the first untranslated exon and the first intron of the β-actin gene. The proximal promoter region contained elements that were critical for transcription activity, including a CCAAT, TATA and CArG box located at –92, –29 and –62 bp upstream of the transcription initiation site, respectively. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector to construct the expressing vector pNA-DsRed. The linearized pNA-DsRed was microinjected into the fertilized eggs of Tanichthys albonubes. The expression of the DsRed2 gene in transgenic fish could be observed under the microfluoroscope and anatomical lens. The results showed that the β-actin gene promoter possessed effective transcription activity.

The present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocyte–cumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14 h after human chorionic gonadotropin (hCG) injection. Cumulus nuclei were injected into ovulated oocytes and then stimulated in the presence of 5 mM SrCl2 for 20 min at various time points (0–3.5 h) after injection. Some of the reconstituted eggs were cultured to observe the pronuclear formation, cleavage, and blastocyst formation. The incidences of eggs forming at least one pronucleus or containing two pronuclei were not significantly different among the periods (82.4–83.5% and 43.4–51.9%, respectively). Nor did the incidences of eggs cleaving (86.7–97.7%) and developing to the blastocyst stage (0–3.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 1–1.5 h after injection, 71.7% of the eggs caused premature chromatin condensation, but only 46.2% of them formed two spindles around each of maternal and somatic chromatins. However, the morphology of the somatic spindles differed from that of the spindles, which formed around the oocyte chromatins. Only 7.5% of the eggs contained the normal chromosomal number. In many reconstituted oocytes, before activation, an abnormal spindle formation was observed in the somatic chromatins. In conclusion, these results show that non-enucleated rat oocytes injected with cumulus nuclei can form pronuclei and cleave following chemical activation, whereas blastocyst formation is very limited, probably caused by abnormalities in the spindle formation and distribution of somatic chromatids.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19–20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94–96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p<0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi-and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p<0.001) and lower total cell number (p<0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

Tubulin from fresh pig brain was extracted through two cycles of polymerization and depolymerization, purified by chromatography on an ion-exchange column and labelled with 5(6)-carboxytetramethylrhodamine succinimidyl ester. We prepared 10.8 mg/ml rhodamine-labelled tubulin probe (dye/protein=0.6) and then microinjected it into living guard cells in leaves of Vicia faba L. After 5–10 min, a network of microtubules (MTs) could clearly be observed in the guard cells under a confocal laser scanning microscope. In opening guard cells, the cortical MTs radiated from the ventral wall to the dorsal wall and in closed guard cells the radial MTs were completely depolymerized into random and diffuse fragments. This shows that pig brain tubulin can easily be incorporated into MTs in guard cells, and that the dynamic behaviour of MTs can be examined directly in vivo during stomatal movement.

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.