Pronuclei Of Zygotes Research Articles

334 EFFECT OF CYTOPLASMIC INJECTION OF dsRNA ON TRANSIENT EXPRESSION OF THE EGFP GENE MICROINJECTED INTO RAT EMBRYOS

RNA interference (RNAi) using double-stranded RNA (dsRNA) is a useful tool to inhibit posttranscriptional specific gene expression in mammalian cells. We investigated the effects of cytoplasmic injection of dsRNAs on transient gene expression in rat embryos when the enhanced green fluorescence protein (EGFP) gene was injected into pronuclei of zygotes. Wistar strain females were superovulated by injections of eCG and hCG and mated with males of the same strain. Pronuclear stage embryos were collected 29 h after hCG injection. The construction of the transgene, the EGFP gene controlled under the CMV-IE promoter, was dissolved at a concentration of 5 μg/mL in DNA injecting buffer (10 mM Tris-HCl, 0.1 mM EDTA), and it (3–5 pL) was microinjected into the pronuclei of embryos. After injection, embryos were cultured in KRB (Toyoda Y and Chang M C. 1974. J. Reprod. Fertil. 36, 9–22) at 37.0°C in 5% CO2 and 95% humidified air until observation. In Experiment I, transient expression of the EGFP gene after microinjection into pronuclei of zygotes was examined. The EGFP expression in the embryos was observed at 6 h intervals until 48 h after the injection using fluorescence microscopy. In Experiment II, the inhibitory effect of dsRNAs targeting the EGFP mRNA was investigated. The dsRNAs for the EGFP were synthesized with T7 RNA polymerase from PCR product amplified with a pair of hybrid primers for the EGFP and T7 RNA polymerase priming site. Then the dsRNAs (1 μg/μL) solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos 1 h after microinjection. As a control, the TE buffer solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos. These embryos were cultured and then observed by fluorescence microscopy 48 h after DNA injection. Fluorescent intensity of the embryos was captured and analyzed with image analyzing software (Scion Image, Scion Corporation, Frederick, MD, USA), and all data were analyzed by χ2 test. In Experiment I, the initiation of the fluorescent embryos was observed 24 h after DNA microinjection, and the proportion of fluorescent embryos in five replicates reached maximum (67%, 106/159) at 48 h after DNA injection. As shown in Table 1, there was a difference (P < 0.05) in the rate of fluorescent embryos between the dsRNA-injected group (18.4%, 33/179; range of 3.0–42.3%) and controls (58.8%, 77/131; range of 42.9–100%) in five replicates of Experiment II. The results of the present study suggest that injection of dsRNAs targeting the EGFP mRNA into the cytoplasm had an inhibitory effect on the transient expression of the EGFP gene microinjected into pronuclei of rat embryos. The inhibition system of the present study provides a powerful tool to study specific gene silencing, gene function, and development of early embryos. Table 1. Effect of cytoplasmic injection of dsRNA on transient expression of the EGFP gene microinjected into rat embryos

331 MODIFIED SINGLE-STRANDED OLIGONUCLEOTIDE-RECOMBINASE COMPLEX MEDIATES GENE TARGETING IN MOUSE EMBRYOS

Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.

131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS

In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.

Localization of Nucleus Organizing Regions (NORs) in One-Cell Mouse Embryos and Oocytes by Fluorescence in situ Hybridization

In mouse zygotes, ribosomal genes (rDNA) are transcriptionally silent and so-called “nucleolar precursor bodies” are present instead of typical nucleoli. However, the functional significance of these structures remains obscure. Specifically, it remains unknown whether structural association between the nucleolar precursor bodies and rDNA are maintained when rDNA synthesis is switched off. Here, we described the rDNA topology in one-cell mouse embryos and MII oocytes using fluorescence in situ hybridization and mouse rDNA probes. Our data suggest that in the pronuclei of one-cell embryos, rDNAs form rather compact clusters, whose number does not exceed that of nucleolus organizing chromosomes characteristic for the haploid set of mouse chromosomes. In zygotic pronuclei, not all nucleolar precursor bodies are associated with rDNA and not all rDNA repeats are attached to the nucleolar precursor bodies. Altogether, these data favor the idea that spatial interactions of nucleolus organizing chromosomes and nucleolar precursor bodies are not obligatory. We assume that associations between nucleolar precursor bodies and nucleolus organizing chromosomal regions are mediated by centromeric heterochromatin. The total numbers of silver stained nucleolus organizing chromosomes in CBA and C57BL mice are different. rDNA genes are unequally distributed among nucleolus organizing chromosomes and nucleolus organizing regions of sister chromatids.

Production of bioproducts through the use of transgenic animal models

Transgenic livestock that produce recombinant proteins in their milk can provide an economic and safe system for production of valuable proteins, such as pharmaceutical proteins for treatment or prevention of human disease or biomaterials for medical use. This method of production is frequently referred to as biopharming. The promise of biopharming, that is the actual commercial production of pharmaceuticals and other bioproducts, is nearing fulfillment. Improvements in molecular and reproductive techniques and strong economic incentives have continued to drive the implementation of transgenic technology to domestic animals. Nuclear transfer using transgenic donor cells is rapidly becoming the predominant technique used in the production of transgenic livestock, replacing the direct injection of DNA into the zygotic pronuclei. Production of transgenic founder animals by nuclear transfer in combination with traditional reproductive technologies can result in the propagation of transgenic herds of sufficient size to meet market demands for commercially important proteins. While some of the companies that have established transgenic programs have run into setbacks owing to a combination of economic, scientific and regulatory difficulties, other companies are continuing to make significant advances. While further improvements are needed to increase efficiencies of production, economically viable production of recombinant proteins using livestock species is not only possible but should be a commercial reality in the very near future.

Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system.

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.

DNA methylation patterns in human tripronucleate zygotes

In mammals, the dynamic reprogramming of DNA methylation begins during gametogenesis and continues through embryogenesis. Recently, immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) has revealed active demethylation of the male pronucleus in zygotes beginning at 4-6 h after fertilization. In this study, we characterized the DNA methylation patterns in mouse zygotes and in human tripronucleate (3 PN) zygotes discarded after conventional fertilization or following ICSI. Pronuclei were subjected to fluorescence in-situ hybridization to identify the X and/or Y chromosomes and then stained with anti-5-MeC. In diandric 3 PN zygotes from conventional IVF, we consistently observed one strongly and two weakly stained pronuclei. In contrast, the majority of 3 PN ICSI zygotes, mainly digynic zygotes, displayed two strongly and one weakly stained pronuclei. Two zygotes from ICSI failed to show any staining difference among the three pronuclei. Our results indicate that the active demethylation of male pronuclei occurs in both mouse and human zygotes. It is possible that the abnormal methylation patterns resulting from a dysfunctional cytoplasm may occur in a small number of oocytes and may affect embryonic viability.

Production of transgenic livestock: promise fulfilled.

The introduction of specific genes into the genome of farm animals and its stable incorporation into the germ line has been a major technological advance in agriculture. Transgenic technology provides a method to rapidly introduce "new" genes into cattle, swine, sheep, and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Methods to produce transgenic animals have been available for more than 20 yr, yet recently lines of transgenic livestock have been developed that have the potential to improve animal agriculture and benefit producers and/or consumers. There are a number of methods that can be used to produce transgenic animals. However, the primary method to date has been the microinjection of genes into the pronuclei of zygotes. This method is one of an array of rapidly developing transgenic methodologies. Another method that has enjoyed recent success is that of nuclear transfer or "cloning." The use of this technique to produce transgenic livestock will profoundly affect the use of transgenic technology in livestock production. Cell-based, nuclear transfer or cloning strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition, and increased disease resistance. One practical application of transgenics in swine production is to improve milk production and/or composition. To address the problem of low milk production, transgenic swine over-expressing the milk protein bovine alpha-lactalbumin were developed and characterized. The outcomes assessed were milk composition, milk yield, and piglet growth. Our results indicate that transgenic overexpression of milk proteins may provide a means to improve swine lactation performance.

Specific Inhibition of Transient and Stable EGFP Gene Expression by Double Stranded RNA Interference in Mouse Preimplantation Embryos

Double stranded RNA (dsRNA) interference is a useful tool for interfering with gene function by promoting the sequence-dependent degradation of targeted mRNA in several organisms. In the present study, in order to confirm and improve the effect of dsRNA, we investigated an inhibitory effect of dsRNA on both transient and stable gene expression of enhanced green fluorescent protein (EGFP) in mouse preimplantation embryos. In the transient expression system, the rates of fluorescent embryos were significantly decreased by co-injection of EGFP dsRNA and EGFP expression vector fragment into the pronucleus of zygotes. In the stable expression system, EGFP expression in transgenic embryos was significantly decreased by injection of EGFP dsRNA into both the pronucleus and cytoplasm of zygotes, but, cytoplasmic injection caused a more significant EGFP inhibition than pronuclear injection. In quantitative PCR analysis, the expression of the EGFP gene was also inhibited by dsRNA injection, whereas the endogenous gene expression was not affected. These data suggest that dsRNA can inhibit the specific gene expression without affecting the development and expression of other genes.

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.

Transgenic Technology in Farm Animals – Progress and Perspectives

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10 −9 to 10 −5 U/nucleus, the amount of 5×10 −8 U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI– Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

Transgenic livestock: premises and promises

Microinjection of DNA constructs into pronuclei of zygotes has been the method of choice for the generation of transgenic livestock. However, this procedure is characterized by low efficiency (1–4% transgenic offspring), random integration and variable expression of the transgene as well as a considerable proportion of mosaicism. Furthermore, it is extremely time consuming and costly. As a consequence, commercial application has focused on the production of recombinant proteins in the mammary gland of transgenic animals and xenotransplantation, e.g. the use of porcine organs in human organ transplantation. In addition, transgenic pigs carrying a modified porcine growth hormone (hMt-pGH) construct show significant improvements in economically important traits without adverse side effects of a GH overproduction. Improvements of transgenic technology will likely come from the generation of appropriate cell lines suitable for transfection or even homologous recombination and their subsequent use in nuclear transfer. Additionally, in the mouse a number of sophisticated molecular tools have been developed that allow precise modifications of the genome. These include the application of artificial chromosomes from yeast (YAC) or bacteria (BAC) for position-independent and copy-number-dependent expression of a transgene, the Tet-system (tetracycline inducible) for a tight temporal control of transgene expression, as well as conditional mutagenesis by applying site-specific DNA recombinases (e.g. Cre, FLP). The successful adaptation of these molecular tools to livestock will enable the fulfilment of many of the promises originally thought to be achievable when transgenic livestock were first reported.

Production of transgenic rabbits using centrifuged pronuclear zygotes.

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.

Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines.

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation.

Removal and restoration of epithelial chloride secretory activity of kinins by gene manipulation

Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract. In this study the chloride secretory activity of lysylbradykinin (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e. which kinin receptors are involved and which chloride channels are responsible for chloride secretion. The approach used was to modify the mice genetically and assess the effects on kinin mediated chloride secretion using voltage clamping at zero potential. Briefly, LBK increased SCC in mouse colon by ∼ 150 μA cm−2 with an EC50 of ∼ 5nM. In null CF mice LBK, 1μM had no effect on chloride secretion, but reduced SCC due to K+ secretion. This effect is normally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion with radioisotopes. Null CF mice produce no cftr, but CFTR was introduced into CF mice by injecting a YAC containing the human CF gene into the pronucleus of CF zygotes. Colonic epithelia from mice with the incorporated YAC showed the same sensitivity to LBK as wild-type tissues and achieved the same maximal chloride secretory response. Colonic epithelia from mice in which the B2r gene had been disrupted showed no response to LBK at normally supramaximally effective concentrations, although responses to other secretagogues were normal. Similarly des-Arg-BK caused no acute chloride secretory response in colonic epithelia from B2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3–6 h. It is concluded that cftr chloride channels and B2rs are required for electrogenic chloride secretion. Further CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion. In vitro, colonic epithelia may generate B2rs which, upon activation, have a minor effect on chloride secretory activity.

Male and Female Genomes Associated in a Single Pronucleus in Human Zygotes

The ploidy of single-pronucleated human zygotes obtained after conventional in vitro fertilization was determined by fluorescent in situ hybridization (FISH) using multiple simultaneous probes for gonosomes and autosomes. After zona removal the single-pronucleated zygotes were exposed to cytochalasin B, and the pronucleus, surrounded by scant cytoplasm and the plasma membrane (karyoplast), was divided from the rest of the egg (cytoplast). The karyoplasts and the corresponding cytoplasts were analyzed separately by FISH. Of the 16 zygotes analyzed, 10 had haploid pronuclei and 6 were diploid. Four diploid pronuclei contained XY chromosomes, and 2 contained XX chromosomes. These results suggest that during the course of their interaction, human gamete nuclei can associate together and form diploid, single-pronucleated zygotes. These findings confirm a newly recognized variation of human pronuclear interaction during syngamy.

Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes1

A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromatic region on the long arm of the human Y chromosome and for unique DNA sequences on human chromosome 19. Chromosome 4 occupied a circumscribed domain in the pronuclei, similar to findings in somatic interphases. Unlike the situation in somatic interphases, the Y heterochromatin was extended throughout the first cell cycle. Pronuclear chromatin was extended 3- to 4-fold compared to somatic interphase chromatin. The extended pronuclear chromatin conformation is likely to affect a zygote's susceptibility to environmental hazards.