Capturing epigenetic dynamics during pre-implantation development using live cell imaging

During mammalian fertilization and pre-implantation development, the highly differentiated gametes revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in male and female gamete chromatin structure, in global epigenetic modifications and in nuclear architecture. We have developed a live cell imaging technique for oocytes and early embryos to understand these series of phenomena. Using this technique, we were able to observe dynamic changes in DNA methylation status in living embryos. Furthermore, epigenetic abnormalities were detected in reconstructed embryos generated by round spermatid injection or by somatic cell nuclear transfer. In this review, I will discuss the usefulness and possibilities of this imaging technique in studies on nuclear dynamics during fertilization and pre-implantation development.     

Mouse offspring after microinjection of heated spermatozoa.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56 degrees C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.     

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.     

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.     

Production of transgenic mice following deoxyribonucleic acid microinjection and embryo freezing

Experiments with mouse embryos were designed to assess the feasibility of freezing embryos after DNA microinjection. One-cell pronuclear stage mouse embryos were microinjected with cloned deoxyribonucleic acid (DNA) and cultured in vitro to the late eight-cell stage. Microinjected and matched control embryos were frozen and stored in liquid nitrogen. Following thawing, embryos were cultured for 8 h and transferred to recipient females. In a separate set of experiments, embryos were transferred to recipients immediately following DNA microinjection. Control (uninjected) embryos developed to the late eight-cell stage significantly better than surviving microinjected embryos. Of the embryos thawed, 76% of the microinjected and 60% of the control embryos survived to be transferred to recipients. Progeny were obtained with similar survival rates from both groups following embryo transfer with transgenic mice identified among the progeny from microinjected embryos. Mouse embryos can be microinjected with DNA, cultured in vitro, frozen, thawed, transferred to recipients and transgenic progeny can be obtained.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.     

Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.     

Qualitative changes in protein synthesis associated with polyspermic fertilization of human eggs

To investigate the early molecular events in human oocytes that are triggered by fertilization, the authors examined the pattern of polypeptides synthesized by unfertilized and dispermic embryos obtained through an in vitro fertilization and embryo transfer (IVF-ET) program. Compared with unfertilized oocytes of the same postovulatory age, the de novo protein synthesis in tripronuclear dispermic zygotes (21 hours postinsemination) was characterized by the appearance of three novel protein bands with molecular weights of 41.2, 35.3, and 26.0 kD. Concomitant with these changes, these zygotes showed the disappearance of bands at 54.0, 36.5, and 28.0 kD, along with the decreased synthesis of a protein band at 42.5 kD. Although 24% of the aged unfertilized oocytes exhibited bands corresponding to 41.2 and 35.3 kD, the 26.0 kD protein is restricted to the tripronuclear embryos. The significance of these results is discussed in relation to the use of polyspermic human oocytes as a model for the study of the early molecular events triggered by fertilization.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

<Emphasis Type="Italic">In vitro</Emphasis> fertilization and preimplantation development in the primate

Nonhuman primates represent a strong model for examining the chromosomal, biochemical, and temporal normality of embryos produced byin vitro fertilization. Morein vitro fertilized embryos from the squirrel monkey(Saimiri sciureus) have been produced and examined than with all other primate species combined. In studies over a 13 year period a fertilization rate approximating 60 % has been developed in this species with 30% of these embryos proceeding to the two cell stage and 50% of these to the three-four cell stage. Chromosomal abnormalities (primarily missing or extra chromosomes) at a level of nine to 16% have been found, a value corresponding to that found inin vivo mating andin vitro fertilization in other species. An incidence of triploidy of 16.7% was observed. RNA and protein synthetic rates appear comparable with those of laboratory species subjected toin vitro fertilization and indicate the initial stages of metabolic activity of the newly formed embryo. Similarly, increases in estrogen incorporation appear after fertilization but no effect is observed in progesterone incorporation. Utilizing 2-deoxy-glucose and insulin, it was determined that the glucose requirement as an energy source for early preimplantationin vitro fertilized primate embryos is very low.Of very great importance is the temporal relationship of the development ofin vitro fertilized squirrel monkey embryos compared with similar development in other primates (including humans) afterin vivo andin vitro fertilization. An analysis of over a decade of work with the squirrel monkey embryos demonstrates a pattern of temporal development that is comparable with all other primate species that have been examined (including the human) and comparable with development afterin vivo fertilization.     

Sperm nuclear decondensation in mammals: role of sperm-associated proteinase in vivo

Previous studies from this (Zirkin et al., '80) and other (Marushige and Marushige, '78) laboratories have shown that proteinase associated with mammalian sperm nuclei is involved in thiol-induced sperm nuclear decondensation and protamine degradation in vitro. The results of these in vitro studies suggested the exciting possibility that the sperm nucleus itself might contribute proteinase involved in its subsequent in vivo decondensation during fertilization. In the present study, microinjection methods were used to test this possibility directly. Control hamster sperm nuclei, which exhibited proteinase activity, decondensed when incubated in vitro with disulfide reducing agent. As expected, these nuclei also decondensed when microinjected into ovulated hamster oocytes and formed morphologically normal pronuclei. When the proteinase associated with isolated sperm nuclei was removed with 0.5 M salt or inhibited with nitrophenyl-p-guanidinobenzoate, the nuclei were rendered incapable of decondensing in response to disulfide reducing agent in vitro. However, when these nuclei were microinjected into eggs, they decondensed and transformed into pronuclei. These results provide direct evidence that sperm-associated proteinase is not required for sperm nuclear decondensation and formation of the male pronucleus during fertilization.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs

The oviducts of both oestrous and pseudopregnant rabbits can be used for the successful culture of mammalian embryos for short periods. This has alowed some selection to be made on the embryos as they are examined on at least two occasions before final transfer. Not only have pregnancy rates been normal, but in some instances they have been higher following a limited period (2–3 days) in the rabbit oviduct. It would appear that these higher pregnancy rates result from a more intensive selection of embryos at the time of transfer rather than from some substance acquired during storage in the oviduct. However, the system is not without disadvantages. There is some loss of embryos (15–30%) in the oviduct and all embryos recovered may not have developed at the normal rate.The rabbit oviduct has been used as a site of xenogenous fertilization. Initial reports indicate that success in that area is lower than when using large animals as the site of fertilization. With more widespread interest in the use of microsurgery in embryos, the rabbit oviduct has been used for the short term storage of agar cylinders and has been found to be unsuitable because of the high rate of degeneration of agar chips. However, the rabbit oviduct is still useful as an experimental tool in the manipulation of embryos from the domestic species.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

The levels and nature of translation in mouse preimplantation embryos with repressed cytokinesis]

The translational activity of embryos cultured up to the 8-cell stage in cytochalasin D-supplemented medium was studied. These embryos remained unicellular during the entire preimplantation period. Blastocoel formation and hatching began in the cytochalasin-treated embryos at the same time as in the control. We have studied the overall translation rates of "one-cell embryos" and estimated the relative translational differences for individual polypeptides compared to the control embryos. Up to the early blastocyst stage, the translation levels in these embryos, measured by 35S-methionine incorporation, were about two times lower than in the control. At later stages, the differences were fivefold. The ppm values of approximately one-third of all individual spots on two-dimensional electrophoregrams differed three times or more between the control and cytochalasin-treated embryos. These results suggest that the first morphogenetic events in mammalian ontogenesis may be controlled autonomously and the timing of morphogenetic transitions is controlled by acquisition of a definite set of stage-specific factors which serves as the signal for subsequent development.     

GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles

BACKGROUND INFORMATION: Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication machinery involved in the process of DNA elongation, recombination, methylation and repair. We have used PCNA fused with green fluorescent protein (GFP-PCNA) as a convenient tool to show the progress of S-phase in single embryos in vivo. Here we make a comparison between Hoechst 33342 and GFP-PCNA as in vivo event markers for DNA synthesis. Hoechst 33342 and DAPI (4,6-diamidino-2-phenylindole) have been used as a simple and rapid method for assessing membrane permeability and staining DNA in mammalian cells. However, it is difficult to use these dyes in living embryos during cell cycle progression studies over long periods of time as they are phototoxic. Moreover, though Hoechst staining reveals nuclear morphology, it gives no information about the progress of S-phase. RESULTS: We have microinjected or expressed a GFP-PCNA chimera to develop a method which enables visualization of S-phase in sea urchin and Caenorhabditis elegans embryos during the first and subsequent embryonic cell cycles and in Drosophila stage 4 embryos during syncytial nuclear divisions. We find that nuclear accumulation of GFP-PCNA correlates with S-phase onset. Loss of the chimera from the nucleus occurs when the nuclear envelope breaks down at mitosis. CONCLUSIONS: GFP-PCNA is a accurate and non-toxic marker of S-phase in embryos during early development.