During the mid-twentieth century Conrad Waddington proposed his ontogenic concept of canalization, and suggested that epigenetic mechanisms might serve as a means of metastable memory imposed on the genetic material during differentiation. Subsequently, Waddington's theories have received substantial acknowledgment during the first decade of the twenty-first century, with numerous studies unraveling the important contributions of epigenetic mechanisms and the epigenomic control of differentiation. The early embryogenesis and primordial germ cell development have received much attention as developmental stages with extensive epigenomic reprogramming. In both developmental phases, the epigenomic landscape of chromosomes is rapidly and dramatically altered, and this goes along with a substantial change in the developmental potential of cells. In this chapter, a summary is provided of the current knowledge of epigenetic reprogramming mechanisms. These are discussed in line with biological concepts, and with the role of DNA methylation in particular.


Keywords:

Zygote;
Chromatin;
Epigenome;
Genomic imprints;
Epigenetic reprogramming

Epigenetic Reprogramming in Mammalian Development

Nuclear Cloning and Epigenetic Reprogramming of the Genome

Recent developments in studies of sperm-mediated gene transfer (SMGT) now provide solid ground for the notion that sperm cells can act as vectors for exogenous genetic sequences. A substantive body of evidence indicates that SMGT is potentially useable in animal transgenesis, but also suggests that the final fate of the exogenous sequences transferred by sperm is not always predictable. The analysis of SMGT-derived offspring has shown the existence of integrated foreign sequences in some cases, while in others stable modifications of the genome are difficult to detect. The appearance of SMGT-derived modified offspring on the one hand and, on the other hand, the rarity of actual modification of the genome, suggest inheritance as extrachromosomal structures. Several specific factors have been identified that mediate distinct steps in SMGT. Among those, a prominent role is played by an endogenous reverse transcriptase of retrotransposon origin. Mature spermatozoa are naturally protected against the intrusion of foreign nucleic acid molecules; however, particular environmental conditions, such as those occurring during human assisted reproduction, can abolish this protection. The possibility that sperm cells under these conditions carry genetic sequences affecting the integrity or identity of the host genome should be critically considered. These considerations further suggest the possibility that SMGT events may occasionally take place in nature, with profound implications for evolutionary processes.

Sperm-mediated gene transfer: Applications and implications

Transgenic rabbits as therapeutic protein bioreactors and human disease models.

Over the past 30 years many cryopreservation procedures have been applied to oocytes, embryos, sperm, ovarian and testicular tissue. Over this time many, often specialized, terms have been developed for all aspects of these procedures. This can make it difficult for readers who are not familiar with the terminology or protocols to compare and evaluate different procedures. This paper describes the main cryopreservation procedures, the terminology associated with them, and briefly explains the underlying physical, chemical and biological processes. The aim is to help readers understand and interpret other papers on slow cooling, rapid cooling, ultrarapid cooling and vitrification.

Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos

Background
Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation.

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Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

Sperm-mediated gene transfer was used to produce transgenic rabbits that expressed the porcine growth hormone gene under the control of a metallothionein promoter. The gene that encodes the selectable marker green fluorescent protein (GFP) was inserted downstream of the transgene. After lipofectin-mediated gene transfer into sperm cells and after subsequent in vitro fertilization using the transfected sperm cells, 32% of the cultured blastocysts exhibited bright green fluorescence when stimulated with blue light. Of the 74 adult rabbits and five fetal rabbits (age, gestational day 15), 2 fetuses and 29 rabbits were GFP-positive as indicated by PCR analysis. Southern blot analysis of their genomic DNA showed that 13 of 21 GFP-positive rabbits were transgenic. GFP expression was observed in different tissues of transgenic rabbits and the growth rate of four GFP-positive rabbits was greater than that of controls. PCR analysis showed that one of six F1 offspring was transgenic. These results suggest that lipofectin-mediated gene transfer into sperm cells can be used to efficiently produce transgenic rabbits.

Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein.

An insertion fragment in porcine FSHbeta subunit gene was cloned by PCR. Sequencing data show that the insertion is a retroposon of 292 bp sitting in intron I at the site between + 809 and +810 base. Based on these results, a PCR programme was created to genotype animal individuals in different pig breeds at FSW locus and polymorphism of FSHbeta gene was analyzed. With the combination of genotype and litter size of sows, it was demonstrated that FSHbeta locus is closely associated with major gene controlling litter size in commercial pig breeds, such as Yorkshire, Landrace, Durco. Averagely the AA sows give more 1.5 piglets than BB sows do per litter.

FSHB subunit gene is associated with major gene controlling litter size in commercial pig breeds.

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization

The ability of rabbit spermatozoa to bind exogenous DNA during sperm-mediated gene transfer (SMGT) was tested in our study. Fresh collected semen, or fully capacitated sperm cells, was co-cultured with plasmid DNA labeled with tetramethyrodamine-6-dUTP. Fluorescent spermatozoa were counted before and after DNaseI treatment. Results showed that fluorescent-labeled plasmid DNA could be taken up by capacitated rabbit sperm cells. 66% spermatozoa carried exogenous DNA in the presence of lipofectin. Bovine serum albumin could block this process effectively. Associated DNA was mainly located in the posterior area of the sperm head. In order to verify whether exogenous DNA was carried into the embryo and expressed in the offspring, further SMGT experiments were carried out using the pHM-CR plasmid which contains LacZ and Neomycin genes. beta-galactosidase was expressed in different stages of embryo development and in the tissues of young rabbit as detected by using X-gal staining. Large portion of embryos survived under the selection pressure in G418 containing medium, after SMGT. Transgene integration was further verified by PCR analysis. These results confirmed the ability of rabbit sperm cells to carry transgene into the embryo during in vitro fertilization.

Y. F. Chen

Papers

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein.

FSHB subunit gene is associated with major gene controlling litter size in commercial pig breeds.

Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization

Association of rabbit sperm cells with exogenous DNA.