184 ALTERNATIVE SPLICING OF DNA METHYLTRANSFERASE 1 IN PORCINE OOCYTES

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as of cloned embryos. The objective of this study was to identify and sequence isoforms of Dnmt1 expressed in porcine oocytes. Total RNA was isolated from pools of 50 denuded mature oocytes as well as from fibroblast cells using the Trizol method. RNA was co-precipitated with glycogen, and residual genomic DNA was removed with DNase I. A RACE System (Invitrogen, Carlsbad, CA, USA) was used to amplify the 5′ cDNA end of Dnmt1. Briefly, the first strand of cDNA was synthesized using SuperScript II and a Dnmt1-specific primer followed by degradation of the RNA strands and incorporation of TdT and dCTP tails to the 3′ ends of the cDNA. Tailed cDNA was amplified by PCR using a forward anchor primer and a reverse Dnmt1-specific primer. PCR products were separated by electrophoresis on an agarose gel. Resulting PCR products were subcloned into a cloning vector, and the cDNA inserts were sequenced. PCR primers capable of amplifying all possible alternatively spliced isoforms of Dnmt1 were used to identify the presence of the RNA sequences found in fibroblasts and oocytes of pigs. Two distinctive bands (290 and 390 bp) were observed after 5′RACE, PCR, and electrophoresis of oocyte Dnmt1 cDNA. Only 1 band of 290 bp was observed after amplification of fibroblast cDNA. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa Dnmt1 genomic sequence located on chromosome 2 (CU462940). The smaller 290-bp band, amplified from both oocytes and fibroblasts, had an identical DNA sequence (EU908731). Structure analysis of the larger 390-bp band indicates that this oocyte Dnmt1 isoform has an additional exon located between exon 1 and 2 of the somatic form of Dnmt1 (EU908730). PCR products amplified using primers specific for the oocyte or somatic transcript verified the presence of the additional exon in the oocyte Dnmt1 splicing variant. In conclusion, this study shows that porcine oocytes express an alternative isoform of Dnmt1 in addition to the somatic transcript. The 2 identified isoforms are produced by alternative splicing of the Dnmt1 gene. Analysis of the oocyte and somatic Dnmt1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

Embryo and Endosperm Development Is Disrupted in the Female Gametophytic capulet Mutants of Arabidopsis

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.

The Mouse Xist Gene: a Model for Studying the Gametic Imprinting Phenomenon

In mammals, normal embryonic development requires differential genomic imprinting of male and female gametes [1, 2]. Many investigations have been directed towards the understanding of the molecular mechanisms of imprinting and the timing of establishment of the imprint during gametogenesis and its erasure during development.Methylation is the focus of many of these studies as it has been known for some time that this epigenetic modification of the DNA correlates with the status of gene activity. So far, five imprinted genes, expressed from only one of the parental alleles, have been found to be differentially methylated in somatic tissue: mouse Igf2 [3] and Xist [4] and human SNRPN [5, 6] expressed from the paternal allele; mouse Igf2r [7] and H19 [8, 9] expressed from the maternal allele. However, so far, a gametic methylation imprint has been detected for only two of these genes: in an intron region of mouse Igf2r [7], and in the promoter region [10] and the first exon [11] of the Xist (X-inactivation-specific transcript [12, 13] gene.The data accumulated for the Xist gene, during different phases of gametogenesis and development, provides the most comprehensive story about the role of methylation as a primary gametic imprint, and on the timing of its establishment during gametogenesis and erasure during development. Methylation studies have now been performed during oogenesis and spermatogenesis [Norris et al., 1994; 11] and in mature gametes and during early stages of development [10, 11]. In addition, expression of the gene has been described during gametogenesis [14-16] and throughout early development [4-17].

Endosperm development in Zea mays; implication of gametic imprinting and paternal excess in regulation of transfer layer development

Fertilisation in maize (Zea mays), in common with most angiosperms, involves two fusion events: one of the two sperm nuclei unites with the egg cell nucleus, while the other sperm nucleus fuses with the two central cell nuclei giving rise to the triploid endosperm. Since deviation from this nuclear ratio (2:1 maternal/paternal) in the endosperm can result in abortion, it has been suggested that the genomes of the sperm and/or central cell are differentially imprinted during sexual development. By crossing a normal diploid maize line as female with its autotetraploid counterpart, an unbalanced genomic ratio (2:2 maternal/paternal) is created in the endosperm which often results in the eventual abortion of the tissue. Detailed structural comparison of these aberrant endosperms with normal endosperms reveals that the formation of the transfer cell layer, a tissue formed some 8 days after pollination and responsible for the transport of nutrients into the endosperm, is almost completely suppressed under conditions of paternal genomic excess. The first structural analysis of the development of this tissue in normal and aberrant endosperms is reported, and the implications of regulating the formation of such a tissue by gametically imprinted genes are discussed in the light of current theories on the consequences of genomic imbalance on early embryonic development.

Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring

Genomic imprinting occurs in both male and female gametes during gametogenesis, but the exact time when imprinting begins and ends is unknown. In the present study we injected nuclei of testicular spermatozoa and round spermatids into mature mouse oocytes to see whether these nuclei are able to participate in syngamy and normal embryonic development. If the injected oocytes develop into normal fertile offspring, imprinting in the male germ cells used must have been completed by the time of injection. Ninety-two percent of mouse oocytes injected with testicular spermatozoa survived and 94% of these were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei). When 44 two-cell embryos so created were transferred to 5 foster mothers, 24 (54.5%) developed into normal offspring. Unlike testicular spermatozoa, round spermatids could not activate the oocytes, and therefore the oocytes had to be activated artificially either before or after spermatid injection. The highest rate (77%) of normal fertilization was obtained when the oocytes were first activated by electric current, then injected individually with a single spermatid nucleus. When 131 two-cell embryos were transferred to 15 foster mothers, 37 (28.2%) reached full term. All but two grew into healthy adults. Thus, it would appear that gametic imprinting in mouse spermatogenic cells is completed before spermiogenesis begins. Under the experimental conditions employed, spermatid nuclei were less efficient than testicular sperm nuclei in producing normal offspring, but perhaps this was due to technical rather than inherent problems.