Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Embryo Mitochondrial DNA Depletion Is Reversed During Early Embryogenesis in Cattle

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA ( mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.

Hydrocoryne iemanja (Cnidaria), a new species of Hydrozoa with unusual mode of asexual reproduction

Hydrocoryne iemanja sp. nov. was found in an aquarium, growing on rhodoliths of coralline algae collected on the southeastern coast of Brazil (20 degrees 40`S 40 degrees 2`W). The colonies were reared through maturity in the laboratory. Each colony had up to 7 sessile, long and thin monomorphic zooids, very extensible and flexible, arising from a chitinous, hard dark-brown plate with minute spines. Medusae budded from near the basal part of hydrocaulus, and were released in immature condition, acquiring fully developed interradial gonads 5-7 days after release. Asexual reproduction by longitudinal fission was observed on the hydrocaulus of the polyps, both for those in normal condition and those with injuries. Fission started at the oral region, extending aborally, with a new hard plate formed in the basal part of hydrocaulus. When fission reached the new hard plate, the new polyp detached, becoming free and sinking to the bottom, starting a new colony. Detached polyps were morphologically indistinguishable from other polyps, being able to produce medusae. Mother and daughter polyps undertook subsequent fissions. This mode of longitudinal fission is distinct from other modes of longitudinal fission, a process known for a few species Of cnidarians. Further studies of this process may shed light on the understanding of the evolutionary pathways in Cnidaria and animals. Hydrocoryne iemanja sp. nov. is distinguishable from its two congeners by the distinct marginal tentacles of the medusae-short and with a median nematocyst knob-an unambiguous character useful even for the identification Of newly liberated medusae.

Effects of competition on sexual and clonal reproduction of a tunicate: the importance of competitor identity

Individual fitness and the structure of marine communities are strongly affected by spatial competition. Among the most common space holders are the colonial ascidians, which have the ability to monopolize large areas of hard substrate, overgrowing most other competitors. The effects of competition on colony growth and on gonad production of the ascidian Didemnum perlucidum were studied in southeastern Brazil by experimentally removing surrounding competitors. Colonies of D, perlucidum competing for space exhibited a growth rate 9 times less than that of colonies that were competitor free. Among the colonies subject to competition, growth rates were unrelated to the percentage of colony border that was free of competitors. However, the identity of the competitor was important in the outcome of border contacts. At the beginning of the experiment, most border encounters of D. perlucidum were with solitary organisms, which in most cases were overgrown. These were progressively replaced by colonial ascidians and bryozoans, resulting mostly in stand-off interactions. Besides reducing asexual growth, spatial competition also affected female gonad production. Colonies free of competitors had a significantly higher proportion of zooids with ovaries. Thus, our findings show that spatial competition reduces both ascidian colony size and gonad production.