Effects of histone deacetylase inhibitors on the early development of bovine androgenetic embryos.

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In our previous study, we enhanced sperm chromatin remodeling within the bovine sperm injection-derived androgenentic (SpI-AG) embryos by sperm pretreatment, and thereby improved their early developmental competence. In this study, we found that blastocyst development of SpI-AG embryos could be elevated by the histone deacetylase inhibitor (HDACi). First, we optimized the efficacy of two histone deacetylase inhibitors [trichostatin A (TSA) and Scriptaid (SCR)] in a dose (0, 5, 10, 20, 50, and 100 nM for TSA; 0, 50, 100, 200, 300, and 500 nM for SCR, respectively) and time-dependent (0, 10, 15, 20, and 25 h) manner on the developmental capacity of these embryos. Furthermore, we quantitatively assessed the alterations in histone H3 and H4 overall acetylation levels and blastocyst quality of SpI-AG embryos by immunofluorescence staining. We found a significantly improved morula and blastocyst development rate of SpI-AG embryos at a mild dose of TSA (20 nM) or SCR (200 nM) for 15 h after embryo activation. Furthermore, both HDACi noticeably increased the levels of acetylated histone H3 and H4 in SpI-AG blastocyst embryos, whereas, SCR treatment improved the quality of blastocysts when compared with control group. In conclusion, HDACi is beneficial for early development of bovine SpI-AG embryos and can be used to improve the efficiency of its in vitro production.

Association analysis of HSP70A1A haplotypes with heat tolerance in Chinese Holstein cattle.

Single-nucleotide polymorphisms (SNPs) in the coding and untranslated regions of heat shock 70 kDa protein 1A (HSP70A1A), an inducible molecular chaperone that is responsible for cellular protection against heat stress, have been reported as being associated with heat tolerance. A fragment of the HSP70A1A gene was amplified in Chinese Holstein cattle and eight novel mutations were found. We performed comprehensive linkage disequilibrium (LD) and haplotype analyses of the eight SNPs of the HSP70A1A gene and examined their involvement in heat resistance in 600 Chinese Holstein cattle. Our results revealed the presence of significant differences between individuals carrying haplotype 1 and those without haplotype 1 for most of the heat-tolerance traits. Haplotype 1 increased the risk of heat stress; however, association analysis of its combination with haplotype 2 showed the lowest rectal temperature and red blood cell K(+) level, moderate respiratory rate, and the highest red blood cell NKA level, suggesting a heterozygote advantage in the penetration of the phenotype. Protein expression levels in white blood cells among haplotype combinations further confirmed the hypothesis that heterozygotes for haplotypes 1 and 2 are more sensitive to heat stress. We presume that these mutations may be useful in the future as molecular genetic markers to assist selection for heat tolerance in cattle.

Sperm capacitation combined with removal of the sperm acrosome and plasma membrane enhances paternal nucleus remodelling and early development of bovine androgenetic embryos.

The androgenetic embryo is a useful model for functional analysis of the paternal genome during embryogenesis. However, few studies have focused on the factors involved in the suppressed developmental competence of such embryos or why sperm cloning-derived androgenetic embryos fail to develop beyond the morula stage in large domestic animals. To overcome this developmental failure, we tried to improve sperm decondensation, as well as to enhance embryonic development by sperm capacitation and removal of the acrosome and plasma membrane before injection of the spermatozoa. Before injection of the spermatozoa, we quantified the effects of sperm capacitation combined with sperm pretreatment on the acrosome and plasma membrane status. We also evaluated sperm decondensation potential, sperm viability and chromatin integrity. Immunostaining data showed that the sperm acrosome and plasma membrane could be more efficiently removed after capacitation. Dithiothreitol-induced sperm decondensation potential was improved with capacitation and removal of the acrosome and plasma membrane. Although most spermatozoa lost viability after pretreatment, their chromatin remained integrated. The patterns of paternal chromatin remodelling within uncleaved androgenetic embryos and the nucleus morphology of cleaved embryos indicated that capacitation combined with membrane disruption could make injected spermatozoa decondense synchronously not only with each other, but also with the developmental pace of the ooplasm. We successfully produced androgenetic blastocysts, and efficiency increased with sperm pretreatment. In conclusion, sperm decondensation and the early development of androgenetic embryos were enhanced with sperm capacitation and removal of the acrosome and plasma membrane prior to sperm injection.

[Active DNA demethylation in mammals].

DNA methylation is a stable and heritable epigenetic mark, and it is one of the best characterized epigenetic modifications. Active DNA demethylation has been reported both in plant and animal cells, and the mechanism behind it is becoming clear in plant. Whereas a bona fide enzyme, which is responsible for active DNA demethylation, have not been identified in mammals, and active demethylation pathway is controversial. In the present review, we described that active DNA demethylation take place in a spatial- and temporal-specific way on the basis of recent literatures. Moreover, several candidate pathways such as oxygenation and deamination of 5-methyl cytosine and DNA repair pathways, which may be responsible for the active process were introduced on a cell- and tissue-specific view. The aim of this paper is to help re-searchers reveal the mechanism underlying this important event during epigenetic reprogramming in mammals.