123 HISTONE H3 MODIFICATIONS IN PIG OOCYTES DURING GROWTH, MATURATION, AND ACTIVATION

Abstract

Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting for producing the mature egg. The first sign of oocyte maturation is phosphorylation of histone H3, which leads to the chromosome condensation (Bui et al. 2004 Biol. Reprod. 70, 1843-1851). The objective of this study was to investigate the change in chromosome morphology in relation to histone modifications in pig oocytes during growth, maturation, and activation. Growing oocytes were collected from follicles at various diameters (from 0.1 to 6 mm) in pig ovaries. For maturation, oocyte-cumulus-granulosa cell complexes (OCGC) were collected from follicles that were 3 to 6 mm in diameter and cultured in modified TCM 199 for different periods of time to obtain meiotic stages of oocytes. For activation, oocytes were cultured for maturation in 42 h and were activated using a protocol that was described previously (Nguyen et al. 2003 Theriogenology 59, 719-734). Then, oocytes were examined by immunostaining with antibodies: anti-phospho-histone H3 at serine 10 or serine 28 (S10 or S28), anti-trimethyl-histone H3 at lysine 9 (K9), and anti-acetyl-histone H3 at lysine 9, 14, or 28 (K9, K14, or K28). Some oocytes were examined for double assay of Cdc2 and H3 kinase, which were measured by phosphorylation of histone H1 and myelin basic protein as their substrates. To examine the effects of histone deacetylase (HDAC) inhibition, OCGC were cultured in maturation medium supplemented with or without 100 nM trichostatin A for 42 h. The results show that, during the growth phase, histone H3 became methylated at K9 and is acetylated at K9, K14, and K18. When the fully grown oocytes start maturation, histone H3 becomes phosphorylated at S28 and then S10 and is deacetylated at K9, K14, and K18. After oocyte activation, reacetylation and dephosphorylation of histone H3 correlates to the decondensation of chromosomes. We also found that the activity of histone H3 kinase occurred at a similar time course to that of phosphorylation of histone H3-S28. This suggests that phosphorylation of H3-S28 might be one of the key events initiating meiotic chromosome condensation. The inhibition of HDAC induces maintenance of acetylation of H3-K14 and dephosphorylation of histone H3 at S10 and S28. Therefore, the chromosome could not condense and affect meiotic progression. It is possible that deacetylation is required for the phosphorylation of histone H3. The results suggest that chromatin morphology of pig oocytes is regulated by acetylation/deacetylation and phosphorylation/dephosphorylation of histone H3 and that histone deacetylase activity is essential for the process of chromatin remodeling in pre-ovulatory oocytes. Although histone acetylation and phosphorylation were reversible, histone methylation has energetic stability and is established during the oocyte growth phase. It is also suggested that the ordered phosphorylation of histone H3 at S10 and S28 is influenced by acetylation of neighboring lysines in the histone H3 molecule.