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.

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The objective of the present study was to gain insight into the epigenetic mechanisms involved in all‐trans retinoic acid (ATRA) and histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu)‐induced Npr1 gene (coding for GC‐A/NPRA) expression. The studies were carried out in rat thoracic aortic smooth muscle cells cultured in DMEM containing 10% FBS and treated with ATRA and NaBu for 24 h. The results showed that ATRA and NaBu induced Npr1 promoter activity by 12‐fold and mRNA levels by 5‐fold in an additive manner. Simultaneous treatment with ATRA and NaBu significantly increased the levels of histones H3K9/14ac, H4K12ac, and H3K4me3 (p<0.001). Overexpression of class I HDACs (HDAC 1 and HDAC 2) reduced Npr1 promoter activity by 50%. ATRA and NaBu inhibited HDAC activity by 50%. Moreover, ATRA and NaBu cooperatively enhanced histone acetyltransferse activity. The results demonstrate that ATRA and NaBu regulate Npr1 gene expression by modulation of histone modifications. The identification of epigenetic signaling as a regulator of Npr1 gene will have important implications in prevention of hypertension and cardiac remodeling.

Atrial natriuretic peptide (ANP) exerts its antihypertensive effects by binding to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which generates the second messenger cGMP. The present study was aimed at understanding the epigenetic signaling governing Npr1 (coding for GC‐A/NPRA) gene transcription. The mouse mesangial cells were cultured in DMEM containing 10% FBS and ITS (insulin, transferrin, and sodium selenite), transfected using Lipofectamine‐2000, and treated with histone deacetylase (HDAC) inhibitors. Luciferase assay showed that trichostatin A (TSA, pan‐inhibitor) and mocetinostat (MGCD0103, class I inhibitor) induced Npr1 promoter activity by 8‐fold and 10‐fold, Npr1 mRNA levels by 4‐ and 5.3‐fold, and protein expression by 2.5‐ and 3‐fold, respectively. TSA and MGCD0103 inhibited HDAC activity by 25% and 50%, respectively. Furthermore, TSA and MGCD0103 enhanced acetylation and binding of H3‐K9/14, H4‐K12, and Sp1 to Npr1 promoter. Taken together, the present results demonstrate that TSA and MGCD0103 enhanced Npr1 gene expression via inhibition of HDAC1 and HDAC2 and increased histone H3, H4, and Sp1 acetylation. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis. This work was supported by the NIH grant (HL57531).

Mice carrying targeted-disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) exhibits hypertension, cardiac, and kidney hypertrophy, and congestive heart failure. The objective of the present study was to determine the role of (pro)renin receptor (P)RR in the kidneys of Npr1 gene-disrupted (-/-; 0-copy), and wild-type (+/+; 2-copy) mice. Disruption of Npr1 gene leads to increased systolic blood pressure by 36mmHg in 0-copy mice (138.4 ± 3.6) as compared with wild-type mice (102.2 ± 3.2 mm Hg). The renal (P)RR protein and mRNA levels were increased by 3-fold (4.8 ± 0.7 vs.1.3±0.4) and 3.5-fold (5.0 ± 0.5 vs.1.9 ± 0.3), respectively, in 0-copy mice compared with 2-copy control mice. To identify the Angiotensin II-independent mechanism of (P)RR, angiotensin converting enzyme (ACE) inhibitor, captopril and angiotensin II receptor-1 (AT1R) antagonist, losartan were administered to all Npr1 mice genotypes. The protein expression of ACE (3.9 ± 0.6 vs.1.9 ± 0.4; 2-fold) and AT1R (4.5 ± 0.4 vs.1.7; 2.5-fold) were decreased in 0-copy treated mice compared with 0-copy control mice, whereas the (P)RR expression was not altered by both inhibitors. Further, expression of phospho-extracellular regulated kinases (p-Erk1/2, 5.3 ± 0.7 vs 1.6 ± 0.5; 3-fold) and phospho-p38 (p-p38, 8.0 ± 0.8 vs. 2.1 ± 0.7; 4-fold) were increased in 0-copy mice as compared with 2-copy control mice. Treatment with Erk1/2 inhibitor U0126 and p38 inhibitor SB203580 attenuated the levels of p-Erk1/2 (6.8 ± 0.5 ± vs.1.1 ± 0.3; 6-fold) and p-p38 (4.7 ± 0.4 vs. 1.6 ± 0.5; 3-fold) in 0-copy treated mice as compared with 0-copy control mice. Captopril and losartan did not alter the expression levels of p-Erk1/2 and p-p38 in 0-copy mice as compared with 0-copy treated mice. The present results suggests that absence of NPRA/cGMP signaling causes increased expression of (P)RR and leads to MAPKs activation in the Npr1 null mutant mice.