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Abstract. The changes in histone acetylation are not always consistent in various cell types and at different developmental stages. We immunostained specific antibodies against acetylated lysine 9 of histone H3 and acetylated lysines 5 and 12 of histone H4 in an effort to understand the detailed changes in histone acetylation during sheep oocyte meiosis. We found that the acetylation fluorescence signals of H3/K9 and H4/K12 on chromatin appeared intensively in the germinal vesicle (GV), late-GV (L-GV), and germinal vesicle breakdown (GVBD) stages and became weak in metaphase I (MI); however staining reappeared in anaphase I-telophase-I (AI-TI) and metaphase II (MII). Furthermore, staining was detected in the first polar bodies. The fluorescence signals of H4/K5 first appeared in the MI stage and became intensive in the AI-TI stage; however they were barely detectable in MII stage chromosomes and first polar bodies. We conclude that the acetylation patterns of H3/K9 and H4/K12 during oocyte meiotic maturation are similar and that the pattern of H4/K5 is unique. Key words: Histone acetylation, Meiotic maturation, Sheep oocyte, Trichostain A (J. Reprod. Dev. 53: [555][556][557][558][559][560][561] 2007) h e b a s i c s t r u c t u r a l u n i t o f e u k a r y o t i c chromosomes is a DNA protein complex called the nucleosome that consists of 147 base pairs of DNA wrapped around an octamer of the H2A, H2B, H3, and H4 histone proteins. The nucleosome histones are thought to play important roles in various cellular functions. It is well known that post-translational acetylation, methylation, phosphorylation, and ubiquitination of histones play an intrinsic role in transcription regulation [1,2]. In these post-translational modifications, h i s t o n e a c e t y l a t i o n a f f e c t s c h r o m a t i o n conformation [3], correlates with gene activity [4,5], and is required for orderly meiosis [6]. The Nterminal tails of histone H3 and H4 have a critical role in the folding of higher order chromatin structure [7][8][9]. The different effects of histone acetylaion on chromatin organization have been analyzed both in vitro and in vivo [10,11] and in mitosis and in meiosis [12]. Recent results show that the acetylation of different lysines of histone is associated with a diversity of chromatin-related processes in mitosis [13] and is necessary for orderly meiosis [6]. The lysine residue-specific changes in histone acetylation during pig oocyte meiosis have distinctive characteristics [6,15] compared with those of mouse oocytes [14].In the present study, we investigated the changes in acetylation of lysine K9 of histone H3, and lysine K5, K12 of histone H4 in sheep oocytes at different stages of maturation.

Podophyllotoxin is used as medical cream which is widely applied to genital warts and molluscum contagiosum. Although previous study showed that podophyllotoxin had minimal toxicity, it was forbidden to use during pregnancy since it might be toxic to the embryos. In present study we used mouse as the model and tried to examine whether podophyllotoxin exposure was toxic to oocyte maturation, which further affected embryo development. Our results showed that podophyllotoxin exposure inhibited mouse oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on oocytes was dose-depended. Further studies showed that the meiotic spindle formation was disturbed, the chromosomes were misaligned and the fluorescence signal of microtubule was decreased, indicating that podophyllotoxin may affect microtubule dynamics for spindle organization. Moreover, the oocytes which reached metaphase II under podophyllotoxin exposure also showed aberrant spindle morphology and chromosome misalignment, and the embryos generated from these oocytes showed low developmental competence. We also found that the localization of p44/42 MAPK and gamma-tubulin was disrupted, which further confirmed the effects of podophyllotoxin on meiotic spindle formation. In all, our results indicated that podophyllotoxin exposure could affect mouse oocyte maturation by disturbing microtubule dynamics and meiotic spindle formation.

Cytoplasmic dynein is a family of cytoskeletal motor proteins that move towards the minus-end of the microtubules to perform functions in a variety of mitotic processes such as cargo transport, organelle positioning, chromosome movement and centrosome assembly. However, its specific roles during mammalian oocyte meiosis have not been fully defined. Herein, we investigated the critical events during porcine oocyte meiotic maturation after inhibition of dynein by Ciliobrevin D treatment. We found that oocyte meiotic progression was arrested when inhibited of dynein by showing the poor expansion of cumulus cells and decreased rate of polar body extrusion. Meanwhile, the spindle assembly and chromosome alignment were disrupted, accompanied by the reduced level of acetylated α-tubulin, indicative of weakened microtubule stability. Defective actin polymerization on the plasma membrane was also observed in dynein-inhibited oocytes. In addition, inhibition of dynein caused the abnormal distribution of cortical granules and precocious exocytosis of ovastacin, a cortical granule component, which predicts that ZP2, the sperm binding site in the zona pellucida, might be prematurely cleaved in the unfertilized dynein-inhibited oocytes, potentially leading to the fertilization failure. Collectively, our findings reveal that dynein plays a part in porcine oocyte meiotic progression by regulating the cytoskeleton dynamics including microtubule stability, spindle assembly, chromosome alignment and actin polymerization. We also find that dynein mediates the normal cortical granule distribution and exocytosis timing of ovastacin in unfertilized eggs which are the essential for the successful fertilization.

Objective To determine the safety and effectiveness of a cross‐linked sodium hyaluronate (CHA) scaffold in cartilage repair. Methods Physicochemical properties of the scaffold were determined. The safety and effectiveness of the scaffold for cartilage repair were evaluated in a minipig model of a full‐thickness cartilage defect with microfracture surgery. Postoperative observation and hematological examination were used to evaluate the safety of the CHA scaffold implantation. Pathological examination as well as biomechanical testing, including Young's modulus, stress relaxation time, and creep time, were conducted at 6 and 12 months postsurgery to assess the effectiveness of the scaffold for cartilage repair. Furthermore, type II collagen and glycosaminoglycan content were determined to confirm the influence of the scaffold in the damaged cartilage tissue. Results The results showed that the routine hematological indexes of the experimental animals were within the normal physiological ranges, which confirmed the safety of CHA scaffold implantation. Based on macroscopic observation, it was evident that repair of the defective cartilage in the animal knee joint began during the 6 months postoperation and was gradually enhanced from the central to the surrounding region. The repair smoothness and color of the 12‐month cartilage samples from the operation area were better than those of the 6‐month samples, and the results for the CHA scaffold implantation group were better than the control group. Greater cell degeneration and degeneration of the adjacent cartilage was found in the implantation group compared with the control group at both 6 and 12 months postoperation, evaluated by O'Driscoll Articular Cartilage Histology Scoring. Implantation with the CHA scaffold matrix promoted cartilage repair and improved its compression capacity. The type II collagen level in the CHA scaffold implantation group tended to be higher than that in the control group at 6 months (2.33 ± 1.50 vs 1.68 ± 0.56) and 12 months postsurgery (3.37 ± 1.70 vs 2.06 ± 0.63). The GAG content in the cartilage of the control group was significantly lower than that of the experimental group (2.17 ± 0.43 vs 3.64 ± 1.17, P = 0.002 at 6 months and 2.27 ± 0.38 vs 4.12 ± 1.02, P = 0.002 at 12 months). Type II collagen and glycosaminoglycan content also demonstrated that CHA was beneficial for the accumulation of both these vital substances in the cartilage tissue. Conclusions The CHA scaffold displayed the ability to promote cartilage repair when applied in microfracture surgery, which makes it a promising material for application in the area of cartilage tissue engineering.