Effect of drug treatment on the α-tubulin acetylation
As indicated by mean pixel intensity after immunofluorescence staining (Fig. 1), the dynamic pattern of α-tubulin acetylation gradually decreased from MI to MII to PN syngamy. The intensity of α-tubulin acetylation treatment with tubacin were stronger than control in MI and MII (P<0.05), but they were not different in PN syngamy (P>0.05).
Effect of increased α-tubulin acetylation on spindle morphology
GV oocytes displayed a large number of α-tubulin in the cortex (Fig. 2A). The normal spindle was barrel shaped or slightly pointed (Fig. 2B,D,E,H), and the abnormal spindle was disorganized, asymmetrical, round or elongated (Fig. 2C,F,G,I). With increased α-tubulin acetylation, the normal spindle was higher than the control in MI and syngamy (87.73 % vs. 70.93 % and 22.22 % vs. 11.94 %, P<0.05; Table 2), and the abnormal spindles were lower than the control in MI and MII (4.94 % vs. 20.25 % and 17.26 % vs. 35.65 %, P<0.05; Table 2). These suggest that the increased α-tubulin acetylation have a critical effect to improve microtubules after meiotic apparatus assembly.
Effect of increased α-tubulin acetylation on mitochondrial distribution and fertilization process
The mitochondria aggregation in the vicinity of treated nucleus was more obvious than the control (47.02 % vs. 25.59 %, P<0.05; Table 3). After in vitro fertilization, The mitochondria aggregation was associated with the pronucleus (Fig. 3B) or syngamy chromosome (Fig. 3C). With increased α-tubulin acetylation, the syngamy proportion was higher than the control (16.04 vs. 6.15, P<0.05; Table 4). These indicated that increased α-tubulin acetylation can promote mitochondria concentration and pronucleus formation.
Effect of increased α-tubulin acetylation on meiotic maturation and IVF embryos development
The rates of maturation and blastocyst on the treated groups were higher than the control (72.90 ± 5.13 % vs. 64.06 ± 2.87 %; 13.28 ± 1.79 % vs. 9.80 ± 1.93 %; P<0.05; Table 5). However, there were not different in 2-cells and total number of blastocyst cells between two groups (P>0.05). These showed that the functional effects of increased α-tubulin acetylation can improve in vitro maturation and fertilization development.
Effect of increased α-tubulin acetylation on the transcription of related genes
From MI to MII, the expression of αTAT1, MAP2, HSP90 and Katanin was significantly up-regulated (P<0.05), but the expression of TUBA1A was significantly down-regulated (P<0.05) (Fig. 5). At the MI stage, the TUBA1A transcription in the treatment had significantly higher levels than the control (P<0.05) (Fig. 5A). The αTAT1 and MAP2 patterns of the MII oocytes in the treatment had significantly higher expression than the control (P<0.05) (Fig.5B,5C). The results support a molecular mechanism of increased α-tubulin acetylation for recruiting MAPs to improve the configuration of microtubule dynamics. The above results indicated that increased α-tubulin acetylation promoted the binding of tubulin to MAPs and motor proteins, which improved microtubule configuration, maturation and fertilization.