鳜仔稚鱼牙齿发育和矿化过程观察

Tooth development and mineralization in mandarin fish(Siniperca chuatsi)at early stage

为了解鳜(Siniperca chuatsi)牙齿的早期发育和矿化过程,采用解剖镜和光学显微镜技术观察了孵化后1~55 d仔稚鱼牙齿出现时间、列数和数目变化,通过软骨-硬骨染色、Von Kossa染色观察了牙齿矿化时间,采用X射线能谱仪测定了牙齿矿化过程中钙、磷元素含量的变化,采用免疫组化方法观察了碱性磷酸酶蛋白表达变化,并用荧光定量PCR方法检测了细胞间隙连接蛋白43(Cx43)和钙结合蛋白-d28k(CaB)基因表达量变化。结果表明:孵化后3 d时鳜颌齿出现,5 d时咽齿出现,8 d时腭齿出现,12 d时犁齿出现;孵化后8 d时下颌齿列数发育完全,11 d时上颌齿列数发育完全;颌齿中,绒毛状齿先发育,犬齿孵化后18 d开始发育;牙齿矿化时间为孵化后11~14 d,矿化过程中钙、磷元素含量显著增加(P<0.05),而钙/磷比值无明显变化(P>0.05);孵化后10、12 d时,碱性磷酸酶蛋白在牙釉质、牙本质中呈阳性反应;Cx43和CaB基因相对表达量均在孵化后10、12 d时有升高,仅CaB基因显著上升(P<0.05),14 d时两基因均显著下降(P<0.05)。研究表明,鳜早期牙齿出现,能直接咬住、固定活饵鱼,防止其逃脱,而钙、磷元素和矿化相关蛋白均参与了牙齿矿化过程。

In order to understand tooth development and mineralization process of mandarin fish(Siniperca chuatsi),the changes in tooth occurrence time,number and column number were observed from 1 day old to 55 days old larva and juveniles after hatching by anatomical microscope and light microscopy at water temperature from 23℃to 25℃.The tooth mineralization time was observed by cartilage bone staining and Von Kossa staining,and the contents of calcium and phosphorus were determined during tooth mineralization by X-ray energy spectrometer.The changes in alkaline phosphatase protein expression were observed by immunohistochemical method,and the expression levels of connexin 43(Cx43)and Calbindin-d28k(CaB)were detected by fluorescence quantitative PCR.The results showed that the maxillary tooth and mandibular tooth were found at 3 days post hatch(dph),the pharyngeal tooth at 5 dph,the palate tooth at 8 dph and the vomerine tooth at 12 dph.The mandibular dentition were fully developed at 8 dph,while maxillary dentition at 11 dph.In the maxillary and mandibular tooth,villous tooth developed firstly,canine tooth began to develop at 18 dph,with completely mineralized teeth from 11 dph to 14 dph.During the tooth mineralization,the contents of Ca and P were shown to be increased significantly(P<0.05),without significant difference in Ca/P(P>0.05).Positive reaction was detected in the enamel and dentin at 10 dph and 12 dph.Relative expression levels of Cx43 and CaB genes were increased at 10 dph and 12 dph,with a significant increase for CaB gene(P<0.05),and significant decrease in expression levels of both genes at 14 dph(P<0.05).The findings indicate that the early teeth of the mandarin fish can directly bite and fix live baitfish to prevent them from escaping.In the process of tooth mineralization,calcium,phosphorus and mineralization-related proteins are all involved.