日本鰻聯接蛋白Je-Cx34.4與Je-Cx43分子結構之分析與表現之研究

Abstract

本論文首次在日本鰻卵巢組織發現兩種聯接蛋白基因，針對基因序列與其在卵巢發育過程表現量進行初探。細胞內離子及小分子經由聯接蛋白 (Connexin；Cx) 組成的間隙連接 (Gap junctions；GJs) 進行傳遞，參與許多生理機制。從硬骨魚到哺乳類的卵巢發育過程皆需要聯接蛋白之參與。然而，硬骨魚的卵巢Cx於前卵黃生成期的表現或參與機制鮮少討論，日本鰻亦復如此。本研究透過日本鰻卵巢轉錄基因體資料庫篩選出一段Cx核酸片段，將基因進行預測胺基酸分析及分子結構分析，與銀鮭Cx34.3屬同源基因，，此支系屬家族分類α子群，其分子量為34.4 kDa，綜合分析結果將其命名為Je-Cx34.4；另外設計Cx43退化性引子進行PCR，篩選片段拼接後預測其胺基酸與其他已知Cx43有高達95% 相似度，又以硬骨魚更為類似 (98%)，屬家族分類α子群且包含一段Cx43特殊domain，我們將其命名為Je-Cx43。兩基因均預測出穿膜區域，其中包含Cx第三穿膜保守區與羧基端保守片段，且均有預測出磷酸化位點，可推定本研究選殖出日本鰻卵巢中兩種Cx基因片段。組織分佈實驗可觀察到Je-Cx34.4僅表現於卵巢，反觀Je-Cx43至少表現於六種組織中。在親緣關係分析中，Je-Cx34.4隸屬一群可能在硬骨魚獨立分支之Cx家族，而Je-Cx43則與其他物種Cx43為一群，兩分群均屬同一大系群，為α子群。欲了解這兩種日本成鰻Cx在卵巢早期發育階段之表現變化，透過外源性鮭魚腦下垂體誘導催熟日本成鰻卵巢至第六週，分別於各週進行採樣，並藉由計算其生殖腺指數 (Gonadosomatic index；GSI%) 作為卵巢發育之指標。卵巢樣本進行cDNA製備並用於即時定量聚合酶鏈鎖反應 (quantitative PCR；qPCR) 測定其相對表現量。結果顯示Je-Cx34.4與 Je-Cx43其相對表現量分別在各週採樣之間沒有顯著差異。雖然在誘導組別中Je-Cx34.4表現量有上升但無顯著差異，而Je-Cx43則在未誘導與誘導組間均無差異。如果以GSI數值高於1.5 %做為性腺發育判斷依據進行分析 (<1.5 % 或 >1.5 %)，結果顯示，Je-Cx34.4在 > 1.5 % 組別裡有較高表現量 (p<0.05)；而Je-Cx43雖然在 > 1.5 % 組別中有趨緩趨勢，但統計上沒有差異。總結以上結果，我們發現在日本鰻卵巢早期發育階段，至少有兩種Cx表現，且1) Je-Cx34.4表現與性腺成熟有密切關係，在進入卵黃生成階段有較高的表現，2) 雖然Je-Cx43的基因表現隨著性腺發育持續表現但變化不大，其生理機制以及與哺乳類之差異有待進一步實驗與討論。

Gap junctions (GJs), composed of connexin (Cx) protein subunits, allow direct communication through conduits between neighboring cells in vertebrate. From teleost to mammals, throughout oogenesis, cell-cell communication via GJs between oocytes and surrounding follicle cells, and/or amongst follicle cells is required for successful follicular development. However, the functions and regulation of ovarian GJs or Cx during earlier stages of oogenesis, such as previtellogenic and vitellogenic stages, mostly remain to explored. To gain a fundamental understanding of ovarian GJs in teleost, we identified 7 Cx genes in our Japanese eel (Anguilla japonica) ovarian cDNA library. One of the gene transcripts designated Je-Cx34.4 were confirmed by polymerase chain reaction (PCR); In addition, Cx43, a highly conserve Cx gene in vertebrates could retrieve partial sequence through PCR with degenerated primers from ovary cDNA, we designated Je-Cx43. Tissue distribution showed that Je-Cx34.4 was only expressed in ovary, by contrast Je-Cx43 were at least expressed in six tissues. Phylogenetic analysis shows that Je-Cx34.4 may be the Cx subfamily only in teleost; Je-Cx43 was similar to other species Cx43, especially. To study early oogenesis of Japanese eel, fish received weekly intraperitoneal injections of salmon pituitary extracts (SPE). Ovarian tissue were sample weekly and gonadosomatic index (GSI%) was used as an ovary development status. Changes in gene expression across the early oogenesis were determined by using quantitative real-time PCR (qPCR). Transcription of Je-Cx34.4 was not different during every week of induction, same results seem in Je-Cx43. Although transcript of Je-Cx34.4 increase in induction group, but was not significantly different to control group. By contrast Je-Cx43 did not have significantly difference between induction group and control group. However fish had various response to SPE induction, in accordance with a dividing point, GSI 1.5%, Je-Cx34.4 was significantly higher in GSI > 1.5% group (p<0.05); Je-Cx43 had a tendency to increase in GSI > 1.5% group, but without statistically significant difference. Our findings suggest that in the early stage of eel ovary, Je-Cx34.4 is an ovary specific Cx and may correlated with folliculogenesis; Je-Cx43 mRNA expression did not change but still had it appearance in early stage of oogenesis.