日本鰻生長分化因子-9 (GDF-9) 基因選殖與其在人工誘導性成熟時卵巢發育的表現

Abstract

在人工養殖下的鰻魚性腺發育會發生停滯，無法進一步發育，加上野生性鰻苗資源量逐年遞減，因此人工誘導日本鰻性腺成熟乃為必要之策略。使用鮭魚腦下垂體研磨液 (SPH) 進行打針誘導鰻魚成熟的方法已經有四十年的歷史。但鰻魚的人工誘導成熟有一個主要的問題，就是以 SPH 進行打針誘導後，卵細胞發育有不同步化的情形。根據本實驗室先前研究發現，甲基睾固酮 (MT) 併用 SPH 的打針處理和單獨使用 SPH 皆能有效推動卵巢發育的進行，但 SPH 併用 MT 的處理較能有效增加多數卵細胞的存活率，且卵發育較能夠同步化。SPH 對於日本鰻卵巢成長發育是必須的，而 MT 在人工誘導過程時的作用是否直接調控卵巢發育則有待釐清。本研究關注於卵細胞自身分泌的生長分化因子-9 (growth differentiation factor 9, GDF-9)，其對於早期濾胞發育相當重要。本研究選殖出日本鰻 GDF-9 的基因序列，得知 cDNA 的序列全長為 2135 個鹼基，可轉譯出 430 個胺基酸，預測出的胺基酸序列與其他硬骨魚類的 GDF-9 相同度約在 38 %~62 % 之間。本研究提出，在 SPH 及 MT 的誘導下，MT 會與 GDF-9 進行交互作用，進而調控 SPH 對日本鰻卵巢發育的影響，以及 GDF-9 可能為雄性素下游調控基因。為驗證此假說，實驗將經過海水馴化後的鰻魚分別以不同外源性激素經兩針及六針的打針誘導，雄性素阻抗劑氟他胺 (flutamide) 則併用外源性激素進行打針處理，並以即時定量反轉錄 PCR (real-time RT-PCR, RT-qPCR) 分析 GDF-9 與濾胞刺激素受體 (follicle stimulating hormone receptor, FSHR) 在卵巢中的基因表現量。研究結果發現，SPH 併用 MT 相較於單獨使用 SPH 能維持 GDF-9 與 FSHR 的基因表現量。單獨使用 MT 也能顯著促進 GDF-9 與 FSHR 的基因表現量，且 GDF-9 和 FSHR 的基因表現量在荷爾蒙誘導下呈現良好正相關，顯示在人工誘導下 GDF-9 與 FSHR 間存在交互作用。氟他胺能顯著抑制由 SPH 所誘導之 GDF-9 的表現。組織切片中發現注射六針 SPH 併用 MT 組中卵徑較大 (280~300μm) 的卵濾胞多數仍停留在油滴期 (oil droplet stage)，反觀 SPH 組在相似的卵徑下 (280μm) 卻已經進入卵黃蓄積期 (vitellogenic stage)。綜合以上結果推論，MT 或許可促進 GDF-9 的表現，並進而提升 FSHR 的表現量，促進卵巢內較小卵濾胞加速濾胞生成，較大的卵濾胞也會因為 GDF-9 的表現量上升而抑制類固醇生合成 (steroidogenesis) 進行，延長前卵黃蓄積期 (pre-vitellogenesis stage)，使油滴得以充分累積，提高卵質，同時使整體卵細胞發育較同步化。此結果顯示 SPH 與 MT 會藉由 GDF-9 的作用而調控日本鰻卵巢發育，然而 GDF-9 在日本鰻卵巢中最早是何時開始表現，以及其對卵濾胞的直接調控機制仍有待更進一步研究。

In captivity, the Japanese eels can not undergo further gonadal development, and the population size of wild elvers had gradually decreased year by year. In the silver Japanese eel, artificial induction of gonadal development and maturation by injection of salmon pituitary homogenate (SPH) has been applied for 40 years. One of the major problems on the eel artificial maturation is that, the development of ovarian follicles are rather asynchronous during artificial maturation by SPH injection. But according to the previous in vivo induction data in our laboratory, although both of 17α-methyltestosterone (MT) combined with SPH (SPH+MT treatment) and SPH alone (SPH treatment) induction could promote ovarian development, SPH+MT enhanced the survival and synchronized ovarian follicles development. Previous results reveal that SPH is essential, and MT may directly regulate ovarian development during induction of sexual maturation. Current research focus on growth differentiation factor 9 (GDF-9) which is one of the oocyte-secreted factors, and it is essential for early follicle development. We have already obtained the cDNA encoding for GDF-9 from female Japanese eel. The overall sequence of GDF-9 cDNA isolated from Japanese eel contained 2135 bp, and this GDF-9 cDNA encodes 430 amino acids of peptide sequence. The deduced amino acid sequence shares 38 %-62 % identity with other teleost GDF-9. We propose that MT would interact with GDF-9 to regulate the SPH action in ovarian development during SPH and MT induction in Japanese eel, and GDF-9 might be regulated by androgen receptor. Cultivated female Japanese eels obtained from local supplier were acclimated to seawater. After acclimation, eels were intraperitoneal injected respectively with different exogenous hormone for 2 times and 6 times at 1-week interval. Another treatment, eels were intraperitoneal injected with exogenous hormone above combined with anti-androgen flutamide. Real-time RT-PCR (RT-qPCR) was employed to investigate the ovarian gene expression of GDF-9 and follicle stimulating hormone receptor (FSHR). Our data showed that the ovarian GDF-9 expression in SPH alone treatment declined significantly after two injections. By contrast, the supplement of MT (SPH+MT) could maintain the expression level after two injections. MT alone treatment could significantly increase the expression level compared with control group (SW control). The gene expression of GDF-9 and FSHR in hormone induction showed the significantly positive correlation, and it implied the interaction between GDF-9 and FSHR. Flutamide could significantly inhibit the SPH-induced GDF-9 expression. In SPH+MT group after six injections, most of larger follicles with larger diameter (280~300μm) were still at the oil droplet stage, on the other hand, those of follicles by SPH tretment with similar oocyte diameter (280μm) had entered the period of yolk accumulation. In conclusion, MT might induce GDF-9 expression and then increase FSHR expression level, and thus promoted folliculogenesis in smaller follicles within the ovary, and the steroidogenesis in larger follicles would be inhibited by the amount of GDF-9 expression. As result, pre-vitellogenic stage could be prolonged, so that oil droplets could be fully cumulated, egg quality could also be improved, and the development of ovarian follicles could be more synchronized. These results imply that the interaction of SPH and MT might be mediated through GDF-9 action to affect ovarian development in Japanese eel. However, when is the initiation of GDF-9 expression in the ovary of Japanese eel, and how does GDF-9 regulate the development of ovarain follicles in Japanese eel needs further elucidation.