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1 中國畜牧學會會誌,39(3):147~164, 2010 生長因子對猪卵母細胞成熟與胚發育之影響 (1) 簡亞嵐 (2) 羅能文 (1)(3) 朱志成 豬胚體外生產系統 (in vitro production, IVP) 包括卵母細胞體外成熟 (in vitro maturation, IVM) 體外受精(in vitro fertilization, IVF) 與卵母細胞激活或受精後胚之體外培養 (in vitro culture, IVC) 目前關於早期豬胚發育之培養系統仍然未臻完善, 而良好之體外培養系統有助於早期胚之生產與研究, 故已有許多研究, 探討添加不同生長因子於培養液中改善卵母細胞與胚體外培養效率 類胰島素生長因子 (insulin-like growth factor, IGF) 與上皮生長因子 (epidermal growth factor, EGF) 之添加可促進卵母細胞進行減數分裂與提高卵母細胞成熟之百分率, 並可改善體外受精豬胚之發育潛能 細胞轉形生長因子 -α (transforming growth factor-α, TGF-α) 則因其構造與 EGF 相似, 可與 EGF 受體結合, 經由激活酪胺酸激酶受體傳導 MAPK AKT 和 JNK 之訊息, 促進豬卵母細胞成熟並提高其囊胚率 TGF-β 超級家族之生長與分化因子 (growth differentiation factor, GDF) 中之 GDF-9 GDF-9B 以及骨質形成蛋白 BMP-6(bone morphogenetic protein) 皆為卵母細胞分泌之旁泌因子 (paracrine factors) 可提升豬卵母細胞之成熟率 近年來之研究顯示, 在豬卵母細胞 卵丘細胞與顆粒性細胞中可發現 Sonic Hedgehog(Shh) 受體蛋白 Ptc Smo 與其下游轉錄因子 Gli1 之表現, 顯示 Shh 訊息傳遞徑路亦與顆粒性細胞之增生和豬卵母細胞成熟有關, 但其確實的調節機制仍需進一步之研究 此外, 經瘦體素 (leptin) 處理過之豬卵母細胞, 可提高其受精後之原核率與改善胚後續之發育能力 綜上所述, 於豬卵母細胞體外成熟過程添加不同調控因子可改善其成熟率與胚後續之發育能力 ( 關鍵語 : 豬卵母細胞 體外成熟 生長因子 瘦體素 ) 豬胚體外生產 (in vitro production, IVP) 系統包括卵母細胞體外成熟 (in vitro maturation, IVM) 體外受精 (in vitro fertilization, IVF) 與卵母細胞經孤雌激活 (parthenogenetic activation) 後胚之體 外培養 良好之體外培養系統有助於胚之早期發育與生產高品質卵與胚之研究 此外, 由於豬之許 (1) (2) (3) (4) 國立中興大學動物科學系,40227 台中市國光路 250 號 東海大學畜產與生物科技學系,40704 台中市西屯區台中港路三段 181 號 國立中興大學農業生物科技中心 通訊作者, jcju@dragon.nchu.edu.tw

2 148 中國畜牧學會會誌第 39 卷第 3 期 多生理構造或現象與人類較為相似, 甚至其卵母細胞體外成熟時間 ( 約 44 h) 亦與人類卵母細胞成熟時間近似 (Suikkari et al., 2000), 故在人類醫療及器官移植之研究常以豬為動物模式 因此, 建立優良之豬卵母細胞體外培養系統不僅可獲致品質優良之卵與胚, 亦可應用於相關之研究 然而, 目前體外培養系統所產製之胚其發育潛能仍然有異於來自體內者, 尤其經體外成熟與培養之豬卵與胚之品質仍然不佳 (Sheerer et al., 2004; Gil et al., 2007) 卵母細胞成熟過程包含核成熟 卵膜與卵質之成熟 (Lonergan et al., 2003); 而卵母細胞減數分裂復甦 (resumption of meiosis) 受到成熟促進因子 (maturation promoting factor, MPF) 與有絲分裂活化蛋白激酶 (mitogen- activated protein kinase, MAPK) 活性的調控 研究顯示影響卵母細胞體外發育的因素很多, 例如, 豬胚發育至桑椹胚與囊胚之能力, 隨著培養液中 NaCl 濃度之提高而降低 (Beckmann and Day, 1993);Nguyen et al.(2003) 指出豬胚於體外培養 48 h 達 4- 細胞期時, 培養液最佳滲透壓為 290 mosm( mosm); 此外,Karja et al.(2004) 亦指出, 豬胚於低氧 (5%) 環境下培養, 其囊胚中之細胞凋亡數顯著較高氧環境下為低 ; 而於不同基礎培養液中添加不同能量物質 ( 醣類 ) 外源性胺基酸 胎牛血清或牛血清白蛋白, 皆能改善豬卵母細胞及胚之體外發育能力 因此於培養液中添加不同生長因子, 為改善胚體外發育之方式之一 本文主要在於探討卵母細胞體外成熟與其相關調控因子, 對卵母細胞成熟與其後續發育之影響, 期能作為改善其體外培養系統之參考 雌性哺乳動物於出生前, 其卵巢內之卵原細胞 (oogonium) 經由有絲分裂增殖, 並形成初級卵母細胞 (primary oocyte), 準備進入第一次減數分裂之前期 (prophase Ⅰ) 此時卵母細胞外圍包被一層扁平之顆粒性細胞 (granulosa cell) 構成始基濾泡 (primitive follicle; Wassarman, 1988) 隨後卵母細胞進入 prophase Ⅰ, 期間歷經細絲期 (leptotene) 偶絲期(zygotene) 粗絲期(pachytene) 與雙絲期 (diplotene), 最後形成核網期 (dictyate) 此時卵母細胞之染色質散佈於一巨大之原泡核 (germinal vesicle, GV) 內, 而處於第一次減數分裂休止期 (Guraya, 1985) 當動物到達發身(puberty) 年齡後, 伴隨每一動情週期便有一批可繼續發育之初級卵母細胞, 分別經由腦下垂體所分泌之激濾泡素 (follicle stimulating hormone, FSH) 與排卵素 (luteinizing hormone, LH) 刺激濾泡之發育與復甦其減數分裂 (Thibault et al., 1975) 減數分裂復甦始於卵母細胞之原泡核崩解(germinal vesicle breakdown, GVBD), 此時染色質開始濃縮而依序進入第一次減數分裂前中期 (prometaphase I) 中期 (metaphase I, M I) 後期(anaphase I) 與末期 (telophase I), 最後排出第一極體 (first polar body), 而停留於第二次減數分裂中期 (mataphase II, MII), 達成細胞核之成熟階段 然而, 卵母細胞之成熟過程, 還包括卵膜及細胞質之成熟 (cytoplasmic maturation) 經此過程後, 卵母細胞即成熟為次級卵母細胞 (secondary oocyte), 再度停滯於 ( 第二次 ) 減數分裂休止期 直到受精後才完成減數分裂所有過程 (Tsafriri et al., 1983) 卵母細胞的生長與成熟, 由第一次減數分裂休止期至復甦而成熟的過程, 受到細胞內外許多分子之調控, 茲分述如下 : 1. 環腺嘌呤核苷單磷酸 (cyclic-adenosine monophosphate, camp) 與卵母細胞成熟抑制因子 (oocyte

3 生長因子對豬卵母細胞成熟與胚發育之影響 149 maturation inhibitor, OMI) 高濃度的 camp 可維持卵母細胞停留於減數分裂的休止狀態 (Conti et al., 2002; Eppig et al., 2004), 其影響徑路可能為顆粒性細胞產生 camp, 經由細胞之間隙聯結 (gap junction) 使卵母細胞內 camp 的濃度提高, 進而抑制 MPF 之活化 (Duckworth et al., 2002) Tsafriri et al.(1985) 指出,OMI 對小型濾泡卵母細胞減數分裂之抑制作用大於對中型與大型濾泡者 LH 潮湧 (surge) 後, 卵丘細胞擴散而使其與卵母細胞之聯結分離, 導致 OMI 無法傳遞至卵母細胞, 因而促使減數分裂復甦 (Tsafriri et al.,1982 b ) 此外,Tsafriri et al.(1997) 發現豬濾泡液亦能抑制大鼠卵母細胞之減數分裂, 顯示 OMI 可能無種别專一性 2. 成熟促進因子 (maturation promoting factor, MPF) Fulka et al.(1985) 發現成熟促進因子為由兩個次單元 (subunit) 組成之蛋白激酶 (protein kinase), 即由 cyclin-dependent kinase 1(CDK1, 34 kda, 亦稱 P34 cdc2 ) 與細胞週期素蛋白 B(cyclin B) 所組成 (Motlik and Kubelka, 1990) 卵母細胞之 MPF 可以活化與不活化之狀態存在, 當 P34 cdc2 與 cyclin B 形成 P34 cdc2 -cyclin B 複合體 ( 或稱 pre-mpf), 經去磷酸化後而活化, 可造成卵母細胞 GV 核膜消失 染色質濃縮與形成紡錘體 (spindle), 並啟動卵母細胞內蛋白質新合成 (Mattioli et al., 1991) MPF 於豬卵母細胞體外成熟培養開始後 8-16 h 可達一閾值, 促使卵母細胞發生 GVBD 而進入 MI 期, 此階段 MPF 之活性亦達細胞周期中之最高值 (Mattioli et al., 1991) 經第一次減數分裂後, MPF 活性暫時下降, 隨即回升, 直至卵母細胞進入成熟階段時到達最高活性, 以維持卵子休止於 MII 期 (Lewin, 1990; Tunquist and Maller, 2003) 3. 有絲分裂活化蛋白激酶 (mitogen-activated protein kinase, MAPK) MAPK 屬絲胺酸 / 酥胺酸蛋白激酶 (serine/threonine- specific protein kinases), 可被其上游 MAPK kinase (MAPKK 或 MEK) 磷酸化而致活 卵母細胞成熟過程亦受 MAPK 上游之 Ras 蛋白激酶經一連串磷酸化反應活化其下游因子所調控, 故 MAPK 亦稱胞外訊息調節蛋白激酶 (extracellular signal regulated protein kinase, ERK)(Zhang et al., 1998) MAPK 通常於 GV 期卵母細胞亦呈不活化狀態, 當 GVBD 或減數分裂復甦時開始被活化, 同樣在 MI 與 MII 期維持高峯至受精後才下降 在減數分裂中期 (metaphase) 之濃縮染色體周圍與細胞質中皆可發現有 p-mapk 表現 減數分裂後期 (anaphase) 則可發現 p-mapk 多集中於豬卵母細胞中央, 當卵母細胞排出第一極體後, 可發現 p-mapk 分佈於極體與核之間, 據此推斷 MAPK 可能亦與極體排出有關 (Lee et al., 2000) Sun et al.(2001) 指出 MAPK 與 γ- 微管 (γ-tubulin) 共存於豬卵母細胞中 因此,MAPK 與微管組織中心 (microtubule organizing center, MTOC) 之形成有關 (Jin et al., 2009) 於培養液中添加不同生長因子, 如 IGF-I 與 EGF 可改善 MAPK 之活化程度, 亦可能改善體外培養卵母細胞與胚之發育能力 幾種常用於添加於培養液中改善培養條件之生長因子及其對於卵母細胞成熟與胚發育的影響, 分述如下 : Silva et al.(2009) 指出,IGF 主要存在兩種形式 (IGF-Ⅰ 與 IGF-Ⅱ), 具有兩種受體 (IGF-ⅠR 與 IGF-ⅡR) 哺乳類動物之 IGF-I( 又稱 somatomedin C) 分子結構與胰島素類似, 主要由生長激

4 150 中國畜牧學會會誌第 39 卷第 3 期 素 (growth hormone, GH) 調控其分泌 GH 由腦下垂體分泌, 經循環系統輸送至肝臟誘導肝臟細胞產生 IGF-Ⅰ IGF-Ⅰ 主要功能為調控體內多種組織細胞與器官的生長, 包含骨骼肌 軟骨 骨骼 肝臟 腎臟 神經 皮膚 造血細胞與肺臟等細胞 此外,IGF-Ⅰ 亦可調控神經細胞與其 DNA 之合成 IGF-Ⅱ 則為懷孕期促進胎兒生長之主要激素之一, 為腦 肝及腎臟等器官生長發育所必需的因子 IGF-Ⅱ 可於濾泡生成時期 (folliculogenesis) 由卵巢鞘膜細胞 (theca cell) 產生, 並作用在鞘膜細胞本身, 亦可經旁泌作用 (paracrine) 影響卵巢顆粒性細胞之增生 ; 在動情週期之濾泡期與 FSH 亦有協同促進顆粒性細胞增生之作用, 在黃體期 (luteal phase) 則與 LH 協同促進孕酮 (progesterone) 分泌 (Silva et al., 2009 ) 在生物體內 IGF-Ⅰ 與 IGF-Ⅱ 分別由六種 IGF 結合蛋白 (IGF binding proteins, IGFBPs) 來調控其在生物體內的利用率 IGFBPs 可分為兩大類 :(1) 小分子 ( 約為 kda) 之結合蛋白, 包括 IGFBP 與 -6;(2) 大分子量 (150 kda) 的結合蛋白, 以 IGFBP-3 為血清中最主要的形式存在 因 IGFBPs 在血液中主司蛋白質運輸功能, 可調節 IGFs 之代謝速率以延長其半衰期 提供辨識標的組織或細胞間之特異性, 並經由影響 IGFs 與其受體之結合, 以直接或間接控制其分子活性 (Monget et al., 1993; Silva et al., 2009 ) 研究指出, 在豬卵巢腔狀濾泡 (antral follicle) 之顆粒性細胞可偵測出 IGF-Ⅰ IGF-Ⅱ 與 IGF-Ⅰ 受體 mrna 之表現 然而女豬卵巢不論處於何種動情週期階段皆無 IGFBP-1 與 -3 之表現, 但 IGFBP-2 與 -4 則大量表現於腔狀和排卵前濾泡中之顆粒性細胞中 IGFBP-5 之 mrna 則集中表現於生殖上皮細胞 (germinal epithelium) 與微血管內皮細胞 (Zhou et al., 1996) 當培養豬腔狀濾泡之鞘膜細胞, 添加 IGF-Ⅰ 可刺激細胞增生並促進分泌雌二醇 (estradiol-17β), 顯示顆粒性細胞及鞘膜細胞皆為 IGF-Ⅰ 作用之標的細胞 (Kolodziejczyk et al., 2003) 當 IGF-Ⅰ 濃度增加時, 可調節濾泡之生成作用, 使多數濾泡同期生長並維持其後續發育, 故 IGF-Ⅰ 可透過自泌 (autocrine) 及旁泌作用刺激濾泡生長 顆粒性細胞增生與卵母細胞之發育 (Mao et al., 2004) 此外,Monget et al.(1993) 指出當直徑 1-2 mm 濾泡發育至排卵前期時, 濾泡內之 IGFBP-2 與 -4 濃度急劇降低 ; 當豬濾泡持續生長時 IGFBP-2-4 與 -5 之降解增加, 當濾泡閉鎖 (atresia) 時, 則減少降解 (Besnard et al., 1997) 由於豬濾泡發育時, 因 IGFBP-4 蛋白質水解 (proteolysis) 而改變其構造, 致使其與 IGF-Ⅰ 或 IGF-Ⅱ 之結合效率提高 (Mazerbourg et al., 2000) IGF1R 為一跨膜受體 (transmembrane receptor), 由二個 α 與 β 次單元聚集組成一四聚體 IGF-Ⅰ 和 IGF-Ⅱ 皆可與 IGF1R 結合, 並激活其酪胺酸激酶 (tyrosine kinase) IGF 與 IGFR 結合後可經由激活 phosphatidylinositol-3-kinase(pi3k) 和 AKT/Protein kinase-b(akt/pkb) 等訊息傳遞徑路減少細胞凋亡, 並透過激活 MAPKs 促進細胞增生和分化 (Vincent and Feldman, 2002) EGF 為由 53 個胺基酸所組成之蛋白質, 在細胞生長減數分裂 腫瘤形成與傷口癒合方面扮演複雜角色 (Ding and Foxcroft, 1994; Singh et al., 1997; Illera et al., 1998; Uhm et al., 1998; Uhm et al., 1999; Sirotkin et al., 2000) 在卵母細胞體外成熟培養系統中添加 EGF 亦可促進卵丘細胞之擴散與卵母細胞減數分裂復甦, 並提高其成熟率 (Singh et al., 1993 a ; Singh et al., 1993 b ; Singh et al., 1995) EGF 受體 EGFR 屬酪胺酸激酶家族 (receptor tyrosine kinase, RTK) 受體, 又稱 ErbB family, 包括四個成員, 分別為 EGFR(ErbB-1) HER2/c-neu(ErbB-2) Her3(ErbB-3) 與 Her 4(ErbB-4) 在卵丘細胞與卵母細胞中皆可發現 EGFR 之表現 (Uhm et al., 1999; Gall et al., 2005); 其表現可能與

5 生長因子對豬卵母細胞成熟與胚發育之影響 151 增進卵母細胞恢復減數分裂之能力有關 (Gall et al., 2004) Chen et al.(2008) 研究指出 EGFR 被 protein kinase C(PKC) 激活, 並能增進激性腺素 (gonadotropin) 誘導豬卵丘卵母細胞恢復減數分裂之作用 此外, 豬卵母細胞體外成熟培養液中額外添加 EGF, 可刺激卵母細胞在 IVM 期間分泌 34 kda 與 45 kda 兩種蛋白質 (Singh et al., 1997), 而此蛋白質構形與 cdc2 和 cyclin 基因之產物一致 (Rime and Ozon, 1990) 據此推測 EGF 可影響成熟卵子胞質內特有蛋白質之表現 (Singh et al., 1997) 若於培養液中同時添加 BSA 及 EGF, 則可改善經體外受精豬胚之發育潛力 (Wei et al., 2001) 當 EGF 與受體結合後, 便可誘導 Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) 磷酸化, 使其與生長因子受體結合蛋白 (growth factor receptor binding protein, Grb2) 以及 SOS 蛋白質 (son of sevenless) 形成複合體, 並致活細胞膜上之 Ras, 因而引發 MAPK 及其下游之訊息傳遞 另外, 當 EGF 與受體結合也可能誘發 AKT 和 JNK 之訊息傳遞, 最後作用於細胞核中之基因如 c-fos 和 AP-1 和 elk-1 等, 此外亦可能致活訊息傳遞與轉錄活化因子 (signal transducers and activators of transcription, STATs)STAT-1 和 STAT-3, 以調控細胞增生 (Downs, 1989; Gall et al., 2005) 1. TGF-α TGF-α 由 50 個胺基酸所組成, 其構造與 EGF 具有 30-40% 相似度 (Marquardt et al., 1983), 亦可與 EGFR 結合, 而表現與 EGF 類似之活性, 經由激活酪胺酸激酶受體啟動 MAPK AKT 和 JNK 之訊息傳遞鏈 (Pike et al., 1982; Massague, 1983; Derynck, 1986; Derynck, 1990) TGF-α 可由豬卵丘細胞及顆粒性細胞合成, 在卵母細胞中並未檢測到 TGF-α mrna 之表現 (Vaughan et al., 1992; Singh and Armstrong, 1995) Coskun and Lin(1994) 指出, 培養液中添加 TGF-α 可促進豬卵母細胞之成熟 ; 然若去除卵丘細胞, 則其對卵母細胞成熟並無顯著影響 故推測 TGF-α 刺激卵丘細胞分泌某些因子, 可能經由卵丘細胞與卵母細胞間之 gap junctions 傳遞至卵母細胞, 以提高卵母細胞之成熟率 Mito et al.(2009) 亦指出, 添加 TGF-α 於 IVM 培養液中, 有利於豬卵母細胞恢復減數分裂時細胞質之重組 (remodeling), 以促進豬卵母細胞成熟, 並提升其後續之囊胚率 2. 細胞轉形生長因子 -β 超級家族 (transforming growth factor-β, TGF-β, superfamily) TGF-β 超級家族為結構 功能相關之多肽生長因子, 包括 TGF-β(TGF-β1 TGF-β2 與 TGF-β3) 活化素 (activin A 與 activin B)/ 抑制素 (inhibin A 與 inhibin B) DVR 家族 (decapentaplegic-vg-related, DVR) 骨質形成蛋白(bone morphogenetic proteins, BMP) 與生長分化因子 (growth differentiation factors, GDF) 等 TGF-β 家族成員皆與兩種膜上蛋白質受體結合, 最初 TGF-β 先與第二型受體 TGF-β-RⅡ (TβRⅡ) 結合 ;TβRⅡ 屬 Serine/threonine 蛋白激酶之家族成員, 當其與 TGF-β 結合後即可磷酸化第一型受體 TGF-β-RI(TβRI, ALK5), 進而活化其下游分子 SMAD(Knight et al., 2006) TβRI 可激活 SMAD2 及 SMAD3,BMP 則激活 SMAD1 SMAD5 與 SMAD8, 隨後再與 Co-SMAD(SMAD4) 結合, 並轉移至細胞核內結合其他轉錄因子調控標的基因之表現 (Knight et al., 2006) TGF-β 家族成員對細胞的增生 分化 胚胎發育 胞外基質形成 骨骼形成及重建皆有影響, 並廣泛存在於多種生物組織中 其中與豬卵母細胞生長發育有關之因子為 GDF-9 GDF-9B( 或 BMP-15) 與 BMP-6 等 這些為卵母細胞所分泌之旁泌因子 (paracrine factor), 可影響顆粒性細胞, 進而達到調節卵母細胞之發育 (Dong et al., 1996; Galloway et al., 2002; Wu and Matzuk, 2002;

6 152 中國畜牧學會會誌第 39 卷第 3 期 Senbon et al., 2003; Hussein et al., 2005); 尤其 GDF-9 與 BMP-15 兩種因子對卵母細胞之成熟與濾泡之形成十分重要 (Elvin et al., 2000; Chang et al., 2002; McNatty et al., 2003; Di Pasquale et al., 2004; Shimizu et al., 2004) 此兩因子主要由卵母細胞分泌而影響其周邊之卵丘細胞(McNatty et al., 2005 b ) 豬卵母細胞於 IVM 培養初期含有較高量之 GDF-9 表現, 至 IVM 經 18 h 後其 GDF-9 表現量降低, 而 BMP-15 之表現則大幅增加 (Li et al., 2008) 由於此時恰為卵丘細胞開始擴散之時, 故推測 BMP-15 與豬卵母細胞卵丘之擴散有關 在哺乳動物生物體中 Hh 蛋白質家族包括三個成員, 分別為 Desert Hedgehog (Dhh) Indian Hedgehog(Ihh) 與 Sonic Hedgehog(Shh); 其構造與功能與最早自果蠅 (Drosophila) 中發現之 Hh 類似 (Echelard et al., 1993; Ingham and McMahon, 2001) 在哺乳動物胚內,Hh 在調控生長過程上扮演著不同角色, 亦與人類多種先天或病原性畸形與癌症有關 (Villavicencio et al., 2000; McMahon et al., 2003) 在此三個成員中,Shh 最廣被研究, 其與早期中央神經系統 左右體軸之建立 神經細胞之分化調節 肢體中軸和器官形成等有關 (Wijgerde et al., 2002; McMahon et al., 2003) Shh 經蛋白質水解後可分裂成 19 kda 之 N 端蛋白 (N-Shh) 與 25 kda 之 C 端蛋白 (C-Shh); 前者為調節脊椎動物與無脊椎動物之所有訊號徑路 (Hammerschimdt et al., 1997), 後者負責維持本身蛋白質之活性 (Porter et al., 1995; Porter et al., 1996 a ),C-Shh 亦可自動誘導膽固醇修飾作用 (Porter et al., 1996 b ) Hh 家族蛋白訊息傳遞是經由細胞膜上之兩種受體蛋白 Patched(Ptc) 和 Smoothened(Smo) 與 Hh 結合以傳導其訊息 ( 圖 1) Ptc 為 12 次穿膜蛋白結合成之 Hh ligand,smo 則為 7 次穿膜蛋白結合而成之訊息傳遞者 未與 Hh 結合前,Ptc 直接或間接抑制 Smo 之作用 (Taipale et al., 2002; Watkins and Peacock, 2004; Hooper and Scott, 2005) 當 Hh 與其受體 Ptc 接合後, 導致 Smo 與 Ptc 分離, 而激活其下游轉錄因子進入細胞核中, 並起始轉錄作用 (Wijgerde et al., 2005; Lee et al., 2006; Russell et al., 2007) 細胞核中的轉錄因子在果蠅為 Cubitus interruptus(ci), 而相對應於脊椎動物者則為 Gli1 Gli2 與 Gli3 研究發現,Ihh 和 Dhh 在小鼠腔前濾泡及腔狀濾泡時期之顆粒性細胞亦有表現, 而其受體 Ptc 和 Smo 則表現於鞘膜細胞, 故 Hh 可能經由旁泌作用聯繫顆粒性細胞與鞘膜細胞 (Wijgerde et al., 2005; Lee et al., 2006), 共同促進顆粒性細胞之增生和卵母細胞成熟 (Russell et al., 2007) 在豬卵母細胞方面, 經免疫染色後發現於小型濾泡 ( 直徑 < 2 mm) 中之卵母細胞 卵丘細胞及顆粒性細胞皆表現 Ptc Smo 和 Gli1(Nguyen et al., 2009) 在此小型濾泡之濾泡液中,Shh 濃度比中 大型濾泡 ( 直徑 >7 mm) 者為高, 可能由於小型濾泡中顆粒性細胞增生較為緻密所致 ; 伴隨著濾泡直徑增加後, 顆粒性細胞增生現象減少或停止, 因而使中 大型濾泡內濾泡液之 Shh 濃度減低 由於 Gli1 是 Hh 訊息傳遞被活化之指標, 又卵母細胞 卵丘細胞與顆粒性細胞皆有 Gli1 之表現, 因此 Hh 在豬卵母細胞成熟期間可能與小鼠中 Hh 訊息傳遞情況類似 另外, 研究亦指出當體外成熟培養液中添加 Shh 可提升豬卵成熟及胚發育率, 然而其詳細作用機制仍需進一步研究 (Ingham and McMahon, 2001; Nguyen et al., 2009) Zhang et al.(1994) 首度發現 leptin(16 kda) 為脂肪細胞所產生的一種激素 (adipocyte-derived hormone), 具 146 個胺基酸 此激素在新陳代謝中扮演控制體內能量平衡之角色 當體脂量多時, 血液中 leptin 量也隨之增高, 藉此提供體脂量的訊息給下視丘, 以調整能量平衡與食物攝取量

7 生長因子對豬卵母細胞成熟與胚發育之影響 153 圖 1. Sonic Hedgehog (Shh) 之訊息傳遞徑路 Fig. 1. The Sonic Hedgehog (Shh) signaling pathway. Hedgehog (Hh) is a ligand of two transmembrane proteins, Patched (Ptc) and Smoothened (Smo). Before binding with Hh, the receptor protein Ptc directly or indirectly inhibits the function of Smo. When Hedgehog binds to Ptc, Ptc interacts with Smo in a way to lift the suppression to Smo. This leads to the Ci/Gli protein entering the nucleus and acting as a transcriptional activator. (Modified from McMahon et al., 2003; Spurgers et al., 2006; Russell et al., 2007) Chehab et al.(1996) 研究指出, 若投予外源性 leptin 給缺乏 leptin 基因 (LEP) 表現之小鼠, 可恢復其體重與生育能力, 乃因 leptin 可刺激下視丘釋放 GnRH 所致 另有研究指出以 leptin 處理可提高 ob/ob 小鼠血清中 LH 與 FSH 之濃度, 並可增加卵巢初級濾泡 (primary follicles) 及葛拉夫濾泡 (Graafian follicle) 的數目 (Barash et al.,1996); 於懷孕第四天時停止 leptin 處理則使其懷孕中止, 胚胎停止發育而被重吸收 (Malik et al., 2001) 在人類方面, 血清中 leptin 濃度亦與排卵有關 ; 又因其濃度在早期濾泡期較低, 而於黃體期表現量提高, 因而推斷 leptin 在早期胚的發育扮演重要角色 (Hardie et al., 1997; Lukaszuk et al., 1998) Leptin 受體 (OB-R) 屬第一型細胞激素受體超級家族 (classⅠcytokine receptor superfamily) 之成員之一, 具有六種異構物 (isoform: OB-Ra OB-Rb OB-Rc OB-Rd OB-Re 與 OB-Rf)(Tartaglia

8 154 中國畜牧學會會誌第 39 卷第 3 期 et al., 1995; Chen et al., 1996; Cioffi et al., 1996; Lee et al., 1996) 在哺乳動物許多組織中皆可測得, 包括肝 心 腎 肺 小腸 睪丸 卵巢 脾臟 胰臟 脂肪組織 下視丘及腦等細胞 (Lee et al., 1996; Lin et al., 2000; Brann et al., 2002) 在 OB-R 六種 isoform 中依其特性可分成短 長與分泌型三 類 OB-Ra OB-Rc OB-Rd 及 OB-Rf 為短型受體, 其中 OB-Ra 和 OB-Rc 在眼睛脈絡叢微血管與腦動 脈中有高表現量, 可能與其調控腦脊髓液中 leptin mrna 表現量與血腦障壁中 leptin 之傳導有關 (Hileman et al., 2002; Bjørbæck and Kahn, 2004) OB-Rb 則屬細胞膜上之長型受體, 主要功能為傳 導 leptin 訊號 圖 2 顯示 leptin 主要藉由 Janus kinases(jaks) 與 STAT 傳遞細胞訊息 (Baumann et al., 1996; Ghilardi et al., 1996; Heinrich et al., 2002) 在 OB-Rb 上具有 box 1 區塊與四個酪胺酸磷酸化位置, 分 別為 Tyr 974 Tyr 985 Tyr 1077 以及 Tyr 1138 當 leptin 與細胞膜上 OB-Rb 結合可導致此四個位置被磷酸化, 並於 box 1 區致活 JAK2(Ihle and Kerr, 1995) JAK 為細胞內酪胺酸激酶, 分子量約 kda, 被 致活後可磷酸化其下游轉錄因子 STAT3 Tyr 1077 與 Tyr 1138 磷酸化位置與不同 STATs 之致活有關 ; 例 如, 當 Tyr 1077 被磷酸化可致活 STAT5,Tyr 1138 則可致活 STAT1/3/5(Hekerman et al., 2005; M unzberg et al., 2005); 而當 Try 1138 磷酸化時, 可導致 STAT3 磷酸化, 並與 JAK2 磷酸化的 STAT3 形成雙體 (dimer), 進入細胞核後可誘導 SOCS3(suppressors of cytokine signaling 3) 和 POMC(pro- opiomelanocortin) 表現, 並抑制 AgRP(agouti-related peptide) 之表現 SOCS 蛋白抑制訊息分子與磷酸化後的 JAKs 鍵結或直接抑制受體上酪胺酸磷酸化之現象 例如,SOCS3 抑制 Tyr 985 位置之磷酸化作用, 進而抑 制 ERK, 為 leptin 訊息傳遞之負迴饋徑路之一 (Bjørbæck et al., 2000) 近來研究發現 LEP 可表現於小鼠 (Antczak and Van Blerkom, 1997; Ryan et al., 2002) 與人類之 卵母細胞 濾泡液 顆粒性細胞及卵丘細胞 (Cioffi et al., 1997) 另外, 於不同發育階段之濾泡 與不同成熟階段之豬卵母細胞中亦皆可偵測到 LEP 之 mrna 與其蛋白質 (Craig et al., 2004); 而 於懷孕 3-4 天之母豬輸卵管內亦有 LEP mrna 之表現 (Craig et al., 2005) 豬顆粒性細胞與鞘膜細 胞中具有 LEP receptor(obr)mrna 之表現 (Zachow and Magoffin, 1997) Craig et al.(2004) 指出來自中型濾泡之豬卵母細胞其 OB-R 表現量相對較多, 而卵母細胞通常於 GVBD 時有最高之 OB-R mrna 表現 在豬卵母細胞成熟時,leptin 可提高卵母細胞 MAPK 的磷酸化程度 ; 若同時亦 添加 MAPK 抑制劑 (U0126) 則 MAPK 磷酸化程度降低 因而推論 leptin 可能經由 MAPK 徑路提 升豬卵母細胞之成熟率 (Craig et al., 2004) 此外, 豬卵母細胞經 leptin 處理過後, 其受精卵內微 管聚集與原核形成率提高, 並有助提升豬胚後續發育能力 (Jin et al., 2009) 在豬胚方面, 活體所 得之四細胞期胚至囊胚可測得 OB-R mrna; 在體外培養系統中, 利用 IVF 與 somatic cell nuclear transfer(scnt) 產製之豬胚亦皆可測得 OB-R 之表現 (Kim et al., 2006) 豬卵母細胞與胚在不同發育階段對不同營養成份之需求不同, 目前已有許多研究探討添加許多不同生長因子, 如 IGF EGF TGF-α TGF-β Shh 與 / 或瘦體素等, 皆可添加於體外培養液中, 以改善體外培養系統, 期能解決體外生產豬胚低品質與細胞數之普遍性問題 然而, 各種生長因子間是否可協同作用於胚之發育, 而使體外胚之生產效率提升, 以及各生長因子影響豬卵母細胞與胚發育之確切機制等, 仍有待進一步釐清

9 生長因子對豬卵母細胞成熟與胚發育之影響 155 圖 2. 瘦體素訊息傳遞徑路 Fig. 2. The leptin signaling pathway. Leptin binds to its receptor (OB-Rb) and activates JAK2, and then phosphorylates the transcription factor STAT3. When phosphorylated by phospho-tyr 985, SHP2 complexes with Grb2 and SOS proteins and then activates Ras, which initiates the signaling cascade involving the MAPK/ERK pathway to activate the target genes for cell proliferation and differentiation. When phospho-tyr 1138 induces STAT3 phosphorylation and dimerisation, the dimerized STAT3 enters the nucleus to initiate the transcription of target genes. (Modified from Ihle and Kerr, 1995; Heinrich et al., 2002; Frühbeck, 2006).

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18 J. Chin. Soc. Anim. Sci. 39(3):147~164, 2010 Effects of growth factors on the maturation of porcine follicular oocytes and their subsequent development Ya-Lan Jian (1), Neng-Wen Lo (2) and Jyh-Cherng Ju (1)(3)(4) ABSTRACT In vitro production (IVP) of embryos includes oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of parthenogenetically activated or fertilized embryos. An optimized IVP system for porcine oocytes and embryos can help improve the quality of IVP embryos as well as facilitate the study of the preimplantation embryo development. Supplementation with different growth factors in the culture medium is one convenient way to enhance the IVP efficiency. For instance, insulin-like growth factor (IGF) and epidermal growth factor (EGF) both promote meiotic resumption and oocyte maturation as well as IVF embryo development when supplemented in culture media. Transforming growth factor-alpha (TGF-α) has a similar structure to EGF. It can bind to EGFR and improve porcine oocyte maturation and blastocyst development by activating the receptor tyrosine kinase, which in turn, triggers MAPK, AKT and/or JNK signaling pathways. TGF-β is a superfamily of growth differentiation factors (GDF) GDF-9, GDF-9B, and bone morphogenetic protein-6 (BMP-6), which are all paracrine factors secreted by oocytes. They enhance the maturation of porcine oocytes by specific oocyte signaling pathways. Additionally, the Shh receptors or its downstream signaling proteins, such as Ptc, Smo and the transcription factor Gli1, are all detectable in porcine oocytes, cumulus cells and granulosa cells, suggesting that Hh signaling pathway is associated with the proliferation of granulosa cells and oocyte maturation. Also, the pronuclear formation rate and the embryo development are improved when porcine oocytes are treated with leptin. This review aims to highlight the effects of those factors associated with oocyte maturation and embryo development, when they are supplemented to animals or culture media. (Key Words: Porcine oocyte, In vitro maturation, Growth factors, Leptin) (1) Department of Animal Science, National Chung Hsing University, No. 250, Kuo Kuang Rd., Taichung City, Taiwan, R. O. C. (2) Department of Animal Science and Biotechnology, Tunghai University, No. 181, Sec. 3, Taichung Port Rd., Situn District, Taichung City 40704, Taiwan, R.O.C. (3) Agricultural Biotechnology Center, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, R. O. C. (4) Corresponding author; jcju@dragon.nchu.edu.tw