GDF9/BMP15對(duì)卵泡發(fā)育的調(diào)控

應(yīng)詩(shī)家，于建寧，施振旦

(江蘇省農(nóng)業(yè)科學(xué)院 畜牧所，江蘇 南京 210014)

卵巢卵泡發(fā)育到排卵需要從原始階段發(fā)育到腔前階段的非促性腺激素依賴階段、早期有腔卵泡階段的促性腺激素反應(yīng)階段和卵泡進(jìn)一步發(fā)育的促性腺激素依賴階段，下丘腦-垂體-性腺軸調(diào)節(jié)促性腺激素反應(yīng)和依賴階段卵泡發(fā)育，而在非促性腺激素依賴階段卵泡發(fā)育受卵巢內(nèi)細(xì)胞因子調(diào)控。生長(zhǎng)分化因子9 (GDF9)和骨形態(tài)發(fā)生蛋白15(BMP15)屬于轉(zhuǎn)化生長(zhǎng)因子β (TGFβ) 超家族，在卵母細(xì)胞中特異表達(dá)，對(duì)早期卵泡發(fā)育、顆粒細(xì)胞和膜細(xì)胞功能起重要調(diào)控作用，是卵巢內(nèi)重要的旁分泌因子[1]。熟悉其信號(hào)通路對(duì)我們進(jìn)一步研究其在卵巢中的功能啟動(dòng)規(guī)律以及促性腺激素低反應(yīng)者繁殖疾病機(jī)理具有重要的意義。本文綜述了GDF9和BMP15的結(jié)構(gòu)，受體和Smads信號(hào)的表達(dá)特性以及在卵巢中的生物學(xué)功能。

1 GDF9/BMP15的結(jié)構(gòu)和表達(dá)

GDF9/BMP15具有與其他TGFβ超家族成員相同的結(jié)構(gòu)特性，但也有其特異性。TGFβ超家族由信號(hào)肽、前導(dǎo)肽和成熟肽構(gòu)成。前導(dǎo)肽與成熟肽由一個(gè)堿性氨基酸酶切位點(diǎn)連接，該位點(diǎn)可被前導(dǎo)肽轉(zhuǎn)化酶家族識(shí)別；成熟區(qū)含有7或9個(gè)半胱氨酸，其中6個(gè)形成胱氨酸節(jié)。大多數(shù)TGFβ超家族成員通過保守的半胱氨酸以共價(jià)鍵結(jié)合形成同質(zhì)或異質(zhì)二聚體，然而，GDF9/BMP15只有6個(gè)半胱氨酸，以非共價(jià)鍵結(jié)合形成同質(zhì)和異質(zhì)二聚體(見圖1)，這種結(jié)構(gòu)差異是GDF9/BMP15與其他成員功能差別的主要原因[2]。

GDF9/BMP15主要在卵母細(xì)胞中特異表達(dá)[3]，但存在種間差異，如綿羊、山羊、牛、負(fù)鼠、倉(cāng)鼠GDF9表達(dá)始于原始卵泡階段，而小鼠、大鼠和人GDF9表達(dá)始于初級(jí)卵泡階段；在綿羊、人、小鼠、大鼠卵巢中，BMP15表達(dá)始于初級(jí)卵泡卵母細(xì)胞，而在負(fù)鼠中表達(dá)始于原始卵泡[2]。種間差異BMP15表達(dá)[4]和GDF9:BMP15比值[5]可能解釋其種間功能差異的原因。另一方面，GDF9/BMP15具有更廣泛的生殖功能，因?yàn)?，其也在顆粒細(xì)胞[6]和睪丸組織[7]中表達(dá)。

2 GDF9/BMP15的信號(hào)通路

2.1 GDF9/BMP15的受體和Smads信號(hào)

目前發(fā)現(xiàn)，有三種受體參與TGFβ超家族信號(hào)轉(zhuǎn)導(dǎo)：I型受體、II型受體和TGFβRIII。I型和II型受體與配體結(jié)合轉(zhuǎn)導(dǎo)信號(hào)，而TGFβRIII主要促進(jìn)紅色表示一個(gè)單體，藍(lán)色表示另一單體，綠色表示鏈間二硫鍵，而黃色表示胱氨酸節(jié)中的半胱氨酸[2]。

圖1 綿羊BMP15和人TGF-β1同質(zhì)二聚體模型。

Fig.1 Model of an ovine bone morphogenetic protein 15 (BMP15) and human transforming growth factor-β1 (TGF-β1) dimmer. For both growth factors, one monomer is in red and the other is in blue. Green: interchain disulphide bond. Yellow: cysteines involved in the cystine knots

TGFβ2與II型受體結(jié)合，增強(qiáng)信號(hào)轉(zhuǎn)導(dǎo)。I型受體包括七個(gè)家族(ALK1-7)，II型受體包括五個(gè)家族 (ActrII、ActRIIB、BMPRII、TGFβII和AMHRII)。TGFβRI(ALK5)為GDF9的I型受體，在人、綿羊和小鼠卵巢各等級(jí)卵泡卵母細(xì)胞中表達(dá)，而只在腔前卵泡的顆粒細(xì)胞中表達(dá)，但其表達(dá)譜存在種間差異，如小鼠表達(dá)始于原始階段，人表達(dá)于原始到初級(jí)階段，綿羊在小腔卵泡顆粒細(xì)胞表達(dá)[8]。BMPRIB(ALK6)是BMP15的I型受體，在大鼠、綿羊和牛卵母細(xì)胞，顆粒細(xì)胞和膜細(xì)胞表達(dá)[9]。BMPRII是GDF9和BMP15的II型受體，在綿羊的原始到有腔階段卵母細(xì)胞及顆粒細(xì)胞表達(dá)。

GDF9/BMP15與其受體復(fù)合物結(jié)合引起Smad信號(hào)分子磷酸化，目前共發(fā)現(xiàn)8種Smad蛋白。GDF9主要激活Smad2和3信號(hào)通路，而BMP15激活Smad 1，5和8信號(hào)通路，Smad6和Smad7是抑制性Smads，阻斷Smads信號(hào)通路[10]。

圖2 TGFβ超家族信號(hào)調(diào)節(jié)簡(jiǎn)化示意圖[2]。從上到下為信號(hào)轉(zhuǎn)導(dǎo)方向Fig.2 Simplified schematic of regulation of transforming growth factor-β (TGF-β) superfamily signaling[2].The orientation of the figure is looking down onto the cell surface.

2.2 卵巢中GDF9/BMP15的信號(hào)通路

GDF9/BMP15通過結(jié)合I型、II型絲氨酸-蘇氨酸激酶受體，啟動(dòng)胞內(nèi)Smads信號(hào)轉(zhuǎn)導(dǎo)，調(diào)控靶基因表達(dá)，發(fā)揮生物學(xué)功能，同時(shí)，某些抑制因子參與調(diào)節(jié)GDF9/BMP15信號(hào)通路(見圖2[2])。TGFβ超家族配體(L)與兩個(gè)I型和兩個(gè)II型結(jié)合，激活I(lǐng)I型受體絲氨酸-蘇氨酸激酶，使II型受體磷酸化，進(jìn)而激活I(lǐng)型受體，激活的I型受體促使Smads受體(rSmad)磷酸化，磷酸化的rSmad與通用Smad(cSmad，Smad4)形成復(fù)合體，進(jìn)入核內(nèi)與特異的DNA識(shí)別位點(diǎn)或調(diào)節(jié)轉(zhuǎn)錄的某些蛋白(X)作用調(diào)控表達(dá)。然而，GDF9/BMP15信號(hào)傳導(dǎo)存在微調(diào)節(jié)機(jī)制，在膜外傳導(dǎo)中，當(dāng)配體與可溶性連接蛋白(BP)，可溶性I型受體或“誘導(dǎo)受體”結(jié)合時(shí)，配體與超家族受體結(jié)合受阻，信號(hào)傳導(dǎo)中斷；在Smad激活階段，Smad抑制物(iSmad)與I型受體作用，阻斷I型受體磷酸化及隨后的rSmad激活，導(dǎo)致信號(hào)中斷。

最新的研究發(fā)現(xiàn)Pin1(一種肽基脯氨酰順反異構(gòu)酶)有助于Smurf2(Smad泛素調(diào)節(jié)因子2)與Smads作用，增強(qiáng)Smad泛素化，下調(diào)Smad2/3蛋白水平，調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)[11]。GDF9和BMP15信號(hào)通路需信號(hào)調(diào)節(jié)激酶(signal-regulated kinase 1 and 2 ；ERK1/2)介導(dǎo)[12]。BMP15的前導(dǎo)肽調(diào)節(jié)其與GDF9間的協(xié)同作用[13]，并且GDF9/BMP15先與BMP II型受體結(jié)合發(fā)揮協(xié)同作用[14]。

3 GDF9/BMP15對(duì)卵泡發(fā)育的作用

GDF9/BMP15是哺乳動(dòng)物[15]和卵生動(dòng)物[16]重要的卵巢內(nèi)調(diào)節(jié)因子，對(duì)早期卵泡發(fā)育，卵泡細(xì)胞增殖，類固醇激素合成和卵丘擴(kuò)展具有重要的作用。

3.1 影響卵泡發(fā)育

研究表明GDF9[17]和BMP15[18]調(diào)節(jié)早期卵泡發(fā)育。敲除GDF9的小鼠卵泡停止發(fā)育[19]，小卵泡膜細(xì)胞比大卵泡膜細(xì)胞對(duì)GDF9更敏感[20]，早期卵泡異常發(fā)育可能與缺少GDF9的轉(zhuǎn)錄無關(guān)[21]，而且，應(yīng)激引起的卵泡發(fā)育抑制與GDF9密切相關(guān)[22]。但是，GDF9和BMP15差異性影響早期卵泡發(fā)育[23]。

GDF9/BMP15影響綿羊排卵率，雜合子促進(jìn)排卵，純合子引起不育。除了早前發(fā)現(xiàn)影響綿羊排卵率的FecXI、FecXH、FecXG、FecXB、FecXL、FecXR和FecG突變[24-26]，近年來發(fā)現(xiàn)GDF9錯(cuò)義突變(c. 1111G>A)引起纈氨酸替換為蛋氨酸，與羅威白羊產(chǎn)羔數(shù)密切相關(guān)[27]；GDF9第一內(nèi)含子A485T和A625T突變與中國(guó)荷斯坦奶牛超數(shù)排卵和可移植胚胎數(shù)密切相關(guān)[28]；GDF9基因成熟肽保守區(qū)苯丙氨酸替換為半胱氨酸導(dǎo)致綿羊產(chǎn)羔數(shù)顯著相關(guān)[29]；而且，排卵率的種間差異與GDF9:BMP15比值密切相關(guān)[5,30]。

3.2 影響卵泡細(xì)胞功能

GDF9/BMP15促進(jìn)顆粒細(xì)胞和膜細(xì)胞增殖分化[20]。GDF9促進(jìn)小鼠[31]、反芻動(dòng)物[20]和禽類[32]顆粒細(xì)胞胸腺嘧啶滲入，促進(jìn)顆粒細(xì)胞增殖，抑制FSH誘導(dǎo)的顆粒細(xì)胞分化。然而，GDF9僅促進(jìn)人膜細(xì)胞數(shù)增殖，而對(duì)顆粒細(xì)胞生長(zhǎng)沒有影響[33]。

卵母細(xì)胞發(fā)育受GDF9/BMP15調(diào)節(jié)。GDF9/BMP15是卵丘擴(kuò)展重要因子[34]，在FSH調(diào)節(jié)[35]下協(xié)同作用發(fā)揮卵丘擴(kuò)展功能[36]。GDF9:BMP15比值通過ALK4/5/7和BMPR2激活SMAD2/3信號(hào)促進(jìn)卵丘擴(kuò)展及相關(guān)基因表達(dá)，如Ptx3、Has2和Ptags2[30]。卵巢BMP15水平與體外受精胚著床率、妊娠率和活胎率密切相關(guān)[37]，BMP15通過調(diào)節(jié)卵丘卵母細(xì)胞氧化磷酸化過程促進(jìn)卵母細(xì)胞發(fā)育能力[38]。

GDF9/BMP15抑制促性腺激素誘導(dǎo)的顆粒細(xì)胞孕酮合成，這種作用可能通過促性腺激素調(diào)控，因?yàn)?，GDF9抑制LHR mRNA表達(dá)[39]，BMP15抑制FSH-R mRNA和LH-R mRNA表達(dá)[40]。GDF9抑制人顆粒細(xì)胞P450arom mRNA表達(dá)，表明GDF9可能影響雌二醇分泌，但BMP15不影響雌二醇分泌。GDF9也促進(jìn)顆粒細(xì)胞抑制素A表達(dá)。

4 結(jié)語與展望

GDF9/BMP15對(duì)哺乳動(dòng)物早期卵泡發(fā)育、卵母細(xì)胞及其緊密連接的體細(xì)胞功能起著重要的作用。盡管其信號(hào)通路在卵巢功能中的作用研究已取得很大進(jìn)展，然而，仍有一些問題尚未解決，如：GDF9/BMP15通過Smads信號(hào)調(diào)節(jié)靶基因表達(dá)，調(diào)節(jié)卵泡功能，何種信號(hào)調(diào)控GDF9/BMP15的表達(dá)；不同綿羊品種高繁殖力性狀與GDF9/BMP15基因不同突變密切相關(guān)，這些突變引起的空間構(gòu)象變化如何促進(jìn)卵泡排卵；GDF9/BMP15協(xié)同作用具有加性效應(yīng)[5]，其信號(hào)通路間是否存在聯(lián)系；GDF9/BMP15是否通過影響卵泡類固醇激素分泌而調(diào)節(jié)卵泡功能。總之， GDF9/BMP15信號(hào)通路及其在卵巢中的功能是一個(gè)重要的研究?jī)?nèi)容，深入研究將為調(diào)控動(dòng)物生殖，治療動(dòng)物繁殖障礙性疾病和人類不孕不育具有重要意義。

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