GDF9/BMP15对卵泡发育的调控

应诗家，于建宁，施振旦

(江苏省农业科学院 畜牧所，江苏 南京 210014)

卵巢卵泡发育到排卵需要从原始阶段发育到腔前阶段的非促性腺激素依赖阶段、早期有腔卵泡阶段的促性腺激素反应阶段和卵泡进一步发育的促性腺激素依赖阶段，下丘脑-垂体-性腺轴调节促性腺激素反应和依赖阶段卵泡发育，而在非促性腺激素依赖阶段卵泡发育受卵巢内细胞因子调控。生长分化因子9 (GDF9)和骨形态发生蛋白15(BMP15)属于转化生长因子β (TGFβ) 超家族，在卵母细胞中特异表达，对早期卵泡发育、颗粒细胞和膜细胞功能起重要调控作用，是卵巢内重要的旁分泌因子[1]。熟悉其信号通路对我们进一步研究其在卵巢中的功能启动规律以及促性腺激素低反应者繁殖疾病机理具有重要的意义。本文综述了GDF9和BMP15的结构，受体和Smads信号的表达特性以及在卵巢中的生物学功能。

1 GDF9/BMP15的结构和表达

GDF9/BMP15具有与其他TGFβ超家族成员相同的结构特性，但也有其特异性。TGFβ超家族由信号肽、前导肽和成熟肽构成。前导肽与成熟肽由一个碱性氨基酸酶切位点连接，该位点可被前导肽转化酶家族识别；成熟区含有7或9个半胱氨酸，其中6个形成胱氨酸节。大多数TGFβ超家族成员通过保守的半胱氨酸以共价键结合形成同质或异质二聚体，然而，GDF9/BMP15只有6个半胱氨酸，以非共价键结合形成同质和异质二聚体(见图1)，这种结构差异是GDF9/BMP15与其他成员功能差别的主要原因[2]。

GDF9/BMP15主要在卵母细胞中特异表达[3]，但存在种间差异，如绵羊、山羊、牛、负鼠、仓鼠GDF9表达始于原始卵泡阶段，而小鼠、大鼠和人GDF9表达始于初级卵泡阶段；在绵羊、人、小鼠、大鼠卵巢中，BMP15表达始于初级卵泡卵母细胞，而在负鼠中表达始于原始卵泡[2]。种间差异BMP15表达[4]和GDF9:BMP15比值[5]可能解释其种间功能差异的原因。另一方面，GDF9/BMP15具有更广泛的生殖功能，因为，其也在颗粒细胞[6]和睾丸组织[7]中表达。

2 GDF9/BMP15的信号通路

2.1 GDF9/BMP15的受体和Smads信号

目前发现，有三种受体参与TGFβ超家族信号转导：I型受体、II型受体和TGFβRIII。I型和II型受体与配体结合转导信号，而TGFβRIII主要促进红色表示一个单体，蓝色表示另一单体，绿色表示链间二硫键，而黄色表示胱氨酸节中的半胱氨酸[2]。

图1 绵羊BMP15和人TGF-β1同质二聚体模型。

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与II型受体结合，增强信号转导。I型受体包括七个家族(ALK1-7)，II型受体包括五个家族 (ActrII、ActRIIB、BMPRII、TGFβII和AMHRII)。TGFβRI(ALK5)为GDF9的I型受体，在人、绵羊和小鼠卵巢各等级卵泡卵母细胞中表达，而只在腔前卵泡的颗粒细胞中表达，但其表达谱存在种间差异，如小鼠表达始于原始阶段，人表达于原始到初级阶段，绵羊在小腔卵泡颗粒细胞表达[8]。BMPRIB(ALK6)是BMP15的I型受体，在大鼠、绵羊和牛卵母细胞，颗粒细胞和膜细胞表达[9]。BMPRII是GDF9和BMP15的II型受体，在绵羊的原始到有腔阶段卵母细胞及颗粒细胞表达。

GDF9/BMP15与其受体复合物结合引起Smad信号分子磷酸化，目前共发现8种Smad蛋白。GDF9主要激活Smad2和3信号通路，而BMP15激活Smad 1，5和8信号通路，Smad6和Smad7是抑制性Smads，阻断Smads信号通路[10]。

图2 TGFβ超家族信号调节简化示意图[2]。从上到下为信号转导方向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的信号通路

GDF9/BMP15通过结合I型、II型丝氨酸-苏氨酸激酶受体，启动胞内Smads信号转导，调控靶基因表达，发挥生物学功能，同时，某些抑制因子参与调节GDF9/BMP15信号通路(见图2[2])。TGFβ超家族配体(L)与两个I型和两个II型结合，激活II型受体丝氨酸-苏氨酸激酶，使II型受体磷酸化，进而激活I型受体，激活的I型受体促使Smads受体(rSmad)磷酸化，磷酸化的rSmad与通用Smad(cSmad，Smad4)形成复合体，进入核内与特异的DNA识别位点或调节转录的某些蛋白(X)作用调控表达。然而，GDF9/BMP15信号传导存在微调节机制，在膜外传导中，当配体与可溶性连接蛋白(BP)，可溶性I型受体或“诱导受体”结合时，配体与超家族受体结合受阻，信号传导中断；在Smad激活阶段，Smad抑制物(iSmad)与I型受体作用，阻断I型受体磷酸化及随后的rSmad激活，导致信号中断。

最新的研究发现Pin1(一种肽基脯氨酰顺反异构酶)有助于Smurf2(Smad泛素调节因子2)与Smads作用，增强Smad泛素化，下调Smad2/3蛋白水平，调节信号转导[11]。GDF9和BMP15信号通路需信号调节激酶(signal-regulated kinase 1 and 2 ；ERK1/2)介导[12]。BMP15的前导肽调节其与GDF9间的协同作用[13]，并且GDF9/BMP15先与BMP II型受体结合发挥协同作用[14]。

3 GDF9/BMP15对卵泡发育的作用

GDF9/BMP15是哺乳动物[15]和卵生动物[16]重要的卵巢内调节因子，对早期卵泡发育，卵泡细胞增殖，类固醇激素合成和卵丘扩展具有重要的作用。

3.1 影响卵泡发育

研究表明GDF9[17]和BMP15[18]调节早期卵泡发育。敲除GDF9的小鼠卵泡停止发育[19]，小卵泡膜细胞比大卵泡膜细胞对GDF9更敏感[20]，早期卵泡异常发育可能与缺少GDF9的转录无关[21]，而且，应激引起的卵泡发育抑制与GDF9密切相关[22]。但是，GDF9和BMP15差异性影响早期卵泡发育[23]。

GDF9/BMP15影响绵羊排卵率，杂合子促进排卵，纯合子引起不育。除了早前发现影响绵羊排卵率的FecXI、FecXH、FecXG、FecXB、FecXL、FecXR和FecG突变[24-26]，近年来发现GDF9错义突变(c. 1111G>A)引起缬氨酸替换为蛋氨酸，与罗威白羊产羔数密切相关[27]；GDF9第一内含子A485T和A625T突变与中国荷斯坦奶牛超数排卵和可移植胚胎数密切相关[28]；GDF9基因成熟肽保守区苯丙氨酸替换为半胱氨酸导致绵羊产羔数显著相关[29]；而且，排卵率的种间差异与GDF9:BMP15比值密切相关[5,30]。

3.2 影响卵泡细胞功能

GDF9/BMP15促进颗粒细胞和膜细胞增殖分化[20]。GDF9促进小鼠[31]、反刍动物[20]和禽类[32]颗粒细胞胸腺嘧啶渗入，促进颗粒细胞增殖，抑制FSH诱导的颗粒细胞分化。然而，GDF9仅促进人膜细胞数增殖，而对颗粒细胞生长没有影响[33]。

卵母细胞发育受GDF9/BMP15调节。GDF9/BMP15是卵丘扩展重要因子[34]，在FSH调节[35]下协同作用发挥卵丘扩展功能[36]。GDF9:BMP15比值通过ALK4/5/7和BMPR2激活SMAD2/3信号促进卵丘扩展及相关基因表达，如Ptx3、Has2和Ptags2[30]。卵巢BMP15水平与体外受精胚着床率、妊娠率和活胎率密切相关[37]，BMP15通过调节卵丘卵母细胞氧化磷酸化过程促进卵母细胞发育能力[38]。

GDF9/BMP15抑制促性腺激素诱导的颗粒细胞孕酮合成，这种作用可能通过促性腺激素调控，因为，GDF9抑制LHR mRNA表达[39]，BMP15抑制FSH-R mRNA和LH-R mRNA表达[40]。GDF9抑制人颗粒细胞P450arom mRNA表达，表明GDF9可能影响雌二醇分泌，但BMP15不影响雌二醇分泌。GDF9也促进颗粒细胞抑制素A表达。

4 结语与展望

GDF9/BMP15对哺乳动物早期卵泡发育、卵母细胞及其紧密连接的体细胞功能起着重要的作用。尽管其信号通路在卵巢功能中的作用研究已取得很大进展，然而，仍有一些问题尚未解决，如：GDF9/BMP15通过Smads信号调节靶基因表达，调节卵泡功能，何种信号调控GDF9/BMP15的表达；不同绵羊品种高繁殖力性状与GDF9/BMP15基因不同突变密切相关，这些突变引起的空间构象变化如何促进卵泡排卵；GDF9/BMP15协同作用具有加性效应[5]，其信号通路间是否存在联系；GDF9/BMP15是否通过影响卵泡类固醇激素分泌而调节卵泡功能。总之， GDF9/BMP15信号通路及其在卵巢中的功能是一个重要的研究内容，深入研究将为调控动物生殖，治疗动物繁殖障碍性疾病和人类不孕不育具有重要意义。

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