【作者】 张美佳；

【导师】 夏国良；

【作者基本信息】 中国农业大学 ， 生理学， 2005， 博士

【摘要】 心钠肽(atrial natriuretic peptide,ANP)主要由心脏分泌的一种小肽类激素,参与了血压调节和机体内环境的动态平衡。近年来研究发现ANP及其受体在哺乳动物卵巢和输卵管中广泛存在。一般认为促性腺激素通过cAMP信使促进卵母细胞的成熟。现在研究表明cGMP也参与卵母细胞的成熟和精子顶体反应。ANP可与受体结合,激活粒性鸟苷酸环化酶,使cGMP水平上升,从而可能在生殖系统中具有重要的生理作用。本文以猪和大熊猫为对象,研究了ANP在卵母细胞成熟和精子功能中的作用及机理。 实验一研究了ANP对猪卵丘细胞包被的卵母细胞(cumulus-enclosed oocytes,CEOs)体外成熟和卵丘扩散的影响。当猪CEOs在添加有卵泡液的培养液中培养时,ANP剂量依赖性负调节FSH诱导的生发泡破裂(在FSH,FSH+10 nM ANP和FSH+1μM ANP处理组中的GVBD分别为:90.1:81.2和68.2%),第一极体排出(在FSH,FSH+1nM ANP和FSH+1μM ANP处理组中的PB1分别为86.1;75.3和53.3%)和卵丘扩散(在FSH,FSH+1nM ANP和FSH+1μM ANP处理组中的卵丘扩散指数分别为3.47;3.16和2.43)。该影响也表现出时间依赖性。当猪CEOs培养在无卵泡液的培养液中时,ANP显著抑制FSH诱导的GVBD(47.6%vs.83.8%)和IPB1的排出(22.4%vs.45.2%).cGMP类似物8-Br-cGMP(10μM-1 mM)可模拟ANP对GVBD、PB1和卵丘扩散的影响。ANP的作用可完全被cGMP依赖性的蛋白激酶(PKG)抑制剂KT5823所抑制,而与ANP受体C特异结合的C-ANP-(4-23)对卵母细胞体外成熟没有影响。ANP和C-ANP-(4-23)对猪卵母细胞自发成熟没有影响。这些结果表明,ANP负调节FSH诱导的猪卵母细胞减数分裂恢复、减数分裂成熟和卵丘扩散。ANP对卵母细胞成熟的影响是通过cGMP依赖性的蛋白激酶(PKG)实现的。 实验二重点研究了ANP在猪卵母细胞成熟过程中的作用机制。结果表明ANP能抑制FSH诱导的猪卵母细胞成熟和卵丘扩散,并阻止卵母细胞和卵丘细胞中有丝分裂原蛋白激酶(mitogen-activated protein kinase,MAPK)的完全磷酸化。ANP的抑制作用可被8-Br-cGMP模拟,而蛋白激酶G(protein kinase G,PKG)抑制剂KT5823却能完全逆转。扎普司特(zaprinast)(cGMP特异的磷酸二酯酶抑制剂)能增强ANP在卵母细胞成熟中的作用。C-ANP-(4-23)(特异地与钠肽受体C(NPRC)结合的类似物)对FSH诱导的和自发的猪卵母细胞成熟没有影响。用毛喉素(forskolin,一种腺苷酸环化酶激动剂)培养猪卵母细胞得到两种不同的结果:培养44h可诱导卵丘扩散但抑制卵丘包被的卵母细胞(cumulus-enclosed oocytes,CEOs)成熟,而培养2h可诱导卵母细胞成熟。ANP和C-ANP-(4-23)都能抑制forskolin对CEOs的成熟影响,而其抑制作用百日咳毒素(pertussis toxin,PT)阻止,表明ANP和C-ANP-(4-23)对forskolin的抑制作用是通过调节卵丘细胞上的Gi蛋白实现的。所有这些结果表明ANP通过两种不同的信号机制调节FSH和forskolin在猪卵母细胞成熟中的作用:一是通过cGMP/PKG通路,另外一种是通过卵丘细胞上的NPRC受体,并激活PT敏感的Gi蛋白通路。 实验三研究了ANP及其受体在猪精子和输卵管中的表达以及其ANP对猪精子功能的影响。RT-PCR分析表明,精子中仅存在钠肽受体A(natriuretic peptide receptors-A,NPRA)基因的表达。ANP mRNA水平在输卵管1期(OT1)、3期(OT3)和5期(OT5)中较高,在2期(OT2)中较低。NPRA mRNA水平在OT1和OT2期较低,OT3期开始上升,到OT4、OT5期达到最高。ANP能诱导猪精子发生顶体反应。当用1nM ANP处理时,其诱导顶体反应的百分率最高(34.1±2.3%)。C-ANP-(4-23)和咖啡因都对猪精子的顶体反应没有影响。ANP对精子顶体反应的影响更多还原

【Abstract】 Atrial natriuretic peptide (ANP) or atrial natriuretic factor (ANF), a small peptide mainly produced in the heart, is involved in the regulation of blood pressure, salt and water excretion, cell proliferation and body fluid homeostasis. Recent reports show that ANP and its receptors are widely present in the ovary and oviduct. Generally, gondaotropins increase cAMP levels to induce oocyte maturation. Recent reports show that cGMP is involved in oocyte maturation. ANP exhibits the high affinity to NPRA thatcan activate particulate guanylate cyclase, which may play an important physiological role in reproductive tissues. In this study, we examined the effect and the mechanism of ANP on porcine oocyte maturation, fertilization and giant panda spermatozoa function.Experiment 1. This study examined the effect of atrial natriuretic peptide (ANP) on porcine cumulus-enclosed oocyte (CEO) maturation and cumulus expansion. ANP negatively regulated follicle-stimulating hormone (FSH)-stimulated germinal vesicle breakdown (GVBD; 90.1, 81.2 and 68.2% for FSH, FSH + 10 nM ANP and FSH +1 μM ANP, respectively), first polar body emission (PB1; 86.1, 75.3 and 53.3% for FSH, FSH + 1 nM ANP and FSH +1 μM ANP, respectively) and cumulus expansion (CEI; 3.47, 3.16 and 2.43 for FSH, FSH + 1 nM ANP and FSH +1 μM ANP, respectively) in a dose-dependently manner when CEOs were cultured in the maturation medium containing porcine follicular fluid (pFF). This negative effect showed a time-dependent manner after preincubation with 100 nM ANP for 5 h (78.4% PB1), 10 h (81.7% GVBD and 74.1% PB1), 20 h (78.5% GVBD and 68.9% PB1), and 44 h (75.3% GVBD and 60.5% PB1), respectively. ANP also significantly inhibited FSH-induced porcine oocyte GVBD (47.6% vs. 83.8%) and PB1 emission (22.4% vs. 45.2%) when CEOs were cultured in pFF-free maturation medium. cGMP analog 8-Br-cGMP (10 μM - 1 mM) mimicked the effects of ANP on GVBD, PB1 and CEI. The negative effect of ANP was completely reversed by KT5823 (a specific inhibitor of cGMP dependent protein kinase), while C-ANP-(4-23) (an analogue of ANP and specific binder for natriuretic peptide receptors-C) was ineffective in oocyte maturation. Both of ANP and C-ANP-(4-23) had no effect on spontaneous porcine oocyte maturation and cumulus expansion. These results suggest that ANP negatively regulates FSH-activated porcine oocyte meiotic resumption, meiotic maturation and cumulus expansion. The function of ANP on porcine oocyte maturation is via the cGMP dependent protein kinase (PKG) pathway.Experiment 2. The present study conducted experiments to examine the mechanism of ANP and forskolin on porcine oocyte maturation. The results revealed that ANP could inhibited follicle-stimulating hormone (FSH)-induced pig oocytes maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase (MAPK) in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cGMP, but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cyclic guanosine 5’-monophosphate (cGMP)-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocytes maturation. A specific analog of ANP, C-ANP-(4-23), which binds to the natriuretic peptide receptor C, had no effect in either FSH-induced or spontaneous oocytes maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocytes maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4-23) could inhibit the effect of forskolin on CEOs maturation, and these inhibitory effects of ANP/C-ANP-(4-23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEOs maturation by two signalling mechanisms respectively: one is via cGMP/PKG pathway; the other is via NPRC receptors in cumulus cells and更多还原