可溶性腺苷酸环化酶介导精子获能及其信号转导通路

研究证明,精子中存在一种可溶性腺苷酸环化酶(sAC),在获能中起重要作用。精子获能过程中存在着Ca2+、cAMP及sAC的调节系统。本文综述sAC的来源、结构及其作用机制,旨在探讨sAC在精子获能过程中的作用及其信号转导通路。     

ZK 93426对小鼠和人精子获能的影响研究

目的探究小分子化合物ZK 93426对小鼠和人精子获能的影响及作用机制。方法采用蛋白质印迹技术(Western Blot)检测ZK 93426对小鼠精子和人精子获能相关的酪氨酸磷酸化的影响;运用Western Blot检测ZK 93426在正常以及缺少HCO3-,BSA,Ca~(2+)的获能缓冲液(modified Krebs-Ringer bicarbonate medium,HMB)中对获能的作用;选取精子特异钙离子通道CatSper抑制剂NNC 55-0396及PKA通路抑制剂H89进一步探索ZK 93426对于精子获能的机制。结果 ZK 93426对小鼠精子和人精子获能相关蛋白酪氨酸磷酸化均有促进作用,且在缺少Ca~(2+)的获能缓冲液中具有更明显的促进蛋白酪氨酸磷酸化作用。NNC 55-0396不会抑制ZK 93426对于获能相关的酪氨酸磷酸化的促进作用,而PKA抑制剂H89可抑制其作用。结论 ZK 93426对精子获能相关的蛋白酪氨酸磷酸化的促进作用不依赖CatSper通道,可能通过作用于PKA通路的上游发挥相应的作用。     

孕酮对正常生育和不明原因不育男性精子细胞内游离Ca~(2+)浓度的影响

目的:比较正常生育男性和不明原因不育男性患者精子细胞内游离Ca2+浓度([Ca2+]i)对孕酮(P)的反应性。方法:选择10例不明原因不育男性患者和9例正常生育男性,用上游法分离活力良好的精子并于体外获能2 h,将获能后精子与8.85μmol/L钙荧光探针Fluo-3/AM避光孵育40 min负载,将负载后的精子悬液与等量20%明胶混匀以制动精子,然后在激光扫描共聚焦显微镜下动态观察单个精子头部的[Ca2+]i基础水平以及10μmol/L P刺激对[Ca2+]i的影响。结果:不育组获能2 h后精子的[Ca2+]i基础水平显著低于生育组(P<0.01)。生育组精子经10μmol/L P刺激后,产生快速而短暂的[Ca2+]i升高,[Ca2+]i峰值水平显著高于其基础水平(P<0.05);而不育组精子经P刺激后,[Ca2+]i仅有微弱增加,其峰值水平与基础水平相比无统计学差异(P>0.05)。生育组由P激发的精子[Ca2+]i峰值水平以及[Ca2+]i升高幅度(峰值水平与基础水平之差)均显著高于不育组(P<0.01)。结论:不明原因不育男性患者精子[Ca2+]i对P的反应性降低,提示这些患者精子膜上负责Ca2+内流的非基因型孕激素受体可能存在功能障碍。     

不同钙浓度透析液对血液透析患者透析前后电解质变化的影响及其相关关系

目的:研究两种不同钙浓度透析液对维持性血液透析患者透析过程中电解质的影响及其相关关系。方法:筛选中山大学附属第五医院和浙江省立同德医院血液净化中心维持性血液透析患者共160人,分别使用两种不同钙浓度(1.75 mmol/L和1.25 mmol/L)的透析液进行透析,即普通钙组和低钙组,检测透析前后血清电解质,并进行部分电解质间的相关分析。结果:(1)两组透析后血清总钙(t Ca)显著升高,血K+、Mg2+、Cl-、P、HCO3-均显著降低(P0.01)。和普通钙组相比,低钙组透后t Ca显著下降,K+、Cl-、HCO3-显著增加(P 0. 01)。透析后血P和Mg2+在两组间无统计学意义(P0.05)。血Na+在普通钙组透后显著降低(P0.01),在低钙组透析前后差异无统计学意义(P=0.71)。(2)血清t Ca与Na+、K+、Cl-均呈显著负相关,而与Mg2+、P无显著相关性,Cl-与HCO3-呈显著正性相关。结论:不同钙浓度透析液影响透后血钙、钠、钾、氯的变化,但不影响血磷和镁水平。1.75 mmol/L钙浓度透析液会增加钙负荷,故临床维持性血透患者应个体化选择不同钙浓度透析液。     

不同吸入麻醉药对人精子运动功能和体外获能的影响

目的 探讨不同吸入麻醉药对人精子运动功能和体外获能的影响.方法 成年男性精液经Percoll梯度离心法处理后,沉淀精子置于人精子体外获能培养基,调节精子密度(30～50)×106/ml,随机分为5组(n=7):对照组(C组)正常培养,2%、4%七氟醚组(SEV1、2组)和1.1%、2.2%异氟醚组(ISO1、2组)在37℃孵箱中分别暴露于2%、4%七氟醚和1.1%、2.2%异氟醚5 h,采用计算机辅助精子分析系统评价精子运动功能,记录精子运动活力[(a+b)%]、曲线速度(VCL)、直线速度(VSL)、平均速度(VAP)和头部侧向运动平均振幅(ALH),采用金霉素荧光染色技术评价精子体外获能情况.计算吸入麻醉药组各指标的相对抑制率.结果 与C组比较,SEV1组、SEV2组和ISO2组(a+b)%、VCL、VSL、VAP降低,SEV2组ALH、SEV2组和ISO1、2组精子获能程度降低(P＜0.05);与SEV1组比较,SEV2组(a+b)%、VCL、VSL、VAP降低,ISO1组(a+b)%、VCL、VSL、VAP的相对抑制率降低(P＜0.05);与ISO1组比较,ISO2组(a+b)%、VCL、VSL、VAP降低(P＜0.05);与SEV2组比较,ISO2组(a+b)%、VCL、VSL、VAP和ALH的相对抑制率降低(P＜0.05).结论 七氟醚和异氟醚均可呈剂量依赖性地抑制人精子运动功能和体外获能;七氟醚抑制精子运动功能的作用较异氟醚强.     

环核苷酸门控通道与精子功能

环核苷酸门控通道(CNG)是非选择性阳离子通道,直接由环核苷酸活化,是Ca2+进入细胞内的主要通道之一。CNG通道蛋白由6个不同基因编码,4个A亚单位和2个B亚单位。CNG通道受Ca2+/钙调蛋白和磷酸化/去磷酸化作用所调控。近年来,CNG通道在生殖系统方面的研究受到了广泛关注。大量研究表明,CNG通道在精子运动、获能和顶体反应中起着重要的作用。本文对CNG通道与精子功能的关系进行综述。     

支持细胞功能和分化的激素调控

支持细胞 (Sertolicell)是启动青春期发育和维持成年精子发生的上皮细胞 ,其功能和分化受多种因素调控 ,包括激素、细胞因子和细胞粘附分子等。卵泡刺激素 (follicle stimulatinghormone ,FSH)是调节支持细胞大部分分化功能的内分泌激素 ,刺激合成雄激素结合蛋白 (androgenbindingprotein ,ABP)、抑制素 (inhibin)和转铁蛋白 (transferrin ,Tf)等 ,主要信使为cAMP和Ca2 + 。另一个重要的调控激素是甲状腺素 (thyroidhormone ,T3 ) ,可调节支持细胞增殖过程、成熟支持细胞数量以及睾丸产生精子的能力。     

血小板活性因子对人精子的体外获能作用

本文应用浊度分析法,放射免疫法和Lectin染色法研究了血小板活性因子对人精液中快速运动相精子的平均运动速度(VRM)。精子内cAMP含量和顶体反应的影响。结果表明,经血小板活性因子作用后,精液中快速运动相精子的平均运动速度,精子内cAMP含量及顶体反应率均增加。因此,本文认为,血小板活性因子具有使人类精子体外获能的作用。     

蛋白激酶C对精子活力和顶体反应的影响

蛋白激酶C(PKC)位于精子头部赤道段和尾部主段。外源性PKC激动剂可增强精子鞭毛的活力 ,而PKC抑制剂如癌基因抑活药 (staurosporine)则能抑制这种作用。精子中PKC的含量与精子活力呈显著正相关 (r =0 .9,P <0 .0 0 1)。精子与透明带 (ZP)结合刺激顶体反应的发生 ,导致顶体释放多种水解酶以及精子暴露出新的膜区域。ZP与精子质膜上的受体结合后 ,可以调节腺苷酸环化酶 (AC)的活性 ,使cAMP浓度升高并激活蛋白激酶A(PKA)。PKA能激活位于顶体外膜的电压依赖性钙通道 ,后者使顶体内部的Ca2 + 释放至胞质中 ,磷脂酶C(PLC)因Ca2 + 浓度升高而激活并水解磷脂酰肌醇二磷酸 ,其水解产物激活PKC ,后者使精子质膜上电压依赖性钙通道 (L型 )开放 ,胞质中第二次较大幅度Ca2 + 浓度的上升导致质膜溶解及顶体反应发生。推测PKC参与调节精子的活力和顶体反应     

环磷酰胺对小鼠精子胞内钙离子浓度的影响

目的 观察腹腔注射环磷酰胺对小鼠精子胞内钙离子浓度的影响.方法 将20只雄性昆明小鼠随机分为对照组和模型组,按60mg/kg体重给对照组小鼠腹腔注射生理盐水(NS),另按60mg/kg体重给模型组小鼠腹腔注射环磷酰胺,每天1次,连续5d,常规饲养观察34d,然后对小鼠精子进行精液常规分析,对精子行FITC-AV/PI染色后用流式细胞仪检测精子凋亡率,用Ca2+荧光探针Fluo-3/AM处理精子后用流式细胞仪检测精子胞内Ca2+浓度.结果 对照组与模型组精子密度分别为(5.20±1.34)×106/ml和(1.73±0.03)×106/ml,精子活力分别为(14.49±0.30)%和(6.64±1.88)%,精子活率分别为(68.39±15.13)%和(39.96±4.89)%,精子凋亡率(AV/PI)分别为(8.38±0.45)和(14.84±1.22),胞内Ca2+浓度分别为(133.14+11.86)和(80.84±6.92),以上项目两组比较差异均有统计学意义(P<0.05).结论 腹腔注射环磷酰胺可使小鼠精子密度、活力、活率降低,精子凋亡增加,精子胞内Ca2+浓度降低.造成精子胞内Ca2+浓度降低的可能机理是环磷酰胺降低了精子特异性Ca2+通道蛋白功能.     

Effects of fertilization promoting peptide,adenosine, and pentoxifylline on thawed human sperm

Fertilization promoting peptide (FPP) and adenosine were demonstrated to be potential modulators of sperm capacitation in mammals. Both FPP and adenosine, by modulating the adenylate cyclase (AC)/cAMP signaling pathway, elicit similar biphasic responses in mammalian sperm (i.e., stimulating capacitation and inhibiting spontaneous acrosome loss). Pentoxifylline, an artificial sperm stimulant, is clinically used to enhance motility of sperm from infertile men. By inhibiting phosphodiesterase, pentoxifylline increases the intracellular cAMP level of sperm, and thus contributes to capacitation, hyperactivation, and acrosome reaction in animal studies. The effects of FPP, adenosine, and pentoxifylline on thawed human sperm are stressed. Chlortetracycline (CTC) fluorescence assessment revealed that none of the 3 reagents improved fertilization ability of post-thawed sperm. Motility studies with computer-aided sperm analyzer (CASA) showed significantly smaller STR (straight-line velocity) and LIN (linearity) in the FPP-treated group at 4 h of incubation p相似文献   

Regulation of mammalian sperm motility

The physiological regulation of sperm motility has become more amendable to investigation since the demonstration that cAMP and calcium play a role in modulating the functioning of the flagellar axoneme. Although the external triggering mechanisms that initiate motility and capacitation are still unknown, evidence supports a modification of the calcium balance by gated Ca2+ channels, accompanied by shifts in the internal pH. Ca2+ and pH may in turn act indirectly through cAMP and cAMP-dependent kinase (kinase(a] to control the phosphorylation state of functional proteins in the flagellar axoneme. The role of calcium is of central importance, but it is clear that several separate Ca2+-dependent mechanisms are involved. Ca2+ controls the curvature of the sperm flagellum and, so, can change the motility of the sperm from progressive swimming to tumbling. Under the appropriate conditions, calcium appears to have the capacity to deactivate motility by activating phosphodiesterase and phosphatase. The deactivating effect of Ca2+ may be offset under some circumstances by coactivation of adenyl cyclase, so phosphorylation of the axoneme and the motility are maintained. The specific factors determining the predominant calcium effect are not yet known, but internal pH of the sperm may play a major role.     

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.     

Implication of calmodulin-dependent phosphodiesterase type 1 during bovine sperm capacitation

Phosphodiesterases (PDEs) are enzymes that degrade cyclic nucleotides. The calcium-calmodulin dependent PDE type 1 (PDE 1) and the cyclic adenosine monophosphate (cAMP)-specific PDE type 4 (PDE 4) have been implicated in sperm function. We tested the hypothesis that specific PDEs regulate capacitation of bovine sperm in a manner independent of those that mediate motility. Our objectives were to determine the effects of inhibiting PDE 1 and PDE 4 on capacitation and motility, and to compare these effects to those of heparin, which is necessary for capacitation of bull sperm in vitro. Fresh sperm were supplemented either with 15 microg/mL heparin (positive control) or the PDE inhibitors vinpocetine (specific for PDE 1) and rolipram (specific for PDE 4), and then incubated for 5 hours. At 0, 3, and 5 hours, samples were assayed for capacitation and motility parameters according to the chlortetracycline (CTC) fluorescent pattern B and computer-assisted sperm analysis, respectively. A higher percentage of CTC pattern B sperm relative to heparin controls was observed at 0 and 3 hours when sperm were incubated with vinpocetine. After 5 hours, the percentage of heparin- and vinpocetine-treated sperm showing pattern B did not differ (P >.05). Rolipram did not affect CTC patterns (P >.05; n = 4). Vinpocetine and heparin both reduced the percentage of progressively motile sperm after 3 and 5 hours, but vinpocetine reduced it more than heparin (P <.05; n = 4). Rolipram transiently increased linearity versus sperm with heparin (P <.05; n = 4). To further test the hypothesis that PDE 1 inhibition permits capacitation, we conducted in vitro fertilization. Vinpocetine did not support the ability of sperm to penetrate homologous oocytes (n = 5). Although cAMP regulation by PDE 1 may occur early during capacitation, downstream events appear to prevent full capacitation from occurring prematurely.     

Activation of protein kinase A during human sperm capacitation and acrosome reaction

Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during sperm capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for capacitation and PKA activation and/or that the cAMP concentration measured in whole spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.     

Ca2+ is Essential for the Motility of Plasma Membrane-Intact, but not of demembranated. Hamster Spermatozoa

Extracellular Ca2+ is essential for the flagellar motility of membrane-intact hamster spermatozoa. When suspended in a medium completely free of Ca2+, most spermatozoa quickly lost their motility, and remained motionless until they were transferred back to Ca2+-containing medium. The motility could not be restored after the spermatozoa had been in Ca2+-free medium for more than 2 hr. Unlike membrane-intact spermatozoa, demembranated spermatozoa (spermatozoa without plasma membranes) exhibited active movement in Ca2+-free medium, and their motility was inhibited by Ca2+. In view of these facts, we suggest that the "hyperactivated motility" which membrane-intact spermatozoa display upon capacitation may be due to the activation of a Ca2+-dependent adenylate cyclase (and the resultant increase in intracellular cAMP), rather than being a direct effect of a rise in the intracellular Ca2+ concentration.     

Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences

Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca(2+)](i). While LY294002 increased [Ca(2+)](i), wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca(2+)](i) operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon.     

Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.

Progesterone induced a rapid, long-lasting, dose-dependent increase of intracellular free calcium concentration ([Ca2+]i) in human sperm capacitated overnight. This effect was not counteracted by the cytosolic progesterone receptor antagonist RU486 (1 mumol/L) nor by the GABA-A receptor antagonists bicuculline (10 mumol/L) and picrotoxin (50 mumol/L). Also, the rank order of potency of several progestative steroids on [Ca2+]i differed from that previously reported for uterine intracellular progesterone receptor or for P-GABA interaction in the central nervous system, indicating a different pathway for progesterone stimulation of human sperm. Modifications of basal and progesterone-stimulated [Ca2+]i during sperm capacitation were also studied. A progressive, parallel increase of basal and progesterone-stimulated [Ca2+]i in capacitating spermatozoa was found. In particular, progesterone-stimulated [Ca2+]i increased from a basal concentration of 147% +/- 17% at 10 minutes to 327% +/- 65% after 120 minutes of incubation in capacitating medium. This increase was well correlated with basal [Ca2+]i (r = 0.93). In contrast, basal and progesterone-stimulated [Ca2+]i concentrations were constantly low in spermatozoa incubated in noncapacitating medium. In capacitated spermatozoa, initial responsiveness to progesterone and basal [Ca2+]i was higher than in capacitating and noncapacitated samples, and remained constant throughout the duration of the experiment. The progressive, parallel increase of [Ca2+]i and response to progesterone observed during in vitro capacitation of human spermatozoa might be physiologically relevant in vivo during capacitation of sperm in the female genital tract.     

Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction

To bind and fertilize the egg, the spermatozoon should undergo few biochemical and motility changes in the female reproductive tract collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, and then undergoes the acrosome reaction (AR), which allows its penetration into the egg. The mechanisms regulating sperm capacitation and the AR are not completely understood. In the present review, we summarize some data regarding the role and regulation of the epidermal growth factor receptor (EGFR) in these processes. In the capacitation process, the EGFR is partially activated by protein kinase A (PKA), resulting in phospholipase D (PLD) activation and actin polymerization. Protein kinase C alpha (PKCα), which is already activated at the beginning of the capacitation, also participates in PLD activation. Further activation of the EGFR at the end of the capacitation enhances intracellular Ca(2+) concentration leading to F-actin breakdown and allows the AR to take place. Under in vivo conditions, the EGFR can be directly activated by its known ligand epidermal growth factor (EGF), and indirectly by activating PKA or by transactivation mediated by G protein-coupled receptors (GPCRs) activation or by ouabain. Under physiological conditions, sperm PKA is activated mainly by bicarbonate, which activates the soluble adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), the activator of PKA. The GPCR activators angiotensin II or lysophosphatidic acid, as well as ouabain and EGF are physiological components present in the female reproductive tract.