钙离子载体及咖啡因对绵羊精子获能的影响

通过添加不同浓度(0.05、0.1、0.2、0.5、1、5、10μmol/L)的钙离子载体(A23187,IA)及2 mmol/L咖啡因,探讨不同作用时间其对绵羊精子体外获能的影响。结果发现,IA浓度越高,精子的体外获能率越高,顶体反应率越低,活力越低,死精子就越多;而延长作用时间对获能、顶体反应的影响不明显。IA和咖啡因共同作用后,咖啡因能够促进IA诱导顶体反应的发生。建议用IA诱导绵羊精子获能时,其浓度以0.05~0.2μmol/L为宜,作用时间1 min。     

不同浓度Ca2+对塔里木马鹿冻融精子体外获能及蛋白酪氨酸磷酸化的影响

为探讨不同浓度Ca²⁺对马鹿精子体外获能的影响,本研究以塔里木马鹿冻融精子为试验材料,将精子分别悬浮于含不同浓度Ca²⁺（0、1.1、2.2、3.5、5.0 mmol/L）的台氏液（sp-TALP液）中,在培养0、2、4 h时,采用金霉素（CTC）染色法评价精子获能状态,采用SDS-PAGE分离精子膜蛋白,进行免疫印迹分析,检测酪氨酸磷酸化蛋白的表达水平。结果表明,Ca²⁺浓度为1.1、2.2 mmol/L有利于精子活力的维持（P<0.05）,精子获能率极显著高于对照组和高浓度组（3.5、5.0 mmol/L;P<0.01）,精子存活时间最长（P<0.01）,但高浓度Ca²⁺（5.0 mmol/L）对精子活力具有显著抑制作用（P<0.05）,精子获能率极显著低于低浓度组（P<0.01）,精子存活时间最短（P<0.01）;另外,随着培养时间的推移精子发生酪氨酸磷酸化蛋白的表达水平有所不同,培养2、4 h时,1.1 mmol/L组精子蛋白磷酸化水平极显著高于其他各组（P<0.01）,高浓度Ca²⁺（3.5、5.0 mmol/L）组酪氨酸磷酸化蛋白的表达水平极显著下降（P<0.01）。结果表明,塔里木马鹿精子体外获能所需的适宜Ca²⁺浓度为1.1 mmol/L,且获能过程中Ca²⁺的存在是必要的。     

CTC法结合体外受精比较不同方法对绵羊精子体外获能的影响

为比较不同获能方法对绵羊精子的获能效果，新鲜绵羊精液分别经肝素上游法(swim-up)、钙离子载体(IA，A23187)诱导法、Percoll离心法和Percoll离心后肝素孵育法进行精子获能的诱导后与体外成熟的绵羊卵母细胞进行体外受精，并利用金霉素荧光染色（chlortetracycline，CTC）法结合各自相应的体外受精结果对其获能效果进行检验。结果显示上游法处理后精子的获能比例和受精后的卵裂率均高于其它组, 说明肝素上游法可以较好的诱导绵羊精子获能。     

不同浓度Ca~(2+)对塔里木马鹿冻融精子体外获能及蛋白酪氨酸磷酸化的影响

为探讨不同浓度Ca~(2+)对马鹿精子体外获能的影响,本研究以塔里木马鹿冻融精子为试验材料,将精子分别悬浮于含不同浓度Ca~(2+)(0、1.1、2.2、3.5、5.0mmol/L)的台氏液(sp-TALP液)中,在培养0、2、4h时,采用金霉素(CTC)染色法评价精子获能状态,采用SDS-PAGE分离精子膜蛋白,进行免疫印迹分析,检测酪氨酸磷酸化蛋白的表达水平。结果表明,Ca~(2+)浓度为1.1、2.2mmol/L有利于精子活力的维持(P0.05),精子获能率极显著高于对照组和高浓度组(3.5、5.0mmol/L;P0.01),精子存活时间最长(P0.01),但高浓度Ca~(2+)(5.0mmol/L)对精子活力具有显著抑制作用(P0.05),精子获能率极显著低于低浓度组(P0.01),精子存活时间最短(P0.01);另外,随着培养时间的推移精子发生酪氨酸磷酸化蛋白的表达水平有所不同,培养2、4h时,1.1mmol/L组精子蛋白磷酸化水平极显著高于其他各组(P0.01),高浓度Ca~(2+)(3.5、5.0mmol/L)组酪氨酸磷酸化蛋白的表达水平极显著下降(P0.01)。结果表明,塔里木马鹿精子体外获能所需的适宜Ca~(2+)浓度为1.1mmol/L,且获能过程中Ca~(2+)的存在是必要的。     

不同浓度肝素对塔里木马鹿精子体外获能效果的影响

为了探讨不同浓度肝素对塔里木马鹿精子体外获能的影响,试验随机取塔里木马鹿冻融精子,添加到SP-TALIP获能液中并添加不同浓度(0,10,20,50,100μg/m L)的肝素,在显微镜下检测0,2,4,6,8小时时的精子活力,试验采用金霉素(CTC)染色法检测精子获能率及顶体反应率等指标,探讨不同浓度肝素对塔里木马鹿精子活力、存活时间、获能率、顶体反应率的影响。结果表明:精子活力随肝素浓度升高而呈下降趋势,4 h后10μg/m L和20μg/m L肝素组精子活力显著高于其他组(P0.05),其中20μg/m L肝素组精子存活时间最长,显著高于其他组(P0.05)。随着培养时间的延长,各试验组精子获能率与对照组相比明显提高,2小时、4小时时20μg/m L肝素组精子获能率显著高于其他组(P0.05);各试验组精子顶体反应率与对照组相比有升高趋势。说明获能液中添加20μg/m L肝素可有效诱导塔里木马鹿精子体外获能。     

不同发情阶段的绵羊血清对绵羊精子获能和体外受精的影响

为比较不同发情阶段和不同浓度的绵羊血清在绵羊体外受精过程中对绵羊精子体外获能及存活时间、受精效果的影响,试验以绵羊输卵管合成液(SOF)为基础获能液,在其中分别添加发情当天、发情结束和发情间期的绵羊血清,血清浓度分别为0%、2%、5%、10%和20%.利用金霉素荧光染色(CTC,chlortetracycline)法及体外受精2种方法检测不同血清及其浓度对绵羊精子获能的影响.结果表明:1)当在获能液中添加发情当天绵羊血清时,在10%和20%血清浓度下作用45 min的获能效果最好,其获能而且未完成顶体反应的精子率(45.28%和44.52%)显著高于0%、2%和5%血清浓度组(30.14%、28.71%和35.49%,P<0.05);2)当在获能液中添加发情结束时或发情间期的绵羊血清时,在含不同浓度的这2种血清的获能液中作用45 min后,各浓度组引起的获能精子百分率变化均不显著(P>0.05);3)在含20%发情当天绵羊血清的获能液中,精子的存活时间(18 h)显著高于含20%发情结束和发情间期的绵羊血清的获能液(11 h;11 h);4)在不同血清诱导获能后进行的体外受精的试验中,添加发情当天绵羊血清组的卵裂率和囊胚率(62.5%和26.25%)均显著高于发情结束时血清组(42.22%和11.11%,P<0.05)和发情间期血清组(37.36%和15.38%,P<005).以上结果表明:在进行绵羊体外受精试验中,采用20%发情当天绵羊血清作用45 min比采用发情结束和发情间期的绵羊血清能更好地诱导精子获能.     

添加咖啡因、亚牛磺酸对牛精子功能的影响

为研究获能液中添加咖啡因和亚牛磺酸对牛精子功能的影响,本研究将荷斯坦牛冻精解冻后分别添加在含不同浓度咖啡因（0、2.5、5.0、7.5 mmol/L）或亚牛磺酸（0、5、10、20、40 μmol/L）的获能处理液中,且每个处理组加入约200 μL的精液,在CO₂培养箱里经上游处理45 min,以评估咖啡因和亚牛磺酸对牛精子活力、顶体及质膜完整性的影响,进而探讨在获能液中亚牛磺酸替代咖啡因的效果。结果显示,添加2.5、5.0 mmol/L咖啡因组经上游法获能处理后的牛精子活力和顶体完整率均显著高于对照组（P<0.05）,且2.5 mmol/L咖啡因组精子活力最高;添加10、20 μmol/L亚牛磺酸组经上游法获能处理后的牛精子活力、顶体完整率和质膜完整率均显著高于对照组（P<0.05）,且20 μmol/L亚牛磺酸组的精子功能参数值最高;将筛选的最佳浓度2.5 mmol/L咖啡因和20 μmol/L亚牛磺酸采用同样的方法处理,发现20 μmol/L亚牛磺酸组的精子顶体完整率和质膜完整率均显著高于2.5 mmol/L咖啡因组和对照组（P<0.05）。因此,20 μmol/L亚牛磺酸可以替代2.5 mmol/L咖啡因用于体外受精体系中的精子获能处理,有助于提高精子功能参数。     

钙离子载体对牛精子体外受精及胚胎发育的影响

在牛精子获能液中添加不同浓度的钙离子载体,应用体外培养技术对牛精子体外获能及早期胚胎发育作了研究。通过对顶体反应、超激活、活率和早期胚胎发育率的观察,筛选出获能液中最适的钙离子载体浓度,作用时间及其有利于早期胚胎发育的最佳浓度。结果表明:钙离子载体在诱导牛精子获能上存在一个浓度-时间阈值,其最佳浓度和时间组合是0.1μM处理2min或者0.15μM处理0.5min,此时的囊胚率最高;钙离子载体的添加不能诱导牛精子获能的发生。     

肝素浓度对辽宁绒山羊精子体外获能的影响

在TALP液中添加不同浓度(25、50、100μg/mL)的肝素对辽宁绒山羊精子进行获能处理,在显微镜下检测处理1、2、3、4、5h后的精子活力,用考马斯亮蓝染色法检测获能处理0.5、1、2、4h后的精子获能状况,探讨肝素浓度对绒山羊精子活力、存活时间、获能率的影响。结果发现,在38.5℃、5%CO2、饱和湿度条件下,精子活力随肝素浓度升高而下降,3 h以后25μg/mL肝素组精子活力显著高于其它肝素组(P<0.05)。添加肝素组获能率显著高于对照组(P<0.05),各肝素组精子获能率差异不显著(P>0.05)。对照组精子存活时间最长,25μg/mL肝素组精子存活时间为10.12 h,显著高于其它两组(P<0.05)。表明25μg/mL肝素处理辽宁绒山羊精子体外获能较为适宜。     

N-乙酰半胱氨酸对绵羊精液低温保存效果的影响

为研究精液稀释液中不同浓度N-乙酰半胱氨酸(NAC)对绵羊精液低温保存效果的影响,试验用含有不同浓度(0,0.5,1.0,1.5,2.0 mmol/L)NAC的绵羊精液稀释液在4℃条件下保存绵羊精液,并对精子活率、有效存活时间以及48小时的精子畸形率和顶体完整率进行了检测。结果表明:在稀释液中添加1.0 mmol/L的NAC能显著提高精子有效存活时间、总存活时间和48小时精子的顶体完整率(P0.05)。说明添加1.0 mmol/L NAC的精液稀释液能显著改善绵羊精液的低温保存效果。     

孕酮和雌二醇对绵羊精子体外获能的影响

试验探讨孕酮和雌二醇对绵羊精子体外获能和顶体反应的影响。将绵羊精子分别加到含不同浓度孕酮(1、10和100μmol/L)和雌二醇(1、10、和100μmol/L)的输卵管合成液(SOF)中,作用不同时间后分别取出部分精子样本进行金霉素荧光染色(chlortetracycline,CTC),通过精子与CTC结合染色的不同类型来评定孕酮和雌二醇对绵羊精子的作用。结果表明:雌二醇对绵羊精子体外获能和顶体反应都没有显著的促进作用(P>0.05);一定浓度的孕酮和雌激素组合抑制绵羊精子体外获能和顶体反应的发生(P<0.05)。     

Signalling Events and Associated Pathways Related to the Mammalian Sperm Capacitation

Capacitation is a biological phenomenon occurring prior to fertilization and is a multiple event process. Many physiological and biochemical changes takes place during the process; these changes are related to lipid composition of membrane, intracellular modulation of ion concentration, protein phosphorylation, sperm movement and membrane permeability. These events occur when the sperm is exposed to the new environment of ion concentration in the female reproductive tract. Ions such as bicarbonate and calcium facilitate capacitation by activating adenylyl cyclase, thus initiating protein kinase A (PKA) signalling cascade. Extracellular‐regulated kinase pathway is activated by ligand binding to the membrane receptors and intracellular activation by reactive oxygen species (ROS). Activation of these pathways leads to the phosphorylation of different proteins, which is associated with events such as capacitation, hyperactivation and acrosome reaction that are essential for successful fertilization. Extensive studies were carried out on protein phosphorylation in relation to capacitation, but its role still remains ambiguous.     

牛卵母细胞体外受精技术研究

本研究从3方面进行了牛卵母细胞的体外受精试验,即用不同离心法、不同温度上浮法、不同精子浓度等方法对COCs进行体外受精,测定其体外受精率。结果表明,两种离心法中,以上清液二次离心法的精子获能效果最好,受精率最高,该法首次采用低速离心技术,突破了离心法的传统方法,离心效果明显提高;不同温度条件下的精子上浮效果以CO2培养箱恒温（38.5℃）上浮法最好,受精率最高;对于选择受精时获能精子的浓度,宜控制在1.0×106～1.5×106个/ml,不仅能提高受精率,还可避免或降低多精受精现象。     

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability

The function of prion‐like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post‐thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim‐up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post‐swim‐up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.     

Angiotensin-II induced nitric oxide production during buffalo sperm capacitation and acrosome reaction

The present study was designed to see the effects of Angiotensin-II (Ang-II) on buffalo sperm capacitation, acrosome reaction (AR), and its relation to nitric oxide (NO()) production. The extent of capacitation or AR was determined by dual staining while the NO() production was determined by spectrophotometry. The results thus obtained revealed that Ang-II induced capacitation in a concentration and time dependent manner and 200 nM Ang-II was found to be optimal for capacitation as it was comparable to heparin treatment (50.7±2.45% vs. 51.66±2.33%). In capacitated cells the extent of AR induced by Ang-II was significantly higher than the untreated control (48.13±2.31% vs. 22.16±2.11%) and comparable to lysophosphatidyl Choline (LPC) treatment (51.56±1.94%). The NO() production during Ang-II induced capacitation and AR was gradual and time dependent. These levels were significantly higher when compared to control (3.65±0.53 nmoles/10(8)cells vs. 9.12±0.30 nmoles/10(8)cells). All the actions of Ang-II were inhibited in the presence of Losartan but not PD123319, indicating the role of AT1 receptors in these actions. Further the NO() production was also significantly inhibited in the presence of neomycin and trifluoperazine pointing towards the role of phosphoinositide pathway in this process. In conclusion, Ang-II has a concentration and time dependent effect on buffalo sperm capacitation and AR, mediated via the AT1 receptors. Its effect on NO() production may be indirect involving the phosphoinositide pathway.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Hyaluronic Acid as Capacitation Inductor: Metabolic Changes and Membrane‐Associated Adenylate Cyclase Regulation

The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane‐associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2′,5′‐dideoxyadenosine was used as a membrane‐associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin‐capacitated spermatozoa was significantly higher than in hyaluronic acid‐treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane‐associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane‐associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin‐capacitated spermatozoa.