DEHP亚慢性暴露对大鼠精子运动参数影响

目的观察邻苯二甲酸(2-乙基已基)酯(DEHP)染毒90 d对大鼠精子运动能力的毒性作用。方法将健康清洁级SD雄性大鼠40只随机分为对照组和3个DEHP染毒组(5、50、500 mg/kg),进行90 d经口染毒;取睾丸、附睾称重,并用扩散法收集大鼠附睾尾精子,应用计算机辅助精子分析系统(CASA)对精子的运动参数进行检测。结果 5 mg/kg DEHP组大鼠精子直线性(LIN)为(44.00±2.52)%,明显低于对照组(50.00±2.71)%(P0.05);500 mg/kg DEHP组大鼠精子鞭打频率(BCF)为(5.8±4.2)Hz,明显低于对照组(10.4±1.7)Hz(P0.05);与对照组比较,500 mg/kg DEHP组大鼠精子的平均路径速度(VAP)、直线运动速度(VSL)、曲线运动速度(VCL)、精子头侧摆幅度(ALH)、BCF明显下降,差异均有统计学意义(均P0.05);随着DEHP染毒浓度增加,大鼠精子各项运动参数逐渐降低,精子的运动能力下降。结论 DEHP对大鼠附睾精子的运动能力有直接毒性作用。     

丙烯酰胺亚慢性染毒大鼠脑和脊髓线粒体某些生化指标的变化

目的 观察丙烯酰胺亚慢性染毒对大鼠脑和脊髓线粒体能量代谢的影响.方法 66只健康雄性Wistar大鼠随机分为0、2、4、6、8、10周对照组和染毒组.染毒组给予丙烯酰胺[生理氯化钠溶液(NS)]40mg/kg腹腔注射,每周3次.对照组同样方式注射NS.观察大鼠的一般情况,每周测量体质量.按设定时间点处死大鼠,留取脑和脊髓组织进行线粒体提取,检测二磷酸腺苷(ADP)和三磷酸腺苷(ATP)的比值、ATP合成酶的活力.结果 染毒组大鼠体质量增长缓慢,染毒第4周后体质量显著低于对照组(P<0.05);脊髓和脑组织线粒体的ADP和ATP比值显著高于对照组(P<0.05,P<0.01).脑和脊髓中ATP合成酶活力分别于第8周和第4周起显著低于对照组(P<0.05),且持续降低至第10周.结论 丙烯酰胺可致染毒大鼠脑和脊髓ADP/ATP比值下降,ATP合成酶活力降低.     

计算机辅助精子分析系统在大鼠精子运动能力测定中的应用

[目的]应用计算机辅助精子分析(computer assisted sperm analysis,CASA)技术研究氯化镉(CdCl_2)不同时间染毒对大鼠成熟精子运动能力的影响。[方法]用扩散法收集大鼠附睾尾成熟精子制成精子悬液,分8组,CdCl_2染毒浓度分别为0,0.5,1.0,2.0,4.0,8.0,16.0,32.0mg/L,每组6个平行样,染毒30min、1h、2h、4h后用CASA仪测定反映大鼠精子运动能力及运动方式的各项参数。[结果]CdCl_2染毒30min后,染毒组精子平均路径速度(VAP)、曲线运动速度(VCL)、直线运动速度(VSL)、直线性(LIN)和精子头侧摆幅度(ALH)等参数与对照组相比差异有统计学意义(P<0.05),鞭打频率(BCF)、前向性(STR)在1.0～32.0mg/L的染毒范围内差异有统计学意义(P<0.05),VAP值随着染毒浓度增加有降低的趋势(r=-0.348,P<0.05)。随着CdCl_2染毒浓度的增加和时间的延长,精子运动能力下降,呈现剂量-效应和时间-效应关系。[结论]体外CdCl_2染毒可以引起成熟精子的运动能力下降。     

雷公藤甲素对雄性大鼠附睾功能与精子动力学参数的影响

目的 研究雷公藤甲素对雄性性成熟期大鼠附睾功能及精子运动的影响,探讨雷公藤甲素生殖毒性的作用机制.方法 将2月龄健康清洁级Sprague-Dawley雄性大鼠40只随机分为对照组(0.5%的羧甲基纤维素钠)及低、中、高雷公藤甲素染毒组(染毒剂量分别为25、50、100 μg/kg),经口灌胃染毒,每天1次.连续染毒50d后,取双侧附睾称重,并计算附睾系数.唾液酸含量的测定采用5-甲基苯二酚法,蛋白含量的测定采用考马斯亮兰法,肉毒碱含量的测定采用酶联免疫吸附法.采用计算机辅助精子分析(CASA)系统评价左附睾尾精子动力学参数.结果 各染毒组大鼠体重间比较,差异无统计学意义(P＞0.05).与对照组相比,中、高剂量组唾液酸含量和高剂量组附睾脏器系数和肉毒碱含量均较低,差异有统计学意义(P＜0.05或P＜0.01).与对照组比较,高剂量组精子曲线速度(VCL)、平均路径速度(VAP)、侧摆幅度(ALH)、鞭打频率(BCF)和中、高剂量组精子直线速度(VSL)、直线性(LIN)、前向性(STR)均较低,差异有统计学意义(P＜0.05或P＜0.01).结论 雷公藤甲素可以引起附睾功能损伤,继而影响附睾尾精子运动速度与线性方向.     

精子形态与精子活力、精子运动参数之间的关系

目的:探讨精子形态与精子活力及精子运动参数之间的关系。方法:847例不育患者采用精子自动分析系统分析精子运动参数及精子活力,采用精子形态检测系统进行精子形态分析。结果:形态异常组精子活率低于形态正常组精子活率(P<0.05);精子形态异常组精子曲线速度(VCL)、直线速度(VSL)及直线性(LIN)均显著低于精子形态正常组(P<0.05);形态正常精子百分率与VSL、LIN及平均移动角度(MAD)有显著正相关性(P<0.01),大头精子百分率与VSL及LIN有显著负相关性(P<0.01),尾部缺陷精子百分率与VSL有显著负相关性(P<0.01)。结论:精子动态和形态异常可能相伴产生,形态异常可能影响精子活力;精液常规与精子形态学分析相结合有助于提高男性不育症的诊断率,为不孕患者选择合适的助孕方式。     

拟除虫菊酯对大鼠皮层腺苷酸环化酶水平影响

目的研究溴氰菊酯(deltamethrin，DM)和氯菊酯(permethrin，PM)对雄性SD大鼠大脑皮层及纹状体腺苷酸环化酶(adenylyl cyclases，AC)蛋白含量和纹状体环磷酸腺苷(cAMP)水平的影响。方法采用不同剂量的溴氰菊酯、氯菊酯对雄性SD大鼠进行经口灌胃给药后，免疫组化的方法测定大鼠大脑皮层及纹状体AC蛋白含量，及放射免疫方法检测DM和PM对大鼠纹状体cAMP水平的影响。结果连续灌胃10d染毒后，大鼠皮层的AC水平和纹状体的cAMP水平明显被抑制。结论拟除虫菊酯能明显降低大鼠皮层AC含量和纹状体cAMP水平。     

妊娠期和哺乳期持续氟铝联合暴露对仔鼠海马环磷酸腺苷-蛋白激酶A-环磷酸腺苷反应元件结合蛋白信号通路的影响

目的探讨妊娠期和哺乳期持续染毒对子二代大鼠海马组织内环磷酸腺苷(cAMP)-蛋白激酶A(PKA)-环磷酸腺苷反应元件结合蛋白(CREB)信号通路的影响。方法将36只健康成年清洁级SD孕鼠随机分为9组,分别为对照(自来水)组和氟化钠(100、200 mg/L)、氯化铝(500、1 000 mg/L)单独染毒组及氟化钠+氯化铝联合染毒组,每组4只。采用自由饮水方式进行染毒,母鼠从妊娠第0天至子一代大鼠出生第21天(postnatal day 21,PND21);子一代大鼠于PND90(性成熟)每组随机选取6只(雌∶雄=2∶1)并进行合笼,从其PND22(断乳后)至子二代大鼠PND21继续染毒;每组随机选取子二代8只(雌雄各半),自PND22(断乳后)~PND60(成年)继续染毒。采用酶联免疫吸附法检测海马组织中cAMP蛋白的含量,采用实时荧光定量聚合酶链式反应(qRT-PCR)方法检测海马组织中PKA和CREB mRNA表达水平,采用免疫组织化学方法检测脑组织中PKA和p-CREB蛋白的表达水平。结果氟铝联合染毒对子二代大鼠海马组织中cAMP蛋白的含量和脑组织中PKA、p-CREB蛋白的表达水平及PKA、CREB mRNA的表达水平均存在拮抗作用(P0.05)。结论氟铝联合暴露可能通过抑制子二代大鼠海马cAMP-PKA-CREB信号通路导致学习记忆能力受损,且具有拮抗作用。     

冷暴露对大鼠黄体细胞与颗粒细胞功能的影响

目的探讨不同冷强度多次冷暴露对大鼠卵巢功能的影响。方法采用体外细胞培养法分离培养大鼠卵巢黄体、颗粒细胞,设37℃对照组与4、0℃冷暴露组,观察人绒毛膜促性腺激素(hCG)诱导黄体与颗粒细胞分泌孕酮及环磷酸腺苷(cAMP)的变化,采用放免分析方法测定孕酮与cAMP生成含量。结果与对照组比较,4℃重复3次冷暴露,黄体细胞与颗粒细胞的孕酮和cAMP生成量呈升高趋势,cAMP升高显著(P0.01);0℃第1次冷暴露,孕酮和cAMP生成量升高,cAMP升高显著(P0.01);第2次和第3次冷暴露,均显著下降(P0.05,P0.01)。结论冷暴露强度、次数对大鼠卵巢黄体细胞与颗粒细胞分泌孕酮及cAMP功能有影响。     

大鼠卵巢细胞低温暴露后分泌功能的变化

目的 探讨不同冷强度多次低温暴露对大鼠卵巢功能的影响.方法 采用体外细胞培养法分离培养大鼠卵巢黄体、颗粒细胞,设37℃对照组与0、-10℃低温暴露组,观察人绒毛膜促性腺激素(hCG)诱导黄体与颗粒细胞分泌孕酮与环磷酸腺苷(cAMP)的变化,采用放免分析方法测定孕酮与cAMP生成含量.结果 0℃第1次低温暴露,孕酮和cAMP生成量升高,cAMP升高显著(P＜0.01),第2次和第3次低温暴露均显著下降(P＜0.05、P＜0.01);-10℃第1次低温暴露,孕酮生成量下降、cAMP生成量升高(P＜0.05),第2次和第3次低温暴露均显著下降(P＜0.01).结论 低温暴露强度、次数对大鼠卵巢黄体细胞与颗粒细胞分泌孕酮及cAMP功能有影响.     

缺氧复合氰化纳中毒对大鼠脑组织线粒体影响

目的 探讨缺氧和氰化物中毒2种因素对大鼠脑线粒体能量储备和线粒体复合体抑制的作用.方法 雄性SD大鼠分为平原组、高原急性缺氧组和阶梯适应组.各组大鼠进行相应的处理后,腹腔注射氰化钠3.6mg/kg,于中毒0,0.5和2h时相点麻醉,断头取脑,提取脑组织线粒体蛋白.高效液相色谱分析大鼠脑线粒体腺苷三磷酸(ATP)、腺苷二磷酸(ADP)和腺苷一磷酸(AMP)含量.常规比色法测定线粒体呼吸链复合体Ⅰ和Ⅳ的活性.结果 平原组、高原缺氧组和阶梯适应组氰化钠中毒脑内线粒体ATP的含量降低,ADP和AMP的含量增加(P<0.05或P<0.01);线粒体复合体Ⅰ和Ⅳ的活性降低,与对照组比较差异有统计学意义(P<0.01);高原缺氧组与相应的平原组比较脑内线粒体ATP的含量降低(P<0.05),ADP和AMP的含世增加(P<0.05).结论 氰化钠中毒可加重急性高原缺氧导致的脑组织线粒体内腺苷酸能量代谢储备障碍和线粒体复合体Ⅰ和Ⅳ活性抑制.     

辛硫磷对大鼠精子生成量和精子运动能力的影响

为探讨有机磷杀虫剂辛硫的雄性生殖毒性,运用动物实验研究了不同剂量辛硫磷径口染毒大鼠６０天对精子生成量和精子运动能力的影响。采用计算机辅助精子分析仪分析精了轨迹。结果显示：辛硫磷２４．５和７３．５ｍｇ／ｋｂＢＷ染毒组,大鼠睾丸精子生成量低于对照组。     

积水输卵管液对精子运动及形态学的影响

目的:通过体外研究积水输卵管液对精子运动及形态学的影响,探讨输卵管积水引起不孕的原因,并为单侧输卵管积水(对侧输卵管通畅)患者的处理提供依据。方法:优化处理后的高质量的精子体外与输卵管积水患者的输卵管液(积水组n=18)及正常输卵管液(对照组n=18)孵育2h、7h和24h后,精子分析仪(CASA)测量精子的各项运动参数,应用精子Diff-Quick涂片染色法分析精子形态。结果:①培养2h、7h和24h后,各时间点积水组精子的前向性运动百分率、平均曲线运动速度(VCL)、平均直线运动速度(VSL)、平均路径运动速度(VAP)、快速直线运动精子活率(MOT)、直线性(LIN)、摆动性(WOB)及前向性(STR)各项精子运动参数均低于对照组(P<0.05),且输卵管积水加快精子的运动速度参数(VCL、VSL、VAP)随时间延长而降低的速度(P<0.05)。②培养2h、7h和24h后,积水组与对照组精子的正常形态率对比无统计学差异,且不随时间延长而改变(P>0.05)。结论:输卵管积水在体外能降低精子的前向性运动百分率、精子运动速度参数(VSL、VCL、VAP)及精子运动方式参数(MOT、LIN、WOB、STR)各项运动指标,并提示输卵管积水不仅干扰了精子活动力,并可能对精子的获能产生不良影响,推测体内输卵管积水对精子产生一定的损害作用,从而影响受孕,但对精子正常形态精子率未有明显影响。     

Antifertility efficacy of twice daily oral administration of 6-chloro-6-deoxy-D-glucose (6 CDG) in male rats

A previous study had shown inability of once daily administration of 6CDG to male rats to completely suppress fertility. Twice daily administration of 12, 24 or 48 mg/kg/day, equivalent total daily doses as in the previous study, now shows dose related suppression of male fertility which is complete at the highest dose level. This dose is also correlated with a significant depression (p < 0.01) in ATP levels of retrograde-flushed epididymal sperm after 21 days of dosing. All dose levels are associated with an accelerated loss of ATP in epididymal sperm over a two-hour post-recovery incubation period relative to controls.In addition twice daily administration of 24 mg/kg shows a duration of dosing, time of incubation interaction on both motility and ATP content of epididymal sperm harvested from the treated rats. Significant effect on both motility and ATP content of these sperm is already apparent after the third dose. Three or more days of dosing results in significant suppression of ATP levels at time of harvest of epididymal sperm.It is suggested that either ATP level within epididymal sperm is a more sensitive index of action of 6CDG than is fertility or alternatively, that the antifertility action of 6CDG is mediated through more than one mechanism.     

Relationship between motility and oxygen consumption of sperm from the cauda epididymides of the rat

The oxygen consumption of rat sperm was low (2.7 microL O2 10(8) sperm(-1) h(-1)) in caudal epididymal semen (CES) when stimulation of motility was avoided. The addition of 1 microL of Krebs Ringer phosphate buffer (KRP) to 40 microL of CES (CES:KRP = 40:1) did not activate motility, but stimulated oxygen consumption 2-fold. Inclusion of 1-5 mM glucose, acetate, pyruvate or lactate in the KRP further stimulated respiration rate (up to 4.3-fold) without activating motility, but respiration was reduced when 2-deoxyglucose replaced energy substrates. Inclusion of dibutyryl cAMP (1 mM) activated sperm motility in all samples and stimulated oxygen consumption 2.9-fold. Dilution of CES at the ratio of CES:KRP = 40:1000 also activated sperm motility and stimulated respiration rate 2.9-fold. The combined effect of dibutyryl cAMP and glucose in stimulating respiration was greater than their individual effects. However, the response to cAMP or substrates was not altered by incubation in KRP containing either 0 or 0.5 mM Ca2+. It was concluded that the motility and metabolism of rat epididymal sperm are suppressed in vivo. Respiration can be stimulated by a small (1.025-fold) dilution and further stimulated by the inclusion of energy substrate, without activating motility. However, a larger dilution or inclusion of cAMP activated motility and simultaneously stimulated metabolism, with exogenous substrate being required to stimulate respiration to the maximum rate. This suggests that prior to activation, the rate of oxygen consumption and sperm motility are not coupled.     

Soluble adenylyl cyclase is required for activation of sperm but does not have a direct effect on hyperactivation

Soluble adenylyl cyclase (SACY) is an essential component of cAMP-signalling cascades that activate sperm motility and capacitate sperm. SACY activity is stimulated by HCO(3)(-) and Ca(2+). Sperm from Sacy(-/-) (null) mice were immotile or weakly motile, but cAMP analogues N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) and adenosine 3',5'-cyclic monophosphate acetoxymethyl ester (cAMP-AM) activated motility. Null sperm activated by dbcAMP quickly developed hairpin bends at the junction of the midpiece and principal piece, which could be prevented by omitting HCO(3)(-). Treating Sacy(-/-) sperm with thimerosal or NH(4)Cl to raise flagellar cytoplasmic Ca(2+) could not substitute for cAMP analogues in activating motility; however, sperm activated with cAMP-AM hyperactivated after thimerosal treatment. Treating activated wild-type sperm with SACY inhibitor KH7 did not prevent hyperactivation from developing during capacitation in vitro, although high doses impaired motility. These results indicate that, while the SACY/cAMP signalling pathway is required for motility activation, it is not directly involved in triggering hyperactivation.     

Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age

Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age:?<35 years, 36-40 years, and 41-44 years. A total of 362 ejaculates from 138 donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and other kinetic parameters did not differ significantly among the groups. The use of CASA allowed the identification of ALH, VCL, LIN, and STR age-related decrease in young men with proven fertility.     

Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age

Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age:?<35 years, 36–40 years, and 41–44 years. A total of 362 ejaculates from 138 donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and other kinetic parameters did not differ significantly among the groups. The use of CASA allowed the identification of ALH, VCL, LIN, and STR age-related decrease in young men with proven fertility.     

Studies of the mechanism of action of gossypol as a male antifertility agent

Gossypol administered orally to male rats at a daily dose of 20 mg/kg body weight for 62 days caused infertility. There were changes in the epididymal epithelium and the sperm were severely damaged and immotile. The sperm head was often detached; other defects were abnormal mitochondria, absence of plasma membranes and axonemal and accessory fibres and a lower oxygen uptake. To study the effect of gossypol on the motor apparatus of sperm, ram sperm were demembranated with the detergent, Triton-X-100. Such sperm models can normally be reactivated with ATP but gossypol (2.5-12.5 microM) decreased reactivation and must have a direct effect on the axoneme. Gossypol also inhibited ram sperm adenyl cyclase which is essential for maintaining high levels of cAMP in sperm and, in turn, motility. Ram sperm adenyl cyclase required Mn2+ for activity and high Mn2+ concentrations protected the enzyme from gossypol inhibition. Electron spin resonance studies proved that gossypol chelated Mn2+ with the formation of a 2:1 complex.     

Kinematic definition of ram sperm hyperactivation

Although it is known that ram spermatozoa exhibit hyperactivated motility under capacitating conditions, quantitative analyses of the head and flagellar movement of washed ram spermatozoa have not been published. Motile spermatozoa were recovered from semen by swim-up into HSOF medium, and their movement in 30-microm-deep chambers was videorecorded. Spermatozoa of interest were identified during tape playback (hyperactivated spermatozoa were identified by visual assessment of flagellar movement) and sequential head and tail images were traced onto overhead projector film attached to the video monitor. The flagellar movement characteristics beat angle (FBA), beat envelope (FBE) and curvature ratio (FCR) were determined by first principles, and head centroid kinematics were determined using Cartesian methods. Hyperactivated spermatozoa had significantly higher FBA and FBE and significantly lower FCR values than non-hyperactivated spermatozoa (all P<0.0001). The centroid kinematic values were also found to be significantly different, and kinematic criteria for ram sperm hyperactivation were developed. These criteria were refined by consideration of 60-Hz CASA-derived trajectories, and ram sperm hyperactivation was defined by: VCL > 250.0 microm s(-1) and VSL < or = 100.0 microm s(-1) and LTN < or = 30% and ALHmax > or = 9.0 microm.     

Effects of electrical stimulation and seminal plasma on the motility of mouse sperm

The effects of electric current (in vivo and in vitro) and seminal plasma on epididymal and ejaculated sperm obtained from C57BL x CBA and C57BL/6J mice were investigated by studying motility parameters, fertilization and embryo development. Electroejaculates were obtained by applying a series of computer-generated sinusoidal alternating currents (0.25-3.0 V at 50 Hz) delivered for 1, 2 and 3 s with 1-s rest periods using a four-electrode rectal probe for 4 min. Epididymal sperm obtained from the same mice were either subjected to electric current in vitro in a Plexiglass chamber or incubated in a medium containing seminal plasma for 2 h. In vitro electric current application and incubation in a medium containing seminal plasma significantly (P < 0.01) decreased sperm motility. Neither electroejaculates nor epididymal spermatozoa incubated with seminal plasma could fertilize oocytes by conventional IVF (P < 0.001), whereas sperm subjected to in vitro electric current had lost little of their ability to fertilize oocytes. Following transfer of embryos generated by intracytoplasmic sperm injection (ICSI), the number of live pups obtained from electroejaculated sperm (10.2%; 6/59) was significantly (P < 0.01) lower than from epididymal sperm (50.0%; 22/42). Electroejaculation using a rectal probe had little effect on motility and fertilization capacity of mouse epididymal sperm, whereas the presence of seminal plasma decreased motility and prevented fertilization.