Follicular fluid stimulates capacitation and acrosome reaction in alpaca sperm (Vicugna pacos)

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (<3 mm), medium (3‐6 mm) and large (>6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa.     

哺乳动物精子质量检测原理及方法

人工授精和体外受精技术的不断发展，以及这些技术在生产实践中如各种濒危野生动物的拯救、哺乳动物繁育、人类不孕症的解决中的应用,使得精子质量评价方法也在不断改善。作者综述了精子的质膜完整性、顶体的状态、染色质状态、线粒体活性、精子DNA损伤检测以及受精能力等指标的检测原理及方法，以便准确预测精子的受精能力。     

棕熊精子体外获能及异种穿卵的超微结构研究

对棕熊精子体外获能前后和异种穿卵的超微结构观察表明,棕熊精子全长77μm,由头、颈和尾3部分组成.头部长7.3μm,宽2.5μm,主要由核、顶体及顶体后区组成;颈部由中心粒和9条纵行分节的节往组成;尾部全长68.2μm,其中中段长13.2μm,线粒体为65～68旋,主段中央为“9 2”的微管结构,其外方被9条致密纤维和纤维鞘包裹.精子获能前群集成簇,运动缓慢;获能后精子呈超激活运动.获能的精子质膜膨胀,顶体外膜囊泡化,引起顶体反应,质膜并未参加囊泡化.顶体反应完成后,仅有顶体内膜包在精子核膜的外面.棕熊精子与仓鼠的卵相互作用,多以赤道段和顶体后区附着于卵膜.     

常用哺乳动物精子质量检测方法

根据精子的特性 ,利用光学显微镜、荧光显微镜、流式细胞记数仪等仪器 ,结合常规染色技术或荧光探针技术 ,通过检测精子染色质的状态、运动能力、质膜的完整性、顶体的状态、线粒体的活性、获能、顶体反应以及与卵子的结合能力等指标来评价精子的功能状态 ,以便准确预测精子的受精能力。     

哺乳动物精子获能机理及体外研究

精子获能是哺乳动物精卵融合之前必须经历的一个阶段。自发现精子获能现象以来,对精子获能机制的研究已取得一些进展,但仍有不少问题需要进一步研究。精子获能为体外受精研究获得突破性进展,为人类直接观察受精过程和研究受精机理奠定了基础。文章主要对精子获能的变化、机制及体外研究进行了综述。     

牛精子体外获能的研究进展

文章从精子体外获能的分子机制和体外获能的影响因素等两个方面对牛精子体外获能的发展现状作了比较全面的概括.同时指出了各种处理精子方法的优缺点.并指出了牛精子体外获能的研究价值和意义.     

The relationship between acrosome reaction and polyunsaturated fatty acid composition in boar sperm

This study investigated the relationship between acrosome reactions and fatty acid composition with respect to fertility in boar sperm. The acrosome reaction was induced more than 85% by 60 mM methyl-beta-cyclodextrin (MBCD), and plasma membrane integrity was significantly reduced dependent on the MBCD level in boar sperm (p < .05). The acrosome-reacted sperm exhibited significantly higher saturated fatty acids (SFAs) and lower polyunsaturated fatty acids (PUFAs) composition compared to the non-acrosome reaction group (p < .0001). In addition, the PUFAs, C22:5n-6 (docosapentaenoic acid [DPA]; p < .01) and C22:6n-3 (docosahexaenoic acid [DHA]; p < .0001) were significantly decreased, and cleavage and blastocyst formation of oocytes were significantly (p < .0001) decreased in acrosome-reacted sperm relative to non-acrosome-reacted sperm. Moreover, acrosome reaction was positively correlated with SFAs, whereas negatively correlated with PUFAs, of the PUFAs, the DPA (p = .0005) and DHA (p = <.0001) were negatively correlated with the acrosome reaction. Therefore, these results suggest that the PUFAs composition of sperm is closely involved in acrosome reaction in pigs.     

猪精子获能前后细胞亚组分蛋白分离及酪氨酸磷酸化蛋白的鉴定

本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础。利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定。结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生酪氨酸磷酸化修饰密切相关;获能精子中126、108、79ku的高分子量蛋白磷酸化程度明显高于未获能精子;分子质量约为25、47、50ku的膜蛋白及47ku胞浆蛋白发生酪氨酸磷酸化,其中25、47ku的膜蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05);分子量约为23、37、42～50ku的核蛋白发生酪氨酸磷酸化,获能精子中23ku的核蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05)。结果提示,猪精子细胞不同亚组分中,发生酪氨酸磷酸化修饰的蛋白以膜蛋白及核蛋白为主,同时有少量的胞浆蛋白。     

酪氨酸磷酸化在精子获能过程中的作用

在精子细胞发生顶体反应并使卵母细胞受精之前,获能是一个重要的生理先决条件。获能是精子在雌性生殖道中进一步成熟的复杂现象,其赋予精子以增强活性的能力,使精子能够与卵母细胞透明带（ZP）相互作用,进行顶体反应并与卵母细胞质膜融合,进而完成受精过程。然而精子获能的分子机制十分复杂,目前还未完全明确,但获能后的精子会有诸多结构及生化方面的变化,如蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化。蛋白质通过磷酸化或去磷酸化调节精子获能和顶体反应等一些重要的现象,这是精子到达、结合、穿透和融合卵母细胞所必需的过程。因此蛋白磷酸化是获能的一个非常重要的过程,尤其是在酪氨酸残基处的磷酸化是获能过程中发生的最重要的事件之一,且酪氨酸磷酸化可能是细胞中信号转导途径的主要甚至是唯一的指标。作者主要针对蛋白磷酸化、精子中酪氨酸磷酸化的发现、作用和定位,以及影响获能过程中酪氨酸磷酸化的几个因素进行阐述。     

Effects of Mifepristone (RU486) on Sperm/Cumulus Penetration in Mice

This study was aimed to investigate the effects of RU486 (mifepristone) on sperm penetration through the cumulus cells layer during fertilization in mice. After 20 μg/mL RU486 was added into the capacitation or the sperm/cumulus penetration medium, respectively, the experiments were conducted to evaluate the ratio of sperm acrosome reaction and ability of sperm/cumulus penetration. The results showed that the addition of RU486 significantly suppressed 5 μg/mL P₄-induced acrosome reaction in the capacitated sperm (P <0.01), and decreased sperm penetrating through the cumulus matrix and reaching the oocyte zona pellucid (ZP) and also remarkably reduced the percentage of acrosome-reacted sperm within the oocyte-cumulus complex (OCC)(P <0.01). Compared with the addition of RU486 alone, P₄ did not reverse the inhibitory effects of RU486 on the acrosome reaction of sperm within the OCC, though it still improved sperm penetration through the cumulus matrix and reaching the ZP (P <0.01). Therefore, RU486 could inhibit P₄-induced acrosome reaction and decrease sperm penetration through the cumulus matrix, which suggested that P₄/progesteron receptor (PGR) pathway might be very important for sperm penetration through the cumulus cell layer.     

米非司酮对小鼠精子穿透卵丘细胞层的影响

本研究旨在探讨米非司酮（mifepristone,RU486）对小鼠精子穿透卵丘细胞层的影响。分别添加20 μg/mL RU486到精子获能液或穿卵培养液,检测小鼠精子顶体反应发生比率和穿透卵丘细胞层能力。结果显示,添加RU486能够极显著抑制孕酮（P₄,5 μg/mL）诱导的精子顶体反应（P ＜0.01）;并极显著减少穿透卵丘细胞层到达卵子透明带的精子数量及在卵子-卵丘细胞复合体（OCC）中精子发生顶体反应的比率（P＜0.01）;同时添加P₄和RU486情况下,相比单独添加RU486,虽然P₄能够极显著地促进精子穿透卵丘细胞层到达透明带,但对OCC中的精子顶体反应没有明显作用。综上表明,RU486能够抑制P₄诱导的顶体反应并影响小鼠精子穿透卵丘细胞层过程,揭示P₄/孕酮受体（PGR）通路在小鼠精子穿卵过程中具有重要作用。     

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。     

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。     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.     

Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

不同培养液对蓝狐精子体外获能效果的比较试验

采用上游法分离优化精子,用mTyrod’s液(T)、BO液(B)以及高渗液(HIS)3种不同培养液,在5%CO2的培养箱中进行获能培养,获能培养时间为6h。利用考马斯亮蓝染色法检测精子顶体反应率,伊红-苯胺黑染色法检测活精子比率以及观测法检测精子活力。在培养的0、1、2、4、6h时分别检测上述指标并统计分析,从而筛选出适合蓝狐精子体外获能的培养液。结果显示:B培养液优于T、HIS培养液,最佳获能培养时间t≥6h;HIS可以提高精子顶体反应率,但较高的渗透压不利于精子保存,不建议使用。     

培养液中不同浓度肝素对蓝狐精子体外获能的影响

采用上游法分离优化精子,以mTyrod's液作为基础培养液检测培养液中不同质量浓度肝素对蓝狐精子体外获能的影响,试验设4个肝素质量浓度:0,10,20,50 μg/mL,38.5℃、5%C02培养箱进行孵育,获能培养时间为6h.利用考马斯亮蓝染色法检测精子顶体反应率,伊红-苯胺黑染色法检测活精子比率,观测法检测精子活力...     

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.