德国牧羊犬冷冻精子超微结构的研究

运用电子显微镜技术观察了２头德国牧羊犬冷冻精子超微结构的变化，结果表明，冷冻的犬精子畸形率明显上升，表现出不同程度的质膜破损或脱落，精核膨胀，中段膨胀，顶体膨胀、部分脱落或全部脱落，螺旋线粒体膨胀、断裂、剥离和丢失。犬精子的原生质膜、顶体、线粒体在冷冻过程中最易受到损害，２头公犬冷冻精子的顶体完整率分别为３６．９％和２５．８％。     

肌醇类冰晶抑制剂和海藻糖对山羊精子冷冻保存效果的影响

为进一步降低冷冻解冻过程对山羊精子的理化损伤,本试验系统检验了肌醇类冰晶抑制剂(SIB)和海藻糖对山羊精子的冷冻保护效果。采用假阴道法采集6只云南黑山羊精液,离心洗涤去除精浆后和冷冻稀释液混合,经4℃平衡、液氮气相预冻后直接投入液氮保存。解冻后检测精子活力、顶体、质膜以及低渗耐受性。同时采用透射电镜(TEM)观察冷冻对精子超微结构的影响。结果表明:1,4-环己二醇(1,4-CHD)和海藻糖处理组精子活力(41.35%±8.14%和43.59%±6.23%)、顶体完整性(62.73%±7.62%和64.29%±7.81%)和低渗耐受性(40.29%±7.41%和39.79%±6.58%)均显著高于对照组(35.97%±6.21%、55.46%±8.91%和34.17%±5.68%,P0.05),且海藻糖可以有效抑制冷冻解冻过程导致的精子凋亡。流式分析结果表明,1,4-CHD不能降低冷冻对山羊精子质膜完整性和磷脂酰丝氨酸(PS)分布的损伤,但是海藻糖可以显著降低冷冻解冻过程中对山羊精子质膜的损伤。电镜结果表明,冷冻对精子质膜和顶体等结构损伤严重,解冻后部分精子质膜发生肿胀、脱落或断裂等现象。而且,顶体外膜存在脱落现象,顶体内容物发生部分甚至完全泄漏。电镜结果进一步证实海藻糖对冷冻精子质膜的保护效果要优于1,4-环己二醇。总之,肌醇类SIB和海藻糖可以降低山羊精子的冷冻损伤,但相对于海藻糖而言,肌醇类SIB并不能降低冷冻对山羊精子质膜的损伤。     

新西兰兔精液冷冻保护剂的筛选

本试验的目的是分析在新西兰兔精液冷冻保存稀释液中分别添加不同浓度的海藻糖、透明质酸、维生素E(Ve)、超氧化物歧化酶(SOD)对兔精液冷冻保存效果的影响,以冻后精子活率、质膜完整性、顶体完整率等作为质量评定指标,筛选出效果较好的新西兰兔精液的冷冻保护剂种类及浓度。结果表明,精液冷冻保存稀释液中添加3种浓度的海藻糖均未提高兔精液冷冻后的精子活率、质膜完整率和顶体完整率(P0.05);添加0.5%和1%的透明质酸提高了兔精液冷冻后的精子活率(P0.05),但各组间质膜完整率和顶体完整率无显著差异;添加2 g/L Ve提高了兔精子4℃平衡后的活率、冷冻后精子活率、质膜完整率和顶体完整率(P0.05);添加4 000 IU的SOD提高了兔精子4℃平衡后的活率、冷冻后精子活率、质膜完整率和顶体完整率(P0.05)。结果证实,在新西兰兔精液冷冻保存稀释液中添加适宜浓度的Ve和SOD可提高兔精液冷冻后的精子活率、质膜完整率和顶体完整率。     

冷冻保存对和田羊精液品质的影响

对和田羊精液进行冷冻保存,以比较冷冻对和田羊精子获能、质膜完整性和顶体完整率的影响。结果表明：①冷冻保存对绵羊精子获能有一定的影响,解冻精子的获能比新鲜精子的获能数量显著增多（P〈O．05）。解冻过程使得显示获能的B型精子的数量显著增加（P〈0．05）。②冷冻使精子质膜破损,致使解冻精子无论是在质膜完整性还是在顸体完整率上都与新鲜精子存在较大差异,说明冷冻过程对精子膜结构造成了严重损伤。     

抗冻蛋白对山羊精子冷冻保护效果的分析

本实验系统评价了抗冻蛋白Ⅲ(AFPⅢ)对山羊精子的冷冻保护效果。采用假阴道法采集6只云南黑山羊精液,离心洗涤去除精浆后和冷冻稀释液(0、0.1、1、10、100μg/m L AFPⅢ)混合,经4℃平衡、液氮气相预冻后直接投入液氮保存。解冻后检测精子活力、顶体、质膜以及早期凋亡等指标。同时采用透射电镜(TEM)观察冷冻对精子超微结构的影响。结果表明:与对照组相比,AFPⅢ并不能显著改善山羊解冻后精子活力、顶体完整性、质膜完整性和低渗耐受性(P0.05)。此外,AFPⅢ并不能抑制解冻后精子的磷脂酰丝氨酸(PS)外翻。电镜实验结果进一步证实,AFPⅢ并不能有效保护冷冻精子质膜。总之,本实验证实,AFPⅢ并不能降低山羊精子的冷冻损伤,相反其可能加重冷冻对精子质膜的损伤,其损伤机制尚需要进一步研究。     

冷冻对种公牛精子损伤的观察与研究

本试验对种公牛新鲜精液和冷冻精液的精子质膜、顶体完整性、线粒体活性、DNA损伤程度以及精子的运动能力等进行检测,比较其差异。结果显示,精液冷冻后含有高能线粒体精子比例为20.1%,质膜完整率为49.8%,显著低于新鲜精子(45.5%,89.7%)。发现采用计算机辅助分析系统(CASA)可以准确客观地对精子运动能力进行检测。结果表明,冷冻会对精子造成损伤,尤其是损伤精子的质膜和线粒体。     

肌醇类SIB对云南半细毛羊精液冷冻保护效果影响的研究

为了进一步降低冷冻过程中冰晶形成对云南半细毛羊精子的机械损伤,实验首次检验了肌醇类冰晶抑制剂(SIB)对绵羊精子的冷冻保护效果。采用的SIB是1,4-环己二醇(1,4-CHD)。电刺激采集精液后,经4℃平衡、液氮气相预冻后直接投入液氮保存。结果表明:1,4-CHD不能提高云南半细毛羊冷冻精子的活力,而且随着1,4-CHD浓度的增加,精子活力呈逐步下降趋势。此外,1,4-CHD也不能改善解冻后云南半细毛羊精子的顶体状态和质膜完整性。解冻后,当用等渗缓冲液稀释精子时,1,4-CHD冷冻组精子的质膜破损率明显高于不含1,4-CHD的对照组(P<0.05)。然而,SIB的存在可能有利于冷冻精子磷脂酰丝氨酸(PS)正常分布的维持。解冻后,1,4-CHD冷冻组精子的PS标记率明显低于对照组(P<0.01)。当分别采用渗透压为404 mOsm,528 mOsm,648mOsm或853mOsm的缓冲液去稀释解冻后的精子时,就移动速度和质膜完整性指标而言,4个处理组之间差异不显著(P>0.05)。总之,肌醇类SIB并不能改善云南半细毛羊冷冻精子的活力、移动速度、顶体以及质膜完整性等指标。然而1,4-CHD可能抑制冷冻解冻过程引起的细胞早期凋亡。     

冷冻保存前平衡温度对猪精子结构的影响

研究冷冻保存前平衡温度对精子结构的影响,为提高猪精子冷冻效果提供参考。新鲜猪精液(Ⅰ组)稀释后于17℃过夜保存(Ⅱ组),然后于冷冻Ⅰ液中4℃平衡1.5h(Ⅲ组),再加入预冷的冷冻Ⅱ液于4℃平衡45min(Ⅳ组)。通过精子形态正常率及活力检测,吖啶橙(AO)染色检测精子DNA完整性,碘化丙啶(PI)染色检测精子头部质膜完整性,低渗肿胀试验(HOS)检测精子尾部质膜完整性,异硫氰荧光素标记的花生凝集素(FITC-PNA)染色检测精子顶体完整性,并用扫描电镜(SEM)观察精子结构,评价冷冻保存前平衡温度对猪精子结构的影响。结果表明,与新鲜精液相比,Ⅱ组精液的精子DNA完整性、质膜完整性、顶体完整性等精子结构检测指标所受影响无统计学意义(P0.05);Ⅲ组和Ⅳ组的精子结构检测指标明显受到影响(P0.05),但Ⅲ组和Ⅳ组间无显著差异(P0.05)。扫描电镜检测结果表明,17℃过夜保存精子未见异常,头、颈、尾结构完整,顶体完整、光滑;Ⅲ组精子头部质膜及头颈结合部有轻微损伤;Ⅳ组精子尾部和头颈结合部有不同程度的断裂。冷冻前从17℃降低至4℃及4℃平衡对猪精子结构有一定程度的损伤。     

马鹿冻精解冻后精子超微结构观察

实验利用透射电镜技术和扫描电镜技术对冷冻/解冻后的塔里木马鹿精子的超微结构进行了观察。结果表明:电镜下观察,塔里木马鹿精子头部呈扁平的卵圆形,长约5.95μm。颈部很短且不明显,长约0.45μm,宽为0.62μm。尾部中段横切面近圆形,直径约为0.58μm,轴丝为9×2+2型;线粒体鞘螺旋段为64～70转。尾部末段仅见质膜包围,9束微管和1对中央微管,形成9+2微管结构。冷冻/解冻后塔里木马鹿部分精子结构发生了变化,一些精子肿胀、质膜皱褶、扭曲,部分质膜损坏或溃散;顶体轻度肿胀,顶体外膜凸凹不平,形成突起。冷冻对头部破坏程度较小。     

使用荧光染色和流式细胞术检测山羊精子

分别使用PI(碘化丙碇),FITC-PSA(异硫氰酸荧光素络合豌豆凝集素)和RH123(罗丹明)对山羊冷冻精子进行染色,再使用流式细胞仪进行检测、分析。试验发现,对于山羊的冷冻精子,PI染色阴性的为质膜完整的精子;FITC-PSA染色阴性的可能为顶体完整的精子,染色FITC-PSA+和FITC-PSA++,可能分别为顶体反应中的精子和顶体破损精子;RH123+应为线粒体无活性精子,有绿色荧光可能是因为染料残留在细胞膜上导致,而RH123++可能应为线粒体有活性的活精子。不同个体羊的精子对于低温敏感度不一样。不同个体羊之间的冻精顶体完整率、质膜完整率、线粒体有活性的精子比率之间差异显著,据此应选择合适的山羊个体进行精液冷冻。     

应用ICSI技术生产转基因水牛胚胎的初步研究

本研究旨在探讨水牛精子完整质膜和破损质膜的方法对水牛单精子注射(ICSI)技术转基因效果的影响。水牛精子经冻融、Triton×-100、超声波3种方法破损精子质膜后与外源质粒DNA混合,采用ICSI的方法将基因转染后的精子注射到水牛体外成熟卵内,观察水牛ICSI卵激活后的发育状态和外源基因的表达效果。结果表明:精子质膜完整性实验中,冻融破损精子质膜组早期胚胎基因表达率为21.8%,显著高于活精子组的5.1%(P<0.01);冻融组与活精子组的卵裂率、囊胚发育率均无显著差异(P>0.05)。以冻融、Triton×-100、超声波3种方法破损精子质膜,冻融组的囊胚发育率(16.7%)最高,显著高于Triton×-100组(P<0.01)。同时,冻融组的早期胚胎基因表达率亦显著高于Triton×-100组和超声断尾组(60.3%vs.31.7%vs.18.2%,P<0.01)。上述结果说明,使用ICSI技术转外源基因可获得表达EGFP基因的水牛早期胚胎;冻融精子质膜破损法能有效破损精子质膜,有利于外源DNA与精子的结合,且提高转基因效率。     

干旱胁迫对圆叶决明叶片超微结构的影响

以豆科牧草圆叶决明CPI86134品系为材料,研究土壤干旱胁迫对其叶片超微结构的影响.结果表明,中度、重度和极度干旱条件下CPI86134叶片细胞中叶绿体和线粒体的超微结构均发生明显变化,线粒体内外膜膨大或破裂,内含物流出;基粒类囊体膨胀,有的严重的细胞中叶绿体膜断裂;另外,随着胁迫程度的增强,淀粉粒的数量减少;叶绿体和类囊体对干旱胁迫最敏感,其次是线粒体,可作为圆叶决明耐旱程度的细胞学指标.     

Dynamics in the membrane organization of the mammalian sperm cell and functionality in fertilization

The capacitation process of sperm cells involves complex changes in the composition and orientation of molecules at the surface of the sperm cell. Here we focus on the lipid architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid organization in living cells and extremely rapid lipid movements were observed. The orientation of lipids in the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for temperature and also changed upon binding of sperm cells to the zona pellucida. The changes in membrane properties coincided with an activation of protein kinases resulting in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these results may provide a physiological basis for new assays, able to discriminate between functional and non-physiological sperm cells.     

Dynamics in the membrane organization of the mammalian sperm cell and functionality in fertilization

Summary

The capacitation process of sperm cells involves complex changes in the composition and orientation of molecules at the surface of the sperm cell. Here we focus on the lipid architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid organization in living cells and extremely rapid lipid movements were observed. The orientation of lipids in the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for temperature and also changed upon binding of sperm cells to the zona pellucida. The changes in membrane properties coincided with an activation of protein kinases resulting in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these results may provide a physiological basis for new assays, able to discriminate between functional and non‐physiological sperm cells.     

马杜霉素对不同虫株柔嫩艾美耳球虫超微结构的影响

鸡体内用5mg/kg马杜霉素作用于柔嫩艾美耳球虫（Eimeria tenella)敏感虫株和抗药虫株，比较裂殖生殖阶段超微结构的差异。敏感株型裂殖体体积变小，所生成裂殖子的数目变小，裂殖子膨胀，裂殖子间（带虫空泡内）微管膨胀，结构模糊，有髓鞘样结构出现；限制膜外突和细胞质分离形成空隙，膜结构模糊，有破损；细胞浆空泡化，出现附加体和髓鞘样结构；少数细胞核的外膜外突和内膜分离，有的部位的核膜模糊、破损。首次发现裂殖子顶体结构发生异常变化，棒状体消失，微线数目减少或者消失；线粒体表现为形态改变，嵴脱落，内部形成空泡、膜状结构和髓鞘样结构，线粒体的嵴与长辆平行排列，且贯穿线粒体。抗药虫株没有发现异常变化。推理抗生素类抗球虫药的作用机理为破坏虫体结构和抑制虫体的生理机能两个方面；球虫对该类药物产生抗药性的机制是基因突变和药物选择的结果。     

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.     

氟对小鼠附睾中成熟精子超微结构的影响

本试验探讨了氟对小鼠附睾中成熟精子超微结构的影响,为氟的生殖毒性研究与检测提供依据。选取8周龄性成熟雄性昆明小鼠20只,随机分为4组,对照组小鼠饮用蒸馏水,低、中和高氟组小鼠分别饮用含25、50和100 mg/L氟化钠的蒸馏水,于45 d后断颈处死小鼠,取小鼠附睾尾,经2.5%戊二醛固定后,采用透射电镜观察小鼠附睾中成熟精子的超微结构变化。与对照组相比,低氟组小鼠精子头部质膜断裂,部分脱落,个别线粒体肿胀、形态模糊;中氟组精子头部质膜脱落,顶体部分缺失,线粒体形状不规则,嵴间腔扩大;高氟组精子头部质膜脱落,线粒体排列不规则,出现空泡化,嵴结构模糊。结果表明,氟暴露小鼠附睾中成熟精子头、尾部中段均有不同程度的结构改变,尤以线粒体出现较明显的异常,且氟浓度越高,精子超微结构损伤越严重。     

Effect of cryopreservation on apoptotic-like events and its relationship with cryocapacitation of buffalo (Bubalus bubalis) sperm

This study investigated the apoptosis-like events associated with cryopreservation process and their relationship with cryocapacitation in buffalo (Bubalus bubalis) sperm. A total of 49 semen ejaculates from seven bulls were studied for structural changes in sperm following cryopreservation. Apoptotic changes were detected by assays specific for translocation of phosphatidylserine (PS) to the cell surface, alterations in membrane permeability and mitochondrial membrane potential (MMP), and DNA integrity. A significant (p < 0.01) percentage of cryopreserved sperm showed externalization of PS and early apoptotic changes and lowered MMP when compared with the fresh sperm. Freezing and thawing of sperm increased permeability to YOPRO-1, an impermeant fluorescent dye. However, on TUNEL staining, cryopreserved sperm showed no breach in DNA integrity. The sperm capacitation status was evaluated by chlortetracycline (CTC) fluorescence pattern, in which a significant (p < 0.01) percentage of cryopreserved sperm were found to be capacitated. The capacitated sperm (Pattern B) was positively correlated with the aforementioned apoptotic events. In conclusion, cryopreservation process induced early apoptosis-like changes in buffalo sperm, and a close link exists between cryocapacitation and apoptosis during cryopreservation of sperm.     

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs

Background: During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis(lipids were extracted, transmethylated and analyzed by chromatography).Results: Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.Conclusions: These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.     

The Use of HOS Test to Evaluate Membrane Functionality of Boar Sperm Capacitated in vitro

The functional and structural integrity of sperm membrane are crucial for the viability of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing sperm membrane measures only its structural integrity. The hypoosmotic swelling test (HOS) originally developed for human sperm ( Jeyendran et al. 1984 ) has been also applied to several species of domestic animals (bull, pig, horse, dog). The test enables to evaluate the functional status of the sperm membrane. The principle of HOS is based on water transport across the sperm tail membrane under hypoosmotic conditions. It has previously been used to assess the semen quality ( Revell and Mrode 1994 ), to analyse fertilizing capacity ( Rota et al. 2000 ; Perez‐Llano et al. 2001 ) and also to detect viable, immotile cells for ICSI (Intra‐cytoplasmic sperm injection) in human ( Zeyneloglu et al. 2000 ). There are two procedures commonly used for sperm capacitation in the pig‐sperm washing and incubation before insemination ( Nagai 1994 ). Capacitation involves several changes like removing molecules coating the sperm head membrane, changes in membrane fluidity and intracellular ion concentration ( Green and Watson 2001 ). Thus the membrane integrity as well as functionality may be affected as shown by Harrison (1996) . The aim of the present study was to analyse changes in sperm membrane integrity after in vitro capacitation by use of the HOS test.