正常精子和圆头精子差异表达蛋白的分离与鉴定

圆头精子症是一种表现为顶体缺失的男性不育症.为了研究正常精子和圆头精子的蛋白质组成差异,用固相pH梯度双向凝胶电泳和质谱分析等蛋白质组学方法,分离了30份正常和3份圆头精子标本的蛋白质.对其中16个在正常精子中有高丰度表达而在圆头精子中缺失,和1个在圆头精子中表达明显下调的蛋白质点,以及在圆头精子中存在而在正常精子中缺失的蛋白质簇W和点X,进行了肽质指纹分析和蛋白质鉴定,获得8个点的肽质量指纹图,经MS-Fit软件搜索SWISS-PROT数据库来鉴定其身分.发现其中3个点与高尔基体相关、2个点与蛋白酶体相关、2个点为锌指蛋白,对其功能和与圆头精子形成的可能关系进行了初步探讨.     

二维电泳分离牛精子蛋白的技术研究

二维电泳是蛋白质分离技术并可由于对精子蛋白的分离。本研究旨在通过对双向电泳条件的研究摸索出一种适用于分离牛精子蛋白的二维电泳技术,并利用其对牛精子蛋白进行分离鉴定。在实验中,优化了等电聚焦程序,研究了精子蛋白的不同制备方法、不同上样量、不同胶条长度对电泳结果的影响。结果表明,采用尿素－盐酸胍两步裂解法裂解精子细胞制备蛋白,使用13cm非线性胶条进行蛋白二维电泳,能获得较好的电泳图谱。图谱经二维电泳软件分析,可检测出约800多个蛋白质点,分子量基本分布在10~100KD、等电点约为4~9的区域内。对精子蛋白二维电泳条件的摸索,为后续牛精子X、Y差异蛋白的检测和分析奠定了理论基础。     

高畸形率水牛精子与正常水牛精子差异表达蛋白的分离和鉴定

旨在分析高畸形率和正常水牛精子的差异表达蛋白.运用双向凝胶电泳以及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)分析鉴定出高畸形率和正常的水牛精子的差异表达蛋白,并对部分蛋白进行生物信息学分析.结果显示,高畸形率和正常水牛精子之间存在16个表达差异明显的蛋白点,与正常水牛精子相比,5个蛋白斑点表达量上调,6个蛋白斑点下调,3个蛋白斑点缺失,2个蛋白斑点在畸形率高的水牛精子特有.质谱鉴定16个差异蛋白,成功鉴定出6个差异蛋白斑点,对应4种蛋白:左旋天冬酰胺酶、热应激蛋白β-9、半乳糖激酶、β-微管蛋白-2C.研究表明,高畸形率和正常水牛精子蛋白质表达存在一定的差异.     

高原脑水肿患者血浆的蛋白质组学研究

目的：高原脑水肿是急性高原病的重症表现之一,其发病机制尚不完全清楚。本实验目的是应用蛋白质组学方法研究高原脑水肿发病时血浆蛋白质表达的变化,为研究高原脑水肿发病分子机制以及高原脑水肿的预防、诊断及治疗提供重要的分子标志物。方法：应用二维凝胶电泳（2D）结合质谱的方法比较了一例高原脑水肿（HACE）患者与高原肺水肿（HAPE）和轻型急性高原反应（mAMS）患者血浆蛋白表达的差异,对差异蛋白进行鉴定,最后用ELISA方法检测载脂蛋白E含量。结果：我们检测到HACE与HAPE患者血浆比较有6个差异蛋白质点,HACE与mAMS患者血浆比较也有6个差异蛋白质点,质谱鉴定结果显示两组比较中都有载脂蛋白E含量变化,ELISA检测结果与二维凝胶电泳结果一致。结论：本实验首次将蛋白质组学技术应用到高原脑水肿研究中,并且发现了可能与高原脑水肿发病密切相关的载脂蛋白E,该蛋白对于研究高原脑水肿发病分子机制以及疾病的预防、诊断及治疗的意义有待进一步研究。     

哺乳动物精子磷酸化蛋白质组学研究进展

蛋白质的表达、修饰及相互作用的研究已成为后基因组学时代蛋白质组学中的重要内容。蛋白质磷酸化和去磷酸化作为最普遍的翻译后修饰之一,是精子细胞信号转导和酶调控、表达的主要分子机制,亦是精子、卵细胞信号识别及完成受精作用的关键环节。对精子磷酸化蛋白功能的研究有助于深入理解精子的获能、超激活运动的维持、发生顶体反应及精卵结合等受精过程的分子调控机理。对哺乳动物精子磷酸化蛋白质组学的研究进展,包括动物精子磷酸化蛋白质组学研究的技术方法、磷酸化蛋白质种类的鉴定、定量及其功能分析进行了综述,为进一步发掘与受精相关的重要生物标志物,揭示精子发育、繁殖潜能变化及受精分子机理奠定基础。     

少弱精子症患者精子候选差异蛋白Trx 2、TrxR 1的验证及在少精、弱精子症中的表达研究

目的:根据TMT技术筛选少弱精子症患者精子差异蛋白的结果,选取硫氧还蛋白2(thioredoxin 2,Trx 2)、硫氧还蛋白还原酶1(thioredoxin reductase 1,TrxR 1)进行验证,探讨二者在少精、弱精和少弱精子症中的表达变化及其意义。方法:收集105例少精子症组(O组)、150例弱精子症组(A组)、50例少弱精子症组(OA组)和106例正常精液男性(N组)精液,分离出精子,对少弱精子症进行串联质谱标签(Tandem Mass Tag,TMT)技术蛋白质组学分析,根据少弱精子症组的精子差异蛋白结果选取Trx 2、TrxR 1,通过免疫荧光和免疫印迹方法检测其在O组、A组、OA组的表达情况。结果:TMT技术蛋白质组学结果显示Trx 2为上调差异蛋白(为N组的1.31倍),TrxR 1为下调差异蛋白(为N组的0.82倍)。免疫荧光和免疫印迹结果显示O组、A组、OA组Trx 2表达显著高于N组(P0.05),O组、OA组TrxR 1的表达显著低于N组(P0.05)。二者在OA组的结果与蛋白质组学结果一致。结论:Trx 2、TrxR 1可能在少精、弱精及少弱精子症的发生中起着重要的作用,并有望成为少弱精子症患者精子的候选标志物及治疗靶点。     

水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。     

双向凝胶电泳在先天性肛门直肠畸形中的初步应用

目的：寻找先天性肛门直肠畸形患儿直肠末端组织中异常表达的蛋白质。方法：通过二维凝胶电泳分离先天性肛门直肠畸形患儿直肠末端组织及正常新生儿直肠末端组织,用Image Master2D Platium6．0软件比较电泳图谱中的异常蛋白质点。结果：筛选出19个表达差异的蛋白质点,其中有12个蛋白质点表达上调,7个蛋白质点表达下调,差异具有统计学意义。结论：先天性肛门直肠畸形可以导致血清中多种蛋白的异常表达。这些差异表达的蛋白可以为先天性肛门直肠畸形的进一步研究提供了依据。     

体外培养大鼠星形胶质细胞液压冲击损伤的研究

目的：研究大鼠星形胶质细胞液压冲击损伤后形态学及蛋白质组学表达变化。方法：建立体外培养星形胶质细胞液压冲击损伤模型。原代培养SD大鼠脑皮质星形胶质细胞,随机分为损伤组与对照组,对照组给予（0.2±0.01）MPa液压冲击损伤,损伤后不同时间点观察细胞形态学变化;双向凝胶电泳技术分析液压冲击损伤后蛋白质组学表达变化。结果：星形胶质细胞在液压冲击损伤后发生了显著的形态学改变,损伤后2h星形胶质细胞出现了细胞水肿、细胞皱缩、细胞连接断开和坏死,损伤后24h、48h细胞胞体肥大、突起增粗明显,部分区域细胞反应性增生明显。液压冲击损伤后,星形胶质细胞蛋白质表达谱发生了显著改变,损伤后有13个蛋白点表达发生显著改变,其中5种蛋白得到质谱鉴定,分别是肌动蛋白结合蛋白、破解蛋白、磷酸甘油酸变位酶1、NADH脱氢酶10亚基和膜联蛋白1。结论：液压冲击损伤能够引起星形胶质细胞发生显著的形态学改变和蛋白质谱表达改变,损伤后表达改变的蛋白质可能与星形胶质细胞的损伤后应激反应相关。     

弱精子症精子中miRNAs特异表达及潜在致病靶点的分析

摘要 目的：弱精子症可见于40%的不育男性,其特征是精子活力低下。微小RNA（MicroRNAs,miRNAs）在精子发生中发挥重要作用,但关于精子中miRNAs在弱精子症中的作用知之甚少。本研究试图初探miRNAs在弱精子症的分子机制。方法：收集了重度弱精子症患者和健康男性的精子样本,采用高通量序列技术来识别差异表达的miRNAs,并对差异显著的miRNAs进行生物信息学分析。通过qRT-PCR 证实了2个特异性改变的 miRNA及其靶基因表达情况。结果：重度弱精子症患者与正常男性相比,共有146 个miRNAs（P＜0.05; |log2 Fold Change|＞1）发生改变,其中表达上调的52个,下调的94个;预测上下调幅度最显著的前10个miRNAs 的靶基因,同时在miRDB和TargetScan 数据库存在的靶基因共有1407个。富集分析结果显示,miRNAs的靶基因富集于精子细胞的生物过程,还参与精子细胞的氧化代谢、刺激反应、增殖和分化以及凋亡等生物过程。通路分析显示,靶基因可能参与细胞自噬、细胞衰老、PI3K-Akt信号通路、MAPK信号通路、HIF-1信号通路、mTOR信号通路等。其中,在弱精子症精子中特异性上调的hsa-miR-371a-5p和hsa-miR-2355-5p,预测靶基因分别为自噬效应蛋白Beclin1和线粒体内膜蛋白抑素2（prohibitin2,PHB2）,二者直接参与线粒体自噬过程。qRT-PCR结果显示随着精子活力的降低,精子中hsa-miR-371a-5p和hsa-miR-2355-5p的表达量升高。结论：本研究发现弱精子症患者精子中特异性失调的miRNAs及其靶基因,为后续深入研究低活力精子中miRNAs参与调控线粒体自噬功能的机制提供新思路和理论依据。     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa

After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.     

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome-bearing spermatozoa sorted by high speed flow cytometry

The objective of this study was to examine preimplantation development and sperm aster characteristics of bovine male and female embryos produced by using spermatozoa sorted for the X or Y chromosome. In vitro matured oocytes were inseminated at 24 h of maturation with sorted X or Y chromosome-bearing spermatozoa, using either fresh or frozen-thawed semen. Samples were taken from each sperm group 12 h post insemination (hpi), fixed, and immunostained for the microtubule cytoskeleton. Confocal microscopy enabled visualization of sperm aster formation and microtubule characteristics of each zygote during early fertilization. Cultured embryos were checked for cleavage at 30, 35, 40 and 45 hpi, embryo development was examined daily until Day 8 of culture. Blastocyst cell numbers were determined at the end of the experiments. Reanalysis of the sorted sperm cells for DNA content showed purity rates of 90.1 and 92.1% for X and Y chromosome-bearing spermatozoa, respectively. Reduced fertilization and development rates were observed when sorted spermatozoa were used compared with fresh and frozen-thawed spermatozoa. Penetration rates at 12 hpi were 39.5, 44.7, 55.9 and 79.0%, while blastocyst formation rates at Day 8 were 26.7, 26.5, 31.7 and 40.7% for X and Y chromosome-bearing spermatozoa, using fresh and frozen-thawed semen groups, respectively. Sperm aster size was larger in males than females, while the size of pronuclei and subjective grade of sperm aster quality showed no differences between sexes. In this study, a greater cleavage rate and sperm aster size in male embryos indicated a dimorphic pattern of development in male and female embryos during fertilization and first cleavage.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Fertility-related proteomic profiling bull spermatozoa separated by percoll

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H(+) transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.     

Challenges facing sex preselection of stallion spermatozoa

Since the production of the first live offspring from sex-sorted spermatozoa in 1989, there have been many developments in the fluorescence-activated cell separation (FACS) procedures to preselect X- and Y-chromosome bearing spermatozoa prior to insemination. During this time, FACS technology has been applied to a range of species and has resulted in offspring from rabbits, cattle, sheep, elk and horses. In horses, satisfactory fertility rates have been achieved after hysteroscopic insemination of 20 x 10(6) fresh or stored, sex-sorted spermatozoa. However, many of the sperm processing protocols are still based on the original protocol and components of these procedures may not necessarily be suitable for the stallion. This review examines the details of FACS protocols that have resulted in the production of live offspring and makes comparisons with the published stallion protocols in an attempt to determine how best to improve the fertility of sorted, frozen-thawed stallion spermatozoa.