Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa

Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integrity to deviations from isotonicity and, in addition, that rhesus sperm motility and membrane integrity were more sensitive to hypertonic than hypotonic conditions. Mitochondrial membrane potential did not explain the lack of sperm motility observed under anisosmolal conditions in our study. Although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility.     

Cryopreservation—induced decrease in heat—shock protein 90 in human spermatozoa and its mechanism

Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.     

Immunolocalization of heat shock protein 70 in bovine spermatozoa

Heat shock protein 70 (HSP70) is part of a superfamily of molecular chaperones, which protect cells from chemical and heat shock. The objectives of this study were to determine the presence of HSP70 in bovine spermatozoa and its subcellular localization during different stages of spermatogenesis. Analysis of sperm proteins by Western blotting using a monoclonal antibody to the inducible form of HSP70 revealed a single immunoreactive band with an estimated molecular weight of 70 kDa in samples from 18 of 18 bulls. Using immunofluorescence microscopy and the same antibody, HSP70 was localized to the cytoplasm of prophase spermatocytes and elongating spermatids, to cytoplasmic droplets of caput epididymal spermatozoa, and to cytoplasmic droplets, acrosome, post-acrosomal region and middle piece of corpus and cauda epididymal spermatozoa. The pattern of distribution changed in freshly ejaculated spermatozoa as HSP70 was detected on the acrosome only. During capacitation and acrosome reaction, HSP70 was once again redistributed, and was localized to the equatorial segment, post-acrosomal region and middle piece. Thus, HSP70 is present in the spermatozoa of mature bulls and redistribution of the protein occurs during capacitation and the acrosome reaction.     

The importance of calcium in the appearance of p32, a boar sperm tyrosine phosphoprotein,during in vitro capacitation

After ejaculation, mammalian sperm must undergo a preparation period known as "capacitation" to become capable of fertilizing the oocyte. Although physiological capacitation occurs in the female genital tract, the process can be reproduced in vitro by incubation in appropriate media. However, the signaling events regulating capacitation are poorly understood, especially in boar sperm. Calcium is thought to be of fundamental importance in capacitation. Our laboratory recently identified a tyrosine-phosphorylated protein of M(r) 32,000 ("p32") from boar sperm, and its appearance is closely related to capacitation. The objective of this study was to understand the mechanism regulating the appearance of our p32 tyrosine phosphoprotein. Since calcium has been linked to sperm capacitation and protein tyrosine phosphorylation in other species, we hypothesized that extracellular calcium is involved in the appearance of the p32. Sperm were incubated in either noncapacitating medium (NCM) or capacitating medium (CM) for various times. Proteins were extracted with sodium dodecyl sulfate (SDS), separated by SDS-polyacrylamide gel electrophoresis (PAGE), and then immunoblotted with an antiphosphotyrosine antibody. To assess intracellular calcium levels, fresh sperm were loaded with the fluorescent calcium indicator indo-1, and relative fluorescence was measured by flow cytometry. Analysis demonstrated that relative intracellular calcium levels increased during incubation in capacitating conditions but not in NCM, which coincides with the appearance of the p32. The p32 tyrosinephosphorylated protein appeared only in the presence of calcium, and the calcium ionophore Br-A23187 accelerated its appearance. Consistent with our hypothesis, the appearance of the p32 was inhibited by extracellular calcium chelators (ethylene glycol-bis(2-aminoethylether)-N,N,N',N',-tetraacetic acid [EGTA], EDTA, and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt [BAPTA-K(+)]), showing the importance of calcium in protein tyrosine phosphorylation related to capacitation in boar sperm.     

Evaluation of HSPA2 in fertile and infertile individuals

Heat‐shock protein A2 (HSPA2) is a testis‐specific member of the HSP70 family known to correlate with sperm maturity, function and fertility. The aim of this study was to compare expression of HSPA2 in fertile and infertile individuals using a recently marketed highly purified polyclonal antibody. Thus, after analysing sperm concentration, motility and morphology in semen sample of 49 individuals with male factor infertility and 47 fertile individuals according to World Health Organization guidelines, we evaluated HSPA2 by microscope florescence, flow cytometry, Western blot and RT‐PCR. We observed higher percentage of sperm expressing HSPA2 in anterior and equatorial regions in fertile than infertile individuals by microscope fluorescence. Percentage of sperm expressing HSPA2 in two conditions (fixed permeabilised and capacitated) by flow cytometry showed that percentage of HSPA2‐positive sperm in fixed permeabilised and also capacitated samples was significantly different between fertile and infertile individuals. Western blot analysis of HSPA2 in semen samples revealed high variation within the fertile and infertile population. The results of RT‐PCR revealed higher expression of HSPA2 in the fertile compared to infertile individuals, but this difference was not significant. According to the results, we suggest that HSPA2 expression is heterogeneously expressed on different part of fixed permeabilised sperm and its expression is significantly higher in fertile compared to infertile individuals. The surface expression of this protein significantly increases following capacitation in both fertile and infertile individuals. HSPA2 expression significantly correlates with sperm concentration and morphology. Therefore, aberrant HSPA2 expression may play an important role in capacitation and fertilisation processes.     

Expression of 60-kDa and inducible 70-kDa stress proteins in gentamicin-induced acute renal failure

Background Heat shock proteins contribute to the survival of cells under stress conditions. We have previously observed the expression of HSP90 and constitutive HSP70 in rat kidneys during stress loading. This study was designed to determine whether HSP60 and HSP70i are induced in rat kidneys during exposure to gentamicin. Methods During the continuous administration of gentamicin (40 mg/kg per day) to rats for 30 days, the changes of HSP60 and HSP70i in these kidneys were quantitatively investigated by immunoblot analyses using specific antibodies. Results The indicators for renal failure,N-acetyl-β-d-glucosaminidase, blood urea nitrogen, and serum creatinine, increased, and reached the maximum levels at day 12. Thereafter, they spontaneously decreased, and were close to their normal levels at day 21, despite continued gentamicin exposure. The time course of HSP60 levels showed a unique 2-peak pattern exclusively in the cytosols of the cortex; the gentamicin target site. The second HSP60 peak occurred during the recovery phase, and coincided with published reports of a second peak of gentamicin accumulation in the kidney. The time course of HSP70i level showed a single peak at day 12 when the renal failure was most intense; the level was similar between the soluble and insoluble fractions in all portions of the kidney, except the inner medulla. Conclusion The increase in cytosolic HSP60 levels, as well as HSP70i levels, may be related to the spontaneous recovery from renal failure at the target site of the drug.     

大鼠烫伤早期肠黏膜组织热休克蛋白HSP70和HSP90的表达

目的 研究烧伤早期肠黏膜组织热休克蛋白HSP70和HSP90的表达、组织含量和分布的变化规律,探讨烧伤早期肠黏膜组织细胞的热休克反应在机体的病理生理反应中的意义。方法 以烫伤大鼠为模型,利用ELISA、免疫印迹分析和免疫组织化学方法,分析和研究肠黏膜组织HSP70和HSP90的表达、组织含量和分布及其功能状态。结果 (1)大鼠烫伤早期肠黏膜游离HSP70的含量有非常显的短暂降低;(2)肠黏膜组织HSP70和HSP90的总体含量在烧伤后有显升高;(3)烧伤早期肠黏膜组织HSP70的分子结构存在显的不均一性。结论 肠黏膜组织细胞中两种热休克蛋白表达、含量和分布的规律性变化,在肠黏膜组织细胞的应激反应、进而在肠道的黏膜屏障机制中,可能有重要的意义。     

Specific order in the appearance of protein tyrosine phosphorylation patterns is functionally coordinated with dog sperm hyperactivation and capacitation

The aims of the present study were to characterize a slow capacitation system that records initial changes in the sperm membrane state, and, using a canine model, to order the specific protein tyrosine phosphorylation signaling in the sequence of capacitational events and to associate them with hyperactivated motility. Dog sperm washed through Percoll were incubated in complete bicarbonate Tyrode medium for 6 hours in 5% CO(2). Capacitation was evaluated using chlortetracycline staining. Tyrosine phosphorylation patterns were assessed by immunocytochemistry. Parallel to this, a computer-assisted motility analysis was performed. Significant changes in the percentage of capacitated and acrosome-reacted cells were first observed after 90 minutes, increasing in a linear manner during further incubation (P <.05). Changes in the percentage of capacitated cells were accompanied by motility changes. During incubation, a strictly sequential phosphorylation of sperm tail (midpiece, principal piece, and end piece) and head proteins was observed. According to an analysis of kinetics, phosphorylation of head proteins occurred after the tail became completely phosphorylated. Changes in head phosphorylation progressed at the same rates as capacitation and acrosome reaction. Sperm motility, curvilinear velocity, average path velocity, straight line velocity, and lateral head displacement were correlated positively or negatively with phosphorylation of midpiece or end piece proteins, respectively. The bicarbonate-stimulated increases in cyclic adenosine monophosphate levels and changes in protein phosphatase activity may be involved in the signaling system that controls membrane changes and motility in dog sperm. Phosphorylation kinetics of sperm proteins are potentially useful for diagnostic purposes to characterize the response of individual males to fertilizing conditions.     

子宫内膜热休克蛋白表达与不明原因不育症的相关性研究

目的 :探讨不明原因不育症与热休克蛋白 ( HSP)表达的关系。方法 :对 72例不明原因不育症 (不育症组 )和 3 4例 (对照组 )子宫内膜作常规染色 ,行组织学分类 ;酶联免疫吸附测定 ( EL ISA)法检测血清抗精子抗体 ( As Ab) ;免疫组织化学染色 ( S-P法 )标记HSP70和 HSP90α在子宫内膜中的表达。结果 :HSP70阳性表达在不育症及对照组子宫内膜分别为 65 %及 3 8% ( P<0 .0 5 ) ;血清 As Ab阳性及阴性患者中分别为 81 %及 3 0 %( P<0 .0 1 )。 HSP90α阳性表达在不育症及对照组子宫内膜分别为 5 4%及 47% ( P>0 .0 5 ) ;血清 As AB阳性及阴性患者中分别为 69%及 2 4% ( P<0 .0 1 )。结论 :不明原因不育症患者子宫内膜 HSPs的高表达可能是一种自身免疫应激反应     

输精管结扎后小鼠睾丸热休克蛋白表达

目的 :探讨热休克蛋白 70 ( HSP70 )在输精管结扎术前后小鼠睾丸中的表达。方法 :60只昆明小鼠行双侧输精管结扎术 ,并设 3 0只假手术小鼠为对照组。分别于术后 1、2、3个月观察。睾丸组织行常规制片后进行免疫组化染色检测 HSP70的表达 ,小鼠血清采用精子凝集和精子制动试验进行抗精子抗体检测。结果 :在结扎组及对照组睾丸生精细胞中均有 HSP70表达。对照组与结扎术后 2个月组 HSP70表达水平均较低 ,两者之间无显著差异 ;结扎术后 2个月组及 3个月组与对照组及结扎术后 1个月组相比 ,HSP70表达水平显著上升 ( P<0 .0 1 )。结论 :输精管结扎术后一段时间内 ,睾丸的生精过程经历从抑制到恢复正常的变化。这一变化可能与 HSP70相关。     

Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO(2) in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO(3). Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 micromol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochemically examined by means of anti-phosphotyrosine (alpha-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P < .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P < .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P < .0001) and a significant increase in the intensity of merocyanine fluorescence (P < .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.     

热休克蛋白在男性不育中的研究进展

热休克蛋白是一类生物进化上高度保守的伴侣蛋白分子,具有多种生物学功能,包括作为分子伴侣、细胞保护、抗凋亡和免疫调节等。近年来研究发现,多种热休克蛋白参与精子发生、精子获能及受精等一系列活动,与男性生殖过程密切相关。因此,进一步研究热休克蛋白在男性不育中的具体机制及作用,可能为男性不育提供新的治疗途径。本文重点对热休克蛋白在男性不育中的研究进展作一综述。     

Biochemical parameters of initiation and regulation of sperm motility

Studies of in vitro models demonstrate that a forward motility protein (FMP) is required for the initiation of forward motility in the immature epididymal spermatozoa. FMP is a heat-stable glycoprotein derived from epididymal plasma. During the epididymal maturation of spermatozoa in vivo, there is a marked increase of intrasperm pH and level of cyclic adenosine monophosphate (cAMP). Several studies suggest that exogenous FMP in concert with elevated intrasperm pH and level of cAMP initiates flagellar motility during the epididymal transit of sperm. cAMP activates sperm cytosolic cAMP-dependent protein kinases, which in turn phosphorylate multiple intrasperm phosphoproteins that may regulate flagellar motility. Exogenous calcium ion activates intact sperm motility, although it inhibits motility of demembranated cells on reactivation. Occurrence of cAMP-dependent type I and II protein kinases, a novel cAMP-independent protein kinase, and a phosphoprotein phosphatase has been demonstrated on the external surface of spermatozoa. The sperm surface has a coupled-enzyme system: ecto-cAMP-independent protein kinase and phosphoprotein phosphatase that regulate the phosphorylation and dephosphorylation of endogenous sperm ectophosphoproteins. The specific activities of these ecto-enzymes increase markedly during forward progression, suggesting that they may have a role in regulating flagellar motility.     

Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences

Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca(2+)](i). While LY294002 increased [Ca(2+)](i), wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca(2+)](i) operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon.     

Redox regulation of mammalian sperm capacitation

Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility.     

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。     

促发精子获能的分子机制

获能是精子在雌性生殖道中转运期间获得受精能力的过程。获能的分子机制相当复杂 ,目前尚未明确 ,但促发获能的起始分子事件已初步确定 ,包括精子细胞膜表面胆固醇外流、膜电位的改变、蛋白磷酸化等。本文综述了促发精子获能的分子机制及其研究前景。     

热休克蛋白70-2基因沉默对大鼠精子附睾成熟的影响

目的 观察热休克蛋白(HSP)70-2基因沉默对大鼠附睾中精子成熟的影响.方法 构建HSP70-2特异的短发夹RNA(shRNA)质粒载体,注入大鼠附睾中,1周后处死大鼠,无菌切取附睾,用逆转录-聚合酶链反应(RT-PCR)方法 检测附睾中HSP70-2 mRNA表达,Western blot观察HSP70-2蛋白表达,应用苏木素-伊红(HE)染色检测附睾管内精子密度.结果 空白对照组HSP70-2蛋白表达量为1.31±0.10,附睾管内成熟精子密度为(63.46±14.43)%.基因沉默组HSP70-2蛋白表达量为0.93±0.13,附睾管内成熟精子密度为(31.51±10.67)%,与对照组比较均显著减少,差异有统计学意义(P＜0.01).结论 HSP70-2基因沉默影响附睾中精子成熟.     

Effects of seminal plasma on cooling-induced capacitative changes in boar sperm

Porcine seminal plasma (SP) has been shown to contain factors that have a decapacitative or capacitation-inhibiting effect on sperm. The objectives of the present study were to compare the capacitative changes observed in cooled sperm with those seen in sperm after in vitro capacitation and to determine whether SP could prevent these changes. Sperm were subjected to incubation or to slow cooling under noncapacitating or capacitating conditions. The effect of SP on protein tyrosine phosphorylation and the ability of the sperm to undergo an acrosome reaction (AR) were determined. Cooled sperm displayed an increased level of tyrosine phosphorylation and a higher percentage of induced AR sperm compared to incubated sperm. The addition of SP inhibited the number of ARs that occurred during incubation and cooling. These results suggest that cooling of sperm augments the capacitative changes in sperm, and that SP contains a factor(s) that effectively prevents these changes.