大熊猫体外受精的初报

我们在大熊猫精子体外获能与异种穿卵成功的基础上,又进行了大熊猫体外受精的首次尝试。 大熊猫卵母细胞取自刚死的育龄雌性大熊猫的卵巢,经体外培养16小时后,加入在体外已获能的大熊猫精子,进行体外受精。发现精子头能穿入卵透明带并进卵膜,继续培养19小时后因污染不幸夭亡。这是大熊猫同种体外受精的第一次报道。这一新进展表明,对研究试管大熊猫又跨近了一步。人们将有可能利用这种生物高技术去主动地拯救大熊猫,为珍稀动物物种的繁殖和保存开劈了一条新途     

大熊猫精液冷冻颗粒的解冻液研究

本研究主要探讨大熊猫(Ailuropoda melanoleucus)精液冷冻颗粒的解冻液。80年代,我们率先研制成功了大熊猫精子体外获能液,继又研究了大熊猫体外受精的培养系统,并     

特定电磁波(TDP)对家兔精子超弱化学发光及体外获能的影响

为了探讨特定电磁波(TDP)对精子超弱化学发光和体外获能的影响,本文用不同剂量的TDP辐照处理家兔精液,对精子超弱化学发光及体外获能进行了研究.实验结果表明,适宜剂量TDP辐照家兔精液,可使其超弱化学发光增强,并对精子体外获能和顶体与反应有诱导和促进作用,大剂量的TDP辐照有促进获能精子死亡的作用.经过对精子超弱化学发光和精子直顶体反应率的相关分析,结果表明,二者呈强正相关(r=0.805,P<0.01).     

用CTC荧光染色法检测添加肝素对牛精子体外获能的影响

本试验在TALP液中添加不同浓度(0、20、40、80μg.ml-1)的肝素对牛精子进行获能,用CTC染色检测精子获能状况,探讨肝素浓度及诱导时间(1、3、6、9h)对牛精子体外获能的影响。结果显示:随着肝素浓度的提高,精子获能效果增强,3 h达到最高,但添加80μg.ml-1的肝素精子的顶体反应率低于其他组。结果表明:在TALP液中增加高浓度的肝素对精子体外获能有促进作用,用高浓度的肝素获能处理3h后的精子进行体外受精可能会得到较好的效果。     

大熊猫精子获能和顶体反应过程中钙分布变化规律的研究

应用焦锑酸钾原位定位法对大熊猫精子获能和顶体反应过程中进行钙定位研究,发现未获能精子的 Ｃａ２＋主要结合于顶体前区和赤道段质膜外侧和顶体内膜内侧（核膜侧）;随着获能的进行,Ｃａ２＋进入精子内部并主要结合于顶体区质膜内侧和顶体外膜外侧;顶体反应的精子,Ｃａ２＋结合于顶体内膜外侧、顶体后区质膜外侧和分散存在于释放的顶体内容物中,有些顶体反应精子的顶体内膜外侧结合的Ｃａ２＋特别丰富。精子尾部的Ｃａ２＋主要分布于中段线粒体内,且其内所含Ｃａ２＋含量随着获能和顶体反应而增加。另外尾部致密纤维和轴丝处也有少量Ｃａ２＋分布。     

猕猴精子优选及体外获能的研究

选择活率高的精子并进行体外获能是开展猕猴体外受精研究的必要程序, 是研究猕猴受精生物学的重要手段。本实验采用上浮法和Percoll 梯度离心法对猕猴精液进行了优选, 并对处理后的精子形态正常率、精子活率、密度及受精率作了比较, 发现二者差异不显著; 用dbcAMP 和咖啡因使精子获能, 发现只有两种获能剂同时存在才能使猕猴精子获能并使卵母细胞受精。结论为: 上浮法和Percoll 法都是有效的精子优选法, 对受精率的影响差异不显著; dbcAMP 和咖啡因在猕猴精子体外获能时缺一不可。     

小鼠卵子在不同条件下的体外受精

本实验比较了小鼠精、卵细胞在不同生理状态下体外的受精能力。结果表明,体内受精率明显地高于体外(p<0.05),自发排出的卵子比超数排出的卵子受精率高(p<0.05),体外获能的附睾精子比体内获能的子宫精子受精率高(p<0.01)。唯独用超数排出的卵子和体外获能的附辜精子体外受精时,其受精率和体内相似。     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

精胺抑制人精子的体外受精能力

以精子穿透去透明带仓鼠卵试验(SPA)为模型,评价了精胺对人精子体外受精能力的影响。精胺(0.25—8.0mmol/L)可抑制人精子体外获能和受精,其抑制作用与精胺浓度呈正相关,此种抑制作用是可逆的。用 HPLC 测定精子精胺含量表明,精子获能后精胺含量明显下降。dbcAMP(0.5—1.0mmol/L)或咖啡因(10mmol/L)可拮抗精胺抑制人精子体外获能。其拮抗作用随 dbcAMP 浓度而增强。钙离子载体 A 23187 2/μmol/L 或胰蛋白酶0.05%均可拮抗精胺抑制人精子穿卵率。上述结果提示,精胺可能通过降低精子 cAMP 含量和抑制钙内流或顶体酶活性,从而阻止人精子体外获能和受精。     

哺乳动物精子获能过程中信号通路研究进展

哺乳动物精子在雌性生殖道内及体外获能培养过程中伴随着胆固醇外流、质膜重组、离子通道调节及获能相关蛋白磷酸化状态改变等相关生理调节过程,其中信号通路及相应信号分子对精子获能及功能修饰起到重要调节作用,成为精子细胞超激活运动及完成受精作用的关键环节。根据近年来的研究报道,对哺乳动物精子获能过程中已知的信号通路、信号分子及调节因子、离子通道、存在的问题及未来研究主要方向进行综述,为精子体外培养及辅助生殖等提供理论参考。     

The role of Ca2+ and protein kinase C in the acrosome reaction of giant panda (Ailuropoda melanoleuca) spermatozoa

Fresh semen was collected from adult male giant pandas and the role of Ca2+, Ca2+ ionophore A23187 and protein kinase C (PKC) in sperm motility and acrosome reaction (AR) was assessed by lens culinaris agglutinin conjugated with fluorescein isothiocyanate (FITC-LCA) labeling and transmission electron microscopy. The AR in giant panda spermatozoa was characterized by vesiculation of the outer acrosomal membrane through its invagination. Both the sperm motility and the AR rate decreased significantly (p < 0.05) in Ca2+-free and low Ca2+ medium. The addition of 10 microM Ca2+ ionophore A23187 potently stimulated AR. After incubation for capacitation, the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated AR in a dose-dependent manner and its effect could be overcome by the PKC inhibitor staurosporine. These results suggest that Ca2+ and PKC play an important role in the sperm acrosome reaction of the giant panda.     

Sperm from beta1,4-galactosyltransferase I-null mice exhibit precocious capacitation

Mammalian sperm must undergo a physiological maturation, termed capacitation, before they are able to fertilize eggs. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. In this paper, we describe the capacitation phenotype of sperm lacking the long isoform of beta1,4-galactosyltransferase I (GalT I), a sperm surface protein that functions as a receptor for the zona pellucida glycoprotein, ZP3, and as an inducer of the acrosome reaction following ZP3-dependent aggregation. As expected, wild-type sperm must undergo capacitation in order to bind the zona pellucida and undergo a Ca(2+) ionophore-induced acrosome reaction. By contrast, GalT I-null sperm behave as though they are precociously capacitated, in that they demonstrate maximal binding to the zona pellucida and greatly increased sensitivity to ionophore-induced acrosome reactions without undergoing capacitation in vitro. The loss of GalT I from sperm results in an inability to bind epididymal glycoconjugates that normally maintain sperm in an 'uncapacitated' state; removing these decapacitating factors from wild-type sperm phenocopies the capacitation behavior of GalT I-null sperm. Interestingly, capacitation of GalT I-null sperm is independent of the presence of albumin, Ca(2+) and HCO(3)(-); three co-factors normally required by wild-type sperm to achieve capacitation. This implies that intracellular targets of albumin, Ca(2+) and/or HCO(3)(-) may be constitutively active in GalT I-null sperm. Consistent with this, GalT I-null sperm have increased levels of cAMP that correlate closely with both the accelerated kinetics and co-factor-independence of GalT I-null sperm capacitation. By contrast, the kinetics of protein tyrosine phosphorylation and sperm motility are unaltered in mutant sperm relative to wild-type. These data suggest that GalT I may function as a negative regulator of capacitation in the sperm head by suppressing intracellular signaling pathways that promote this process.     

Suppression effect of seminal vesicle autoantigen on platelet-activating factor-induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.     

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility

When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.     

Decrease in order of human sperm lipids during capacitation

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of the sperm sterols, cholesterol and desmosterol, is an obligatory step in the capacitation of human sperm. Because sterols can increase the order of membrane phospholipids, it has been suggested that the importance of sterol loss is that it decreases membrane lipid order. The present study tested the hypotheses that sterol loss decreases sperm membrane lipid order during capacitation and that lipid disorder is a sufficient stimulus for capacitation. Steady-state fluorescence anisotropy of the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, decreased during capacitation, indicating a decrease in lipid order. The decrease was dependent on the loss of sperm sterols, suggesting that it reflected diminished sterol-mediated phospholipid ordering. However, the lipid-fluidizing agents, benzyl alcohol and 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, did not cause sperm capacitation or overcome inhibition by cholesterol. In summary, loss of sperm sterols caused a significant decline in lipid order during capacitation; however, decreased bulk lipid order was not sufficient to trigger the subsequent events that complete capacitation.     

Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: A flow cytometric study using lectins

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 μg/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. © 1996 Wiley-Liss, Inc.     

Sphingomyelin modulates capacitation of human sperm in vitro

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.     

Mice carrying two t haplotypes: sperm populations with reduced Zona pellucida binding are deficient in capacitation.

Capacitation is the unique process by which mammalian sperm become capable of undergoing the acrosome reaction (AR). An approach to studying sperm capacitation is to identify mutations altering this process. Male mice carrying two t haplotypes are sterile, with poor sperm motility, reduced zona pellucida binding, and an inability to penetrate zona-free oocytes. The objective of this study was to examine sperm capacitation and its potential relationship to zona pellucida binding in mice of the same genetic strain carrying none, one, or two t haplotypes. Sperm capacitation was assessed by the B pattern of staining by chlortetracycline (CTC) and by the ability of sperm to undergo the lysophosphatidylcholine (LPC)-induced AR. The CTC assay demonstrated that sperm capacitation from t/+ mice was similar to that from +/+ mice, but sperm from t/t mice were deficient. LPC induced the AR of capacitated sperm, but not noncapacitated sperm, in a concentration-dependent manner. Sperm from t/t mice were also deficient in the LPC-induced AR. Thus, by two independent assays, sperm from t/t mice were shown to be deficient in capacitation. To determine whether a deficiency in capacitation could influence zona binding, the ability of capacitated versus noncapacitated sperm to bind to the zona pellucida was tested. The mean numbers of sperm bound per oocyte were significantly greater for capacitated sperm than for noncapacitated sperm. These results suggest that the deficient capacitation of sperm from t/t mice could be responsible for, or at least contribute to, their reduced ability to bind to the zona pellucida.     

Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions

Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl beta-cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.