“益精方”对小鼠精子尾部特异性钙通道CatSper1的影响

目的:观察"益精方"对环磷酰胺所致少弱精子症模型小鼠精子尾部特异性钙通道CatSper1的影响。方法:将40只雄性昆明小鼠随机分为对照组(CG)、模型组(MG)、小剂量中药治疗组(SG)和大剂量中药治疗组(LG),按60mg/kg体重给CG小鼠腹腔注射生理盐水(NS),给MG、SG、LG小鼠腹腔注射环磷酰胺,每天1次,连续5d,第6d开始按体重分别给SG和LG小鼠灌服"益精方",剂量分别为人类常规用量(以60kg为标准)的1倍和5倍,MG小鼠给予等体积的NS灌胃,每天1次,连续34d,CG常规饲养,然后对小鼠进行精液常规分析,并用RT-PCR法检测小鼠精子CatSper1的表达。结果:CG、MG、SG和LG组小鼠附睾精子密度分别为(5.20±1.34)、(1.73±0.03)、(2.08±0.01)、(3.31±0.56)×106/ml,a+b级精子百分率分别为(14.49±0.30)%、(6.64±1.88)%、(11.99±1.01)%、(19.40±3.13)%,a+b+c级精子百分率分别为(68.39±15.13)%、(39.96±4.89)%、(62.28±4.43)%、(73.61±5.05)%,MG组精子密度、a+b级精子百分率、a+b+c级精子百分率显著低于CG组(P<0.05),经治疗后LG组精子密度、a+b级精子百分率、a+b+c级精子百分率明显增加,与MG组比较有显著性差异(P<0.05),而与CG组在a+b级精子百分率和a+b+c级精子百分率上没有明显区别。CatSper1的表达量在CG、MG、SG和LG组分别为0.76±0.05、0.73±0.03、0.75±0.12、0.85±0.04,LG组显著高于MG组(P<0.05),而与CG组比较没有明显区别。结论:腹腔注射环磷酰胺可使小鼠精子密度、a+b级精子百分率、a+b+c级精子百分率降低,CatSper1表达量下降,大剂量"益精方"可以通过提高CatSper1的表达量,增加小鼠精子密度、a+b级和a+b+c级精子百分率,从而达到治疗少弱精子症的目的。     

Effects of dioxin on testicular histopathology,sperm parameters,and CatSper2 gene and protein expression in Naval Medical Research Institute male mice

The CatSper gene family is known to be solely expressed in sperm cells and is possibly associated with sperm motility and penetration through the zona pellucida. Despite its vital role in male fertility, factors regulating its expression are not widely known. The present study aimed at evaluating the effects of dioxin on CatSper2 gene and protein expression, testicular histopathology, sperm quality and biochemical parameters in a mice model. The experiments were performed on 32 Naval Medical Research Institute male mice (2–3 months). The animals were divided into four groups in a random manner: (a) control; (b) dioxin 1; (c) dioxin 2; and (d) dioxin 3. The treatment groups received 0.1, 0.5 and 1 µg/kg of dioxin intraperitoneally every day for 2 weeks. Administration of dioxin significantly downregulated the CatSper2 gene and protein expression. A greater reduction in gene and protein expression was found at higher doses of dioxin. At the same time, sperm parameters, especially sperm motility and count, decreased in mice exposed to dioxin. The results of testicular histopathology showed necrotic degeneration and epithelium thickness reduction in the dioxin groups in comparison with the controls. Besides, oxidative stress increased in seminiferous tubules.     

Movement characteristics and hyperactivation of human sperm on different epithelial cell monolayers

Studies of sperm movement characteristics concern mainly sperm swimming between two glass surfaces (as in the Makler chamber). Using automated videomicrography, (CellSoft, Cryo Resources, New York, USA), we have analysed the movements of human sperm swimming on monolayers of different origins: monkey kidney (Vero) cells, bovine oviduct cells, and human endometrial cells. About 10(5) sperm were deposited upon preparations consisting of monocellular layers adhering to a coverglass, and placed in a deep slide-coverglass system. Experiments were first performed at room temperature then at 37 degrees C. At room temperature, motion characteristics on Vero cell layers (six samples) were not different from those measured in either the conditioned or corresponding non-conditioned media, except for the amplitude of lateral head displacement (ALH) which was significantly lower. Comparison of the three different cell monolayers showed no difference between them for the corresponding motion parameters. The data were dramatically different at 37 degrees C: sperm swimming on cell monolayers of genital origin (oviduct or endometrium) exhibited high rates of hyperactivation (HA: 36.7% and 38.6% respectively), which was significantly more than on either Vero cells (10.9%) or in a control medium (12.6%). Moreover, HA rates were significantly higher on genital cell monolayers than in the corresponding conditioned medium. Hyperactivated sperm exhibited lasting 'star-spin' trajectories rather than 'transitional phases'. It is concluded that passage of sperm on either oviduct or endometrial epithelial cell monolayers can induce sperm hyperactivation and improve their fertilizing capacity.     

CatSper基因家族在人和小鼠组织中的表达特征

目的研究CatSper基因家族在人和小鼠组织中的分布特点。方法将不同发育阶段的小鼠睾丸组织cDNA与Affymetrix全基因组芯片探针进行杂交,筛选出差异表达基因CatSper基因家族。RT—PCR验证差异表达基因在不同发育阶段的小鼠睾丸组织的表达特征,及其在人和小鼠不同组织中的分布。结果基因芯片分析发现CatSper1、CatSper2和CatSper3在小鼠睾丸的表达呈阶段特异性。RT-PCR结果表明该基因家族在睾丸的表达丰度明显高于其它组织;CatSper1和CatSper4在人体睾丸组织中特异性表达。结论CatSper基因家族在人和小鼠中呈现睾丸特异性表达或高表达,可能在精子发生中发挥重要功能。     

用SYBR Green I实时逆转录-聚合酶链反应定量检测人、小鼠精子中CatSper1 mRNA

目的:建立并优化SYBR GreenI实时RT-PCR体系,定量检测人、小鼠成熟精子中的CatSper1 mRNA。方法:用TRIzol分别提取人、小鼠成熟精子中的总RNA,逆转录后用SYBR GreenI实时PCR定量检测CatSper1 mRNA。SYBR GreenI实时PCR采用普通PCR试剂,加入SYBR GreenI染料,优化退火温度、Mg2+浓度及上、下游引物比例,并在PCR循环时采用四步法以消除引物二聚体的影响。优化完成后用不同浓度的精子cDNA为模板做标准曲线,以检测SYBR GreenI实时PCR的扩增效率。结果:定量检测CatSper1 mRNA的SYBR GreenI实时PCR体系适宜退火温度、Mg2+浓度及上、下游引物比例分别为63℃、3.0mmol/L和1∶1,四步法中采集荧光的温度为88℃。优化后用人和小鼠精子cDNA为模板做标准曲线分别为Y=-3.402log(X)+25.99和Y=-3.409log(X)+24.09,扩增效率分别为96.8%和96.5%,可定量检测人、小鼠成熟精子中的CatSper1 mRNA。结论:用普通的逆转录及PCR系统和试剂,建立了一种方便、廉价、可靠的SYBR GreenI实时荧光定量RT-PCR系统,可用于人、小鼠精子中CatSper1 mRNA定量检测。     

人精子RNA的提取及含量分析

目的建立可靠的提取人精子RNA的方法,分析其含量并用于检测基因mRNA。方法9例正常精液标本液化后,经Percoll纯化,用体细胞裂解液(含0.1%十二烷基硫酸钠和0.5%Triton X-100的水溶液)于0℃处理15 min去除精子以外的细胞。用RNeasy Kit提取精子RNA,微量核酸测定仪测定RNA量。RNA用于逆转录-聚合酶链反应(RT-PCR),检测β-Actin、精子特异性阳离子通道2(CatSper2)、鱼精蛋白2(Protamine-2)mRNA,同时,检测c-Kit、CD4、上皮细胞钙粘蛋白(E-Cad-herin)mRNA,分别排除生精细胞、白细胞和上皮细胞的污染。结果体细胞裂解液于0℃处理15 min能去除精子以外的细胞,使精子RNA提取量提高(2.2~4.9倍)。9例正常精液标本RNA量为(233.5±75.3)ng/106精子。所有标本RNA用RT-PCR,均成功扩增β-Actin、CatSper2、Protamine-2,但扩增c-Kit、CD4、E-Cadherin未见目的条带。结论人精子中含微量RNA,本提取方法能提取人精子中微量RNA,且避免其他细胞污染,可供人精子RNA研究和临床检测选用。     

Effects of a micronutrient supplementation combined with a phosphodiesterase type 5 inhibitor on sperm quantitative and qualitative parameters,percentage of mature spermatozoa and sperm capacity to undergo hyperactivation: A randomised controlled trial

The main objective of this study was to evaluate the effects of a micronutrient supplementation (MS) combined with avanafil on sperm function. Oligoasthenospermic men (n = 217) were treated daily for 90 days with either an MS (45 men, Group A), l ‐carnitine (44 men, Group B), MS plus avanafil (43 men, Group C) or avanafil (43 men, Group D); another group of 42 men with oligoasthenospermia (Group E) received no treatment. Sperm parameters were evaluated before and after the end of treatment in each Group A, B, C and D respectively. The same sperm parameters were measured in each participant of Group E before and at the 90‐day experimental period. Within Groups A, C or D, the total percentage of motile spermatozoa, the hypoosmotic swelling test (HOST) result and the percentage of hyperactivated spermatozoa after incubation under conditions known to induce sperm capacitation were significantly greater after MS or MS plus avanafil treatment, or avanafil treatment than before the respective treatment. We suggest that MS or MS plus avanafil combined administration or avanafil alone improves sperm membrane permeability with an overall result improvement in sperm motility, outcome of HOST and increase in the percentage of hyperactivated spermatozoa.     

CatSper1在精索静脉曲张模型大鼠精子中的差异表达及左卡尼汀对其的保护作用

目的:通过制备精索静脉曲张大鼠模型,检测其附睾精子中精子特异性钙通道(CatSper1)的表达,观察左卡尼汀对大鼠精子CatSper1的影响。方法:70只雄性大鼠随机分为7组,每组10只。分别为对照组(A组),精索静脉曲张组(B组),精索静脉曲张+生理盐水组(C组),精索静脉曲张+小、中、大剂量左卡尼汀组(D、E、F组)和F+延长喂养14 d组(G组)。手术结扎部分左肾静脉制备精索静脉曲张大鼠模型,12周后,C组给予生理盐水1 ml/d,D、E、F组分别用左卡尼汀小[0.05 g/(kg·d)]、中[0.1 g/(kg·d)]、大[0.2 g/(kg·d)]剂量灌胃35 d,G组在F组基础上延长灌胃14 d;实验结束后处死大鼠,进行精子参数分析,应用RT-PCR以及Western印迹分别检测精子中CatSper1 mRNA及蛋白表达情况。结果:与A组比较,B组a+b级精子百分率、精子活率及浓度均不同程度下降(P0.01),B组精子CatSper1 mRNA及蛋白相对表达量均明显下降(1.44±0.67 vs 0.71±0.38;1.87±0.67 vs 0.84±0.42,P0.01)。与C组比较,应用左卡尼汀后,各组精子浓度无明显增加, a+b级精子百分率、精子活率以及精子CatSper1 mRNA及蛋白表达含量明显增高,尤其大剂量组F、G组增高更加明显(P0.01)。与F组比较,G组延长应用2周左卡尼汀,精子CatSper1 mRNA及蛋白表达量无明显增加(P0.05)。结论:精索静脉曲张模型大鼠精子中CatSper1表达下降,应用左卡尼汀后,CatSper1的表达量及a+b级精子百分率、精子活率明显增加。     

Panax ginseng improves survival and sperm quality in guinea pigs exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin

OBJECTIVES: To further assess the effect of Panax ginseng on survival and sperm quality of guinea pigs exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). MATERIALS AND METHODS: Eighty male guinea pigs were divided into eight equal groups. The normal control (NC) group received vehicle and saline; one dose of 1 micro g/kg body weight TCDD was injected intraperitoneally into the single TCDD-treated (TT) and test groups (P100, P200, C100, C200); G and NC groups received vehicle instead of TCDD. P. ginseng water extract (PG-WE) was injected intraperitoneally at daily doses of 100 (G100, P100, C100) or 200 mg/kg body weight (G200, P200, C200). The PG-WE was administered to the P and G groups for 12 weeks from 1 week before TCDD exposure, and to the C groups for 10 weeks from 1 week after TCDD exposure. After a 4-week discontinuation of PG-WE treatment after the 13th week the surviving males were then tested for fertility by mating them with females. The litter size, death rate, male/female birth ratio and physical abnormalities of the progeny were investigated. After confirming delivery of the offspring, the parent males were killed at 40 weeks, their testes weighed and sperm quality assessed. RESULTS: All TT animals died within 18 days after TCDD exposure, but 40-70% of the PG-WE-treated groups, depending on the group, survived until death at 40 weeks. All the surviving males were fertile regardless of TCDD exposure; there was no difference in litter size between the NC and test groups. Notably the death rate of progeny born to PG-WE-treated groups was lower than that of progeny born to the NC group. The progeny born to TCDD-exposed groups (P200 and C groups) had a preponderance of females. G Group animals had higher sperm quality than that of NCs even long after discontinuing PG-WE. CONCLUSION: P. ginseng improves the survival rate and sperm quality in guinea pigs exposed to TCDD.     

Functional motion changes during sperm transit to the site of fertilization and in-vitro applications: a review

Continued research to define the parameters of sperm function should aid the evaluation of various approaches in infertility management as well as the efficacy of contraceptives for men which do not necessarily achieve azoospermia.
Many treatment forms have been advocated for male factor infertility but have yielded little effect. These include, for example, gonadotrophins, clomiphene citrate, the weakly androgenic steroid, mesterolone. Often, improvements in oligoasthenozoospermia that are not related to genital infection, do not attain normozoospermic levels.
Owing to lack of success with the various treatment modalities, assisted reproductive technology encompassing artificial insemination by husband or donor following in vitro enhancement of sperm function have assumed an important role in male infertility. Agents that have been shown to induce and support sperm capacitation processes such as hyperactivation, could serve an important role. These include human follicular fluid (HFF), maternal serum, fetal cord serum and methyl xanthine derivatives.     

环核苷酸门控通道与精子功能

环核苷酸门控通道(CNG)是非选择性阳离子通道,直接由环核苷酸活化,是Ca2+进入细胞内的主要通道之一。CNG通道蛋白由6个不同基因编码,4个A亚单位和2个B亚单位。CNG通道受Ca2+/钙调蛋白和磷酸化/去磷酸化作用所调控。近年来,CNG通道在生殖系统方面的研究受到了广泛关注。大量研究表明,CNG通道在精子运动、获能和顶体反应中起着重要的作用。本文对CNG通道与精子功能的关系进行综述。     

Hyperactivated motility of sperm from fertile donors and asthenozoospermic patients before and after treatment with ionophore

Hyperactivated motility of capacitated sperm was studied before and after contact for 30 min with the Ca2+ ionophore A23187. Sperm from fertile donors and asthenozoospermic infertile patients were incubated in the presence of increasing concentrations of A23187 in the medium. At 5-10 microM, the ionophore induced a significant increase (P less than 0.05) in the percentage of hyperactivation of sperm from asthenozoospermic subjects. Higher concentrations (20 and 30 microM) were needed to enhance hyperactivation of sperm in the fertile population. In the latter, extended contact with the ionophore induced no significant change compared to a 30-min incubation period. Since this type of movement is an essential feature of the fertilizing gamete, a low hyperactivation rate may partly explain fertilization failure in the asthenozoospermic group.     

STIM1和Orail在骨肉瘤细胞HOS增殖和凋亡中的作用

目的：探讨钙库操纵的钙内流（ SOCE）组成蛋白STIM1和Orai1蛋白在骨肉瘤细胞系HOS增殖和凋亡中的作用。方法用shRNA干扰技术抑制STIM1和Orai1蛋白的表达。 Western blot测定STIM1和Orai1的蛋白表达;激光共聚焦检测细胞Ca2＋内流变化;噻唑蓝（ MTT）比色法检测HOS细胞的增殖能力;流式细胞技术检测HOS 细胞的凋亡变化。结果与空白对照组和转染negative-shRNA的对照组相比,转染48 h后STIM1-shRNA 组STIM1蛋白的表达均明显降低（ P＜0.01）,Orai1-shRNA组Orai1蛋白的表达也明显降低。抑制STIM1和Orai1蛋白后,细胞Ca2＋内流减少。转染24、48和72 h后,STIM1-shRNA组及Orai1-shRNA组HOS细胞的增殖能力明显下降（ P＜0.01）。流式细胞检测显示,与control组和negative-shRNA组相比,转染48 h后细胞的周期受到明显抑制,而凋亡水平明显升高（P＜0.01）。结论 STIM1和Orai1在骨肉瘤细胞HOS增殖中有重要作用,抑制STIM1和Orai1蛋白表达可以抑制HOS细胞增殖、促进凋亡。     

Human sperm movement assessed with the Hamilton-Thorn motility analyzer and in vitro fertilization

Summary. The relationship between sperm movement characteristics obtained by computerized analysis and the in vitro fertilization rates of human oocytes was studied. In 144 consecutive in vitro fertilization treatments a sample of prepared semen was analysed by a Hamilton-Thorn Motility Analyzer. In addition a visual estimation of sperm count and motility was made. Significant correlations with the fertilization rate were found for all visual parameters. Of the computerized measurements, the mean velocities of motile spermatozoa and the concentration of motile cells were significantly correlated. The average path velocity correlated best ( r = 0.42, P < 0.001). There was no relationship between the percentage of motile sperm showing hyperactivated movement and the fertilization rate. A forward stepwise logistic regression analysis selected the following variables of predictive value for fertilization: average path velocity, male factor infertility as indication for in vitro fertilization, motility and concentration, as measured by the Hamilton-Thorn analyzer. A logistic regression model to predict the cases with low (< 0.2) or high fertilization rates, included the average path velocity as a significant variable and classified the samples with 90% overall accuracy. In conclusion: movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates.     

Effects of piperine on hamster sperm capacitation and fertilization in vitro

The effect of piperine on the fertilizing ability of hamster sperm was investigated in vitro . Sperm were incubated in a capacitation medium for 3 h prior to co-incubation with hamster eggs in a fertilization medium for another 3 h. Addition of 0.18–1.05 mM piperine to the capacitation medium reduced both the percentage of eggs fertilized and the degree of polyspermia in a dose-dependent manner. When piperine was added to the fertilization medium alone, a significant reduction in fertilization was observed only at high doses (0.70–1.05 mM). The presence of piperine in the capacitation medium inhibited the acrosome reaction in a dose-dependent manner but had no effect on sperm motility, whether this was measured quantitatively or qualitatively. Piperine also inhibited the influx of calcium into sperm during capacitation. It is suggested that such an inhibition might be a major cause of a reduction of the acrosome reaction and the subsequent impairment of fertilizing ability of sperm.     

三七总皂苷对5/6肾切除大鼠肾脏皮质细胞外基质积聚的影响

目的：观察三七总皂苷（PNS）对5/6肾切除大鼠肾皮质细胞外基质（ECM）积聚的影响,并探讨其肾脏保护机制。方法：将45只Wistar大鼠按体重随机取8只为正常对照组,余用5/6肾切除法建立慢性肾衰竭（CRF）动物模型。将造模成功后的大鼠随机分为模型组、百令胶囊组（简称对照组）、PNS低剂量组（低剂量组）、PNS高剂量组（高剂量组）,分别给与相应浓度和剂量的药物,实验期间测定大鼠的尿蛋白、肾功能,治疗12周后观察其肾脏病理改变,用半定量方法计算肾小球硬化指数（GSI）和肾小管损伤指数,免疫组化法检测肾小球Ⅳ型胶原（ColⅣ）、纤连蛋白（FN）和肾皮质基质金属蛋白（MMP-2）和金属蛋白酶组织抑制物（TIMP-2）蛋白表达。结果：PNS能明显减少CRF大鼠尿蛋白的排出（P〈0.05）,有一定改善肾功能的作用;PNS能减轻肾脏病理损害,减轻肾小球硬化、肾小管损伤程度;PNS能下调TIMP-2蛋白表达,增加MMP-2活性,抑制了FN、ColⅣ在肾组织的表达（P〈0.01）,减轻ECM积聚。结论：PNS可能通过减少5/6肾切除大鼠尿蛋白的排出,增加肾脏MMP-2活性,降低TIMP-2表达,增加ECM降解,减轻肾小球硬化,从而起到治疗作用。     

Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component

We evaluated antihyperglycemic and anti-obese effects of Panax ginseng berry extract and its major constituent, ginsenoside Re, in obese diabetic C57BL/6J ob/ ob mice and their lean littermates. Animals received daily intraperitoneal injections of Panax ginseng berry extract for 12 days. On day 12, 150 mg/kg extract-treated ob/ob mice became normoglycemic (137 +/- 6.7 mg/dl) and had significantly improved glucose tolerance. The overall glucose excursion during the 2-h intraperitoneal glucose tolerance test decreased by 46% (P < 0.01) compared with vehicle-treated ob/ob mice. The improvement in blood glucose levels in the extract-treated ob/ ob mice was associated with a significant reduction in serum insulin levels in fed and fasting mice. A hyperinsulinemic-euglycemic clamp study revealed a more than twofold increase in the rate of insulin-stimulated glucose disposal in treated ob/ ob mice (112 +/- 19.1 vs. 52 +/- 11.8 micromol x kg(-1) x min(-1) for the vehicle group, P < 0.01). In addition, the extract-treated ob/ob mice lost a significant amount of weight (from 51.7 +/- 1.9 g on day 0 to 45.7 +/- 1.2 on day 12, P < 0.01 vs. vehicle-treated ob/ob mice), associated with a significant reduction in food intake (P < 0.05) and a very significant increase in energy expenditure (P < 0.01) and body temperature (P < 0.01). Treatment with the extract also significantly reduced plasma cholesterol levels in ob/ob mice. Additional studies demonstrated that ginsenoside Re plays a significant role in antihyperglycemic action. This antidiabetic effect of ginsenoside Re was not associated with body weight changes, suggesting that other constituents in the extract have distinct pharmacological mechanisms on energy metabolism.     

Effect of Panax ginseng extract on the activity of diabetic fibroblasts in vitro

Numerous studies have demonstrated the various medicinal properties of Panax ginseng, including angiogenic, immuno‐stimulating, antimicrobial, and anti‐inflammatory activities, which can be helpful in chronic wound healing. However, a direct role for P. ginseng in chronic wound healing has not been demonstrated. The present study was designed to evaluate the effects of P. ginseng extract on diabetic fibroblasts in vitro. Human diabetic fibroblasts were cultured in the presence of Ginsenoside Rb1 (G‐Rb1), the active component in P. ginseng (10 ng/mL), and untreated diabetic fibroblasts were used as controls. Cell proliferation, collagen synthesis, the production of various growth factors (basic fibroblast growth factor [bFGF]; vascular endothelial growth factor [VEGF]; and transforming growth factor‐β1 [TGF‐β1]), and the synthesis of matrix metalloproteinase 1 (MMP‐1) and tissue inhibitor of metalloproteinases 1 (TIMP‐1) were compared using enzyme‐linked immunosorbent assay and immunofluorescence staining. Compared with the control group, G‐Rb1‐treated fibroblasts showed significantly (P < 0.05) higher levels of cell proliferation, collagen synthesis, VEGF, TGF‐β1, and TIMP‐1. However, no significant differences in bFGF and MMP‐1 levels were observed between the two groups. These results suggest that P. ginseng treatment may stimulate the wound‐healing activity of diabetic fibroblasts in vitro.