Ropporin基因在人睾丸和精子中的鉴定与蛋白定位

目的:对人睾丸和精子中的Ropporin基因表达进行鉴定和蛋白定位,为进一步研究其在精子中的作用奠定基础。方法:应用免疫组织化学Western blotting和RT-PCR及间接免疫荧光方法检测Ropporin在正常人睾丸组织射出精子中的表达特征。结果:在睾丸组织中,Ropporin主要表达在圆形精子细胞阶段;在精液精子中,mRNA和蛋白水平上均检测出Ropporin的表达,且蛋白主要定位在精子鞭毛的主段和末段上。结论:人睾丸圆形精子细胞和精液精子中均有Ropporin mRNA和蛋白的表达,其可能与精子运动有关。     

哺乳动物P1精蛋白基因表达的研究

目的:研究哺乳动物P1精蛋白基因在大鼠和小鼠中的表达。方法:用RT-PCR法制备大鼠P1精蛋白(rP)cDNA,用小鼠P1精蛋白(mP1)DNA重组质粒(pUC8)制备mP1cDNA,rPcDNA和mP1cDNA经迪高辛标记后作为探针,用Northern杂交等技术分析基因表达情况。结果:rPcDNA与大鼠睾丸RNA有杂交反应,而与肝、脑RNA无交叉反应;rPcDNA和小鼠睾丸RNA有交叉反应;mP1cDNA探针与性成熟不同阶段小鼠睾丸RNA杂交结果表明:小鼠性成熟过程中,在睾丸出现圆形精子细胞后mP1基因开始转录,而翻译成蛋白质是在性成熟小鼠睾丸内出现长形精子细胞后。结论:哺乳动物P1精蛋白的表达有器官特异性,是睾丸特异表达的基因,P1精蛋白基因在进化上保守,因而在大、小鼠睾丸中均能检测到杂交讯号;精蛋白基因的表达表现为翻译延迟,该基因在圆形精子细胞阶段开始转录,而在长形精子细胞阶段翻译成蛋白质。     

不育病人血清中精子反应性抗体对雌兔生育力的影响

抗精子抗体在人的不育症中有重要意义,而且其针对多种精子抗原。近期应用单克隆抗体研究证实哺乳动物精子有多种与受精有关的抗原,并在兔、小鼠等多种属间有交叉反应。如受精抗原(FA-1,为精子表面糖蛋白)、鱼精蛋白(精子核抗原)等。作者对免疫不育病人血清中的抗鱼精蛋白抗体、抗FA-1抗体及与不育症的关系进行了研究。以期了解这些抗体的存在型式及对精子有损伤作用的抗体是否损伤兔精子的生育能力,试图得出研究和治疗人类免疫不育的动物模型。应用的抗原包括人精子浸出物(HSE)、鱼精蛋白及FA-1。因酶联免疫吸附试验(ELISA)检测抗体。共检     

体外培养方法诱导大鼠精母细胞减数分裂

目的:探讨大鼠睾丸精母细胞在体外减数分裂、分化为精子细胞或精子的可能性。方法:取出生后20-22dWistar大鼠睾丸组织,经胶原酶消化分离细胞,分别用含2%胎牛血清(FBS)的DMEM/F12培养基(对照组)和含2%FBS的DMEM/F12培养基+多种性激素+多种细胞生长因子等(实验组)作体外培养,于培养后d3、d7、d14收集细胞,采用流式细胞技术,检测培养前后生精细胞染色体倍体的变化;RT-PCR法检测精子细胞特异性基因过渡蛋白1(TP1)mRNA的表达。结果:培养d2,可见支持细胞贴壁生长,生精细胞粘附于支持细胞表面,实验组生精细胞存活率>90%,对照组<5%;培养14d,实验组可见长有鞭毛的长形精子细胞。流式细胞检测结果显示,培养前生精细胞为二倍体和四倍体细胞群,培养d14实验组单倍体细胞占总细胞数的6.2%,对照组未见单倍体峰。RT-PCR结果显示与流式细胞技术检测结果一致:培养前生精细胞未检测到TP1mRNA的表达,培养7d后实验组可检测到该基因的表达,培养14d,其表达量显著增加。结论:采用生精细胞与支持细胞混合培养并在培养液中添加性激素、细胞生长因子等的方法,大鼠精母细胞可以在体外进行减数分裂,分化成长形精子细胞。     

一种多价精子抗原表位肽的设计与合成

目的 :开发可供两性使用的多价合成嵌合肽疫苗。方法 :选择小鼠精子 /睾丸特异性抗原 SP1 7、Cyritestin中特异性氨基酸序列与牛核糖核酸酶非限制性 T细胞表位作为骨架 ,按一定连接方式设计、合成新抗原 3 5肽 ,并与等量弗氏佐剂混悬后免疫雌性 BALB/c小鼠。结果 :在 43 0 A固相自动肽合成仪上成功地合成了 3 5肽 ,并经 HPLC纯化、质谱鉴定证实其纯度达 95 %以上 ;新抗原免疫小鼠后获得的特异性抗血清可识别小鼠、大鼠及人睾丸组织蛋白提取物中相应分子量的蛋白质 ,且血清中特异性 Ig G抗体滴度最高达 1∶ 6 0 0 0 ,阴道粘膜冲洗液中特异性 Ig A抗体滴度最高达 1∶ 3 0 0。结论 :由含有两种特异性精子抗原、具有特异性氨基酸序列的新抗原所制备的抗血清可识别两种天然抗原 ,并在小鼠体内激发出较理想的特异性体液免疫 ,从而为研制适宜于两性的多价抗生育疫苗提供了实验基础     

HPV58型E2蛋白表达纯化与多克隆抗体制备

目的:表达纯化重组人乳头瘤病毒(human papillomavirus,HPV)58型E2蛋白,制备多克隆抗体。方法:构建重组质粒p ET-28b-E2,转化至BL21(DE3)p Lys S中诱导表达,包涵体洗涤后经镍柱亲和层析分离得到纯化目的蛋白。用纯化的重组E2蛋白免疫新西兰白兔,制备兔抗HPV58型E2蛋白多克隆抗体,ELISA分析多克隆抗体的效价,免疫印迹检测抗体的特异性。结果:表达纯化了重组HPV58型E2蛋白,制备了高滴度和高特异性的多克隆抗体。结论:制备的多克隆抗体可用于对HPV58型E2蛋白进行精细B细胞线性表位鉴定。     

人白细胞抗原B7和BW35与精子抗体的关系

正常时精子抗原被隔离在血睾屏障之后,破坏此屏障完整性的因素如睾丸活检,输精管结扎或感染可引起精子抗体产生。无此种因素而发生精子免疫的男子提示遗传因素起作用,本文研究人白细胞抗原(HLA)与精子抗体所致不育相关联的可能性。 103对不育失妇和50对生育夫妇用被动血凝试验和微量精子细胞毒试验测定血清、精浆、宫颈和阴道分泌物中的抗精子抗体,用标准National Instituts of Health(NIH)淋巴细胞毒技术作A、B位点HLA定型。结果发现不育组87个男子和76个妇女有全身和/或局部抗体,50对生育夫妇和12对不育夫妇无抗体。有抗体的夫妇中54%男子和43%妇女     

精子顶体小泡蛋白-1(ACRV1)在小鼠睾丸组织中的表达与定位

目的:研究精子顶体小泡蛋白-1(ACRV1)在小鼠睾丸组织发育过程中的表达特征。方法:将4 d、9 d、18 d、35 d、54 d和6月龄小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交,筛选出差异表达的ACRV1基因。采用RT-PCR方法检测ACRV1基因在小鼠不同日龄和不同组织中的表达情况,采用免疫组织化学方法检测ACRV1蛋白在小鼠睾丸组织中的定位。结果:对Affymetrix全基因组芯片杂交结果分析后,筛选得到1个差异表达杂交点,通过在NCBI网站与小鼠全基因组序列Blast分析可知该差异表达基因是ACRV1基因。RT-PCR结果表明ACRV1 mRNA呈小鼠睾丸特异性表达,在出生31 d小鼠睾丸开始高表达,在成年前达到高峰。ACRV1蛋白主要定位在睾丸圆形精子和长形精子细胞。结论:ACRV1基因存在发育的表达调控,为小鼠年龄依赖性表达基因,其表达与小鼠精子发生的过程有很强的一致性,且具有睾丸特异性表达的特征,因此推测该基因可能在精子发生中具有关键作用。     

84KD精子膜蛋白与抗精子抗体

为了进一步研究精子抗原及其相应抗体在不育中的作用,本文用SDS-PAGE和免疫印迹法检查了一对长期不育夫妇之间的抗精子免疫应答。该夫妇双方均含高滴度抗精子抗体。丈夫精子膜蛋白提取液中分子量84KD蛋白含量显著增加,为正常男性的3～5倍。该成份在免疫印迹中可被夫妇双方血清特异性识别。提示不育男性精子抗原成份与生育力正常男子有量或质的差异,可能是产生抗精子抗体并造成不育的一个原因。     

NO对抗精子抗体阳性大鼠精子自发顶体反应的影响

目的 :探讨 NO对抗精子抗体 ( As Ab)阳性大鼠精子自发顶体反应的影响。方法 :采用主动免疫法建立 As Ab阳性大鼠动物模型 ;浅盘凝集实验和 ELISA法检测大鼠血清 As Ab;考马斯亮蓝染色法进行精子顶体染色 ,以观察大鼠自发精子顶体反应率。结果 :As Ab阳性大鼠精子自发 AR%下降 ,且内源性 NO含量、精子内 SOD和 Na+ -K+ ATP酶活性明显低于对照组 ;低剂量 NO( SNP1 0 - 9～ 1 0 - 8mol/L)可提高 As Ab阳性大鼠精子自发顶体反应率、SOD活性 ,但对 Na+ -K+ATP酶活性无影响 ( P>0 .0 5 )。高剂量 NO( SNP1 0 - 6 ～ 1 0 - 4mol/L)则进一步降低上述三项指标。结论 :As Ab阳性大鼠自发顶体反应率降低可能与精子内 NO生成减少、O2 - ·产生增多 ( SOD活性降低 )有关。低浓度 NO可以灭活过量的超氧化物而提高 As Ab阳性大鼠精子自发顶体反应率 ;但过高浓度 NO则损害精子的功能     

Studies of the nature of extracellular components of rat seminiferous tubular wall. II. Immunological characterization of basement membrane antigen.

An antiserum was obtained in rabbits using as antigen a preparation rich in basement membranes isolated from normal rat testis. By passive hemagglutination test a titer of 1/20,000 was obtained when the antiserum was reacted with the specific antigen; no cross reactions were detected with other fractions extracted from the germinal cells of rat testis. A weak cross reaction was obtained when the antiserum was reacted with collagen extracted from rat testis. Moreover, the antiserum cross reacted with other isolated basement membranes such as glomerular basement membrane of rat kidney and sheep lens capsule. By the indirect immunofluorescent technique the localization of the antigen antibody reaction was detected at the seminiferous tubular wall and vessels. No reaction was observed in other structures of the testis except for a faint reaction at the interstitial collagen fibers. Both, serological and immunohistochemical techniques demonstrated: (1) the existence of common antigenic determinants among basement membranes of different organs of the rat, (2) a partial immunological identity among basement membranes of different species.     

Modification of the penetration rate of human spermatozoa in the zona pellucida free hamster oocyte system by the use of polyclonal and monoclonal antibodies to human sperm

Incubation of human spermatozoa with polyclonal anti-sperm antibodies from sterile females reduced the penetration rates in the zona-pellucida-free hamsteroocyte assay significantly. Twelve sera having titers from 1:4,096 to 1:64 were used. Fifty-three ejaculates from men with normozoospermia were tested. A few of the ejaculates used were classified as slightly pathological showing asthenospermia. At an average titer 1:860 the reduction of penetration rate was about 49% (range 9-90%). The degree of reduction was dependent on the agglutination titer of the test serum, the presence of immobilizing antibodies, the amount of antiserum added, and on the quality of the ejaculates to a minor extent. The penetration rate of human spermatozoa in zona-pellucida-free hamster-oocytes was reduced by 4 monoclonal anti-sperm-antibodies (A-24, B-20, III,3, and VII-5) out of 6 tested. The other 2 (VI-1 and VI-16) left the penetration rates virtually unchanged. The extent of penetration rate reduction depended on the concentrations of antibodies A-24, B-20, III-3, and VII-5; if undiluted, they produced a significant reduction of penetration rates, the largest average reduction being almost 50% with undiluted antibody A-24. Decreasing concentrations of the monoclonal anti-sperm-antibodies resulted in significantly smaller reductions of sperm penetration rates. The reason for the reduced penetration rates observed may be a blockage of receptors on the sperm surface by the named monoclonal anti-sperm-antibodies. This can inhibit enzyme reactions of the spermatozoa, or interfere with the interaction between oocyte and spermatozoa membranes. It is noted that only the antibody III-3 caused agglutination of human spermatozoa in the micro-sperm-agglutination and immobilization test. Therefore, the agglutination or immobilization of spermatozoa may not be held responsible for the reduced penetration rates after addition of monoclonal antibodies, as opposed to the findings with polyclonal antibodies.     

Spermatozoal antibodies in cervical mucus

The indirect immunofluorescent technique performed on spermatozoa provides a suitable method for detecting spermatozoal antibodies in cervical mucus. Blood sera and preovulatory cervical mucus samples from 13 women with infertility of unknown origin were tested extensively with this technique. Spermatozoal antibodies in cervical mucus were detected in 3 out of 13 patients. The specificity of these antibodies was shown to be IgA in two cases and IgG in the third case. One IgA antibody was also present in the blood serum of this particular patient, however in a low titer (1:4). Blood serum from the patient with an IgG antibody in cervical mucus contained the same antibody in a high titer (1:64). The results werecompared to the concentration of immunoglobulins in cervical mucus. In view of present knowledge our data are consistent with the following theories about circulating spermatozoal antibodies. IgA antibodies result from local immunization in the female genital tract and may leak to the general circulation. IgG and IgM antibodies result mainly from general immunization. IgG antibodies may diffuse to female genital tract secretions when present in a sufficiently high concentration. IgM antibodies are rarely found in the female reproductive tract.     

Decrease of sperm antibody titer in males, and conception after treatment of chronic prostatitis.

Although autoimmunization to spermatozoa is a cause of male infertility, the cause of antibody formation is unknown in most cases. It has been shown that the titer is usually unchanged for as much as 16 years in the same individual. Trials to reduce the titer with varying methods have not been successful. A new possibility of treatment was indicated by the finding of a higher incidence of prostatitis in men with sperm antibodies than in a control group. Following treatment of prostatitis we observed a reduction of the antibody titer in eight cases. In five cases the cervical mucus penetrating capacity of the spermatozoa improved, and conception occurred.     

An objective sperm cytotoxicity assay for male-specific antisera based on ATP levels of unlysed cells: application to assay of H-Y antigen

We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-in-activated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influence of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1 : 8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.     

CLINICAL ASSISTED REPRODUCTION: Development of Blastocyst-Stage Embryos After Round Spermatid Injection in Patients with Complete Spermiogenesis Failure

Purpose: Our purpose was to evaluate the progression of embryos derived from round spermatid injection to the blastocyst stage and compare the results with those obtained by the use of testicular or epididymal spermatozoa.Methods: Thirty-eight patients with azoospermia enrolled in this study. In 29 patients with obstructive or nonobstructive azoospermia, spermatozoa were recovered from epididymis or testis. In the remaining nine cases with nonobstructive azoospermia, only round spermatids were found in seven, whereas in two of the patients, there were no elongated or round spermatids. Six of these cases underwent round spermatid injection.Results: Twenty-one of 29 patients with injection of spermatozoa underwent embryo transfer on day 3, and 10 pregnancies (47.6%) were obtained. In eight cycles, embryos were further cultured for delayed transfer. In six cases undergoing round spermatid injection, no transfer was performed on day 3 and extended culture with delayed embryo transfer was applied. The mean number of fertilized oocytes and mean number of embryos on day 3 and also the fertilization rate and mean number of good-quality embryos on day 3, mainly grade 1 or 2, were statistically significantly higher in the spermatozoa group than the round spermatid injection group. Compared to the spermatozoa group, the number of arrested embryos was significantly higher and the number of blastocyst-stage embryos and number of good-quality blastocysts were significantly lower in the spermatid injection group. No blastocysts developed in two spermatid cycles and embryo transfer was not possible, and in the remaining four cycles, after at least one blastocyst transfer, no pregnancies were achieved. However, in eight cycles with extended culture in the spermatozoa group, embryo transfers were achieved in all and three pregnancies, for a pregnancy rate of 37.5%, were obtained after blastocyst transfer.Conclusions: Our preliminary results showed that round spermatid injection was associated with a significantly lower fertilization and embryo development rate and a significantly higher developmental arrest rate compared with the injection of spermatozoa. Extended culture and delayed embryo transfer did not improve the clinical outcome after round spermatid injection, and these results suggested a developmental failure in embryos preventing successful implantation after round spermatid injection.     

Isolation of a human spermatozoal hapten "II2.2' and its reaction with naturally occurring human sperm-immobilizing sera from infertile patients

An extract of human spermatozoa was prepared using Hyamine 2389 and Triton X-100. With gel and ion-exchange chromatography several fractions were obtained of which are reacted specifically with sperm-immobilizing antibodies of infertile females and males in an immune inhibition test. This fraction showed haptenic properties, ahd a molecular weight of 1600 and was excluded as an anticomplement factor. After conjugation with cytochrome c the hapten II2.2 formed precipitation reactions with 3 out of 4 sperm-immobilizing sera. The titer reduction in sperm-immobilizing sera after adsorption with II2.2 may represent an in vitro model for a possible treatment of infertility in cases of a humoral sensitization against spermatozoa. On the other hand, the hapten might easily be synthesized and could, after conjugation to an appropriate carrier, serve as a contraceptive vaccine.     

Frequency of anti-sperm antibodies demonstrated by classical tests for sperm antibodies in patients with unexplained infertility

The aim of the present investigation was to establish the frequency of sperm antibodies in patients with etiologically unexplained infertility, and to compare the demonstrated frequencies with the results from y control group of y healthy blood donors, as well as with the results of other investigators. The gelatin agglutination test of Kibrick and the tray agglutination test of Friberg were applied to test 244 sera from infertile patients and 50 sera from healthy blood donors at the Laboratory of Immunology of Reproduction, Department of Biology, Medical University of Sofia. For the infertile patients, relevant sperm antibody titers were demonstrated in 2.5% (titer > or = 16) for the Kibrick method, and in 7% (titer > or = 32) for the Fiberg method. The test of Kibrick did not reveal significant antibody titers in the healthy controls, while the test of Friberg showed sperm antibodies in 2% of the blood donors. Fisher's exact test demonstrated extremely significant correlation (p < 0.0001) between the presence of sperm antibodies in sers of patients with unexplained infertility as revealed by the tests of Kibrick and Friberg. Most often mixed agglutinates were demonstrated in the Friberg test. In contrast with the results of other investigators head-to-head agglutinins were observed more often in male sera, while tail-to-tail agglutinins--in female sera. Finally, the results from the present investigation, as well as the analyzed literature data showed a low frequency of anti-sperm immunity in the Bulgarian population. The established high degree of correlation between the tests of Kibrick and Friberg, the good reproducibility of the results and the low cost of these methods confirm their appropriate use for the diagnosis of sperm antibodies in patients with unexplained infertility.     

Effect of antisperm sera on the acrosomal proteolytic activity in vitro

The effect of isoimmune antisperm sera from heifers and the effect of sera from cows and women with unexplained infertility were studied on the sperm acrosomal proteolytic activity (SAPA) of bull and human spermatozoa. All the sera were preliminarily tested by the method of Kibrick, and the cow sera were additionally tested by method of Isojima et al. Ejaculated bull and human spermatozoa were incubated in a medium containing antisera. SAPA was monitored on KODAK AR-10 autoradiographic plates according to Wendt et al. In some experiments the bull spermatozoa were preincubated in a medium containing 30% follicular fluid to induce capacitation and acrosomal reaction. The incubation of spermatozoa in isoimmune sera led to a marked inhibition of SAPA. A similar effect was observed when sera from cows and women with unexplained infertility were used. Preincubation of bull spermatozoa in follicular fluid intensified SAPA. The subsequent treatment with sera from infertile cows with high titres of spermagglutinins strongly inhibited this activity.     

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with na?ve females and female immune rats with na?ve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.