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1.
用中药复方免不1号,免不2号治疗免疫性不育雄鼠,观察睾丸,附睾组织学和免疫组化的变化。用精子抗原免疫昆明种雄性小白鼠,建立免疫性不育动物模型。同时分别饲喂中药复方免不1号,免不2号,醋酸强的松,生理盐水;从组织学和免疫组化等方面观察免疫性不育症的变化。结果显示免疫性不育雄鼠血清,精囊液抗精子抗体高,睾丸间质,睾丸曲细精管界膜,精原细胞,附睾管上皮细胞免疫复合物沉积多,睾丸每曲细精管精子和晚期精子细胞减少,中药免不1号和2号能降低抗精子抗体,清除免疫复合物的沉积,恢复曲细精管精子和晚期精子细胞数。结果表明:免不1号和2号通过调节全身免疫系统,清除循环和局部的抗精子抗体,免疫复合物,提高精子和精子细胞数,从而提高小鼠的受孕率。  相似文献   

2.
本文采用免疫组织化学和免疫电镜对正常人精子中尿激酶型纤溶酶元激活因子(UPA)的分布进行了定位观察。结果发现尿激酶型纤溶酶元激活因子分布在精子顶部体内外膜和精子头部浆膜上,且精子尾部浆膜上也有阳性着色,提示尾部浆膜上也有尿激酶型纤溶酶元激活因子的存在。推测精子中尿激酶型纤溶酶元激活因子可能与精子的运行、获能、顶体反应及受精有关联。  相似文献   

3.
精子特异性乳酸脱氢酶是精子能量代谢的一个关键酶,具有很强的组织特异性和免疫原性.在本研究中,将人和小鼠(Mus musculus)的精子特异性乳酸脱氢酶编码序列分别与真核表达载体pVAX1连接,构建了两个DNA疫苗:pVAX1-hLDHC和pVAX1-mLDHC.将这两个DNA疫苗通过黏膜表面滴注的方式分别免疫雌雄两性BALB/c小鼠,在实验小鼠的血清和雌性实验小鼠的阴道分泌液中均检测到特异性抗体的存在,滴度随免疫次数的增加而升高.交配实验中,实验组出生子代的数量明显减少,有的小鼠甚至未生育,与未免疫组和pVAX1空质粒免疫组在统计学上有显著性差异.精子凝集实验表明,实验小鼠血清和雌性实验小鼠阴道分泌液均可引起正常雄性小鼠的精子悬液发生凝集反应.免疫组化结果显示:实验小鼠血清和雌性实验小鼠阴道分泌液中的抗体与位于正常精子胞质、顶体外膜及顶体囊中的乳酸脱氢酶抗原发生特异性结合.本研究初步显示了人用避孕DNA疫苗免疫效果,为以后可能进行的临床实验奠定了基础.  相似文献   

4.
在Balb/c、C57和昆明鼠的消化道不同部位以人精子抗原免疫后,取其脾、肠系膜淋巴结、Peyer氏淋巴小结及子宫或附睾中的淋巴细胞进行培养。以ELISA法测定了这些部位的淋巴细胞所分泌的以及免疫小鼠肠道冲洗液和血清中的抗精子抗体。结果提示,Balb/c和昆明鼠抗精子免疫反应较强,而且雌性强于雄性。免疫部位以小肠及Peyer氏淋巴小结内注射的免疫效果较好,不仅可引起消化道局部免疫反应,而且子宫、附睾等生殖道组织也有淋巴细胞分泌抗精子抗体。  相似文献   

5.
异精效应在雌核发育彭泽鲫胚胎发育中的同工酶证据   总被引:11,自引:0,他引:11  
采用聚丙烯酰胺凝胶垂直板电泳技术 ,对同源和异源精子激发雌核发育彭泽鲫子代胚胎发育过程中四种同工酶 (EST ,LDH ,MDH ,ME)的表达情况进行了比较研究。结果表明同源和异源精子激发的胚胎在MDH和LDH同工酶表达上存在明显差异。MDH同工酶的差异主要表现为 :在孵出期 ,异源精子激发的胚胎比同源精子激发的胚胎多出两条谱带MDH4’和MDH5’ ;LDH同工酶的差异表现为 :在原肠中期至肌肉收缩期的五个时期中 ,异源精子激发的胚胎比同源精子激发的胚胎多出 4条谱带 (LDH9’— 12’)。这种差异说明同源与异源精子对子代胚胎发育过程中同工酶表达的影响不同 ,可能属于“异精生物学效应”的一种表现形式  相似文献   

6.
陈炯  冯巍  詹飞 《生物工程学报》2019,35(1):150-158
hLCN6 (Human lipocalin 6)是附睾特异性分泌蛋白,它能与精子结合,对精子的成熟发挥着重要作用。为了探究应用抗hLCN6单克隆抗体偶联免疫磁珠技术分离混合细胞中精子的可行性,建立混合斑中精子细胞分离的新方法,制备了不同比例的精子-上皮细胞混合悬液及混合斑样本,以生物素标记的hLCN6单克隆抗体孵育样品,再用亲和素包被的免疫磁珠捕获分离精子细胞,提取精子DNA进行PCR-STR (Short tandem repeat)分型,同时以差异裂解法提取精子,比较两者的差异。经ELISA检测,hLCN6单克隆抗体与抗原的亲和力解离常数(Kd)为3.47×10~–9 mol/L。免疫印迹和免疫荧光结果显示,hLCN6在精子中可检测到,主要定位于精子头部的顶体后区域,但不能在上皮细胞中检测到。hLCN6抗体偶联的免疫磁珠复合物能够实现精子细胞的捕获和分离,显微镜观察显示免疫磁珠能够与精子头部特异性结合。对精子个数为10~3/mL的混合悬液,STR分型成功率(正确分型13个以上,RFU200)为90%。当精子数量≥10~4/mL时,分型成功率达10~0%;对精子数分别为10~3/mL、10~4/mL、10~5/mL的混合斑STR分型成功率分别为40%、90%和10~0%。综上,hLCN6抗体偶联免疫磁珠法可以有效分离混合细胞中的精子,分型成功率高于传统的差异裂解法。该方法简单高效,可作为性侵案件中法医混合斑检验的有效补充手段。  相似文献   

7.
为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和/或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。  相似文献   

8.
5只性成熟成年雄性比格犬被施行附睾尾逆向注射10%精氨酸锌0.5毫升。一般饲料饲养两月后处死作病理检查,发现精子在睾丸曲细精管内的生成及在附睾管内的成熟均受到影响,附睾尾管腔内精子降到了极少程度且多已变性。免疫组织化学反应发现睾丸间质及附睾管上皮间隙内,附睾间质中均有大量免疫复合物的沉积,而其它主要生命器官均未发现异常。结果表明,精氨酸锌附睾尾注射的绝育作用是肯定的,其伴随出现的抗精子抗体及免疫复合物仅对睾丸、附睾及生育功能产生影响,对机体主要生命器官并无损害。  相似文献   

9.
免疫不育疫苗主要以哺乳动物的精子或卵子蛋白以及在受精和胚胎早期发育过程中发挥重要作用的激素为靶抗原。以激素为抗原的不育疫苗产生的不育效果多为不可逆的,且对机体损伤较大。以精子表面抗原制备的疫苗能够诱导产生精子抗体和不育效果,目前已成为避孕研究的一个热点。哺乳动物卵透明带(zona pellucida,ZP)是覆盖于卵母细胞及着床前受精卵外的一层基质,其在调节精卵特异性结合、诱导获能精子发生顶体反应和阻止多精受精等方面发挥着重要作用。ZP相对分子质量较小且免疫原性强,是免疫不育疫苗理想的靶抗原,抗ZP抗体可阻断精卵结合,故可被用作人类避孕和免疫不育控制有害动物种群数量的靶抗原,但人用ZP疫苗免疫机体后造成的卵巢功能损伤和免疫抑制等问题尚有待明确。  相似文献   

10.
人体精子LDH酶谱中一条新酶带的发现和探讨   总被引:2,自引:0,他引:2  
用paragon电泳技术在人精子LDH酶谱中发现一条新的酶带,暂称y酶带。该y酶带的位置在LDH-4和LDH-5之间,含重为LDH的1.57%±0.82%,与LDH-4比较其相对迁移率(RF值)为1.25-1.35。 试验证明y酶带的存在与否与精子密度和LDH、CPK总活力有明显相关性。  相似文献   

11.
Immunity to sperm-specific lactate dehydrogenase C4 (LDH-C4) results in reduction of fertility in females. Stimulation of a local mucosal immune response to LDH-C4 in the reproductive tract would guarantee the presence of antibodies at the site of fertilization, which should enhance suppression of fertility. After intrauterine immunization with LDH-C4, SJL/J female mice secrete immunoglobulin (Ig)A antibodies specific for LDH-C4 into their uterine fluids. Furthermore, these animals demonstrate a lower pregnancy rate than controls receiving an intrauterine immunization without LDH-C4. Thus, induction of a local immune response is an effective alternative to systemic immunization for administering a contraceptive vaccine.  相似文献   

12.
中国林蛙乳酸脱氢酶多基因系统及基因间连锁关系的研究   总被引:4,自引:0,他引:4  
张辉  吴清江 《遗传学报》1996,23(1):11-17
(1)用聚丙烯酰胺凝胶电泳对山西产中国林蛙4个地理群的333只林蛙进行了分析,结果表明:中国林蛙的LDH由LDH-A,LDH-B和LDH-C3个基因决定。LDH-A是单态座位,LDH-B和LDH-C均为多态座位,每个多态座位均有两个等位基因。LDH-B与LDH-C呈紧密连锁关系。认为LDH-C是LDH-B的重复产物。(2)热稳定性、尿素处理稳定性及组织特异性研究表明:LDH对温度和尿素处理稳定性顺序为A4>B’4>B4>C4。A4在骨骼肌和肝脏等组织中活力最大,B4在心肌和卵巢中活力最强,LDH-C主要在眼球和卵巢中表达。(3)Ldh-b和Ldh-b'在不同地理群间呈差异分布,随着纬度的增高,Ldh-b在种群中的频率增大。  相似文献   

13.
The lactate dehydrogenase (LDH) has been studied widely because it exists in various isozymic forms. The association of A and B subunits of LDH can generate five tetrameric isozymes, but the finding of the sixth isozyme in mature human testis and sperm indicated the presence of an additional subunit of LDH, designated as LDH-X (also termed LDH-C4 due to tetrameric nature of C-subunit). LDH-C4 isozyme is an iso-, allo-, and auto-antigen present in mammalian sperm cells. The synthesis of LDH-C4 in the testis takes place during sexual maturation, and it is the predominant fraction in mature spermatozoa. Though, originally considered to be testis specific, LDH-C or Ldh3 in mice was later detected in the murine oocyte and early embryo. Ldh3 in mouse supports its role in energy production in spermatids that favor lactate as substrate and in spermatozoa with a characteristic aerobic glycolytic path to yield ATP. During last two decades, cancer/testis-associated genes (CTAs) which are expressed only in the germinal epithelium of the testis are also expressed in some cancer cells, but not in non-cancerous somatic tissues. The CTAs are considered promising candidates for diagnosis and immunotherapy of cancer. The sperm-specific Ldh-c gene has been shown to express in a broad spectrum of human tumors, with high frequency in lung cancer, melanoma, and breast cancer; the protein being expressed virtually in all tumor types tested. Accordingly, LDH-C4 is the unique target for contraception in both males and females and offers potential future for immunotherapy of different types of cancers. As LDH-C has a preference for lactate as a substrate, LDH-C activation in cancer may depend on lactate for ATP production. The major aim of this article is to review the salient features of LDH-C subunit and the immune responses of LDH-C4 in homologous and heterologous species in relation to its role in acceptance or rejection of the allograft and its application in contraception and immunotherapy of cancer, directly or indirectly through the regulation of its substrate, the lactate.  相似文献   

14.
Non-cross-reactivity of antibodies to murine LDH-C4 with LDH-A4 and LDH-B4   总被引:1,自引:0,他引:1  
The induction of infertility by immunization with the sperm-specific lactate dehydrogenase, LDH-C4, suggests its use in a contraceptive vaccine. Development of an immunological contraceptive for human use, however, requires that there be no cross-reactions with somatic tissues. We have demonstrated, using enzyme-linked immunoabsorbence, solid-phase radioimmunoassay, and competitive inhibition radioimmunoassay, that antisera to LDH-C4 is specific and does not cross-react with the somatic isozymes, LDH-A4 and LDH-B4.  相似文献   

15.
Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.  相似文献   

16.
SJL/J female mice were tested for a cell-mediated cytotoxic response to sperm-specific lactate dehydrogenase C4 (LDH-C4). We demonstrated this response with a 51chromium release assay. Mouse LDH-C4 was coupled to EL-4 tumor cells. These cells were then labeled with 51Cr and mixed with splenocytes from LDH-C4-immune and -nonimmune mice. Specific lysis of the tumor cells by splenocytes from LDH-C4-immune mice was detected at 7 days and 14 days following a single footpad immunization. Since LDH-C4 is present on the surface of sperm, these results support the suggestion that cytotoxic removal of sperm from the female reproductive tract is one of the mechanisms whereby fertility is reduced following immunization with this enzyme.  相似文献   

17.
We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.  相似文献   

18.
Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.  相似文献   

19.
Spermatogenesis is a programmed developmental process characterized by the inactivation of certain genes and the activation of other, testis-specific genes. Synthesis of unique gene products such as LDH-C4 and cytochrome ct occurs only at precise stage of germ cell formation. The developmental sequences of gene activation for these proteins was observed by immunohistochemical procedures. LDH-C4 is first detectable during mid-pachytene of the primary spermatocyte. The C subunits appear to be uniformly distributed throughout the cytoplasm of the spermatocyte. The mid-pachytene stage also marks the first appearance of cytochrome ct. The association of this electron transport protein with spermatocyte mitochondria is reflected in a granular fluorescence of specific antibody-treated sections of testis. Neither the C subunit of LDH, nor cytochrome ct appear in leptotene or early pachytene primary spermatocytes. These analyses indicate that there is stage-specific protein synthesis in the primary spermatocyte which is characterized by differential activation of the LDH-C locus and of the gene coding for cytochrome ct.  相似文献   

20.
Lactate dehydrogenase-C (LDH-C) mRNA was purified from DBA2 mouse testes and translated invitro. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  相似文献   

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