Immunogenicity of synthetic peptides corresponding to flexible and antibody-accessible segments of mouse lactate dehydrogenase (LDH)-C4

The immunological properties of a panel of synthetic peptides that represent the most accessible and mobile segments of the lactate dehydrogenase (LDH)-C4 molecule were characterized. Peptides corresponding to mouse LDH-C4 amino acid sequences: 1-14b, 5-15, 49-58, 97-110, 211-220, 231-243, 274-286, 304-316, and 318-330 were synthesized and compared in terms of binding antibodies raised in rabbits against the intact protein. Six of these sequences were covalently coupled to diphtheria toxoid and used to immunize groups of rabbits. LDH-C4-specific antibodies were detectable in immune sera by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation assays. The immunogenicity of the mouse LDH-C4 peptides in rabbits could be ranked in the following order: 5-15, 304-316 greater than 211-220, 274-286 greater than 49-58, 97-110. The immunological properties of these short synthetic peptides did not correlate with features of the mouse LDH-C4 structure except that the most active sequences appeared to be those that differed from the somatic isozymes to the greatest extent. These results have direct bearing on the selection of immunogenic LDH-C4 peptides for contraceptive vaccine studies in humans and non-human model systems.     

Local reproductive tract immunity to sperm-specific lactate dehydrogenase-C4

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.     

Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.     

Binding of antibodies against antigenic domains of murine lactate dehydrogenase-C4 to human and mouse spermatozoa

Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing two of these sequences. Rabbits were immunized with peptides designated MC5-15 and MC211-220, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are first absorbed with the native LDH-C4 molecule, significant inhibition of binding to sperm results. Antisera to both DT-MC5-15 and DT-MC211-220 bind to human sperm with similar but weaker patterns than seen with mouse sperm. These data indicate that the immune response to synthetic peptides containing antigenic sequences of LDH-C4 includes antibodies that specifically bind to this enzyme on the surface of sperm. In addition, there are shared antigenic sequences between mouse and human LDH-C4, including the MC5-15 and MC211-220 peptides.     

LDH-C4: a target with therapeutic potential for cancer and contraception

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.     

精子特异性乳酸脱氢酶的免疫学特性及其应用

精子特异性乳酸脱氢酶特异地存在于鸟类和哺乳类动物的成熟睾丸和精子中,为精子的运动和存活提供能量,它是一种自身抗原,其天然抗体不与体细胞LDH同工酶发生交叉反应,用LDH－C4免疫小鼠或免疫等能够诱导免疫应答,导致生育率的降低,因此在人类避免和鼠害控制方面将有较好的应用前景。     

Oxamic acid analogues as LDH-C4-specific competitive inhibitors

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.     

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

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.     

Induction of cell-mediated cytotoxic immunity to sperm-specific lactate dehydrogenase-C4 in SJL/J female mice

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.     

乳酸脱氢酶C4的精子免疫荧光定位特性

用１４个抗小鼠ＬＤＨ－Ｃ４的单克隆抗体对小鼠、树鼯和人精子的ＬＤＨ－Ｃ４进行了间接免疫荧光定位。结果显示：不同的单抗可以结合到小鼠精子表面不同的位置，其中ＰＡ１（Ｋ０２１）在尾部，ＰＡ２（Ｋ０２２）、ＰＡ４和ＰＡ５（Ｋ０２３）在中段和尾部，ＳＭ１、ＳＭ２和ＰＧ２的中段，ＳＭ３和ＳＭ４在顶体，ＳＭ５在顶体和赤道板。人和树鼯精子表面的ＬＤＨ－Ｃ４也呈现类似的情况。这些研究表明精子表面的ＬＤＨ－Ｃ４呈现     

Lactate dehydrogenase C4 in male sex accessory glands of normal mice and in testes of sex-reversed mice.

Lactate dehydrogenase (LDH) C4 activity was observed in testis extracts of sex-reversed mice (Sxr) and in male sex accessory gland (seminal vesicle and prostate) extracts from C3H/He and C57BL/Go mice. These results reflect either (1) the presence of low concentrations of germinal cells in these tissues; or (2) the synthesis of LDH-C4 by somatic cells in Sxr testes and normal male sex accessory glands.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

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.     

抗小鼠精子特有乳酸脱氢酶C4的与分泌片装配或未装配的单克隆抗体IgA的生物学功能

为了测定抗精子ＩｇＡ在免疫不育和抗精子避孕疫苗研制方面的生物学作用,用肠道内免疫的方法制备了一组抗乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）的单克隆ＩｇＡ抗体（ｍｏＩｇＡ）。以免疫印迹证实了它们的异质同形体。大部分ｍｏＩｇＡ（ＰＡ１－ＰＡ５）是用肠道内免疫和以派依尔氏淋巴细胞作为亲本细胞进行融合来获得的。在豚鼠血清补体存在的情况下,小鼠精子可以被ｍｏＩｇＡＰＡ１、ＰＡ２和ＰＡ４所制动。高浓度ＰＡ４和ＰＡ５可凝集小鼠精子。小鼠体外受精率可被３个ｍｏＩｇＡ（ＰＡ２、ＰＡ３和ＰＡ４）显著降低,但用ＰＡ１、ＰＡ２和ＰＡ５被动免疫之后,小鼠体内受精无明显变化。纯化的小鼠胆汁分泌片可同纯化的ｍｏＩｇＡ或腹水中的ｍｏＩｇＡ在体外组装起来。同分泌片结合之后,ｍｏＩｇＡ对精子的制动、凝集和体外受精无明显变化。这些研究结果提供了抗ＬＤＨ－Ｃ４的ｍｏＩｇＡ和分泌性ＩｇＡ对精子功能和体外受精的生物学作用的直接证据,在免疫不育的防治,避孕疫苗的研制以及性传播疾病的防治方面均有一定的指导意义。     

Electrophoretic analyses of lactate dehydrogenase C4 in testes and vesicular glands of normal and male sterile translocation mice

Lactate dehydrogenase (LDH) C, activity was observed in testis extracts from normal mice but was progressively reduced in mice carrying the male-sterile translocations T31H, T32H, T37H, T38H, T40H and T42H, with no detectable activity being observed in the last two mice. None of the vesicular gland extracts from these male-steriles showed LDH-C4 activity, unlike normal mice. The differential LDH-C4 activity in male-sterile testes is interpreted as reflecting the varying stages of the spermatogenic defect during meiosis. In general, early meiotic defects exhibited no LDH-C4 activity whereas late stage (usually after metaphase-1 stage) defect animals exhibited some activity. The results also provide evidence for contaminating sperm being the source of normal vesicular gland LDH-C4 activity.     

Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4

After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process.     

Inhibition of lactate dehydrogenase C4 (LDH-C4) blocks capacitation of mouse sperm in vitro

Lactate dehydrogenase C4 (LDH-C4) is a tissue-specific enzyme in the mammalian testis and the only lactate dehydrogenase isozyme of sperm. Inhibitors of LDH activity were used to determine whether this enzyme plays a role in sperm capacitation, the acrosome reaction and/or fertilization. Oxamate or its derivative was used to inhibit sperm LDH activity in a medium promoting capacitation. Complete inhibition of LDH activity blocked capacitation. This effect could be reversed partially by the addition of dbcAMP or pentoxifylline to the culture medium. Western blotting showed that oxamate and N-isopropyl oxamate inhibited the tyrosine phosphorylation of proteins during the sperm capacitation process. Presumably, glycolysis is the primary energy pathway for sperm metabolism. The oxidation of reduced NAD with the conversion of pyruvate to lactate by LDH provides ATP necessary for protein kinase A (PKA) activity. Our data indicate that LDH-C4 plays an important metabolic role in sperm capacitation.     

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

（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。     

不明原因男性不育人群中睾丸特异性乳酸脱氢酶基因突变的发现及其意义

为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.     

Modulation of T cell functions by sperm-specific lactate dehydrogenase: fluorescence analysis of immune competent cells and local graft versus host reaction.

Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.