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.     

Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse.

Mouse sperm-specific lactate dehydrogenase-C (LDH-C) cDNA was cloned and sequenced from lambda gt11 expression library. The LDH-C cDNA insert of 1236 bp consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (113 bp) non-coding regions, and the poly(A) tail (70 bp). The Northern blot analysis of poly(A)-containing RNAs from mouse testes and liver indicates that the LDH-C gene is expressed in testes but not in liver, and that its mRNA is approx. 1400 nucleotides in length. The nucleotide and amino acid sequences of the mouse LDH-C cDNA show 73% and 72% homologies, respectively, with those of the mouse LDH-A. The Southern blot analysis of genomic DNAs from mouse liver and human placenta indicates the presence of multiple LDH-C gene-related sequences.     

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.     

Modulation of allo-immune responsesin vivo andin vitro by sperm specific lactate dehydrogenase-C4

The role of sperm specific lactate dehydrogenase-C₄ (LDH-C₄) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generationin vitro and local graft versus host (LGVH) reaction and allograft enhancementin vivo has been ascertained. LDH was purified from testes (LDH-C₄) and kidney (LDH-B₄) of C57 Bl/Ks mice. MLC and CTL were performed using C57 Bl/Ks-anti A/J lymphocytes in presence of 10^–3-1 g LDH-B₄ or LDH-C₄ per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C₄. Early MLC culture gave significantly low stimulation index at 10^–2–10^–1 g LDH-C₄ as compared to non-treated control cultures. However, the MLC response in presence of LDH-C₄ was not different from the LDH-B₄ treated one which showed a similar biphasic trend. On the other hand,⁵¹Cr release from YAC-222 target cells was practically abolished by LDH-C₄ at 10^–3–1^–1 g, and this was strikingly different from LDH-B₄ or non-treated cultures. LGVH reactivity as performed by using C57 Bl/Ks lymphocytes along with LDH-C₄ in (C57 Bl/Ks x A/J) F₁ hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C₄ or LDH-B₄) LGVH. Allograft enhancement of Sa I (A/J) in C57 Bl/Ks mice in presence of LDH-C₄, was delayed slightly but significantly during primary or secondary transplantation reaction. The reaction of LDH-C₄ in the modulation of allo-immune responses was more specificin vivo thanin vitro since the B₄ isozyme did not modify LGVH and Sa1 allograft rejection. Resultsper se suggest that LDH-C₄ is immunosuppressive for cell mediated allo-immune responsesin vivo andin vitro.     

An evaluation of heterologous antibodies to lactate dehydrogenase-C in the detection of mutations

We report that we are unable to repeat consistently the results published by Ansari et al. (1980) using antibodies to detect mutations in lactate dehydrogenase-C (LDH-C, previously called LDH-X) directly in sperm of mice exposed to the mutagen procarbazine. The approach made use of the interspecies differences in the antigenic sites between the LDH-C of the rat and mouse in sperm. The visualization of mutations in mouse LDH-C was based on the detection of alterations in antigenic sites of mouse LDH-C such that mouse sperm would bind the antibody that was specific for rat LDH-C (presumptive mutants); the antibody was termed specific when it immunofluorescently labeled rat sperm but not mouse sperm. The original work reported increases in the frequency of occurrence of mouse sperm that would bind rat-specific antibody from mice treated with procarbazine as compared control mice; a single absorbed antiserum was used throughout the experiments. In this study, we found that there is too much variation in the frequency of mouse sperm that react with rabbit antibodies to purified rat LDH-C for the system to be useful in mutagenesis studies. The fundamental criterion of antibody specificity was maintained as in the original work. The frequency of labeled mouse sperm depended on the absorption of the antibody on mouse proteins, indicating that the factors denoting a presumptive mutant were associated with the mouse proteins. In some experiments, the frequency of labeled mouse sperm was higher among sperm from procarbazine-treated mice than among sperm from control mice. This increase, however, was not consistently reproducible. After extensive absorption of the antibody on mouse proteins, no presumptive mutants were observed in sperm from treated and control animals; these antibodies continued to immunofluorescently label rat sperm. The absence of presumptive mutants with highly absorbed antibody suggests that natural variation between species may not be appropriate as markers for the detection of mutations without a thorough knowledge of the number of independent events at the DNA level required to produce a change in antigenic recognition.     

Strain differences in the immune response of mice to homologous sperm-specific lactate dehydrogenase (LDH-C4).

The sperm-specific isozyme of murine lactate dehydrogenase (LDH-C4) was injected into female mice of various strains. Two regulatory phenotypes characterize the resultant immunity to LDH-C4: one is manifested by high, intermediate or low levels of response, the other by the immediate or delayed maturation of peak titer. The response of several strains can be classified as high (SWR, SJL, BABL/c, C3H/He) and intermediate to low (A, CBA, DBA/2, DBA/1, C57BL/6) according to the level of antibody production and cell mediated immunity. BALB/c, SJL and SWR strains are immediate responders while DBA/2 and C3H/He mice are clearly delayed responders. Maturation and magnitude of response do not appear to be related. Both the antibody and cell mediated responses are T-dependent, but are not obviously associated with Ig allotype or H-2 regulation.     

高原鼠兔脑组织中精子特异性乳酸脱氢酶的作用

高原鼠兔对高原低氧环境有很强的适应性。研究发现,精子特异性乳酸脱氢酶(LDH-C4)基因在高原鼠兔脑组织中表达,为阐明LDH-C4在高原鼠兔脑组织中的作用,应用荧光定量PCR和Western blot方法,测定了Ldh-c基因在高原鼠兔脑组织中的表达水平;应用对精子特异性乳酸脱氢酶(LDH-C4)特异性的抑制剂(N-isopropyl oxamate),通过肌肉注射后,研究抑制剂对高原鼠兔脑组织中LDH比活力、乳酸和ATP含量的影响。结果表明,在mRNA和蛋白水平,Ldh-c基因在高原鼠兔脑组织中均有表达,相对表达水平分别为0.38±0.05和0.74±0.13;当肌肉注射1 m L 1 mol/L的抑制剂30 min后,血液中抑制剂浓度为0.08 mmol/L;与对照组相比,抑制剂组脑组织中LDH比活力、乳酸和ATP含量显著下降,抑制剂对LDH、乳酸和ATP的抑制率分别为30.78%、46.47%和21.04%。结果表明,精子特异性乳酸脱氢酶基因在高原鼠兔脑组织中表达。LDH-C4通过催化无氧糖酵解过程,为其脑组织生命活动提供至少20%的ATP,可能使高原鼠兔减小在低氧环境中对氧气的依赖,增强对低氧环境的适应能力。     

Misoprostol impairs female reproductive tract innate immunity against Clostridium sordellii

Fatal cases of acute shock complicating Clostridium sordellii endometritis following medical abortion with mifepristone (also known as RU-486) used with misoprostol were reported. The pathogenesis of this unexpected complication remains enigmatic. Misoprostol is a pharmacomimetic of PGE(2), an endogenous suppressor of innate immunity. Clinical C. sordellii infections were associated with intravaginal misoprostol administration, suggesting that high misoprostol concentrations within the uterus impair immune responses against C. sordellii. We modeled C. sordellii endometritis in rats to test this hypothesis. The intrauterine but not the intragastric delivery of misoprostol significantly worsened mortality from C. sordellii uterine infection, and impaired bacterial clearance in vivo. Misoprostol also reduced TNF-alpha production within the uterus during infection. The intrauterine injection of misoprostol did not enhance mortality from infection by the vaginal commensal bacterium Lactobacillus crispatus. In vitro, misoprostol suppressed macrophage TNF-alpha and chemokine generation following C. sordellii or peptidoglycan challenge, impaired leukocyte phagocytosis of C. sordellii, and inhibited uterine epithelial cell human beta-defensin expression. These immunosuppressive effects of misoprostol, which were not shared by mifepristone, correlated with the activation of the G(s) protein-coupled E prostanoid (EP) receptors EP2 and EP4 (macrophages) or EP4 alone (uterine epithelial cells). Our data provide a novel explanation for postabortion sepsis leading to death and also suggest that PGE(2), in which production is exaggerated within the reproductive tract during pregnancy, might be an important causal determinant in the pathogenesis of more common infections of the gravid uterus.     

An allelic variant of the sperm-specific lactate dehydrogenase C4 (LDH-X) isozyme in humans

An unusual pattern of LDH isozymes was observed by gel electrophoresis of an extract of a human testis. This isozyme composition is consistent with an allelic variant of Ldh-c.     

Implication of tissue expression of lactate dehydrogenase-C gene in phylogenetic study of euteleosts

An electrophoretic investigation of the lactate dehydrogenase isozymes in twelve euteleostean species was conducted. Expression of the LDH-C locus and association of the A and B subunits in these fishes is discussed. InChanos chanos this locus is found prevalent in the liver suggesting a close relation to otophysans. Presence of four iso-spaced A-B polymers in this species is a character different from otophysans which are provided with five iso-spaced A-B tetramers. Absence of tissue specificity of C4 band in all holocentrid species suggests a possible primitive phylogenetic status of this family in the Beryciformes. However, expression of the LDH-C locus provides no strong evidence for the resolution of the phylogenetic positions of the Polymixiidae and the other groups examined.     

Status of local reproductive tract immunity in men with inflammatory diseases of the reproductive system

The clinico-immunological study of 80 patients with chronic prostatitis of different etiology was carried out. As controls, 20 healthy adult males were used. Mucous membranes of the reproductive tract were found to have different mechanisms of antimicrobial protection whose disturbances led to the inflammatory process. The clinico-immunological analysis carried out in the course of this study made it possible to state that, in contrast to healthy adults, pronounced changes in the characteristics of the local immunity status of the reproductive system occur in chronic prostatitis patients.     

Local immunity factors in the reproductive system of women with Chlamydia infection

A total of 50 healthy women and 184 women of reproductive age with Chlamidia infection, complicated by candidiasis, mycoplasmosis and bacterial vaginosis were under examination. The local infectious immunity indices of cervical mucous were detected. The investigation of cell-mediated and humoral factors of cervical secretions revealed the dysfunction of local infectious protection in women with Chlamydia infection.     

Local anti-infective protection of the reproductive tract in women of different ages

Healthy girls and women of the reproductive age, as well as women immediately before and after menopause, were examined. Neutrophils and immunoglobulins of cervical and vaginal secretions were studied and, as a result, age-dependent differences in the activity of the anti-infectious protection of the reproductive tract of women were found.     

Gene transfer to the mammalian reproductive tract

This review summarizes the results of research on gene transfer to the mammalian genital tract. Gene transfer experiments have been developed during the last 2 decades and have been applied using in vitro, ex vivo and in vivo procedures. (i) In vitro methods have been applied to the uterine epithelial cells with the principal purpose of analysing some pathological change occurring in the uterus. In the male tract, epididymal cell lines have been used to evaluate the expression of particular genes and the function of specific proteins. (ii) Ex vivo methods have been applied to both the uterus and the vas deferens in humans, and good transgene expression has been recorded. (iii) In vivo gene transfer in the female tract has been employed in the uterus and oviduct using gene injections or electroporation methods. The glandular epithelium of both organs can be transfected efficiently, and transfection efficiency depends on the hormonal stage of the animal. The best expression occurred during pseudopregnancy and meta-estrus periods, when high progesterone and low estradiol concentrations occur. In the male tract, in vivo methods have been applied to mouse vas deferens and epididymis. In both organs, patches of epithelial regions appeared to express the transgenes. Furthermore, the secretions of both organs were also modified using gene constructions that led to the expression of some secretory proteins. In summary, gene modifications in the epithelium of the mammalian reproductive tract have been successful employing different technologies. Further improvements in transfection efficiency would help provide new insights into the physiology of these reproductive organs. Furthermore, the use of these methods could also be used to modify the fertility of mammals.     

Locus determining the human sperm-specific lactate dehydrogenase, LDHC, is syntenic with LDHA

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldh1, Ldh2, and Ldh3, respectively.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.     

cDNA cloning of pig testicular lactate dehydrogenase-C, thermal stability of the expressed enzyme, and polymorphism among strains

Pig testicular lactate dehydrogenase-C (LDHC) cDNA was cloned and sequenced. The deduced sequence of 332 amino acids from pig LDHC shows 73% and 67% identity with that of pig LDHA (muscle) and LDHB (heart) respectively, whereas pig LDHA and LDHB isozymes shows 74% sequence identity. Pig and mouse LDHC cDNAs were subcloned into bacterial expression vector, and the expressed pig LDHC isozyme was shown to be as thermally stable as mouse LDHC isozyme. Pig genomic DNAs from Chinese Meishan, English Yorkshire, Danish Landrace and American Duroc were shown to exhibit polymorphic sites for restriction enzymes EcoRI, BamHI and PstI.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.