Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.     

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.     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica)

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.     

Coexpression of distinct eye- and liver-specific LDH isozymes in cichlid fish

In a recent electrophoretic survey of lactate dehydrogenase (LDH) in neotropical cichlid fishes (Perciformes, Cichlidae) we have discovered several species in which a cathodal liver-specific isozyme is expressed along with the highly-anodal eye-specific isozyme (LDH-C4) typically encountered in perciform fishes. We believe this fourth, liver-specific LDH isozyme to be real and not artifactual since homogenization of fresh liver from one of these species, the Basketmouth cichlid (Acaronia nassa), in either of two nondenaturing detergents or in the presence of the protease inhibitor phenylmethylsulfonylfluoride affects neither the presence nor mobility of this cathodal band. Moreover, it continues to be expressed in the captively bred F1 of these same wild fish. The discovery of several fish species, like the Basketmouth, in which biochemically distinct eye- and liver-specific LDH isozymes are coexpressed, is discussed in light of the currently accepted hypothesis that these two isozymes are encoded by a single locus (LDH-C) which has undergone divergent tissue expression in several other major teleost groups. Preliminary characterization of the liver-specific isozyme relative to the eye-specific LDH-C4 in the Basketmouth cichlid with respect to thermolability and NADH-induced binding to oxamate-sepharose columns suggests that the eye- and liver-specific LDH isozymes are biochemically quite distinct in this fish and that they are probably encoded by two distinct loci.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

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.     

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.     

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.     

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.     

Lactate dehydrogenase in teleosts. The role of LDH-C4 isozyme.

1. Lactate dehydrogenase (LDH) occupies an important position in cell metabolism. 2. Teleosts possess at least three genetic loci coding for lactate dehydrogenase subunits, Ldh-A, Ldh-B and Ldh-c. LDH exists in most tissues in several isozymic forms. 3. The isozyme LDH-C4 is synthesized predominantly in regions of the nervous system concerned with the eye.     

An immunochemical analysis of the novel liver-restricted LDH activity from cichlid fishes (Cichlidae: Teleostei) confirms its non-orthology with piscine LDH-C

In an electrophoretic survey of lactate dehydrogenase (LDH) isozymes in neotropical cichlid fishes (Perciformes: Cichlidae) several species were discovered in which a cathodal liver-restricted isozyme is expressed along with the highly anodal eye-restricted isozyme (LDH-C₄) typically encountered in perciform fishes. Biochemical characterization of these two isozymes from the basketmouth cichlid, Acaronia nassa (Heckel), strongly suggested that they were non-orthologous and challenged the accepted view that eye- and liver-restricted LDH isozymes are alternative expressions of the same (LDH-C *) gene. In this study, antiserum raised against cypriniform (goldfish) liver-restricted LDH-C₄ failed to cross-react with the basketmouth liver-restricted analogue while effectively titrating the eye-restricted, anodal isozyme and, at higher titres, the LDH-B₄, heart-restricted isozyme from all cichlid species. Anti-serum raised against basketmouth muscle-restricted LDH-A₄ failed to titrate any of the eye- and liver-restricted isozymes. These data confirm the orthology of eye- and liver-restricted LDH isozymes in Cypriniform and Perciform fishes as originally proposed, suggest that the liver-restricted isozyme of cichlid fishes is non-orthologous and further raise the question of identity and evolutionary origin of this anomalous LDH activity.     

Heparin- and superoxide anion-dependent capacitation of cryopreserved bovine spermatozoa: requirement of dehydrogenases and protein kinases

Capacitation is part of an oxidative process necessary for bovine spermatozoa to acquire fertilizing capacity. This process includes the generation of reactive oxygen species (ROS) and the participation of protein kinases such as A (PKA), C (PKC) and tyrosine kinase (PTK). A redox status is required to support both sperm motility and capacitation. Our aim was to determine the requirement of lactate dehydrogenase C4 (LDH-C4) and isocitrate dehydrogenase (NADP-ICDH) and of protein kinases in cryopreserved bovine sperm capacitation. The presence of inhibitors of both LDH-C4 and NADP-ICDH prevented the heparin-induced capacitation. H89, GF109203X or genistein blocked capacitation triggered by heparin or the superoxide (O(-*)(2))generator system xanthine-xanthine oxidase-catalase (XXOC) suggesting the requirement of PKA, PKC and PTK in this process. Taken together these results suggest that LDH-C4 and NADP-ICDH contribute with the redox status to support bovine sperm capacitation and that PKA, PKC and PTK are involved in different mechanisms induced by different inducers that lead bovine spermatozoa to be capacitated.     

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.     

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.     

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。     

Comparison of urinary creatine with other biomarkers for the detection of 2-methoxyethanol-induced testicular damage

This study has compared different biomarkers of testicular damage, in particular evaluating urinary creatine as a non-invasive marker. Male rats were exposed to various doses of 2-methoxyethanol, a known testicular toxicant. Pathological damage, testis weight, urinary creatine and creatinine, serum lactate dehydrogenase, isozyme C4 (LDH-C4), and serum testosterone were determined. 2-Methoxyethanol caused dose-dependent pathological damage to the testes which was detectable at the lowest dose (100 mg kg^-1). Urinary creatine excretion was significantly raised at all doses but testis weight was only significantly decreased at the highest two doses (500, 750 mg kg^-1). Serum testosterone was only significantly decreased at 500 mg kg^-1 and LDH-C4 was not significantly increased at any dose. Therefore urinary creatine was the most sensitive marker of 2-methoxethanol-induced testicular damage and dysfunction.     

Localization and catalytic properties of lactate dehydrogenase in different sperm models

A study of the intracellular localization and catalytic properties (effects of substrates and products) on lactate dehydrogenase has been carried out on a series of spermatozoa endowed with mitochondria and characterized by aerobic metabolism of fatty acids (sea urchin); aerobic metabolism of fatty acids and exogenous carbohydrates and lactic acid (bull); metabolism of glycogen connected with mitochondria (Lebistes); metabolism of periaxonemal glycogen (Octopus). The data obtained indicate that LDH is present only when carbohydrates are metabolized; its localization follows the glycogen stores; in the Octopus sperm where mitochondria are poorly developed relative to the flagellar length, and exogenous lactate is not available, LDH catalyses predominantly the reduction of pyruvate; in Lebistes and trout sperms, where mitochondria are better developed, LDH is more similar to the mammalian heart and sperm isozymes.