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.     

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.      

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。     

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.     

Non-cross-reactivity of antibodies to murine LDH-C4 with LDH-A4 and LDH-B4

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.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

Lactate dehydrogenase expression at the onset of altered loading in rat soleus muscle.

Both functional overload and hindlimb disuse induce significant energy-dependent remodeling of skeletal muscle. Lactate dehydrogenase (LDH), an important enzyme involved in anaerobic glycolysis, catalyzes the interconversion of lactate and pyruvate critical for meeting rapid high-energy demands. The purpose of this study was to determine rat soleus LDH-A and -B isoform expression, mRNA abundance, and enzymatic activity at the onset of increased or decreased loading in the rat soleus muscle. The soleus muscles from male Sprague-Dawley rats were functionally overloaded for up to 3 days by a modified synergist ablation or subjected to disuse by hindlimb suspension for 3 days. LDH mRNA concentration was determined by Northern blotting, LDH protein isoenzyme composition was determined by zymogram analysis, and LDH enzymatic activity was determined spectrophotometrically. LDH-A mRNA abundance increased by 372%, and LDH-B mRNA abundance decreased by 43 and 31% after 24 h and 3 days of functional overload, respectively, compared with that in control rats. LDH protein expression demonstrated a shift by decreasing LDH-B isoforms and increasing LDH-A isoforms. LDH-B activity decreased 80% after 3 days of functional overload. Additionally, LDH-A activity increased by 234% following 3 days of hindlimb suspension. However, neither LDH-A or LDH-B mRNA abundance was affected following 3 days of hindlimb suspension. In summary, the onset of altered loading induced a differential expression of LDH-A and -B in the rat soleus muscle, favoring rapid energy production. Long-term altered loading is associated with myofiber conversion; however, the rapid changes in LDH at the onset of altered loading may be involved in other physiological processes.     

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.