Erwin Goldberg

Expression of the Gene for Mouse Lactate Dehydrogenase C (Ldhc) Is Required for Male Fertility

Abstract The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc−/− mice but not in female Ldhc−/− mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc−/− sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C4) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc−/− sperm than in wild-type sperm. Moreover, Ldhc−/− sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.

Testis-Specific Cytochrome c-Null Mice Produce Functional Sperm but Undergo Early Testicular Atrophy

ABSTRACT Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt c T) that is distinct from the cytochrome c expressed in somatic cells (Cyt c S). To examine the role of Cyt c T in germ cells, we generated mice null for Cyt c T. Homozygous Cyt c T −/− pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt c T-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt c T-null mice contained a greater number of immotile spermatozoa than did samples from control mice, i.e., 53.1% ± 13.7% versus 33.2% ± 10.3% (P < 0.0001) for vas deferens sperm and 40.1% ± 9.6% versus 33.2% ± 7.5% (P = 0.0104) for epididymal sperm. Cyt c T-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt c T −/− testes and controls. Our data indicate that the Cyt c T-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation.

Immunohistochemical localization of LDH-X during spermatogenesis in mouse testes☆

Abstract Testis-specific lactate dehydrogenase, LDH-X, was localized spatially and temporally in the germinal epithelium of the mouse by the indirect fluorescent antibody technique. The enzyme was first detected in midpachytene primary spermatocytes and appeared to increase in concentration as spermatogenesis progressed to the spermatid. LDH-X synthesis, therefore, is initiated in the primary spermatocyte and continues at least until sometime during spermiogenesis. The uniform fluorescence of the cells containing LDH-X suggests that it is distributed throughout the cytoplasm rather than restricted to organelles such as mitochondria.

Selective active site inhibitors of human lactate dehydrogenases A4, B4, and C4

Human lactate dehydrogenases (LDH-A4, -B4, and -C4) are highly homologous with 84-89% sequence similarities and 69-75% amino acid identities. Active site residues are especially conserved. Gossypol, a natural product from cotton seed, is a non-selective competitive inhibitor of NADH binding to LDH, with K(i) values of 1.9, 1.4, and 4.2 microM for LDH-A4, -B4, and -C4, respectively. However, derivatives of gossypol and structural analogs of gossypol in the substituted 2,3-dihydroxy-1-naphthoic acid family exhibited markedly greater selectivity and, in many cases, greater potency. For gossypol derivatives, greater than 35-fold selectivity was observed. For dihydroxynaphthoic acids with substituents at the 4- and 7-positions, greater than 200-fold selectivity was observed. Inhibition was consistently competitive with the binding of NADH, with dissociation constants as low as 30 nM. By comparison, a series of N-substituted oxamic acids, which are competitive inhibitors of the binding of pyruvate to LDH, exhibited very modest selectivity. These results suggest that substituted dihydroxynaphthoic acids are good lead compounds for the development of selective LDH inhibitors. Selective inhibitors of LDH-C4 targeted to the dinucleotide fold may hold promise as male antifertility drugs. Selective inhibitors of LDH-A4 and -B4 may be useful for studies of lactic acidemia associated with ischemic events. More broadly, the results raise the question of the general utility of drug design targeted at the dinucleotide binding sites of dehydrogenases/reductases.

Lactate dehydrogenase ontogeny, paternal gene activation, and tetramer assembly in embryos of brook trout, lake trout, and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.

Infertility in female rabbits immunized with lactate dehydrogenase X

Immunization of female rabbits with the sperm-specific lactate dehydrogenase (LDH-X) resulted in a highly significant reduction of pregnancies compared to nonimmunized controls. This is the first demonstration of immunosuppression of fertility by a crystalline protein shown to be strictly homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, immunodiffusion, and micro complement fixation.

Lactate dehydrogenases in spermatozoa: Subunit interactions in vitro

Abstract The sperm-specific lactate dehydrogenase (LDH) isozyme from human, bovine, and murine sources has been dissociated, and its subunits have been recombined with subunits from the other forms of the enzyme. New LDHs are generated and have been characterized on the basis of substrate and coenzyme specificity. The properties of these new molecules are a reflection of their subunit composition, which, in turn, must determine either directly or indirectly the characteristics of the catalytic site(s) of the enzyme. The observations reported here demonstrate that the sperm-specific LDH contains polypeptide subunits which differ from those in LDH 1, 2, 3, 4, and 5.

Lactate Dehydrogenase C and Energy Metabolism in Mouse Sperm

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. Lack of LDHC impairs mouse sperm glycolysis but not pyruvate to lactate conversion.

Transgenic mice demonstrate a testis-specific promoter for lactate dehydrogenase, LDHC

The mammalian genome encodes a family of lactate dehydrogenase (LDH) isozymes. Two of these, ldha andldhb, are expressed ubiquitously. The ldhc gene is active only in the germinal epithelium during spermatogenesis. In our analysis of ldhc gene regulation, we found that a 60-base pair promoter sequence was sufficient for testis-specific expression in an in vitro transcription assay. To confirm these findings, a genomic fragment containing 100 base pairs overlapping the transcription start site was isolated and linked to theEscherichia coli lacZ gene. We report that this genomic fragment drives testis-specific expression in transgenic mice. We conclude that transcription of the transgene and possibly of the endogenous ldhc gene is restricted to leptotene/pachytene primary spermatocytes.

Lactate Dehydrogenase of Trout: Hybridization in vivo and in vitro

Speckled trout and lake trout contain five forms of lactate dehydrogenase, but a diflerent electrophoretic distribution of isozymes characterizes each species. The hybrid splake, which is produced artificially by fertilizing lake trout eggs with speckled trout sperm, contains nine isozymes. This comnplement of isozymeg In vivo could be produced in vitro by recombination of subunits from tissues of the parent species. In the splake trout, this complement is the result of heterozygosity at the gene locus responsible for synthesis of LDH-5. Extracts of trout eyes contain at least two additional forms of LDH which could not be demonstrated in other tissues.