Ldhc Gene Research Articles

Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

Cancer-Testis Antigen LDH-C4 in Tissue, Serum, and Serum-Derived Exosomes Serves as a Promising Biomarker in Lung Adenocarcinoma.

ObjectiveAs a cancer-testis antigen (CTA), human lactate dehydrogenase C4 (LDH-C4) enzyme protein encoded by the LDHC gene has been reported to be involved in the occurrence and development of various malignancies, while its expression and clinical significance in lung adenocarcinoma (LUAD) remain unclear. This study aims to investigate the expression of LDH-C4 in LUAD and its diagnostic and prognostic value.MethodsThe mRNA and protein levels of LDH-C4 in LUAD and adjacent normal tissues were analyzed based on the UALCAN database, and the prognostic significance was assessed using the LOGpc database. The LDHC mRNA level in serum and serum secretion of LUAD patients was determined by quantitative real-time PCR (qRT-PCR). Based on the high-throughput LUAD tissue chip combined with immunohistochemistry (IHC), the protein level of LDH-C4 in LUAD tissues was measured, and its correlation with clinicopathological features and prognosis was analyzed.ResultsLDHC expression was upregulated in LUAD, which was related to the clinical stage and poor prognosis of patients. The positive rates of LDHC mRNA expression in serum and exosome of LUAD patients were 78.3% and 66.7%, respectively. The area under the curve (AUC) of serum and exosomal LDHC in the diagnosis of LUAD was 0.8121 and 0.8925, respectively. The expression of LDHC in serum and serum-derived exosomes from LUAD patients was negatively correlated with medical treatment and positively correlated with the recurrence of LUAD. The positive expression rate of LDH-C4 in LUAD tissues was 96.7% (89/92), which was significantly higher than that in adjacent normal tissues 22.6% (19/84) (p < 0.001). The median overall survival (OS) time of patients with a high expression of LDH-C4 was significantly shorter than that of patients with low expression (34 months versus 62 months) (p = 0.016). Further relative risk analysis exhibited that the expression of LDH-C4 was an independent prognostic factor of OS in patients with LUAD.ConclusionsLDHC/LDH-C4 expression was upregulated in LUAD, and LDH-C4 could be used as a molecular indicator of the prognosis of LUAD. Serum and serum-derived exosomes of LDHC can be used as an important biomarker for the diagnosis, efficacy evaluation, and recurrence monitoring of LUAD.

Allele-specific methylation of type 1 diabetes susceptibility genes

The susceptibility to autoimmune diseases is influenced by genes encoding major histocompatibility complex (MHC) proteins. By examining the epigenetic methylation maps of cord blood samples, we found marked differences in the methylation status of CpG sites within the MHC genes (cis-metQTLs) between carriers of the type 1 diabetes risk haplotypes HLA-DRB1*03-DQA1*0501-DQB1*0201 (DR3-DQ2) and HLA-DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8). These differences were found in children and adults, and were accompanied by reduced HLA-DR protein expression in immune cells with the HLA-DR3-DQ2 haplotype. Extensive cis-metQTLs were identified in all 45 immune and non-immune type 1 diabetes susceptibility genes analyzed in this study. We observed and validated a novel association between the methylation status of CpG sites within the LDHC gene and the development of insulin autoantibodies in early childhood in children who are carriers of the highest type 1 diabetes risk genotype. Functionally relevant epigenetic changes in susceptibility genes may represent therapeutic targets for type 1 diabetes.

Lactate Dehydrogenase C Produces S-2-Hydroxyglutarate in Mouse Testis.

Metabolomics is a valuable tool for studying tissue- and organism-specific metabolism. In normal mouse testis, we found 70 μM S-2-hydroxyglutarate (2HG), more than 10-fold greater than in other tissues. S-2HG is a competitive inhibitor of α-ketoglutarate-dependent demethylation enzymes and can alter histone or DNA methylation. To identify the source of testis S-2HG, we fractionated testis extracts and identified the fractions that actively produced S-2HG. Through a combination of ion exchange and size exclusion chromatography, we enriched a single active protein, the lactate dehydrogenase isozyme LDHC, which is primarily expressed in testis. At neutral pH, recombinant mouse LDHC rapidly converted both pyruvate into lactate and α-ketoglutarate into S-2HG, whereas recombinant human LDHC only produced lactate. Rapid S-2HG production by LDHC depends on amino acids 100-102 being Met-Val-Ser, a sequence that occurs only in the rodent protein. Other mammalian LDH can also produce some S-2HG, but at acidic pH. Thus, polymorphisms in the Ldhc gene control testis levels of S-2HG, and thereby epigenetics, across mammals.

Characterization and Inhibitor Screening of Plateau Zokor Lactate Dehydrogenase C4.

Lactate dehydrogenase C4 (LDH-C4) is considered to be a target protein for the development of contraceptives. In this work, the characterization of plateau zokor LDH-C4 and the screening of a series of N-substituted oxamic acids as inhibitors against zokor LDH-C4 were reported. The cDNA of zokor LDH-C gene was cloned and expressed in Escherichia coli, from which the protein was purified and further characterized. The protein was a tetramer (LDH-C4) and thermally stable up to 62°C with a K m of 63.9μM for pyruvate and with optimal pH values of 7.95 and 10.1 for the forward and backward reactions respectively. Virtual and in vitro screening against zokor LDH-C4 revealed eight N-substituted oxamic acids with IC50s ranging from 198 to 2513μM, higher than that of oxamic acid (150μM) and (ethylamino)(oxo)acetic acid (59μM). The inhibition potencies of N-substituted oxamic acids tested are in the micromolar range, and the increase in the length of substituting chain seems not to increase inhibition potency.

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 RACE method. The cDNA was 1498 bp in length containing 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 degrees 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 microM 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.

Testis-specific lactate dehydrogenase is expressed in somatic tissues of plateau pikas

LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion of pyruvate with lactate. In mammals the, Ldh-c gene was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), belonging to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living at 3000–5000 m above sea levelon the Qinghai-Tibet Plateau. We found that the expression pattern of six LDH isoenzymes in the somatic tissues of female and male plateau pikas to be the same as those in testis and sperm, suggesting that LDH-C4 was expressed in somatic tissues of plateau pika. Here we report the detection of LDHC in the somatic tissues of plateau pika using RT-PCR, Western blotting and immunohistochemistry. Our results indicate that Ldh-c mRNA is transcribed in the heart, liver, lung, kidney, brain, skeletal muscle and testis. In somatic tissues LDHC was translated in the cytoplasm, while in testis it was expressed in both cytoplasm and mitochondria. The third band from cathode to anode in LDH isoenzymes was identified as LDH-C4. The finding that Ldh-c is expressed in both somatic tissues and testis of plateau pika provides important implications for more in-depth research into the Ldh-c function in mammals.

Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak

Huang, L., Jin, S-Y., Xu, Y-O., Li, Y-P., Lin, Y-Q. and Zheng, Y-C. 2012. Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak. Can. J. Anim. Sci. 92: 291–296. The main objective of the present study was to analyze quantitatively the alternative splicing of the lactate dehydrogenase C (ldhc) gene in the testes of yak (Bos grunniens) and male sterile yak hybrid. RT-PCR amplification of ldhc cDNA in the testes revealed eight splice variants formed by the deletion of one or more exons in the mRNA transcripts. The deleted exons occur mostly in exons 7 and 4. The deletion of exons caused reading frame shift and formation of stop codon in some variants. Quantitative real-time RT-PCR analysis using ldhc variant-specific primers showed that the mRNA level of full length ldhc decreased dramatically in the testes of sexually immature yak calf (n=6) and male sterile hybrid cattle–yak (n=4) compared with that of adult yak (n=14). The proportions of the ldhc variants assayed differed significantly among adult yak, yak calf and cattle–yak; more ldhc transcripts were spliced in immature or sterile testes. Our results suggest that the alternative splicing could play a role in the regulation of ldhc expression in testes, and could be one factor that plays a role in infertility of yak hybrids.

Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak

Huang, L., Jin, S-Y., Xu, Y-O., Li, Y-P., Lin, Y-Q. and Zheng, Y-C. 2012. Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak. Can. J. Anim. Sci. 92: 291-296. The main objective of the present study was to analyze quantitatively the alternative splicing of the lactate dehydrogenase C (ldhc) gene in the testes of yak (Bos grunniens) and male sterile yak hybrid. RT-PCR amplification of ldhc cDNA in the testes revealed eight splice variants formed by the deletion of one or more exons in the mRNA transcripts. The deleted exons occur mostly in exons 7 and 4. The deletion of exons caused reading frame shift and formation of stop codon in some variants. Quantitative real-time RT-PCR analysis using ldhc variant-specific primers showed that the mRNA level of full length ldhc decreased dramatically in the testes of sexually immature yak calf (n=6) and male sterile hybrid cattle-yak (n=4) compared with that of...

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.

Alternative Splicing of Testis-Specific Lactate Dehydrogenase C Gene in Mammals and Pigeon

The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.

A Mutation of Testis-specific Lactate Dehydrogenase Gene Found in Male Patients With Unexplained Infertility*

To investigate the role played by mutations of LDHC gene in male infertility, 100 male patients with unexplained infertility were screened by activity staining of LDH-C4 on sperm smears and those with lowered or no LDH-C4 activity in their sperms were subject to DNA analysis. The encoding exons were PCR amplified and the PCR products were screened by DHPLC for mutations. The PCR products with abnormal elution peaks were subject to DNA sequencing. In 14 infertile male patients with lowered LDH-C4 activity in sperms, it was identified a heterozygous T A mutation in base 115 of exon 5 of LDHC gene(GenBank accession number: GU479375), changing codon 178 (TTG, encoding leucine) to a stop codon (TAG). This nonsense mutation (L178X) was predicted to result in a severely truncated protein. The mutation and the heterozygosity were further confirmed by T-cloning. To the best of the knowledge, this is the first mutation found in human LDHC gene, substantiating that mutations in LDHC gene might cause male infertility.

급격한 환경변화에 대한 angelfish (Pterophyllum scalare) 젖산탈수소효소 동위효소의 변화

열대 저산소 환경에 적응되어 있는 angelfish (Pterophyllum scalare)를 급격한 온도변화(<TEX>$27{\pm}0.5{\rightarrow}18{\pm}0.5^{\circ}C$</TEX>) 및 DO 변화(<TEX>$6{\pm}1{\rightarrow}18\;ppm$</TEX>)에 2시간 동안 적응시킨 후 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) 동위효소의 특성 및 유전자발현을 연구하였다. LDH 동위효소의 특성은 native-polyacrylamide gel 전기영동, Western blot 분석 및 효소활성 측정으로 확인하였다. 전기영동 결과 liver- 및 eye-specific Ldh-C 유전자는 간, 눈 및 뇌 조직에서 발현되었다. Western blot 분석 결과 LDH <TEX>$A_4$</TEX> 동위효소는 <TEX>$B_4$</TEX> 동위효소보다 음극 쪽에 나타났다. 간 조직에서 온도 저하 시 LDH <TEX>$A_4$</TEX> 동위효소가 증가하고 <TEX>$B_4$</TEX> 동위효소는 감소하였으며, DO 증가 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$C_4$</TEX> 동위효소가 증가하고 <TEX>$B_4$</TEX> 동위효소는 감소하였다. 눈 조직에서는 온도 저하 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소가 증가하고 <TEX>$C_4$</TEX> 동위효소는 감소하였으며, DO 증가 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소는 증가하지만 <TEX>$C_4$</TEX> 동위효소 및 하부단위체 C를 포함하는 동위효소는 감소하였다. 심장 조직에서는 DO 증가 시 LDH 활성이 증가하였고, LDH <TEX>$B_4$</TEX> 동위효소가 증가하였다. 뇌 조직에서는 온도 저하 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소가 증가하였고, DO 증가 시 LDH <TEX>$B_4$</TEX> 동위효소는 증가하였다. 따라서 liver- 및 eye-specific Ldh-C는 DO 변화에 의해 영향을 받으며 간 및 눈 조직에서 LDH <TEX>$B_4$</TEX> 및 <TEX>$C_4$</TEX> 동위효소는 서로 상대적으로 조절되므로 <TEX>$C_4$</TEX> 동위효소는 lactate oxidase로서 기능을 나타내는 것으로 사료된다. In this study, the properties and gene expression of the lactate dehydrogenase (EC 1.1.1.27, LDH) isozyme were studied in angelfish (Pterophyllum scalare) - known for their adaptation to the low oxygen environment of the tropics - which were acclimated to acute temperature change (<TEX>$27{\pm}0.5{\rightarrow}18{\pm}0.5^{\circ}C$</TEX>) and dissolved oxygen (DO) change (<TEX>$6{\pm}1{\rightarrow}18\;ppm$</TEX>) for 2 hours. The properties of the LDH isozymes were confirmed in the native-polyacrylamide gel electrophoresis, Western blot analysis and enzyme activity measurement. Liver- and eye-specific Ldh-C gene were expressed in liver, eye and brain tissues. Through Western blot analysis, the LDH <TEX>$A_4$</TEX> isozyme was shown to have a more cathodal mobility relative to the <TEX>$B_4$</TEX> isozyme. In the liver tissue, the LDH <TEX>$A_4$</TEX> isozyme increased with temperature drop while the <TEX>$B_4$</TEX> isozyme decreased. The LDH <TEX>$A_4$</TEX> and <TEX>$C_4$</TEX> isozymes increased with DO increment, while the <TEX>$B_4$</TEX> isozyme decreased. In the eye tissue, the LDH <TEX>$A_4$</TEX> and B4 isozymse increased with temperature drop while the <TEX>$B_4$</TEX> isozyme decreased. The LDH <TEX>$A_4$</TEX> and <TEX>$B_4$</TEX> isozymes increased with DO increment, but the <TEX>$C_4$</TEX> isozyme and isozymes including the subunit C decreased. In the heart tissue, LDH activity increased with DO increment, as well as the LDH <TEX>$B_4$</TEX> isozyme. In the brain tissue, the LDH <TEX>$A_4$</TEX> and <TEX>$B_4$</TEX> isozymes increased with temperature drop. The LDH <TEX>$B_4$</TEX> isozyme increased with DO increment. Accordingly, since the liver- and eye-specific Ldh-C are influenced by changes in DO and the LDH <TEX>$B_4$</TEX> and <TEX>$C_4$</TEX> isozymes are relatively controlled in the liver and eye tissues, the <TEX>$C_4$</TEX> isozyme can be considered to have a lactate oxidase function.

Computational analyses of mammalian lactate dehydrogenases: Human, mouse, opossum and platypus LDHs

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase ( LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 ( LDHA and LDHC) or 8 ( LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

Homo sapiens Lactate Dehydrogenase c (Ldhc) Gene Expression in Cancer Cells Is Regulated by Transcription Factor Sp1, CREB, and CpG Island Methylation

The human testis-specific lactate dehydrogenase c gene (hLdhc) is transcribed only in cells of the germinal epithelium. Recently hLdhc was reported to express in a broad spectrum of tumors with relatively high frequency in lung cancer, melanoma, and breast cancer, and in some prostate cancers. Two melanoma cell lines that express the hLdhc gene, A375M and C81-61, were identified and were used to characterize the hLdhc promoter and explore transcriptional regulation of this gene. A 110-bp core promoter, including a conserved GC box and cyclic adenosine monophosphate-responsive element (CRE), were identified as essential for basal promoter activity. The methylation status of the CpG island (CGI) in the hLdhc core promoter sequence was analyzed in hLdhc-expressing and nonexpressing cells and human prostate tumor tissues. The CGI in 2 cell lines expressing the gene was hypomethylated whereas the DNA from cells that did not express hLdhc was hypermethylated. The role of methylation in regulating this promoter was confirmed by experimental induction of hLdhc transcription with the methylation inhibitor 5'aza-deoxycytidine. Quantitative analyses of the methylation level in the CGI were performed in prostate tumor tissues by pyrosequencing. Overall, these experiments demonstrated that hLdhc expression in cancer cells was regulated by transcription factor Sp1 and CREB and promoter CGI methylation. In addition, these findings suggest the possibility of developing a biomarker for cancer diagnosis/prognosis based on DNA methylation of the Ldhc gene.

Expression of the Gene for Mouse Lactate Dehydrogenase C (Ldhc) Is Required for Male Fertility1

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-C(4)) 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 Lactate Dehydrogenase (LDH‐C4; Ldh3) in Murine Oocytes and Preimplantation Embryos

LDH-C(4) (Ldh3) is a member of the lactate dehydrogenase family of isozymes that catalyze the terminal reaction in the glycolytic pathway. In mammals, 3 genes, ldha, ldhb, and Ldhc, encode the subunits that assemble into catalytically active homo- and heterotetramers. Differential expression of these genes determines the lactate dehydrogenase (LDH) isozyme composition of tissues, and, as is well known, A subunits predominate in skeletal muscle and B subunits are abundantly produced in brain and heart, with the Ldh2 isozyme the most abundant form in oocytes. The C peptide can be detected first in pachytene spermatocytes and constitutes the primary LDH of spermatozoa. Originally the Ldhc gene (Ldh3 in terminology applied to murine cells) was considered to be testis specific on the basis of immunochemical, enzymatic, and molecular analyses. Here we report the detection of this isozyme in the murine oocyte and early embryo. Our results indicate that Ldh3 mRNA is transcribed in oocytes and cannot be detected in fertilized eggs. Ldh3 protein, however, persists to the blastocyst stage of embryonic development localizing mainly to the cortex region of oocytes, eggs, zygotes, and embryonic blastomeres.

Heat-induced apoptosis of mouse meiotic cells is suppressed by ectopic expression of testis-specific calpastatin.

Calpastatin is a naturally occurring inhibitor of calpain, a protease involved in apoptotic cell death. A testis-specific isoform of calpastatin (tCAST) has been identified that is transcribed in haploid germ cells but not in spermatocytes. To investigate the possible function(s) of tCAST, we tested the hypothesis that the ectopic expression of calpastatin in spermatocytes would suppress the death of these cells in response to an apoptosis-inducing stimulus in vivo. To this end, the 5'-flanking region of the mouse ldhc gene was linked to tCAST, and transgenic mice were generated. Immunohistochemical analysis revealed that, in contrast to control sections in which the signal for tCAST was seen in round spermatids, intense staining was visualized in pachytene spermatocytes in the transgenic animals, indicating that the strategy we used to generate the transgenic animals resulted in the ectopic expression of tCAST in spermatocytes. We then tested the effect of a short period of heating on germ cell apoptosis in the testes of wild-type and transgenic mice. Pachytene spermatocytes were the major germ cell type seen to undergo apoptosis after heat treatment. There were no differences in the number of apoptotic germ cells per seminiferous tubule between wild-type and tCAST transgenic control mice; thus, there was no apparent effect of the transgene on normal apoptosis. Heating resulted in increased numbers of TUNEL-positive germ cells in both wild-type and tCAST transgenic mice, as well as increased testicular DNA fragmentation. Heating the tCAST transgenic mouse testes resulted in significantly fewer apoptotic cells per seminiferous tubule than in wild-type mice at both 8 and 24 hours after treatment. Thus, as hypothesized, the ectopic expression of tCAST in pachytene spermatocytes suppressed germ cell apoptosis.

Abundance of repetitive sequence elements in the mouse testis-specific lactate dehydrogenase-C gene.

We have cloned and sequenced the entire mouse ldhc gene and mapped it physically in relation to the somatic ldha gene. The 2 genes were found to be oriented in head-to-tail fashion with about a 6-kilobase (kb) distance between the 3' end of ldha and the 5' end of ldhc. The ldhc gene is composed of 43% repetitive elements compared to only 16% in the ldha gene. Despite the close physical distance of mouse ldha and ldhc, the 2 genes have a very different content of repetitive elements, and this most likely reflects different levels of selective pressure.

Ldhc Expression in Non-germ Cell Nuclei Is Repressed by NF-I Binding

Developmental and testis-specific expression of the mouse lactate dehydrogenase C (mldhc) gene requires mechanisms for activation in germ cells and repression in somatic cells. Promoter activity restricted to the testis has been demonstrated using in vitro transcription assays with a 60-base pair promoter sequence upstream of the transcription initiation site. This promoter fragment has a TATA box and an overlapping 31-base pair palindromic sequence. Here we have explored the role of the palindrome as a silencer of the ldhc gene in somatic tissues. A gel retardation assay detected two sites within the palindrome that were important for protein binding. A member of the NF-I/CTF family was identified as the protein binding to one of the sites. In transiently transfected mouse L cells, a promoter fragment in which the NF-I site was mutated showed a 4-fold greater activity as compared with the wild-type sequence. Overexpression of the four NF-I proteins, NF-IA, -B, -C, or -X, in mouse L cells transiently transfected with an ldhc promoter-reporter construct resulted in a 20-50% decrease in activity of the wild-type promoter but had no effect when the NF-I binding element in the palindrome was mutated. These results indicate a role for the NF-I proteins in regulation of the mldhc gene.