Expression Of LDHC Research Articles

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.

Aberrant expression of sperm‑specific glycolytic enzymes are associated with poor sperm quality.

The energy required for normal sperm function is mainly generated by glycolysis and oxidative phosphorylation. Testis-specific glycolytic enzymes are expressed in germ cells and present in mature spermatozoa. The objective of the present study was to investigate the association between aberrant expression of glycolytic enzymes and human sperm quality. In silico analysis of glyceraldehyde-3-phosphate dehydrogenase, testis-specific (GAPDHS), phosphoglycerate kinase 2 (PGK2) and lactate dehydrogenase (LDHC) identified that they were exclusively expressed in post-meiotic germ cells and this was validated in human testes using immunohistochemistry. Compared with the testes of young adults, markedly lower expression levels of these glycolytic enzymes were observed in the testes of elderly adults. Similarly, low levels were observed in immature and asthenozoospermic spermatozoa. The expression levels of GAPDHS, PGK2 and LDHC in the spermatozoa were closely correlated with progressive sperm motility. The results indicated that the expression of GAPDHS, PGK2 and LDHC in sperm may be associated with sperm quality, and there may be a similar molecular mechanism underlying sperm quality in immature and asthenozoospermic spermatozoa. This study reveals a close association of glycolytic enzymes with sperm quality. The data may greatly contribute to the molecular evaluation of sperm quality and the diagnosis and treatment of asthenozoospermia.

N-acetylcysteine modulates lipopolysaccharide-induced intestinal dysfunction

The gastrointestinal epithelium functions in nutrient absorption and pathogens barrier and its dysfunction directly affects livestock performance. N-Acetylcysteine (NAC) improves mucosal function, but its effects on intestinal functions at the molecular level remain unclear. Here, we performed gene expression profiling of the pig small intestine after dietary NAC treatment under LPS challenge and investigated the effects of NAC on intestinal epithelial cells in vitro. Dietary NAC supplementation under LPS challenge altered the small intestine expression of 959 genes related to immune response, inflammatory response, oxidation-reduction process, cytokine-cytokine receptor interaction, and the cytokine-mediated signalling, Toll-like receptor signalling pathway, Jak-STAT signalling pathway, and TNF signalling pathway. We then analysed the expression patterns of the top 10 altered genes, and found that NAC markedly stimulated HMGCS3 and LDHC expression in IPEC-J2 cells. In vitro, NAC pre-treatment significantly reduced TNF-α and NF-κB, TNF-α, IFN-γ, and IL-6 expression in LPS-induced IPEC-J2 cells. NAC treatment also significantly reduced oxidative stress in LPS-induced IPEC-J2 cells and alleviated intestinal barrier function and wound healing. Thus, NAC as a feed additive can enhance livestock intestinal health by modulating intestinal inflammation, permeability, and wound healing under LPS-induced dysfunction, improving our molecular understanding of the effects of NAC on the intestine.

The expression of Ldh-c in the skeletal muscle of plateau pika (Ochotona curzoniae) enhances adaptation to a hypoxic environment.

ABSTRACTThe plateau pika (Ochotona curzoniae) is a species of sprint-running alpine animals in the Qinghai-Tibet Plateau, which is a harsh highland hypoxic environment. Ldh-c is expressed in the testis, sperm and somatic tissues of plateau pika. To reveal the role and physiological mechanisms of sperm-specific lactate dehydrogenase (LDH-C4), in plateau pika to adapt to hypoxic environment, an adenoviral line of pMultiRNAi-Ldhc was constructed and injected into the bilateral biceps femoris of the hind legs. The swimming times of the pikas, and the Ldh-c expression levels, total LDH activities and ATP levels in skeletal muscle, were measured after the pikas were raised in the trapped site for 5 days. Our results showed that after Ldh-c was silenced, the sprint-running ability (swimming time) of the plateau pikas was significant decreased, and the total LDH activities and ATP levels were reduced by 28.21% and 27.88%, respectively. Our results indicated that expression of Ldh-c in the skeletal muscle of plateau pika increased anaerobic glycolysis and enhanced adaptation to highland hypoxic environments.

Expression of Ldh-c (Sperm-Specific Lactate Dehydrogenase Gene) in Skeletal Muscle of Plateau Pika, Ochotona curzoniae, and its Effect on Anaerobic Glycolysis

Expression of Ldh-c (Sperm-Specific Lactate Dehydrogenase Gene) in Skeletal Muscle of Plateau Pika, Ochotona curzoniae, and its Effect on Anaerobic Glycolysis

Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

Sperm specific lactate dehydrogenases (LDH-C4) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000–5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika’s heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C4, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment.

Human Lactate Dehydrogenase A (LDHA) Rescues Mouse Ldhc-Null Sperm Function1

By targeted disruption of the lactate dehydrogenase c (Ldhc) gene, we demonstrated that spermatozoa require Ldhc for capacitation, motility, and fertilizing capacity. Ldhc expression is restricted to the developing germ cells that, however, are apparently not compromised by the lack of the LDHC isozyme. Because LDHC is abundant in spermatozoa that utilize aerobic glycolysis for energy requirements, its main function was presumed to be the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD(+). We found that sperm without LDHC were still able to convert lactate to pyruvate as mediated by LDHA that is tightly bound to the fibrous sheath. It was assumed that the level of glycolysis was insufficient to power motility and the subsequent fertilizing capacity of the mutated sperm. To investigate whether LDHC possesses certain unique characteristics essential for fertility, human LDHA was introduced as a transgene to Ldhc-null mice. We report here that the exogenous LDHA rescued the phenotype of the Ldhc-null males. Sperm from the LDHA transgenic males with the Ldhc deletion (LDHA(+)/Ldhc(-/-)) are motile, capable of protein tyrosine phosphorylation, and able to fertilize, thus restoring these properties to LDHC-null sperm. However, the lactate and ATP levels in the rescued sperm did not differ significantly from sperm lacking LDHC. We suggest that it is the localization of the transgene to the sperm cytosol that is mainly responsible for restoration of sperm function and fertility.

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.

A-MYB (MYBL1) Stimulates Murine Testis-Specific Ldhc Expression via the cAMP-Responsive Element (CRE) Site1

Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.

LDH-C can be differentially expressed during fermentation of CHO cells

Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO) Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore(http://hugroup.cems.umn.edu/CHO/cho_index.html). Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate.The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

Transactivation Function of A-MYB Regulates Testis Specific Ldhc Expression via the CRE Cis Element.

The mechanism regulating gene expression is largely unknown for primary spermatocytes. In spermatids however, there is a specific co-activator ACT (Activator of CREM in testis) directing expression. In our model system a 100-bp core ldhc sequence proved to be sufficient to drive spermatocyte-specific reporter gene expression. The core promoter is composed of two essential elements, a GC-box and CRE site, both conserved in murine and human testes. Here we report that transcription factor A-Myb acts as a co-activator directing tissue-specific expression via the CRE cis element in primary spermatocytes. AMYB was localized in the same cell type as the reporter gene driven by ldhc promoter. AMYB was able to induce Ldhc promoter activity 4-8 folds. However a gel shift assay showed that A-Myb did not bind to the myb consensus sequences in the ldhc promoter. Instead, A-myb interacted with a synthetic promoter containing CRE elements only. A co-activator CBP was able to enhance promoter activity in a dosage-dependent manner. A ChIP experiment confirmed the binding of AMYB to the CRE-containing promoter in vivo. A testis-specific PGK2 gene containing CRE promoter element was also activated 3 folds by A-Myb. These observations support a novel mechanism that the interaction of A-Myb with CREB directs a tissue-specific activation in the primary spermatocyte. (poster)

가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성

가물치(Channa argus) 조직의 젖산탈수소효소 동위효소(EC 1.1.1.27, lactate dehydrogenase, LDH)를 정제하고 생화학적, 면역학적 및 역학적 방법으로 특성을 연구하였다. LDH 활성은 골격근이 380.4 units로 가장 높고 심장 13.4, 눈 3.5, 뇌 조직 5.4 units이었으며, 심장의 CS 활성은 20.7 unit로 가장 높고, LDH/CS는 골격근 172.9, 심장 0.6, 눈 0.32, 뇌 0.47이고, 단백질 양은 골격근 14.7 mg/g이며, 특이활성(units/mg)은 골격근 25.88, 심장 0.79, 눈 0.31, 뇌 1.38 units/mg이었으므로 골격근은 혐기적이고, 심장은 호기적이었다. LDH <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>에 대한 항혈청을 사용한 Western blot, 면역침강반응 및 native-polyacrylamide gel electrophoresis에 의해 <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX> 및 <TEX>$B_4$</TEX>가 모든 조직에서 확인되었고, 눈 조직에서 <TEX>$C_4$</TEX>와 <TEX>$AC_3$</TEX>, <TEX>$A_2C_2$</TEX>, <TEX>$AC_3$</TEX>, 뇌 조직에서 <TEX>$A_3C$</TEX>도 확인되었다. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX> 동위효소는 affinity chromatography와 Preparative PAGE Cell에 의해 정제되었다. LDH <TEX>$A_4$</TEX> 동위효소는 <TEX>$NAD^+$</TEX> 유입 후 정제되었고, eye-specific <TEX>$C_4$</TEX>는 <TEX>$A_4$</TEX>에 이어 용출되기 시작하였으며 <TEX>$B_4$</TEX>는 buffer 유입 후 용출되었다. 정제한 결과 <TEX>$A_4$</TEX>는 <TEX>$B_4$</TEX> 및 eye-specific <TEX>$C_4$</TEX>와 분자구조의 일부가 유사하였지만 <TEX>$B_4$</TEX>와 <TEX>$C_4$</TEX>는 서로 다른 것으로 나타났으므로, 하부단위체 A는 보존적이고, 하부단위체 B는 A보다 더 빠르게 진화된 것으로 보인다. 피루브산 10 mM에서 <TEX>$A_4$</TEX> 동위효소 39.98%, <TEX>$A_2B_2$</TEX> 21.28%, <TEX>$B_4$</TEX> 19.67% 및 eye-specific <TEX>$C_4$</TEX> 16.87%의 활성이 남아있었고, 피루브산에 대한 <TEX>$Km^{PYR}$</TEX>은 <TEX>$A_4$</TEX> 0.17 mM, <TEX>$B_4$</TEX> 0.27 mM, eye-specific <TEX>$C_4$</TEX> 0.133 mM였다. <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$A_3B$</TEX> 및 <TEX>$AB_3$</TEX>의 최적 pH는 각각 pH 6.50, pH 8.5, pH 5.5, pH 6.0-6.5, 5.0 및 pH 7.5였고, 동질사량체 <TEX>$A_4$</TEX>와 이질사량체 동위효소들은 넓은 pH 영역에서 안정하였다. 특히 골격근은 LDH 활성이 크므로 활동성이 크며, 눈조직에서 피루브산 친화력이 강한 eye-specific <TEX>$C_4$</TEX>에 의해 피루브산 대사가 빠르게 일어나고, 이어서 <TEX>$A_4$</TEX>에 의해 젖산이 산화되어지는 것으로 사료되므로, 종의 생태환경 및 먹이 획득 양식에 따라 LDH-C 발현, 기질에 대한 친화도 및 대사 시간이 다른 것으로 사료된다. The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, and eye-specific <TEX>$C_4$</TEX>. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>. <TEX>$AB_3$</TEX> and <TEX>$B_4$</TEX> isozymes were detected in every tissue, <TEX>$C_4$</TEX>, <TEX>$AC_3$</TEX>, <TEX>$A_2C_2$</TEX> and <TEX>$A_3C$</TEX> were detected in eye tissue, and <TEX>$A_3C$</TEX> was found in brain tissue. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX> isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH <TEX>$A_4$</TEX> isozyme was purified in the fraction from elution with <TEX>$NAD^+$</TEX> containing buffer of affinity chromatography. Eye-specific <TEX>$C_4$</TEX> isozyme was eluted right after <TEX>$A_4$</TEX>, after which <TEX>$B_4$</TEX> isozyme was eluted with plain buffer. As a result, one part of molecular structures in <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX> and eye-specific <TEX>$C_4$</TEX> were similar, but were different from each other in <TEX>$B_4$</TEX> and <TEX>$C_4$</TEX>. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH <TEX>$A_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$B_4$</TEX>, and eye-specific <TEX>$C_4$</TEX> isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The <TEX>$Km^{PYR}$</TEX> values were 0.17, 0.27 and 0.133 mM in <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX> and eye-specific <TEX>$C_4$</TEX> isozymes, respectively. The optimum pH of LDH <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$A_3B$</TEX>, and <TEX>$AB_3$</TEX> were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The <TEX>$A_4$</TEX> and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific <TEX>$C_4$</TEX> isozyme as eye-specific <TEX>$C_4$</TEX> have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by <TEX>$A_4$</TEX> isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of <TEX>$C_4$</TEX> isozyme were different according to the ecological environmental and feeding capturing patterns.

Regulation of Murine Lactate Dehydrogenase C (Ldhc) Gene Expression1

Expression of Ldhc begins with the onset of meiosis in male germ cells and continues throughout spermatogenesis. Transcriptional regulatory mechanisms, especially in primary spermatocytes, are poorly described because of the lack of a reliable cell culture system. We constructed mouse transgenics and transfected germ cells in situ to study expression of the testis-specific isozyme of lactate dehydrogenase (LDH). From previous work, we determined that a 100-bp Ldhc core promoter contained potential cis regulatory elements, including a palindrome (-21 to +10), GC box (-70 to -65), and cAMP-responsive element (CRE) sites (-53 to -49, -39 to -35). We provide here the demonstration of a functional role for these sequences by expression of mutated transgenes in vivo. Our results reveal for the first time that mutation of the GC box does not abolish promoter activity, which remains testis-specific. Mutation of GC box or CRE sites resulted in a 73% and 74% reduction in promoter activity, respectively, in a transient transfection of germ cells in vivo by electroporation; the combination of GC box and CRE site mutations eliminates promoter activity. Therefore, we conclude that simultaneous occupancy of the GC box and CRE sites in the core promoter is necessary for full expression of Ldhc in the testis.

A transgenic analysis of mouse lactate dehydrogenase C promoter activity in the testis.

Transcription of the mouse testis-specific lactate dehydrogenase c (mldhc) gene is limited to cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-bp core promoter was able to regulate testis-specific transcription in vitro and in transgenic mice. Surprisingly, expression of the reporter in transgenic testes was limited to pachytene spermatocytes, whereas native LDH-C(4) was detected in pachytene and all later germ cells. To locate additional regulatory sequence that could recapitulate the native LDH-C(4) distribution pattern, we investigated the contribution of 5' and 3' flanking sequences to the regulation of LDH-C(4) expression. We found that transcription factor YY1 binds to the mldhc promoter, that the mldhc 3' untranslated sequence does not permit a postmeiotic expression of a beta-galactosidase reporter in transgenic mice, and that native mldhc mRNA is predominately meiotic, with only a low level of postmeiotic distribution. Our results suggest that the high level of LDH-C(4) in postmeiotic cells results from mRNA and protein stability.

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.

Deoxyribonucleic acid-protein interactions and expression of the human testis-specific lactate dehydrogenase promoter: transcription factor Sp1 plays a major role.

The human testis-specific lactate dehydrogenase c gene (ldh-c) shows an exceptionally large window of expression throughout pre- and postmeiotic stages of the male germ cell lineage. In order to characterize the multiple stage-specific transcription factors necessary for ldh-c expression, we previously characterized the human ldh-c core promoter. Here, we used a combination of gel retardation assays and an in vitro transcription system derived from human tissues to better define the elements that govern ldh-c transcription. Three classes of transcriptional regulators were defined by these experiments. 1) The Sp1 transcription factor is a testis-"enriched" protein that is absent from most somatic tissues and that appears to play a major role in determining ldh-c expression in the testis. Highest levels of Sp1 during spermatogenesis correlate with maxima of ldh-c expression. 2) The testis-specific cAMP response element modulator (CREM) transcription factor binds a cAMP response element (CRE)-like sequence located at position -433. This transcriptional activator might contribute to postmeiotic transcription of ldh-c. 3) Factors present in tissues negative for ldh-c expression appear to bind both the CRE-like sequence and an adjacent hormone response element. The presence of this element could be involved in regulating ldh-c through the glucocorticoid/androgen pathways at the early stages of ldh-c expression.

Posttranscriptional regulation of primate Ldhc mRNA by its AUUUA-like elements.

The Ldhc locus encodes the testis-specific isozyme of lactate dehydrogenase in mammals. In our efforts to understand the regulatory mechanisms involved in expression of Ldhc, we recognized the possibility that this gene could be post-transcriptionally regulated in certain species as the 3'-untranslated region (3'-UTR) of Ldhc in primates, but not rodents, contains a number of AU-rich motifs and is conserved. To determine whether the primate Ldhc mRNA is posttranscriptionally regulated, comparison of baboon and mouse Ldhc mRNA stability was made in a cell-free system. The results indicated that the baboon mRNA is labile, while that of mouse, which does not contain the AU-rich motifs, is highly stable. Consistent with these results, the steady state level of primate Ldhc was found to be 8 to 12 fold lower than that of the mouse. We show that in a transformed murine germ cell line, the human Ldhc mRNA is moderately unstable, and removal of its 3'-UTR leads to stabilization of the mRNA. Mutations disrupting the AU-rich motifs of human Ldhc result in stabilization of the mRNA in vitro. On the basis of these observations, we conclude that stability of the primate Ldhc transcript is regulated by dispersed AU-rich elements found in its 3'-UTR. Because AU-rich motifs similar to these are found in many mRNAs, these findings may have broad implications.

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.