Lactate Dehydrogenase C Research Articles

Inhibiting Human Lactate Dehydrogenase-C for Male Fertility Control; Initial Hits

Lactate dehydrogenase-C (LDH-C) is an oxido-reductive enzyme of LDH (EC 1.1.1.27) family which is present abundantly in testes, spermatocytes, spermatids and sperms. It plays an important role in maintaining glycolysis and ATP production in the flagellum during sperm capacitation, therefore essential for male fertility. Profuse expression exclusively in these tissues makes LDH-C a potential target for male contraceptive development. However, creating a specific inhibitor for LDH-C is challenging due to its extensive sequence similarity (84-89%) with somatic isozymes LDH-A and B.

Cloning and Characterization of Multiple RNA Splicing Variants of LDH-C Gene in Human and Rat

The expression of LDH-C (Lactate dehydrogenase C) gene is restricted in mature germ cells; however multiple splice variants of LDH-C expressed in human cancers and yak normal testes were reported recently. In order to know if there are any LDH-C splice variants in human normal testes, we set out to clone the putative variants in human and rat. Four splicing variants in human testes, 1 splicing variant in human spermatozoa, 6 splicing variants in rat testes and 1 splicing variant in rat non-testes tissues (liver, heart and muscle) were cloned. The putative polypeptides encoded by these variants were compared with the full-length LDH-C protein, the results showed that these putative polypeptides were truncated LDH-C proteins or truncated LDH-C proteins with a few amino acid residues different at N or C terminal. This suggested that these variants are possibly not used for translation, but targets of nonsense-mediated mRNA decay. Western blotting did not detect any bands with similar molecular weight as the putative polypeptides. RT-PCR showed that the expression levels of the splicing variants were significant during development of rat testes. The results indicate that LDH-C was not silenced by transcriptional repression in non-mature germ cells, but significantly transcripted and alternatively spliced.

Identification of developmental competence‐related genes in mature porcine oocytes

Oocyte competence is a key factor limiting female fertility, yet the underlying molecular mechanisms that contribute to oocyte competence remain unclear. The objective of this study was to elucidate specific genes whose function contributes to oocyte competence. We observed that 6 of 20 target genes examined were differentially expressed between adult (more competent) and prepubertal (less competent) porcine in vitro matured (IVM) oocytes. These genes were the cholesterol synthesis-related gene HMG-CoA reductase (HMGCR), fatty acid oxidation genes acyl-CoA synthetase long-chain family member 3 (ACSL3) and long-chain acyl-CoA dehydrogenase (ACADL), glycolytic genes fructose 1,6 bisphosphate aldolase (ALDOA) and lactate dehydrogenase C (LDHC), and tumor necrosis factor-α (TNF). These 6 genes, as well as 3 other genes [porcine endogenous retrovirus (PERV), transcribed loci 10 (TL10), serine/arginine-rich splicing factor 1 (SRSF1)], were further analyzed by comparing transcript abundance in IVM and in vivo matured (VVM) prepubertal and adult porcine oocytes. Among these 9 target genes, 5 were differentially expressed between IVM and VVM prepubertal oocytes, while 8 genes were differentially expressed between IVM and VVM adult oocytes. No genes were differentially expressed between VVM prepubertal and adult oocytes. A functional study of TNF demonstrated that depletion of endogenous TNF decreased oocyte competence and TNFAIP6 expression in cumulus cells, while TNF in IVM medium regulated TNFAIP6 expression in cumulus cells. Differential expression of the genes identified in this study suggests that these genes may be functionally relevant to oocyte competence.

Genome-Wide Association Study Identifies Two Novel Regions at 11p15.5-p13 and 1p31 with Major Impact on Acute-Phase Serum Amyloid A

Elevated levels of acute-phase serum amyloid A (A-SAA) cause amyloidosis and are a risk factor for atherosclerosis and its clinical complications, type 2 diabetes, as well as various malignancies. To investigate the genetic basis of A-SAA levels, we conducted the first genome-wide association study on baseline A-SAA concentrations in three population-based studies (KORA, TwinsUK, Sorbs) and one prospective case cohort study (LURIC), including a total of 4,212 participants of European descent, and identified two novel genetic susceptibility regions at 11p15.5-p13 and 1p31. The region at 11p15.5-p13 (rs4150642; p = 3.20×10−111) contains serum amyloid A1 (SAA1) and the adjacent general transcription factor 2 H1 (GTF2H1), Hermansky-Pudlak Syndrome 5 (HPS5), lactate dehydrogenase A (LDHA), and lactate dehydrogenase C (LDHC). This region explains 10.84% of the total variation of A-SAA levels in our data, which makes up 18.37% of the total estimated heritability. The second region encloses the leptin receptor (LEPR) gene at 1p31 (rs12753193; p = 1.22×10−11) and has been found to be associated with CRP and fibrinogen in previous studies. Our findings demonstrate a key role of the 11p15.5-p13 region in the regulation of baseline A-SAA levels and provide confirmative evidence of the importance of the 1p31 region for inflammatory processes and the close interplay between A-SAA, leptin, and other acute-phase proteins.

LDHC: The Ultimate Testis‐Specific Gene

Lactate dehydrogenase C (LDHC) was, to the best of our knowledge, the first testis-specific isozyme discovered in male germ cells. In fact, this was accomplished shortly before "isozymes or isoenzymes" became a field of study. LDHC was detected initially in human spermatozoa and spermatogenic cells of the testes by gel electrophoresis. Immunohistochemistry was used to localize LDHC first in early-pachytene primary spermatocytes, with an apparent increase in quantity after meiosis, to its final localization in and on the principal piece of the sperm tail. After several decades of biologic, biochemical, and genetic investigations, we now know that the lactate dehydrogenase isozymes are ubiquitous in vertebrates, developmentally regulated, tissue and cell specific, and multifunctional. Here, we will review the history of LDHC and the work that demonstrates clearly that it is required for sperm to accomplish their ultimate goal, fertilization.

Placental gene expression is related to glucose metabolism and fetal cord blood levels of insulin and insulin-like growth factors in intrauterine growth restriction

Despite the importance of glucose for fetal growth, placental gene expression related to the glycolytic pathway has not been studied. Insulin-like growth factors (IGFs) and insulin are known to play a critical role in fetal growth. In our study, we identified differentially expressed genes related to the glycolytic pathway using oligonucleotide microarray analysis and confirmed these genes with quantitative real-time PCR between uncomplicated pregnancies and pregnancies with intrauterine growth restriction (IUGR). We also compared the concentrations of insulin and IGFs in cord blood between the two groups. Microarray experiments identified increased expression of glycolytic enzyme-related genes, including lactate dehydrogenase C (LDHC), dihydrolipoamide S-acetyltransferase (DLAT), 6 phosphofructo-2-kinase/fructose-2, 6-biphosphatase 2 (PFKFB2), oxoglutarate dehydrogenase, phosphorylase, and insulin-like growth factor (IGF)-II and decreased expression of IGF-I in placentas from pregnancies with IUGR ( p < 0.05). There were significantly lower concentrations of glucose, insulin, IGF-1, and IGF-II in the fetal cord blood of pregnancies with IUGR ( p < 0.05). Microarray analysis revealed increased expression of enzyme genes related to the tricarboxylic acid cycle pathway in placentas from pregnancies with IUGR; the cause of hypoglycaemia in IUGR is attributed to increased glycolytic pathway activity in placentas from pregnancies with IUGR.

Oxidation of Exogenous Lactate by Lactate Dehydrogenase C in the Midpiece of Rat Epididymal Sperm is Essential for Motility and Oxidative Activity

Problem statement: To identify the metabolic reaction-glycolysis or oxidative phosphorylation that is mainly involved in the production of energy required for rat sperm mobilization. Approach: Epididymal sperm were collected from Wistar rats and extended in lactate-containing or lactate-free raffinose-modified Krebs-Ringer Bicarbonate solution (mKRB)-egg yolk medium supplemented with 0, 1, 2, or 3 mM 2-Deoxy-D-Glucose (2 DG) and 1, 2, or 3 mM sodium Oxamate (OX). Sperm motility, straight-line velocity (VSL) and oxygen consumption were evaluated. Further, immunofluorescent localization of Lactate Dehydrogenase C (LDH-C) in sperm was also performed. Results: Low concentrations of 2DG (1 and 2 mM) did not significantly affect motility, VSL and oxygen consumption of sperm extended in the lactate-containing medium. While sperm motility and oxygen consumption were significantly inhibited by even 1mM 2DG in sperm extended in lactate-free medium. Sperm motility significantly inhibited in the case of sperm extended in lactate-containing and free-medium with 1 mM OX. We also found that sperm motility was not maintained in the absence of lactate throughout the 3 h incubation period. Immunofluorescence study revealed that mainly LDH-C was may be localized in the intramitochondrial region of the sperm. Conclusion: These results suggest that exogenous lactate enhances lactate oxidation by LDH-C, thereby promoting mitochondrial oxidative reactions in the midpiece and maintaining the mobilization of rat epididymal sperm.

Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue

Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered. The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.

Cloning and Sequence Analysis of Multiple Splice Variants of Lactate Dehydrogenase C in Yak Testes

Lactate dehydrogenase C (LDH-C) has been reported to play a role in the energy metabolism of mammal spermatozoa. However, the functions and expression patterns of LDH-C still remain unclear. In order to elucidate the functions and expression patterns of LDH-C, we cloned the cDNA of yak LDH-C. Total RNA was extracted from yak testes and reverse transcribed and amplified by PCR. The full length open reading frame (ORF) of LDH-C and its five splice variants were obtained. The full length ORF contained 999 bp encoding a 332-amino-acid protein that showed 100% identity with bovine LDH-C. Compared with the full length ORF of LDH-C, the five variants used the same start codon as the full length ORF and encoded 5 putative proteins. In detail, variants 1 (missing the coding sequence of exon 6 and 7) and 2 (missing the coding sequence of exon 7) bear the entire nicotinamide-adenine dinucleotide (NAD) binding domain and an active site. Variants 3 (missing the first 42 nuleotides of exon 4) and 4 (missing the coding sequences of exons 5, 6 and 7) lack part of the NAD binding domain but contained the entire active site. Variant 5 (missing the coding sequence of exons 4 and 7) lacks a large part of the NAD binding domain and the entire active site. Native polyacrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from yak testes and bovine spermatozoa. This study suggests the alternative splicing of LDH-C is ubiquitous in bovine testes and might be involved in regulation of LDH-C expression. The findings also help to elucidate the functions of LDH-C.

Chromosomal aberrations of malignant pleural effusions of lung adenocarcinoma: Different cytogenetic changes are correlated with genders and smoking habits

Chromosomal aberrations of malignant cells from pleural effusions of 31 cases of lung adenocarcinoma were analyzed. Pooled CGH results showed frequent amplifications on chromosome arms 1p (22.6%), 1q (35.5%), 2q (25.8%), 3q (38.7%), 4q (41.9%), 5p (41.9%), 5q (51.6%), 6p (19.4%), 6q (25.8%), 7p (41.9%), 7q (35.5%), 8q (32.3%), 12q (38.7%), 13q (22.6%), 14q (35.5%), 17q (19.4%), Xp (22.6%), and Xq (38.7%). Frequent deletions were found on 1p (19.4%), 3p (16.1%), 4q (16.1%), 8p (25.8%), 9p (22.6%), 9q (29.0%), 10q (22.6%), 13q (22.6%), 16p (19.4%), 16q (22.6%), 17p (29.0%), 18q (16.1%), 19p (41.9%), 19q (32.3%), 20p (19.4%) and 22q (29%). These genomic changes were generally found consistent with previous reports of CGH analysis of primary tumors of lung adenocarcinoma. Loss of 19q and 22q were more frequently found in our studies (32.3% and 29.0%, respectively) than studies of primary tumors (less than 7% for both genetic changes). Gain of 11p, although not a frequent finding, was relatively more common in this (16%) than other studies (range, 2.9-11.8%). Interestingly, occurrences of 3p loss and 11p gain were higher in smokers than non-smokers, and deletion of 3p and increased copy number of 11p and Xp appeared more often in male than female patients. Among 17 male patients, gain of chromosomal 11p was a frequent aberration in tumors of smokers, while gain of Xp was more easily found in tumors of non-smokers. One candidate gene located within 11p15, lactate dehydrogenase C (LDHC), was selected for further study. Three cases with 11p gain had amplified FISH signals of LDHC. Also tumors from smokers or male had significantly higher transcript level of LDHC than non-smokers or female, respectively. The results demonstrate that different cytogenetic changes of malignant pleural effusions from lung adenocarcinoma are correlated with genders and smoking habits. The role of LDHC in the carcinogenesis of smoking-related lung adenocarcinoma, especially in male patients with pleural effusions, deserves further investigations.

CXCR3 is inolved in tubulointerstitial injury in human glomerulonephritis

Chromosomal aberrations of malignant cells from pleural effusions of 31 cases of lung adenocarcinoma were analyzed. Pooled CGH results showed frequent amplifications on chromosome arms 1p (22.6%), 1q (35.5%), 2q (25.8%), 3q (38.7%), 4q (41.9%), 5p (41.9%), 5q (51.6%), 6p (19.4%), 6q (25.8%), 7p (41.9%), 7q (35.5%), 8q (32.3%), 12q (38.7%), 13q (22.6%), 14q (35.5%), 17q (19.4%), Xp (22.6%), and Xq (38.7%). Frequent deletions were found on 1p (19.4%), 3p (16.1%), 4q (16.1%), 8p (25.8%), 9p (22.6%), 9q (29.0%), 10q (22.6%), 13q (22.6%), 16p (19.4%), 16q (22.6%), 17p (29.0%), 18q (16.1%), 19p (41.9%), 19q (32.3%), 20p (19.4%) and 22q (29%). These genomic changes were generally found consistent with previous reports of CGH analysis of primary tumors of lung adenocarcinoma. Loss of 19q and 22q were more frequently found in our studies (32.3% and 29.0%, respectively) than studies of primary tumors (less than 7% for both genetic changes). Gain of 11p, although not a frequent finding, was relatively more common in this (16%) than other studies (range, 2.9–11.8%). Interestingly, occurrences of 3p loss and 11p gain were higher in smokers than non-smokers, and deletion of 3p and increased copy number of 11p and Xp appeared more often in male than female patients. Among 17 male patients, gain of chromosomal 11p was a frequent aberration in tumors of smokers, while gain of Xp was more easily found in tumors of non-smokers. One candidate gene located within 11p15, lactate dehydrogenase C (LDHC), was selected for further study. Three cases with 11p gain had amplified FISH signals of LDHC. Also tumors from smokers or male had significantly higher transcript level of LDHC than non-smokers or female, respectively. The results demonstrate that different cytogenetic changes of malignant pleural effusions from lung adenocarcinoma are correlated with genders and smoking habits. The role of LDHC in the carcinogenesis of smoking-related lung adenocarcinoma, especially in male patients with pleural effusions, deserves further investigations.

Vitamin A: Overlapping Delivery Pathways to Tissues from the Circulation

Although retinol bound to retinol-binding protein (RBP) is the most abundant retinoid form present in the circulations of humans and most mammals, other retinoid and proretinoid forms are also present in the blood. We are interested in understanding to what extent each of these circulating retinoid forms contributes towards retinoid actions within cells and tissues. Here we report two studies focused on this question. First, we examined retinoid transport and storage in RBP-deficient mice that lack circulating RBP. These mice under normal laboratory conditions are phenotypically normal except for a visual impairment early in life that is corrected if the mice are maintained on a vitamin A-sufficient diet throughout life. The RBP-deficient mice take up vitamin A from the diet into most tissues at least as well as wild type mice. Compared to wild type mice, mice lacking RBP accumulate excess vitamin A in the liver, since there is no RBP to facilitate mobilization of stored retinol from hepatic stores. In a second study, we explored in vitro the actions of carotene cleavage enzyme (CCE) in facilitating beta-carotene cleavage to retinoid in the testis. CCE is most highly expressed in the testis. Pull-down experiments coupled with MALDI-MS analysis showed that mouse testis CCE is able to interact with the testis-specific lactate dehydrogenase-C (LDH-C) isoform. This may suggest that CCE and LDH-C act in concert to catalyze beta-carotene cleavage.

Alteration Of Cyclic Adenosine 3’,5’-Monophosphate Signaling In Rat Testicular Cells After Spinal Cord Injury

Introduction: Earlier studies demonstrated that the effects of spinal cord injury (SCI) on spermatogenesis were associated with altered Sertoli cell responses to treatment with follicle-stimulating hormone (FSH) and/or testosterone (T). Because of the importance of the cyclic adenosine 3’,5’-monophosphate (cAMP) signal pathway in hormonal actions on Sertoli cells and spermatogenesis, the purpose of this study was to determine whether cAMP signaling in testicular cells is altered after SCI.Methods: Rats with SCI were treated with FSH, T, or FSH + T for 7 or 14 days. Northern blot cDNA hybridization was used to measure testicular levels of Sertoli and germ cell-specific transcripts encoded by genes that contain cAMP responsive element (CRE) and/or steroid hormone responsive element (HRE). Cellular distribution of CRE modulator (CREM) was determined by immunohistochemistry.Results: Treatment of sham control rats with FSH or T + FSH for 2 weeks resulted in decreases in mRNAs for CREM and CRE binding protein (CREB). Concomitantly, levels of mRNA for Sertoli cell inhibin a and germ cell-specific protamine 1 (Pm- 1), transition protein 2 (TP-2), and lactate dehydrogenase C (LDHC) were all reduced. In contrast, identical FSH and/or T treatments resulted in increases in levels of CREM and CREB mRNAs in the testes of SCI rats; these effects were associated with similar changes in mRNAs for inhibin α, Pm- 1, TP-2, and LDHC. The effects of SCion CREM expression were corroborated by similar changes in its distribution in testicular cells.Conclusion: SCI is associated with changes in FSH and/or T regulation of cAMP/CRE and HRE signaling in testicular cells. These effects may mediate the effects of SCion spermatogenesis.} Spinal Cord Med. 2003;26:69–78

Long-lasting effects of lindane on mouse spermatogenesis induced by in utero exposure

Long-lasting effects on mouse spermatogenesis induced by prenatal exposure to the insecticide lindane have been investigated by conventional reproductive endpoints complemented by the flow cytometric (FCM) DNA content analysis of testis cells and by the Sperm Chromatin Structure Assay (SCSA). Two lindane dose levels, 15 and 25 mg/kg bw, and diethylstilboestrol (DES, 10 μg/kg bw) as positive control, were administered daily by gavage to pregnant CD1 mice on gestation days (GD) 9–16. Reproductive endpoints were evaluated on F1 male mice on postnatal day (PND) 60; additionally, animals treated with lindane 25 mg/kg per day and DES were examined on PND 100 to evaluate the possible reversibility of the effects. On PND 60, lindane and DES caused a reduction in the sperm head count and concentration, with recovery in older lindane 25 mg/kg per day animals (PND 100). By contrast, the DES group exhibited a greater reduction in the sperm head count on PND 100 than on PND 60. Changes in biochemical parameters in the testes, lactate dehydrogenase-C 4 (LDH-C 4), and sorbitol dehydrogenase (SDH) activities, were also observed in adult treated F1 mice. Furthermore on PND 60, the FCM analysis revealed changes in the pattern of testicular germ cell distribution, especially in the haploid subcompartment, in the lindane 25 mg/kg per day group. A dose-dependent increase in chromatin abnormalities of the epididymal sperm was also shown by SCSA. These changes recovered on PND 100. Preliminary qualitative examination did not reveal any significant difference in the structure of testicular tissue; however, there were suggestions of a moderate increase in number and size of Leydig cells in both DES- and lindane-treated animals. The partial reversibility of these effects and the lack of structural modification of the testicular tissue as evidenced by histopathologic assessment suggest a functional impairment of sperm production and maturation, possibly associated with changes induced by lindane on factors affecting intratesticular steroidogenesis.

CDNA cloning of pig testicular lactate dehydrogenase-C, thermal stability of the expressed enzyme, and polymorphism among strains

Pig testicular lactate dehydrogenase-C (LDHC) cDNA was cloned and sequenced. The deduced sequence of 332 amino acids from pig LDHC shows 73% and 67% identity with that of pig LDHA (muscle) and LDHB (heart) respectively, whereas pig LDHA and LDHB isozymes shows 74% sequence identity. Pig and mouse LDHC cDNAs were subcloned into bacterial expression vector, and the expressed pig LDHC isozyme was shown to be as thermally stable as mouse LDHC isozyme. Pig genomic DNAs from Chinese Meishan, English Yorkshire, Danish Landrace and American Duroc were shown to exhibit polymorphic sites for restriction enzymes EcoRI, BamHI and PstI.

Cell-type-specific transcription factor interactions with cis-elements present in the mouse LDH/C proximal promoter region

The major aim of this study was to identify cell-type-specific transcription factors present in somatic and germ cells that interact at cis-elements within the lactate dehydrogenase C (LDH/C) proximal promoter region. Electrophoretic mobility shift assays (EMSAs) were performed using wild-type and mutagenized GC-box LDH/C double-stranded (ds) oligonucleotides to probe nuclear extracts from primary germ cell populations, and an LDH/C nonexpressing cell line (Hela cells). Significant differences were observed in the composition of nuclear transcription factors bound to cis-elements within the LDH/C proximal promoter region in somatic versus germ cells. Using the wild-type LDH/C ds oligonucleotide to probe germ cell nuclear extracts, we consistently observed two DNA-protein complexes, one formed by Sp1 proteins and one formed by another uncharacterized germ cell nuclear transcription factor. Methylation interference analysis identified a unique 5'G residue within the GC-box motif as an integral part of the cis-element recognized by this germ cell nuclear transcription factor. In contrast, at least six major DNA-protein complexes were observed in EMSA studies using Hela cell nuclear extracts probed with the LDH/C wild-type oligonucleotide. In these EMSA studies, supershift analyses with antibodies recognizing the somatic Sp1 protein and the GC-box mutated LDH/C ds oligonucleotide demonstrated that only one of the six complexes formed was due to binding of Sp1 proteins. These in vitro EMSA studies have identified several somatic nuclear factors and at least one other germ cell nuclear transcription factor, which in addition to members of the Sp1 protein family, recognized cis-elements within the LDH/C proximal promoter region. Observations presented in this report strongly suggest that these somatic and germ cell nuclear transcription factors play central roles in modulating transactivation or suppression of the mouse LDH/C subunit gene in the respective cell types.

Human testis-specific lactate dehydrogenase-C promoter drives overexpression of mouse lactate dehydrogenase-1 cDNA in testes of transgenic mice

The three isozymes of lactate dehydrogenase, each encoded by a separate gene, are developmentally regulated and differentially expressed in tissue-specific patterns. The lactate dehydrogenase-C (LDHC, mouse Ldh3) gene is temporally expressed exclusively in the germ line during spermatogenesis, whereas lactate dehydrogenase-A (LDHA, mouse Ldh1) and B (LDHB, mouse Ldh2) genes are active in somatic tissues. To determine, therefore, whether overexpression of Ldh1 would perturb spermatogenesis, we constructed a transgene in which a sequence from the promoter region of human LDHC was coupled with mouse Ldh1 cDNA. Among nine (three males, six females) founder lines that were identified as being transgenic for the construct, one male transmitted the gene through its germ line. Homo- and heterotetramers containing the LDH-A subunit were detected in homogenates of testes from transgenic animals. We conclude that the human LDHC promoter contains the necessary regulatory sequence(s) for specific expression of mouse Ldh1 as a transgene during spermatogenesis. The fertility of the founder animal was not impaired.

Lactic dehydrogenase-C4 activity in seminal plasma and male infertility

To determine the lactate dehydrogenase C4 (LDH-C4) isoenzyme activity in seminal fluid with the purpose of studying its clinical use as marker of seminiferous epithelium activity. A total of 224 semen samples were classified into the following four groups: [1] men with proven fertility (n = 27); [2] oligozoospermic patients (n = 34); [3] normozoospermic subjects with infertile couple (n = 120); and [4] vasectomized patients (n = 22). It was found that LDH-C4 activity discriminates well between the infertile groups (groups 2 and 3) with the fertile group (group 1). We correlated LDH-C4 activity with the different parameters of the seminogram, the best correlation being obtained with the sperm count. Lactate dehydrogenase-C4 activity:sperm count ratio showed a high correlation with the "vital" variables of spermatozoon. The logistic regression analysis gave a mathematical model that only includes viability and percent C4. This model accurately classifies 88.4% of the cases. In vasectomized individuals, we proved an earlier disappearance of the LDH-C4 activity, than that of the sperm count. Our results confirm the clinical utility of this parameter as a good marker for both germinal activity and spermatozoid quality.

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-β-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome c t and lactate dehydrogenase-C 4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.