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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 following meiosis to its final localization in and on the principle piece of the sperm tail. After several decades of biological, biochemical and genetic investigations we now know that the lactate dehydrogenase isozymes are ubiquitous in vertebrates, developmentally regulated, tissue and cell specific and multi-functional. 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.

The testis chromatin undergoes profound structural alterations and functional changes during spermatogenesis. Changes in DNA methylation have been correlated with gene expression in a number of systems, but the relationship between methylation and gene expression for testicular genes is unclear. To address this question, DNA methylation patterns and mRNA expression for a somatic form of lactate dehydrogenase (LDH), LDH-A, were compared with those of the testis-specific form, LDH-C, in preparations from testes of prepubertal and sexually mature mice, from isolated testicular cells, and from somatic tissues. At specific sites, LDH-A was less methylated in adult testis than in spleen DNA; the decreased methylation in the testicular DNA occurred as early as type A spermatogonia. In contrast, DNA methylation patterns for LDH-C did not differ between spleen and testis DNAs. In Northern blots, the levels of LDH-A transcripts were low in total testis RNA obtained from 6-12-day-old mice, and in type A and B spermatogonia from 8-day-old mice. LDH-A mRNA levels increased gradually in testes from 16-45-day-old mice. LDH-C transcripts were first detectable in the testes of 12-day-old mice and increased as spermatogenesis proceeded. Both LDH-A and LDH-C mRNA levels were low in preleptotene spermatocytes and leptotene/zygotene spermatocytes and increased substantially in pachytene spermatocytes and round spermatids. Reduced levels of LDH-A and LDH-C mRNAs were found in the residual bodies/cytoplasts fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH.

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.

Members of the transforming growth factor-beta (TGF-beta) superfamily play a variety of important roles in testicular development and function. The tumor suppressor gene, Smad4, is a common mediator of TGF-beta, activin, and bone morphogenetic protein-mediated signaling pathways. To investigate the role of the Smad4 gene during testicular development and function, transgenic mice were generated using a Flag-tagged Smad4 gene driven by 180-bp fragment of the Mullerian inhibiting substance upstream promoter sequence. Three Smad4 transgenic founders (A, B, and G) were detected by Southern blot analysis; line B showed the highest expression of the Smad4 transgene and was further studied. The fertility in F1 generation (B) and F2 generation (BB) of the Smad4 transgenic mice was not impaired. However, in the F3 generation (B2x) all animals were impacted by the overexpression of the Smad4 transgene and two kinds of phenotypes were observed. In one group animals were completely infertile, while in the other group animals were fertile and sired the normal number of pups/litter. These groups are designated as infertile and fertile in the text. Histological evaluation of the testes from the infertile group showed variable degrees of Leydig cell hyperplasia, apoptosis of germ cells, spermatogenic arrest, seminiferous tubule degeneration, and infertility. In the fertile group, there was no apparent change in the histology of the testis except for a slight increase in the number of Leydig cells. Serum follicle-stimulating hormone levels in the adult animals of both groups of Smad4 transgenic male mice were not significantly different from normal littermates; however, testosterone levels in both groups were significantly (P0.05) increased. These results suggest that overexpression of Smad4 leads to testicular abnormalities and infertility supporting the hypothesis that the TGF-beta signaling pathways are carefully orchestrated during testicular development. In the absence of normal levels of Smad4 testicular function is compromised.

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

Four sexually mature male baboons ( Papio sp.) were immunized with a chimeric peptide containing a B-cell epitope of the testis-specific lactate dehydrogenase (LDH-C 4 ) and a promiscuous T-cell epitope of tetanus toxin. LDH-C 4 is the testis-specific isozyme of lactate dehydrogenase, and antibodies to this protein reduce fertility significantly in female nonhuman primates. Animals were immunized on Day 0 and received booster injections at Days 29, 61, and 344 after priming. Serum specific antibodies were determined at regular intervals during the initial 6 months and after the last booster. Testis biopsies were taken at Days 61, 127, and 183 after the primary immunization. Sperm-zona binding was assessed prior to and three times after the last booster. The present study demonstrated that this epitope of LDH-C 4 did not cause autoimmune disease and that sperm from these immunized males had a diminished zona binding capacity. These results suggest that a safe male immunocontraceptive based on development of anti-sperm antibodies may be feasible.

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.

This study developed a baboon in vitro system that allows transport of sperm from a treatment facility to an off-site location for subsequent evaluation of sperm functional capacity. We further described a sperm functional assay that evaluates baboon sperm binding to homologous zona pellucida, a baboon hemizona assay (HZA). Semen samples were collected from baboons via electroejaculation directly into refrigeration transport buffer. Postshipment semen characteristics were analyzed and each specimen prepared for assessment of sperm-zona pellucida interaction. Optimization of the baboon HZA included determination of the relationship between motile sperm concentration and zona pellucida binding. The effect of the sperm activators, caffeine and dbcAMP, on computerized sperm motion characteristics and HZ binding was also determined. A significant motile sperm concentration dependent increase was observed in sperm-zona pellucida binding. Maximal binding was observed at approximately 1-2 million motile sperm/mL. Treatment with the sperm activators, caffeine and dbcAMP, resulted in a significant increase in sperm progressive motility, straightline velocity (VSL), and amplitude of lateral head displacement (ALH), p0.05 and a highly significant increase in curvilinear velocity (VCL), p0.01. Treatment with caffeine and dbcAMP was not an absolute requirement for sperm-zona pellucida binding, inasmuch as binding did occur in the absence of activators. However, treatment with the two activators, caffeine and dbcAMP, resulted in a highly significant increase in HZ binding, p0.0001. This system allows for the short-term maintenance of baboon sperm in a semiquiescent state until stimulation with the activators, caffeine and dbcAMP. It further provides a novel approach to delineating a contraceptive regimen's or agent's (ie, sperm vaccine) impact on specific cellular events occurring in the male gamete during fertilization.

A 39 amino acid peptide consisting of a B-cell epitope of the sperm specific lactate dehydrogenase (LDH-C4) and a promiscuous T-cell epitope of Tetanus toxin (TT) was tested as a contraceptive vaccinogen in female baboons. In a series of three trials significant and reversible suppression of fertility was observed. This synthetic peptide construct is the first sperm antigen proposed for Phase I trials in human subjects.p

The promoter of the mouse lactate dehydrogenase c (Ldh-c) gene was assayed in an in vitro transcription system. Of the nuclear extracts isolated from several mouse tissues, only testis supported transcription from a 710 Ldh-c promoter fragment. Mutation analysis indicated that a canonical TATA box and a 30-bp palindromic sequence are both required for promoter activity. Liver nuclear extract (LN) can reduce transcriptional activity significantly when added to testis nuclear extract (TN). Deletion of the 3' palindromic sequence resulted in a low level of transcriptional activity in LN. Competition with a double-stranded synthetic palindromic oligonucleotide inhibited transcription in TN and rescued a low level of transcription in LN. Our results strongly indicate that the palindromic sequence contains both a negative element for repression in somatic tissues and a positive element for activation of the Ldh-c gene in testis.

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.

Expression of Ldh-a and Ldh-c mRNAs was examined in the rat testis. The mRNA levels of both Ldh-a and Ldh-c increase during testicular maturation. In the adult testis, Ldh-a mRNA is expressed maximally in primary spermatocytes. Comparison of the Ldh-a mRNA expression profile with its translation product suggests that this gene is translationally down-regulated during spermatogenesis.

Sera from patients with known or suspected immunological infertility were used to screen a human testis cDNA library. A total of 59 sera detected 38 unique cDNA inserts of which four were testis specific by Northern blot analyses. One of these is a testis-specific isoform of calpastatin. Five additional clones, although not testis specific, were found to be testis abundant. The number and type of clones identified by these human sera suggests a possible aetiology for immunologic infertility. The testis-specific clones will be further characterized to establish their usefulness as contraceptive vaccine candidates.

Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate that transgene expression is repressed in all somatic tissues examined even when heavy metals are administered. Nuclear run-on assays indicate that failure of expression in the liver (in which the metallothionein I promoter is highly active) occurs at the transcriptional level. In contrast, the transgene mRNA is transcribed in male germ cells and is developmentally regulated during spermatogenesis. Examination of the transgene methylation status reveals that expression is inversely correlated with hypermethylation of the locus; all CpG dinucleotides examined in the promoter region were found to be fully methylated in kidney and liver but were undermethylated in testis. Since methylation of the murine metallothionein I promoter is sufficient to inhibit its activity, it is likely that suppression of the transgene in somatic tissues is mediated by methylation.

Publisher Summary This chapter discusses the use of molecular modeling to delineate B-cell and T-cell epitopes of human sperm-specific LDH-C4. The sperm-specific isozyme of lactate dehydrogenase (LDH-C4) has been purified from mouse testes, and has been shown to suppress fertility in female mice, rabbits, and baboons. The results of a study described in the chapter have suggested that LDH-C4 might form the basis of an immunocontraceptive vaccine that would not rely on disrupting the endocrine milieu of the recipient. A combination of immunoaffinity purification of tryptic peptides and epitope mapping using synthetic peptides has identified several peptides that can elicit LDH-C4 reactive antibodies. Molecular modeling has been used to localize these peptides within the crystal structure of the protein. This study used computer-assisted secondary structure analyses together with experience with mouse LDH-C4 to predict an immunodominant B-cell epitope of human LDH-C4. This peptide, when conjugated to diphtheria toxoid (DT-hC9-20), was highly immunogenic in rabbits. In addition, similar analyses enabled to identify a T-cell epitope of human LDH-C4 that could substitute for the normally required carrier molecule in eliciting an immune response to hC9-20.

Open-air hydrophobic interaction chromatography with alkyl carbon functional groups coupled to agarose beads has been used to desalt large volumes of ammonium sulfate-fractionated bacterial cell lysates. The protein of interest can be simultaneously desalted and concentrated in less than 4 h, and the yield is significantly better than that obtained by the standard technique of precipitation, centrifugation, and dialysis.

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

Current molecular biology technology provides an increased level of sensitivity for the analysis of sperm structure and function. Excellent laboratory manuals are available that provide familiarity with the few basic concepts required for mastery of these techniques (Maniatis et al.,1982; Ausubel et al.,1987; Berger and Kimmel, 1987). Therefore, this chapter does not detail such laboratory procedures but rather attempts to review the recent literature in which sperm function has been studied by using the tools of molecular biology. Basically, I focus on the investigations that involve DNA and RNA isolation, cloning, and analysis to shed light on the mechanisms whereby the spermatozoan acquires the unique properties necessary to achieve success in accomplishing its primary function, fertilization of the egg. This overview is intended to be instructive rather than exhaustive, and I hope it will provide a resource with which to initiate further study of fertilization.

The goals of this study were to determine the site at which fertility is impaired in mice immunized with LDH-C4 and to determine whether immunization of both males and females would have a greater antifertility effect than immunization of one sex alone. Mice were immunized with LDH-C4 in two systemic doses in Freund's adjuvants and two gastric doses in bicarbonate buffer. The presence of anti-LDH-C4 antibodies in uterine fluid was confirmed. Male and female mice were assigned to four blocks in which either the males, the females, both, or neither were immunized. Oviducts were viewed directly 1 h after mating for the presence of sperm. No significant effect of immunization on sperm transport to the oviduct could be demonstrated. Fertilization was evaluated 4 h after mating. It was found that immunization of males, but not females, impaired fertilization (19.6% versus 50.8% of the oocytes penetrated in 17 and 19 females, respectively). Orchitis was found histologically in 43.8% of the immunized males and 10% of the control males.

Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.

The orientation of the molecular 2-fold axes of mouse testicular lactate dehydrogenase (LDHase ‡ -C 4 ) was determined by a rotation function search. These were subsequently identified with the P , Q , and R axes of dogfish LDHase-M 4 . Since LDHase-C 4 crystallized with one molecule in a triclinic cell, the origin of the co-ordinate system was arbitrarily fixed at the molecular center. Structure factor phases were derived from an appropriately oriented dogfish apo LDHase-M 4 phasing model and combined with the observed structure amplitudes to produce a hybrid electron density map. Density points related by the molecular 222 point symmetry were averaged so as to remove the bias of the phasing model. At 7.5 A resolution, the structure of the crystallized mouse LDHase-C 4 was found to be without coenzyme, with a conformation indistinguishable from that of dogfish apo LDHase-M 4 .

Human sera from the WHO Reference Bank were tested by a liquid phase radioimmunoassay for antibodies to the sperm-specific form of lactate dehydrogenase (LDH-C4; LDH-X). Modestly elevated levels of antibodies were detected in about 10% of infertile males and females and in about 20% of vasectomized males, as well as in a few cases in other categories. The occurrence of this anti-LDH-C4 activity was without overt correlation with other anti-sperm antibody assays. The primary advantage of the radioimmunometric assay is its absolute specificity for a sperm antigen. These findings lend support to contraceptive vaccine development based on LDH-C4 antigenicity.

Lactate dehydrogenase C, (LDH-C4) is an antigenic protein that occurs only in spermatozoa and the mature testis. The antibody-combining sites of this enzyme were mapped by measuring the binding of anti-LDH-C4 by isolated peptides. Pure mouse LDH-C4 was digested with trypsin, and the resulting fragments were fractionated by reverse-phase high-pressure liquid chromatography. Rabbit anti-mouse LDH-C4 bound to 13 pure peptides. Amino acid compositions and partial or complete sequencing by the Edman degradation was used to identify eight of these fragments in the complete structure of the molecule. The relationship between structure and antigenicity of these peptides is discussed in detail. These data fit best to the domain model of protein antigenicity. This antigenic map of LDH-C4 will be useful in the design of a synthetic contraceptive vaccine.

The immunological properties of a panel of synthetic peptides that represent the most accessible and mobile segments of the lactate dehydrogenase (LDH)-C4 molecule were characterized. Peptides corresponding to mouse LDH-C4 amino acid sequences: 1-14b, 5-15, 49-58, 97-110, 211-220, 231-243, 274-286, 304-316, and 318-330 were synthesized and compared in terms of binding antibodies raised in rabbits against the intact protein. Six of these sequences were covalently coupled to diphtheria toxoid and used to immunize groups of rabbits. LDH-C4-specific antibodies were detectable in immune sera by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation assays. The immunogenicity of the mouse LDH-C4 peptides in rabbits could be ranked in the following order: 5-15, 304-316 greater than 211-220, 274-286 greater than 49-58, 97-110. The immunological properties of these short synthetic peptides did not correlate with features of the mouse LDH-C4 structure except that the most active sequences appeared to be those that differed from the somatic isozymes to the greatest extent. These results have direct bearing on the selection of immunogenic LDH-C4 peptides for contraceptive vaccine studies in humans and non-human model systems.

LDH-X from spermatozoa can be demonstrated within the perivitelline space and zona pellucida of the preimplantation mouse embryo. Peroxidase reaction product is deposited in these areas of blastocysts incubated with rabbit anti-LDH-X IgG and then with peroxidase labelled goat anti-rabbit IgG. Unfertilized ova do not bind antibody to LDH-X.

Immunity to sperm-specific lactate dehydrogenase C4 (LDH-C4) results in reduction of fertility in females. Stimulation of a local mucosal immune response to LDH-C4 in the reproductive tract would guarantee the presence of antibodies at the site of fertilization, which should enhance suppression of fertility. After intrauterine immunization with LDH-C4, SJL/J female mice secrete immunoglobulin (Ig)A antibodies specific for LDH-C4 into their uterine fluids. Furthermore, these animals demonstrate a lower pregnancy rate than controls receiving an intrauterine immunization without LDH-C4. Thus, induction of a local immune response is an effective alternative to systemic immunization for administering a contraceptive vaccine.

The sequence and structure of human testis-specific L-lactate dehydrogenase [LDHC4, LDHX; (L)-lactate: NAD+ oxidoreductase, EC 1.1.1.27] has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC4 is as different from rodent LDHC4 (73% homology) as it is from human LDHA4 (76% homology) and porcine LDHB4 (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC4 and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete antigenic determinants of mouse and human LDHC4 reveals significant differences. Knowledge of the human LDHC4 sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.

Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) that contain antigenic sequences of the native protein have been identified. The present study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing two of these sequences. Rabbits were immunized with peptides designated MC515 and MC211320, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are first absorbed with the native LDH-C4 molecule, significant inhibition of binding to sperm results. Antisera to both DT-MC5,5 and DT-MC211220 hind to human sperm with similar but weaker patterns than seen with mouse sperm. These data indicate that the immune response to synthetic peptides containing antigenic sequences of LDH-C4 includes antibodies that specifically bind to this enzyme on the surface of sperm. In addition, there are shared antigenic sequences between mouse and human LDH-C4, including the MC,15 and MC211.220 peptides.

The atomic structure of mouse testicular apolactate dehydrogenase C4 has been refined to 3.0-A resolution yielding a final crystallographic R-factor of 0.256. Comparison with the refined structure of dogfish apolactate dehydrogenase A4 shows that equivalent secondary structure elements are essentially in the same position relative to the molecular 2-fold axes, except for the helices alpha D, alpha E, and alpha 2G in the vicinity of the active center, and the carboxyl-terminal helix alpha H. The positions of antigenic peptides correlate best with surface accessibilities of the monomer rather than of the full tetrameric molecule.

LDH isoenzyme patterns were determined in prostatic fluid from 286 patients with a prostate gland judged to be normal, the site of benign hyperplasia on clinical evidence or the site of benign hyperplasia or carcinoma on the basis of histologic evidence. Fluid from 12 of 15 patients (80 per cent) with carcinoma representing all stages and grades of disease showed an LDH-V/I ratio of more than 3. This ratio was exceeded in only 6 of 57 (11.8 per cent) and 14 of 97 (14.4 per cent) patients in whom the diagnosis of benign prostatic hyperplasia without evidence of infection was established histologically or clinically, respectively. An LDH-V/I ratio exceeding 3 was seen commonly in patients with a diagnosis of benign prostatic hyperplasia with a recent history suggesting infection and more than 10 WBCs per high power field on microscopic examination of the prostatic fluid. These observations suggest that an LDH-V/I ratio exceeding 3 in the prostatic fluid in the absence of a history of infection and more than 10 WBCs per high power field on microscopic examination should be regarded as an indication of a high risk of the presence or development of a malignancy of the prostate. The results obtained also support the concept that, in the presence of malignancy, expressed prostatic fluid provides an adequate sample of cells with altered metabolism.

Female rabbits were immunized systemically with purified sperm specific lactate dehydrogenase (LDH-C4). Some of these were treated additionally by depositing sperm (as a source of LDH-C4) directly into various regions of their reproductive tract. During these treatments, reproductive tract fluids were collected and the amount of immunoglobulin present that was specific for LDH-C4 was determined. In immunized females without sperm, the concentration of anti-LDH-C4 in follicular fluid was the same as in serum, whereas in oviducal fluid, the amount ranged from 3-27% of serum levels. Sperm deposition in the tract provoked a local increase in fluid levels of anti-LDH-C4. Thus rises in uterine or oviducal fluid titers occurred following the direct introduction of sperm into the uterine horns or oviducts, respectively. Although IgA was present in reproductive tract fluids, the anti-LDH-C4 was always of the IgG class. These reproductive tract immune responses have direct bearing on the reduction in fertility previously demonstrated for rabbits immunized against LDH-C4.

Lactate dehydrogenase C4 (LDH-C4) is an antigenic protein found only in spermatozoa and the mature testis. Synthetic peptides containing the amino acid sequences of the C-subunit, designated MC5-15, MC97-110 and MC211-220, were each conjugated to the carrier proteins diphtheria toxoid or bovine serum albumin. Rabbits immunized with these peptide-carrier conjugates produced antibodies to the peptide that cross-reacted with native LDH-C4. These data support our map that identifies antigenic domains of LDH-C4. Such synthetic peptides will be useful in the design of a contraceptive vaccine.

SJL/J female mice were tested for a cell-mediated cytotoxic response to sperm-specific lactate dehydrogenase C4 (LDH-C4). We demonstrated this response with a 51chromium release assay. Mouse LDH-C4 was coupled to EL-4 tumor cells. These cells were then labeled with 51Cr and mixed with splenocytes from LDH-C4-immune and -nonimmune mice. Specific lysis of the tumor cells by splenocytes from LDH-C4-immune mice was detected at 7 days and 14 days following a single footpad immunization. Since LDH-C4 is present on the surface of sperm, these results support the suggestion that cytotoxic removal of sperm from the female reproductive tract is one of the mechanisms whereby fertility is reduced following immunization with this enzyme.

Temperature and starvation were found to be factors which affected the PPP dehydrogenase activities in brook trout liver. Fish acclimated at 5 °C possessed greater levels of G6PD, H6PD, and 6PGD activity than those fish maintained at 10 or 15 °C. This phenomenon was probably associated with increased lipogenesis during cold acclimation. During starvation hepatic G6PD and 6PGD activities decreased, whereas H6PD activity increased slightly. Upon refeeding, the G6PD level gradually increased, but the “overshoot” in enzyme activity reported in mammalian studies was not observed. When both cold acclimation and starvation were studied simultaneously, regulation by temperature was initially the dominant control factor. After 6 wk at 5 °C, there was no difference in specific activities between starved and fed fish. However, fish maintained at 5 °C for longer than 2 mo did show the normal response to starvation and refeeding. Therefore, regulation of the PPP by temperature appears to be a transitory phenomenon and may be associated with temporary metabolic reorganization in the fish.

A cDNA clone complementary to the mRNA encoding the sperm-specific lactate dehydrogenase, LDHC, has been used to map the LDHC locus to the short arm of human chromosome 11. In situ hybridization data and analysis of mouse/human somatic cell hybrids carrying deletions of human chromosome 11 suggest that the gene is localized at p15.3-p15.5 close to the LDHA gene.