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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 isozyme concept owes its development to Clement Markert (Markert and Appella, 1961) who was one of the first to recognize the significance of multiple molecular forms of enzymes, and since this time, isozymes have been extensively studied or used as markers for normal and abnormal cell function. One of the earliest studied and best examples is the lactate dehydrogenase (LDH) enzyme family which catalyzes the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD+ (Everse and Kaplan, 1973). The first evidence of the presence of multiple forms of LDH was found using the technique of protein electrophoresis (at this time in a starch matrix) elaborated by Smithies (1959) coupled with cytochemical visualization of LDH activity (Allen, 1961; Dewey and Conklin, 1960). We learned that LDH consists of A and B subunits that assemble into homo- or heterotetramers that are distributed in the body in combinations reflecting the metabolic requirements of different tissues and consistent with the catalytic properties of the isozymes. For example, LDHA is most abundant in skeletal muscle where oxygen deficiency from exercise requires glycolysis to satisfy metabolic needs, while LDHB is expressed abundantly in cardiac muscle that is dependent upon aerobic metabolic pathways.
Our entrance into the field in its infancy was when we asked whether a single cell type, the spermatozoan, contained one or multiple forms of lactate dehydrogenase (Goldberg, 1963). That question began this journey with the discovery of LDH-X (now known as LDHC). This unique band corresponding to the homotetramer LDH-C4 was only revealed in testis and spermatozoa and not in other tissues or cells. It was to our knowledge the first testis specific isozyme described (Blanco and Zinkham, 1963; Goldberg, 1963; Goldberg, 1964). Since that time, the question of why testes and sperm need this unique form of LDH remains to be discovered.
We learned that different forms of LDH are the product of 3 different genes: Ldha, Ldhb, and Ldhc which encode A, B and C subunits (Li, 1989). In the human and mouse genomes, Ldha and Ldhc genes are located in tandem on chromosomes 11 and 7 respectively (Edwards et al, 1989), and Ldhb gene on chromosomes 12 (human) and 6 (mouse) (Takeno and Li, 1989). In terms of gene evolution, the current model is that in vertebrate evolution, the original Ldh gene soon duplicated, giving rise to Ldha and Ldhb genes (Markert et al, 1975). The Ldhc gene then arose from a second independent gene duplication event, by duplication of the Ldhb gene in fish and columbid birds (Mannen et al, 1997; Zinkham et al, 1969), and by duplication of the Ldha gene during mammalian evolution (Li et al, 1983b; Li et al, 2002; Millan et al, 1987). Divergences in the structure, function and localization of the protein and in gene regulation must have conferred a genetic advantage since LDHC was conserved from its appearance throughout evolution. Finally, direct evidence of the importance of LDHC was demonstrated by the generation of an Ldhc knockout mouse model, but even though this model gave us some answers, it also raised new questions. LDHC still did not reveal all of its mysteries.
The LDH isozymes differ in net charge which gave us the ability to electrophoretically distinguish them. In terms of activity, earlier studies showed that the kinetics of catalysis were different between the LDH isozymes (Goldberg, 1972). Mouse LDH-C4 structure was characterized by crystallography (Goldberg, 1972; Musick and Rossmann, 1979) and complete sequences for LDH-C4 became available (Pan et al, 1983). These structural studies confirmed that LDHC is homologous to the LDHA and LDHB subunits (72.5% and 75.3% identity with LDHA, and 64.5% and 69.8% identity with LDHB respectively in mouse and human). However, LDH-C4 displays substitutions in amino acid sequence and some modifications in three-dimensional structure which gives LDH-C4 unique structural (Hogrefe et al, 1987) and functional properties (Blanco et al, 1976; Li et al, 1983a). LDH-C4 has high thermostability (Goldberg, 1972) and has a broader range of substrates; for example LDH-C4 is able to metabolize α-hydroxyvalerate (Allen, 1961; Blanco et al, 1976; Goldberg, 1965). However the differences between LDH isozymes are not in the orders of magnitude sufficient to confer advantage to one of the three relative to germ cell metabolism (Goldberg, 1972).
We showed the presence of LDHC protein in the testis using gel electrophoretic (Goldberg and Hawtrey, 1967) and immunohistochemical (Hintz and Goldberg, 1977) techniques (Figure 1). By using isolated fractions of mouse germ cells, the protein was detected first in preleptotene spermatocytes and was abundant in spermatids and spermatozoa (Li et al, 1989). LDHA also is present in pachytene spermatocytes, but the majority of LDH activity in male germ cells is from LDH-C4 (Erickson et al, 1975a; Li et al, 1989; Odet et al, 2008), and heterotetramers containing both A and C subunits are not detected in murine or human testes (Goldberg and Hawtrey, 1967; Li et al, 1989).
Our initial experiments suggested that LDHC was the only LDH isozyme present in spermatozoa (Goldberg, 1965). However, recent studies have shown that LDHA also is present in spermatozoa (Krisfalusi et al, 2006; Odet et al, 2008; Sleight et al, 2005). However, LDH-C4 is responsible for more than 80% of the total LDH activity in mouse spermatozoa (Odet et al, 2008).
Originally, LDH-C4 was considered specific to male germ cells (Blanco and Zinkham, 1963; Goldberg, 1963), while LDHB was described as the predominant LDH isozyme in oocytes (Roller et al, 1989). However, Coonrod et al. (2006) showed recently that LDHC protein can be detected by IHC in germinal-vesicle stage oocytes and fertilized eggs, and persists to the preimplantation blastocyst. However the enzymatic activity of LDH-C4 was not detectable in egg extracts, presumably because of its low level, and Ldhc transcript levels are substantially lower in oocytes (102 per million transcripts) than in whole testis (2844 per million transcripts) (UniGene build #168, UniGene accession number for Ldhc: Mm.16563). The function(s) of LDH-C4 during oogenesis, oocyte maturation, or early development is (are) still unclear. We do know that Ldhc null females are fertile (Odet et al, 2008).
Subcellular localization of a protein results in exposure to a specific microenvironment that might affect its function. Immunohistochemical studies have shown that LDHC is present in the cytosol of spermatocytes and spermatids, and in the principal piece of spermatozoa (Alvarez and Storey, 1984; Blanco et al, 1976; Burgos et al, 1995; Wheat and Goldberg, 1984) (Figure 2). In the sperm principal piece, LDHA but not LDHC was bound tightly to the fibrous sheath (Krisfalusi et al, 2006). This difference of intracellular localization between LDHA and LDHC might explain the absence of in vivo heterotetramers of A-C whereas different tetramer combinations were seen in vitro (Goldberg, 1965).
The relative hydrophobicity of LDHC (Millan et al, 1987) suggested that LDHC might be located close to the plasma membrane. Localization of LDHC in the cortical region in oocytes and preimplantation embryos (pronuclear zygotes to blastocysts) was observed (Coonrod et al, 2006). This is difficult to determine in sperm because of the small volume of the cytosol. However, by isolating different subcellular fractions of rabbit spermatozoa, Alvarez and Storey (1984) found 10 % of LDHC in the plasma membrane fraction. Consistent with this result, LDHC was also found at the surface of the spermatozoa (Beyler et al, 1985; Erickson et al, 1975a) possibly because of the high diffusibility of LDHC (Zinkham et al, 1964). However, the presence of extracellular LDHC might also have resulted from the release of LDHC during the isolation procedure from damaged cells.
Several studies have provided evidence that LDHC might be localized in the matrix of sperm type mitochondria (STM) (Alvarez and Storey, 1984; Burgos et al, 1995; Montamat et al, 1988). However, most of these data were based on indirect methods of detection and because of the high quantity of LDHC proteins present in spermatozoa, cross contamination between subcellular fractions is quite possible. Immunohistofluorescence studies with antibodies produced with peptides designated MC5-15 and MC211-220 (Beyler et al, 1985) detected strong signals in the principal piece of the spermatozoa (Duan and Goldberg, 2003; Goldberg, 1975; Odet et al, 2008), but only a weak signal in the midpiece region where the STM are localized (Figure 2). This weak signal was also found in Ldhc null sperm suggesting that it was only background (Odet, unpublished observation). However, LDHC localized in STM might have been lost due to the permeabilization and other treatments during preparation of the sperm smear. High resolution localization experiments using gold immunolabeling, a specific antibody and spermatozoa from Ldhc null mice as negative controls are needed to resolve this controversy. However, it is worth mentioning that mitochondrial activity as measured by JC-1 staining and oxygen consumption (in medium with lactate as the only substrate) is normal in Ldhc null spermatozoa (Odet, unpublished observations); therefore, even if a small amount of LDHC is present in the matrix of STM, we were unable to detect any function for LDHC in the respiration process.
The level of Ldhc transcripts is higher in whole testis (2844 per million transcript; Unigene Mm.16563) than Ldha transcripts (584 per million; Mm.29324) and Ldhb transcripts (160 per million; Mm.9745). This is consistent with LDHC being the major LDH in germ cells and with germ cells being the major component of adult testis (>80%, mainly in the meiotic and postmeiotic phase). Like the protein, Ldhc transcripts were found in mouse testis only after 11 days post partum with the appearance of spermatocytes and then further increased with the appearance of spermatids (Alcivar et al, 1991; Goldberg, 1990; Thomas et al, 1990). This specific time course expression pattern of Ldhc was confirmed by microarray studies in the Griswold laboratory (Shima et al, 2004) (Figure 3). By using isolated testicular cell types they were also able to confirm that the Ldhc gene is expressed first in leptotene-zygotene spermatocytes with the highest level of Ldhc mRNA being present in spermatids (Figure 4). While Ldhb and Ldha transcripts were found in germ cells, they were at considerably lower levels than Ldhc. Ldhb was found mostly in spermatogonia.
The human LDHC and murine Ldhc genes were cloned and sequenced (Cooker et al, 1993; Millan et al, 1987) and their promoter sequences defined (Zhou et al, 1994). Transcriptional analysis of these and other genes expressed in germ cells is compromised by the lack of a reliable cell culture system. Nevertheless, we have made progress in elucidating the regulation of Ldhc gene expression. Our approaches, construction of transgenic mice (Li et al, 1998; Markert et al, 1998), transfection of heterologous cell systems and injection of expression plasmids directly into seminiferous tubules via the rete testes have defined both cis and trans acting factors responsible for Ldhc transcription (Kroft et al, 2003; Tang and Goldberg, 2009; Tang et al, 2008). We confirmed and extended prior reports that transcription of the Ldhc gene begins with the onset of meiosis in male germ cells. The potential cis regulatory elements identified in studies using transgenic animals include a palindrome (-21 to +10) (PAL), GC box (-70 to -65) and CRE sites (-53 to -49, -39 to -35). We described a functional role for these sequences by expression of mutated transgenes in vivo. Our results (Tang et al, 2008) revealed for the first time that mutation of the GC box did not abolish promoter activity which remained testis-specific. Mutation of GC box or CRE sites resulted in a 73% and 74% reduction in promoter activity respectively in transient transfection of germ cells and in vivo by electroporation; the combination of GC box and CRE site mutations eliminated promoter activity whereas mutation of PAL had no effect. Therefore, we concluded that simultaneous occupancy of the GC box and CRE sites in the core promoter was necessary for full expression of Ldhc in the testis. Our working hypothesis proposes a partnership between the Sp and CREB factors mediated by an activator protein. We have been able to extend our studies to the human Ldhc promoter because it is activated in a malignant melanoma cell line. We determined that the murine and human promoters are only partially conserved since the latter sequence lacks a consensus TATA element upstream of the transcriptional start site. However, regulatory factors are similar for the murine and human Ldhc promoters (Tang and Goldberg, 2009). These findings are significant for developing an understanding of gene regulation in the human testis. Human material would be difficult, if not impossible to obtain for such research. Overall, it appears that the role of the CRE site is essential for spermatocyte-specific gene expression.
It has been demonstrated that the high level of Ldhc mRNA in germ cells is also the consequence of mRNA stability due to longer polyadenylation tails appearing with the meiotic divisions (Fujimoto et al, 1988). However, it seems that Ldhc mRNA stability is also species-specific and dependant on the presence of an AU-rich motif in the 3’ untranslated region (3’UTR) (Salehi-Ashtiani and Goldberg, 1995). This information leads one to wonder if LDHC might be post-transcriptionally regulated by an inhibition of translation or an increase of mRNA stability by the new and exciting way of regulating transcript levels with non coding RNAs as microRNAs.
In the last decade, the importance of epigenetic regulation has emerged, especially during the highly regulated process of spermatogenesis (Zamudio et al, 2008). Genes frequently are hypomethylated in the testis (Oakes et al, 2007). In earlier studies, no differences were found in the methylation patterns of the Ldhc gene between somatic and germ cells (Alcivar et al, 1991) but the technique of restriction analysis of DNA methylation overlooked many potential methylation sites (Alcivar et al, 1991). Other studies demonstrated that the human promoter contains a mini-CpG island (Bonny and Goldberg, 1995), and that its methylation in non-expressing cells serves as the likely mechanism to suppress Ldhc gene activation (Tang and Goldberg, 2009).
LDHC was the first germ cell specific glycolytic isozyme discovered, but subsequent studies revealed that it was not the only one. Two other genes encoding isozymes for glyceraldehyde-3-phophate dehydrogenase type S (GAPDHS) (Welch et al, 2000; Welch et al, 1992) and phosphoglycerate kinase type 2 (PGK2) (Boer et al, 1987; McCarrey and Thomas, 1987) in mouse and human were found to be expressed only in spermatogenic cells. In addition, several glycolytic enzymes with unique properties were detected in male germ cells (reviewed in Miki, 2007) (Figure 5).
Several studies have shown that meiotic and post-meiotic male germ cells preferentially use lactate and pyruvate over glucose as an energy substrate (reviewed in Boussouar and Benahmed, 2004; Grootegoed et al, 1984; Jutte et al, 1982; Mita and Hall, 1982) suggesting that lactate oxidation by LDH isozymes has a significant role in energy metabolism during the middle and later stages of spermatogenesis. While these observations would fit with the abundance of LDHC in these stages of spermatogenesis, there is no direct evidence to support this contention. On the other hand, it has been shown that glycolysable substrates are essential for sperm motility (Mukai and Okuno, 2004; Williams and Ford, 2001), for the protein tyrosine phosphorylation indicative of capacitation (Travis et al, 2001; Urner et al, 2001; Urner and Sakkas, 2003; Williams and Ford, 2001) and for fertilization (Bone et al, 2000; Urner and Sakkas, 1996). This was proven when inactivation of the gene for the sperm-specific glycolysis pathway enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDHS) (Miki et al, 2004) dramatically reduced the level of ATP in sperm, caused severe defects in progressive sperm motility, and resulted in male infertility.
These data were consistent with our hypothesis that LDHC is essential for lactate metabolism during spermatogenesis, and for the conversion of pyruvate to lactate accompanied by the generation of reduced NAD+ (coenzyme essential for GAPDHS activity) in spermatozoa.
One approach to assess LDHC function in spermatozoa was the use of an LDH inhibitor. In bovine sperm, O’Flaherty et al. (2002) demonstrated that treatment with sodium oxamate (NaOx, pyruvate analog and LDH inhibitor) suppressed sperm capacitation. Similar results were obtained with mouse sperm (Duan and Goldberg, 2003). However, in addition to concerns about the specificity of the inhibitor, we found that it inhibited LDH activity efficiently in protein extracts, but not in intact sperm (Odet, unpublished observations). An alternative and ultimately successful approach was to assess LDHC function by targeted disruption of the Ldhc gene (Odet et al, 2008). Initial attempts were not successful, probably because of the abundance of repetitive sequence elements in the Ldhc gene (Olsson et al, 2003). However, when the Ldhc null mouse was generated, it proved to be an exciting model of male infertility and a unique biological resource that could lead to more complete understanding of the biochemical mechanisms underlying sperm function.
Our first observation was that spermatogenesis appeared normal in Ldhc null mice. The testis histology looked unaffected and daily sperm production and sperm counts in the cauda epididymis were normal. To identify potential transcriptional modifications in Ldhc null testis, a microarray was performed in collaboration with Dr. Michael Griswold. However, preliminary results indicate that only Ldhc transcript levels were significantly different between WT and null testes by this method and by real-time PCR (Odet, unpublished observations). Although these results suggest that LDH-C4 does not play an essential role in the maintenance of spermatogenesis, they do not rule out that the absence of LDH-C4 has a subtle effect on spermatogenesis that subsequently compromises sperm function.
Although spermatogenesis appeared normal, the fertility of Ldhc null males was severely compromised. Our initial study was performed with animals from the F3 and F5 back-cross generations on the C57Bl/6N background (predicted 12.5% and 3.13% residual 129svEv background, respectively). From a total of 11 different Ldhc null males mated with more than 44 WT females over a 2 to 4 months period, only 2 litters of 1 and 3 pups were recovered (Odet et al, 2008).
Other studies demonstrated that this extreme subfertility was a consequence of a defect(s) in sperm function. Motility of Ldhc null sperm was impaired and their progressive motility decreased over time suggesting that Ldhc null sperm are unable to swim through the female tract (Odet et al, 2008). Moreover, even if Ldhc null sperm were able to reach the eggs, IVF experiments indicated that they were unable to fertilize oocytes. Ldhc null sperm did not undergo the protein tyrosine phosphorylation changes characteristic of capacitation and were unable to develop hyperactivated motility, processes essential for fertilization. When the zona pellucida was removed, Ldhc null sperm fertilized 29% of eggs. This was a significantly lower percentage of fertilization than occurred with sperm from heterozygote (HET) mice suggesting that, in addition to a defect in the mechanical process of oocyte penetration through the zona pellucida, there is also a defect in fusion to oocytes by Ldhc null sperm.
While it was clear that sperm defects were responsible for the infertility, the causes of the defects remained to be determined. A high rate of ATP production in sperm is known to be essential for maintaining a high level of motility for a prolonged period of time, and to induce sperm capacitation and hyperactivity. We hypothesized that lack of LDHC would induce a defect in ATP production by inhibition of glycolysis, the principal source of ATP in most mammalian sperm. Consistent with this hypothesis, we found rapid decreases in progressive motility and ATP levels over time in Ldhc null sperm than in WT sperm (Odet et al, 2008). By following the transformation of 13C-labelled glucose using NMR spectroscopy, we found that unlike WT sperm, Ldhc null sperm were unable to consume glucose (Odet, unpublished observations). However, by using direct (consumption of 13C-pyruvate, NMR spectroscopy) and indirect methods (amount and rate of lactate production from pyruvate) we also observed an unexpected result: Ldhc null sperm were able to convert pyruvate into lactate with the same rapid kinetics as WT sperm (Odet, unpublished observations). Because LDHA has been found in association with the isolated fibrous sheath, these results strongly suggest that LDHA is responsible for some or most of the LDH activity in Ldhc null sperm.
It is worth mentioning that sperm from mice heterozygous for the Ldhc mutation were fertile, whereas Ldhc transcript levels were reduced by 40% in the testes of these mice, and global LDH activity was reduced by 19.1% in testis and 24.7% in sperm. These observations indicate that sperm contain substantially more LDH-C4 than is required to maintain normal fertility.
Finding that the function of sperm from Ldhc null mice is severely compromised confirms the importance of this isozyme and strongly justifies further study of the role it plays in conferring or maintaining sperm fertilizing capacity. As has been noted previously, these studies are of translational relevance to problems of male infertility (Gavella and Cvitkovic, 1985; Orlando et al, 1988) and to the development of a male contraceptive (Erickson et al, 1975b; Goldberg, 1973; Goldberg et al, 2001; Mahi-Brown et al, 1990; O’Hern et al, 1995).
Therefore, we still must ask why sperm contain such a large quantity of LDHC. What could be the function of LDHC if LDHA alone can provide the terminal reaction of glycolysis? Many questions about LDHC remain to be elucidated.
This research was supported in part by NIH HD05863 (EG) and in part by the Intramural Research Program of the NIH, National Institutes of Environmental Health Sciences (EME).