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Gamete Biology; |
Laboratory of Reproductive and Developmental Toxicology,4 National Institute of Environmental Health Sciences, National Institutes of Health (NIH), Research Triangle Park, North Carolina 27709
Department of Biochemistry, Molecular Biology, and Cell Biology,5 Northwestern University, Evanston, Illinois 60208
ABSTRACT
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-C4) 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.
capacitation, fertilization, gamete biology sperm, gene targeting, glycolysis, mouse, sperm capacitation, sperm motility and transport, testis
Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate with the concomitant oxidation of NADH to NAD+ [1]. Catalytically active LDH consists of A and B subunits that assemble into homotetramers or heterotetramers and are distributed in the body in combinations reflecting the metabolic requirements of different tissues. For example, LDHA is most active in skeletal muscle, where oxygen deficiency requires glycolysis to satisfy metabolic needs, whereas LDHB is abundantly expressed in cardiac muscle that is dependent upon aerobic metabolic pathways. Exquisite tissue specificity is exemplified by the Ldhc gene, the third member of the family; it is expressed only in male [2, 3] and female [4] germ cells, and its protein product forms the enzymatically active homotetramer, classically referred to as LDH-C4.
Originally, LDH-C4 was considered specific to male germ cells [5], whereas LDHB was described as the predominant LDH isozyme in oocytes [6]. However, Coonrod et al. [4] showed recently that Ldhc transcripts are present in germinal vesicle-stage oocytes and not in fertilized eggs, whereas LDHC protein is present in germinal vesicle-stage oocytes and fertilized eggs, and persists to preimplantation blastocyst development. Because of these observations, the function of LDH-C4 during oogenesis, oocyte maturation, or early development is unclear.
However, Ldhc transcript levels are substantially lower in oocytes than in male germ cells. The National Center for Biotechnology Information UniGene expression profiles indicate that 102 per one million transcripts present in oocytes are for Ldhc, whereas 9340 per one million transcripts present in pachytene spermatocytes and round spermatids are for Ldhc (UniGene build 168; UniGene accession number for Ldhc: Mm.16563). In addition, Ldhc transcripts are more abundant in whole testis (2844 per million) than Ldha transcripts (584 per million; Mm.29324) and Ldhb transcripts (160 per million; Mm.9745). Several studies have shown that isolated meiotic and postmeiotic male germ cells preferentially use lactate and pyruvate over glucose as an energy substrate [7–9] (reviewed in Boussouar and Benahmed [10]), suggesting that lactate oxidation by LDH isozymes might be significant in energy metabolism during the middle and later stages of spermatogenesis. In male germ cells, Ldhc expression is activated with the onset of meiosis. The LDHC protein can be detected first in preleptotene spermatocytes and is abundant in spermatids and spermatozoa [11, 12]. Heterotetramers containing LDHA and LDHB also were present in spermatogonia, spermatocytes, and spermatids [13]. However, LDH-C4 was the major LDH isozyme in male germ cells, and heterotetramers containing LDHC and either LDHA or LDHB were not detected in murine or human testes [13].
It was thought that LDH-C4 is the only member of the LDH family active in spermatozoa [5, 13]. However, recent studies have shown that LDHA also is present in spermatozoa [14, 15]. LDHA was localized to the principal piece of the sperm flagellum where LDHC [16, 17] and other glycolytic enzymes also are concentrated [14]. However, LDHA but not LDHC was bound tightly to the fibrous sheath [14]. In contrast to spermatogenic cells, sperm exhibit high levels of glycolysis. Several in vitro studies provided evidence that glucose metabolism has an important role in production of the ATP required for sperm motility, hyperactivation, and capacitation [18–21]. This was proven when inactivation of the gene for the sperm-specific glycolysis pathway enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDHS) [22] dramatically reduced the level of ATP in sperm, caused severe defects in progressive sperm motility, and resulted in male infertility. Why Ldhc gene expression has been conserved in mammalian germ cells where Ldha and Ldhb genes also are transcribed remains an enigma. Kinetics of catalysis between the isozymes differ, but not by the orders of magnitude sufficient to confer advantage to one of the three relative to germ cell metabolism [23]. However, LDH-C4 has high thermostability [23] and is able to metabolize 
-hydroxyvalerate [24, 25], indicating that it has unique structural and functional properties [26]. In this study, we have disrupted the Ldhc gene to address the role of LDH-C4 in male and female germ cell development, gamete function, and fertility.
All materials and reagents were of the highest quality available and were purchased from Sigma-Aldrich (St. Louis, MO) or Mallinckrodt Baker (Phillipsburg, NJ) unless designated otherwise.
Targeting Construct and Targeted Disruption of the Ldhc Gene
Mice with exon 3 of the Ldhc gene deleted were generated using 129SvEv embryonic stem cells and C57BL/6N blastocysts to produce chimeras, Lhdctm1Erg (Supplemental Fig. 1 available online at www.biolreprod.org). All wild-type animals were purchased from Charles River (Raleigh, NC). This study was performed with animals from the third and fifth backcrossed generations with wild-type C57BL/6N mice.
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Phenotype Determination: Body and Reproductive Organ Weights, Sperm Number, and Daily Sperm Production
All animal procedures were performed in accordance with National Institutes of Health guidelines and approved by the National Institute of Environmental Health Sciences (NIEHS) and/or Northwestern University Animal Care and Use Committee. Mice from 12 to 32 wk of age were killed by CO2 asphyxiation, followed by cervical dislocation. Total body, testis, epididymis, and seminal vesicle weights were determined. Sperm numbers were determined using a hemocytometer. Daily sperm production was estimated as described previously [27, 28]. Briefly, testes were weighed and homogenized in an extraction buffer (0.15 M NaCl, 0.1 mM NaN3, 0.05% Triton X-100) to yield homogenization-resistant condensed spermatid heads. The number of spermatid heads was determined using a hemocytometer [27, 28].
Cauda epididymides collected in 1x PBS (Ca2+/Mg2+-free) were carefully dissected to remove blood vessels and fat, several small cuts were made with iridectomy scissors, and sperm were allowed to swim out into the medium. Capacitating medium was a modified Krebs-Ringer bicarbonate solution (119.37 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2 • 2H2O, 1.19 mM MgSO4 • 7H2O, 1.19 mM KH2PO4, 25.07 mM NaHCO3, and 5.6 mM glucose) with 4 mg/ml BSA [17, 29], or human tubular fluid medium (HTF) supplemented with 4 mg/ml BSA (Millipore, Bedford, MA). Sperm were incubated for selected lengths of time at 37°C in 5% CO2 in humidified air.
For some experiments, sperm isolated from cauda epididymides as described above were purified with the PureSperm 40/80 kit (Nidacon International) following the manufacturer's instructions. Briefly, the sperm suspension was layered onto the PureSperm gradient and centrifuged at 300 x g for 20 min. The sperm pellet was washed in PureSperm Wash buffer. Sperm purity was approximately 99%–100%, with negligible contamination by somatic cells.
Histology and Immunodetection of LDHC
Testes were fixed in Bouin solution (Polysciences Inc., Warrington, PA), dehydrated, and embedded in paraffin using standard methods. Sections 5 µm thick were stained with hematoxylin and eosin for histological analysis. The antibody to LDHC was diluted 1:10 000 in 1x PBS and 1% BSA for immunohistochemistry (IHC) and 1:4000 for indirect immunofluorescence (IIF) [16, 17]. Immunohistochemistry on paraffin sections was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. For IIF, sperm suspensions were pipetted onto slides (Superfrost/Plus; Fisher Scientific), allowed to settle and attach at 4°C for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 2 min, followed by 1 min in cold methanol 100% (–20°C). After incubation in antibody to LDHC for 2 h at room temperature, sperm were incubated with Alexa Fluor 488 donkey anti-rabbit immunoglobulin G (dilution 1:200; Invitrogen, Carlsbad, CA) for 1 h and mounted with Vectashield (Vector Laboratories). Samples were prepared for transmission and scanning electron microscopy as described previously [19].
RNA Extraction, RT-PCR, and Real Time RT-PCR
RNA was extracted with TRIzol reagent (Invitrogen). Specific primers were designed using the Primer3 processor (http://frodo.wi.mit.edu) [30], and the optimal temperature of annealing was defined for each primer pair (Supplemental Table 1 available online at www.biolreprod.org). Primers for Ldhc RT-PCR were chosen in different exons, and PCR products were sequenced using a Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).
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Real-time RT-PCR was performed using the SybrGreen kit (Applied Biosystems). All reactions were repeated three times, and 5S rRNA was used as an internal control to ensure equal amplification efficiency.
Homogenized testis fragment or purified sperm was suspended in buffer (0.1 M Tris-HCl, pH 7) and subjected to three 10-sec ultrasonic pulses. The suspension was centrifuged at 14 000 x g for 10 min at 4°C. The supernatant was collected, and protein levels were measured using the BCA protein assay kit (Pierce Biotechnology, Indianapolis, IN). Lactate dehydrogenase activity was determined as previously described [31]. Briefly, proteins were loaded on a 10% native polyacrylamide gel and separated at 100 V for 5 h at 4°C. Lactate dehydrogenase isozyme activity was visualized by incubating the gel for 15 min in the dark in a staining mix (0.05 M Tris-HCl, pH 8.4; 2 mM NAD; 0.8 mM NBT; 62.5 mM DL-lactate, and a few crystals of phenazine methosulfate). Global LDH activity was assayed in solution by spectrophotometry [17]. Protein extract (10 µg) was added to 1 ml reaction buffer (0.05 M Na2HPO4, pH 7; 0.1 mg/ml NADH; and 27.5 µg/ml pyruvate). Lactate dehydrogenase activity was calculated as the change in absorbance at 340 nm over a period of 1 min and expressed as U/min/µg of protein.
Purified sperm were washed in capacitating medium without lactate and pyruvate (present in the PureSperm buffer) and then incubated in capacitating medium at a concentration of 4 x 106 spermatozoa/ml at 37°C in 5% CO2 in humidified air. An aliquot was taken at time 0 to measure the residual levels of lactate and then after 0.5, 1, 2, and 4 h of incubation. After centrifugation for 5 min at 10 000 x g, duplicate 5-µl aliquots of medium were collected. Lactate levels were determined using a commercial kit (BioVision, Mountain View, CA) based on an enzymatic reaction by lactate oxidase and interaction of the product with a probe to produce fluorescence (at excitation/emission = 535/587 nm). The concentration of each sample was calculated using a standard curve. The detailed protocol for this assay is available on the manufacturer's website.
Proteins were either extracted as previously described (see LDH activity section) for LDHC immunodetection, or in a denaturating extraction buffer (125 mM Tris-HCl, pH 7.5; 4% SDS; complete cocktail inhibitors 1x [Roche Applied Science, Indianapolis, IN]) for immunodetection of phosphotyrosine-containing proteins. Proteins were separated on a 10% polyacrylamide gel and transferred onto an Immobilon membrane (Millipore). The membrane was incubated in blocking buffer (PBS with 0.1% Tween-20 [PBS-T] plus 5% nonfat dry milk for anti-LDHC or 1% BSA for anti-phosphotyrosine) for 1 h at room temperature with shaking. After three washes in 1x PBS-T, the membrane was incubated with the primary antibody against either LDHC (diluted 1:10 000 and incubated overnight at 4°C) or phosphotyrosine (clone PT-66; diluted 1:1000 and incubated for 1 h at room temperature; Sigma-Aldrich). After several stringency washes, the membrane was incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody. The Amersham ECL+ kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) was used to detect the signal.
Assessment of Fertility by Natural Mating
To assess male fertility, individual Ldhc–/– males were mated continuously for two or more 4-wk periods with two C57BL/6N wild-type females. Individual Ldhc–/– females were mated with one wild-type C57BL/6N male for 8 wk. All females were maintained for 4 wk after the mating period to assess possible pregnancy.
Individual Ldhc–/– (n = 10) and wild-type males (n = 6) were mated with one superovulated wild-type female overnight to assess male mating ability and to determine the percentage of eggs fertilized in vivo. Superovulated females that were not mated were used to determine the number of unfertilized two-cell eggs resulting from either parthenogenetic activation or oocyte fragmentation. In the morning following mating, females were examined for copulatory plugs and killed (experiment repeated four times). Zygote-cumulus complexes were isolated from the swollen ampullae. Zygotes were denuded by a brief exposure to hyaluronidase (200 IU) and then washed thoroughly in M2 medium (Millipore). Zygotes were cultured in groups of 10 in 20-µl drops of KSOM+AA medium (Millipore) in 60-mm dishes covered with light mineral oil in an atmosphere of 5% CO2 in humidified air at 37°C. They were examined at 4 h for the presence of pronuclei, and two-cell and four-cell embryos were counted 1 and 2 days later, respectively. Blastocysts were collected from superovulated females killed on Day 4.5 postinsemination (experiment repeated three times). Uteri were flushed with M2 medium, and expanded blastocyst morulae or degenerated eggs were washed in M2 medium and counted.
Cauda epididymal sperm were capacitated in HTF medium supplemented with 4 mg/ml BSA for 1.5 h at 37°C in 5% CO2 in humidified air. Oocytes from superovulated CD-1 mice were treated with hyaluronidase to remove cumulus cells. In some cases, zonae pellucidae were removed by chymotrypsin treatment (10 µg/ml, less than 5 min). Cumulus- and zona pellucida-free eggs were incubated in groups of 20 in 100-µl droplets for 4 h and 1 h, respectively, with Ldhc+/– and Ldhc–/– sperm at a concentration of 0.2 x 103 sperm/µl in 5% CO2 in humidified air at 37°C. The eggs were washed (to remove cellular debris and surplus sperm) and incubated overnight in KSOM+AA medium. The following morning, two-cell embryos were counted and transferred to fresh KSOM+AA medium. The eggs were considered fertilized if they developed to blastocysts after 4 days in culture.
Sperm were incubated in capacitating medium (described above) at 37°C in 5% CO2 in humidified air and assayed after 10 min, 90 min, and 4 h. Sperm ATP levels were measured as previously described [22]. After centrifugation at 1000 x g for 3 min, the pellet was resuspended in 100°C buffer (100 mM Tris-HCl, 4 mM EDTA, pH 7.8) and incubated for another 2 min at 100°C. Samples were centrifuged at 10 000 x g for 5 min, and aliquots of the supernatant were analyzed in duplicate. Adenosine triphosphate was measured using a luciferase bioluminescence assay according to the manufacturer's protocol (ATP Bioluminescence Assay kit CLS II; Roche Applied Science).
Sperm motility was observed on a Leica DM IRB inverted phase microscope with a 20x lens, and movies were taken with a Leica DFC420 digital camera mounted on this microscope and captured using Leica LAS software version 2.8.1.
Quantitative parameters of sperm motility were determined as described by a computer-assisted sperm analysis (CASA) instrument (software version 12; Hamilton Thorne Research, Beverly, MA) after incubation for 0.5, 1.5, and 4 h in capacitating medium. Median values of each of the kinematic parameters were obtained for each sample (for definitions, see Mortimer [32]). Kinematic parameters measured included curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of the lateral displacement of sperm head (ALH), beat-cross frequency (BCF), linearity (LIN = 100% x VSL/VCL), and straightness (STR = 100% x VSL/VAP) [32–34]. Sperm were counted as motile with any type of movement and progressively motile when VAP > 50 µm/sec and STR > 50%. Hyperactive sperm were identified with the "sort fraction" function of the Hamilton Thorne analyzer using these criteria: VCL > 240 µm/sec, ALH > 18 µm and BCF < 40 Hz.
All results are represented as the mean values of each group ± SEM. The significance of the results was determined using one-way ANOVA, followed by the Mann-Whitney U test. Differences were considered significant at P < 0.05.
Generation and Fertility of LDHC-Deficient Mice
The Ldhc locus was inactivated by gene targeting to determine whether LDH-C4 activity is required for gametogenesis and/or gamete function in male and female mice. Exon 3, which encodes both the coenzyme-binding domain and part of the substrate-binding domain, was flanked by loxP sites (Supplemental Fig. 1 available online at www.biolreprod.org). The targeted allele was transmitted through the germline. Offspring heterozygous for the targeted floxed allele were mated with transgenic mice expressing Cre to produce heterozygous Ldhc+/– mice, and these were mated to produce homozygous Ldhc–/– male and female mice. Both Ldhc–/– male and female mice were generated in expected Mendelian ratios (data not shown).
The Ldhc–/– female mice exhibited normal fertility, producing approximately one litter of 8 ± 3 pups per month, which was not significantly different from control C57BL/6N wild-type mice (7 ± 3 per month). The fertility of Ldhc+/– males also was comparable to that of control wild-type males (8 ± 4 pups per month). In their fertility trials, each of the eleven 60-day-old Ldhc–/– males (third backcross generation, n = 8; fifth backcross generation, n = 3) was mated with four different C57BL/6N females over 2- to 4-mo periods. Two of the Ldhc–/– males (one third and one fifth backcross generation) sired single litters of one and three pups, respectively, during the first trial. To extend this analysis, these two males were kept for two additional trials (total of six different females over a 4-mo period), but they did not sire litters in these subsequent matings. No other Ldhc–/– males have produced litters. Genotyping confirmed that these two males were Ldhc–/– and that their offspring were Ldhc+/–.
Morphology of Testes and Sperm from Ldhc–/– Mice Appears Normal
There were no significant differences in body weights of wild-type, Ldhc+/–, and Ldhc–/– males. The same was true for testis, epididymis, and seminal vesicle weights (Table 1). Sperm production was unaffected, with no differences seen in daily sperm production or epididymal sperm counts (Table 1). Sperm from Ldhc–/– mice examined by light and scanning electron microscopy also exhibited normal morphology (data not shown).
Testis morphology as assessed by histological analysis and transmission electron microscopy was comparable in wild-type, Ldhc+/–, and Ldhc–/– animals (data not shown). Using both IHC and IIF, we confirmed that LDHC is present in the cytoplasm of meiotic and postmeiotic germ cells and in the sperm flagellum of wild-type and Ldhc+/– mice (Fig. 1, A and B). As expected, no LDHC was detectable in Ldhc–/– testes and sperm (Fig. 1, A and B) or oocytes (Supplemental Fig. 2 available online at www.biolreprod.org). The absence of LDHC in the testes of Ldhc–/– mice also was confirmed by Western blotting (Fig. 1C).
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Ldha and Ldhb Transcript Levels Are Not Changed in Ldhc–/– Testes
We used real-time RT-PCR and gene-specific primers (Supplemental Table 1 available online at www.biolreprod.org) to measure the Ldha, Ldhb, and Ldhc transcript levels in the testes from wild-type, Ldhc+/–, and Ldhc–/– mice. No significant differences were observed in Ldha and Ldhb transcript levels in Ldhc+/– or Ldhc–/– mice compared with wild-type mice (Fig. 2A). However, with PCR primers specific for exon 8, Ldhc transcript levels were 5.2% and 60% in testes from Ldhc–/– and Ldhc+/– mice, respectively, of the levels in testes from wild-type mice (Fig. 2A), rather than 0% and 50%, as expected, indicating that transcription of the mutant allele was not inhibited completely. Sequencing of the PCR product confirmed that it was amplified from transcripts containing exon 8 of Ldhc (data not shown). To determine whether these results were due to the presence of variant transcripts lacking exon 3, PCR assays were performed with primers in exons 3 and 4 (E3-E4) and primers in exons 2 and 4 (E2-E4). With RNA from wild-type testes, we observed a unique PCR product with both primer pairs. While the E3-E4 primer pair did not yield a PCR product from Ldhc–/– testis RNA (Fig. 2B, row E3-E4), the E2-E4 primer pair generated a second PCR product from Ldhc–/– and Ldhc+/– testis RNA (Fig. 2B, row E2-E4). Sequence analysis of the E2-E4 primer pair PCR product from Ldhc–/– testis RNA determined that it lacked nucleotides 161–288 (data not shown), which correspond to exon 3. However, no full-length transcripts were found in Ldhc–/– testis. The transcripts detected at low levels were always missing exon 3. Because exon 3 encodes both the coenzyme-binding domain and part of the substrate-binding domain, it is not likely that the variant transcript produces a functional LDH-C4 enzyme.
Total LDH Activity Is Lower in Ldhc–/– Testes and Sperm
Lactate dehydrogenase activity differed significantly in testis and sperm extracts from wild-type, Ldhc+/–, and Ldhc–/– mice (Fig. 3, A and B). Total LDH activity was reduced by one half in Ldhc–/– testis extracts compared with wild type. While total LDH activity from Ldhc+/– testis extracts appeared to be reduced by approximately one quarter compared with wild type, this was not statistically significant. A similar pattern was observed in spermatozoa, with significant reductions of 24.7% and 82.2% in LDH activity, respectively, in Ldhc+/– and Ldhc–/– sperm compared with wild-type sperm (Fig. 3A). However, the total LDH activities in extracts of Ldhc–/– testis and sperm were 50% and 17.8% of the total LDH activity in testis and sperm from wild-type mice. This is to be expected, since both LDHA and LDHB are present in testes [13], and LDHA has been found in sperm by proteomic screens [14, 15].
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The activity of the different LDH isozymes was visualized after tissue extracts were separated by electrophoresis on a nondenaturing polyacrylamide gel. Proteins extracted from heart were used as a control to detect isozymes containing LDHA and LDHB. As previously reported [5, 13], bands corresponding to homotetramers and heterotetramers of LDHA and LDHB activity and a strong band corresponding to LDH-C4 homotetramer activity were visualized in proteins extracted from wild-type testis. In spermatozoa, only the LDH-C4 activity band was observed (Fig. 3B). As expected, no LDH-C4 activity was detected in testis and sperm of Ldhc–/– mice, whereas the other LDH isozymes activities in testis extract appeared comparable to those in the testes of wild-type mice (Fig. 3B). However, consistent with our previous observation on total LDH activity, a minor band on an LDH activity gel corresponding to LDH-A4 was observed in extracts of sperm and testis from Ldhc–/– mice. This band was not visualized in wild-type sperm, presumably because the strong band of LDH-C4 obscured that of LDH-A4.
Lactate Production by Ldhc–/– Sperm Is Compromised
We measured lactate production by wild-type, Ldhc+/–, and Ldhc–/– sperm. Pyruvate is the end product of glycolysis, and LDH-C4 converts the pyruvate into lactate. Lactate can diffuse across the sperm plasma membrane, and thus the accumulation of lactate in the medium indicates LDH activity. Lactate was found to accumulate over time in media containing wild-type and Ldhc+/– sperm. After incubation of wild-type sperm for 30 min, 61.4 nmol/ml lactate was present. In contrast, after incubation of Ldhc–/– sperm for 30 min, there was little, if any, lactate production (Fig. 4). However, Ldhc–/– sperm were capable of lactate production, and after 4 h of incubation, 46.6 nmol/ml lactate was present. Nevertheless, this was less than 10% of the amount of lactate in media containing wild-type sperm after the same time period (576.6 nmol/ml).
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In Vivo and In Vitro Fertilization by Ldhc–/– Sperm Is Defective
Copulatory plugs were produced by Ldhc–/– males mated overnight with superovulated wild-type female mice. We found that 62.8% of the eggs recovered from matings with wild-type males were fertilized (presence of pronuclei), but fewer than 0.5% from Ldhc–/– males appeared to be fertilized (Table 2). The number of one-cell eggs developing to the two-cell stage was similar for eggs recovered from females bred to Ldhc–/– males and eggs recovered from females not bred, and none of these divided to the four-cell stage in culture (control; Table 2). A similar result was found when female reproductive tracts were flushed 4.5 days after mating. No morulae or blastocysts were recovered from females mated with Ldhc–/– males, whereas 75.3% of the embryos recovered from females mated with wild-type males had developed to the morula stage (16.9%) or the blastocyst-stage (58.4%; Table 2).
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The in vitro fertilization assay results were consistent with the results of the mating studies. Only 3% of cumulus-free eggs incubated with sperm from Ldhc–/– males became two-cell embryos, none of which developed into blastocysts. In contrast, 51% of eggs fertilized by Ldhc+/– sperm became two-cell embryos, and 39% became blastocysts (Table 3). However, 29% of eggs with the zona pellucida removed and incubated with sperm from Ldhc–/– males became two-cell embryos, and 20% developed into blastocysts (Table 3). Even so, the number of zona pellucida-free eggs fertilized was only one third of the 87% observed with Ldhc+/– sperm. These in vitro fertilization assays were performed with a small number of sperm (20 000 sperm per 100-µl droplet per 20 eggs) to avoid polyspermy. However, some tests were performed with a larger number of sperm (2 and 10 times more), and the same results were observed (data not shown).
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Lack of LDH-C4 Alters Sperm Motility
Maintenance of progressive motility and acquisition of hyperactivated motility by sperm are required for fertility. Motility parameters of Ldhc–/– sperm were analyzed by CASA after 30 min, 90 min, and 4 h of incubation in capacitating medium (Fig. 5). Initially (t0.5), the motility of Ldhc–/– sperm was comparable to wild-type sperm (Fig. 6 and Supplemental Movies 1 and 2 available online at www.biolreprod.org). After 4 h of incubation, the percentage of Ldhc–/– sperm that were motile was reduced, and there was an even greater decrease in the percentage with progressive motility (Figs. 5 and 6; Supplemental Movies 3 and 4 available online at www.biolreprod.org). Moreover, Ldhc–/– sperm failed to develop the hyperactivity pattern characterized by vigorous flagellar movements. This was identified by CASA as showing a high VCL and ALH and lower BCF [33]. Only 1.93% of Ldhc–/– sperm were hyperactive, whereas 46.9% of Ldhc+/– sperm were hyperactive (t1.5 h; Fig. 5).
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Loss of LDH-C4 Alters Protein Tyrosine Phosphorylation in Sperm
A hallmark of sperm capacitation is an increased level of protein tyrosine phosphorylation [35–37]. The level of tyrosine phosphorylation in sperm from wild-type, Ldhc+/–, and Ldhc–/– mice was evaluated by Western blotting to determine whether LDH-C4 is required for this process. The extent of tyrosine phosphorylation was substantial in wild-type and Ldhc+/– sperm incubated in capacitating medium for 90 min, but was negligible in Ldhc–/– sperm (Fig. 7). The constitutively phosphorylated hexokinase (molecular mass 116 kDa) served as an internal positive control for the antibody and demonstrated that the samples contained equal amounts of protein.
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LDH-C4 Is Required to Maintain Sperm ATP Levels
High ATP levels are required for sperm motility, hyperactivity, and capacitation [19, 21]. There were no differences in the ATP levels in sperm from wild-type, Ldhc+/–, and Ldhc–/– mice after incubation for 10 min in capacitating medium (Fig. 8). However, the ATP level was 52.3% lower at 90 min in Ldhc–/– sperm than in wild-type sperm and even lower (81.8%) after 4 h. There was not a significant difference in ATP levels between Ldhc+/– and wild-type sperm at any of the time points (Fig. 8).
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This study confirms reports that LDHC is present in both spermatogenic cells and oocytes (Fig. 1 and Supplemental Fig. 2 available online at www.biolreprod.org) [4, 5]. Disruption of the Ldhc gene caused male but not female infertility and established that LDHC is essential for sperm function but unlikely to have a significant role in oocyte function. As expected, native transcripts, LDHC protein, and LDH-C4 enzymatic activity were not detected in Ldhc–/– mice. However, a variant transcript lacking the exon 3 coding region was present at low levels in Ldhc–/– and Ldhc+/– testes. The low transcript level might suggest that sequences in exon 3 and/or the intronic regions flanking the exon that are deleted during the Cre-mediated LoxP recombination event might contain regulatory region(s) that have effects on transcription or Ldhc mRNA stability.
The level of Ldhc transcript levels in testis from Ldhc+/– mice was reduced by 40%. We also found a decrease in global LDH activity of 19.1% in testis and 24.7% in sperm from Ldhc+/– mice. However, the fertility of Ldhc+/– mice was comparable to the fertility of wild-type mice. Moreover, no differences were observed between wild-type and Ldhc+/– sperm in ATP levels, motility, hyperactivation, or capacitation-related changes in phosphorylation. These observations indicate that sperm contain substantially more LDH-C4 than is needed to maintain normal fertility.
The total LDH activity was reduced to 50% in the testis and 17.8% in sperm from Ldhc–/– mice compared with the total LDH activity in testis and sperm from wild-type mice. Although LDHA and LDHB heterotetramers are present in testis, and LDHA homotetramers are present in sperm [16, 17], these reductions indicate that a substantial amount of the LDH activity in testis and most of the LDH activity in sperm are due to LDH-C4.
The substantial level of LDH-C4 activity in pachytene spermatocytes and spermatids might indicate that this isozyme has a significant role in spermatogenesis. However, based on histology and sperm production numbers, spermatogenesis appears to progress normally in the Ldhc–/– testis. This seems not to be due to functional compensation by the other LDH isozymes, because Ldha and Ldhb transcript levels were comparable in testes of Ldhc–/– and wild-type mice. Although these results suggest that LDH-C4 does not play an essential role in the maintenance of spermatogenesis, it does not rule out that the absence of LDH-C4 has a subtle effect on spermatogenesis that subsequently compromises sperm function.
The fertility of Ldhc–/– males was severely compromised, even though the sperm morphology, numbers, and motility observed soon after isolation appeared normal. The assessment of male fertility by natural mating showed that the majority of Ldhc–/– males were infertile, but two males were subfertile, each producing one small litter. However, no morulae or blastocysts were recovered from the reproductive tracts of superovulated females mated with Ldhc–/– males, and no blastocyst embryos were produced by in vitro fertilization using sperm from Ldhc–/– males and eggs with an intact zona pellucida. Furthermore, the absence of LDH-C4 in sperm resulted in a decline in progressive motility compared with wild-type sperm and a failure to achieve hyperactivated motility. These findings strongly suggest that the qualitative and quantitative effects that the absence of LDH-C4 has on sperm motility contribute to the severely compromised fertility of Ldhc–/– males. Nevertheless, the mating studies indicated that there is a small probability that Ldhc–/– sperm can reach and fertilize the oocyte. This presumably occurs quite soon after mating, before sperm motility declines, and suggests that the in vivo conditions are more conducive to fertilization by these sperm than the in vitro conditions.
Our results parallel clinical observations that have been made on sperm from patients. It was reported that LDHC could not be detected in spermatozoa from some men presenting at a fertility clinic [38]. These men were infertile and produced normal numbers of sperm that initially were motile and soon became poorly motile or nonmotile. Thus, a more complete understanding of the role of LDH-C4 will be important for diagnosing some causes of infertility and for advancing the development of new approaches to controlling fertility in men.
Lactate dehydrogenase is the terminal enzyme of glycolysis with the usual role of reducing pyruvate to lactate when oxygen is limiting. However, aerobic glycolysis is the main source of ATP in sperm of mice [22] and, presumably, of many other mammalian species to provide energy required for the flagellar activity that produces sperm motility. The failure of Ldhc–/– sperm to acquire hyperactivated motility and to fertilize eggs in vitro with an intact zona pellucida is quite likely a consequence of ATP deficit. However, higher levels of ATP and sperm motility were observed in Ldhc–/– sperm than in Gapdhs–/– sperm [22]. This suggests that the LDHA activity present in Ldhc–/– sperm might generate enough ATP to support motility initially, but cannot compensate sufficiently for the lack of LDH-C4 sufficiently to allow later processes required for normal fertility to occur, such as hyperactivation and capacitation.
However, when the zona pellucida was removed, Ldhc–/– sperm fertilized 29% of eggs. Because this is a lower percentage of fertilization than occurred with Ldhc+/– sperm, it implies a defect in sperm-egg fusion by Ldhc–/– sperm. These results are consistent with previous studies indicating that glucose is necessary for sperm capacitation, protein tyrosine phosphorylation, and sperm-oocyte fusion [18, 39]. In addition, the Ldhc–/– sperm did not undergo the protein tyrosine phosphorylation changes characteristic of capacitation, suggesting that defects in phosphorylation also contributed to the failure to fertilize zona pellucida-intact eggs.
Our results suggest that the fertility defect in Ldhc–/– males has more than one cause. These appear to include a more rapid decline in motility than in sperm from wild-type males, a defect in capacitation, as indicated by a failure in phosphorylation events that are hallmarks of this process, and a fertilization defect. All of these probably are due to compromised ATP production caused by the disruption of glycolysis. However, the underlying defects that lead to these changes remain to be determined. Because LDH catalyzes the reduction of pyruvate to lactate with a concomitant oxidation of NADH to NAD+, accumulation of pyruvate or other upstream metabolites that inhibit glycolysis and/or the effects of disruption of NADH oxidation may be involved. The effects of a lack of LDH-C4 on these parameters currently are being studied. In addition, we cannot exclude that LDH-C4 may have another essential function that remains to be determined.
ACKNOWLEDGMENTS
We thank Dr. Philip Leder for providing TC1 embryonic stem cells, Linwood Koonce from the NIEHS for excellent technical assistance, John Otstot from the NIEHS for sequencing, Drs. Kiyoshi Miki and Masuo Goto for helpful suggestions, and Drs. Christopher Geyer, Michael G. O'Rand, and Brigitte Le Magueresse for critically reading this manuscript.
FOOTNOTES
1Supported in part by NIH HD05863 to E.G., and in part by the Intramural Research Program of the NIH, National Institutes of Environmental Health Sciences to E.M.E. 
Correspondence: 2E. Goldberg, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208. FAX: 847 467 1380; e-mail: erv{at}northwestern.edu
3These authors contributed equally to this work.
Received: 13 February 2008.
First decision: 28 February 2008.
Accepted: 10 March 2008.
REFERENCES
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