Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility

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-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.

Fig.1

(A) Immunohistochemical localization of LDHC in testis from Ldhc^+/- (HET) and Ldhc^-/- (KO) mice. Arrows indicate positive areas and the asterisk indicates the absence of LDHC. Bar = 100 μm. (B) Immunofluorescence of LDHC on sperm from wild type (WT) and Ldhc^-/- (KO) mice. (C) Western blot analysis of testis extracts from wild type (WT), Ldhc^-/- (KO), heterozygote type 1 (HET^f; floxed), and Ldhc^+/- (HET) mice, probed with anti-LDHC.

Fig.2

RT-PCR analysis of Ldha, Ldhb and Ldhc transcript levels. Total RNAs were extracted from whole testis from wild type (WT; n=8), Ldhc^+/- (HET; n=7), and Ldhc^-/- (KO; n=9) mice. (A) Real-time RT-PCR analysis of Ldha, Ldhb and Ldhc (E8-E8 primers). Each reaction was done in triplicate for each gene and the amount of cDNA was determined by using a standard curve. Data are means ± SEM and are expressed in arbitrary units after normalization with 5S rRNA. *p < 0.05; **p < 0.01 compared to wild type. (B) RT-PCR products were separated on 2% agarose gels and their sizes were confirmed with a DNA ladder (not shown). Primer pair E3-E4 for Ldhc identifies the native transcript and will not produce a band when Exon 3 is deleted; primer pair E2-E4 amplifies transcripts containing exon 3 with a 254 bp product, and the truncated transcript with a 127 bp product; primer pair E8-E8 amplifies both Ldhc transcripts without size distinction.

Fig.3

LDH activity analysis in sperm from wild type (WT), Ldhc^+/- (HET) and Ldhc^-/- (KO) mice. (A) Global LDH activity was measured by spectrophotometry as the decrease in absorbance at 340 nm over 1 min (OD_t0-OD_t1min). Values are mean ± SEM (n=6). * p<0.05, ** p < 0.01, compared to wild type mice. (B) LDH activity visualized on an activity staining gel. A representative gel is shown (n=3). Subunit composition of LDH isozymes are indicated on the left.

Fig.4

Lactate level in medium containing wild type (WT), Ldhc^+/- (HET) and Ldhc^-/- (KO) sperm (4 ×10⁶ /ml) after incubation in capacitating medium at different times. Values are the mean lactate levels ± SEM (n=3). * p < 0.001 compared to wild type.

Fig.5

Sperm motility assessed by CASA. Sperm were collected in capacitating medium and incubated at 37°C in 5% CO₂ in humidified air. Motile sperm, progressively motile sperm, and hyperactived sperm are expressed in percentage of sperm presenting indicated characteristic (see Materials and Methods) of total sperm counted. VCL = curvilinear velocity, ALH = amplitude of the lateral displacement of sperm head, BCF = beat-cross frequency. Median values ± SEM (minimum 200 sperm recorded) of each of the kinematic parameters were obtained for each sample (n=3). Letters (a, b) above the means denote no significant difference if identical or significant difference if not identical. * p< 0.05 compared to wild type.

Fig.6

Still images taken from the supplemental movies (available online at www.biolreprod.org). Sperm from Ldhc^+/- (HET) and Ldhc^-/- (KO) mice were collected in capacitation medium and incubated 1 h or 4 h at 37°C in 5% CO₂ in humidified air. Movies were taken with a Leica DFC420 digital camera mounted on a Leica DM IRB inverted phase microscope with a 20X lens and captured using Leica LAS software version 2.8.1.

Fig.7

Protein tyrosine phosphorylation occurring in wild type (WT), Ldhc^+/- (HET) and Ldhc^-/- (KO) sperm assayed by western blotting with an antibody against phosphotyrosine. Proteins were extracted immediately after sperm collection or after 90 min incubation in capacitating medium. The band at 116kDa corresponds to hexokinase. The example shown is representative of three experiments.

Fig.8

Time-dependent decrease in ATP levels in Ldhc^-/- sperm (A). Values are the mean ATP levels ± SEM from wild type (WT; n=7), Ldhc^+/- (HET; n=8), and Ldhc^-/- (KO; n=10) mice. * p < 0.05, **p < 0.001 compared to wild type.