Intracellular Localization of the Testicular and Sperm-Specific Lactate Dehydrogenase Isozyme C4 in Mice1

Abstract

The proposed dual intracellular distribution of the sperm-specific lactate dehydrogenase (EC 1.1.1.27) isozyme C4 (LDH C4) has been based on indirect evidence. In order to obtain direct evidence on the localization of this LDH isozyme in mice, postembedding immunocytochemistry at ultrastructural level was performed on testes, epididymal spermatozoa, and isolated testicular mitochondria. The immunogold technique was applied to thin sections incubated first in partially purified specific anti-LDH C4 rabbit IgG, and immunoreactive sites were detected with colloidal gold adsorbed to anti-rabbit IgG. In the testis, immunostaining was found in the cytoplasm of spermatocytes and spermatids and in the principal and middle pieces of differentiating spermatozoa. Spermatozoa from epididymis also exhibited heavy labeling of colloidal gold in their middle and principal pieces, but the immunostaining was weak in the special type of mitochondria present in spermatocytes, spermatids, and spermatozoa (sperm-type mitochondria, STM). The isolation of STM produced several morphological changes in comparison with those in situ, including an enhancement of the LDH C4 labeling in the mitochondrial matrix. The other type of mitochondria (non-STM) from spermatocytes and nonspermatogenic cells were not immunostained and served as background control. The results presented here confirm previous findings, gathered by indirect methods, indicating a dual localization of LDH C4 in the cytosol of spermatocytes, spermatids, and spermatozoa, as well as in the matrix of sperm-type mitochondria.

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

“…Several ATP production-related proteins have been identified in the mitochondria of spermatozoa. For example, both spermspecific isoforms of lactate dehydrogenase catalyze the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD + ; hexokinase catalyzes the conversion of glucose to glucose-6-phosphate in the initial step of glycolysis and is expressed at the mid-piece of mouse sperm [45,46]. HIBADH is an NAD + -dependent mitochondrial enzyme that catalyzes oxidation of 3-hydroxyisobutyrate, an intermediate of valine catabolism, to methylmalonate semialdehyde [33,34].…”

Moving to the beat: a review of mammalian sperm motility regulation

“…However, sperm mitochondria possess several unique proteins or protein isoforms that are not found in somatic mitochondria. In mice, these include sperm-specific isoforms of lactate dehydrogenase and hexokinase (Burgos et al 1995;Bunch et al 1998;Mori et al 1998;Travis et al 1998). Thus, defects in these spermspecific proteins may result only in problems with sperm mitochondrial function, rather than with mitochondrial dysfunction in all cells.…”

Yak Lactate Dehydgogenase A4: Purification, Properties, and cDNA Cloning

“…[8][9][10][11][12] In this study, we purified and characterized LDH-A4 from skeletal muscle of yak and cattle, and found that yak LDH-A4 Km for pyruvate was much higher than that of cattle. Yak LDH-A cDNA was cloned and analyzed in order to elucidate the amino acid substitutions between yak and cattle.LDH-A4 was purified from skeletal muscles of yak and Chinese yellow cattle by modification of the methods reported by Dissing et al (1998) and Burgos et al (1995). 13,14) Skeletal muscle tissues were homogenized in cold phosphate buffered saline (PBS), incubated for 4 h at 4 C, and centrifuged at 10,000 g for 20 min at 4 C. The supernatant was used as the crude extract, and ammonium sulfate was added to 80% saturation.…”

“…LDH-A4 was purified from skeletal muscles of yak and Chinese yellow cattle by modification of the methods reported by Dissing et al (1998) and Burgos et al (1995). 13,14) Skeletal muscle tissues were homogenized in cold phosphate buffered saline (PBS), incubated for 4 h at 4 C, and centrifuged at 10,000 g for 20 min at 4 C. The supernatant was used as the crude extract, and ammonium sulfate was added to 80% saturation.…”

“…13,14) Skeletal muscle tissues were homogenized in cold phosphate buffered saline (PBS), incubated for 4 h at 4 C, and centrifuged at 10,000 g for 20 min at 4 C. The supernatant was used as the crude extract, and ammonium sulfate was added to 80% saturation. The precipitate was obtained by centrifugation, as described above, suspended in 0.02 mol/l PBS, and applied to a HiTrapÔ Desalting column.…”