Intracellular localization of the testicular and sperm-specific lactate dehydrogenase isozyme C4 in mice

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.