Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4.

Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner.

Distinct expression and localization of the type II diacylglycerol kinase isozymes δ, η and κ in the mouse reproductive organs.

BACKGROUND: We have revealed that the type II diacylglycerol kinases (DGKs) δ, η and κ were expressed in the testis and ovary. However, these enzymes' functions in the reproductive organs remain unknown. RESULTS: In this study, we first identified the expression sites of type II DGKs in the mouse reproductive organs in detail. Reverse transcription-polymerase chain reaction and Western blotting confirmed that DGKδ2 (splicing variant 2) but not DGKδ1 (splicing variant 1) and DGKκ were expressed in the testis, ovary and uterus. DGKη1 (splicing variant 1) but not DGKη2 (splicing variant 2) was strongly detected in the ovary and uterus. Interestingly, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the testis. Moreover, we investigated the distribution of type II DGKs in the testis, ovary and uterus through in situ hybridization. DGKδ2 was distributed in the primary spermatocytes of the testis and ovarian follicles. DGKη1 was distributed in the oviductal epithelium of the ovary and the luminal epithelium of the uterus. Intriguingly, DGKη3 was strongly expressed in the secondary spermatocytes and round spermatids of the testis. DGKκ was distributed in the primary and secondary spermatocyte of the testis. CONCLUSION: These results indicate that the expression patterns of the type II DGK isoforms δ2, η1, η3 and κ differ from each other, suggesting that these DGK isoforms play specific roles in distinct compartments and developmental stages of the reproductive organs, especially in the processes of spermatogenesis and oocyte maturation.

Expression and localization of type II diacylglycerol kinase isozymes δ and η in the developing mouse brain.

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II-VI of the cerebral cortex; CA-CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II-VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.