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

Abstract

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.

Figure 1

Expression of type II DGK mRNAs (A) and proteins (B) in the reproductive organs: testis, ovary and uterus. (A) RT-PCR analysis of the mRNA from the reproductive organs was performed: testes from 12-week-old male mice and ovaries and uteruses from 12-week-old female mice. The panels show that 999-bp cDNA fragments were amplified for DGKδ (35 cycles), 826-bp cDNA fragments were amplified for DGKη (35 cycles), 842-bp cDNA fragments were amplified for DGKκ (35 cycles) and 978-bp cDNA fragments were amplified for mouse glyceraldehyde phosphate dehydrogenase (GAPDH, 35 cycles), as determined through agarose gel electrophoresis. The asterisk indicates a ~730-bp band that was amplified from the testis mRNA. Representatives of five independent experiments with five male and five female mice are shown. (B) The protein samples (30 μg) from the indicated tissues of 10-week-old male and female mice were detected by Western blotting using anti-DGKδ and DGKη antibodies. Representatives of five independent experiments with five male and five female mice are shown.

Figure 2

Alternative splicing products of DGKη. (A) The nucleotide sequences and deduced amino acid sequences of DGKη1, DGKη2 and DGKη3 are shown. The nucleotide and amino acid numbers are indicated in the left in italic and plain text, respectively. (B) The exon-intron structure of the DGKη gene (exons 25–27) and the alternative splicing that yields the two different forms (DGKη1/2 and DGKη3) are shown.

Figure 3

In situ hybridization of the type II DGK mRNAs in the testis of a 12-week-old male mouse. (A) The type II DGK mRNAs were hybridized and detected with the antisense probe of the type II DGK mRNAs. The testis sections were also hybridized with the sense probes as controls. (B) High-magnification image of the seminiferous tubules. Representatives of three independent experiments with three male mice are shown. Sg, spermatogonium; Ps, primary spermatocyte; Ss, secondary spermatocyte; Rs, round spermatid; Es, elongated spermatid; Lc, Leydig cell. The scale bars in (A) represent 200 μm, and the scale bars in b represent 40 μm.

Figure 4

In situ hybridization of the type II DGK mRNAs in the ovary of a 12-week-old female mouse. (A) The type II DGK mRNAs were hybridized and detected with the antisense probes of the type II DGK mRNAs. The ovary sections were also hybridized with the sense probes as controls. (B) High-magnification image of the ovarian follicles (secondary follicles). Although we observed histological specimens from five female mice in different estrous cycles, essentially the same results were obtained. Representatives of five independent experiments with five female mice are shown. Pf, primary follicle; Sf, secondary follicle; Mf, mature follicle; Cl, corpus luteum; Gc, granulosa cell; M, medulla. The scale bars in (A) represent 200 μm, and the scale bars in (B) represent 40 μm.

Figure 5

In situ hybridization of the type II DGK mRNAs in the oviduct of a 12-week-old female mouse. (A) The type II DGK mRNAs were hybridized and detected with the antisense probes of the type II DGK mRNAs. The oviduct sections were also hybridized with the sense probes as controls. (B) High-magnification image of the ovarian oviduct. Representatives of four independent experiments with four female mice are shown. Oe, oviductal epitherium. The scale bars in (A) represent 100 μm, and the scale bars in (B) represent 40 μm.

Figure 6

In situ hybridization of the type II DGK mRNAs in the uterus of a 12-week-old female mouse. (A) The type II DGK mRNAs were hybridized and detected with the antisense probes of the type II DGK mRNAs. The uterus sections were also hybridized with the sense probes as controls. (B) High-magnification image of the endometrial epithelium. Representatives of four independent experiments with four female mice are shown. L, luminal epithelium; E, endometrium; Ug, uterine gland. The scale bars in (A) represent 200 μm, and the scale bars in (B) represent 40 μm.