ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

Abstract

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

Fig. 1.

Generation of Elovl2-ablated mice. A: Elovl2 gene targeting construct. The neomycin resistance (neo^r) gene was used to replace a major portion of exon 3 in the ELOVL2 gene. The mutated Elovl2 allele differs from that of the wild-type by the addition of an internal NsiI site in the neo^r gene. Black boxes represent exons and are numbered (–8) above. TK, thymidine kinase. Genomic DNA from embryonic stem cells (B) and tail DNA (C) was digested with NsiI and analyzed with Southern blotting for the mutated Elovl2 allele. Digestion yielded the 12.8 kb wild-type band and the 9.6 kb targeted band detected by the probe indicated in panel A. Elovl2 expression analysis using real-time PCR is shown in liver (D) and testis (E). Results are means ± SEM of 7 wild-type (Wt) heterozygous (Hz) mice and 2 knockout (KO) animals. *, P < 0.05; **, P < 0.01.

Fig. 2.

Histological analysis of testes in wild-type, Elovl2^+/−, and Elovl2^−/− mice. Mature spermatozoa are shown with elongated heads in seminiferous tubules (A–C, arrows) and epididymes (D) of wild-type mice. Elovl2^+/− testis are seen with numerous spermatozoa displaying rounded, condensed heads (E–G, arrows), and the condensed nucleus frequently displays a small rounded vacuole about 1–2 µm (G, arrowhead). Epididymes of wild-type (D) and Elovl2^+/− (H) mice display spermatozoa with rounded condensed heads compared with those of wild-type epididymis. Elovl2^−/− mice displayed only spermatogonia and primary spermatocytes (I, J) with multinucleate giant cells in the lumen (K, L, arrows). (M) Spermatogenesis within the seminiferous tubules and blockage by Elovl2 ablation in Elovl2^−/− and Elovl2^+/− mice. Sg, spermatogonia; pSc, primary spermatocytes; sSc, secondary spermatocytes; BTB, blood testis barrier; St, spermatides; eSt, elongated spermatides; Sz, spermatozoa.

Fig. 3.

Testicular gene expression. Analysis of the AKAP3 (A), TISP50 (B), TISP69 (C), Tnp (D) and Cttn (E) genes were examined by real-time PCR in wild-type (Wt), Elovl2 ^+/−(Hz [heterozygous]), and Elovl2^−/− (KO [knockout]) mice. Results shown are the means ± SEM of 7 Wt and Hz animals and 2 KO animals. *, P < 0.05; **, P < 0.01.

Fig. 4.

Biosynthesis of VLC-PUFAs in wild-type, Elovl2^+/−, and Elovl2^−/− mice. The composition of the testicular acyl-CoA pool was determined by extraction, derivatization, and HPLC analysis of acyl-etheno-CoAs. Carbon 17:0 istd is the internal standard acyl-CoA. A: Acyl-CoA composition of wild-type and Elovl2^−/− testes. Blue trace corresponds to Elovl2^+/+, and the red trace corresponds to Elovl2^−/− mice. *, indicates differences between the genotypes. B: Acyl-CoA composition of wild-type and Elovl2^+/− testes. Blue trace corresponds to Elovl2^+/+ mice, and the red trace corresponds to Elovl2^+/− mice. *, indicates differences between the genotypes. Results shown are pooled samples from 7 wild-type and heterozygous mice and 2 knockout animals.

Fig. 5.

In vivo VLC-PUFA biosynthesis by ELOVL2, ELOVL4, and ELOVL5. The 18:3n-3 and 18:2n-6 PUFAs were obtained from the diet and were subsequently elongated and desaturated by the indicated enzymes into longer PUFAs. ELOVL5 is involved in the elongation of PUFAs up to 22 carbons in length. ELOVL2 elongates from 20:4n-6 and 20:5n-3 series. Specifically, in the testis, ELOVL2 is involved in the biosynthesis of VLC-PUFA (≥C24) up to 30 carbons in length of the n-6 family (gray area). ELOVL4 elongates up to C38 in length. C26FADS, fatty acid desaturase.

Fig. 6.

Total fatty acid composition of liver and testis. Fatty acid composition up to C24, analyzed by gas chromatography of liver (A) and testis (B) of wild-type (wt) and Elovl2^+/− (Hz [heterozygous]) male mice and testis of two pooled wild-type and Elovl2^−/− male mice (C). Bars indicate means ± SEM from 6 animals. *, P < 0.05; **, P < 0.01.

Fig. 7.

Differences in total PUFA composition of serum. Fatty acid composition up to C24, by gas chromatography analysis of serum in wild-type and Elovl2^−/− male mice. Bars indicate means ± SEM from 3 animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.