Elovl4 and Fa2h expression during rat spermatogenesis: a link to the very-long-chain PUFAs typical of germ cell sphingolipids.

The sphingolipids (SLs) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa contain nonhydroxylated and 2-hydroxylated versions of very-long-chain (C26-C32) PUFAs (n-V and h-V, respectively) not present in Sertoli cells (SCs). Here, we investigated the expression of selected fatty acid elongases [elongation of very-long-chain fatty acid protein (Elovl)], with a focus on Elovl4, and a fatty acid 2-hydroxylase (Fa2h) in rat testes with postnatal development and germ cell differentiation. Along with Elovl5 and Elovl2, Elovl4 was actively transcribed in the adult testis. Elovl4 mRNA levels were high in immature testes and SCs, though the protein was absent. The Elovl4 protein was a germ cell product. All cells under study elongated [3H]arachidonate to tetraenoic and pentaenoic C24 PUFA, but only germ cells produced C26-C32 PUFAs. Spermatocytes displayed the highest Elovl4 protein levels and enzymatic activity. Fa2h mRNA was produced exclusively in germ cells, mostly round spermatids. As a protein, Fa2h was mainly concentrated in late spermatids, in the step of spermiogenesis in which they elongate and their heads change shape. The expression of Elovl4 and Fa2h thus correlate with the abundance of n-Vs and h-Vs in the SLs of rat spermatocytes and spermatids, respectively.

Lipid Biochemical and Biophysical Changes in Rat Spermatozoa During Isolation and Functional Activation In Vitro.

In spermatozoa isolated from rat epididymis, lipids are differentially modified after in vitro induction of capacitation (Cap) and the acrosomal reaction (AR). This study uses Laurdan fluorescence generalized polarization values (GPv) to evaluate the effect of lipid changes occurring after isolation and functional activation on sperm membrane biophysical properties. In gametes isolated in the presence of a divalent cation chelator, no lipid changes occurred and the GPv were the lowest recorded, indicating maximal membrane lipid mobility. In sperm isolated as rapidly and gently as possible in the absence of chelator, part of the sphingomyelins (SM) were converted into ceramides (Cer), giving rise to higher GPv. In samples incubated as controls for Cap and AR, unchanged cholesterol and reduced glycerophospholipid levels were accompanied by the accumulation of free fatty acids (FFA), leading to even higher GPv. After completion of Cap, the GPv returned to lower levels as a result of the spermatozoa losing part of their cholesterol and FFA. Cap samples became relatively enriched in polyunsaturated fatty acids-containing plasmalogens because hydrolysis affected phosphatidyl rather than plasmenyl glycerophospholipid subclasses. The highest Cer:SM ratio and the highest GPv were found after completion of AR induced by A23187. The degree of SM → Cer conversion among the samples, including controls, correlated with the extent of AR. FFA and Cer augmented GPv when added to liposomes prepared from the membrane lipid of intact sperm. Our results underscore the importance of hydrolytic changes that affect sperm lipids, especially the decisive lipid SM and Cer pair, not only after inducing sperm functional changes such as Cap and AR, but also under control conditions.

Differentiation-related changes in lipid classes with long-chain and very long-chain polyenoic fatty acids in rat spermatogenic cells.

In rat seminiferous tubules (ST), cells that contain polar and neutral lipids with long-chain polyenoic fatty acids (PUFA) and sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA of the n-6 series coexist. In this study, pachytene spermatocytes and round spermatids were isolated to determine how these lipids change during spermatogenesis. As the amount per cell of PUFA-rich glycerophospholipids (GPL) decreased with cell size, the 22:5/20:4 ratio increased with cell differentiation. The elovl2 and elovl5 genes, required for 22:5 formation, were expressed (mRNA) in both cell types. Residual bodies- particles with compacted organelles and materials discarded from late spermatids-concentrated cholesterol, 22:5-rich triacylglycerols, and GPL, including plasmalogens and phosphatidylserine. Species of SM and Cer with nonhydroxylated (n-) VLCPUFA (28:4, 30:5, and 32:5) predominated in pachytene spermatocytes, whereas species with the corresponding 2-hydroxy (2-OH) VLCPUFA prevailed in round spermatids. Thus, a dramatic increase in the 2-OH/n-VLCPUFA ratio in SM and Cer was a hallmark of differentiation. A substantial decrease of 2-OH SM occurred between spermatids and mature spermatozoa and 2-OH SM species were collected in residual bodies "en route" to Sertoli cells. Notably, spermatids and spermatozoa gained a significant amount of ceramides devoid of n-VLCPUFA but having 2-OH VLCPUFA as their main fatty acids.

Mild testicular hyperthermia transiently increases lipid droplet accumulation and modifies sphingolipid and glycerophospholipid acyl chains in the rat testis.

Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigate the effects on testicular lipids of the transient germ cell loss that is induced by mild testicular hyperthermia. Adult rat testes were exposed once a day to 43 °C for 15 min for 5 days and the effects were followed for several weeks. Two week after the last heat exposure, spermatocytes and early spermatids had virtually disappeared and the seminiferous tubules were populated mostly by mature spermatids and spermatozoa. One week later, the latter were also absent and mostly Sertoli cells populated the tubules. During these 3 weeks, glycerophospholipids (Gpl) and triacylglycerols with long-chain polyunsaturated fatty acids (PUFA) (e.g., 22:5n-6) and species of sphingomyelin and ceramide with nonhydroxy and 2-hydroxy very long-chain (VLC) PUFA (e.g., 28:4n-6, 2-OH 30:5n-6) decreased alongside the germ cells. Concomitantly, the amounts of cholesteryl esters and ether-linked triglycerides increased, both lipids accumulating long-chain and very-long-chain polyenes. This concurred with a considerable buildup of lipid droplets in Sertoli cells, evidently containing these neutral lipids, apparently formed during germ cell-derived membrane lipid catabolism. Between week 4 and week 6, new cohorts of spermatocytes appeared, and by week 12 most cell changes were reversed. Accordingly, as germ cell differentiation proceeded, 22:5n-6-rich Gpl augmented and spermatocyte-associated sphingolipids with nonhydroxy VLCPUFA appeared before their 2-hydroxy counterparts. The unique fatty acids of rat testicular lipids after mild hyperthermia reveal lipid catabolic and biosynthetic reactions that occur in normal spermatogenesis.

Uneven distribution of ceramides, sphingomyelins and glycerophospholipids between heads and tails of rat spermatozoa.

Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.