Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa.

Polyenoic very-long-chain fatty acids (VLCFA) have been shown to be localized in unusual molecular species of sphingomyelin in the testes and spermatozoa of the ram, bull, rat, and boar and in the spermatozoa of man. The composition of polyenoic VLCFA-sphingomyelin was comparable in the testes and spermatozoa of each mammalian species; however, the sphingolipid was more concentrated in spermatozoa. The composition of testicular and spermatozoan polyenoic VLCFA-sphingomyelin differed considerably between animal types. Human spermatozoa mainly contained n-6 polyenoic VLCFA with two to four double bonds and even-carbon chain lengths up to 32. In ram and bull testes and spermatozoa, n-3 and n-6, tetra-, penta-, and hexaenoic VLCFA with even-carbon chain lengths up to 34 predominated. In rat and boar testes and spermatozoa, the polyenoic VLCFA were mainly n-6 derivatives with three to five double bonds and even- and odd-carbon chain lengths up to 34. The testes and spermatozoa of the latter two animal species contained 2-hydroxylated, in addition to non-hydroxylated, polyenoic VLCFA in sphingomyelin. This is the first time that 2-hydroxylated polyenoic VLCFA have been recognized in biological systems. Non-hydroxylated polyenoic VLCFA were initially observed in the sphingomyelin of rat testes 25 days after birth, followed by 2-hydroxylated derivatives at 30 days. The total amount of polyenoic VLCFA associated with rat testicular sphingomyelin increased dramatically from 25 to 40 days of postnatal life and then remained constant to 60 days (sexual maturity). The ratio of 2-hydroxylated to non-hydroxylated polyenoic VLCFA increased during this period. Polyenoic VLCFA-sphingomyelin seems to occur exclusively in the testes and spermatozoa of mammals, and it is postulated that this lipid plays a role in reproduction.

Although most studies on the polyenoic fatty acids of mammalian testes and spermatozoa have focused on those with carbon chain lengths of 22 or less, polyenoic very-long-chain fatty acids (VLCFA) with more than 22 carbon atoms are also present. Bridges and Coniglio (20) observed that tetracosatetraenoic acid (c,&4,".6) and tetracosapentaenoic acid (C245.n.6) are formed from linoleic acid (C182,n"j) and arachidonic acid (c204,n.6) in rat testes. Grogan and Lam (22) and Grogan and Huth (23) showed that cultured mouse spermatocytes and spermatids can synthesize tetracosatetraenoic acid (Czk4,n.e) and tetracosapentaenoic acid (c24:+6) from arachidonic acid (c2@,,.6). It was subsequently demonstrated that arachidonic acid (c,04,n.6) is the precursor of even longer chain polyenoic fatty acids such as hexacosatetraenoic acid (C264,n.6), hexacosapentaenoic acid (c26:5,n.6), octacosapentaenoic acid (c28,&), and triacontapentaenoic acid (CW,,~.~) in rat testes (25). Poulos et al. (26) reported that polyenoic VLCFA represent a significant component of the total fatty acids (approximately 5%) in the spermatozoa of a number of mammalian species, and that the composition varies markedly according to the species. Human spermatozoa contain predominantly di-, tri-, and tetraenoic fatty acids with up to 32 carbon atoms, while boar, ram, and bull spermatozoa also contain pentaenoic and/or hexaenoic fatty acids with up to 34 carbon atoms. In ram and bull spermatozoa the polyenoic VLCFA belong to both the n-3 and n-6 series; however, in human and boar spermatozoa these fatty acids are mainly n-

Novel Molecular Species of Sphingomyelin in
Testes and Spermatozoa 1747 6 derivatives. It has been shown that the polyenoic VLCFA in ram spermatozoa are enriched in molecular species of sphingomyelin (27).
In the present paper we have investigated the lipid distribution of polyenoic VLCFA in the testes and spermatozoa of several mammalian species. We were curious to know whether the polyenoic VLCFA content varies with testicular development. During the course of this work we have identified a unique group of 2-hydroxylated polyenoic VLCFA.

RESULTS AND DISCUSSION
Fractionation of lipids isolated from mature ram, bull, boar, and rat testes and spermatozoa and adult human spermatozoa using a combination of silicic acid and DEAE-cellulose DE52 column chromatography and preparative TLC indicated that polyenoic VLCFA were mainly present in sphingomyelin (80-go%), with the remainder in ceramide (10-20%) and phosphatidylcholine (less than 5%). This confirms and extends our earlier report that polyenoic VLCFA are concentrated in molecular species of sphingomyelin in ram spermatozoa (27). Sphingomyelin containing polyenoic VLCFA (designated polyenoic VLCFA-sphingomyelin) was found to be a minor component of seminal plasma and was undetectable in the accessory glands (prostate gland and/or seminal vesicles) of male reproductive tracts. This phospholipid was absent from the ovaries of female animals. It is noteworthy that brain and retina are the only other tissues reported to contain significant amounts of polyenoic VLCFA, but these compounds are localized in phosphatidylcholine, rather than sphingomyelin (33,34,(39)(40)(41). Polyenoic VLCFA-sphingomyelin was found to be exclusively present in the seminiferous tubules of dissected adult rat testes. This suggests that the polyenoic VLCFA-sphingomyelin in spermatozoa originates from testicular germinal cells and/or Sertoli cells.
Polyenoic VLCFA represented approximately 15% of the total fatty acids in testicular and spermatozoan sphingomyelin of adult animals. The polyenoic VLCFA composition of sphingomyelin was similar in the testes and spermatozoa of a particular mammalian species, but there was a higher concentration of the polyenoic VLCFA-sphingolipid in spermatozoa. The polyenoic VLCFA composition of testicular and spermatozoan sphingomyelin varied between animal types. In human spermatozoa, the polyenoic VLCFA were mainly n-6 derivatives with two to four double bonds and even-carbon chain lengths up to 32. Polyenoic VLCFA belonging to the n-3 and n-6 series with four, five, and six double bonds and even-carbon chain lengths up to 34 predominated in ram and bull testes and spermatozoa. Rat and boar testes and spermatozoa contained principally n-6 polyenoic VLCFA with three to five double bonds and even-and odd-carbon chain lengths up to 34.
The total ion chromatograms produced by GLC-MS analysis of polyenoic VLCFA isolated from the sphingomyelin of rat and boar testes and spermatozoa, contained some additional, previously unreported, broad peaks. Their mass spectra contained ions at m/z 79,91, and 150, which are characteristic of n-6 polyenoic VLCFA (36,37). Each had a strong high mass ion which was smaller than that in the mass spectrum of the adjacent polyenoic VLCFA. From their mass spectra * Portions of this paper (including "Experimental Procedures," Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. and their chromatographic behavior, these peaks were tentatively assigned as hydroxylated polyenoic VLCFA. Fig. 1 represents the partial total ion chromatograms of the fatty acid methyl esters obtained from the sphingomyelin of adult bull and boar testes, indicating the hydroxylated fatty acids.

Figs. 1-4, and
Further confirmation that these broad peaks were hydroxylated polyenoic VLCFA was obtained after preparation of the PFP derivatives. Partial total ion chromatograms of derivatized polyenoic VLCFA methyl esters are presented in Fig. 2 for rat and boar testicular sphingomyelin. The PFP derivatives chromatographed as sharp peaks, and their mass spectra contained strong molecular ions as well as ions from the loss of pentafluoropropionic acid. These chromatograms show that for every polyenoic VLCFA with 28 or more carbon atoms there was a hydroxylated analogue. There were only minor differences in the composition of the fatty acids in the samples derived from the rat and boar.
The position of hydroxylation was not directly apparent from the mass spectra. Accordingly, a sample of the fatty acids from the sphingomyelin of rat testes was hydrogenated. Mass spectra were obtained of the hydroxylated CZ8 and Cz0 methyl esters that were produced. Fig. 3 shows the mass spectrum of hydroxylated C28:4,n.6 methyl ester and its reduction product, hydroxylated Czk0 methyl ester. The McLafferty rearrangement ions of m/z 74 and 87, observed in the mass spectra of saturated methyl esters, were not present, but instead ions at m/z 90 and 103. These ions were used by Carballeira and Lopez (42) to identify 2-hydroxylated saturated fatty acids (as methyl esters) in marine sponges. The hydroxylated polyenoic VLCFA isolated from the sphingomyelin of rat and boar testes and spermatozoa were thus assigned as 2-hydroxylated. Unfortunately, the optical isomerism of the testicular and spermatozoan 2-hydroxylated polyenoic VLCFA could not be determined because the amount of sample available was too low. Naturally occurring 2-hydroxylated fatty acids have previously been found to possess the R configuration (42), and presumably this also applies to the 2-hydroxylated derivatives isolated in the present study.
To provide additional evidence that these 2-hydroxylated polyenoic VLCFA were associated with sphingomyelin, a FAB mass spectrum was obtained on a sample of the boar testicular sphingomyelin region after isolation by TLC. Protonated molecular ions of sphingomyelin containing saturated fatty acids were observed, but none for the hydroxylated polyenoic VLCFA species. The low mass region contained ions at mlz 125,166, and 184 which are characteristic of a phosphocholine skeleton (38). Ions from other phospholipid skeletons were not detected in the spectrum. FAB-MS is often an unpredictable technique and there may be one of several reasons for the nonappearance of the molecular ions. The hydroxy group may inhibit the formation of protonated molecular ions, the protonated molecular ions may fragment before detection, or the protonated molecular ions of the saturated fatty acid species may suppress the formation of ions of other species present.
Hydrolysis of the boar testicular sphingomyelin fraction with sphingomyelinase, to remove the phosphocholine, gave a product with the same TLC relative retention as ceramide. A portion of the product was transesterified, treated with pentafluoropropionic anhydride, and the fatty acid methyl esters analyzed by GLC-MS. 2-Hydroxylated fatty acids with zero to six double bonds and odd-and even-carbon chain lengths from 16 to 34 were identified. The remainder of the product was converted to a trimethylsilyl derivative and analyzed by GLC-MS. A series of ceramides containing fatty acids such as c16:0, Cleo, and Czk0 were recognized by their molecular ions and characteristic fragmentation ions as described by Samuelsson and Samuelsson (43). Ceramides containing hydroxylated fatty acids including hydroxylated Ciao (mass spectrum mlz 769 (M)+, 458, 283, 731, Cleo, C20:o, Gz0, C2z1, and small amounts of C24:3,,.6 (mass spectrum m/z 785 (M-TMSOH)', 592, 283, 73) and C26:3,,.6 (mass spectrum m/z 813 (M-TMSOH)', 620, 283, 73) were identified. Unfortunately, the higher homologues above c 2 6 were involatile, even with a short, lightly loaded capillary chromatography column operating at its temperature limit.
Acid hydrolysis of the boar testicular sphingomyelin gave a product with the same TLC mobility as lysosphingomyelin. The FAB mass spectrum (shown in Fig. 4) contained ions at m/z 437 and 465 which can be rationalized to belong to lysosphingomyelin containing two long chain bases, i.e. Clel and C1el (sphingosine).
Together these separate analyses confirm that the 2-hydroxylated polyenoic VLCFA are indeed incorporated in molecular species of sphingomyelin found in rat and boar testes. T o our knowledge, this is the first report that 2-hydroxylated polyenoic VLCFA exist in nature. 2-Hydroxylated VLCFA have been detected in the sphingolipids of mammalian brain (44, 45) and in the phospholipids of marine sponges (42,46); however, these are saturated and monoenoic derivatives.
The polyenoic VLCFA content of sphingomyelin in rat testes varied from birth to sexual maturity, as depicted in Table 1. Polyenoic VLCFA-sphingomyelin was not present in the testes in 10-and 20-day-old animals. Sphingomyelin containing non-hydroxylated polyenoic VLCFA (C28:4,n.6, C30:5,n.6, and c30,,.3) was initially detected in rat testes 25 days after birth. At 30 days of postnatal life, 2-hydroxylated polyenoic VLCFA (C28:4,n.6, C30:5,n.6, and c30,,.3) were observed in addition to the non-hydroxylated derivatives in the sphingomyelin. From 35 to 50 days, the sphingomyelin contained non-hydroxylated and 2-hydroxylated polyenoic VLCFA which were n-3 hexaenoic and n-6 tetraenoic and pentaenoic derivatives with carbon chain lengths from 24 to 32. The nonhydroxylated and 2-hydroxylated n-3 hexaenoic VLCFA were no longer detectable in the testicular sphingomyelin of 55and 60-day-old animals, unlike the corresponding n-6 tetraenoic and pentaenoic VLCFA. Table I indicates that the total amount of polyenoic VLCFA associated with sphingomyelin in rat testes increased approximately 19-fold from 25 to 40 days after birth (mainly due to an increase in non-hydroxylated and 2-hydroxylated C28:4,n.6 and c30,,,.6) and then remained static to 60 days. The total amount of 2-hydroxylated polyenoic VLCFA relative to non-hydroxylated polyenoic VLCFA in the testicular sphingomyelin increased with maturation ( Table I). Differentiation of testicular germinal cells (spermatogonia -P spermatocytes --.* round spermatids + condensing spermatids + spermatozoa) in rats is complete about 50 days after birth, with the formation of round and condensing spermatids at approximately 30 and 40 days, respectively (47,48). The production of non-hydroxylated and 2-hydroxylated polyenoic VLCFA-sphingomyelin in rat testes after 25 days of life may be associated with spermatid development. It is also feasible that Sertoli cells have the capacity to produce polyenoic VLCFA-sphingomyelin at this time. There is evidence that polyenoic VLCFA are synthesized by chain elongation and desaturation of shorter-chain n-3 and n-6 precursors in rat testes (20,22,23,25) and other tissues (49-52). Polyenoic VLCFA are presumably introduced into the sphingomyelin molecule via the direct transfer of ceramide to phosphocholine from phosphatidylcholine by phosphatidylcholine-ceramide cholinephosphotransferase (53,54). It is not known whether the polyenoic VLCFA become 2-hydroxylated in the unesterified form or after incorporation into ceramide or sphingomyelin. The synthesis of non-hydroxylated and 2-hydroxylated polyenoic VLCFA-sphingomyelin in the testes clearly warrants further investigation. Sphingomyelin containing polyenoic VLCFA is likely to play a significant role in male reproductive physiology because of its exclusive presence in the testes and spermatozoa and its unique structure. This phospholipid may be crucial in maintaining membrane integrity of spermatozoa and facilitate their survival in the female reproductive tract. There is considerable evidence that sphingolipids and their breakdown products are important in cellular regulation (55)(56)(57)(58)(59). It is therefore tempting to speculate that polyenoic VLCFA-sphingomyelin and/or its polyenoic VLCFA-catabolism products (unesterified fatty acids and ceramide) may be metabolically active compounds and involved in the regulation of spermatozoan events such as motility, the acrosome reaction or membrane fusion during fertilization. The presence of 2hydroxylated polyenoic VLCFA in the testicular and spermatozoan sphingomyelin of some animal species is an additional complicating factor.

EXPERlllENTN PROCEDURES
~--S l l ? c l c acid (325 mesh). In some c a l e~. rat testes were