Long and Very Long Chain Polyunsaturated Fatty Acids of the n-6 Series in Rat Seminiferous Tubules ACTIVE DESATURATION OF 24:4n-6 TO 24:5n-6 AND CONCOMITANT FORMATION OF ODD AND EVEN CHAIN TETRAENOIC AND PENTAENOIC FATTY ACIDS UP TO C,,*

The formation of long and very long chain (VLC) n-6 polyunsaturated fatty acids (PUFA) in isolated rat seminiferous tubules was investigated by following the metabolism of three l-14C-labeled n-6 tetraenoic fatty acids (20:4, 24:4, and 324) and [U-“Clacetate. In contrast to [I4C]32:4, which was poorly incorporated and altered, [‘4C]20:4 and [14C]24:4 were efficiently taken up by the tubules, esterified into lipids, elongated to VLCPUFA, and desaturated to pentaenoic fatty acids; the rate of [14C]24:4 desaturation to [14C]24:5 was notably high. The main products with [I4C]acetate as precursor were labeled saturates and VLCPUFA, most of the label in tetraenoic and pentaenoic acids appearing in 24:4 and 24:5, respectively. These two Car polyenes, that a testis We that a- role the PUFA.


1) Recipient of a Charles John Everard Postdoctoral Scholarship
from the University of Adelaide, Australia.
The fatty acids are abbreviated by the convention, number of carbon atoms and number of double bonds; n-3 or n-6 denote the position of the first double bond counting from the methyl end. The abbreviations used are: VLC, very long chain; PUFA, polyunsaturated fatty acids; CGP, EGP, choline and ethanolamine glycerophospholipids respectively; PC, PE, PS, PI, phosphatidyl-choline, ethanola-(PUFA) in the lipids of the male reproductive tract in many mammalian species. In rat testis, where these two fatty acids are major PUFA constituents, n-6 pentaenoic and tetraenoic fatty acids with chain lengths in excess of 22 carbons, such as 24:5n-6 and 24:4n-6, have been isolated and characterized as normal products of the elongation and desaturation of 204n-6 (1-3), in turn known to derive from the essential fatty acid 182n-6. Also in rat testis, Grogan (4) characterized n-6 tetraenes and pentaenes with very long chains (VLC), such as 28:4n-6 and 30:5n-6, which were labeled with 14C 2 days after intratesticular injections of [ 1-14C]20:4n-6. Work from our own laboratories has shown that n-6 tetraenoic and pentaenoic as well as n-3 pentaenoic and hexaenoic fatty acids with chain lengths from 24 to 34 carbons (VLCPUFA) occur in mammals. They have been characterized in the spermatozoa of a variety of species including man (5) and in the retina photoreceptor cells of several vertebrates (6, 7 ) . Whereas the VLCPUFA of retina are specifically concentrated in phosphatidylcholine (PC) ( 7 ) , those of spermatozoa are important components of sphingomyelin (SM) (8). Recent reports show that in several mammals spermatozoon and testis SM also contains 2-hydroxy derivatives of n-3 and n-6 VLCPUFA, those of the n-6 series predominating in rat (9).
Much is stilt to be learned on the biochemistry of VLCPUFA and their derivatives. In the present work, the metabolism of these polyenes has been studied in isolated seminiferous tubules from rat testis using three l-14C-labeled n-6 tetraenoic fatty acids and [U-14C]acetate as precursors. Our purpose was to compare how tetraenoic fatty acids of different chain lengths, 204n-6, 244n-6, and 32:4n-6, are processed in the testicular tissue and to determine whether seminiferous tubules are able to produce labeled n-6 tetraenes and pentaenes from [14C]acetate, in the manner very long (n-3) pentaenes and hexaenes were shown to be synthesized in mammalian retina (10). During the course of our research into this specific area, we have come up with interesting findings on more general aspects of PUFA metabolism. We report results which are relevant to the mechanism of A4 desaturation of n-6 PUFA, suggest the involvement of triacylglycerols in PUFA metabolism, and show the occurrence of unusual odd chain tetraenoic and pentaenoic fatty acids which reveal the existence of non-conventional pathways in PUFA metabolism. mine, serine, and inositol respectively; SM, sphingomyelin; TG, triacylglycerols; FAME, fatty acid methyl esters; MS, mass spectrometry; TLC, GC, HPLC, thin-layer, gas-liquid, and high performance liquid chromatography, respectively; DMEM, Dulbecco's modified Eagle's medium.
Male Porton rats aged 6 weeks were killed by C02 asphyxiation and the testes rapidly excised. Seminiferous tubules were obtained from the testes essentially as described in Refs. 13 and 14, except that the medium used was Dulbecco's modified Eagle's medium (DMEM), high glucose (25 mM), containing glutamine and sodium pyruvate (GIBCO-BRL), to which 50 and 100 mg/liter of penicillin and streptomycin, respectively, and 3.7 g/liter of sodium bicarbonate had been added. Gently decapsulated testes were incubated in standard plastic culture flasks for 15 min at 30 "C in 10 ml of the above medium containing collagenase (1 mg/ml), using an agitation of 50 cycles/min. The flask caps were pierced with syringe needles to allow continuous in-and out-flow of gases during incubation. The gasphase consisted of a water-saturated medical air atmosphere (5% C0.r). The incubations with the enzyme were followed by two successive washings, each with 10 ml of fresh DMEM medium. The long stretches of seminiferous tubules obtained were separated by decantation from the cells released from the testicular interstitium, predominantly composed of blood cells, macrophages, and Leydig cells (13,14). The tubules thus prepared were incubated for 1 or 2 h with the radioactive precursors in 10 ml of DMEM containing the l-"Clabeled fatty acids (1 pCi/sample dissolved in 100 pl of ethanol), or for 1-4 h with [U-"Clacetate (62 pCi/sample dissolved in water). Each sample contained the tubules from one testis in 10 ml of medium (536 f 37 pg of total lipid phosphorus/flask). Incubations proceeded at 35 "C, under the mentioned gas atmosphere, with an agitation of 60 cycles/min. The effluent gas was bubbled into KOH to trap the ["C]C02 produced.
After incubation, tubules were separated from incubation media by brief centrifugation. In the case of ["Clacetate, the collected tubules were resuspended in fresh DMEM and centrifuged again to reduce water-soluble metabolites present in the samples before lipid extraction. Lipid extracts from tubules or media were prepared and partitioned into organic and aqueous phases according to the procedure of Folch et al. (15). When ["Clacetate was present, three successive washings of the extracts with Folch's upper phase solvents were also performed to reduce the level of water-soluble radioactivity in the organic phases. Lipid classes were separated by standard thin layer chromatography (TLC), using precoated, 250-pm thick silica gel plates (Merck). Polar lipids were resolved on magnesium acetateimpregnated TLC plates using chloroform/methanol, 28% ammonia (65:35:5) for the first, and chloroform/acetone/methanol/acetic acid/ water (50201010:5) for the second dimension (16). Neutral lipids were separated on Silica Gel G in two steps, using hexane/ether/ acetic acid (60401) up to the middle and then hexane/ether/acetic acid (901O:l) up to the top of the TLC plates. After preparative isolation, the choline and ethanolamine glycerophospholipids (CGP, EGP) were resolved into their major subclasses by TLC after briefly exposing the lipids to HC1 (17).
Fatty acid methyl esters (FAME) were prepared from lipids by treatment with 14% BF3 in methanol (18) or with 1.5% HzSO4 in previously degassed, HPLC grade methanol (4 h at 75 "C) in screwcap sealed tubes under N2 (19), both procedures yielding similar fatty acid patterns from lipids. FAME were purified by TLC on Silica Gel G using dichloromethane or hexane/ether (95:5) and resolved into groups of similar unsaturation on plates impregnated with AgN03 (4 g/plate), using chloroform/methanol (95:5) for the resolution of polyenoic fractions, and chloroform/methanol(99:1) for that of dienoic, monoenoic, and saturated ones. To separate FAME according to chain length, the major fractions were located, scraped from the plates, eluted, and subjected to reverse-phase TLC using KCIS plates (Whatmann) as support and acetonitrile/tetrahydrofuran (9O:lO) as solvent (19). After development, the reverse-phase plates were subjected to autoradiography (Hyperfilm, Amersham) as previously described (19) for the location of radiolabeled areas. The fatty acid bands were scraped directly from the plates into liquid scintillation vials. The small fraction of 2-hydroxy FAME was isolated by similar procedures, except that chloroform/methanol (9010) was used for the argentation TLC of its polyenoic constituents.
After TLC, lipid zones were located by exposing the plates to iodine vapor, for phosphorus and radioactivity measurements, or to UV light after spraying with 2',7'-dichlorofluorescein for further analytical procedures. Elution of lipid compounds from the TLC supports was done with chloroform/methanol/acetic acid/water (5039:1:10), the eluates being then partitioned with ammonia solutions and washed (20). Phospholipids were quantified by phosphorus analysis (21). The fatty acid composition of lipids was determined by gas-liquid chromatography (GC) after conversion of the lipids to FAME, using a polar stationary phase and NZ as the carrier gas. Methyl heneicosanoate was used as an internal standard for the quantification of lipids not containing phosphorus. Capillary GC-MS was used to confirm the identification of components of the FAME fractions separated by argentation TLC essentially as described in previous work (5,8). HPLC of the same fractions was performed using acetonitrile as solvent (0.5 ml/min, room temperature) and a glass-lined 0.4 X 25cm stainless steel column packed with 5-pm particle size octadecylsilane (SGE, Victoria, Australia). Fatty acids were detected by their absorption at 192 nm (22) and collected for radioactivity measurements, this procedure yielding similar "C distributions to reversephase TLC. Radioactivity was measured in glass vials by standard liquid scintillation counting techniques. When lipids were in the presence of silica gel, water was added, the vial contents were thoroughly mixed with the scintillation mixture, and the support was allowed to settle before counting. In the case of FAME separated by argentation TLC, 2 M NaCl was used instead of water to trap the silver as AgCl and then the mixture. When the support was octadecylsilane, no water was added to the system.

RESULTS
Metabolic Transformations of [l -14C]20:4n-6, 24:4n-6, and 324n-6-When incubated with similar amounts of these fatty acids (-1 pCi, -1.7 p~) , rat seminiferous tubules took up from the media more of the total [l4C]20:4 than of the [1-14C] 24:4 initially provided, and much more of these two than of the [1-I4C]32:4 (90.5, 76.7, and 25.5%, respectively, percentages which were the same after 1 and 2 h of incubation). Most of the label remaining in the media could be accounted for by I4C-fatty acids not utilized by the tissue (20:4 < 24:4 < 32:4), but a fraction was also recovered as labeled water-soluble metabolites. In lipid extracts prepared from both media and tubules, this water-soluble fraction was the largest for The incorporation and distribution among lipids of the three polyenes was similar after 1 and 2 h of incubation, indicating that a plateau of incorporation had been reached by 1 h (Table I). Most of the label esterified in neutral lipids was concentrated in triacylglycerols (TG). These were in fact the most highly labeled lipids in all three cases, especially taking into consideration that they amounted to only 15.0 * 0.6 mo1/100 mol of lipid phosphorus. Another highly labeled non-polar lipid was a minor fraction tentatively identified as 1-@alkyl, 2,3-diacylglycerols Distribution of esterified label (W) Phosphatidylcholine (Table IIA) and then resolved according to chain length (Table IIB). Prior to this separation it had been observed that most of the radioactivity was in the three cases in regular fatty acids (>99.8%); however negligible, the conversion into 2-hydroxy-fatty acid derivatives was relatively more active for   partly "recycled," in the sense that it was oxidized and the products reutilized, as was evident from the formation of 14Csaturated fatty acids, the main one being palmitate. This process was most active for ["C]24:4, as was the case with the desaturation and the formation of 14C02 and water-soluble products.
Utilization of fU-14C]Acetate-The utilization of this precursor by seminiferous tubules increased continuously between 1 and 4 h of incubation. In samples incubated for 4 h, nearly 9% of the total radioactivity initially provided, in water, was recovered in the form of lipid-associated *' C, and as much as 5% in the form of 14C02. The 14C present in lipids was mostly in esterified form, less than 1% of the label appearing as free fatty acids throughout the period in vitro. Of the total 14C in fatty acids, only a small percentage (0.19 f 0.02%) was in 2-hydroxy derivatives. As much as 75% of the label in fatty acids was in saturated components, the rest being mostly concentrated in fatty acids with 1-6 double bonds.
The incorporation of [U-14C]acetate into lipids increased continuously between 1 and 4 h of incubation, the rate tending to reach a plateau between 3 and 4 h. A large proportion of the esterified label was in non-polar lipids. As with 1-I4Cpolyenes, the triacylglycerols (Table I) constituted the most highly labeled lipid class, alone making up for -49 and -41% of the total lipid label after 1 and 4 h of incubation, respectively. These decreasing percentages were not due to a decrease in TG labeling; they only reflect the fact that increases in the labeling of polar lipids were relatively slower. Also as with 14C-PUFA, with [14C]acetate the ether-linked TG were highly labeled in proportion to their mass.
Despite the increasing I4C incorporation over 4 h of incubation, the percentage distribution of label among lipid fatty acid types remained remarkably constant, as indicated by the relatively small deviations in the data in Table 111. There were, nevertheless, some significant differences among lipid classes in the proportions of saturated and polyenoic fatty acids incorporated into the various lipid classes. TG contributed most, not only to the actual amounts of radioactivity but also to the pattern of labeling observed in the total lipid. These lipids concentrated the largest share of the total saturated and polyenoic fatty acids synthesized from [14C]acetate and were the constituents in which the percentage of label in polyenes was the largest.
The distribution of acetate-derived I4C among individual fatty acids separated by chain length (Table IV) was also relatively constant between 1 and 4 h of incubation. More than 80% of the total 14C in saturates was in palmitate. The higher the degree of unsaturation, the larger the proportion of radioactivity in components having longer chains. Tetraenes from Czo to Czs and pentaenes from C22 to C30 all had some 14C from acetate; albeit with meagre labeling, 30:4 and 325 were also detected (Table IV and Fig. 2). Of the total 14C in tetraenes and pentaenes, the largest proportions were in 24:4 and 245, respectively. Comparing the distribution of radioactivity and the composition of the fractions (also shown in Table IV), it was apparent that the lowest relative specific radioactivities among tetraenoic and pentaenoic fatty acids corresponded to the major components, 20:4 and 225, and the highest to the pairs 22:4/24:4 and 24:5/26:5, respectively.

Very Long Polyenoic Fatty Acids of Rat Seminiferous Tu-
bules-In seminiferous tubule total lipid, 22:5n-6 accounted for 22%, and the sum of VLCPUFA for about 6% of the fatty acids, Almost 90% of this sum was made up of 24:4n-6 and 24:5n-6, and the rest of polyenes up to (232. None of the major glycerophospholipids contained significant amounts of VLCPUFA longer than 24:4 and 245. PUFA with chain lengths longer than c 2 4 were found to be concentrated in specific neutral lipids and in sphingomyelin. l'riacylglycerols were considerably rich in PUFA, with nearly 5% 20:4, 5% 22:4, and 26% 225; almost 10% of TG fatty acids was accounted for by VLCPUFA, most (90%) of this being 24:4 and 245. The minor lipid in the band of ether-linked TG contained similar percentages of 20:4 and 22:4 to TG, had somewhat less 2 2 5 (22%), and was even richer in VLCPUFA. We were surprised to find that as much as 34% of the fatty acids of this lipid were VLCPUFA, of which 70% were the c 2 4 polyenes, and the rest longer. Similarly unexpected was the fact that VLCPUFA accounted for as much as 33% of the acyl chains of seminiferous tubule cholesteryl esters, this minor lipid class (0.5 -t 0.05 mo1/100 mol lipid phosphorus)

TABLE IV
Distribution of radioactivity from ['4C]acetate (disintegrationslmin %) compared with the % distribution Of (weight %) amW? the fatty acids of the total lipid of rat seminiferous tubules Fatty acid methyl ester fractions were obtained by argentation TLC and resolved by means of reverse-phase TLC according to chain lengths. &&oactivity was located by autoradiography and measured by liquid scintillation counting. Even when the amount of radioactivity increased from 1 to 4 h incubation, the distribution of label among fatty acids did not vary significantly with time, for the percentages were averaged (mean values f S.D. are shown). The corresponding distribution of mass, obtained in separate analyses by GLC, is given in parentheses.

Fraction
Dienes Trienes being the richest in, and the main source of, very long chain pentaenoic fatty acids such as 26:5, 2 8 5 , and 305 (5, 14, and 1%, respectively, of cholesteryl ester fatty acids). SM (4.9 & 0.7 mo1/100 mol of lipid P) was the lipid with the lowest percentage of Cz4 polyenes, but was the main source of 284n-6 (alone, 5.0 f 0.5% of SM fatty acids) among other VLCPUFA (total, -7%). This phospholipid was also accountable for the 2-hydroxy derivatives of 284,30:4,305, and 32:5 shown in Fig. 2 (9). Odd Chain Polyenes-Along with the regular even chain fatty acids, all fatty acid fractions of rat seminiferous tubules, from saturates to pentaenes, were found to contain a series of minor odd chain components (Table IV). We first observed the odd chain PUFA as 14C-FAME on the autoradiograms we employed to locate compounds labeled with [14C]acetate on reverse-phase TLC plates, which prompted us to examine them by HPLC (Fig. 2 ) . By separating the acids first by degree of unsaturation and then by chain length, we were able to spot unusual odd chain n-6 tetraenoic and pentaenoic fatty acids of diverse chain lengths. Their identity was confirmed by capillary GC-MS as described for other PUFA (5, 8). In fact, some odd chain polyenes had previously been noticed along with the fatty acids of spermatozoa (5,9). On reversephase columns, the odd chain tetraenes and pentaenes of seminiferous tubules conformed to the chromatographical behavior expected of a complete homologous series, covering the asterisks denote an increase in detector sensitivity (at 190 nm). On the right, the retention of all compounds, expressed as the logarithm of their capacity factors (k') has been plotted as a function of their carbon atoms. Circles, FAME; triangles, 2-hydroxy FAME.

0 q 24
the whole range of chain lengths from 21 to 31 carbon atoms (Fig. 2).

DISCUSSION
The results presented here show that rat seminiferous tubule cells are capable of synthesizing tetraenoic and pentaenoic fatty acids of the n-6 series covering a whole range of chain lengths up to 32 or more carbons. The use of The pattern of fatty acid labeling observed in this work suggests that the synthesis of very long tetraenes and pentaenes up to C32 occurs through sequential addition of two carbon units to pre-existing fatty acids, starting from 204n-6 and 22:5n-6, respectively, in analogy to the usual, malonyl CoA-dependent elongations (23)  that the latter was labeled, at least, at its C3. Although on the basis of the present results option ( a ) cannot be entirely ruled out, the evidence is on the whole against it: microsomes from rat testis have been shown not to produce such a desaturation using [3H]22:4 as substrate (24), and the very existence of this putative A4 desaturase activity has been not only questioned for some time, but has recently been disproven (25 (24): this apparent discrepancy may be due to the fact that fatty acid desaturation in vitro is normally assayed using a system that precludes fatty acid elongation. A third desaturase activity possibly interconnecting n-6 tetraenoic and pentaenoic fatty acids could be that involved in the desaturation of [14C]26:4 to ["C] 26:5. Although proven to be absent in liver (26), a A8 desaturase has been proposed as responsible for the conversion of [14C]202n-6 to [14C]20:3n-6 in rat testis (27). Even though such desaturation cannot be ruled out with the present data, the elongation of ["C]24:5 does appear to be more prominent quantitatively as a source of the available [14C]26:5.
Our data suggest that of all seminiferous tubule lipid classes, it is triglycerides which have a special role to play in PUFA metabolism. These lipids contain substantial proportions of PUFA and VLCPUFA, particularly the c 2 4 tetra-and pentaenoic fatty acids, and concentrate the highest levels of 14C-PUFA, whether exogenously provided or endogenously synthesized from ["Clacetate. These observations agree with earlier work showing that intratesticular injection of l-14Clabeled 204n-6 and 22:4n-6 results in the incorporation of a large proportion of the 14C into TG (1,24). Significant labeling of TG has also been observed in testicular slices incubated with [l-'4C]acetate (28). Far from constituting a mere "reservoir" for the accumulation of spare fatty acids, the triglycerides of seminiferous tubules appear to be involved actively in the metabolic transformations affecting PUFA. With both labeled PUFA and acetate as precursors, it is clearly apparent that most of the changes observed in the total lipid of seminiferous tubules take place in fatty acids that are esterified to TG. Whether PUFA are transformed metabolically and immediately transferred to TG, or whether intact TG are used directly as substrates for changes affecting their acyl chains remains to be elucidated and provides a challenging area for future research.
The presence of odd chain tetra-and pentaenoic PUFA in seminiferous tubule lipids raises several questions, of which one of the most intriguing relates to the possible metabolic origin of these components. Odd chain straight saturates and, through desaturation, the corresponding monoenes, are known to be formed ( a ) by de m u 0 synthesis through successive additions of 2-carbon units to propionyl CoA, ( b ) by 8oxidation of the odd chain fatty acids thus synthesized; and (c) by one-carbon shortening of even numbered fatty acids by a-oxidation (29,30). Much less is known on the formation of odd chain PUFA, although it is unlikely that they are formed ( a ) during the process of their biosynthesis, since all n-6 and n-3 PUFA are known to originate in the stepwise addition of 2-carbon units to two even-carbon essential fatty acids, 18:2n-6 and 18:3n-3, or ( b ) during the /3-oxidation of any of such even-carbon PUFA, since this would be bound to result only in even-carbon (n-2) homologues. Odd chain PUFA are most likely to arise ( c ) via oxidation of even carbon PUFA, 1carbon unit at a time, converting each to its corresponding (n-1) homologue and releasing a 1-carbon unit molecule, such as COZ, in the process. We speculate that such a-oxidation is an important mechanism in the shortening of fatty acids and that odd chain PUFA are metabolic intermediates.
The presence of label from [14C]acetate in odd chain polyenes including 21:4 and 23:5 supports the view that, in addition to one step of P-oxidation, two steps of a-oxidation of  (31) after injection of [5-l4c] 22:5n-6. Retroconversion of n-6 and n-3 Cz2 PUFA has also been documented in a number of animal tissues and cells (32) using 3-14C-labeled Cz, PUFA. Although fatty acid retroconversion was not specifically addressed in the present work ( a ) the active production of 14C-labeled l4COZ and water-soluble metabolites from [1-14C]24:4; ( b ) the continuously increasing and parallel patterns of 14C-fatty acid and l4CO2 production during incubation with [U-14C]acetate; (c) the lack of timedependent changes in the distribution of label among acetatederived fatty acids from 1 to 4 h of incubation; and ( d ) the formation of labeled odd-chain PUFA, are consistent with the possibility of VLCPUFA retroconversion. In our view it is probable that PUFA undergo a continuous process of elongation, the resulting VLCPUFA being continuously chain shortened or retroconverted to maintain a concerted, balanced equilibrium in the proportion of PUFA and to enable cells to conserve fatty acids with metabolically costly double bonds. a-Oxidation could play a specific role in fatty acid metabolism, providing an alternative mechanism to @-oxidation for the formation of shorter fatty acids from longer ones, including the major Czo and C 2 2 polyenes.