EFFECT OF DIETARY LINOLEIC AND LINOLENIC ACIDS ON TESTICULAR DEVELOPMENT IN THE RAT

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1 Quarterly Journal of Experimental Physiology (1983), 68, Printed in Great Britain EFFECT OF DIETARY LINOLEIC AND LINOLENIC ACIDS ON TESTICULAR DEVELOPMENT IN THE RAT W. M. F. LEAT, CHRISTINE A. NORTHROP, F. A. HARRISON AND R. W. COX* A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4A T and *Institute of Ophthalmology, University of London, 17/25 Cayton Street, London EC] V 9A T (RECEIVED FOR PUBLICATION 25 AUGUST 1982) SUMMARY In two experiments a total of twelve male rats were reared from weaning for up to 63 weeks on an essential fatty acid (EFA)-deficient diet alone (2 x two animals) or supplemented with the methyl esters of linoleic acid (18: 2w6) (2 x two animals) or linolenic acid (18: 3w3) (2 x two animals). Testicular development was normal in rats given 18:2w6, but in rats fed the EFAdeficient diet alone, and in those supplemented with 18:3w3 the testes were reduced in size. Histologically, a degeneration of the seminiferous tubules was noted, with progressive loss of the germinal cells, and with an absence of spermatozoa in the lumina of the seminiferous tubules and epididymides. Leydig cells appeared unaffected, and were prominent. The six rats in Experiment 1 were capable of mating with females reared on commercial diets, but only the two 18: 20)6 supplemented animals were fertile. There was a marked reduction in the percentage of arachidonic acid (20:4wj6) and docosapentaenoic acid (22: 516) in the total fatty acids of the atrophic testes. There was no compensatory increase in long-chain derivatives of 18: 3w3 in the 18: 3w3 fed rats and it is concluded that linolenic acid cannot replace linoleic acid in the development of the rat testis. INTRODUCTION The essential fatty acids (EFA) comprise two series of fatty acids of which linoleic acid (cis,cis-9,12-octadecadienoic acid [18:2wo6]) is the parent acid of the first series (w06), and linolenic acid (cis,cis,cis-9,12,15-octadecatrienoic acid [18:3w3]) is the parent acid of the second series (w3). These fatty acids undergo alternate desaturation and elongation to form a series of long chain polyunsaturated fatty acids some of which are precursors of prostaglandins (see Table 1). The non-essential fatty acid, oleic acid (cis-9-octadecenoic acid [18: 1 w9]), parent acid of the w9 series of fatty acids, is also metabolized by similar pathways but neither the parent acid nor its metabolites have any EFA activity. Each series is structurally distinct and fatty acids of the different series that have common designations (Table 1) are positional isomers. Linoleic acid is generally considered to be the major essential fatty acid and it satisfies the requirements of growth and reproduction of rats through two generations (Tinoco, Babcock, Hincenbergs, Medwadowski & Miljanich, 1978). Linolenic acid has only partial EFA activity (see Holman, 1968) and the extent to which it can replace linoleic acid is poorly defined (Food and Agriculture Organization (FAO) Report, 1978; Leat, 1981). However, the presence of long-chain derivatives of linolenic acid in the cerebral cortex and retina (Svennerholm, 1968; Daemen, 1973) suggests that the w3 series of EFA may have specific functions in these tissues. During the course of an investigation into the possible role of linolenic acid in the rat we observed that the testes of rats, reared for over 30 weeks with linolenic acid as the sole source of EFA, showed marked atrophy when compared with

2 222 W. M. F. LEAT, C. A. NORTHROP, F. A. HARRISON AND R. W. COX Table 1. Metabolism of the w9, w6 and w3 series offatty acids w9 series (oleic acid)c18: 1 C18:2 C20:2 C20: 3 C22:3 C22:4 w6 series (linoleic acid)c18:22 C18:3 C20:3 C20:4 C22:4 - C22:5 1 1 PGE, PGE2 PGF1, PGF21 w3 series (linolenic acid) C18:3 - C18:4 -C20:4 - C20:5 -. C22:5 - C22:6 1 PGE3 PGF3ot those of rats reared with linoleate supplements (Cox, Harrison, Leat & Northrop, 1981). These observations have now been confirmed and extended, and the results are presented in this paper. A preliminary communication has been made (Leat, Clarke, Harrison & Cox, 1982). METHODS Animals. Rats of the Wistar strain that had been bred at the Institute were used. They were the male progeny, weaned at 4 weeks of age, from dams fed a purified diet deficient in EFA and supplemented with either 18: 2w6 or 18: 3w3 (Leat & Northrop, 1981). The rats were housed in pairs in galvanized grill-bottomed cages in a room maintained at IC and lit from h daily. Food and water were provided ad libitum. Diet. The EFA-deficient diet consisted of (g/kg): sucrose, 666; casein, 160; hydrogenated coconut oil, 53 4; DL-methionine, 3 2; vitamin premix, 10 7; mineral premix, 42 7; kaolin, 21 3; methyl cellulose, 42 7; (see Leat & Northrop, 1981). This diet contained 40 mg 18:2 and 20 mg 18: 3/kg diet. Methyl linoleate or methyl linolenate (99% pure: Sigma Chemical Company Ltd) were fed daily at 1,ul/g body-weight/d for 5 consecutive d/week to a maximum of 150,ul/rat. Design of experiment (a) Experiment I (six rats). (i), A pair of weanlings from an 18: 3w3-supplemented dam was reared on the EFA-deficient diet (Nil group); (ii) a pair of weanlings from an 18: 2w6-supplemented dam was reared on the EFA-deficient diet and supplemented with 18: 2w6; (iii), a pair of weanlings from a second 18:3w3-supplemented dam was reared on the EFA-deficient diet and supplemented with 18:3w3. Between 15 and 32 weeks of age each male was placed with a mature female reared on a commercial diet, and was removed when a copulatory plug was found. The female rats used in the mating experiments had a mean age of 28 weeks (± 2-2S.E.M.) and a mean weight of 273 g (± 4-8 g S.E.M.). In each rat between weeks of age a unilateral orchidectomy was performed under general anaesthesia (see below), and the remaining testis was removed when the animals were killed at weeks of age. (b) Experiment 2 (eight rats). Eight weanlings from two dams supplemented with 18: 3C3 were reared as four pairs on: (i) EFA-deficient (Nil group); (ii) EFA-deficient diet supplemented with 18:2wo6; (iii), EFA-deficient diet supplemented with 18:3w3 and (iv), a commercial diet. In this experiment a unilateral orchidectomy was performed on one animal from each group at 11 weeks and on the second animal at 22 weeks of age. The remaining testis from each rat was removed when the animals were killed at weeks of age. The testes and epididymides obtained at biopsy and at death were dissected from adhering tissue and weighed separately. Each testis was cut into two portions, one half being retained for chemical analysis and the other half, together with the epididymis, being fixed in Bouin fluid for histology. At death a record was also made of the weights of the seminal vesicles and the ventral prostate. Surgical procedures. Anaesthesia was induced by i.p. injection of pentobarbitone solution ('SAGATAL' 60 mg/ml; May & Baker Ltd, Dagenham) at a dosage of 0 1 ml/100 g body weight and given about 45 min before surgery. Using aseptic procedures one testicle was exposed by incision

3 ESSENTIAL FATTY ACIDS AND TESTICULAR DEVELOPMENT 223 of the scrotum and splitting of the cremaster muscle. The vas deferens and the pampiniform plexus were separately ligated and the testis and epididymis removed. The cremaster muscle and peritoneum were closed with continuous catgut mersuture (3/0) and the skin incision was closed with single interrupted silk mersutures (3/0). Chemical methods. The portion of testis for analysis was immediately extracted with chloroform/methanol (2:1) by the method of Folch, Lees & Sloane-Stanley (1951), and an aliquot of the lower chloroform phase was transmethylated with 5% sulphuric acid/methanol. The methyl esters were separated on a 10% Silar-1OC column (Applied Science Laboratories Inc.) using a Pye 104 Chromatograph linked to a DP88 Integrating Computer. Peaks were identified by comparison with standards of mixed methyl esters. RESULTS Experiment 1. The general health of all experimental animals was good, although the rats fed the EFA-deficient diet alone grew more slowly than those supplemented with 18: 2c6 or 18: 3w3. For example, at 20 weeks of age the body-weights of the EFA-deficient males were 309 g and 268 g compared to 323 g and 363 g for the 18: 2w6 supplemented rats and 344 g and 333 g for those supplemented with 18:3w3. At about weeks of age the scrotal development of rats fed the EFA-deficient diet alone and of those supplemented with 18: 3w3 appeared less marked than in those given 18: 2w6. On palpation, the testes of the EFA-deficient and 18: 3w3 supplemented rats were smaller and less firm than those of the rats supplemented with 18: 2w6. However, each of the six males left a copulatory plug when placed with a mature female fed a commercial diet. The eleven females which copulated with the two EFA-deficient males did not become pregnant; neither did the nine females mated with the 18:3w3 supplemented males. However, ten of the thirteen mature females placed with the 18: 2w6 supplemented males became pregnant and gave birth to a total of eighty-eight pups. Four of these females, producing forty-four pups, had previously copulated with the 18: 3w3 supplemented males in their mating trials which did not produce any pregnancies. The 18: 2o6 and 18: 3w3 supplemented males were subsequently placed again with mature females, which were killed immediately after copulatory plugs were found. The female reproductive tract was removed and flushed out with a small volume of saline which was examined microscopically for the presence of spermatozoa. Spermatozoa were found only in the reproductive tract of the females mated with 18: 2w6 supplemented males, suggesting that the males supplemented with 18: 3w3 were azoospermic. Experiment 2. The second experiment was carried out to investigate testicular changes occurring before 35 weeks of age in rats of comparable nutritional status. It was again apparent from palpation of the scrotum that at both 11 weeks and 22 weeks of age the testes of the rats fed the EFA-deficient diet alone and of those supplemented with 18: 3w3 were smaller than those of the rats supplemented with 18: 2w6 or of those fed a commercial diet. Weights of reproductive organs Statistical analyses of the weights of the reproductive organs experiments (see Table 2) were carried out using Student's t test: from rats in both (a) Weight of organ (g/100 g body weight). Compared with rats fed 18:2w06, significant differences were found in: (a) testis: nil group 4 (***P < 0-001), 18:3 group 4 (***P < 0-001); (b), epididymis: nil group 4 (**P < 0 01), 18:3 group I (***P < 0-001).

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5 ESSENTIAL FATTY ACIDS AND TESTICULAR DEVELOPMENT 225 I..m. I- mm Fig. 1. A, testis of a 38 week old rat (no. 9; Experiment 1), fed an EFA-deficient diet supplemented with linoleate (18:2wo6), showing well developed seminiferous tubules. B, testis of a 37 week old rat (no. 49, Experiment 1), fed with an EFA-deficient diet alone, showing patchy atrophy of the seminiferous tubules. Stained with Haematoxylin and Eosin. Scale bar: 1 mm. A

6 226 W. M. F. LEAT, C. A. NORTHROP, F. A. HARRISON AND R. W. COX (b) Absolute weight of organ (g). Compared with rats fed 18: 2w6, significant differences were found in (a), testis: nil group I(**P < 0-01), 18:3 group I(***P < 0 001); (b), epididymis: nil group I(***P < 0-001), 18:3 group I(***P < 0001). The weights of the testes and epididymides from the rats of the 18:3w03 group were significantly less (*P < 0 05) than those from the nil group when compared on a g/100 g body weight basis. The number of observations for the weights of the other reproductive organs was insufficient for statistical analyses but there did not appear to be any marked difference between the groups in the weights of the prostate or seminal vesicles. Histology of testis Experiment 1. The rats fed 18:2w6 supplements revealed normal testicular structure (Fig. 1 A) with active spermatogenesis. The epididymis was teeming with spermatozoa. By comparison, the rats on the EFA-deficient diet (Fig. 1 B) and those on the diet supplemented with 18: 3w3 (Fig. 2 A) showed testicular atrophy with merely debris in the epididymis. The seminiferous tubules were small, most containing Sertoli cells only, but the Leydig cells were prominent (Fig. 2 B). In many tubules even the Sertoli cells were unusual. They had lost their peripheral arrangement, and formed irregular groups and cellular patterns within the tubules. Their nuclei were often small and distorted. Multinucleated cells were visible in some tubules and spermatogonia and primary spermatocytes were seen occasionally. In the testes removed at operation, the more severely damaged tubules were separated by less affected ones. Nevertheless, the changes were progressive so that after 61 weeks of 18: 3w3 supplementation, most tubules were badly damaged. The testicular atrophy in rats fed the EFA-deficient diet appeared less severe than that in rats supplemented with 18: 3w3 (Fig. 1 B and 2 A), and spermatozoa were sometimes recognizable. Experiment 2. At 11 weeks of age there was a decrease in the size of many of the tubules in rats fed 18: 3w3. These tubules were occupied by irregular arrays of Sertoli cells and by a few multinucleated cells. In other tubules, spermatogonia and primary spermatocytes were discernible and, infrequently, late spermatids or spermatozoa. Leydig cells tended to be conspicuous near the most markedly disordered tubules. By 22 weeks the alterations were more marked with a greater number of tubules that were shrunken and filled with displaced and abnormal Sertoli cells only. The Leydig cells were well defined, but only rarely could late spermatids and spermatozoa be observed. At 36 weeks the atrophy had progressed even further. Again, the EFA-deficient rats showed similar, but slightly less notable, degenerative changes when compared with rats on the 18:3w3 supplement. The rats on the 18:2w)6 supplement and on the commercial diet showed normal testicular structure at all ages. Fatty acid composition of testicular lipids The testicular fatty acids of rats fed 18 :2w)6 as the sole source of EFA were similar in composition to those of stock rats fed the commercial ration, the major fatty acids being 20: 4)6 (arachidonic acid) and 22: 5)6 (Table 3). The content of these long-chain polyunsaturated fatty acids was very much reduced in the testes of rats fed the EFA-deficient diet and in those supplemented with 18: 3w3. In the EFA-deficient rats there were compensatory increases, mainly in the proportion of 18: 1 and of 20:3w09, a fatty acid that accumulates specifically in EFA deficiency. In the 18: 3w3 fed rats the percentages of both

7 ESSENTIAL FATTY ACIDS AND TESTICULAR DEVELOPMENT 227 I- 1 mm mm Fig. 2. Testis of a 35 week old rat (no. 29; Experiment 1), fed an EFA-deficient diet supplemented with linolenate, (18: 303) showing: A, extensive atrophy of the seminiferous tubules (Scale bar: 1 mm), B, presence of Sertoli cells and absence of germinal cells in the seminiferous tubules, and prominence of the interstitial (Leydig) cells. Stained with Haematoxylin and Eosin. Scale bar 0 1 mm.

8 228 W. M. F. LEAT, C. A. NORTHROP, F. A. HARRISON AND R. W. COX Table 3. Major fatty acids (% by weight) of the total lipids of the testes of rats fed an EFAdeficient diet alone (Nil) and when supplemented with linoleate (18:2w6) or linolenate (18:3w3) Experiment 1 Experiment 2 Rat no Age (weeks) Diet Nil 18:2w6 18:303 Nil 18:2wo6 18:3w3 Commercial Fatty acid 16: : : : :2w : :3w :3w) :4o :5w :4wo : 5w) :5w :6w others : 3wj9 and 22: 5w6 were lower than in the EFA-deficient rats, which is probably a consequence of the inhibition of the metabolism of w9 and w6 fatty acids by the w3 fatty acid. DISCUSSION It is known that rats fed diets deficient in essential fatty acids (EFA) show testicular degeneration which can be prevented by the inclusion of linoleic acid in the diet (Burr & Burr, 1930; Evans, Lepkovsky & Murphy, 1934). Whether 18:3w3 can prevent testicular degeneration in the rat is not known; and evidence is conflicting concerning the effect of the long-chain polyunsaturated fatty acids derived from 18: 3w3. Nicolaysen (1962) commented that rats fed cod liver oil, which is rich in derivatives of 18: 3w3, were sterile, but experimental details were scanty. Ayala, Brenner & Dumm (1977), however, reported normal testicular development in rats fed from weaning to 7-9 weeks of age on fish oil containing high concentrations of derivatives of 18:3w3. The results of our experiments show that when male rats were reared from weaning for 7-63 weeks on 18: 3w3 as the sole source of essential fatty acid, degeneration of the seminiferous tubules occurred and increased in severity with time. In younger animals the degeneration was patchy with some tubules being more severely affected than others and with a marked reduction in the number of spermatids and spermatozoa. In older animals degeneration of the seminiferous tubules was extensive with eventual complete atrophy, spermatozoa being absent from both testis and epididymis. However, degeneration did not occur in the Leydig cells which appeared more prominent. The histological changes occurring in the testes of the EFA-deficient animals were similar to those reported earlier (Evans et al. 1934; Panos & Finerty, 1954). Possible reasons for the discrepancy between our results and those of Ayala et al. (1977) are: (a), the rats used in our investigations would have had very low reserves of 18:2W6, since they were born to mothers fed 18:3w3 (Leat & Northrop, 1981), and this may have

9 ESSENTIAL FATTY ACIDS AND TESTICULAR DEVELOPMENT resulted in a more rapid degeneration of the testes; (b), the duration of the spermatogenic cycle in the rat is 7-8 weeks (see Clermont, 1972) and any marked changes in testicular morphology might not become apparent in experiments lasting less than 8 weeks. The atrophy in the testes and epididymides ofthe EFA-deficient and 18: 3w3-supplemented animals noted on histological examination was reflected in the smaller weights of these organs when compared with those of the rats fed 18:2wo6. In addition, the mean weight (g±s.e.m.) of the testes of the 18: 3w3 fed animals ( ) was also lower than that of the EFA-deficient animals ( ) suggesting that the addition of 18: 3w3 to the EFA-deficient diet exacerbated the testicular degeneration occurring in the EFA-deficient animals. Supporting evidence is provided by (a), histology and (b), fatty acid composition of the testes. The degenerative changes in the tubules appeared more severe in the 18:3w03 rats than in the EFA-deficient animals. The percentage of docosapentaenoic acid (22: 5w6) in testicular lipids was lower in the 18: 3w3 rats than in the EFA-deficient animals and, since 22:5w6 is associated with spermatids and spermatozoa (Davis, Bridges & Coniglio, 1966), this could imply a decrease in the number of these germinal cells in the 18:3w3 fed rats. Competition between the 18: 3w3 and the very small amounts of 18: 2w6 in the EFA-deficient diet may explain this observation. The testicular degeneration in EFA deficiency may be (a), a primary hypogonadism resulting from a direct lack of 18:2w)6 or its metabolites in the germinal cells, or (b), a secondary dysfunction due to failure of the hypophysis to secrete gonadotrophins (Panos, Klein & Finerty, 1959). If the degeneration is primary any of the metabolites of 18:2w6 could be involved; but since 20:44w6 and 22: 5w6 are the acids found in the highest concentration (Table 3) it is most likely that one or both of these fatty acids could be responsible. The possibility should also be considered that the regression of spermatogenesis may be the result of malfunctioning Sertoli cells, which is often seen in various types of intoxications. However, 18:3w3 (linolenic acid) is a naturally occurring fatty acid (e.g. in grass and flax) and is unlikely to be toxic per se. A number of explanations are possible for the inability of 18: 3w3 to replace 18: 2W6 in testicular function, namely: (1) the degeneration of the testis may be indirect in origin. For example, the greater unsaturation of 18: 3w3 compared to 18: 2w6 may antagonize or inhibit the action of another nutrient such as vitamin E which is known to be required for testicular development (Mason, 1954). However, this would seem unlikely from the adequate quantities of vitamin E (64 mg/kg) in the diet and the small amounts (750,ul per week) of unsaturated fatty acids administered (see Moore, Sharman & Ward, 1959). (2) There may be a requirement in spermatogenesis for a specific configuration of fatty acid unsaturation. The major fatty acid of rat testis is a pentaenoic acid (Aaes-Jorgensen & Holman, 1958), and in the developing rat there is a close relation between the increase in the concentration of 22:5wo6 and the appearance of spermatids and spermatozoa (Davis et al. 1966). Our findings show that in the testes of the rats fed 18: 3w3 the concentrations of all long-chain derivatives of 18: 3w3 are low, suggesting, perhaps, that 22: 5w3 cannot replace 22: 5o6 in function. (3) One or more of the prostanoid derivatives of 18: 2w6, but not of 18: 3w3, may be involved in testicular development and spermatogenesis. For example, prostaglandins are known to be produced by the testis (Carpenter, 1974) and prostaglandin E2 (from metabolism of the w6 series) has been shown to prevent the impairment of male fertility associated with the feeding of EFA-deficient diets (Hafiez, 1974). If prostaglandins of the 2 series are a major factor controlling testicular development the absence of 22: 5w6 from the atrophic testes may merely reflect the resultant absence of spermatids and spermatozoa which, in the rat, are rich in 22: 5A6. Our results indicate, therefore, that in the rat linolenic acid (18: 3w3) cannot replace 229

10 230 W. M. F. LEAT, C. A. NORTHROP, F. A. HARRISON AND R. W. COX linoleic acid (18: 2w6) in testicular development. Structurally, linolenic acid can be considered to be linoleic acid with an additional double bond between carbon 15 and 16 (counting from the carboxyl end, or at position 3 counting from the methyl end), and it is of interest to determine why this additional bond converts a fatty acid that is active in spermatogenesis to one that is inactive. Whereas 22: 5w6 predominates in the testis of the rat, long-chain metabolites of 18: 3w03 are major components of the testis and spermatozoa of some other species, e.g. man, bull and ram (Bieri & Prival, 1965; Neill & Masters, 1972; Evans & Setchell, 1979). In other species, such as the mouse and guinea-pig, metabolites of both series of essential fatty acids are present in testis (Bieri & Prival, 1965). Whether 18:3w3 plays any role in testicular development in these species is as yet unknown. REFERENCES AAES-JORGENSEN, E. & HOLMAN, R. T. (1958). Essential fatty acid deficiency: 1. Content of polyenoic acids in testes and heart as an indicator of EFA status. Journal of Nutrition 65, AYALA, S., BRENNER, R. R. & DUMM, C. G. (1977). Effect of polyunsaturated fatty acids of the a-linolenic series on the development of rat testicles. Lipids 12, BIERI, J. G. & PRIVAL, E. L. (1965). Lipid composition of testes from various species. Comparative Biochemistry and Physiology 15, BURR, G. 0. & BURR, M. M. (1930). On the nature and role of the fatty acids essential in nutrition. Journal of Biological Chemistry 86, CARPENTER, M. P. (1974). Prostaglandins of rat testis. Lipids 9, CLERMONT, Y. (1972). Kinetics of spermatogenesis in Mammals: Seminiferous epithelium cycle and spermatogonial renewal. Physiological Reviews 52, Cox, R. W., HARRISON, F. A., LEAT, W. M. F. & NORTHROP, C. A. (1981). Essential fatty acids and testicular development in the rat. Journal of Physiology 313, 52P. DAEMEN, F. J. M. (1973). Vertebrate rod outer segment membranes. Biochimica et Biophysica Acta 300, DAVIS, J. T., BRIDGES, R. B. & CONIGLIO, J. G. (1966). Changes in lipid composition of the maturing rat testis. Biochemical Journal 98, EVANS, H. M., LEPKOVSKY, S. & MURPHY, E. A. (1934). Vital need of the body for certain unsaturated fatty acids. VI. Male sterility on fat-free diets. Journal of Biological Chemistry 106, EVANS, R. W. & SETCHELL, B. P. (1979). Lipid changes during epididymal maturation in ram spermatozoa collected at different times of the year. Journal ofreproduction and Fertility 57, FAO Food and Nutrition Paper No. 3. (1978). Dietary fats and oils in human nutrition. Rome: Food and Agriculture Organization of the United Nations. FOLCH, J., LEES, M. & SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry 226, HAFIEZ, A. A. (1974). Prostaglandin E2 prevents impairment of fertility in rats fed a diet deficient in essential fatty acids. Journal of Reproduction and Fertility 38, HOLMAN, R. T. (1968). Essential fatty acid deficiency. In Progress in the Chemistry of Fats and other Lipids, vol. 9, pp Oxford: Pergamon Press. LEAT, W. M. F. (1981). Man's requirement for essential fatty acids. Trends in Biochemical Science 6, IX-X. LEAT, W. M. F. & NORTHROP, C. A. (1981). Effect of dietary linoleic and linolenic acids on gestation and parturition in the rat. Quarterly Journal of Experimental Physiology 66, LEAT, W. M. F., CLARKE, N. G. E., HARRISON, F. A. & Cox, R. W. (1982). Testicular lipids of rats given an EFA deficient diet supplemented with linoleate or linolenate. Proceedings of the Nutrition Society 41, 59A. MASON, K. E. (1954). Effects of deficiency in animals. In The Vitamins, vol. 3, ed. SEBRELL, W. H. & HARRIS, R. S., pp Academic Press: London. MOORE, T., SHARMAN, I. M. & WARD, R. J. (1959). Cod-liver oil as both source and antagonist of vitamin E. British Journal of Nutrition 13, NEILL, A. R. & MASTERS, C. J. (1972). Metabolism of fatty acids by bovine spermatozoa. Biochemical Journal 127,

11 ESSENTIAL FATTY ACIDS AND TESTICULAR DEVELOPMENT 231 NICOLAYSEN, R. (1962). Lipids and diet. Proceedings of Royal Society, Series B 156, PANOS, T. C. & FINERTY, J. C. (1954). Effects of a fat-free diet on growing male rats with special reference to the endocrine system. Journal of Nutrition 54, PANOS, T. C., KLEIN, G. F. & FINERTY, J. C. (1959). Effects of fat deficiency and pituitary-gonad relationships. Journal of Nutrition 68, SVENNERHOLM, L. (1968). Distribution and fatty acid composition of phosphoglycerides in normal human brain. Journal of Lipid Research 9, TINOCO, J., BABCOCK, R., HINCENBERGS, I., MEDWADOWSKI, B. & MILJANICH, P. (1978). Linolenic acid deficiency: Changes in fatty acid patterns in female and male rats raised on a linolenic acid-deficient diet for two generations. Lipids 13, 6-17.