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A time-course study of the effects of gonadal hormones on avian liver fatty acid composition
ht. J.
Biochem., 1975, Vol. 6, pp. 25 to 30.
A TIME-COURSE HORMONES
STUDY
ON AVIAN
Pergamon Press.
OF THE
LIVER
D. BALNAVE
EFFECTS
FATTY
AND
J.
Printed in Great Britain
ACID
25
OF GONADAL COMPOSITION
PEARCE
Department of Agricultural and Food Chemistry, Queen’s University, Newforge Lane, Belfast BTg 5PX, U.K., and Department of Agriculture, Northern Ireland
(Rzceived7 3uncI974) ABSTRACT I. An investigation was carried out to study the effects of gonadal hormone administration, over a period of 1-g days, on liver fatty acid composition in immature pullets. 2. The only treatments which substantially influenced liver fatty acid composition were those containing oestradiol. 3. When compared to the changes observed under oestradiol treatment, testosterone and progesterone exerted little effect, although considerable changes occurred at early time intervals following testosterone treatment. 4. The variations in liver fatty acid composition are compared with the changesin hepatic lipogenesis and fatty acid oxidation observed in these experiments.
IT ISknown that the pattern of lipid changes, similar to those occurring at the onset of lay in the pullet, can be induced in immature birds by any one of a range of oestrogenic compounds (Lorenz, 1954). There is also much evidence to suggest that androgens and progesterone are involved in the natural and physiological changes encountered at point-of-lay in the fowl (Domm, 1987 ; Common, Bolton & Rutledge, 1948; Brant & Nalbandov, 1956). Although, in contrast to oestrogen, neither aqlrogen nor progesterone have any effect on liver lipid content it has recently been suggested that these hormones may exert a significant influence on the metabolic processes of hepatic lipid biosynthesis and degradation in the pullet (Balnave, 1968; Pearce & Balnave, 1978; Balnave & Pearce, 1974). In addition, Balnave (1969) has observed that testosterone, but not progesterone, significantly influences liver fatty acid composition in immature chicks, the changes resulting from testosterone treatment contrasting with the patterns of change observed as a result of oestrogen treatment. The analyses described in this paper were undertaken in an attempt to clarity the roles of oestrogen, androgen and progesterone in influencing avian liver fatty acid composition
and to confirm previous studies on this topic (Balnave, 1969). The present work is a timecourse study ofthe effects ofgonadal hormones on liver fatty acid composition to determine whether changes in composition could be related to differences in the specific activities of hepatic lipogenic enzymes (Balnave & Pearce, I 974). MATERIALS
AND
METHODS
Day-old Thomber Superwhite pullets were obtained from a local hatchery and reared to four weeks of age on a standard chick mash. They were then randomly distributed, four birds per treatment, into the following treatment groups Treatment Treatment Treatment Treatment Treatment
I
: 0.3 ml. maize oil
mg. oestradiol dipropionate in 0.3 ml. maize oil 3 : 2 mg. testosterone propionate in 0.3 ml. maize oil 4: 2 mg. progesterone in 0.3 ml. maize oil 5 : 2 mg. oestradiol dipropionate and I mg. testosterone propionate in 0.3 ml. maize oil 2 : 2
These hormone treatments were administered by intramuscular injection on alternate days and the birds killed I, 2, 4 or g days after the commencement of hormone administration. The birds were killed by decapitation and exsanguination. The livers were rapidly excised and the liver lipids determined gravimetrically after extraction by the method of Folch, Lees &
26
Id.
BALNAVEANDPEARCE
The fatty acids were Sloane-Stanley ( 1957). methylated using BFs-methanol reagent and the resulting esters separated by gas-liquid chromatography on a Perkin-Elmer F30 chromatograph using columns packed with 6% DEGS on 8o-100 mesh Chromosorb W (acid-washed, DMCS treated). Peak areas were determined using a Vidar 6300 Digital Integrator. In a second experiment the above procedures were again carried out except that the combined oestrogen plus testosterone treatment was excluded. The physiological responses to gonadal hormone administration and the enzymatic data from these two experiments have been reported separately (Balnave & Pearce, 1974; Pearce & Balnave,
‘974).
The observed fatty acid percentages were transformed bv the annular transformation method and analysis ‘of variznce carried out. The backtransformed data are given in the tables along with the S.E.M. of the transformed data. All statistical significances quoted are at the 5 ‘A level. RESULTS
The effects of gonadal hormone treatment on liver lipid content in these experiments are shown in Table I. There were no significant differences in liver lipid content at any time between the control, testosteroneand progesterone-treated pullets. The overall mean liver lipid contents of the birds receiving oestradiol and the double hormone treatment were significantly increased compared with the other treatment groups. In the first experiment this effect of oestradiol was observed as early as the second day although no statistically significant differences in liver lipid content were observed until the fourth
Table L-THE
Control Testosterone Progesterone Oestrogen Oestrogen + Testosterone SEM
(60df) (48df)
Biochem.
day of hormone treatment. In the second experiment the effect of oestradiol on liver lipid content attained significance on the day following the initial hormone administration. The influence of gonadal hormones on the liver percentage palmitic and palmitoleic acid contents is shown in Table II. The overall mean palmitic acid level in the oestradioltreated birds was significantly greater than in the control group in both experiments. In addition, in the first experiment the overall mean liver percentage palmitic acid in the oestradiol-treated birds was significantly greater than for testosteroneand progesterone-treated birds, but no significant differences between the oestradiol and double hormone-treated groups were observed. The maximum relative differences between the oestradiol-treated birds and the control group was observed on the day following the initial oestradiol treatment. In both experiments the increase in the overall mean liver palmitoleic acid level in the oestradiol-treated birds was significantly greater than for the controls, testosteroneand progesterone-treated birds (Table II). In the first experiment the overall mean palmitoleic acid level in the double hormone-treated birds was significantly greater than for the controls, but no significant overall differences were observed between the oestradiol and double hormone treatments or between the control, testosterone- and progerterone-treated birds. In the first experiment, substantial, though non-
INFLUENCE OF GONADAL HORMONESON LNER LIPID CONTENT (g/xoo g liver) DAYS
TREATMENT
3.
EXPERIMENT
I
2
I 2 I 2 I 2
5’27 4’37 4’97 428 5’15 4.01
4’75 3’47 4’94 409 488 3.61
I 2
5’33 6.46
I
6.00
I 2
4
9
OVERALL MEAN
4.65
480
6.82 5’39
4’52 3’77 5.58 3’71 454 3’49 12’01 8.19
4’19 4.64 4’57 5.11 4’04 6.41 7.66
3’95 5’03 4.16 4’92 3’79 7.64 6.92
6.28
8.15
8.02
7.11
0.130 0’529
0.565 0.264
1975,6
HORMONES
AND FATTY
ACIDS
27
significant, increases in palmitoleic acid levels resulting from oestradiol and double hormone administration were attained on the second day of the experiment and these increases were maintained at subsequent time intervals. In the second experiment a significant response to oestradiol treatment was observed on the first day and these effects were again maintained throughout the subsequent time intervals. The effects of gonadal hormones on the stearic and oleic acid contents from both experiments are shown in Table III. The overall mean percentage stearic acid contents of the oestradiol- and double hormonetreated chicks were significantly reduced compared with the other treatment groups and no significant difference between the oestradiol and double hormone treatments was observed. No significant differences were observed between the control, testosteroneand progesterone-treated pullets. There was a rapid reduction in the stearic acid levels of birds receiving oestradiol or the double hormone treatment. In the first experiment the reduction in the stearic acid levels on day I were significantly greater for the double hormone-treated birds than for those receiving oestradiol alone but thereafter no significant differences between these treatment groups could be observed. In the first experiment the stearic acid content in the double hormone-treated pullets was significantly reduced compared with the control group on day I whereas in the oestradiol-treated birds the reduction in stearic acid levels only attained significance, compared with contro1 birds, on the second day. In the second experiment the rapid reduction in the stearic acid levels of the oestradiol-treated chicks only attained statistical significance on days 4 and g. On day g the stearic acid levels in the liver of the testosterone- and progesteronetreated birds were significantly reduced compared with the controls. In the first experiment the overall mean amounts of oleic acid in the livers of both the oestradiol and double hormone-treated birds were significantly greater than for control, testosteroneand progesterone-treated birds (Table III). There was no significant differ-
28
BALNAVE
AND
ence between the oestradiol and double hormone-treated groups. In the second experiment the overall mean oleic acid level in the oestradiol-treated pullets was significantly greater than for the other treatment groups. It should also be noted that in both experiments the livers of the testosteronetreated birds contained greater quantities of oleic acid than the control birds at all time intervals, although this increase only attained statistical significance on days 4 and g of the second experiment. At all time intervals, except day 4 of the first experiment, the livers ofthe testosterone-treated birds also contained greater amounts of oleic acid than the livers of birds receiving progesterone. Although there was a substantial increase in the percentage oleic acid content in the liver of the birds receiving oestradiol and the double hormone treatment in experiment I, these differences only attained statistical significance from day 2, whereas in the second experiment the differences resulting from oestradiol treatment attained statistical significance on day I. The effects of gonadal hormones on the liver percentage levels of the essential fatty acids, linoleic and arachidonic, are shown in Table IV. The overall mean values for the first experiment indicate that the level of linoleic acid in the oestradiol-treated chicks was significantly less than for control and testosterone-treated birds. The overall mean linoleic acid level in the double hormonetreated pullets was also significantly less than for controls and no significant difference between the oestradiol and double hormone treatments was observed. In the second experiment no significant differences in the overall means were observed although that of the oestradiol-treated birds was again the lowest of any treatment group. Generally, the linoleic acid contents in the oestradioltreated birds were lower than in control birds at any time interval in either experiment and the decrease in linoleic acid content was observed as early as one day after the initial hormone administration. In the first experiment the double hormone-treated birds showed the same pattern as birds receiving oestradiol alone.
PEARCE
Int.
3.
Biochetn.
HORMONES AND FATTY
ACIDS
29
No significant overall effects of hormones on the arachidonic acid levels in the first experiment were observed although in the second experiment the overall mean arachidonic acid level for the testosterone-treated birds was significantly reduced compared with either the control or progesterone-treated birds (Table IV). It was again difficult to observe any specific trends when the data were compared on a daily basis although various differences did attain statistical significance in both experiments. DISCUSSION The changes in fatty acid composition obtained through administering gonadal hormones to immature pullets are similar to those reported previously (Balnave, I 969, 1971; Balnave & Brown, 1967). When the overall mean data and the data for the later time intervals are considered it is apparent that the only treatments which substantially influenced liver fatty acid composition were those containing oestradiol. When compared to the extent of the changes observed under oestradiol treatment, testosterone and progesterone exerted little effect and the liver fatty acid compositions of birds treated with these hormones were little different from those of the controls. When the day I data from the first experiment are examined it is apparent that oestradiol had a substantial influence on liver fatty acid composition before any marked increase in liver lipid content was observed. This effect is particularly noteworthy as enzymatic studies in this experiment (Balnave & Pearce, 1974) have shown that the maximum specific activities of the two hepatic lipogenic enzymes, ATP-citrate lyase and NADP-malate dehydrogenase, were attained only on the second day following the initial oestradiol administration. These results suggest that liver lipid metabolism is modified in response to oestradiol before lipogenic enzyme concentrations are altered. In this connection it is interesting that Yeh & Leveille (1970) have reported that changes in lipogenesis occur in fasted chicks without any change in lipogenic enzyme activity. At early time intervals considerable changes
30
BALNAVEANDPEARCE
in the percentages of the individual fatty acids occurred in the livers of the testosteronetreated birds relative to controls, whereas no such effect was observed in birds receiving progesterone. These variations in the fatty acid composition of the testosterone-treated birds may be related to variations in lipogenesis and fatty acid oxidation observed one day after testosterone treatment of immature pullets (Balnave 8z Pearce, 1974; Pearce & Balnave, 1974). In these studies no significant variations in lipogenesis or fatty acid oxidation resulted from progesterone treatment and this would perhaps confirm the lack of any significant effect of progesterone on liver fatty acid composition in this and earlier work (Balnave, I 969). The principal effects of oestradiol administration to immature pullets were to increase both the 16-carbon fatty acids, palmitic and palmitoleic, as well as oleic acid. Corresponding reductions in stearic and linoleic acids were observed. The reduction in linoleic acid is probably the result of increased fatty acid synthesis since this is an essential fatty acid and must be supplied in the diet. The increase in the percentages of the monounsaturated fatty acids may presumably be a reflection of increased desaturation activity. Balnave (1971) has observed that most of the changes in blood and liver fatty acids resulting from the attainment of sexual maturity in pullets may be reproduced by administering exogenous oestrogens to immature birds. However, in the case of the immature birds receiving oestradiol there was a substantial increase in the percentage blood and liver palmitic acid levels which was not reflected in the mature bird. This presumably reflects an abnormal utilization of palmitic acid in the immature bird treated with oestradiol. A probable explanation for this difference is the fact that the laying hen requires lipid for egg production purposes and so lipid is continually removed from the body. Clearly no such mechanism operates in the immature pullet and the only likely possibilities of decreasing blood and liver palmitic acid are through increased deposition of body fat or oxidation to carbon dioxide.
Int.
3.
Biochem.
ACKNOWLEDGEMENT The authors thank Mr T. W. Oliver and Mr A. H. Johnson for skilled technical assistance. REFERENCES BALNAVE,D. (rg68), ‘ The influence of gonadal hormones on the uptake of [r4C]acetate by liver linid fractions in the immature male chick ‘, B.?&?riD”;. . trc?690St27’.Theeffects of certain gonadal hormone& the content and composition of lipids in the blood and liver of immature male chicks ‘, Camp. Biochem. Physiol., 28, 709-7 16. BALNAVP., D. (I 97 I), ‘ The influence of exogenous oestrogens and the attainment of sexual maturity on fatty acid metabolism in the immature pullet ‘, Camp. Biochem. Physiol., 40, 189-197. BALNAVE,D., & BROUTN,W. 0, (1g67), ‘ Liver fatty acid composition of normal and gonadal hormone-treated chicks ‘, Camp.Biochem. Physiol., 2% 313-317. BALNAVE,D., & PEARCE,J. (tgj4), ‘ Induction by gonadal hormones of hepatic lipogenic enzyme activity in immature pullets ‘, 3. Endow., 61, 29-43. BRANT,J. W. A., & NALBANDOV, A. V. (rg56), ‘ Role of sex hormones in albumen secretion by the oviduct of chickens ‘, Pot&. Sci., 35,6g2-Too. COMMON, R. H.. BOLTON,W., & RUTLEDGE,W. A. ( I 948); ‘ The* influence of gonadal hormones on the comnosition of the blood and liver of the domestic’fowl ‘, 3. E&r., 5,263-273. DOMM, L. V. (tg37), ‘ Observations concerning anterior pituitary-gonadal interrelations in the fowl ‘, Cold. Spring. Harb. Spp. &ant. Biol., 5, Fo1”,‘,;‘5”7,. LEES, N., & SLOANE-STANLEY, G. H. (1g57), ‘ A simple method for the isolation and purification of total lipids from animal tissues ‘, 3. Bial. Gem., 226,4gj-309. LOFZENZ, F. W. (rg34), ‘ Effects of oestrogens on the domestic fowl and applications in the poultry industrv ‘. vitams. Harm.. 12. 215-275. PEARCE,Jl, 6 BALNAVE,D.*(I g$; A’ &ne-course study of the effects of gonadal hormone administration on lipogenic enzyme activity in the liver of the immature pullet ‘, Biochem. Sot. Trans., I,
769-n-I.
PEARCE, J., & BALNAVE,D. (rg74), ‘Effects of gonadal hormones on the hepatic oxidation of fatty acids in immature pullets ‘, 3, Endow., 62, 425-426. YEH, Y. Y., & LEVEILLE, G. A. (igjo), ‘ Hepatic fatty acid synthesis and plasma free fatty acid levels in chicks subiected to short periods of food restriction and refeeding ‘, J.- Nutr., NW, 1389-1398. Key Word Index: Immature hormones, liver fatty acids.
pullets, gonada
Biochem., 1975, Vol. 6, pp. 25 to 30.
A TIME-COURSE HORMONES
STUDY
ON AVIAN
Pergamon Press.
OF THE
LIVER
D. BALNAVE
EFFECTS
FATTY
AND
J.
Printed in Great Britain
ACID
25
OF GONADAL COMPOSITION
PEARCE
Department of Agricultural and Food Chemistry, Queen’s University, Newforge Lane, Belfast BTg 5PX, U.K., and Department of Agriculture, Northern Ireland
(Rzceived7 3uncI974) ABSTRACT I. An investigation was carried out to study the effects of gonadal hormone administration, over a period of 1-g days, on liver fatty acid composition in immature pullets. 2. The only treatments which substantially influenced liver fatty acid composition were those containing oestradiol. 3. When compared to the changes observed under oestradiol treatment, testosterone and progesterone exerted little effect, although considerable changes occurred at early time intervals following testosterone treatment. 4. The variations in liver fatty acid composition are compared with the changesin hepatic lipogenesis and fatty acid oxidation observed in these experiments.
IT ISknown that the pattern of lipid changes, similar to those occurring at the onset of lay in the pullet, can be induced in immature birds by any one of a range of oestrogenic compounds (Lorenz, 1954). There is also much evidence to suggest that androgens and progesterone are involved in the natural and physiological changes encountered at point-of-lay in the fowl (Domm, 1987 ; Common, Bolton & Rutledge, 1948; Brant & Nalbandov, 1956). Although, in contrast to oestrogen, neither aqlrogen nor progesterone have any effect on liver lipid content it has recently been suggested that these hormones may exert a significant influence on the metabolic processes of hepatic lipid biosynthesis and degradation in the pullet (Balnave, 1968; Pearce & Balnave, 1978; Balnave & Pearce, 1974). In addition, Balnave (1969) has observed that testosterone, but not progesterone, significantly influences liver fatty acid composition in immature chicks, the changes resulting from testosterone treatment contrasting with the patterns of change observed as a result of oestrogen treatment. The analyses described in this paper were undertaken in an attempt to clarity the roles of oestrogen, androgen and progesterone in influencing avian liver fatty acid composition
and to confirm previous studies on this topic (Balnave, 1969). The present work is a timecourse study ofthe effects ofgonadal hormones on liver fatty acid composition to determine whether changes in composition could be related to differences in the specific activities of hepatic lipogenic enzymes (Balnave & Pearce, I 974). MATERIALS
AND
METHODS
Day-old Thomber Superwhite pullets were obtained from a local hatchery and reared to four weeks of age on a standard chick mash. They were then randomly distributed, four birds per treatment, into the following treatment groups Treatment Treatment Treatment Treatment Treatment
I
: 0.3 ml. maize oil
mg. oestradiol dipropionate in 0.3 ml. maize oil 3 : 2 mg. testosterone propionate in 0.3 ml. maize oil 4: 2 mg. progesterone in 0.3 ml. maize oil 5 : 2 mg. oestradiol dipropionate and I mg. testosterone propionate in 0.3 ml. maize oil 2 : 2
These hormone treatments were administered by intramuscular injection on alternate days and the birds killed I, 2, 4 or g days after the commencement of hormone administration. The birds were killed by decapitation and exsanguination. The livers were rapidly excised and the liver lipids determined gravimetrically after extraction by the method of Folch, Lees &
26
Id.
BALNAVEANDPEARCE
The fatty acids were Sloane-Stanley ( 1957). methylated using BFs-methanol reagent and the resulting esters separated by gas-liquid chromatography on a Perkin-Elmer F30 chromatograph using columns packed with 6% DEGS on 8o-100 mesh Chromosorb W (acid-washed, DMCS treated). Peak areas were determined using a Vidar 6300 Digital Integrator. In a second experiment the above procedures were again carried out except that the combined oestrogen plus testosterone treatment was excluded. The physiological responses to gonadal hormone administration and the enzymatic data from these two experiments have been reported separately (Balnave & Pearce, 1974; Pearce & Balnave,
‘974).
The observed fatty acid percentages were transformed bv the annular transformation method and analysis ‘of variznce carried out. The backtransformed data are given in the tables along with the S.E.M. of the transformed data. All statistical significances quoted are at the 5 ‘A level. RESULTS
The effects of gonadal hormone treatment on liver lipid content in these experiments are shown in Table I. There were no significant differences in liver lipid content at any time between the control, testosteroneand progesterone-treated pullets. The overall mean liver lipid contents of the birds receiving oestradiol and the double hormone treatment were significantly increased compared with the other treatment groups. In the first experiment this effect of oestradiol was observed as early as the second day although no statistically significant differences in liver lipid content were observed until the fourth
Table L-THE
Control Testosterone Progesterone Oestrogen Oestrogen + Testosterone SEM
(60df) (48df)
Biochem.
day of hormone treatment. In the second experiment the effect of oestradiol on liver lipid content attained significance on the day following the initial hormone administration. The influence of gonadal hormones on the liver percentage palmitic and palmitoleic acid contents is shown in Table II. The overall mean palmitic acid level in the oestradioltreated birds was significantly greater than in the control group in both experiments. In addition, in the first experiment the overall mean liver percentage palmitic acid in the oestradiol-treated birds was significantly greater than for testosteroneand progesterone-treated birds, but no significant differences between the oestradiol and double hormone-treated groups were observed. The maximum relative differences between the oestradiol-treated birds and the control group was observed on the day following the initial oestradiol treatment. In both experiments the increase in the overall mean liver palmitoleic acid level in the oestradiol-treated birds was significantly greater than for the controls, testosteroneand progesterone-treated birds (Table II). In the first experiment the overall mean palmitoleic acid level in the double hormone-treated birds was significantly greater than for the controls, but no significant overall differences were observed between the oestradiol and double hormone treatments or between the control, testosterone- and progerterone-treated birds. In the first experiment, substantial, though non-
INFLUENCE OF GONADAL HORMONESON LNER LIPID CONTENT (g/xoo g liver) DAYS
TREATMENT
3.
EXPERIMENT
I
2
I 2 I 2 I 2
5’27 4’37 4’97 428 5’15 4.01
4’75 3’47 4’94 409 488 3.61
I 2
5’33 6.46
I
6.00
I 2
4
9
OVERALL MEAN
4.65
480
6.82 5’39
4’52 3’77 5.58 3’71 454 3’49 12’01 8.19
4’19 4.64 4’57 5.11 4’04 6.41 7.66
3’95 5’03 4.16 4’92 3’79 7.64 6.92
6.28
8.15
8.02
7.11
0.130 0’529
0.565 0.264
1975,6
HORMONES
AND FATTY
ACIDS
27
significant, increases in palmitoleic acid levels resulting from oestradiol and double hormone administration were attained on the second day of the experiment and these increases were maintained at subsequent time intervals. In the second experiment a significant response to oestradiol treatment was observed on the first day and these effects were again maintained throughout the subsequent time intervals. The effects of gonadal hormones on the stearic and oleic acid contents from both experiments are shown in Table III. The overall mean percentage stearic acid contents of the oestradiol- and double hormonetreated chicks were significantly reduced compared with the other treatment groups and no significant difference between the oestradiol and double hormone treatments was observed. No significant differences were observed between the control, testosteroneand progesterone-treated pullets. There was a rapid reduction in the stearic acid levels of birds receiving oestradiol or the double hormone treatment. In the first experiment the reduction in the stearic acid levels on day I were significantly greater for the double hormone-treated birds than for those receiving oestradiol alone but thereafter no significant differences between these treatment groups could be observed. In the first experiment the stearic acid content in the double hormone-treated pullets was significantly reduced compared with the control group on day I whereas in the oestradiol-treated birds the reduction in stearic acid levels only attained significance, compared with contro1 birds, on the second day. In the second experiment the rapid reduction in the stearic acid levels of the oestradiol-treated chicks only attained statistical significance on days 4 and g. On day g the stearic acid levels in the liver of the testosterone- and progesteronetreated birds were significantly reduced compared with the controls. In the first experiment the overall mean amounts of oleic acid in the livers of both the oestradiol and double hormone-treated birds were significantly greater than for control, testosteroneand progesterone-treated birds (Table III). There was no significant differ-
28
BALNAVE
AND
ence between the oestradiol and double hormone-treated groups. In the second experiment the overall mean oleic acid level in the oestradiol-treated pullets was significantly greater than for the other treatment groups. It should also be noted that in both experiments the livers of the testosteronetreated birds contained greater quantities of oleic acid than the control birds at all time intervals, although this increase only attained statistical significance on days 4 and g of the second experiment. At all time intervals, except day 4 of the first experiment, the livers ofthe testosterone-treated birds also contained greater amounts of oleic acid than the livers of birds receiving progesterone. Although there was a substantial increase in the percentage oleic acid content in the liver of the birds receiving oestradiol and the double hormone treatment in experiment I, these differences only attained statistical significance from day 2, whereas in the second experiment the differences resulting from oestradiol treatment attained statistical significance on day I. The effects of gonadal hormones on the liver percentage levels of the essential fatty acids, linoleic and arachidonic, are shown in Table IV. The overall mean values for the first experiment indicate that the level of linoleic acid in the oestradiol-treated chicks was significantly less than for control and testosterone-treated birds. The overall mean linoleic acid level in the double hormonetreated pullets was also significantly less than for controls and no significant difference between the oestradiol and double hormone treatments was observed. In the second experiment no significant differences in the overall means were observed although that of the oestradiol-treated birds was again the lowest of any treatment group. Generally, the linoleic acid contents in the oestradioltreated birds were lower than in control birds at any time interval in either experiment and the decrease in linoleic acid content was observed as early as one day after the initial hormone administration. In the first experiment the double hormone-treated birds showed the same pattern as birds receiving oestradiol alone.
PEARCE
Int.
3.
Biochetn.
HORMONES AND FATTY
ACIDS
29
No significant overall effects of hormones on the arachidonic acid levels in the first experiment were observed although in the second experiment the overall mean arachidonic acid level for the testosterone-treated birds was significantly reduced compared with either the control or progesterone-treated birds (Table IV). It was again difficult to observe any specific trends when the data were compared on a daily basis although various differences did attain statistical significance in both experiments. DISCUSSION The changes in fatty acid composition obtained through administering gonadal hormones to immature pullets are similar to those reported previously (Balnave, I 969, 1971; Balnave & Brown, 1967). When the overall mean data and the data for the later time intervals are considered it is apparent that the only treatments which substantially influenced liver fatty acid composition were those containing oestradiol. When compared to the extent of the changes observed under oestradiol treatment, testosterone and progesterone exerted little effect and the liver fatty acid compositions of birds treated with these hormones were little different from those of the controls. When the day I data from the first experiment are examined it is apparent that oestradiol had a substantial influence on liver fatty acid composition before any marked increase in liver lipid content was observed. This effect is particularly noteworthy as enzymatic studies in this experiment (Balnave & Pearce, 1974) have shown that the maximum specific activities of the two hepatic lipogenic enzymes, ATP-citrate lyase and NADP-malate dehydrogenase, were attained only on the second day following the initial oestradiol administration. These results suggest that liver lipid metabolism is modified in response to oestradiol before lipogenic enzyme concentrations are altered. In this connection it is interesting that Yeh & Leveille (1970) have reported that changes in lipogenesis occur in fasted chicks without any change in lipogenic enzyme activity. At early time intervals considerable changes
30
BALNAVEANDPEARCE
in the percentages of the individual fatty acids occurred in the livers of the testosteronetreated birds relative to controls, whereas no such effect was observed in birds receiving progesterone. These variations in the fatty acid composition of the testosterone-treated birds may be related to variations in lipogenesis and fatty acid oxidation observed one day after testosterone treatment of immature pullets (Balnave 8z Pearce, 1974; Pearce & Balnave, 1974). In these studies no significant variations in lipogenesis or fatty acid oxidation resulted from progesterone treatment and this would perhaps confirm the lack of any significant effect of progesterone on liver fatty acid composition in this and earlier work (Balnave, I 969). The principal effects of oestradiol administration to immature pullets were to increase both the 16-carbon fatty acids, palmitic and palmitoleic, as well as oleic acid. Corresponding reductions in stearic and linoleic acids were observed. The reduction in linoleic acid is probably the result of increased fatty acid synthesis since this is an essential fatty acid and must be supplied in the diet. The increase in the percentages of the monounsaturated fatty acids may presumably be a reflection of increased desaturation activity. Balnave (1971) has observed that most of the changes in blood and liver fatty acids resulting from the attainment of sexual maturity in pullets may be reproduced by administering exogenous oestrogens to immature birds. However, in the case of the immature birds receiving oestradiol there was a substantial increase in the percentage blood and liver palmitic acid levels which was not reflected in the mature bird. This presumably reflects an abnormal utilization of palmitic acid in the immature bird treated with oestradiol. A probable explanation for this difference is the fact that the laying hen requires lipid for egg production purposes and so lipid is continually removed from the body. Clearly no such mechanism operates in the immature pullet and the only likely possibilities of decreasing blood and liver palmitic acid are through increased deposition of body fat or oxidation to carbon dioxide.
Int.
3.
Biochem.
ACKNOWLEDGEMENT The authors thank Mr T. W. Oliver and Mr A. H. Johnson for skilled technical assistance. REFERENCES BALNAVE,D. (rg68), ‘ The influence of gonadal hormones on the uptake of [r4C]acetate by liver linid fractions in the immature male chick ‘, B.?&?riD”;. . trc?690St27’.Theeffects of certain gonadal hormone& the content and composition of lipids in the blood and liver of immature male chicks ‘, Camp. Biochem. Physiol., 28, 709-7 16. BALNAVP., D. (I 97 I), ‘ The influence of exogenous oestrogens and the attainment of sexual maturity on fatty acid metabolism in the immature pullet ‘, Camp. Biochem. Physiol., 40, 189-197. BALNAVE,D., & BROUTN,W. 0, (1g67), ‘ Liver fatty acid composition of normal and gonadal hormone-treated chicks ‘, Camp.Biochem. Physiol., 2% 313-317. BALNAVE,D., & PEARCE,J. (tgj4), ‘ Induction by gonadal hormones of hepatic lipogenic enzyme activity in immature pullets ‘, 3. Endow., 61, 29-43. BRANT,J. W. A., & NALBANDOV, A. V. (rg56), ‘ Role of sex hormones in albumen secretion by the oviduct of chickens ‘, Pot&. Sci., 35,6g2-Too. COMMON, R. H.. BOLTON,W., & RUTLEDGE,W. A. ( I 948); ‘ The* influence of gonadal hormones on the comnosition of the blood and liver of the domestic’fowl ‘, 3. E&r., 5,263-273. DOMM, L. V. (tg37), ‘ Observations concerning anterior pituitary-gonadal interrelations in the fowl ‘, Cold. Spring. Harb. Spp. &ant. Biol., 5, Fo1”,‘,;‘5”7,. LEES, N., & SLOANE-STANLEY, G. H. (1g57), ‘ A simple method for the isolation and purification of total lipids from animal tissues ‘, 3. Bial. Gem., 226,4gj-309. LOFZENZ, F. W. (rg34), ‘ Effects of oestrogens on the domestic fowl and applications in the poultry industrv ‘. vitams. Harm.. 12. 215-275. PEARCE,Jl, 6 BALNAVE,D.*(I g$; A’ &ne-course study of the effects of gonadal hormone administration on lipogenic enzyme activity in the liver of the immature pullet ‘, Biochem. Sot. Trans., I,
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PEARCE, J., & BALNAVE,D. (rg74), ‘Effects of gonadal hormones on the hepatic oxidation of fatty acids in immature pullets ‘, 3, Endow., 62, 425-426. YEH, Y. Y., & LEVEILLE, G. A. (igjo), ‘ Hepatic fatty acid synthesis and plasma free fatty acid levels in chicks subiected to short periods of food restriction and refeeding ‘, J.- Nutr., NW, 1389-1398. Key Word Index: Immature hormones, liver fatty acids.
pullets, gonada