Lipid and Fatty Acid Composition of Testes of Quaking Mice J.G. C O N I G L I O , W.M. GROGAN,JR.,D.G. HARRIS, and M.L. F I T Z H U G H , Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 METHODS
ABSTRACT
The mice were C 57BL/6J males obtained from the Jackson Laboratory, Bar Harbor, Maine. Controls were furnished by the same supplier (qk/+ or +/+, indistinguishable). The animals were maintained on Purina chow until their death by decapitation. Testes were removed quickly and chilled in ice, after which they carefully were cleaned of all adhering tissue and weighed. A small amount of tissue was used from each of several testes from the various groups of quaking and control mice for histological examination. These were fixed in Bouin's media and stained with periodic Schiff stain (PAS) and counter-stained with Wright's iron hematoxylin. The balance of the tissues was either hydrolyzed with alcoholic potassium hydroxide for total fatty acid separation by methods reported previously (5) or homogenized in Folch mixture (chloroform:methanol 2/1) for extraction of total lipid. Aliquots of the extracts were used for determinations of triglyceride (6), phospholipid phosphorus (7), and of free and esterified cholesterol (8). Classes of lipids wre separated by thin layer chromatography (TLC) using the solvent system: petroleum ether-ethyl ether-glacial acetic acid, 80:20:1 (v/v/v). Individual phosphatides were separated using Supelcosil 42A (Supelco, Bellefonte, Pa.) for two dimensional TLC using the solvent systems: (A) chloroform-methanol28% ammonia, 65:25:5 (v/v/v) and (B) chloro-
Testes of quaking mice (sterile mutants) and of controls were analyzed for major lipid classes and fatty acid composition. Of the main lipid classes, only cholesterol esters differed significantly in concentration between the two groups (1.01 for quakers vs 0.69 mg/g wet wt of tissue for controls). The concentration of triglycerides was 4.5-5.0, that of total phosphatides 18-19, and that of free cholesterol 1.9-2.0 mg/g for mutants and controls. The concentrations of phosphatidyl ethanolamine and of sphingomyelin were both lower in quaking than in normal mice, but only the change in the former was statistically significant. Phosphatidyl choline was the major phosphatide (43-45% of total phosphatides) followed by phosphatidyl ethanolamine (24-26%) and sphingomyelin, phosphatidyl serine, and phosphatidyl inositol (all ca. 7% of total phosphatides). Minor differences between the mutants and controis were observed in concentrations of fatty acids of major lipid classes. The mutants, sterile because of-faulty spermarid differentiation, had normal quantities of 22:6 w3 and 22:5 w6. These data are consistent with the hypothesis that the 22-carbon polyenes are associated with the formation of spermatids, rather than with their final differentiation into spermatozoa.
TABLE I Fatty Acid Composition of Testicular Total Lipids of Quaking Mice
I NTR OD UCTI ON
The male quaking mouse (qk/qk) has been reported to be sterile because of faulty spermatid differentiation (1). Lipid abnormalities have been found in the central nervous system (2,3) and in organs, such as thymus, spleen, and kidney (4) of this animal. However, lipids or the testis of the quaking mouse have not been studied. Our interest in the role of lipids and fatty acids in male reproductive tissue led us to consider the possibility of a defect in lipid composition and metabolism in the testis of this mutant. Accordingly, we have determined the major lipid classes and fatty acid composition of testes of the quaking mouse and compared the values with those of controls.
Fatty acid
Percent of total fatty acids
16:0 a 16:1 18:0 18:1 18:2 20:3 20:4 22:4 22:5 22:6 24:4 t 24:5
29.9 + 0.78 b 1.48 + 0.09 6.95 +- 0.22 15.8 -+0.82 8.11 + 0.89 1.17 + 0.04 11.9 + 0.37 1.67 + 0.21 11.0 +- 0.43 7.01 + 0.26 2.79 + 0.25
aNumber of carbons in chain: number of double bonds. bMean of 6 samples -+ standard error of the mean; testes of 2-5 mice/sample.
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J.G. CONIGLIO, W.M. GROGAN JR., D.G. HARRIS AND M.L. FITZHUGH
reanalyzed by GLC. The completely hydrogenated samples had amounts of saturated fatty acids consistent with the amounts of individual components of the same carbon number in the nonhydrogenated samples. Lipid composition was determined in 2 sets of mice of ages 83-111 days. The results are given in Table II. Most of the lipid was phosphatide, and free fatty acids were present only in trace quantities. There were no significant differences between controls and quakers in the concentrations of phosphatide, triglycerides, or free cholesterol. However, there was a statistically significant greater amount of esterifled cholesterol in testes of the quaking mice. Ca. 90% of the total phosphatide phosphorus was accounted for in 5 identified phosphatides given in Table II. Most of the phosphatides in testes was phosphatidyl choline and phosphatidyl ethanolamine, but considerable concentrations of phosphatidyl serine, phosphatidyl inositol, and sphingomyelin also were present. No significant differences between controls and mutants were found for any of these, except phosphatidyl ethanolamine. Although the difference in concentration of sphingomyelin was not statistically significant, it is important to indicate that in every case except one the concentration was greater in the testes of the controls than in the quakers. In addition to these major phosphatides, small amounts of cardiolipin and lysophosphatides were present. R ESU LTS The fatty acids obtained from each of the The ages of the mice in the first set ranged main classes of testicular lipids of mice aged from 41-124 days. During this time, the body 83-111 days were analyzed by GLC. The results wt of controls increased from 16-27 g, while are given in Table III. Very few differences that of the quaking mice increased from 13-21 were found between controls and quakers g. Wt of the testes of control mice increased which proved statistically significant. These from 0.07 at 41 days to 0.11 g/testis but that included 18:1 of phosphatides; 20:4 and 22:4 of testes of quakers showed little or no increase of cholesterol esters; and 14:0, 18:0, 18:2, and from the 41 day wt of 0.06 g/testis. Total fatty 22:5 w3 of triglycerides. In addition to the acid concentration was constant throughout fatty acids reported in Table III, there were this period for both groups (14.7 + 1.26 mg/g small amounts of numerous components among for controls vs. 13.7 -+ 0.57 mg/g for quaking which were tentatively identified 15:0, 20- and mice). The fatty acid composition of total 22-carbon mono-, di-, and trienes; and 18:3. tipids did not differ significantly between the There were also small but significant amounts two groups. In Table I is given the composition of components with retention times beyond for testes of quaking mice. The means of pools 24:5, but these were not identified or studied of all ages are given, since there was no further. consistent change in the concentration of any Histological examination of the sections fatty acid during this entire age period. Only revealed that very few spermatids with tails those fatty acids present in a concentration of were present in the testes of the quaking mice, 1% or greater are listed. Among those fatty although these were abundant in those of acids present in concentrations less than 1% controls. were 14:0, 18:3, 20:2, 20:5, 22:3, and 22:5 w3 (the isomer reported in the table is 22:5 w6). DISCUSSION Ca. twice as much 24:5 as 24:4 was present. Several of the samples were hydrogenated and The role of lipids, and particularly polyenoic
f o r m - a c e t o n e - m e t h a n o l - acetic acid-water, 6:8:2:2:1 (all on a volume basis). After developing and charring lightly with sulfuric acid, the identified spots were scraped quantitatively into test tubes and phosphorus quantitated (7). All analyses of lipid fractions were done at least in duplicate. Fatty acids from total lipids and from separated lipid classes were methylated with boron trifluoride-methanol (9) for analysis by gas liquid chromatography (GLC). GLC analyses were done as reported previously (5). Standard methyl esters obtained from Applied Science Laboratories, State College, Pa., were used to establish retention times and for calibration of the flame ionization detectors. Hydrogenation of methyl esters was done (10), and the hydrogenated derivatives were reanalyzed by GLC to establish the total amount of fatty acids of a particular Chain length. Further identifications were made by reference to fatty acid methyl esters obtained from rat and human testes and previously characterized by chemical means. Testes from 2-5 mice were pooled for each sample analyzed to obtain sufficient lipid. Four different sets of mice were obtained for these studies, but, in most cases, the results from more than one set have been grouped in a table for calculation of means and for statistical evaluation. The ages of the animals are given with each set of data.
LIPIDS, VOL. 10, NO. 2
111
T E S T I C U L A R L I P I I ) S O1: Q U A K I N G MICE T A B L E II Lipid a n d P h o s p h a t i d e C o m p o s i t i o n o f Testes o f Q u a k i n g a n d C o n t r o l Mice m g / g Wet w t testis a Lipid class
Controls
Triglycerides Phosphatides Free c h o l e s t e r o l Esterified cholesterol Ratio: e s t e r i f i e d c h o l e s t e r o l
Quaking
4 . 8 0 -+ 0 . 5 2 19.0 +- 0 . 5 4 1.98 :~: 0 . 0 3 0 . 6 9 • 0.07 0.35-+ 0.04
free c h o l e s t e r o l
4 . 5 9 -+ 0 . 3 0 18.2 +- 0 . 6 0 1.90 -+ 0.2 ! 1.01 • 0 . 0 9 ( P = 0 . 1 7 ) 0.53-+ 0 . 0 7 ( P = 0 . 0 6 1 )
P e r c e n t o f total p h o s p h a t i d e s a Controls
Phosphatidyl choline b Phosphatidylethanolamine b Sphingomyelin P h o s p h a t i d y l serine P h o s p h a t i d y l inositol
42.7 26.4 7.24 7.69 6.88
Quaking
• 1.3 -+ 0.52 • 0.43 • 0.33 -+ 0 . 3 5
45.7 24.3 6.41 7.63 6.80
-+ 1.2 -+ 0 . 8 9 0 a = . 0 5 ) • 0.30 -+ 0 . 1 9 -+ 0 . 5 9
a M e a n -+ s t a n d a r d e r r o r o f the m e a n o f 8 s a m p l e s ; testes f r o m 2-5 m i c e w e r e p o o l e d for each s a m p l e . b l n c l u d e s p l a s m a l o g e n s , if p r e s e n t .
"fABLE
III
F a t t y Acids o f P h o s p h o l i p i d s , T r i g l y c e r i d e s , a n d C h o l e s t e r o l Esters f r o m Testes o f C o n t r o l a n d Q u a k i n g Mice P e r c e n t o f total f a t t y acids Phosphatides a F a t t y acid 12:0
Controls
Trigly cerides a
Quaking
tr
tr
Controls
Quaking
1.1 • 0 . 2 8 (P
0.89-+ 0 . 2 0
14:0 16:0 16:1
0.81 :~: 0.13 29.2 • 0 . 9 4 1.3 •
0.80-+ 0.07 30.4 • 1.3 1.5 •
18:0
8.0
• 0.47
18:1
12.2
• 0.40
14.3
:~. 0.52
25.0-+ 1.4
2.7 1.4
-2- 0.1 I -+ 0.1 I
3.0 1.3
• 0.26 • 0.06
22.3• 0.73•
/(P
:
-+ 0 . 4 4
=
Quaking
2.3 • 0 . 3 8
3.0 +- 0 . 5 9
+ 0.14 -+.0.35 -+0.17
4.1 • 0.31 15.3 • 6.8-+.0.35
4.1 -+ 0.79 14.8+- 1.6 6.4-+0.32
.001)
2.7•
4.2
•
7.2•
6.3-+0.36
.008)\
/ I 8:2 20:3 w6
Controls
.001)
1.3 • 0.09 2.1 1 6 . 7 - + 0 . 9 3 18.2 5.1 -+0.30 4.8 (P
8.9
:
C h o l e s t e r o l esters a
(P
22.7
-+ 1.0
.03) \ 16.2 • 0.86-+0.11
18.4-+ 1.1
17.8-+ 1.3
3.5-+0.28 0.67•
3.1-+0.3 0.44•
=
20:4
14.2 • 0.52
13.0
• 0.79
1.7 , 0 . 1 8
1.7•
(P = 0 . 0 0 8 ) f 1.7-+0.12 1.2-+0.13
22:4 22:5w6
1.4 • 12.3 •
1.4 10.8
-+0.10 •
1.6• 5.9•
2.1 • 6.7•
2.5-+0.18 3.9•
1.6-+0.24 3.2•
(P : / 1.1 • 2.9• 0.8 • 0 . 1 7 1.3•
.05)\ 2.7• 6.7~0.90 2.7-+0.40 3.9-+0.49
1.88-+0.31 5.1• 2.7• 3.0-+0.31
/(P
22:5w3 22:6 24:4 24:5
0.51 -+0.06 8.50-+0.80 l.I -+0.11 1.5 -+0.16
0.55• 7.0 ~ 0.62 I . I • 0.13 1.5 -+0.13
1.7• 3.7• l . l -+ 0.22 1.7•
: .Ol)\
a M e a n -+ s t a n d a r d e r r o r o f the m e a n o f 8 s a m p l e s for p h o s p h a t i d e s a n d t r i g l y c e r i d e s a n d o f 10 s a m p l e s for c h o l e s t e r o l esters; testes f r o m 2-5 mice w e r e p o o l e d for each s a m p l e . L I P I I ) S , VOL. 10, NO. 2
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J.G. CONIGL10, W.M. GROGAN JR., D.G. HARRIS AND M.L. FITZHUGH
f a t t y acids, in t h e male r e p r o d u c t i v e s y s t e m has a t t r a c t e d increasing a t t e n t i o n . In t h e rat, it was shown that the testicular concentration of 22:5 w6 i n c r e a s e d d r a m a t i c a l l y w i t h sexual m a t u r a t i o n o f t h e a n i m a l (1 1). F u r t h e r m o r e , in c o n d i t i o n s in w h i c h t h e testes were caused t o d e g e n e r a t e by m e a n s o f surgical c r y p t o r c h i d i s m or b y use of c a d m i u m c h l o r i d e , t h e c o n c e n t r a t i o n o f this p o l y e n e d e c r e a s e d t o low values (12). Because the q u a k i n g m o u s e has a d e f e c t in spermiogenesis, it m i g h t be a useful a n i m a l in t h e s t u d y o f t h e role of lipids in s p e r m a t o g e n esis. Our studies s h o w o n l y m i n o r d i f f e r e n c e s in lipid a n d f a t t y acid c o m p o s i t i o n o f testes o f q u a k i n g c o m p a r e d t o c o n t r o l mice. In t h e m o u s e , 2 2 : 6 w3 is a p r o m i n e n t c o m p o n e n t of t e s t i c u l a r f a t t y acids in a d d i t i o n t o 2 2 : 5 w 6 ( w h i c h is t h e chief 2 2 - c a r b o n p o l y e n e in rat testes). B o t h o f these p o l y e n e s were p r e s e n t in testicular lipid f r a c t i o n s o f q u a k i n g mice in c o n c e n t r a t i o n s n o t significantly d i f f e r e n t f r o m c o n t r o l s . B e n n e t t , et al., (1) has p r o p o s e d t h a t t h e defect in testis of the q u a k i n g m o u s e is o n e of spermatid differentiation, and our limited histological e x a m i n a t i o n s c o n f i r m t h i s m o r p h o logical o b s e r v a t i o n . Our results are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t e s t i c u l a r 2 2 : 5 w6 a n d 2 2 : 6 w3 are r e l a t e d t o t h e p r o d u c t i o n o f s p e r m a t i d s , r a t h e r t h a n t o t h e final differentiation into spermatozoa. Some d i f f e r e n c e s b e t w e e n testicular lipids of q u a k i n g a n d c o n t r o l mice were n o t e d in t h e s e studies. M i n o r differences were f o u n d in f a t t y acid c o n c e n t r a t i o n s i n p a r t i c u l a r lipid classes (for e x a m p l e , 14:0, 18:0, a n d 1 8 : 2 in triglycerides), b u t t h e s e c a n n o t be r e l a t e d to a n y m o r p h o l o g i c a l changes. Moreover, t h e s e differences were small a n d m a y n o t r e p r e s e n t i m p o r t a n t a l t e r a t i o n s . A m e a n i n g f u l c h a n g e was a n increase in t h e a m o u n t o f esterified cholesterol ( b u t n o t free c h o l e s t e r o l ) in testes of q u a k i n g mice. Generally, esterified c h o l e s t e r o l increases d u r i n g i m p a i r m e n t o f t h e s p e r m a t o genic process (13). Sphlngolipids in t h e c e n t r a l n e r v o u s s y s t e m were r e p o r t e d r e d u c e d c o m p a r e d t o o t h e r s t r u c t u r a l c o m p o n e n t s (2,3). In o u r studies, p h o s p h a t i d y l e t h a n o l a m i n e was the o n l y p h o s p h a t i d e w h i c h was decreased signific a n t l y in testes o f q u a k i n g mice, a n d t h i s decrease was o n l y b o r d e r l i n e (P = 0.05). The c o n c e n t r a t i o n o f s p h i n g o m y e l i n was l o w e r in t h e m u t a n t s t h a n in c o n t r o l s in all cases b u t one, b u t t h e difference in t h e m e a n s was n o t statistically significant (P = 0.1). A greater n u m b e r of analyses e v e n t u a l l y m i g h t show t h i s difference to be significant. Analysis o f t h e
LIPIDS, VOL. 10, NO. 2
f a t t y acid p a t t e r n o f i n d i v i d u a l p h o s p h a t i d e s a n d p a r t i c u l a r l y of s p h i n g o m y e l i n of t e s t e s of q u a k i n g a n d c o n t r o l mice m a y s h o w a l t e r a t i o n s n o t seen in t h e f a t t y acid p a t t e r n of t o t a l p h o s p h a t i d e s , b u t t h e s e s t u d i e s have n o t y e t b e e n done. A r e d u c t i o n in t h e C 2 0 - C o A elongat i o n s y s t e m has b e e n s h o w n in m i c r o s o m e s f r o m b r a i n of q u a k i n g mice (14). A glycerol-base ( r a t h e r t h a n a s p h i n g o s i n e base) glycolipid p r e s e n t in t e s t e s of various animals was r e p o r t e d t o be r e d u c e d greatly in a m o u n t i n testes o f t h e w / w v m o u s e , a sterile m u t a n t deficient in g e r m i n a l cells (15). T h e c o n c e n t r a t i o n o f tiffs glycolipid in testes of t h e q u a k i n g m o u s e has n o t y e t b e e n d e t e r m i n e d .
ACKNOWLEDGMENTS R.F. Sellers, Department of Anatomy, oreparedthe histological sections, and M.-C. Orgebin-Crist, Department of Obstetrics-Gynecology, interpreted the histological studies. This work was supported by Research Grant 1 RO1-HD 07694 from the U.S. Public Health Service. REFERENCES 1. Be/~nett, W.I., A.M. Gall, J.L. Southard, and R.L. Sidman, Biol. Reprod. 5 : 30 (1971 ). 2. Baumann, N.A., C.M. Jacque, S.A. Pollett, and M.L. Harpin, Eur. J. Biochem. 4:340 (1968). 3. Bowen, D.M., and N.S. Radin, J. Neurochem. 16:457 (1969). 4. Kishimoto, Y., Fed. Proc. Fed. Amer. Soc. Exp. Biol. 29:894 (1970). 5. Bridges, R.B., and J.G. Coniglio, J. Biol. Chem. 245:46 (1970). 6. Sardesai, V.M., and J.A. Manning, Clin. Chem. 14:156 (1968). 7. Doizaki, W.M., and L. Zieve, Proc. Soc. Exp. Biol. Med. 113:91 (1963). 8. Sperry, W.M., and M. Webb, J. Biol. Chem. 187:97 (1950). 9. Metcalfe, L.D., and A.A. Schmitz, Anal. Chem. 33:363 (1961). 10. Farquhar, J.W., W. Insull, Jr., P. Rosen, W. Stoffel, and E.H. Ahrens, Nutr. Rev. 17 (supplement 2):1 (1959). 11. Davis, J.T., R.B. Bridges, J.G. Coniglio, Biochem. J. 98:342 (1966). 12. Davis, J.T., and J.G. Coniglio, J. Reprod. Fert. 14:407 (1967). 13. Johnson, A.D., in "The Testis," Vol. II, Edited by A.D. Johnson, W.R. Gomes, and N.L. Vandemark, Academic Press, New York, N.Y., 1970, p. 252. 14. Goldberg, I., I. Schechter, and K. Bloch, Science 182:497 (1973). 15. Kornblatt, M.M., H. Schachter, and R.K. Murray, in the "Ninth International Congress of Biochemistry Abstract Book, Aktiebotaget Egnellska Boktryckeriet, Stockholm, Sweden, 1973, p. 396. [Received O c t o b e r 7, 1 9 7 4 ]
ABSTRACT
The mice were C 57BL/6J males obtained from the Jackson Laboratory, Bar Harbor, Maine. Controls were furnished by the same supplier (qk/+ or +/+, indistinguishable). The animals were maintained on Purina chow until their death by decapitation. Testes were removed quickly and chilled in ice, after which they carefully were cleaned of all adhering tissue and weighed. A small amount of tissue was used from each of several testes from the various groups of quaking and control mice for histological examination. These were fixed in Bouin's media and stained with periodic Schiff stain (PAS) and counter-stained with Wright's iron hematoxylin. The balance of the tissues was either hydrolyzed with alcoholic potassium hydroxide for total fatty acid separation by methods reported previously (5) or homogenized in Folch mixture (chloroform:methanol 2/1) for extraction of total lipid. Aliquots of the extracts were used for determinations of triglyceride (6), phospholipid phosphorus (7), and of free and esterified cholesterol (8). Classes of lipids wre separated by thin layer chromatography (TLC) using the solvent system: petroleum ether-ethyl ether-glacial acetic acid, 80:20:1 (v/v/v). Individual phosphatides were separated using Supelcosil 42A (Supelco, Bellefonte, Pa.) for two dimensional TLC using the solvent systems: (A) chloroform-methanol28% ammonia, 65:25:5 (v/v/v) and (B) chloro-
Testes of quaking mice (sterile mutants) and of controls were analyzed for major lipid classes and fatty acid composition. Of the main lipid classes, only cholesterol esters differed significantly in concentration between the two groups (1.01 for quakers vs 0.69 mg/g wet wt of tissue for controls). The concentration of triglycerides was 4.5-5.0, that of total phosphatides 18-19, and that of free cholesterol 1.9-2.0 mg/g for mutants and controls. The concentrations of phosphatidyl ethanolamine and of sphingomyelin were both lower in quaking than in normal mice, but only the change in the former was statistically significant. Phosphatidyl choline was the major phosphatide (43-45% of total phosphatides) followed by phosphatidyl ethanolamine (24-26%) and sphingomyelin, phosphatidyl serine, and phosphatidyl inositol (all ca. 7% of total phosphatides). Minor differences between the mutants and controis were observed in concentrations of fatty acids of major lipid classes. The mutants, sterile because of-faulty spermarid differentiation, had normal quantities of 22:6 w3 and 22:5 w6. These data are consistent with the hypothesis that the 22-carbon polyenes are associated with the formation of spermatids, rather than with their final differentiation into spermatozoa.
TABLE I Fatty Acid Composition of Testicular Total Lipids of Quaking Mice
I NTR OD UCTI ON
The male quaking mouse (qk/qk) has been reported to be sterile because of faulty spermatid differentiation (1). Lipid abnormalities have been found in the central nervous system (2,3) and in organs, such as thymus, spleen, and kidney (4) of this animal. However, lipids or the testis of the quaking mouse have not been studied. Our interest in the role of lipids and fatty acids in male reproductive tissue led us to consider the possibility of a defect in lipid composition and metabolism in the testis of this mutant. Accordingly, we have determined the major lipid classes and fatty acid composition of testes of the quaking mouse and compared the values with those of controls.
Fatty acid
Percent of total fatty acids
16:0 a 16:1 18:0 18:1 18:2 20:3 20:4 22:4 22:5 22:6 24:4 t 24:5
29.9 + 0.78 b 1.48 + 0.09 6.95 +- 0.22 15.8 -+0.82 8.11 + 0.89 1.17 + 0.04 11.9 + 0.37 1.67 + 0.21 11.0 +- 0.43 7.01 + 0.26 2.79 + 0.25
aNumber of carbons in chain: number of double bonds. bMean of 6 samples -+ standard error of the mean; testes of 2-5 mice/sample.
109
ll0
J.G. CONIGLIO, W.M. GROGAN JR., D.G. HARRIS AND M.L. FITZHUGH
reanalyzed by GLC. The completely hydrogenated samples had amounts of saturated fatty acids consistent with the amounts of individual components of the same carbon number in the nonhydrogenated samples. Lipid composition was determined in 2 sets of mice of ages 83-111 days. The results are given in Table II. Most of the lipid was phosphatide, and free fatty acids were present only in trace quantities. There were no significant differences between controls and quakers in the concentrations of phosphatide, triglycerides, or free cholesterol. However, there was a statistically significant greater amount of esterifled cholesterol in testes of the quaking mice. Ca. 90% of the total phosphatide phosphorus was accounted for in 5 identified phosphatides given in Table II. Most of the phosphatides in testes was phosphatidyl choline and phosphatidyl ethanolamine, but considerable concentrations of phosphatidyl serine, phosphatidyl inositol, and sphingomyelin also were present. No significant differences between controls and mutants were found for any of these, except phosphatidyl ethanolamine. Although the difference in concentration of sphingomyelin was not statistically significant, it is important to indicate that in every case except one the concentration was greater in the testes of the controls than in the quakers. In addition to these major phosphatides, small amounts of cardiolipin and lysophosphatides were present. R ESU LTS The fatty acids obtained from each of the The ages of the mice in the first set ranged main classes of testicular lipids of mice aged from 41-124 days. During this time, the body 83-111 days were analyzed by GLC. The results wt of controls increased from 16-27 g, while are given in Table III. Very few differences that of the quaking mice increased from 13-21 were found between controls and quakers g. Wt of the testes of control mice increased which proved statistically significant. These from 0.07 at 41 days to 0.11 g/testis but that included 18:1 of phosphatides; 20:4 and 22:4 of testes of quakers showed little or no increase of cholesterol esters; and 14:0, 18:0, 18:2, and from the 41 day wt of 0.06 g/testis. Total fatty 22:5 w3 of triglycerides. In addition to the acid concentration was constant throughout fatty acids reported in Table III, there were this period for both groups (14.7 + 1.26 mg/g small amounts of numerous components among for controls vs. 13.7 -+ 0.57 mg/g for quaking which were tentatively identified 15:0, 20- and mice). The fatty acid composition of total 22-carbon mono-, di-, and trienes; and 18:3. tipids did not differ significantly between the There were also small but significant amounts two groups. In Table I is given the composition of components with retention times beyond for testes of quaking mice. The means of pools 24:5, but these were not identified or studied of all ages are given, since there was no further. consistent change in the concentration of any Histological examination of the sections fatty acid during this entire age period. Only revealed that very few spermatids with tails those fatty acids present in a concentration of were present in the testes of the quaking mice, 1% or greater are listed. Among those fatty although these were abundant in those of acids present in concentrations less than 1% controls. were 14:0, 18:3, 20:2, 20:5, 22:3, and 22:5 w3 (the isomer reported in the table is 22:5 w6). DISCUSSION Ca. twice as much 24:5 as 24:4 was present. Several of the samples were hydrogenated and The role of lipids, and particularly polyenoic
f o r m - a c e t o n e - m e t h a n o l - acetic acid-water, 6:8:2:2:1 (all on a volume basis). After developing and charring lightly with sulfuric acid, the identified spots were scraped quantitatively into test tubes and phosphorus quantitated (7). All analyses of lipid fractions were done at least in duplicate. Fatty acids from total lipids and from separated lipid classes were methylated with boron trifluoride-methanol (9) for analysis by gas liquid chromatography (GLC). GLC analyses were done as reported previously (5). Standard methyl esters obtained from Applied Science Laboratories, State College, Pa., were used to establish retention times and for calibration of the flame ionization detectors. Hydrogenation of methyl esters was done (10), and the hydrogenated derivatives were reanalyzed by GLC to establish the total amount of fatty acids of a particular Chain length. Further identifications were made by reference to fatty acid methyl esters obtained from rat and human testes and previously characterized by chemical means. Testes from 2-5 mice were pooled for each sample analyzed to obtain sufficient lipid. Four different sets of mice were obtained for these studies, but, in most cases, the results from more than one set have been grouped in a table for calculation of means and for statistical evaluation. The ages of the animals are given with each set of data.
LIPIDS, VOL. 10, NO. 2
111
T E S T I C U L A R L I P I I ) S O1: Q U A K I N G MICE T A B L E II Lipid a n d P h o s p h a t i d e C o m p o s i t i o n o f Testes o f Q u a k i n g a n d C o n t r o l Mice m g / g Wet w t testis a Lipid class
Controls
Triglycerides Phosphatides Free c h o l e s t e r o l Esterified cholesterol Ratio: e s t e r i f i e d c h o l e s t e r o l
Quaking
4 . 8 0 -+ 0 . 5 2 19.0 +- 0 . 5 4 1.98 :~: 0 . 0 3 0 . 6 9 • 0.07 0.35-+ 0.04
free c h o l e s t e r o l
4 . 5 9 -+ 0 . 3 0 18.2 +- 0 . 6 0 1.90 -+ 0.2 ! 1.01 • 0 . 0 9 ( P = 0 . 1 7 ) 0.53-+ 0 . 0 7 ( P = 0 . 0 6 1 )
P e r c e n t o f total p h o s p h a t i d e s a Controls
Phosphatidyl choline b Phosphatidylethanolamine b Sphingomyelin P h o s p h a t i d y l serine P h o s p h a t i d y l inositol
42.7 26.4 7.24 7.69 6.88
Quaking
• 1.3 -+ 0.52 • 0.43 • 0.33 -+ 0 . 3 5
45.7 24.3 6.41 7.63 6.80
-+ 1.2 -+ 0 . 8 9 0 a = . 0 5 ) • 0.30 -+ 0 . 1 9 -+ 0 . 5 9
a M e a n -+ s t a n d a r d e r r o r o f the m e a n o f 8 s a m p l e s ; testes f r o m 2-5 m i c e w e r e p o o l e d for each s a m p l e . b l n c l u d e s p l a s m a l o g e n s , if p r e s e n t .
"fABLE
III
F a t t y Acids o f P h o s p h o l i p i d s , T r i g l y c e r i d e s , a n d C h o l e s t e r o l Esters f r o m Testes o f C o n t r o l a n d Q u a k i n g Mice P e r c e n t o f total f a t t y acids Phosphatides a F a t t y acid 12:0
Controls
Trigly cerides a
Quaking
tr
tr
Controls
Quaking
1.1 • 0 . 2 8 (P
0.89-+ 0 . 2 0
14:0 16:0 16:1
0.81 :~: 0.13 29.2 • 0 . 9 4 1.3 •
0.80-+ 0.07 30.4 • 1.3 1.5 •
18:0
8.0
• 0.47
18:1
12.2
• 0.40
14.3
:~. 0.52
25.0-+ 1.4
2.7 1.4
-2- 0.1 I -+ 0.1 I
3.0 1.3
• 0.26 • 0.06
22.3• 0.73•
/(P
:
-+ 0 . 4 4
=
Quaking
2.3 • 0 . 3 8
3.0 +- 0 . 5 9
+ 0.14 -+.0.35 -+0.17
4.1 • 0.31 15.3 • 6.8-+.0.35
4.1 -+ 0.79 14.8+- 1.6 6.4-+0.32
.001)
2.7•
4.2
•
7.2•
6.3-+0.36
.008)\
/ I 8:2 20:3 w6
Controls
.001)
1.3 • 0.09 2.1 1 6 . 7 - + 0 . 9 3 18.2 5.1 -+0.30 4.8 (P
8.9
:
C h o l e s t e r o l esters a
(P
22.7
-+ 1.0
.03) \ 16.2 • 0.86-+0.11
18.4-+ 1.1
17.8-+ 1.3
3.5-+0.28 0.67•
3.1-+0.3 0.44•
=
20:4
14.2 • 0.52
13.0
• 0.79
1.7 , 0 . 1 8
1.7•
(P = 0 . 0 0 8 ) f 1.7-+0.12 1.2-+0.13
22:4 22:5w6
1.4 • 12.3 •
1.4 10.8
-+0.10 •
1.6• 5.9•
2.1 • 6.7•
2.5-+0.18 3.9•
1.6-+0.24 3.2•
(P : / 1.1 • 2.9• 0.8 • 0 . 1 7 1.3•
.05)\ 2.7• 6.7~0.90 2.7-+0.40 3.9-+0.49
1.88-+0.31 5.1• 2.7• 3.0-+0.31
/(P
22:5w3 22:6 24:4 24:5
0.51 -+0.06 8.50-+0.80 l.I -+0.11 1.5 -+0.16
0.55• 7.0 ~ 0.62 I . I • 0.13 1.5 -+0.13
1.7• 3.7• l . l -+ 0.22 1.7•
: .Ol)\
a M e a n -+ s t a n d a r d e r r o r o f the m e a n o f 8 s a m p l e s for p h o s p h a t i d e s a n d t r i g l y c e r i d e s a n d o f 10 s a m p l e s for c h o l e s t e r o l esters; testes f r o m 2-5 mice w e r e p o o l e d for each s a m p l e . L I P I I ) S , VOL. 10, NO. 2
112
J.G. CONIGL10, W.M. GROGAN JR., D.G. HARRIS AND M.L. FITZHUGH
f a t t y acids, in t h e male r e p r o d u c t i v e s y s t e m has a t t r a c t e d increasing a t t e n t i o n . In t h e rat, it was shown that the testicular concentration of 22:5 w6 i n c r e a s e d d r a m a t i c a l l y w i t h sexual m a t u r a t i o n o f t h e a n i m a l (1 1). F u r t h e r m o r e , in c o n d i t i o n s in w h i c h t h e testes were caused t o d e g e n e r a t e by m e a n s o f surgical c r y p t o r c h i d i s m or b y use of c a d m i u m c h l o r i d e , t h e c o n c e n t r a t i o n o f this p o l y e n e d e c r e a s e d t o low values (12). Because the q u a k i n g m o u s e has a d e f e c t in spermiogenesis, it m i g h t be a useful a n i m a l in t h e s t u d y o f t h e role of lipids in s p e r m a t o g e n esis. Our studies s h o w o n l y m i n o r d i f f e r e n c e s in lipid a n d f a t t y acid c o m p o s i t i o n o f testes o f q u a k i n g c o m p a r e d t o c o n t r o l mice. In t h e m o u s e , 2 2 : 6 w3 is a p r o m i n e n t c o m p o n e n t of t e s t i c u l a r f a t t y acids in a d d i t i o n t o 2 2 : 5 w 6 ( w h i c h is t h e chief 2 2 - c a r b o n p o l y e n e in rat testes). B o t h o f these p o l y e n e s were p r e s e n t in testicular lipid f r a c t i o n s o f q u a k i n g mice in c o n c e n t r a t i o n s n o t significantly d i f f e r e n t f r o m c o n t r o l s . B e n n e t t , et al., (1) has p r o p o s e d t h a t t h e defect in testis of the q u a k i n g m o u s e is o n e of spermatid differentiation, and our limited histological e x a m i n a t i o n s c o n f i r m t h i s m o r p h o logical o b s e r v a t i o n . Our results are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t e s t i c u l a r 2 2 : 5 w6 a n d 2 2 : 6 w3 are r e l a t e d t o t h e p r o d u c t i o n o f s p e r m a t i d s , r a t h e r t h a n t o t h e final differentiation into spermatozoa. Some d i f f e r e n c e s b e t w e e n testicular lipids of q u a k i n g a n d c o n t r o l mice were n o t e d in t h e s e studies. M i n o r differences were f o u n d in f a t t y acid c o n c e n t r a t i o n s i n p a r t i c u l a r lipid classes (for e x a m p l e , 14:0, 18:0, a n d 1 8 : 2 in triglycerides), b u t t h e s e c a n n o t be r e l a t e d to a n y m o r p h o l o g i c a l changes. Moreover, t h e s e differences were small a n d m a y n o t r e p r e s e n t i m p o r t a n t a l t e r a t i o n s . A m e a n i n g f u l c h a n g e was a n increase in t h e a m o u n t o f esterified cholesterol ( b u t n o t free c h o l e s t e r o l ) in testes of q u a k i n g mice. Generally, esterified c h o l e s t e r o l increases d u r i n g i m p a i r m e n t o f t h e s p e r m a t o genic process (13). Sphlngolipids in t h e c e n t r a l n e r v o u s s y s t e m were r e p o r t e d r e d u c e d c o m p a r e d t o o t h e r s t r u c t u r a l c o m p o n e n t s (2,3). In o u r studies, p h o s p h a t i d y l e t h a n o l a m i n e was the o n l y p h o s p h a t i d e w h i c h was decreased signific a n t l y in testes o f q u a k i n g mice, a n d t h i s decrease was o n l y b o r d e r l i n e (P = 0.05). The c o n c e n t r a t i o n o f s p h i n g o m y e l i n was l o w e r in t h e m u t a n t s t h a n in c o n t r o l s in all cases b u t one, b u t t h e difference in t h e m e a n s was n o t statistically significant (P = 0.1). A greater n u m b e r of analyses e v e n t u a l l y m i g h t show t h i s difference to be significant. Analysis o f t h e
LIPIDS, VOL. 10, NO. 2
f a t t y acid p a t t e r n o f i n d i v i d u a l p h o s p h a t i d e s a n d p a r t i c u l a r l y of s p h i n g o m y e l i n of t e s t e s of q u a k i n g a n d c o n t r o l mice m a y s h o w a l t e r a t i o n s n o t seen in t h e f a t t y acid p a t t e r n of t o t a l p h o s p h a t i d e s , b u t t h e s e s t u d i e s have n o t y e t b e e n done. A r e d u c t i o n in t h e C 2 0 - C o A elongat i o n s y s t e m has b e e n s h o w n in m i c r o s o m e s f r o m b r a i n of q u a k i n g mice (14). A glycerol-base ( r a t h e r t h a n a s p h i n g o s i n e base) glycolipid p r e s e n t in t e s t e s of various animals was r e p o r t e d t o be r e d u c e d greatly in a m o u n t i n testes o f t h e w / w v m o u s e , a sterile m u t a n t deficient in g e r m i n a l cells (15). T h e c o n c e n t r a t i o n o f tiffs glycolipid in testes of t h e q u a k i n g m o u s e has n o t y e t b e e n d e t e r m i n e d .
ACKNOWLEDGMENTS R.F. Sellers, Department of Anatomy, oreparedthe histological sections, and M.-C. Orgebin-Crist, Department of Obstetrics-Gynecology, interpreted the histological studies. This work was supported by Research Grant 1 RO1-HD 07694 from the U.S. Public Health Service. REFERENCES 1. Be/~nett, W.I., A.M. Gall, J.L. Southard, and R.L. Sidman, Biol. Reprod. 5 : 30 (1971 ). 2. Baumann, N.A., C.M. Jacque, S.A. Pollett, and M.L. Harpin, Eur. J. Biochem. 4:340 (1968). 3. Bowen, D.M., and N.S. Radin, J. Neurochem. 16:457 (1969). 4. Kishimoto, Y., Fed. Proc. Fed. Amer. Soc. Exp. Biol. 29:894 (1970). 5. Bridges, R.B., and J.G. Coniglio, J. Biol. Chem. 245:46 (1970). 6. Sardesai, V.M., and J.A. Manning, Clin. Chem. 14:156 (1968). 7. Doizaki, W.M., and L. Zieve, Proc. Soc. Exp. Biol. Med. 113:91 (1963). 8. Sperry, W.M., and M. Webb, J. Biol. Chem. 187:97 (1950). 9. Metcalfe, L.D., and A.A. Schmitz, Anal. Chem. 33:363 (1961). 10. Farquhar, J.W., W. Insull, Jr., P. Rosen, W. Stoffel, and E.H. Ahrens, Nutr. Rev. 17 (supplement 2):1 (1959). 11. Davis, J.T., R.B. Bridges, J.G. Coniglio, Biochem. J. 98:342 (1966). 12. Davis, J.T., and J.G. Coniglio, J. Reprod. Fert. 14:407 (1967). 13. Johnson, A.D., in "The Testis," Vol. II, Edited by A.D. Johnson, W.R. Gomes, and N.L. Vandemark, Academic Press, New York, N.Y., 1970, p. 252. 14. Goldberg, I., I. Schechter, and K. Bloch, Science 182:497 (1973). 15. Kornblatt, M.M., H. Schachter, and R.K. Murray, in the "Ninth International Congress of Biochemistry Abstract Book, Aktiebotaget Egnellska Boktryckeriet, Stockholm, Sweden, 1973, p. 396. [Received O c t o b e r 7, 1 9 7 4 ]
