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Experimental Studies |
Division of Endocrinology, Diabetes, and Clinical Nutrition, Department of Medicine, Oregon Health Sciences University, Portland, Oregon 97201; and Oregon Regional Primate Research Center, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: William E. Connor, M.D., Department of Medicine, L-465, Oregon Health Sciences University, Portland, Oregon 97201-3098.
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Abstract |
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Although the cholesterol content in the testis remained relatively unchanged throughout life, the desmosterol content first decreased from 59 µg/g in infants to 6 µg/g in prepubertal monkeys, increased to 83 µg/g during puberty, and reached a plateau of 248 µg/g in the young adult, where it remained into old age. The polyunsaturated fatty acid composition of the testis also changed markedly. Docosahexaenoic acid (22:6 n-3) increased from 5.1% of total fatty acids in infants and juveniles to 18.1% in postpubertal young adults. Although some n-6 fatty acids, arachidonic (20:4 n-6) and linoleic (18:2 n-6), decreased from 16.0% and 10.0% in prepubertal juveniles, respectively, to 7.1% and 3.3% in young adults; dihomogamma-linolenic acid (20:3 n-6), the precursor of 1 series PGs, increased greatly from 1.8% to 10.3%. Similar changes occurred in both membrane and storage lipids (phospholipids and triglycerides), respectively. After puberty, the testicular fatty acid pattern remained stable into old age.
Our data demonstrated that puberty is accompanied by substantial changes in the lipid composition of the primate testis. These changes suggest that desmosterol and both n-3 and n-6 polyunsaturated fatty acids may have important roles in sexual maturation.
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Introduction |
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In most previous studies, rodents were used as the animal model. However, the maturation process between birth and complete sexual maturity occurs very rapidly in laboratory rodents. In the rat, spermatogonia are actively dividing as early as the fourth postnatal day. Between days 9–12, meiotic development starts, and spermatogenesis is completed by about 45 days of age (8). In contrast, spermatogenic development in humans does not begin until 9–13 yr of age, and another 3–4 yr are necessary before complete sexual maturation is attained (9, 10). Nonhuman primates also have a lengthy period of development before sexual maturity is attained. For example, in the rhesus monkey spermatogenesis does not occur until approximately 3–4 yr of age, and full sexual maturity is not achieved until 5–6 yr (11, 12).
In a second homology, the sperm of both rhesus monkeys and humans contain high concentrations of desmosterol and docosahexaenoic acid (DHA; 22:6 n-3) (13) (Connor, W. E., and D. S. Lin, unpublished data). In contrast, the sperm and testis of rabbits and rats have little desmosterol and have docosapentaenoic acid (22:5 n-6) as the major fatty acid (14, 15, 16). Thus, nonhuman primates would provide a more appropriate model to study the developmental changes in the testis with reference to human physiology. However, there has been an absence of information about developmental changes in lipid composition in the primate testis. To provide such information, we analyzed testicular sterols and fatty acids from monkeys of different ages from midgestation (prenatal) to 30 yr of age.
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Materials and Methods |
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Testicular sterols and fatty acids were analyzed as previously described (13, 17). The lipids of the testes were extracted by standard methods (18), aliquots of lipid extract were saponified with alcoholic KOH, and the sterols were extracted with hexane. The sterol content was determined by gas-liquid chromatography (model 8500, Perkin-Elmer, Norwalk CT) on a 30-m SE-30 capillary column with column, detector and injection port temperatures of 260, 300, and 300 C, respectively. Helium was the carrier gas, and cholestane was used as the internal standard.
For analysis of free and esterified sterols in testis, aliquots of the lipid extracts were plated on silica gel G thin layer chromatography (TLC) plates after \[4-14C\]cholesterol and cholesteryl [14C]oleate (New England Nuclear Corp., Boston, MA) were added as internal standards. The plates were developed in hexane-chloroform-ether-acetic acid (80:10:10:1). The free sterol band containing both cholesterol and desmosterol was removed and extracted with ether. Sterol esters were saponified with alcoholic KOH, and the sterols were extracted with hexane. The sterol content was determined by the gas-liquid chromatography method described above. The sterol ester fatty acids were recovered by acidifying the aqueous phase and reextracting with hexane for fatty acid analysis.
For the analysis of sterol sulfates, another aliquot of lipid extract was applied on silica gel H TLC plates. Cholesterol \[4-14C\] sulfate was added as an internal standard. The TLC plates were developed in chloroform-methanol-acetic acid (80:20:2) (19). Sterol sulfates were extracted from the TLC gel by chloroform and solvolyzed to liberate free sterols (20). The quantity of sterols was determined by gas-liquid chromatography as described above.
The fatty acid compositions of the four major lipid classes (phospholipids, free fatty acids, triglycerides, and sterol esters) of testicular lipids were determined. Lipid classes were separated by the TLC system described above for free and esterified sterols. Fatty acids of each lipid class were transmethylated with boron trifluoride-methanol (21). Fatty acid methyl esters were analyzed by gas chromatography on an instrument equipped with a hydrogen flame ionization detector (model Sigma 3B, Perkin-Elmer) and a 30-m SP-2330 fused silica capillary column (Supelco, Bellefonte, PA). The temperatures of the column, detector, and injection port were 195, 250, and 250 C, respectively. Helium was the carrier gas. The split ratio was 1:170. The retention time and area of each peak were measured by an HP-3390 integrator, and a computer (HP85, Hewlett Packard, Palo Alto, CA) identified and quantified each individual fatty acid. A mixture of fatty acid standards was run daily.
Statistics were calculated by one-way ANOVA with Newman-Keuls pairwise post-hoc testing.
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Results |
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). The total sterols of the testis varied from
1540 µg/g tissue in mature adults to 2028 µg/g tissue in subadults
and were not different between the various groups. The concentration of
cholesterol in the testes remained relatively unchanged during
different stages of development, with only a small increase in the
subadult group relative to all other ages. In contrast, the desmosterol
content varied greatly. This sterol decreased from 59 µg/g testis in
infancy to the lowest level of 6 µg/g in the prepubertal stage. It
then increased to 248 µg/g in the testis of young adult monkeys and
remained high into old age (Table 1 and Fig. 1).
Desmosterol made up only 0.4% of testicular sterols in prepubertal
males, but was approximately 12% in adults. The ratio of desmosterol
to cholesterol (Table 1) decreased from 0.037 in infant monkeys to
0.004 in prepubertal monkeys, and then increased to 0.147 in young
adults.
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). The majority was in the free form
(84–91%) at all ages. Cholesterol ester contributed 7–18% of the
total sterols and was present from birth to old age. Only a small
fraction of the cholesterol was in sulfate form (0.2–2.5%).
Desmosterol was present in only two forms, free and esterified, but in
contrast to cholesterol, there was only free desmosterol in the testes
of infant and juvenile monkeys. In the testes of older monkeys,
(subadult, young adult, and old), 80–92% of the desmosterol was free,
and 8–20% was esterified.
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). During puberty, the DHA (22:6 n-3) content increased
from 6.3% of the total fatty acids in prepubertal subjects to 14.8%
in young adults (Fig. 2). At the same time,
dihomogamma-linolenic acid (20:3 n-6), the precursor of the 1 series of
PGs, increased from 1.8% to 9.6%. Interestingly, this increase
occurred despite decreases in the immediate precursor of 20:3 n-6,
linoleic acid (18:2 n-6), and decreases in the other major n-6 fatty
acid, arachidonic acid (20:4 n-6). The ratio of total n-6 to total n-3
fatty acids decreased by half, from 4.25 to 1.70. Two major saturated
fatty acids changed in opposite directions; palmitic acid increased
from 20.4% to 34.4%, and stearic acid decreased from 15.7% to 8.8%.
Oleic acid, the major monounsaturated fatty acid, decreased from 12.0%
to 5.5%.
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The fatty acid composition of the total lipids in the testes was similar to that of the testicular phospholipids. The same shifts in several individual fatty acids during sexual maturity were observed in total lipids as well (data not shown). For example, DHA was 4.7% of the total fatty acids in the total lipids of juvenile monkey testis. It increased to 18.1% of the total fatty acids in young adult monkey testes. At the same time, arachidonic acid decreased from 16% of the total fatty acids to 7.1% of the total fatty acids.
The fatty acid composition of testicular triglycerides is presented in
Table 4. As in testicular phospholipids, there were
shifts in the concentrations of DHA, 20:3 n-6, 18:2 n-6, 18:0, and 16:0
during sexual maturation. DHA increased from 4.3% of the total fatty
acids in prepubertal juvenile monkeys to 16.1% in young adults. At the
same ages, dihomogamma-linolenic acid (20:3 n-6) increased from 2.3%
to 12.2% of the total fatty acids, whereas 18:2 n-6 decreased from
7.9% to 3.7% of the total fatty acids. For the two major saturated
fatty acids, 16:0 increased from 24.4% to 33.6% of the total fatty
acids, and 18:0 decreased from 9.6% to 6.4% of the total fatty acids.
However, unlike the phospholipid fraction, testicular triglycerides
showed no changes in the level of arachidonic acid.
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Discussion |
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Testicular lipids consist of glycerides, which form the energy source of germ cells, and sterols and phospholipids, which are the primary structural components of membranes. In addition, cholesterol serves as the precursor for testicular steroidogenesis. The precise function of the other sterol, desmosterol, is unknown, but it may be involved with sperm motility because it is present in high concentration in the tails of sperm (22). Phospholipids comprise nearly 80% of the total lipids in the testis, with glycerides and sterols accounting for the remaining 20% (23).
The mature testis contains a high concentration of desmosterol, which
is not found in any other tissue of the body except its product, sperm.
The concentration of desmosterol in sperm is 58.6% of the total
sterols (13), considerably higher than that in the adult testes, where
its concentration is only 12%. Desmosterol is an intermediate compound
in the cholesterol synthetic pathway, and testicular cholesterol is
mostly synthesized locally (24, 25, 26); therefore, high levels of
desmosterol may result from low levels of the 
24-reductase enzyme,
which converts desmosterol to cholesterol, and/or the presence of an
inhibitor of this enzyme in the testis. In that context, the drug
triparanol, which was used as a cholesterol-lowering agent in humans
many years ago, inhibited this same enzyme, so that considerable
quantities of desmosterol accumulated in the body. Although the plasma
cholesterol level was lower, the total sterol content may not have been
decreased, particularly as desmosterol replaced the cholesterol that
was not being synthesized (27, 28). Desmosterol is, of course, found in
the brain during its development, particularly in chick brain on day
13, and then it disappears from the brain before hatching (29).
Desmosterol is also present in nature in the earthworm and in snails
and slugs (30, 31). Its presence suggests that these lower forms have
some capacity to synthesize cholesterol because there is no foodstuff
containing desmosterol.
Although testicular cholesterol remained relatively unchanged throughout the lifespan, the desmosterol concentration fluctuated markedly. It first fell by a factor of 10 from the fetal and newborn monkey to the prepubertal animal, and then increased 40-fold to the young adult level. As testosterone is synthesized from cholesterol in the Leydig cells of the testis, cholesterol synthesis may increase during puberty to provide an adequate substrate for increasing testosterone production. In the rat testis, Ness reported that 3-hydroxy-3-methyl glutaryl-coenzyme A reductase activity was highest at 21 days of age, the time of weaning, when sexual maturation had already begun (32). In monkeys, testosterone production occurs prenatally, is minimal after birth, and then increases again at puberty (12, 33, 34). The fluctuation, therefore, of desmosterol levels in the testis parallels changes in testosterone production and may reflect the activity of the cholesterol synthetic pathway stimulated by testosterone.
The possible sources of cholesterol substrate for testosterone production could be either plasma lipoprotein cholesterol or cholesterol synthesized in situ. In a previous study, we fed isotopic cholesterol daily to a human volunteer to reach an isotopic steady state and found that approximately 45% of sperm cholesterol was derived from plasma and 55% from de novo synthesis (35). Presumably, both of these sources would be available as cholesterol substrates for testosterone production.
Puberty was also accompanied by a variety of changes in fatty acid composition. There was a great increase in n-3 polyunsaturated fatty acids, but a decrease in total n-6 polyunsaturated fatty acids. Although the increase in n-3 fatty acids occurred mainly from an increase in DHA, the decrease in n-6 fatty acids was not uniform. There was a substantial increase in dihomogamma-linolenic acid (20:3 n-6) despite significant decreases in its precursor, linoleic acid (18:2 n-6), and in the other major n-6 fatty acid, arachidonic acid (20:4 n-6). The monounsaturated fatty acid oleic acid decreased, and two major saturated fatty acids, palmitic acid and stearic acid, changed in opposite directions.
In the rat, Oshima and Carpenter reported that the fatty acid compositions of the young and mature testes were similar (23); however, Davis et al. (36) reported an increase in docosapentaenoic acid (22:5 n-6), the predominant polyunsaturated fatty acid in the rat testis, from 4 to 9 weeks of age, a period that coincided with the appearance and maturation of spermatids. Their data thus suggested a changing lipid metabolism during sexual maturation. Coniglio et al. (37) found that the testes of human infants had a lower content of DHA and dihomogamma-linolenic acid (20:3 n-6) compared to adult testes. These data are similar to our findings in rhesus monkeys. However, they did not detect differences in other fatty acids. The increased DHA in the mature testis may be derived directly from the blood circulation or by de novo synthesis from linolenic acid (18:3 n-3) in situ. Davis and Coniglio have shown in rats that docosapentaenoic acid (22:5 n-6) was derived by testicular synthesis from linoleate and arachidonate (38).
The importance of the n-6 and n-3 fatty acids in reproductive physiology is illustrated by the following studies. Dietary deficiency of all polyunsaturated fatty acids results in degeneration of the seminiferous tubules and failure of spermatogenesis, effects that can be reversed by supplementing the diet with n-6 fatty acids (3, 39). In females also, essential fatty acid deficiency delays the onset of puberty and results in reproductive failure (3, 40).
Evidence is growing that the 22-carbon long chain polyunsaturated fatty acids of the n-6 and/or n-3 fatty acid families play a specific role in spermatogenesis in many species (5). The sharp increase in DHA in the testis during puberty may be in response to the special need for spermatogenesis. In addition, as DHA is an important component of cell membranes, the increase in DHA may also reflect the change in membrane composition of the spermatogonia reaching maturity. In our previous study of the lipid composition of monkey sperm, we found that DHA was almost the sole n-3 fatty acid (95% of the total n-3 fatty acids) and also the predominant polyunsaturated fatty acid (58% of the total polyunsaturated fatty acids) (13). Of the principal cell types in the testis, spermatogonia, Leydig cells, and Sertoli cells, it is the spermatogonia in various stages of maturity that make up the bulk of the tissue. In sperm, DHA makes up 23.9% of the total phospholipid fatty acids compared to 15.1% in the adult testes. This implies the probable concentration of DHA in the various stages of sperm formation to the mature product. The n-6 fatty acid, dihomogamma-lenolenic acid (20:3 n-6), is present in a relatively high concentration in the adult testis (9.6%), but accounts for only 3.7% of the total phospholipid fatty acids in sperm. Thus, the composition of the testis and that of ejaculated sperm is different. Like desmosterol, DHA is present in a high concentration in the tails of sperm (19.6% of the total fatty acids), but in a very low concentration in the heads of sperm (1.1% of fatty acids) (22). Perhaps membrane fluidity is important for the motile tails; desmosteral has two double bonds, and DHA has six double bonds.
Dihomogamma-linolenic acid (20:3 n-6) and arachidonic acid (20:4 n-6) are the precursors of 1 and 2 series PG, respectively (41, 42, 43, 44). It has been suggested that PGs may play a role in the maturation process of spermatozoa (45, 46, 47). Interestingly, during puberty, the concentrations of the precursors of these two PG precursors in monkey testis changed in opposite directions: 20:3 n-6 increased, and 20:4 n-6 decreased. In most species, the 20:3 n-6 content of tissue phospholipids was far lower than the content of 20:4 n-6, and PGs of the 2 series predominated; however, human and monkey seminal vesicles contain high levels of the 1 series PGs, especially 19-hydroxy-PGE1, derived from 20:3 n-6 (48, 49). In rhesus monkey semen, 19-hydroxy-PGE1 is present at 5 times the level of 19-hydroxy-PGE2. Thus, the specific increase in 20:3 n-6 at puberty may reflect the importance of 1 series PGs in mature testicular function.
In the phospholipid molecular species of testicular and other tissue membranes, polyunsaturated fatty acids occupy the sn-2 position, whereas oleic, palmitic, and stearic acids generally occupy the sn-1 position (13). Thus, the changes observed in the latter three fatty acids as well as various polyunsaturated fatty acids in the testis during puberty suggest possible changes in the composition of membrane phospholipid molecular species. In our previous studies, we found that the membranes of each tissue (brain, retina, sperm, and erythrocytes) have a specific and individualized profile of the phospholipid molecular species (13, 50, 51, 52). Changes in phospholipid molecular species composition have also been found to coincide with dietary and functional changes. It would be informative to examine the changes in the phospholipid molecular species of the testicular membranes during puberty.
In conclusion, the adult primate testis has a unique lipid composition with high concentrations of desmosterol, DHA, and dihomogamma linolenic acid. These three lipids increase greatly during puberty and provide biochemical markers for sexual development in the testis.
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Footnotes |
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Received November 19, 1996.
Revised February 26, 1997.
Accepted March 4, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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