Physiology&Behavior.Vol. 52, pp. 13-16, 1992
0031-9384/92 $5.00+ .00 Copyright © 1992PergamonPressLtd.
Printed in the USA.
Sexual Behavior and Serum Levels of Prolactin, Testosterone, and Estradiol in Young and Old Rhesus Males K A T H L E E N C. C H A M B E R S *l A N D C H A R L E S H. P H O E N I X t
*University of Southern California, Department of Psychology, SGM 501, Los Angeles, CA 90089-1061, and ?Oregon Regional Primate Research Center, Division of Reproductive Biology and Behavior, 505 N W 185th Avenue, Beaverton, OR 97006 Received 9 N o v e m b e r 1990 CHAMBERS, K. C. AND C. H. PHOENIX. Sexual behaviorand serum levelscf prolactin, testosterone, and estradiol in young and old rhesusmales.PHYSIOL BEHAV 52( 1) 13-16, 1992.--It has been suggestedthat increasedprolactin levelsmay contribute to decreased libido in aging male primates. To test this hypothesis, the association of sexual performance and serum prolactin levels was determined in young ( 10 year) and old (25.7 year) male rhesus macaques. Old males displayedsignificantlylower levels of sexual behavior than young males but their serum prolactin levels were not significantly higher. The correlation between prolactin levels and different measures of sexual behavior also were not significant for either old or young males. These data suggest that elevations in prolactin levels do not significantlycontribute to the age-related decline in sexual performance in rhesus males. Aging Males Sexual behavior
Rhesusmacaques
Serumestradiol
Serumprolactin
Serumtestosterone
Because a significant increase in prolactin secretion is reported to occur in old male rats and in old men (17), we undertook this study to determine whether PRL levels in old rhesus males were different than those of young males. We also set out to determine whether the decreased level of sexual performance in old males might be associated with higher levels of serum PRL.
IT is clear that the decline in sexual behavior of rhesus macaques is not caused by a decrease in blood levels of testosterone (T). Although there are changes in the puisatile release of T at night in aged males, there are no changes in mean 24-h levels, and the sexual behavior of old males whose performance levels have declined is not improved by treatment with exogenous testosterone propionate (15,23). Some investigators have suggested that prolactin deserves consideration as a hormone influencing libido in aging primates (11). It has been well documented that hyperprolactinemia has a suppressive effect on reproductive behavior in rats and mice (10,17). In addition, sexually impotent men with hyperprolactinemia induced by pituitary tumors have reported increased potency after tumor removal or after treatment with the prolactin-secretion inhibitor, bromocriptin ( 11). Although elevated levels of prolactin (PRL) have been associated with aging in males of several species, the relationship between these increases and age-related decreases in sexual performance is unknown. Levels of reproductive behavior decline in both rhesus macaques and men (6,8,12,16,20). However, there are no published data on circulating PRL levels in old rhesus males or in any other old nonhuman primate males, and the reports on changes in PRL levels in aged men are inconsistent (7,17).
METHOD
Subjects The subjects consisted of 13 rhesus (Macaca mulatta) males whose ages were estimated to be between 21.0 and 30.9 years (mean 25.0 years) and l0 younger rhesus males whose ages ranged between 10.4 and 11.8 years (mean 10.9 years). Eight of the old males were wild-born, whereas five of the old and all of the young males were captive-born. The procedure for estimating ages and a discussion of maximum life span and mean life expectancy of rhesus macaques has been presented elsewhere (4). Only six of the old and six of the young males were tested behaviorally since only these males had been tested in the past and were thoroughly familiar with the behavioral testing procedures. The old males had displayed high levels of sexual behavior in similar tests when younger. Four ovariectomized females (wild-
1Requests for reprints should be addressed to Kathleen C. Chambers.
13
14
born), estimated to be between the ages of 15.5 and 19.9 years, served as sexual partners.
lhlsbandr 3, The animals were housed individually in temperature- andlight-controlled rooms (lights on from 0700 to 1900). They were tested in an adjacent sound-attenuated room that was also temperature- and-light-controlled. Animals were fed commercially prepared food twice each day and fresh fruit daily. Water was always available. Details of husbandry, and animal care have been presented elsewhere (5,20),
General Procedures and Behaviors The cage in which the monkeys were paired measured 1.81 × 0.71 × 1.22 m. It was constructed of stainless steel and had a Plexiglas front. Shortly after the female was introduced into the test cage, a male was permitted to enter, a computer clock was started, and behavior was recorded directly into a computer and stored for later analysis. The observer viewed the animals from an adjacent room through a one-way glass that was tilted in such a way that the animals were not able to view themselves in the glass. Tests lasted 10 min or until the male ejaculated. Because test duration varied when ejaculation occurred, frequency measures were converted to rates (in min). In computing latency measures, the default value of 600 s (length of test) was used in instances when the behavior in question failed to occur at least once during the test. The behaviors and latencies recorded are commonly used in describing rhesus sexual behavior. They include contact, mount, intromission, ejaculation, latencies to the first mount, intromission and ejaculation, sidle, female present to male contact (P/C ratio), female initiated present (FIP), prox (sit within 1 ft of a seated partner), groom, yawn, threaten partner, and aggression. These behaviors and other derived scores have been described in detail elsewhere (2,19,21,22). Males were given tests of sexual behavior either on Monday and Wednesday or Tuesday and Thursday of each week for 2 consecutive weeks. Each test was with a different female partner. Every male was tested once with each of the four females. The female partners were injected for 12 consecutive days with 10 /~g of estradiol benzoate and served as test partners on days 11 and 12 of injection.
('~IAMBERS '\NI) I'tlOENIX
~alues were I 1.2 pg and 7.5 pg, respectively: and the intrassa,~ coefficients of variation were 6.4q~ and 9.YY,, respectively. Prolactin was measured using the M2 IGB monkey prolactin standard and the methods of Bethea and Papkofl'(I I. Dattl .4 nal.l'si,~
Mean values tbr each behavioral measure tbr each male were calculated and Wilcoxon-Mann-Whitncy tests were used to determine the significance of the differences in pertbrmance between old and young males. Wilcoxon-Mann-Whitney tests also were used to analyze differences in serum hormone levels between old and young males and Wilcoxon Signed Ranks tests were used to analyze differences between hormone levels at 0900 h and 2100 h. The Spearman rank correlation coetficient, r, was used to measure the degree of association between levels of PRL and T, E, and those measures of sexual behavior tor which there were age differences. For all tests, one-tailed probabilities were used to assess significance. RESUI TS
Behavior Young males had significantly higher rates of contacting, mounting, and intromitting than old males [W(6, 6) = 53, 53, and 55, respectively, p - 0.013 and 0.004, respectively] and their latencies to the first mount, intromission, and ejaculation were significantly shorter than those of the old males [W(6, 6) = 27, 25, and 26, respectively, p = 0.032, 0.013, and 0.021 respectively; Fig. 1]. Old and young males did not differ in the percentage of tests in which they developed a penile erection and displayed at least one contact. But the percentages of tests in which old and young males displayed at least one mount and intromission and an ejaculation were significantly greater among young males [W(6, 6) = 50, 53, and 52, respectively, p = 0.046, 0.013. and 0.021, respectively]. The number of contacts, mounts,
30 2 5
12 10
~
Blood Colh,ction and ttormone Assay Approximately 5 ml ofsaphenous vein blood was drawn from the restrained, unanesthesized males at approximately 0900 h and at 2100 h for 2 consecutive days beginning 4 days after the last behavioral test. We, thus, obtained two morning and two night samples from six old and six young males. Samples were taken within 2-3 min after the subjects had been taken from their home cage, and all of the males were adapted to the bleeding procedure. The blood was allowed to clot at 4°C. It was centrifuged at 2000 rpm (4000 × g) and 4°C for 30 min. The serum was then removed and stored in 1 ml aliquots at - 2 0 ° C until assayed for PRL, T, and estradiol (E). Testosterone and E concentrations were estimated in 0.01 and 0.5 ml of serum, respectively, after chromatographic purification on LH-20 Sephadex columns (26,27). The hormones were quantified by radioimmunoassay. For T and E, the percentages of recovery were 86.5% and 67.4%, respectively; the mean of the water blanks were 5.0 pg and 6.4 pg, respectively; the mean of the gonadectomized male serum
05
2 CT
MT
80 +
:~
MT
IT
a
a
IT
EJ
[ ] Old
°o 40
20 CT
MT
IT
EJ
PE
FIG. 1. Mean (+SE) of several measures of sexual behavior of old and young males when paired with female partners, a Differed significantly from old males (p < 0.05). Abbreviations: CT, contact; EJ, ejaculation: IT, intromission; MT, mount; PE, penile erection.
PRL IN RHESUS MALES and intromissions preceding ejaculation for the old males (5.25, 5.25, and 2.92, respectively) was not significantly different than that of the young males (3.58, 3.00, and 2.23, respectively). The rates of proxing, yawning, and the time spent grooming were relatively low for both groups of males and differences were not statistically significant (p > 0.05). None of the males threatened or displayed aggression toward their female partners in any of the tests. The rate at which females sidled and the ratio of female presents to male contacts tended to be higher when the females were paired with young males (0.76 and 0.95, respectively) than when they were paired with old males (0.48 and 0.84, respectively), but the differences only approached significance [ W(6, 6) = 48 and 49, p = 0.09 and 0.066, respectively). However, the FIP rate was significantly lower when the females were with the young males, W(6, 6) = 24, p = 0.008; none of the females initiated a present to a young male. Females proxed old males at about the same rate as they proxed young males and differences in the time spent grooming the two groups of males were low and not statistically significant (p > 0.05).
15 bb
5O
!: A
]Old
IIv,,~
0900 h
2100 h
,,,
Hormone Levels" None of the differences between young and old males in serum levels of PRL, T, and E at 0900 was statistically significant (p > 0.05, Fig. 2). At 2100, only the T levels of young and old males were significantly different (Z = 1.89, p = 0.029). For both young and old males, serum T levels were significantly higher at 2100 h than at 0900 h [T(10) = 53, p = 0.0029 and T(13) = 87, p = 0.0009, respectively) and PRL levels also were significantly higher at night [T(10) = 45, p = 0.042 and T(13) = 85, p = 0.0017, respectively). Serum levels of E did not differ significantly with time of day. Serum levels of PRL were not correlated significantly with T levels at 2100 h, but a significant negative correlation was found at 0900 h (rs -- -0.449, p < 0.025). PRL levels were not correlated significantly with levels of E at 0900 or 2100 h. None of the correlations of sexual behavior and PRL levels at 0900 and 2100 were statistically significant (p > 0.05). DISCUSSION Finding lower performance levels in old males replicates previous findings but extends those observations to very old males. The old males ranged in age from 21 to 31 years and the mean age was 25 years. Reported maximum longevity records for rhesus macaques (sex not specified) range from 19-1/2 years to 27 years, and the mean life expectancy of males born and reared at the Oregon Regional Primate Research Center is 15-18 years (4,14,18). It is unlikely that we will ever test the sexual behavior of a group of healthy males with a higher mean age than those in the present study, It is interesting to note that all of the old males mounted, 67% of them intromitted and 33% ejaculated. The frequency of ejaculation in one of the 27-year-old males was greater than or equal to that of 50% of the young males. Thus, there are striking individual differences in the performance levels of old males, and for some old males performance levels do not diminish substantially with time. In previous studies, we failed to find age-related differences in T levels in the morning (0900 h) or at night (2100 h) (3,6,21). Mean T levels, however, generally tended to be higher in young males at 2100 h. With a larger number of young and old subjects in the present study then in previous studies, this apparent agerelated difference achieved significance. This result is similar to
0900 h
2100 h
ILl
0900 h
2100 h
FIG. 2. Mean (+SE) serum hormone levels of old and young rhesus males at 0900 and 2100 h. aDiffered significantlyfrom old males (p = 0.029). bDiffered significantlyfrom the corresponding 0900 h value (p < 0.05). that reported by Kaler et al. (15). Mean T concentration and mean T pulse numbers were higher in young males than old males at night when blood samples were taken every 20 min for 24 h. No differences in PRL levels were reported in Japanese macaque males (Macacafuscata) that ranged in age from infant to full adult (9). Prolactin had not been measured previously in old males. The serum PRL levels at 0900 and 2100 in old and young adult males did not differ. We also found a significant diurnal variation in PRL for both old and young males. This replicates the finding of Plant (24) who observed diurnal variations in the PRL levels of adult rhesus males. A significant positive correlation between PRL levels and T in socially living Japanese macaques has been noted (9) and it has been suggested that there is an association between the nadir in PRL and T levels (24). We found elevations in both PRL and T levels at night, but we did not find a significant correlation between serum PRL levels and T levels in old or young rhesus males. Although our samples were taken within 2-3 min after the subjects had been taken from their home cage, and all of the males were thoroughly adapted to the bleeding procedure, the possibility exists that stress associated with obtaining a blood sample could have affected levels of PRL. However, Herndon et al. (13) have found that PRL and T levels from samples taken remotely by a venous indwelling catheter did not differ from those subsequently obtained by venipuncture. Also, no changes in PRL levels were found in samples taken at 20-min intervals for 60 min from rhesus males restrained in their cages with a pulling device (25). One of our interests in measuring PRL levels was to determine whether an increase in prolactin might be associated with the lower level of sexual performance associated with
16
t ItAMBERS
old age. Since P R L levels were no h i g h e r in old t h a n in y o u n g males a n d there was no c o r r e l a t i o n between P R L levels a n d p e r f o r m a n c e levels, it is unlikely that alterations in PRL levels play a role in the decline in sexual b e h a v i o r associated with aging.
A N D PH()EN1X
:',.CKNOWI.I 1)( ;[!MI-N1 S I-he work described in this article, Publication No. 1843 of the Oregon Regional Primate Research Center, was supported by Grants AG-0161)8, RR-00163, and HD- 18185 from the National Institutes of health. We thank J. N. Jensen and A. M. Walther lbr their valuable technical assistance.
REFERENCES 1. Bethea, C. L.; Papkoff, H. Purification of monkey prolactin from culture medium: Biochemical and immunological characterization. Soc. Exp. Biol. Med. 182:23-33; 1986. 2. Chambers, K. C.; Hess, D. L.: Phoenix, C. H. Relationship of free and bound testosterone to sexual behavior in old rhesus males. Physiol. Behav. 27:615-620; 1981. 3. Chambers, K. C.; Phoenix, C, H. Diurnal patterns of testosterone, dihydrotestosterone, estradiol, and cortisol in serum of rhesus males: Relationship to sexual behavior in aging males. Horm. Behav. 15: 416-426; 1981. 4. Chambers, K. C.: Phoenix, C. H. Sexual behavior in response to testosterone in old long-term-castrated rhesus males. Neurobiol. Aging 4:223-227; 1983. 5. Chambers, K. C.; Phoenix, C. H. Restoration of sexual perlbrmance in old rhesus macaques paired with a preferred female partner. Int. J. Primatol. 5:287-297; 1984. 6. Chambers, K. C.; Resko, J. A.; Phoenix, C. H. Correlation of diurnal changes in hormones with sexual behavior and age in male rhesus macaques. Neurobiol. Aging 3:37-42; 1982. 7. Davidson, J. M.; Chen, J. J.; Crapo, L.; Gray, G. D.; Greenleaf. W. J.; Catania, J. A. Hormonal changes and sexual function in aging men. J. Clin. Endocrinol. Metab. 57:71-77: 1983. 8. Davidson, J. M.; Gray, G. D.: Smith, E. R. The sexual psychoendocrinology of aging, In: Meites, J., ed. Neuroendocrinology of aging. New York: Plenum Press; 1983:221-258. 9. Dessi-Fulgheri, F. P.; Messeri, P.; Lupo Di Prisco, C. A study of testosterone, estradiol, cortisol and prolactin in a socially intact group of Japanese macaques. Antropol. Contemp. 4:123-127; 1981. 10. Doherty, P. C.; Bartke, A.; Smith, M. S. Differential effects of bromocriptine treatment on LH release and copulatory, behavior in hyperprolactinemic male rats. Horm. Behav. 15:436-450; 1981. 11. Donovan, B. T. Hormones and male sexual behaviour. In: Hormones and human behavior. Cambridge, MA: Cambridge University Press; 1985:121-130. 12. Harman, S. M. Relation of the neuroendocrine system to reproductive decline in men. In: Meites, J., ed. Neuroendocrinology of aging, chapter 11. New York: Plenum Press; 1983:203-219. 13. Herndon, J. G.; Turner, J. J.; Perachio, A. A.; Blank, M. S.; Collins, D. C. Endocrine changes induced by venipuncture in rhesus monkeys. Physiol. Behav. 32:673-676; 1984.
14. Jones, M. L. Mammals in captivity--Primate longevity. Lab. Primate Newslett. 1:3-14: 1962. 15. Kaler, L W.: Gliessman, P.: Hess, D. L.: Hill, J. l h e androgen status of aging male rhesus macaques. Endocrinology 119:566-571 : 1986. 16. Kinsey, C. A.; Pomeroy, W. B.: Martin, C. E. Sexual behavior in the human male. Philadelphia, PA: W. B. Saunders Co.: 1948. 17. Meites, J.; Steger, R. W.: Huang, H. H. H. Relation ofneuroendocrine system to the reproductive decline in aging rats and human subjects. Fed, Proc. 39:3168-3172:1980. 18. Napier, J. R.; Napier, P. H. A Handbook of living primates. New York: Academic Press: 1967. 19. Phoenix, C. H. Effects ofdihydrotestosterone on sexual behavior of castrated male rhesus monkeys. Physiol. Behav. 12:1045-1055: [974. 20. Phoenix, C. H. Factors influencing sexual performance in male rhesus monkeys. J. Comp. Physiol. Psychol. 91:697-710; 1977. 21. Phoenix, C. H.: Chambers, K. C. Sexual deprivation and its influence on testosterone levels and sexual behavior of old and middle-aged rhesus males. Biol. Reprod. 31:480-486: i984. 22. Phoenix, C. H,: Chambers, K. C. Sexual solicitation by middle-aged and old rhesus females. Neurobiol. Aging 7:173-177: 1986. 23. Phoenix, C. H.: Chambers, K. C. Testosterone therapy in young and old rhesus males that display low levels of sexual activity. Physiol. Behav. 43:479-484: 1988. 24. Plant, T. M. Time courses of concentrations of circulating gonadotropin, prolactin, testosterone, and cortisol in adult male rhesus monkeys (Macaca mulatta) throughout the 24 h light-dark cycle. Biol. Reprod. 25:244-252: 1981. 25. Purl, C. P.: Puri, V.: Anand Kumar, T. C. Serum levels of testosterone, cortisol, prolactin and bioactive luteinizing hormone in adult male rhesus monkeys following cage-restraint or anaesthetizing with ketamine hydrochloride. Acta Endocrinol. 97:118-124:1981. 26. Resko, J. A.: Ellinwood, W. E.: Pasztor, L. M.: BuM, A. E. Sex sleroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation. J. Clin. Endocrinol. Metab. 51): 900-905:198(/. 27. Resko, J. A.: Ploem, J. G.; Stadelman, H. L. Estrogens in tbtal and maternal plasma of the rhesus monkey. Endocrinology 97:425-43(/: 1975.
0031-9384/92 $5.00+ .00 Copyright © 1992PergamonPressLtd.
Printed in the USA.
Sexual Behavior and Serum Levels of Prolactin, Testosterone, and Estradiol in Young and Old Rhesus Males K A T H L E E N C. C H A M B E R S *l A N D C H A R L E S H. P H O E N I X t
*University of Southern California, Department of Psychology, SGM 501, Los Angeles, CA 90089-1061, and ?Oregon Regional Primate Research Center, Division of Reproductive Biology and Behavior, 505 N W 185th Avenue, Beaverton, OR 97006 Received 9 N o v e m b e r 1990 CHAMBERS, K. C. AND C. H. PHOENIX. Sexual behaviorand serum levelscf prolactin, testosterone, and estradiol in young and old rhesusmales.PHYSIOL BEHAV 52( 1) 13-16, 1992.--It has been suggestedthat increasedprolactin levelsmay contribute to decreased libido in aging male primates. To test this hypothesis, the association of sexual performance and serum prolactin levels was determined in young ( 10 year) and old (25.7 year) male rhesus macaques. Old males displayedsignificantlylower levels of sexual behavior than young males but their serum prolactin levels were not significantly higher. The correlation between prolactin levels and different measures of sexual behavior also were not significant for either old or young males. These data suggest that elevations in prolactin levels do not significantlycontribute to the age-related decline in sexual performance in rhesus males. Aging Males Sexual behavior
Rhesusmacaques
Serumestradiol
Serumprolactin
Serumtestosterone
Because a significant increase in prolactin secretion is reported to occur in old male rats and in old men (17), we undertook this study to determine whether PRL levels in old rhesus males were different than those of young males. We also set out to determine whether the decreased level of sexual performance in old males might be associated with higher levels of serum PRL.
IT is clear that the decline in sexual behavior of rhesus macaques is not caused by a decrease in blood levels of testosterone (T). Although there are changes in the puisatile release of T at night in aged males, there are no changes in mean 24-h levels, and the sexual behavior of old males whose performance levels have declined is not improved by treatment with exogenous testosterone propionate (15,23). Some investigators have suggested that prolactin deserves consideration as a hormone influencing libido in aging primates (11). It has been well documented that hyperprolactinemia has a suppressive effect on reproductive behavior in rats and mice (10,17). In addition, sexually impotent men with hyperprolactinemia induced by pituitary tumors have reported increased potency after tumor removal or after treatment with the prolactin-secretion inhibitor, bromocriptin ( 11). Although elevated levels of prolactin (PRL) have been associated with aging in males of several species, the relationship between these increases and age-related decreases in sexual performance is unknown. Levels of reproductive behavior decline in both rhesus macaques and men (6,8,12,16,20). However, there are no published data on circulating PRL levels in old rhesus males or in any other old nonhuman primate males, and the reports on changes in PRL levels in aged men are inconsistent (7,17).
METHOD
Subjects The subjects consisted of 13 rhesus (Macaca mulatta) males whose ages were estimated to be between 21.0 and 30.9 years (mean 25.0 years) and l0 younger rhesus males whose ages ranged between 10.4 and 11.8 years (mean 10.9 years). Eight of the old males were wild-born, whereas five of the old and all of the young males were captive-born. The procedure for estimating ages and a discussion of maximum life span and mean life expectancy of rhesus macaques has been presented elsewhere (4). Only six of the old and six of the young males were tested behaviorally since only these males had been tested in the past and were thoroughly familiar with the behavioral testing procedures. The old males had displayed high levels of sexual behavior in similar tests when younger. Four ovariectomized females (wild-
1Requests for reprints should be addressed to Kathleen C. Chambers.
13
14
born), estimated to be between the ages of 15.5 and 19.9 years, served as sexual partners.
lhlsbandr 3, The animals were housed individually in temperature- andlight-controlled rooms (lights on from 0700 to 1900). They were tested in an adjacent sound-attenuated room that was also temperature- and-light-controlled. Animals were fed commercially prepared food twice each day and fresh fruit daily. Water was always available. Details of husbandry, and animal care have been presented elsewhere (5,20),
General Procedures and Behaviors The cage in which the monkeys were paired measured 1.81 × 0.71 × 1.22 m. It was constructed of stainless steel and had a Plexiglas front. Shortly after the female was introduced into the test cage, a male was permitted to enter, a computer clock was started, and behavior was recorded directly into a computer and stored for later analysis. The observer viewed the animals from an adjacent room through a one-way glass that was tilted in such a way that the animals were not able to view themselves in the glass. Tests lasted 10 min or until the male ejaculated. Because test duration varied when ejaculation occurred, frequency measures were converted to rates (in min). In computing latency measures, the default value of 600 s (length of test) was used in instances when the behavior in question failed to occur at least once during the test. The behaviors and latencies recorded are commonly used in describing rhesus sexual behavior. They include contact, mount, intromission, ejaculation, latencies to the first mount, intromission and ejaculation, sidle, female present to male contact (P/C ratio), female initiated present (FIP), prox (sit within 1 ft of a seated partner), groom, yawn, threaten partner, and aggression. These behaviors and other derived scores have been described in detail elsewhere (2,19,21,22). Males were given tests of sexual behavior either on Monday and Wednesday or Tuesday and Thursday of each week for 2 consecutive weeks. Each test was with a different female partner. Every male was tested once with each of the four females. The female partners were injected for 12 consecutive days with 10 /~g of estradiol benzoate and served as test partners on days 11 and 12 of injection.
('~IAMBERS '\NI) I'tlOENIX
~alues were I 1.2 pg and 7.5 pg, respectively: and the intrassa,~ coefficients of variation were 6.4q~ and 9.YY,, respectively. Prolactin was measured using the M2 IGB monkey prolactin standard and the methods of Bethea and Papkofl'(I I. Dattl .4 nal.l'si,~
Mean values tbr each behavioral measure tbr each male were calculated and Wilcoxon-Mann-Whitncy tests were used to determine the significance of the differences in pertbrmance between old and young males. Wilcoxon-Mann-Whitney tests also were used to analyze differences in serum hormone levels between old and young males and Wilcoxon Signed Ranks tests were used to analyze differences between hormone levels at 0900 h and 2100 h. The Spearman rank correlation coetficient, r, was used to measure the degree of association between levels of PRL and T, E, and those measures of sexual behavior tor which there were age differences. For all tests, one-tailed probabilities were used to assess significance. RESUI TS
Behavior Young males had significantly higher rates of contacting, mounting, and intromitting than old males [W(6, 6) = 53, 53, and 55, respectively, p - 0.013 and 0.004, respectively] and their latencies to the first mount, intromission, and ejaculation were significantly shorter than those of the old males [W(6, 6) = 27, 25, and 26, respectively, p = 0.032, 0.013, and 0.021 respectively; Fig. 1]. Old and young males did not differ in the percentage of tests in which they developed a penile erection and displayed at least one contact. But the percentages of tests in which old and young males displayed at least one mount and intromission and an ejaculation were significantly greater among young males [W(6, 6) = 50, 53, and 52, respectively, p = 0.046, 0.013. and 0.021, respectively]. The number of contacts, mounts,
30 2 5
12 10
~
Blood Colh,ction and ttormone Assay Approximately 5 ml ofsaphenous vein blood was drawn from the restrained, unanesthesized males at approximately 0900 h and at 2100 h for 2 consecutive days beginning 4 days after the last behavioral test. We, thus, obtained two morning and two night samples from six old and six young males. Samples were taken within 2-3 min after the subjects had been taken from their home cage, and all of the males were adapted to the bleeding procedure. The blood was allowed to clot at 4°C. It was centrifuged at 2000 rpm (4000 × g) and 4°C for 30 min. The serum was then removed and stored in 1 ml aliquots at - 2 0 ° C until assayed for PRL, T, and estradiol (E). Testosterone and E concentrations were estimated in 0.01 and 0.5 ml of serum, respectively, after chromatographic purification on LH-20 Sephadex columns (26,27). The hormones were quantified by radioimmunoassay. For T and E, the percentages of recovery were 86.5% and 67.4%, respectively; the mean of the water blanks were 5.0 pg and 6.4 pg, respectively; the mean of the gonadectomized male serum
05
2 CT
MT
80 +
:~
MT
IT
a
a
IT
EJ
[ ] Old
°o 40
20 CT
MT
IT
EJ
PE
FIG. 1. Mean (+SE) of several measures of sexual behavior of old and young males when paired with female partners, a Differed significantly from old males (p < 0.05). Abbreviations: CT, contact; EJ, ejaculation: IT, intromission; MT, mount; PE, penile erection.
PRL IN RHESUS MALES and intromissions preceding ejaculation for the old males (5.25, 5.25, and 2.92, respectively) was not significantly different than that of the young males (3.58, 3.00, and 2.23, respectively). The rates of proxing, yawning, and the time spent grooming were relatively low for both groups of males and differences were not statistically significant (p > 0.05). None of the males threatened or displayed aggression toward their female partners in any of the tests. The rate at which females sidled and the ratio of female presents to male contacts tended to be higher when the females were paired with young males (0.76 and 0.95, respectively) than when they were paired with old males (0.48 and 0.84, respectively), but the differences only approached significance [ W(6, 6) = 48 and 49, p = 0.09 and 0.066, respectively). However, the FIP rate was significantly lower when the females were with the young males, W(6, 6) = 24, p = 0.008; none of the females initiated a present to a young male. Females proxed old males at about the same rate as they proxed young males and differences in the time spent grooming the two groups of males were low and not statistically significant (p > 0.05).
15 bb
5O
!: A
]Old
IIv,,~
0900 h
2100 h
,,,
Hormone Levels" None of the differences between young and old males in serum levels of PRL, T, and E at 0900 was statistically significant (p > 0.05, Fig. 2). At 2100, only the T levels of young and old males were significantly different (Z = 1.89, p = 0.029). For both young and old males, serum T levels were significantly higher at 2100 h than at 0900 h [T(10) = 53, p = 0.0029 and T(13) = 87, p = 0.0009, respectively) and PRL levels also were significantly higher at night [T(10) = 45, p = 0.042 and T(13) = 85, p = 0.0017, respectively). Serum levels of E did not differ significantly with time of day. Serum levels of PRL were not correlated significantly with T levels at 2100 h, but a significant negative correlation was found at 0900 h (rs -- -0.449, p < 0.025). PRL levels were not correlated significantly with levels of E at 0900 or 2100 h. None of the correlations of sexual behavior and PRL levels at 0900 and 2100 were statistically significant (p > 0.05). DISCUSSION Finding lower performance levels in old males replicates previous findings but extends those observations to very old males. The old males ranged in age from 21 to 31 years and the mean age was 25 years. Reported maximum longevity records for rhesus macaques (sex not specified) range from 19-1/2 years to 27 years, and the mean life expectancy of males born and reared at the Oregon Regional Primate Research Center is 15-18 years (4,14,18). It is unlikely that we will ever test the sexual behavior of a group of healthy males with a higher mean age than those in the present study, It is interesting to note that all of the old males mounted, 67% of them intromitted and 33% ejaculated. The frequency of ejaculation in one of the 27-year-old males was greater than or equal to that of 50% of the young males. Thus, there are striking individual differences in the performance levels of old males, and for some old males performance levels do not diminish substantially with time. In previous studies, we failed to find age-related differences in T levels in the morning (0900 h) or at night (2100 h) (3,6,21). Mean T levels, however, generally tended to be higher in young males at 2100 h. With a larger number of young and old subjects in the present study then in previous studies, this apparent agerelated difference achieved significance. This result is similar to
0900 h
2100 h
ILl
0900 h
2100 h
FIG. 2. Mean (+SE) serum hormone levels of old and young rhesus males at 0900 and 2100 h. aDiffered significantlyfrom old males (p = 0.029). bDiffered significantlyfrom the corresponding 0900 h value (p < 0.05). that reported by Kaler et al. (15). Mean T concentration and mean T pulse numbers were higher in young males than old males at night when blood samples were taken every 20 min for 24 h. No differences in PRL levels were reported in Japanese macaque males (Macacafuscata) that ranged in age from infant to full adult (9). Prolactin had not been measured previously in old males. The serum PRL levels at 0900 and 2100 in old and young adult males did not differ. We also found a significant diurnal variation in PRL for both old and young males. This replicates the finding of Plant (24) who observed diurnal variations in the PRL levels of adult rhesus males. A significant positive correlation between PRL levels and T in socially living Japanese macaques has been noted (9) and it has been suggested that there is an association between the nadir in PRL and T levels (24). We found elevations in both PRL and T levels at night, but we did not find a significant correlation between serum PRL levels and T levels in old or young rhesus males. Although our samples were taken within 2-3 min after the subjects had been taken from their home cage, and all of the males were thoroughly adapted to the bleeding procedure, the possibility exists that stress associated with obtaining a blood sample could have affected levels of PRL. However, Herndon et al. (13) have found that PRL and T levels from samples taken remotely by a venous indwelling catheter did not differ from those subsequently obtained by venipuncture. Also, no changes in PRL levels were found in samples taken at 20-min intervals for 60 min from rhesus males restrained in their cages with a pulling device (25). One of our interests in measuring PRL levels was to determine whether an increase in prolactin might be associated with the lower level of sexual performance associated with
16
t ItAMBERS
old age. Since P R L levels were no h i g h e r in old t h a n in y o u n g males a n d there was no c o r r e l a t i o n between P R L levels a n d p e r f o r m a n c e levels, it is unlikely that alterations in PRL levels play a role in the decline in sexual b e h a v i o r associated with aging.
A N D PH()EN1X
:',.CKNOWI.I 1)( ;[!MI-N1 S I-he work described in this article, Publication No. 1843 of the Oregon Regional Primate Research Center, was supported by Grants AG-0161)8, RR-00163, and HD- 18185 from the National Institutes of health. We thank J. N. Jensen and A. M. Walther lbr their valuable technical assistance.
REFERENCES 1. Bethea, C. L.; Papkoff, H. Purification of monkey prolactin from culture medium: Biochemical and immunological characterization. Soc. Exp. Biol. Med. 182:23-33; 1986. 2. Chambers, K. C.; Hess, D. L.: Phoenix, C. H. Relationship of free and bound testosterone to sexual behavior in old rhesus males. Physiol. Behav. 27:615-620; 1981. 3. Chambers, K. C.; Phoenix, C, H. Diurnal patterns of testosterone, dihydrotestosterone, estradiol, and cortisol in serum of rhesus males: Relationship to sexual behavior in aging males. Horm. Behav. 15: 416-426; 1981. 4. Chambers, K. C.: Phoenix, C. H. Sexual behavior in response to testosterone in old long-term-castrated rhesus males. Neurobiol. Aging 4:223-227; 1983. 5. Chambers, K. C.; Phoenix, C. H. Restoration of sexual perlbrmance in old rhesus macaques paired with a preferred female partner. Int. J. Primatol. 5:287-297; 1984. 6. Chambers, K. C.; Resko, J. A.; Phoenix, C. H. Correlation of diurnal changes in hormones with sexual behavior and age in male rhesus macaques. Neurobiol. Aging 3:37-42; 1982. 7. Davidson, J. M.; Chen, J. J.; Crapo, L.; Gray, G. D.; Greenleaf. W. J.; Catania, J. A. Hormonal changes and sexual function in aging men. J. Clin. Endocrinol. Metab. 57:71-77: 1983. 8. Davidson, J. M.; Gray, G. D.: Smith, E. R. The sexual psychoendocrinology of aging, In: Meites, J., ed. Neuroendocrinology of aging. New York: Plenum Press; 1983:221-258. 9. Dessi-Fulgheri, F. P.; Messeri, P.; Lupo Di Prisco, C. A study of testosterone, estradiol, cortisol and prolactin in a socially intact group of Japanese macaques. Antropol. Contemp. 4:123-127; 1981. 10. Doherty, P. C.; Bartke, A.; Smith, M. S. Differential effects of bromocriptine treatment on LH release and copulatory, behavior in hyperprolactinemic male rats. Horm. Behav. 15:436-450; 1981. 11. Donovan, B. T. Hormones and male sexual behaviour. In: Hormones and human behavior. Cambridge, MA: Cambridge University Press; 1985:121-130. 12. Harman, S. M. Relation of the neuroendocrine system to reproductive decline in men. In: Meites, J., ed. Neuroendocrinology of aging, chapter 11. New York: Plenum Press; 1983:203-219. 13. Herndon, J. G.; Turner, J. J.; Perachio, A. A.; Blank, M. S.; Collins, D. C. Endocrine changes induced by venipuncture in rhesus monkeys. Physiol. Behav. 32:673-676; 1984.
14. Jones, M. L. Mammals in captivity--Primate longevity. Lab. Primate Newslett. 1:3-14: 1962. 15. Kaler, L W.: Gliessman, P.: Hess, D. L.: Hill, J. l h e androgen status of aging male rhesus macaques. Endocrinology 119:566-571 : 1986. 16. Kinsey, C. A.; Pomeroy, W. B.: Martin, C. E. Sexual behavior in the human male. Philadelphia, PA: W. B. Saunders Co.: 1948. 17. Meites, J.; Steger, R. W.: Huang, H. H. H. Relation ofneuroendocrine system to the reproductive decline in aging rats and human subjects. Fed, Proc. 39:3168-3172:1980. 18. Napier, J. R.; Napier, P. H. A Handbook of living primates. New York: Academic Press: 1967. 19. Phoenix, C. H. Effects ofdihydrotestosterone on sexual behavior of castrated male rhesus monkeys. Physiol. Behav. 12:1045-1055: [974. 20. Phoenix, C. H. Factors influencing sexual performance in male rhesus monkeys. J. Comp. Physiol. Psychol. 91:697-710; 1977. 21. Phoenix, C. H.: Chambers, K. C. Sexual deprivation and its influence on testosterone levels and sexual behavior of old and middle-aged rhesus males. Biol. Reprod. 31:480-486: i984. 22. Phoenix, C. H,: Chambers, K. C. Sexual solicitation by middle-aged and old rhesus females. Neurobiol. Aging 7:173-177: 1986. 23. Phoenix, C. H.: Chambers, K. C. Testosterone therapy in young and old rhesus males that display low levels of sexual activity. Physiol. Behav. 43:479-484: 1988. 24. Plant, T. M. Time courses of concentrations of circulating gonadotropin, prolactin, testosterone, and cortisol in adult male rhesus monkeys (Macaca mulatta) throughout the 24 h light-dark cycle. Biol. Reprod. 25:244-252: 1981. 25. Purl, C. P.: Puri, V.: Anand Kumar, T. C. Serum levels of testosterone, cortisol, prolactin and bioactive luteinizing hormone in adult male rhesus monkeys following cage-restraint or anaesthetizing with ketamine hydrochloride. Acta Endocrinol. 97:118-124:1981. 26. Resko, J. A.: Ellinwood, W. E.: Pasztor, L. M.: BuM, A. E. Sex sleroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation. J. Clin. Endocrinol. Metab. 51): 900-905:198(/. 27. Resko, J. A.: Ploem, J. G.; Stadelman, H. L. Estrogens in tbtal and maternal plasma of the rhesus monkey. Endocrinology 97:425-43(/: 1975.
