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ExperimentalGerontology, Vol. 17, pp. 437--443, 1982
0531-5565/82/060437-07503.00/0 Copyright ©'1983 Pergamon Press Ltd
Printed in the USA. All rights reserved.
PLASMA AND PITUITARY CONCENTRATIONS OF LUTEINIZING HORMONE, FOLLICLE-STIMULATING H O R M O N E A N D P R O L A C T I N IN A G E D , O V A R I E C T O M I Z E D CD-1 AND C57BL/6 MICE
T.A. PARKENING, T.J. COLLINS and E.R. SMITH* Departments of Anatomy and *Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, TX 77550
(Received 26 January 1982) Abstract-Plasma and pituitary concentrations of luteinizing hormone (LID, follicle-stimulating hormone (FSID, and prolactin (Prl) were determined by ratioimmunoassay in young (2-4 monthsold) and aged CD-I (14--18 months-old) and C57BL/6 (16-22 months-old) mice one month after ovariectomy. In young, ovariectomized mice, plasma and pituitary concentrations of LH and FSH were significantly higher, whereas concentrations of Prl were significantly lower than those in control mice (sham-operated). In contrast, plasma concentrations of LH, FSH and Prl were not statistically different in aged, ovariectomized mice and aged, control mice. There were also no differences in pituitary concentrations of the three hormones when comparing the same aged C57BL/6 mice, although the aged, ovariectomized CD-I mice exhibited higher pituitary levels of each hormone than those of their controls. The pituitary of the aged mouse responds differently to ovariectomy than that of the young mouse because of age-related changes in the ovary and/or hypot halamic-h ypophyseal complex.
INTRODUCTION AOE-RELATED changes in the hypothalamic-hypophyseal-ovarian complex are in part responsible for the loss of reproductive capacity in laboratory rodents. Radioimmunoassay studies conducted in our laboratory on aged oestrous and constant dioestrous C57BL/6 mice (Parkening et al., 1980a) and on anoestrous Syrian hamsters (Parkening et al., 1982) have shown that with advancing age a significant increase occurs in the circulating plasma concentrations of gonadotrophins and a decrease occurs in the plasma concentrations of prolactin (Prl). It is not known if these hormonal changes result from a dysfunctioning ovary or an increasing insensitivity of the pituitary-hypothalamic complex to gonadal feedback. In aged, ovariectomized, anoestrous hamsters, an insensitivity of the pituitary-hypothalamic complex appeared to be the primary reason for age-relat.ed changes in the secretion of pituitary hormones (Parkening et al., 1982). To better determine what influence the ovary has on the pituitary-hypothalamic complex in aged mice, plasma and pituitary concentrations of gonadotrophins and Prl were measured in bilaterally ovariectomized mice. Address all correspondence to: T.A. Parkening, Ph.D., Dept. of Anatomy, The University of Texas Medical Branch at Galveston, Galveston, TX 77550 437
438
PARKENING, COLLINS AND SMITH
MATERIALS AND METHODS Aged CD-1 Swiss albino (14-18 months-old) and C57BL/6 06--22 months-old) mice and younger mature females (2-4 months-old) of both strains were obtained from Charles River Laboratories, Wilmington, Mass. The aged CD-1 females were acquired at 6 weeks of age and bred continuously until used in the experiment. The aged C57BL/6 mice were virgin females acquired from the NIH colony maintained by Charles River Laboratories. The latter females ranged from 15-20 months-of-age and were used in the experiment within 6-8 weeks of their delivery date. The mice were housed under uniform temperature and lighting conditions (14 h light/10 h darkness) and provided with water and mouse chow ad libitura. Young mature and aged females of both strains were either bilaterally ovariectomized or sham-operated. One month following surgery, the ovariectomized mice were bled unanesthetized between 1000-1200 h by cardiac puncture using a heparinized syringe. Control females were checked after one month and bled as above on the day they exhibited a dioestrous vaginal smear. All of the younger sham-operated mice exhibited a 4-day oestrous cycle, and most of the aged, sham-operated mice an irregular oestrous cycle or a constant leukocytic vaginal smear (only C57BL/6 mice showed the latter). None of the smears of the aged mice showed more than 2 days of cornified cells. After bleeding, the pituitary gland was removed, weighed and cut in half. One-half was fixed for future immunocytochemical studies and the other half was reweighed and homogenized in 0.5 ml of 0.01 M phosphatebuffered saline (pH 7.6) containing 0.05 M EDTA and 0.1°70 sodium azide. The blood plasma and supernatant of the pituitary homogenates were stored at - 8 5 " C to permit an analysis of all samples in a given assay. Procedures for the radioimmunoassay (RIA) of LH, FSH and Prl have been presented in detail elsewhere (Parkening et aL, 1980a, b; Collins et al., 1980). NIAMDD rat-LH and rat-FSH RIA kits and a NIAMDD mouse-Prl RIA kit were used in determining concentrations of LH, FSH and Prl. In 90°/0 of the mice, sufficient plasma was obtained from each animal to assay for all three hormones (single 100-200 #l samples for LH and FSH and duplicate 50 td samples for Prl). Pituitary hormonal concentrations were determined in duplicate (200 #1 of a I/100 dilution o f the supernatant from the pituitary homogenate for LH, 200 #l of a l / l 0 dilution for FSH and 200 #l of a 1/20 dilution for Prl). No interassay variances occurred because all samples for an individual hormone were analyzed in a single assay. All intraassay variances were less than 10g0. A comparison of mean hormonal concentrations was made using the Student's t test. Statistical significance was determined at the 0.05 probability level.
RESULTS The concentrations of LH in the plasma of the young mature CD-1 and C57BL/6 mice that had been ovariectomized were at least twice as great as those found in the shamoperated controls (Table 1). In contrast, LH concentrations in aged, ovariectomized mice of both strains were similar to those in the aged controls. Aged, control C57BL/6 mice, however, were found to have plasma LH concentrations that were more than twice as great as those in young mature mice (p < 0.001). In the pituitary, the concentrations of LH were much higher for the ovariectomized mice (except aged C57BL/6 mice) than for their controls (Table I). The concentrations of FSH in the plasma of young CD-1 and C57BL/6 mice that had been ovariectomized were at least 5 times as great as those in their controls; however, no differences were found between the FSH concentrations in aged, ovariectornized mice and aged, control mice (Table 1). The aged, sham-operated mice, therefore, had significantly higher concentrations of circulating FSH than the sham-operated, younger mice (p < 0.001). In contrast, young, ovariectomized mice had significantly higher concentrations of FSH than aged, ovariectomized mice (p < 0.001). Greater concentrations of FSH were found in the pituitary of the young, ovariectomized mice (p < 0.001) than in their controls, and in the aged, ovariectomized CD-1 mice (p < 0.01) than in their aged controls (Table 1). No differences existed between concentrations of FSH in aged, control and aged, ovariectomized C57BL/6 mice. The concentrations of Prl that were found in the plasma of ovariectomized, young mice were considerably lower than those in the young controls (p < 0.001), whereas no differences were found in the concentrations of Prl in the aged, ovariectomized and aged,
439
LH, FSH & PROLACTIN iN AGED, OVARiECTOMIZED MiCE TABLE 1. PLASMA AND PITUITARY LEVELS OF LH, F S H AND PRL IN YOUNG MATURE AND AGED OVARIEGTOMIZED C D - I AND G 5 7 B L / 6 MICE.
CD-1 Mice 2 - 4 months-old Plasma ( n g / m l ± S.E.M.) LH Sham-operated Ovariectomized FSH Sham-operated Ovariectomized Prl Sham-operated Ovariectomized Pituitary L H (ng/p.g 4- S.E.M.) Sham-operated Ovariectomized FSH ( n g / # g 4- S.E.M.) Sham-operated Ovariectomized Prl ( n g / m g 4- S.E.M.) Sham-operated Ovariectomized
14-18 m o n t h s - o l d
C 5 7 B L / 6 Mice 2 - 4 months-old
16-22 months-old
26.0 4- 0.8 (16)* 54.3 4- 3.44 (20)
30.7 -4- 2.1 (I0) 35.0 4- 3.Y (10)
24.2 -4- 1.1 (17) 66.3 4- 3.74 (22)
55.9 4- 5.8 h (14) 51.0 .4- 2.7 I ( 11)
284.7 -4- 16.6 (16) 1545.2 4- 76.54 (20)
1102.3 4- 168.2 h (9) 939.2 -~ 93.5 k (10)
373.8 4- 16.9 (15) 1939.6 4- 88,14 (17)
973.5 4- 63.4 h (15) 1135.3 4- 84.4 ~ (I 1)
75.4 4- 5.5 (16) 36.5 4- 3.24 (20)
61.3 4- 4.3 (9) 70.5 4- 5.0 k (10)
62.4 4- 4.8 (14) 33.6 4- 3.3 d (17)
71.0 4- 3.2 (15) 68.5 4- 5.4 ~ (11)
4.6 4- 0.4 (16) 9.4 4- 0.5" (20)
5.2 4- 0.7 (13) 8.2 4- 1.0 / ( l 0)
4.4 4- 0.3 (18) 6.5 4- 0.4 d (22)
5.4 ± 0.6 (15) 6.9 4- 0.6 ( l l)
5.6 4- 0.5 h (13) 7.9 4- 0.Y .h (10)
3.7 4- 0.2 (18) 9.9 -4- 0.5 d (22)
4.8 4- 0 . ~ (15) 5.5 4- 0.3 k (1 l)
778.9 4- 44.4 (18) 281.4 4- 23.6 d (22)
532.0 4- 37.2 h (15) 495.5 4- 29.8 ~ ( I 1)
3.2 4- 0.3 (16) 15.0 ± 0.94 (20) 597.5 4- 24.1 (16) 510.0 4- 20.0 b (20)
373.9 4- 34.5 h (13) 467.0 4- 22.2" (10)
*Numbers o f females assayed are in parentheses. p < 0.05 b, p < 0.01 c, p < 0.001d: significantly different when c o m p a r i n g younger mature sham-operated and ovariectomized mice. p < 0.05 °, p < 0.0V: significantly different when c o m p a r i n g aged sham-operated a n d ovariectomized mice. p < 0.05,, p < 0.001h: significantly different when c o m p a r i n g younger mature a n d aged sham-operated mice. p < 0.05', p < 0.01~, p < 0.001~: significantly different when comparing younger m a t u r e and aged ovariectomized mice,
control mice. Aged, ovariectomized and aged, control mice had concentrations of Prl in their plasma that were similar to those in the younger, control mice (Table 1). Concentrations of pituitary Prl were lower in ovariectomized, young CD-1 (p < 0.05) and C57BL/6 (p < 0.001) mice than in their controls. Aged, ovariectomized CD-I mice had higher pituitary concentrations of Prl than their aged controls (p < 0.05), but no differences were discovered between pituitary Prl concentrations in aged, ovariectomized and aged, con-
440
PARKENING,COLLINSANDSMITH
trol C57BL/6 mice. The aged, control mice of both strains had lower concentrations of pituitary Prl than the young controls (p < 0.001). DISCUSSION A number of radioimmunoassay studies have shown that plasma concentrations of LH and FSH rise following gonadectomy in rats (Howland and Preiss, 1975; Gosden and Bancroft, 1976; McPherson et al., 1977) and mice (Kovacic and Parlow, 1972). Gosden et al. (1978) have reported, however, that levels of serum LH in 3-6 months-old CBA mice, one month after ovariectomy, were similar to those in sham-operated controls. In contrast, Kovacic and Parlow (1972) showed that mice ovariectomized for 50 days had serum levels of LH that were significantly greater (approximately 2 ½ times higher) than those found in intact mice during dioestrus. In the present study, both strains of young mature mice had concentrations of LH that were approximately twice those found in shamoPerated control, dioestrous animals. The results that were obtained by Gosden et aL, (1978) may have been influenced by the strain of mouse they used. The CBA mouse is precocious in its loss of follicles and it is not known what causes Such a loss. An early onset of acyclicity in this strain has been reported (Jones and Krohn, 1961) which implies premature hormonal changes. Serum concentrations of Prl in C3H/St mice that had been ovariectomized for 2 weeks have been shown to be lower than those in sham-operated controls (Sinha et al., 1972), although the difference was not significant. Pituitary concentrations of Prl in these same mice, however, were significantly less than those in ovariectomized animals. The discrepancies between that study and the present investigation in which significantly lower plasma Prl concentrations were found in younger ovariectomized CD-I and C57BL/6 mice when compared to intact females, may result from the longer period the animals were ovariectomized (1 month) prior to bleeding or from strain differences. Female C57BL/St mice were shown to possess three times more serum Prl than C3H/St mice, but significantly less pituitary Prl (Sinha et al., 1972). The present study indicates that the hypophyseal-hypothalamic complex of the aged female mouse has little capacity to respond to ovariectomy. The response that does exist, as reflected by the concentrations of pituitary hormones that were found in 14-18 monthsold CD-1 mice, apparently declines even further with increasing age, since no statistical differences were found in concentrations of pituitary hormones in Qvariectomized and control 16-22 months-old C57BL/6 mice. The pattern of hormonal secretion in young mature, ovariectomized mice in the present study was similar to that reported for young mature, ovariectomized rats (Huang et al., 1976). Plasma concentrations of LH, FSH and Prl in aged CD-1 and C57BL/6 mice, ovariectomized for one month, remained similar to those in aged sham-operated mice. In contrast, aged rats (22-24 months-of-age) that either were in contrast oestrus or repetitive pseudopregnancy before ovariectomy, exhibited a rise in the levels of serum LH, FSH and Prl when compared with those levels found in younger, intact rats (Huang et al., 1976). Moreover, even ovariectomized anoestrous rats (26-30 months-of-age) had significantly higher levels of FSH and Prl than intact, aged anoestrous rats (Huang et al., 1976). The pituitary of aged, anoestrous Syrian hamsters is also capable of responding to ovariectomy by increasing LH and FSH concentrations, although not to the same degree as the pituitary of younger, ovariectomized hamsters (Parkening et al., 1982). While the
LH, PSH & PROLACTIN IN AGED, OVARIECTOMIZED MICE
441
hypothalamic-hypophyseal complex of anoestrous rats and hamsters appear to retain some ability to respond to the absence of negative feedback, the hypothalamichypophyseal complex of the aged mouse does not. The possibility exists, however, based on the elevated concentrations of LH in aged intact C57BL/6 mice and the higher concentrations of FSH in aged intact mice of both strains, that a poorly functioning ovary in these animals has already created a hormonal environment similar to that found in a gonadectomized mouse. Aged mice given steroid injections should provide answers as to whether the hypothalamic-hypophyseal complex is still capable of responding. In just such a study, aged C57BL/6J mice given exogenous oestradiol did not produce an LH surge comparable to that of young or middle-aged mice (Finch et al., 1980). Younger mice in this same study exhibited a considerably higher LH surge than middle-aged mice, implying that the hypothalamus is responsible to a greater degree for changes in gonadotrophin secretion than are the ovarian steroids. Aged rats and mice do not respond to exogenous GnRH in the same manner as younger animals. Serum concentrations of LH that were measured after an initial injection of GnRH were uniformly lower in aged rats of both sexes in comparison to those in younger rats (Miller and Riegle, 1978). The pituitaries of 16-22 months-old female C57BL/6 mice were also found to be considerably less sensitive than those of 2--4 months-old mice (Collins et aL, 1981). In another study, medial basal hypothalamic concentrations of GnRH in repetitively pseudopregnant 8-12 months-old rats (ovariectomized for 3 weeks) dropped appreciably from those of an intact group (Wise and Ratner, 1980). In the intact group, however, concentrations of GnRH were lower than those of younger rats with regular oestrous cycles. From this data it was concluded that the anterior pituitary was largely responsible for such changes in the older rats. In contrast, hypothalamic concentrations of GnRH in intact and ovariectomized rats exhibiting constant oestrus (8-12 months-old) were not statistically different from one another (although both were lower than those in intact, younger rats with regular oestrous cycles) and it was suggested that the major defect in these rats resided within the hypothalamus (Wise and Ratner, 1980). It is possible therefore, that the stage of irregular cyclicity exhibited by some older laboratory rodents is the direct result of age-related changes in either the anterior pituitary gland or the hypothalamus, rather than the entire complex. In view of the many changes reported to be occurring in the concentrations of hypothalamic catecholamines, dopamine and serotonin in aged male and female rats (Clemens et al., 1969; Quadri et al., 1973; Meites et al., 1979; Walker et al., 1980) and aged male mice (Finch, 1973; Jonec and Finch, 1975); studies of a similar nature directed toward the hypothalamus of the older female mouse should add to our understanding of the age-related loss of reproductive capability by this species. Recent studies (Banerji et al., 1982), conducted in this laboratory, on the levels of hypothalamic dopamine-flhydroxylase (a norepinephrine-synthesizing enzyme), in four age groups of female C57BL/6 mice, have indicated that age-related differences occur in the levels of this enzyme during the evening of the proestrous LH surge. In mice 2-4, 7-8 and 12-13 monthsof-age there was a significant increase of dopamine-/3-hydroxylase activity at the same time there was a marked increase in circulating concentrations of LH. In contrast, 16-20 months-old mice which showed no appreciable increase in hypothalamic dopamine-flhydroxylase activity from that on the morning of proestrus, contained less than half the concentrations found in 2-4 and 7-8 months-old mice. These data are consistent with studies (Meites et aL, 1979) done on the aging rat which suggested that age-related alterna-
442
PARKENING,COLLINSAND SMITH
tions in. hypothalamic biogenic amines may be one of the primary causes of loss of reproductive capacity. Since the pituitaries of aged mice do not respo,nd to ovariectomy in the same manner as those of younger mice, the mechanisms for controlling the secretion of gonadotrophins and Prl may be less efficient in older mice. This deficiency may result from a reduction in hypothalamic and pituitary sensitivity to steroid feedback, changes in biogenic amine turnover, impairment in the release of GnRH or Prl-inhibiting factor (PIF), decreased binding of GnRH or PIF to pituitary cell receptors, or internal cellular alterations in the production and release of pituitary hormones. All of the above factors may be involved and research to elucidate the role each plays in the aging of the hypothalamic-pituitary complex is essential for understanding the causes of reproductive senescence. SUMMARY Concentrations of LH, FSH, and prolactin (Prl) in the plasma and pituitaries of young (2-4 months-old) and aged CD-1 (14-18 months-old) and C57BL/6 (16-22 months-old) mice, were determined by radioimmunoassay, one month after ovariectomy. Plasma concentrations of LH and FSH in young, ovariectomized mice were 2-5 times greater than those in sham-operated control mice. Plasma concentrations of Prl in young, ovariectomized mice were significantly lower than those in young controls. Pituitary concentrations of LH and FSH were also statistically higher, whereas Prl concentrations were significantly lower in young, ovariectomized mice than in intact, control mice. In contrast, the plasma concentrations of LH, FSH, and Prl were not significantly different in aged, ovariectomized mice and aged, sham-operated mice. No differences existed between pituitary concentrations of LH, FSH, or Prl in aged, ovariectomized and aged, control C57BL/6 mice, although the pituitaries of aged, ovariectomized CD-I mice had statistically higher levels of each hormone than those of their controls. Aged, shamoperated mice had significantly higher plasma concentrations of LH (C57BL/6 mice only) and FSH (both CD-1 and C57BL/6 mice) than young, control mice. No differences in plasma concentrations of Prl were found between young and aged, control mice. These data indicate that either the aged mouse ovary is no longer capable of producing adequate steroids to act on the hypothalamic-hypophyseal complex or (more probably) that the hypothalamic-hypophyseal complex in the aged mouse is less sensitive to gonadal feedback. Acknowledgements--This research was supported in part by a NIH Grant (AG-00892) awardedto E.R. Smith.
REFERENCES
Neuroendocrinology 34, 14. CLEMENS,J.A., AMENOMOm,Y., JENKn~S,T. and MEITES,J. (1969) Proc. Soc. Exp. Biol. Med. 132, 561. COLLINS,T.J., P~tCENINO,T.A. and Sun'n, E.R. (1980) Exp. Geront. 15, 209. Cou.rNs, T.J., PARKEmNC,T.A. and SmTH,E.R. (1981) Neurobiol. Aging 2, 125. FI~CH, C.E., FEL~C~O,T.L.S., FL~RK~Y,K., GEE, D.M., MOBaS,C., NELSON,J.F. and OSTEREURO,H.H. (1980) Peptides, 1 (Suppl. 1), 163. GOSD~N,R.G. and B^NCROrr,L. (1976) Exp. Geront. 11, 157. Gospel, R.G:i JONES,E.C. and JACKS,F. (1978) Exp. Geront. 13, 159. Fn~CH,C.E. (1973) Brain Res. 52, 261. HOWt~ND,B.E. and PREISS,C. (1975) Fertil. Steril. 26, 271. HUANO,H.H., MARSHALL,S. and MEIT~, J. (1976) Biol. Reprod. 14, 538. BA~m~rl, T.K., PARKENINQ,T.A. and COLLINS, T.J. (1982)
LH. FSH & PROLACTIN IN AGED, OVARIECTOMIZED MICE
443
JOr~EC, V.G. and Fn~crl, C.E. (1975) Brain Res. 91, 197. JONES, E.C. and KROaN, P.L. (1961) J. Endocrinol. 21,469. KovAcic, N. and PARLOW, A.F. (1972) Endocrinology 91, 910, McPnERSOr~, J.C., III, COSTOFF,A. and MAHESH,V.B. (1977) Fertil. SteriL 28, 1365. MEITES, J., SIMPKINS,J.W. and HUANO, H.H. (1979) In: Physiology and Cell Biology o f Aging (Edited by A. CHERKIN, N. KHARASCH,F.L. SCOTT, C.E. FINCH, T. MAKINODANand B. STREHLER),Aging Vol. 8, P, 87. Raven Press, New York. MILLER, A.E. and RmGLE, G.D. (1978) Proc. Soc. Exp. Biol. Med. 157, 494. PARKEt~raG, T.A., COLtI~S, T.J. and StarrH, E.R. (1980a) J. Reprod. Fertil. 58, 377. PARKEr,S(3, T.A., COLLn~S,T.J. and Sm'r~, E.R. (1980b). Biol. Reprod. 22, 513. PARKEr~rnG, T.A., COLLINS,T.J., LAU, I.F. and S~SENA, S.K. (1982) J. Reprod. Fert. 64, 37. QUADR1, S.K., KLEDZm, G.S. and ME~TES,J. (1973) Neuroendocrinology 11,248. SI~HA, Y.N., SELBY,F.W., LEWIS, U.J. and VA~DERLAAN,W.P. (1972) Endocrinology 91, 1045. WALKER, R.F., COOPER, R.L. and TIMmAS, P.O. (1981) Endocrinology 107, 249. WISE, P.M. and RATNER,A. (1980) Neuroendocrinology 30, 15.
0531-5565/82/060437-07503.00/0 Copyright ©'1983 Pergamon Press Ltd
Printed in the USA. All rights reserved.
PLASMA AND PITUITARY CONCENTRATIONS OF LUTEINIZING HORMONE, FOLLICLE-STIMULATING H O R M O N E A N D P R O L A C T I N IN A G E D , O V A R I E C T O M I Z E D CD-1 AND C57BL/6 MICE
T.A. PARKENING, T.J. COLLINS and E.R. SMITH* Departments of Anatomy and *Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, TX 77550
(Received 26 January 1982) Abstract-Plasma and pituitary concentrations of luteinizing hormone (LID, follicle-stimulating hormone (FSID, and prolactin (Prl) were determined by ratioimmunoassay in young (2-4 monthsold) and aged CD-I (14--18 months-old) and C57BL/6 (16-22 months-old) mice one month after ovariectomy. In young, ovariectomized mice, plasma and pituitary concentrations of LH and FSH were significantly higher, whereas concentrations of Prl were significantly lower than those in control mice (sham-operated). In contrast, plasma concentrations of LH, FSH and Prl were not statistically different in aged, ovariectomized mice and aged, control mice. There were also no differences in pituitary concentrations of the three hormones when comparing the same aged C57BL/6 mice, although the aged, ovariectomized CD-I mice exhibited higher pituitary levels of each hormone than those of their controls. The pituitary of the aged mouse responds differently to ovariectomy than that of the young mouse because of age-related changes in the ovary and/or hypot halamic-h ypophyseal complex.
INTRODUCTION AOE-RELATED changes in the hypothalamic-hypophyseal-ovarian complex are in part responsible for the loss of reproductive capacity in laboratory rodents. Radioimmunoassay studies conducted in our laboratory on aged oestrous and constant dioestrous C57BL/6 mice (Parkening et al., 1980a) and on anoestrous Syrian hamsters (Parkening et al., 1982) have shown that with advancing age a significant increase occurs in the circulating plasma concentrations of gonadotrophins and a decrease occurs in the plasma concentrations of prolactin (Prl). It is not known if these hormonal changes result from a dysfunctioning ovary or an increasing insensitivity of the pituitary-hypothalamic complex to gonadal feedback. In aged, ovariectomized, anoestrous hamsters, an insensitivity of the pituitary-hypothalamic complex appeared to be the primary reason for age-relat.ed changes in the secretion of pituitary hormones (Parkening et al., 1982). To better determine what influence the ovary has on the pituitary-hypothalamic complex in aged mice, plasma and pituitary concentrations of gonadotrophins and Prl were measured in bilaterally ovariectomized mice. Address all correspondence to: T.A. Parkening, Ph.D., Dept. of Anatomy, The University of Texas Medical Branch at Galveston, Galveston, TX 77550 437
438
PARKENING, COLLINS AND SMITH
MATERIALS AND METHODS Aged CD-1 Swiss albino (14-18 months-old) and C57BL/6 06--22 months-old) mice and younger mature females (2-4 months-old) of both strains were obtained from Charles River Laboratories, Wilmington, Mass. The aged CD-1 females were acquired at 6 weeks of age and bred continuously until used in the experiment. The aged C57BL/6 mice were virgin females acquired from the NIH colony maintained by Charles River Laboratories. The latter females ranged from 15-20 months-of-age and were used in the experiment within 6-8 weeks of their delivery date. The mice were housed under uniform temperature and lighting conditions (14 h light/10 h darkness) and provided with water and mouse chow ad libitura. Young mature and aged females of both strains were either bilaterally ovariectomized or sham-operated. One month following surgery, the ovariectomized mice were bled unanesthetized between 1000-1200 h by cardiac puncture using a heparinized syringe. Control females were checked after one month and bled as above on the day they exhibited a dioestrous vaginal smear. All of the younger sham-operated mice exhibited a 4-day oestrous cycle, and most of the aged, sham-operated mice an irregular oestrous cycle or a constant leukocytic vaginal smear (only C57BL/6 mice showed the latter). None of the smears of the aged mice showed more than 2 days of cornified cells. After bleeding, the pituitary gland was removed, weighed and cut in half. One-half was fixed for future immunocytochemical studies and the other half was reweighed and homogenized in 0.5 ml of 0.01 M phosphatebuffered saline (pH 7.6) containing 0.05 M EDTA and 0.1°70 sodium azide. The blood plasma and supernatant of the pituitary homogenates were stored at - 8 5 " C to permit an analysis of all samples in a given assay. Procedures for the radioimmunoassay (RIA) of LH, FSH and Prl have been presented in detail elsewhere (Parkening et aL, 1980a, b; Collins et al., 1980). NIAMDD rat-LH and rat-FSH RIA kits and a NIAMDD mouse-Prl RIA kit were used in determining concentrations of LH, FSH and Prl. In 90°/0 of the mice, sufficient plasma was obtained from each animal to assay for all three hormones (single 100-200 #l samples for LH and FSH and duplicate 50 td samples for Prl). Pituitary hormonal concentrations were determined in duplicate (200 #1 of a I/100 dilution o f the supernatant from the pituitary homogenate for LH, 200 #l of a l / l 0 dilution for FSH and 200 #l of a 1/20 dilution for Prl). No interassay variances occurred because all samples for an individual hormone were analyzed in a single assay. All intraassay variances were less than 10g0. A comparison of mean hormonal concentrations was made using the Student's t test. Statistical significance was determined at the 0.05 probability level.
RESULTS The concentrations of LH in the plasma of the young mature CD-1 and C57BL/6 mice that had been ovariectomized were at least twice as great as those found in the shamoperated controls (Table 1). In contrast, LH concentrations in aged, ovariectomized mice of both strains were similar to those in the aged controls. Aged, control C57BL/6 mice, however, were found to have plasma LH concentrations that were more than twice as great as those in young mature mice (p < 0.001). In the pituitary, the concentrations of LH were much higher for the ovariectomized mice (except aged C57BL/6 mice) than for their controls (Table I). The concentrations of FSH in the plasma of young CD-1 and C57BL/6 mice that had been ovariectomized were at least 5 times as great as those in their controls; however, no differences were found between the FSH concentrations in aged, ovariectornized mice and aged, control mice (Table 1). The aged, sham-operated mice, therefore, had significantly higher concentrations of circulating FSH than the sham-operated, younger mice (p < 0.001). In contrast, young, ovariectomized mice had significantly higher concentrations of FSH than aged, ovariectomized mice (p < 0.001). Greater concentrations of FSH were found in the pituitary of the young, ovariectomized mice (p < 0.001) than in their controls, and in the aged, ovariectomized CD-1 mice (p < 0.01) than in their aged controls (Table 1). No differences existed between concentrations of FSH in aged, control and aged, ovariectomized C57BL/6 mice. The concentrations of Prl that were found in the plasma of ovariectomized, young mice were considerably lower than those in the young controls (p < 0.001), whereas no differences were found in the concentrations of Prl in the aged, ovariectomized and aged,
439
LH, FSH & PROLACTIN iN AGED, OVARiECTOMIZED MiCE TABLE 1. PLASMA AND PITUITARY LEVELS OF LH, F S H AND PRL IN YOUNG MATURE AND AGED OVARIEGTOMIZED C D - I AND G 5 7 B L / 6 MICE.
CD-1 Mice 2 - 4 months-old Plasma ( n g / m l ± S.E.M.) LH Sham-operated Ovariectomized FSH Sham-operated Ovariectomized Prl Sham-operated Ovariectomized Pituitary L H (ng/p.g 4- S.E.M.) Sham-operated Ovariectomized FSH ( n g / # g 4- S.E.M.) Sham-operated Ovariectomized Prl ( n g / m g 4- S.E.M.) Sham-operated Ovariectomized
14-18 m o n t h s - o l d
C 5 7 B L / 6 Mice 2 - 4 months-old
16-22 months-old
26.0 4- 0.8 (16)* 54.3 4- 3.44 (20)
30.7 -4- 2.1 (I0) 35.0 4- 3.Y (10)
24.2 -4- 1.1 (17) 66.3 4- 3.74 (22)
55.9 4- 5.8 h (14) 51.0 .4- 2.7 I ( 11)
284.7 -4- 16.6 (16) 1545.2 4- 76.54 (20)
1102.3 4- 168.2 h (9) 939.2 -~ 93.5 k (10)
373.8 4- 16.9 (15) 1939.6 4- 88,14 (17)
973.5 4- 63.4 h (15) 1135.3 4- 84.4 ~ (I 1)
75.4 4- 5.5 (16) 36.5 4- 3.24 (20)
61.3 4- 4.3 (9) 70.5 4- 5.0 k (10)
62.4 4- 4.8 (14) 33.6 4- 3.3 d (17)
71.0 4- 3.2 (15) 68.5 4- 5.4 ~ (11)
4.6 4- 0.4 (16) 9.4 4- 0.5" (20)
5.2 4- 0.7 (13) 8.2 4- 1.0 / ( l 0)
4.4 4- 0.3 (18) 6.5 4- 0.4 d (22)
5.4 ± 0.6 (15) 6.9 4- 0.6 ( l l)
5.6 4- 0.5 h (13) 7.9 4- 0.Y .h (10)
3.7 4- 0.2 (18) 9.9 -4- 0.5 d (22)
4.8 4- 0 . ~ (15) 5.5 4- 0.3 k (1 l)
778.9 4- 44.4 (18) 281.4 4- 23.6 d (22)
532.0 4- 37.2 h (15) 495.5 4- 29.8 ~ ( I 1)
3.2 4- 0.3 (16) 15.0 ± 0.94 (20) 597.5 4- 24.1 (16) 510.0 4- 20.0 b (20)
373.9 4- 34.5 h (13) 467.0 4- 22.2" (10)
*Numbers o f females assayed are in parentheses. p < 0.05 b, p < 0.01 c, p < 0.001d: significantly different when c o m p a r i n g younger mature sham-operated and ovariectomized mice. p < 0.05 °, p < 0.0V: significantly different when c o m p a r i n g aged sham-operated a n d ovariectomized mice. p < 0.05,, p < 0.001h: significantly different when c o m p a r i n g younger mature a n d aged sham-operated mice. p < 0.05', p < 0.01~, p < 0.001~: significantly different when comparing younger m a t u r e and aged ovariectomized mice,
control mice. Aged, ovariectomized and aged, control mice had concentrations of Prl in their plasma that were similar to those in the younger, control mice (Table 1). Concentrations of pituitary Prl were lower in ovariectomized, young CD-1 (p < 0.05) and C57BL/6 (p < 0.001) mice than in their controls. Aged, ovariectomized CD-I mice had higher pituitary concentrations of Prl than their aged controls (p < 0.05), but no differences were discovered between pituitary Prl concentrations in aged, ovariectomized and aged, con-
440
PARKENING,COLLINSANDSMITH
trol C57BL/6 mice. The aged, control mice of both strains had lower concentrations of pituitary Prl than the young controls (p < 0.001). DISCUSSION A number of radioimmunoassay studies have shown that plasma concentrations of LH and FSH rise following gonadectomy in rats (Howland and Preiss, 1975; Gosden and Bancroft, 1976; McPherson et al., 1977) and mice (Kovacic and Parlow, 1972). Gosden et al. (1978) have reported, however, that levels of serum LH in 3-6 months-old CBA mice, one month after ovariectomy, were similar to those in sham-operated controls. In contrast, Kovacic and Parlow (1972) showed that mice ovariectomized for 50 days had serum levels of LH that were significantly greater (approximately 2 ½ times higher) than those found in intact mice during dioestrus. In the present study, both strains of young mature mice had concentrations of LH that were approximately twice those found in shamoPerated control, dioestrous animals. The results that were obtained by Gosden et aL, (1978) may have been influenced by the strain of mouse they used. The CBA mouse is precocious in its loss of follicles and it is not known what causes Such a loss. An early onset of acyclicity in this strain has been reported (Jones and Krohn, 1961) which implies premature hormonal changes. Serum concentrations of Prl in C3H/St mice that had been ovariectomized for 2 weeks have been shown to be lower than those in sham-operated controls (Sinha et al., 1972), although the difference was not significant. Pituitary concentrations of Prl in these same mice, however, were significantly less than those in ovariectomized animals. The discrepancies between that study and the present investigation in which significantly lower plasma Prl concentrations were found in younger ovariectomized CD-I and C57BL/6 mice when compared to intact females, may result from the longer period the animals were ovariectomized (1 month) prior to bleeding or from strain differences. Female C57BL/St mice were shown to possess three times more serum Prl than C3H/St mice, but significantly less pituitary Prl (Sinha et al., 1972). The present study indicates that the hypophyseal-hypothalamic complex of the aged female mouse has little capacity to respond to ovariectomy. The response that does exist, as reflected by the concentrations of pituitary hormones that were found in 14-18 monthsold CD-1 mice, apparently declines even further with increasing age, since no statistical differences were found in concentrations of pituitary hormones in Qvariectomized and control 16-22 months-old C57BL/6 mice. The pattern of hormonal secretion in young mature, ovariectomized mice in the present study was similar to that reported for young mature, ovariectomized rats (Huang et al., 1976). Plasma concentrations of LH, FSH and Prl in aged CD-1 and C57BL/6 mice, ovariectomized for one month, remained similar to those in aged sham-operated mice. In contrast, aged rats (22-24 months-of-age) that either were in contrast oestrus or repetitive pseudopregnancy before ovariectomy, exhibited a rise in the levels of serum LH, FSH and Prl when compared with those levels found in younger, intact rats (Huang et al., 1976). Moreover, even ovariectomized anoestrous rats (26-30 months-of-age) had significantly higher levels of FSH and Prl than intact, aged anoestrous rats (Huang et al., 1976). The pituitary of aged, anoestrous Syrian hamsters is also capable of responding to ovariectomy by increasing LH and FSH concentrations, although not to the same degree as the pituitary of younger, ovariectomized hamsters (Parkening et al., 1982). While the
LH, PSH & PROLACTIN IN AGED, OVARIECTOMIZED MICE
441
hypothalamic-hypophyseal complex of anoestrous rats and hamsters appear to retain some ability to respond to the absence of negative feedback, the hypothalamichypophyseal complex of the aged mouse does not. The possibility exists, however, based on the elevated concentrations of LH in aged intact C57BL/6 mice and the higher concentrations of FSH in aged intact mice of both strains, that a poorly functioning ovary in these animals has already created a hormonal environment similar to that found in a gonadectomized mouse. Aged mice given steroid injections should provide answers as to whether the hypothalamic-hypophyseal complex is still capable of responding. In just such a study, aged C57BL/6J mice given exogenous oestradiol did not produce an LH surge comparable to that of young or middle-aged mice (Finch et al., 1980). Younger mice in this same study exhibited a considerably higher LH surge than middle-aged mice, implying that the hypothalamus is responsible to a greater degree for changes in gonadotrophin secretion than are the ovarian steroids. Aged rats and mice do not respond to exogenous GnRH in the same manner as younger animals. Serum concentrations of LH that were measured after an initial injection of GnRH were uniformly lower in aged rats of both sexes in comparison to those in younger rats (Miller and Riegle, 1978). The pituitaries of 16-22 months-old female C57BL/6 mice were also found to be considerably less sensitive than those of 2--4 months-old mice (Collins et aL, 1981). In another study, medial basal hypothalamic concentrations of GnRH in repetitively pseudopregnant 8-12 months-old rats (ovariectomized for 3 weeks) dropped appreciably from those of an intact group (Wise and Ratner, 1980). In the intact group, however, concentrations of GnRH were lower than those of younger rats with regular oestrous cycles. From this data it was concluded that the anterior pituitary was largely responsible for such changes in the older rats. In contrast, hypothalamic concentrations of GnRH in intact and ovariectomized rats exhibiting constant oestrus (8-12 months-old) were not statistically different from one another (although both were lower than those in intact, younger rats with regular oestrous cycles) and it was suggested that the major defect in these rats resided within the hypothalamus (Wise and Ratner, 1980). It is possible therefore, that the stage of irregular cyclicity exhibited by some older laboratory rodents is the direct result of age-related changes in either the anterior pituitary gland or the hypothalamus, rather than the entire complex. In view of the many changes reported to be occurring in the concentrations of hypothalamic catecholamines, dopamine and serotonin in aged male and female rats (Clemens et al., 1969; Quadri et al., 1973; Meites et al., 1979; Walker et al., 1980) and aged male mice (Finch, 1973; Jonec and Finch, 1975); studies of a similar nature directed toward the hypothalamus of the older female mouse should add to our understanding of the age-related loss of reproductive capability by this species. Recent studies (Banerji et al., 1982), conducted in this laboratory, on the levels of hypothalamic dopamine-flhydroxylase (a norepinephrine-synthesizing enzyme), in four age groups of female C57BL/6 mice, have indicated that age-related differences occur in the levels of this enzyme during the evening of the proestrous LH surge. In mice 2-4, 7-8 and 12-13 monthsof-age there was a significant increase of dopamine-/3-hydroxylase activity at the same time there was a marked increase in circulating concentrations of LH. In contrast, 16-20 months-old mice which showed no appreciable increase in hypothalamic dopamine-flhydroxylase activity from that on the morning of proestrus, contained less than half the concentrations found in 2-4 and 7-8 months-old mice. These data are consistent with studies (Meites et aL, 1979) done on the aging rat which suggested that age-related alterna-
442
PARKENING,COLLINSAND SMITH
tions in. hypothalamic biogenic amines may be one of the primary causes of loss of reproductive capacity. Since the pituitaries of aged mice do not respo,nd to ovariectomy in the same manner as those of younger mice, the mechanisms for controlling the secretion of gonadotrophins and Prl may be less efficient in older mice. This deficiency may result from a reduction in hypothalamic and pituitary sensitivity to steroid feedback, changes in biogenic amine turnover, impairment in the release of GnRH or Prl-inhibiting factor (PIF), decreased binding of GnRH or PIF to pituitary cell receptors, or internal cellular alterations in the production and release of pituitary hormones. All of the above factors may be involved and research to elucidate the role each plays in the aging of the hypothalamic-pituitary complex is essential for understanding the causes of reproductive senescence. SUMMARY Concentrations of LH, FSH, and prolactin (Prl) in the plasma and pituitaries of young (2-4 months-old) and aged CD-1 (14-18 months-old) and C57BL/6 (16-22 months-old) mice, were determined by radioimmunoassay, one month after ovariectomy. Plasma concentrations of LH and FSH in young, ovariectomized mice were 2-5 times greater than those in sham-operated control mice. Plasma concentrations of Prl in young, ovariectomized mice were significantly lower than those in young controls. Pituitary concentrations of LH and FSH were also statistically higher, whereas Prl concentrations were significantly lower in young, ovariectomized mice than in intact, control mice. In contrast, the plasma concentrations of LH, FSH, and Prl were not significantly different in aged, ovariectomized mice and aged, sham-operated mice. No differences existed between pituitary concentrations of LH, FSH, or Prl in aged, ovariectomized and aged, control C57BL/6 mice, although the pituitaries of aged, ovariectomized CD-I mice had statistically higher levels of each hormone than those of their controls. Aged, shamoperated mice had significantly higher plasma concentrations of LH (C57BL/6 mice only) and FSH (both CD-1 and C57BL/6 mice) than young, control mice. No differences in plasma concentrations of Prl were found between young and aged, control mice. These data indicate that either the aged mouse ovary is no longer capable of producing adequate steroids to act on the hypothalamic-hypophyseal complex or (more probably) that the hypothalamic-hypophyseal complex in the aged mouse is less sensitive to gonadal feedback. Acknowledgements--This research was supported in part by a NIH Grant (AG-00892) awardedto E.R. Smith.
REFERENCES
Neuroendocrinology 34, 14. CLEMENS,J.A., AMENOMOm,Y., JENKn~S,T. and MEITES,J. (1969) Proc. Soc. Exp. Biol. Med. 132, 561. COLLINS,T.J., P~tCENINO,T.A. and Sun'n, E.R. (1980) Exp. Geront. 15, 209. Cou.rNs, T.J., PARKEmNC,T.A. and SmTH,E.R. (1981) Neurobiol. Aging 2, 125. FI~CH, C.E., FEL~C~O,T.L.S., FL~RK~Y,K., GEE, D.M., MOBaS,C., NELSON,J.F. and OSTEREURO,H.H. (1980) Peptides, 1 (Suppl. 1), 163. GOSD~N,R.G. and B^NCROrr,L. (1976) Exp. Geront. 11, 157. Gospel, R.G:i JONES,E.C. and JACKS,F. (1978) Exp. Geront. 13, 159. Fn~CH,C.E. (1973) Brain Res. 52, 261. HOWt~ND,B.E. and PREISS,C. (1975) Fertil. Steril. 26, 271. HUANO,H.H., MARSHALL,S. and MEIT~, J. (1976) Biol. Reprod. 14, 538. BA~m~rl, T.K., PARKENINQ,T.A. and COLLINS, T.J. (1982)
LH. FSH & PROLACTIN IN AGED, OVARIECTOMIZED MICE
443
JOr~EC, V.G. and Fn~crl, C.E. (1975) Brain Res. 91, 197. JONES, E.C. and KROaN, P.L. (1961) J. Endocrinol. 21,469. KovAcic, N. and PARLOW, A.F. (1972) Endocrinology 91, 910, McPnERSOr~, J.C., III, COSTOFF,A. and MAHESH,V.B. (1977) Fertil. SteriL 28, 1365. MEITES, J., SIMPKINS,J.W. and HUANO, H.H. (1979) In: Physiology and Cell Biology o f Aging (Edited by A. CHERKIN, N. KHARASCH,F.L. SCOTT, C.E. FINCH, T. MAKINODANand B. STREHLER),Aging Vol. 8, P, 87. Raven Press, New York. MILLER, A.E. and RmGLE, G.D. (1978) Proc. Soc. Exp. Biol. Med. 157, 494. PARKEt~raG, T.A., COLtI~S, T.J. and StarrH, E.R. (1980a) J. Reprod. Fertil. 58, 377. PARKEr,S(3, T.A., COLLn~S,T.J. and Sm'r~, E.R. (1980b). Biol. Reprod. 22, 513. PARKEr~rnG, T.A., COLLINS,T.J., LAU, I.F. and S~SENA, S.K. (1982) J. Reprod. Fert. 64, 37. QUADR1, S.K., KLEDZm, G.S. and ME~TES,J. (1973) Neuroendocrinology 11,248. SI~HA, Y.N., SELBY,F.W., LEWIS, U.J. and VA~DERLAAN,W.P. (1972) Endocrinology 91, 1045. WALKER, R.F., COOPER, R.L. and TIMmAS, P.O. (1981) Endocrinology 107, 249. WISE, P.M. and RATNER,A. (1980) Neuroendocrinology 30, 15.