neuroendocrine dysfunction, independent of ovarian secretions, sets an upper limit on the ameliorative influence of longterm ovariectomy.

Transcription

1 Proc. Nat!. Acad. Sci. USA Vol. 80, pp , October 1983 Medical Sciences Restoration of ovulatory cycles by young ovarian grafts in aging mice: Potentiation by long-term ovariectomy decreases with age (reproductive aging/ovulation/vaginal cyclicity/c57bl/6j mice) L. S. FELICIO*t, J. F. NELSON*t, R. G. GOSDENt, ANID C. E. FINCH* tdepartment of Physiology, Universits Medical School, Teviot Place. Edinburgh, United Kingdom, and *Departmelst of Biological Sciences and Andros Gerontology Center, Universit of Solrhern California, Los Angeles, California Communicated by Elizabeth S. Russell, June 17, 1983 ABSTRACT The relative contributions of ovarian and hy- -iuk ha,.. >itt~->; Cetors to the anovulatorv status of aging mice were evaluated by measuring the capacity of mice to resume ovulatory cyclicity after receiving young ovaries under the renal capsule. Young grafts partially restored cyclicity if old hosts were acutelv ovariectomized but almost fully restored cyclic ovulatory function if the old hosts had been ovariectomized early in adulthood. With advancing age, however, the efficacy of the grafts declined progressively in both acute and long-term ovariectomized groups. These data show that both ovarian and hypothalamic-pituitary aging contribute to the etiology of anovulation. Although chronic withdrawal from ovarian secretions retards the age of onset of hypothalamic-pituitary aging, the duration of this ameliorative effect is limited by progressive ovary-independent neuroendocrine dysfunction. The acyclic anovulatorv status of aging mammals is well-docunmented (1), yet the relative importance of ovarian versus hypothalamic-pituitary factors has not been established. Although steadily declining oocvte reserves clearly place an upper limit on the ovulatory life span (2), the reduced capacity of the aging hypothalamic-pituitary axis to release preovulatory surges of luteinizing hormone is also implicated in some rodent species (3-8). The possibility that this hypothalamic-pituitary dysfunction is a consequence of long-term exposure to ovarian steroids was first indicated by Aschheim's observation in a small group of old rats that young ovarian grafts restored vaginal cyclicity only if these rats had been ovariectomized at an early age (9). Subsequent studies showed that chronic treatment of young rodents with estradiol (10) led to a premature onset of degenerative changes in the hypothalamic arcuate nucleus, a region involved in the regulation of gonadotropin secretion. However, crucial questions bearing on the etiology of ovulatory failure in rodents remain unanswered. Namely, is hvpothalamic-pituitary dysfunction solely responsible for the anovulatory condition, or does primary ovarian failure contribute as well? What is the duration of the ameliorative effect of long-term ovariectomy on reproductive function? Do factors other than exposure to ovarian secretions contribute to the aging of the reproductive hypothalamic-pituitary axis? To answer these questions, we examined the ability of young ovarian grafts to restore ovulatory cyclicity in a longitudinal study of mice at ages spanning the adult life span. WVe report that the anovulatory condition is a consequence of both ovarian and hypothalamic-pituitary insufficiency. Although long-term withdrawal from endogenous ovarian secretions dramatically potentiates the restoration of ovulatory cyclicity in aging animals with young ovarian grafts, this effect is transitory. Progressive The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement' in accordance with 18 U.S.C solely to indicate this fact. neuroendocrine dysfunction, independent of ovarian secretions, sets an upper limit on the ameliorative influence of longterm ovariectomy. MATERIALS AND METHODS Animals and Vaginal Cycdicit. Virgin female C57BL/6J mice (The Jackson Laboratory) were kept in a limited-access aging colony (11). Mice intended for long-term ovariectomized groups were ovariectomized at age 5 months. Ovaries from 5-monthold donor mice were grafted under the renal capsules of acutely or long-term ovariectomized mice aged 17 and 25 months; donors for the 30-month-old group were 3 months old. Cycle frequency (cycles/month) and cycle length were evaluated from daily vaginal smears by a program developed for an Apple II microcomputer (11). Monthly cycle frequency was plotted as a function of age, and the integrated areas under the respective cycle frequency profiles were used to compare the effectiveness of ovarian grafts among the various experimental groups. Statistical significance of these data was determined by analysis of variance (ANOVA) followed by Duncan's multiple-range test for comparisons among group means. Differences with P values < 0.05 were considered significant. Ovarian Grafting. Donor and host mice were anesthetized with 2,2,2-tribromoethanol (12). One of the ovaries of the donor was gently pulled out from a dorso-lateral incision and reflected onto a piece of sterile gauze. Under a dissecting microscope, the ovarian bursa was slit and the hilum cut to release the ovarv. Simultaneously, the kidney of the host was exposed by a second individual while the ovary was cut into three segments to facilitate insertion under the renal capsule. The interval between removal and transplantation was less than 1 min. Although some mice developed self-inflicted wounds at the incision sites and were culled, >7 5% of the animals in all treatment groups recovered completely. At all ages, >90% of the fully recovered mice with ovarian grafts exhibited vaginal cornification, an index of estrogenic support and, thus, of a functioning ovarian graft. Only fully recovered mice exhibiting vaginal cyclicity or cornification were included in the study. Histology. Ovarian grafts were removed from some mice on the third day after vaginal proestrus, fixed in Bouin's solution, embedded in paraffin, serially sectioned at 7 rem, and stained with hemotoxylin and eosin. Sections were searched microscopically for recently formed corpora lutea (these appear more eosinophilic and with a higher nuclear/cytoplasmic ratio than previous sets of corpora lutea) and for oocytes that had been shed into the fluid-filled space between the ovarian graft and 6076 Abbreviations: LH, luteinizing hormone; ANOVA, analysis of variance. t Present address: Centre for the Study of Reproduction, McGill University, Roval Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A lal, Canada.

2 Medical Sciences: Felicio et al. Proc. Natl. Acad. Sci. USA 80 (1983) ) Ei4 An, 3 Q Age, months the renal capsule. Total oocyte number was estimated based on counts of every 20th- section. Luteinizing Hormone (LH) Radioimmunoassay. Plasma was frozen and assayed for LH by a double antibody radioimmunoassay (rabbit anti-ovine LH) with NIH-LH-RP1 standard as described in detail (6). RESULTS Young (5 month) acutely ovariectomized mice bearing young (5 month) ovarian grafts served as controls (designated Y-to-Y) for the efficacy of the grafting procedure. During the initial 3-month period after grafting, the cycle frequency and cycle length of these Y-to-Y controls were indistinguishable from those of intact mice (P > 0.5, ANOVA; Fig. 1 and Table 1). Thereafter, cycle frequency declined more rapidly in grafted mice com- Table 1. Estrous cyclicity in acute and long-term ovariectomized mice of different ages bearing young ovarian grafts % of cycles with length Age at of grafting, % Total mo Group* n cyclingt cyclest days days 5 Intact ± Acute-OVX ± 1.8a Acute-OVX ± 2.9b 76 24c,d 17 Long-OVX ± 1.7a Acute-OVX ± eef 25 Long-OVX ± 2.1b 73 27g 30 Long-OVX * Acutely ovariectomized (acute-ovx) mice were ovariectomized immediately prior to receiving ovarian grafts. Long-term ovariectomized (long-ovx) mice were ovariectomized at age 5 months. Donor age was 5 months, except for 30-month-old hosts that received 3-monthold ovaries. tshowing >3 cycles. ti ± SEM (values based on cycling subpopulation). The effect of the group was significant (P < , ANOVA): a, significantly different from age-matched groups; b, significantly less than 5-month-old acute- OVX hosts; P < 0.05 by Duncan's multiple range test. During the fist 3 months after surgery. Frequency distributions were significantly different from that of the 5-month-old intact mice (c, P < 0.05; g, P < 0.01; and e, P < 0.001) and significantly different from the age-matched long-ovx group (d, P < 0.01; f, p < 0.05; x2 test). FIG. 1. Effect of long-term ovariectomy on the ability of young ovarian grafts to restore vaginal cyclicity in acyclic mice. Long-term ovariectomized mice(----e) were ovariectomized at age 5 months, and ovaries from 5-month-old donors were grafted under their-renal capsules at ages 17, 25, or 30 months. Control mice were either intact (. ) or acutely ovariectomized (o-o) at ages 5, 17, or 25 months and immediately were given 5- month-old ovaries. Data are expressed as Ftx + SEM. Sample sizes ranged between 11 and 22, with the exception of.the 31-month- 30 old group (n = 4). Statistical analyses are described in the text. pared to intact controls, probably because of oocyte losses (about 50%) consequent to the initial ischaemia of grafting (Table 2). In 17-month-old acutely ovariectomized mice, cyclicity was maintained for 3 months after grafting at 25% of the Y-to-Y control level (Fig. 1); thereafter all the mice were acyclic. By contrast,.y-to-y controls cycled for 4 more months. The incomplete restoration of function in 17-month-old acutely ovariectomized mice was primarily due to two factors: (i) half-of the population' failing to resume cycling and (ii) fewer cycles in the cycling subpopulation. In addition, the frequency of long cycles was higher in acutely ovariectomized mice (Table 1). In 17-month-old longterm ovariectomized hosts, cycle frequency during the first 3 months after grafting was 3-fold greater than in acutely ovariectomized mice (P < 0.001, ANOVA; Fig. 1). The frequency and duration of cyclicity were >80% of that of Y-to-Y controls (Fig. 1 and Table 1). In hosts-aged 25 months at grafting, the efficacy of the grafts declined proportionately in both acutely ovariectomized and long-term ovariectomized groups to only 30% of their values in 17-month-old hosts (P < 0.02 and.p < 0.001, respectively, ANOVA). In hosts aged 30 months at grafting, cycle frequency in long-term ovariectomized mice was restored to only 5% of that in 17-month-old long-term ovariectomized mice. The ovulatory potential of the ovarian grafts was assessed in 5-month acutely ovariectomized and 32-month long-term ovariectomized mice that had received 3-month-old ovaries 2 monthsearlier. All cycling mice in both groups had fresh corpora lutea with ova present in the subcapsular space. 3 days after showing vaginal proestrus (Fig. 2). Moreover, ovulation rate, assessed The onset of declining cycle frequency was defined as the earliest age at which cycle frequency fell significantly below the value for the month of maximal cycling frequency (P < 0.05, Duncan's multiple range test; ref. 11). By this criterion, cycle frequency began to decline at 8 months in young mice bearing ovarian grafts-2 months earlier than in intact controls (Fig. 1). Table 2. Comparison of follicular and ovulatory status of grafted and in situ ovaries Age of host at Age at grafting,* autopsy, Primordial Corpora Mice with mo mo n folliclest luteat ova, % Intact 5 4 1,355 ± 228t 8.2 ± ± ± ± ± * Ovarian grafts were obtained from 3-month-old hosts. t i ± SEM. tsignificantly greater than hosts (P < 0.05, ANOVA).

3 6078 Medical Sciences: Felicio et al. Proc. Natl. Acad. Sci. USA 80 (1983) RC V.:,?Pra. S -z-s Akr - 4 i.%.wl A B,I 0 RC C D...,,,2R FIG. 2. Representative examples of follicular and luteal development in intact ovaries and in ovarian grafts under the renal capsule. (H and E; x32.) (A) Five-month-old ovary from a cycling intact mouse. Several growing follicles and corpora lutea are present. (B) Five-month-old ovarian graft in a cycling 5-month-old host, which was ovariectomized at 3 months and given ovarian grafts from a 3-month-old donor. Growing follicles and corpora lutea are present. Also visible are the underlying cortex (C) of the kidney to which the graft is attached and the renal capsule (RC), which is typically separated from the graft by a fluid-filled space (F). (C) Five-month-old graft in a cyclic 32-month-old host that was ovariectomized at age 3 months and received ovarian grafts from a 3-month-old host at 30 months. Several primary and secondary follicles are present. Recent ovulation is indicated by corpora lutea and the presence of several ova (*) in the fluid-filled space between the graft and the renal capsule (see B for symbol legend). (D) Five-month-old graft in an acyclic, 32-month-old host showing persistent vaginal cornification. The host retained its ovaries until age 30 months, when it received 3-month-old ovarian grafts. Corpora lutea are absent, although antral follicles are present (see B for symbol legend).

4 Medical Sciences: Felicio et al. Intact Y to Y Y to 10V, Acyclic FIG. 3. Plasma LH concentrations in 11- to 14-month-old acutely ovariectomized (Y to Y) or 23- to 25-month-old long-term ovariectomized (Y to O%,) mice bearing young ovarian grafts since age 5 or 17 months, respectively. Intact mice (13-15 months) served as controls. Cycling mice (f) (n = 9-12 per treatment) were sampled 3 hr after lights were out on the evening of proestrus, when LH levels are elevated (4). Acyclic mice (ES) showing persistent vaginal cornification were sampled atthe same time (acyclic mice from all treatment groups were pooled; n = 12). LH levels were significantly increased in cycling mice compared to acyclic mice (P < 0.005, ANOVA with logarithm-transformed data to eliminate heteroscedasticity, inequality of variances among samples), but there was no effect of treatment on the proestrus levels in cycling mice. by the number of fresh corpora lutea, was equivalent to that of intact mice (Table 2). These data indicate that some long-term ovariectomized mice retain the potential to ovulate a full complement of ova at an age twice their normal ovulatory life span (13-16 months; refs. 11 and 13) and confirm previous observations that regular vaginal cyclicity of singly housed mice is a reliable correlate of ovulatory cyclicity (13, 14). The efficacy of long-term ovariectomy in prolonging normal reproductive hypothalamic-pituitary function was further demonstrated by preovulatory increases of LH in old long-term ovariectomized mice that were indistinguishable from those in intact and acutely ovariectomized, middle-aged animals (Fig. 3). DISCUSSION This study establishes that both ovarian insufficiency and hypothalamic-pituitary dysfunction contribute to the anovulatory condition of aging mice. The data also show that the retarding effect of long-term ovariectomy on hypothalamic-pituitary aging is not indefinite, indicating that one or more factors in addition to exposure to ovarian secretions contribute to the aging of the reproductive hypothalamic-pituitary axis. The ability of young ovarian grafts to partially restore cyclicity in aging acutely ovariectomized mice establishes that ovarian Proc. Natl. Acad. Sci. USA 80 (1983) 6079 agizig is a significant factor in the etiology of acyclicity in this strain. In old acyclic rats, similar efforts to restore cyclicity with young ovarian grafts have failed (9, 15, 16). Although this failure has been interpreted to disclaim a role for ovarian aging in the loss of cyclicity in the rat, the possibility should be considered that the different outcomes of grafting studies in rats and mice reflect experimental rather than species differences. For example, the importance of the age of the host on the success of the graft has not been taken into account previously. In this study, virtually no restoration of cyclicity was achieved when young ovaries were grafted in 25-month-old acutely ovariectomized mice, whereas partial success was achieved in 17-monthold mice. Although the ages of the rats used in earlier studies are quite disparate, most animals were over 18 months. The possibility of graft rejection also deserves consideration because animals used in earlier studies were outbred. By contrast, C57BL/6J mice have been inbred for over 40 years (17), and there was no histological evidence of graft rejection (e.g., lymphocytic infiltration) in this study. The age-related depletion of oocyte reserves probably accounts for the implicit failure of the old ovary to support cycles. Oocyte reserves are nearly exhausted in these mice at the age of onset of acyclicity (13-16 months), and the anovulatory subpopulation of mice at this age has half the reserves of mice still ovulating (13). Although the oocyte pool is not completely depleted, the reduced pool of growing follicles may be too small to maintain the estrogenic stimulation of the hypothalamic-pituitary axis required for preovulatory surges of LH. The failure of young ovarian grafts to completely restore cyclicity in acutely ovariectomized acyclic mice shows that extraovarian loci, presumably hypothalamic or hypophyseal, also contribute to the anovulatory state. Long-term ovariectomy followed by young ovarian grafts at 17 months permitted nearly complete restoration of ovulatory cycles and preovulatory LH surges for a duration equivalent to that of young controls. Thus, one component of the age-related loss of hypothalamic-pituitary function can be attenuated by long-term withdrawal of ovarian secretions. This dysfunction may be due to refractoriness of the hypothalamic-pituitary axis to hormonal stimulation, as suggested by the impaired preovulatory surge of LH in aging rodents with regular cycles (3-6) or after stimulation with exogenous steroids (7, 8). The reactive gliosis in the hypothalamic arcuate nucleus of spontaneously acyclic aging rodents (18) has been offered as evidence for a lesion deafferentating the medial basal hypothalamus from the medial preoptic area and, thereby, impairing the induction of a preovulatory LH surge (19). It is noteworthy that these glial changes can also be delayed by long-term ovariectomy (18) and that their age of onset can be accelerated by chronic treatment with physiological levels of estradiol (10). Prolactin-secreting pituitary tumors are common after 24 months in female C57BL/6J mice (20-22). Because these tumors are often estrogen-dependent (23, 24) and hyperprolactinemia can interfere with ovulatory cyclicity (25-27), the greater ability of long-term ovariectomized hosts to support cycles with ovarian grafts might be a result of reduced tumor incidence, particularly in the hosts aged 25 and 30 months. In the present study, when mice that received young ovaries at 17 months were autopsied at 25 months, tumor incidence in the long-term ovariectomized hosts was only 35% of that of the acutely ovariectomized hosts (22). However, we consider it unlikely that pituitary tumors influenced the ability of acutely or long-term ovariectomized hosts aged 17 months to initiate estrous cycicity with young ovarian grafts. First, tumors are small and incidence is low (:20%) in 17-month-old mice (20). Second, in the present study there was no difference in the incidence or

5 6080 Medical Sciences: Felicio et al. size of pituitary tumors between cycling and acyclic acutely ovariectomized hosts (unpublished data). Further studies will be needed to resolve conclusively the relative roles of pituitary pathology and primary impairment of gonadotropin secretion in the loss of ovulatory cycling potential. The progressive decline in the effectiveness of long-term ovariectomy suggests that neuroendocrine impairments independent of ovarian influence ultimately limit the extension of the ovulatory life span achievable by ovarian hormonal withdrawal. Whether extraovarian estrogens or other hormones, acting analogously to the debilitating ovarian secretions, contribute to this ultimate neuroendocrine failure remains to be determined. Studies also are needed to identify the endogenous ovarian hormone(s), their duration of exposure, and the specific secretory profiles that lead to dysfunction of the reproductive hypothalamic-pituitary axis. Elucidation of these issues may provide insight not only into the age-related disruption of the hypothalamic-pituitary-ovarian axis but also into the aging of other endocrine-dependent systems. Note Added in Proof. Additional evidence has been obtained recently (28) for the retarding effect of long-term ovariectomy on the age-related decline of the estradiol-induced LH surge in C57BL/6J mice. We thank K. Grant, B. Jacobson, and H. Osterburg for their expert technical assistance and G. Kendrick and M. Young for excellent animal care. We also thank Drs. J. Brawer, C. Holinka, H. Huberman, B. Robaire, and B. Schachter for their critical review of this manuscript. This work was supported by National Institutes of Health Grants AG-00117, AG 00446, and AG to C. E. F.; an Eli Lilly Fellowship and Medical Research Council of Canada Grant MA-7739 to J. F. N.; and a Wellcome Trust Travel Grant to R.G.G. 1. Talbert, G. (1977) in Handbook of the Biology of Aging, eds. Finch, C. E. & Hayflick, L. (Van Nostrand-Reinhold, New York), pp Zuckerman, S. (1951) Rec. Prog. Horm. Res. 6, van der Schoot, P. (1976) J. Endocrinol. 69, Cooper, R. L., Conn, P. M. & Walker, R. F. (1980) Biol. Reprod. 23, Proc. Natl. Acad. Sci. USA 80 (1983) 5. Wise, P. M. (1982) Proc. Soc. Exp. Biol. Med. 169, Flurkey, K., Gee, D. M., Sinha, Y. N., Wisner, J. R., Jr., & Finch, C. E. (1982) Biol. Reprod. 26, Steger, R. W., Huang, H. H., Chamberlain, D. S. & Meites, J. (1980) Biol. Reprod. 22, Gray, G. D., Tennent, B., Smith, E. R. & Davidson, J. M. (1980) Endocrinology 107, Aschheim, P. (1964/1965) Gerontologia 10, Brawer, J. R., Schipper, H. & Robaire, B. (1983) Endocrinology 112, Nelson, J. F., Felicio, L. S., Randall, P. K., Sims, C. & Finch, C. E. (1982) Biol. Reprod. 27, Nelson, J. F., Felicio, L. S., Osterburg, H. H. & Finch, C. E. (1981) Biol. Reprod. 24, Gosden, R. G., Laing, S. C., Felicio, L. S., Nelson, J. F. & Finch, C. E. (1983) Biol. Reprod. 28, vom Saal, F. & Bronson, F. H. (1980) Biol. Reprod. 22, Zeilmaker, G. H. (1969) J. Endocrinol. 43, XXI-XXII. 16. Peng, M. T. & Huang, H. 0. (1972) Fertil. Steril. 23, Staats, J. (1976) Cancer Res. 36, Schipper, H., Brawer, J. R., Nelson, J. F., Felicio, L. S. & Finch, C. E. (1981) Biol. Reprod. 25, Brawer, J. R., Ruf, K. B. & Naftolin, F. (1980) Neuroendocrinology, 30, Felicio, L. S., Nelson, J. F. & Finch, C. E. (1980) Exp. Gerontol. 15, Schechter, J. E., Felicio, L. S., Nelson, J. F. & Finch, C. E. (1981) Anat. Rec. 199, Nelson, J., Felicio, L., Sinha, Y. N. & Finch, C. (1980) Gerontologist 20 (II), 171 (abstr.). 23. Brawer, J. R., Naftolin, F., Martin, J. & Sonnenschein, C. (1978) Endocrinology 103, Duchen, L. W. & Schurr, P. H. (1976) in Hypothalamus Pituitary and Aging, eds. Everitt, A. V. & Burgess, J. A. (Thomas, Springfield, IL), pp Everett, J. W. (1980) Endocrinology 106, McNeilly, A. S. (1980) J. Reprod. Fertil. 58, Evans, W. S., Cronin, M. J. & Thorner, M. 0. (1982) in Frontiers in Neuroendocrinology, eds. Ganong, W. F. & Martini, L. (Raven, New York), Vol. 7, pp Mobbs, C. V., Gee, D. M. & Finch, C. E. (1982) Abstr. Soc. Neurosci. 8, 79.