|Home | About | Journals | Submit | Contact Us | Français|
Previously we reported that prepubertally ovariectomized mice that received young transplanted ovaries at a postreproductive age showed a 40% increase in life expectancy. To study this phenomenon in greater detail, 11-month-old ovariectomized and ovary-intact CBA/J mice underwent ovarian transplantation with 60-day-old ovaries or a sham surgery. Results from observations on transplant recipients in the current study extended our previous results. Whereas intact control mice lived an average of 726 days, transplant recipients lived an average of 770 days (i.e., 780 days for intact recipients and 757 days for ovariectomized recipients). If intact recipients had ceased reproductive cycling by the time of transplant, we observed a further increase in mean life span to 811 days. These results demonstrate that young ovaries enhanced longevity when transplanted to old mice and that ovarian status, examined by means of ovariectomy and ovarian transplantation, clearly influenced the potential of young transplanted ovaries to positively impact longevity.
GONAD manipulation in model organisms provides strong evidence for a direct link between reproduction and longevity (1–3). In the hermaphroditic worm Caenorhabditis elegans, neonatal ablation of the gonadal germ line cells while leaving the somatic gonad intact resulted in increased life span, but removal of the entire gonad yielded no change in life span (4). In both worms and flies, germ line stem cells act in the adult to influence life span (2,5). Gonadal manipulation has also been used as a tool to study reproductive senescence in mammals. Mobbs and colleagues (6) transplanted young ovaries into old C57BL/6J mice. The old mice resumed reproductive cycling but at a lower frequency compared with the cycle frequency in young mice. When the old mice were ovariectomized 2 months before ovarian transplantation, they resumed cycling but at a higher cycle frequency than in the previous experiment. This suggests that gonadal removal has a positive effect on resumption of reproductive cycling.
In addition to reproductive cycling effects, ovaries transplanted from a young mouse to an old mouse can also have a positive effect on survival. In a previous study in our laboratories, Cargill and colleagues (7) prepubertally ovariectomized CBA/J mice at 21 days. They next transplanted 60-day-old ovaries to the ovariectomized mice when the mice reached 5, 8, and 11 months. Mice that received young ovaries at 5 and 8 months showed no significant increase in remaining life expectancy. However, mice that received young ovaries at 11 months showed a 40% increase in life expectancy, relative to intact controls. The 11-month-old recipient females resumed estrus and continued to cycle for several months past the normal point of reproductive senescence for this strain of mice. Ovariectomized sham mice that did not receive new ovaries showed a decreased life expectancy, compared with intact control mice. The decreased life expectancy effects observed in ovariectomized sham mice in the study by Cargill and colleagues (7), combined with the lack of life-span benefit in gonadectomized C. elegans (4), suggests that a gonad-intact transplant model might show increased longevity compared with an ovariectomized transplant model. We conducted a study parallel to the work of Cargill and colleagues (7) with additional treatments that included ovarian transplantations and sham surgeries in mice that were gonad intact at the time of transplant.
Adult (40 g) CBA/J strain female mice (Jackson Laboratory, Bar Harbor, ME) were provided ad-libitum access to feed (Purina Mouse Chow 5008: 23.5% protein, 6.5% fat; Purina Mills, St Louis, MO) and water (deionized) and were housed under conditions of constant temperature (21°C ± 2°C), humidity (minimum 50%), and lighting (14L:10D, lights on at 0700 hours). Individual pups were weaned and ear notched at 21 days (day of birth = 0 days). All female weanlings were housed individually in a 26 × 17 × 13–cm shoebox cage in a specific pathogen–free colony where pathology on sentinel mice was done quarterly and pathological results showed no breach in this status. Mice were maintained in an American Association for Accreditation of Laboratory Animal Care–approved facility in accordance with National Institutes of Health animal use guidelines. Animal care and use protocols were developed under National Research Council guidelines found in the Guide for the Care and Use of Laboratory Animals. This project was approved by the University of California, Davis Institutional Animal Care and Use Committee.
Animals were randomly assigned to control, sham transplant, or transplant group as follows (Figure 1):
Controls consisted of the following:
Shams consisted of the following:
Transplants consisted of the following:
Bilateral ovariectomies at 21 days and ovarian transplantation and sham surgeries at 11 months were carried out as previously described (8) with the following exceptions: The ovarian bursa was closed with one to three sutures of 10-0 Ethilon monofilament (Ethicon, Inc., Somerville, NJ) instead of the 6-0 suture described previously. Intact sham animals underwent sham ovarian transplant surgery in which their endogenous ovaries were removed, placed in cold sterile saline, and returned to the original bursae. Data on vaginal cytology were collected for 10 consecutive days before and after surgery to ensure (a) complete removal of the ovarian tissue and (b) success of the ovarian transplantation procedure.
Presumptive ovariectomized mice that showed signs of gonadal input before surgery at 11 months were excluded from analysis. Ovarian transplant recipients that failed to show evidence of gonadal input after transplant surgery were also excluded from analysis. Gonadal input was determined by vaginal cytology analysis, as described in the Surgical Procedures section. Mice that showed no cyclic activity for a 10-day period before and/or after surgery were determined to have no gonadal input for said period. Mice that showed at least one full estrous cycle in a 10-day period before and/or after surgery were determined to have gonadal input for said period.
Experimental data were analyzed preliminarily by one-way analysis of variance and linear regression analysis. Significance tests for each predictor in the multiple linear regression model were implemented with the Student's t test, two tailed, unequal distribution of variance assumed, and chi-square (χ2) analysis where appropriate. Test results were considered significant for p values <.05.
Mice that received new ovaries (intact + ovariectomized) lived significantly longer than sham transplant groups (intact + ovariectomized; Figure 2). Transplantation of young ovaries to old mice had a positive effect on life span, producing a 6.2% increase in life span in transplant recipients compared with control mice (770 vs 726 days, respectively, p = .0538) and a 6.7% increase compared with sham mice (722 days, p = .0029).
When the transplanted groups were split into intact and ovariectomized recipients, the life-span advantage was significant in intact recipients (780 days, p = .0310) but not in ovariectomized recipients (757 days, p = .1950; Figure 3). The mean life span of intact transplant recipients was 7.6% greater than that of the controls, as reflected in the survival curves of these and the other groups presented in Figure 4. The increased survival of the intact transplant group is clearly evident from this graph.
When the intact transplant group was divided based on reproductive cycling status at the time of transplantation, mice that had ceased cycling before ovarian transplantation showed an increase in life span compared with mice still cycling at transplantation (811 vs 754 days; Figure 5). Non-cycling recipients increased their mean life span by 11.7%, relative to controls. This cycling effect was not seen in sham groups. The pattern of survival decline in the longest lived group (non-cycling, IT-TX) was similar to that in the other groups, but occurred later (Figure 6).
Our laboratories previously reported a 40% increase in remaining life expectancy at 11 months, when young ovaries were transplanted to postreproductive-aged mice that had been ovariectomized at 21 days. The current study was conducted to examine this phenomenon in greater detail, including whether the longevity-enhancing effect of young transplanted ovaries, seen in ovariectomized mice, would manifest differently in gonad-intact mice. A significant number of comparisons are reported in the current study. Some comparisons appeared to produce physiologically significant results but included too few animals to reach statistical significance. Therefore, we have chosen to present our data without including a correction for multiple comparisons, leaving the interpretation of significance to the reader.
Our current results confirmed and extended the previous results of Cargill and colleagues (7). Although the study by Cargill and colleagues reported a significant increase in life expectancy in ovariectomized 11-month transplant recipients, the intact 11-month transplant recipients in the current study extended those results by showing an increase in life span, relative to mice in which ovaries were not transplanted.
The relationship between reproduction and chronological life span is well established (9). In C. elegans, life span can be altered by manipulating the germ line stem cells (4). In Mediterranean fruit flies, selecting individuals with extended reproductive potential also selects for increased longevity (3). Guppies subject to increased predation pressure show increased reproductive and chronological life span (10). Due to the increased duration of ovarian influence provided by the young transplanted ovaries, it may be implied that reproductive life span was increased in old mice receiving transplants. This implication would suggest that, in our mice, the observed increase in chronological life span positively corresponds to an increased reproductive life span, similar to the observations in C. elegans, Mediterranean fruit flies, and guppies, as previously mentioned.
Although ovarian transplantation tended to increase longevity in all recipients, removal of gonadal input through prepubertal ovariectomy reduced the degree to which longevity was enhanced by the young ovaries. The observation that the greatest life-span increase was observed in mice with increased reproductive exposure is inconsistent with the often-cited theory that increased reproductive influence has a negative effect on longevity (1,11,12). In our study, decreased reproductive influence through prepubertal gonad removal had a dampening effect on the ability of the young transplanted ovaries to increase life span. Providing a gonad-intact female with a second set of ovaries at 11 months greatly increased her exposure to reproductive influence and greatly increased her life span. In contrast to experiments in C. elegans, where neonatal removal of gonadal germ line cells increased survival (4), on its own, prepubertal gonad removal had no significant effect on life span in our ovariectomized sham mice. However, in our ovariectomized transplant recipients, prepubertal gonad removal decreased the life-span–extending ability of new ovaries, relative to intact recipients. Our gonad-intact transplant recipients endured increased germ line and somatic gonadal exposure and showed the greatest life-span increases of all mice receiving transplants.
In the current study, we observed life-span differences in two groups of transplant recipients: (a) between intact and ovariectomized transplant recipients and (b) between cycling and non-cycling transplant recipients. In previous studies that focused on reproductive cycling, young ovaries transplanted to middle-aged mice extended the host's duration of reproductive cycling by 50% in cycling mice but only 25% in non-cycling mice (5,13,14). In these studies, the duration of reproductive cycling was increased to the greatest degree in cycling recipients. In our study, the greatest life-span extensions were observed in non-cycling ovary recipients. Because we followed reproductive cycling for only a short period after ovarian transplantation, the degree to which life span was affected by reproductive status is unknown. However, our ovariectomized recipient group was comparable with the recipient mice in the study by Cargill and colleagues (7), which were followed throughout their life span for reproductive cycling. Many of these ovariectomized transplant recipients continued to cycle for several months past the normal point of reproductive senescence. Although this provided support for the level of reproductive influence in our ovariectomized mice, a direct extrapolation to our intact recipients is not possible.
Reproductive status of the recipient at the time of transplantation was a dominant influence on the life-span–extending effect of the new ovaries, as non-cycling mice showed the greatest increase in life span (11.7%). Interestingly, the trend for life-span increase in cycling mice closely paralleled the trend seen in ovariectomized recipients (754 and 757 days, respectively). Although these groups showed only a trend toward increased life span compared with controls, they were distinctly different from non-cycling recipients (811 days), suggesting an important non-gonadal influence on longevity. Further support for a non-gonadal influence on longevity is often seen in calorie-restricted and hypophysectomized mice and many transgenic mouse longevity models (10,15–17). A non-gonadal influence on longevity is also suggested in the study by Cargill and colleagues (7), where ovariectomized mice that received ovarian transplantations at a diverse range of ages showed diverse longevity effects.
Results from this study support our previous observations that young ovaries transplanted to postreproductive-aged mice provide a longevity advantage in transplant recipients. However, prepubertal ovariectomy dampened this longevity-enhancing effect. The reproductive status of recipients at the time of transplantation (intact or ovariectomized, cycling or non-cycling) influenced the extent of the life-span–extending benefit extracted from the young ovaries. With respect to longevity extension, prepubertally ovariectomized mice benefited the least from transplanted young ovaries and non-cycling intact mice benefited the most. Although the postreproductive period no longer involves the natural production of offspring, the reproductive axis still appears to be vitally important for subsequent survival.
Research was funded, in part, by grants to J.R.C., National Institute on Aging/National Institutes of Health grants PO1 AG022500-01 and PO1 AG08761-10, and a grant from the Center for the Economics and Demography of Aging, University of California, Berkeley. J.B.M. was supported, in part, by an American Physiological Society Porter Physiology Fellowship and a University of California, Davis Lyons Fellowship.
We gratefully acknowledge Thomas Famula, Alice Moyer, and Sandra Weisker for their intellectual support and technical assistance.