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PohnB6F1: A Cross of Wild and Domestic Mice That Is a New Model of Extended Female Reproductive Life Span

¹ The Jackson Laboratory, Bar Harbor, Maine.
² College of the Atlantic, Bar Harbor, Maine.
³ University of Texas Health Science Center, Barshop Institute for Longevity and Aging Study, San Antonio.
⁴ University of Michigan Geriatrics Center, Ann Arbor.

Address correspondence to David E. Harrison, PhD, The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609. E-mail: david.harrison{at}jax.org

	Abstract

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In the search for novel genetic diversity that affects the timing of life history traits, we investigated a wild-derived stock of mice (Pohn). Early generations showed extended reproductive life span; however, this phenotype diminished with propagation of the stock. Out-crossing latter generation Pohn mice to C57BL/6J (B6) mice produced PohnB6F1 hybrids with remarkably extended reproductive life spans—mean age at last litter of 647 ± 32 days—longer than for the parental strains (70% longer than Pohn, 88% longer than B6) and longer than for highly heterogeneous crosses of laboratory mice. Litter size among young PohnB6F1 adults was similar to parental stocks, but their age-related decline in litter size was delayed by 150–200 days, resembling the earlier Pohn generations. Possibly, out-crossing Pohn mice unmasked cryptic alleles that promote extended female reproduction. This work establishes the PohnB6F1 hybrid as a new model for delayed reproductive aging.

Mice derived, without domestication, from wild-trapped progenitors may provide new resources for studying genes that affect life history and aging (6–8). In crosses between wild-derived and standard laboratory Mus musculus inbred strains, we identified genetic loci that regulate maximum life span (Leg1 and Leg2). Only wild-derived strains carried alleles that significantly increased maximum life span (1). Furthermore, Miller and colleagues (9) observed that some stocks of wild-derived Mus musculus exhibited longer life spans and other traits associated with delayed aging, compared to a genetically segregating stock generated from standard inbred strains of mice.

Three decades ago, another group reported that breeding pairs from a stock of wild-derived Mus musculus had a mean reproductive life span of 21.5 months, longer than the mean reproductive life spans of most F1 hybrids of standard laboratory inbred strains, which are about 16–18 months (10). More recently, Zitnik and coworkers (11) described a wild-derived stock of Mus caroli that maintained its reproductive effort to a maximum of 22 months. Unfortunately, these populations are no longer available, and we cannot assume that such long reproductive life spans will routinely be found among new stocks generated from wild-derived Mus musculus [e.g., Wallace (12)].

In the present study, we evaluated life history traits related to female reproductive senescence in a stock, Pohn, recently derived from mice trapped on the island of Pohnpei (13). Early generations of Pohn mice exhibited increased female reproductive life span and a shift in reproductive potential to later ages that was not evident in later generations. However, out-crossing Pohn mice from the later generations to B6 mice produced F1 hybrids with extraordinarily long reproductive life spans. Thus, the PohnB6F1 hybrid must carry alleles for delayed female reproductive senescence, providing a valuable new model for studies of the genetics of aging.

	MATERIALS AND METHODS

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Mice
Pohn mice.-- Free-living mice were trapped on Pohnpei Island (7N lat., 158E long.) in the Federated States of Micronesia, using Sherman traps (8 cm x 8 cm x 16 cm). These mice were trapped by the same person (S. N. Austad), with the same methods, on the same island as were the PoG0 mice described by Miller and colleagues (7). One female and three males from Pohnpei survived shipping to The Jackson Laboratory, and were housed in a Plexiglas enclosure (isolator) using protocols that microbiologically separate the mice from the barrier facility, but permit access to the mice through rubber gloves attached to the wall of the isolator. The Pohn female produced at least one litter with each of the three males. As soon as new pups were observed during daily inspection, we transferred them to a foster mother, which was kept under specific pathogen-free (SPF) conditions. Using these half siblings, we set up five initial breeding pairs (C1, A2, E3, D4, and F5) to establish a stock of Pohn mice. We maintained the stock by crossing offspring from these breeding pairs, avoiding sibling crosses, and, after the fourth generation, avoiding first cousin crosses, to minimize inbreeding. When setting up breeders for each generation, we were careful not to exclude offspring from parents that began breeding late.

In this study, we analyzed data from two cohorts of Pohn mice and an F1 hybrid. The first cohort (Pohn 2–5) comprises the five initial breeding pairs of the second generation and 22 breeding pairs from the third, fourth, and fifth generations. (We selected only breeding pairs that produced at least three litters; we deemed the criterion of three litters as sufficient evidence, in all groups of this study, that fertility was normal for a breeding pair.) Mean reproductive life span (age of the dam at the birth of her last litter) was not different across these generations. The second cohort of Pohn mice comprises the 20 breeding pairs of the ninth and tenth generations that each produced at least three litters (Pohn 9–10). To produce the F1 hybrids (PohnB6F1), we mated Pohn females from the Pohn 9–10 population with C57BL/6J males. All these mice were housed in the same animal room throughout their lives.

Control populations.-- Because no formal control stock exists for Pohn mice, and because heterosis and inbreeding have major effects on reproductive potential, we used three long-lived control populations—two highly heterogeneous 4-way cross stocks (1ML and 3ML) and an inbred line (C57BL/6J [B6])—to represent the reproductive potential of Mus musculus in general. The 1ML and 3ML groups were part of a genetic study of life span (1,8). We generated the 1ML stock by crossing (ST/bJ x C57BL/6J) F1 females with (CAST/Ei x DBA/2J) F1 males, and the 3ML stock by crossing (SJL/J x YBR/Ei) F1 females with (RIII/DmMob x CE/J) F1 males. The 1ML stock was the more diverse because we used a wild-derived strain (CAST) in its production, whereas, for the 3ML stock, we used standard laboratory strains.

The 1ML stock had greater reproductive life spans than the 3ML stock, perhaps due to the use of a wild-derived strain in the 1ML stock. Therefore, to provide the most stringent comparison to the Pohn 2–5 group, we used the 1ML stock as the control group in statistical tests. We specifically used the second generation of the 1ML stock, which we designate 1MLS1, to compare to the Pohn 2–5 group for two reasons: (i) Unlike the first generation (designated 1MLS0), which comprises the immediate offspring of the cross of two different F1 hybrids, the S1 generation permits the expression of all recessive alleles at polymorphic loci in the cross, as does each generation of the Pohn stock, and (ii) compared to the subsequent generations (S2, S3, etc.), the S1 generation maximizes heterosis. Thus, the effects of inbreeding depression, which can diminish female reproductive fitness, are minimized in the S1 generation, providing a conservative control. We maintained all 1MLS1 breeders in the same animal room as the Pohn 2–5 breeders. The Pohn 2–5 breeders were born over a 3-year period, whereas the 1MLS1 breeders were born over a 1-month period, 1 year earlier than the Pohn 2 (second generation) breeders.

To further characterize the genetics of reproductive aging in Pohn stocks, we evaluated female reproductive life span and related variables in PohnB6F1 hybrids. Because we wanted to evaluate effects of heterosis, our primary comparison was to the parental stocks. However, we also performed a "historical" comparison to the generation of the control stocks that is genetically most comparable to an F1—the S0 generation. We chose the S0 groups for comparison because, as with an F1, the S0 mice would have had the greatest heterosis of any generation of the stock. We chose 1MLS0 for comparison because it included, among the parental strains, B6 and a wild-derived strain; we chose 3MLS0 because it included only domesticated inbred strains as founders.

As an additional control, we used the B6 inbred strain, which is widely used for studies of senescence. We obtained data for B6 breeders from two groups: One (B6-ref) was from a B6 colony that we maintained in the same mouse room as the Pohn 2–5 and 1ML breeders, but 3 years earlier; the other (B6-rb) was from B6 breeding pairs that we transferred intact from the production facilities at The Jackson Laboratory to our research colony when they were 8 months old. The birth dates of the B6-rb group overlapped those of the Pohn 9–10 and the PohnB6F1 females. Because our Pohn stock was generated from a single female, and because the four progenitors may be closely related, the Pohn stock may be relatively inbred. Therefore, we chose control groups to "bracket" the range of genetic heterogeneity in Mus musculus, from completely inbred (B6) to extremely heterogeneous (1ML and 3ML).

For analysis of ovarian oocytes, we compared ovaries from Pohn females to ovaries from B6 females and from females of the eighth generation of the 1ML cross (1MLS7). The 1MLS7 generation had been developed for a selection study on reproductive and total life span (14) as follows: Beginning with the S1 generation, each generation was selected for female reproductive longevity (dams that remained fertile after 12 months), and beginning with the S2 generation, each generation was simultaneously selected for grandparental longevity and female reproductive longevity. To avoid inbreeding when propagating the 1ML stock, siblings and first cousins were not mated; the inbreeding coefficient for the S7 generation was 0.4 (Roderick TH, Harrison DE, 2003, unpublished data). The 1MLS7 mice provide a useful comparison for the following reasons: They are the progeny of the 1MLS1 group that was used for comparison to the progenitors of the Pohn 9–10 group; both the 1ML and the Pohn lines were propagated during the same time period for about 5–7 generations; and, the selection procedures used for the 1ML line should minimize loss of reproductive performance during propagation, providing a conservative comparison.

Husbandry
We housed all mice in standard polycarbonate double "shoebox" cages with filter-hooded tops. We provided mice with autoclaved NIH-31 chow with 4% fat (Purina, Richmond, IN) and tap water acidified to pH 2.8–3.0 (to minimize Pseudomonas) ad libitum. Room temperatures were 23–25°C; the light cycle was 14:10 (light/dark). SPF procedures have been described (15). Pathogen testing was performed monthly by culturing swab samples taken from various sites in the animal room, and quarterly by direct culture of tissue samples taken from sentinel mice. The colony remained free of all tested pathogens except Pasteurella, which had a low incidence in the colony. The colony was not evaluated for helicobacter.

Reproductive Characteristics
We set up breeding pairs for the 1MLS0, 3MLS0, 1MLS1, Pohn 2–5, and Pohn 9–10 populations with males from the same stock as the females, but from different parents. We paired PohnB6F1 females at weaning with either proven breeder B6 males (n = 7) or Pohn males (n = 3). We set up breeding pairs within 5 weeks of weaning for the 1MLS0, 3MLS0, 1MLS1, and B6-rb groups (Table 1). We checked breeding pairs twice weekly for litters and recorded litter size and the estimated date of birth. To avoid complicating the pedigrees for the 1ML, 3ML, and Pohn stocks, we did not replace males if they died or if the female stopped reproducing. Also, we did not replace males for the B6-ref mice of the reference colony. But, because we initially paired the PohnB6F1 females with older proven breeder B6 males, when these males were 14–16 months old, we replaced them with young B6 males. Similarly, we replaced the B6 males in the B6-rb breeding pairs (the group that was contemporaneous with the PohnB6F1 mice) when these males were 14–16 months old.

Reproductive life span (age at last litter).-- For determination of reproductive life span, our objective was to analyze data that most closely reflected normal age-related female infertility. We considered breeding data as incomplete for a pair if a female might have died from pregnancy-related or nursing-related complications, or if the death of the male might have precluded the expression of full female reproductive potential. Therefore, we censored data for calculation of reproductive life span if the dam died before the last litter was weaned or if the sire died within 50 days of the birth of the last litter. Treating such data as censored for Kaplan–Meier plots and Mantel–Cox log-rank tests retains the information that the mice were fertile up until the age at which they were censored, then treats the value for the censored mice as unknown afterward. Table 1 provides the number of mice censored for each reason. For the calculation of the mean reproductive life span (Tables 2 and 3), we excluded censored data.

Total litter number and total pup number.-- We counted all litters and pups, including those that were dead at the time of observation. When calculating total pup number and total litter number (Tables 2 and 3), we excluded data from breeding pairs that were censored (above). In addition, we also excluded breeding pairs that were set up when females were older than 10 weeks of age (Table 1) because data on their early breeding potential were incomplete. Data from these two subsets of breeding pairs were used for the other reproductive variables because, for these variables, there was no statistical difference between breeders that were set up early and those that were set up later.

Age-specific litter size and age-specific interlitter interval.-- We calculated litter sizes and interlitter intervals within 50-day and 150-day periods, respectively, for each female. If a female had more than one litter within a 50-day period, we used the mean of the litter size as the value of that female during that period. If a female had no litters within a 50-day period, we entered no data for that female during that period. Because we checked breeders only twice weekly, it is possible that some newborn litters were completely cannibalized before they were recorded.

We scored the interlitter interval as the number of days between the births of consecutive litters The value for the interlitter interval within each 150-day period was the mean of all intervals that ended within that period. We used 150-day periods to calculate interlitter interval because the relative variance was quite high when we calculated the interval per 50-day period. We excluded interlitter intervals in which the breeder male was replaced, because infertility of the pair during that interval could have resulted from infertility of the previous male.

Ovarian Oocyte Number
For determination of oocyte numbers, we used virgin females to avoid potential confounds from individual differences in reproductive history. We fixed both ovaries from each female in Bouin's solution, then serially sectioned (5 µm) and stained them with hematoxylin and eosin. We examined every third section. To eliminate double counting, we counted an oocyte only if its cross-section contained a nucleolus. We determined total number of oocytes using standard methods (16). Other details, including ages of the mice, are given in Table 4.

	RESULTS

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Reproductive Life Span
Pohn 2–5 breeding pairs reproduced for a longer period than 1MLS1 breeding pairs (Figure 1A). For both groups, age-related infertility first appeared between 251 and 300 days. However, for 1MLS1 mice, the subsequent infertility rate was about three times greater than for Pohn 2–5 mice (Figure 1A). Three Pohn 2–5 females bred for an unusually long time: Two delivered their last litters in their 22^nd month (649 and 651 days), and the best performer in her 25th month (743 days). Both Pohn 2–5 and 1MLS1 groups reproduced for longer periods than we observed for the long-lived, inbred B6 strain (B6-ref) in our colony (Figure 1A).

View larger version (22K): [in this window] [in a new window]   Figure 1. Reproductive life span in Pohn mice. Kaplan–Meier plots are shown for reproductive life span, as indicated by age at birth of the last litter. A, A greater proportion of breeding pairs of Pohn 2–5 mice (from a stock of wild-derived mice) remained fertile as they aged compared to breeding pairs of mice from a 4-way cross of inbred strains (1MLS1) (p =.039, Mantel–Cox log-rank test). The long-lived inbred strain C57BL/6J is included for comparison without statistical evaluation. (See Table 1 for the numbers of breeding pairs.) In all studies, we defined reproductive life span as the age at the birth of the last litter for breeding pairs in which the dam survived weaning this litter and the sire survived 50 days past the birth of this litter. B, Reproductive longevity is diminished for Pohn mice by generations 9–10 in the laboratory compared to generations 2–5 (p =.019, Mantel–Cox). In both (A) and (B), open symbols represent censored data. Data from these breeding pairs, for which the total reproductive life span was not known, are censored for statistical analyses (details in Materials and Methods)

Because we set up Pohn breeding pairs over a range of ages (26–160 days; Table 1), we tested whether the age at which a breeding pair was set up affected reproductive life span among Pohn mice. We divided the Pohn groups into halves: those first paired when young (26–91 days) and those first paired when mature (92–160 days). Within the Pohn 2–5 group, the age at first pairing had no effect on reproductive life span (young: 488 ± 31 days [mean ± standard error of the mean {SEM}, n = 12] vs mature: 477 ± 54 days [n = 8]). Within the Pohn 9–10 group, breeding pairs that were set up when they were young became infertile sooner than the breeding pairs that were set up when they were mature (young: 293 ± 32 days [n = 7] vs mature: 435 ± 42 days [n = 11], p = 0.011, ANOVA with a Bonferroni–Dunn correction). Thus, had we set up all Pohn breeding pairs when the mice were young, as we did for all other groups of mice, the reproductive life span of the Pohn 9–10 mice may have been even shorter than we report here. This effect of age at first pairing in the Pohn 9–10 mice suggests that the diminished reproductive potential that can occur when propagating wild mice may result in part from some altered maturational function that limits reproductive life span when breeding begins at a young age. Drickamer (18) similarly observed that manipulation of age at sexual maturation, using olfactory cues, affected the total number of litters that wild Mus musculus females produce; later maturing mice produced more litters. Some Pohn 9–10 breeding pairs did remain fertile for a very long time; interestingly, the 15% (3 pairs) of the breeding pairs that were still fertile after 19 months were first paired when the female was older than 91 days. In both groups of Pohn mice, age at first pairing did not affect other variables—the age-specific litter size or age-specific interlitter interval.

PohnB6F1 breeding pairs reproduced much longer than breeding pairs of either parental genotype (Figure 2A), demonstrating that inheritance for reproductive life span for this cross shows considerable heterosis. By the age at which the first PohnB6F1 breeding pair became infertile (469 days), 75% of the Pohn 9–10 pairs and all of the B6-rb breeding pairs were infertile. This extremely long reproductive life span was unusual even for hybrid crosses; PohnB6F1 mice bred far longer than mice of the S0 generation of either of the 4-way crosses we used for comparison (Figure 2B), even though each of these stocks was produced by crossing two different F1 hybrids. Four of ten PohnB6F1 females weaned litters past their 23rd month; the oldest mother weaned litters of two and three in her 25th and 26th months—at 753 and 777 days of age. For comparison, the maximum female reproductive life span among the 1MLS0 mice (of 22 breeding pairs) was 615 days.

View larger version (22K): [in this window] [in a new window]   Figure 2. Reproductive life span in PohnB6F1 mice. Kaplan–Meier plots are shown for reproductive life span, as indicated by age at birth of the last litter. A, Reproductive longevity is enhanced in PohnB6F1 females, compared to parental Pohn 9–10 and C57BL/6J-rb groups, demonstrating heterosis (p <.0001, Mantel–Cox test for both comparisons). B, Reproductive longevity is greater in PohnB6F1 females than in other highly heterogeneous stocks of mice (1MLS0 and 3MLS0) (p <.0001, Mantel–Cox test for both comparisons). Compared to the 3MLS0 breeding pairs, a greater proportion of 1MLS0 breeding pairs remained fertile as they aged (p =.0004, Mantel–Cox test). In both (A) and (B), open symbols represent censored data

Reproductive Cycle Length (Age-Specific Interlitter Interval)
As expected, reproductive cycle length—measured as interlitter interval—increased with age in both the Pohn and the control groups, indicating that this is a valid biomarker of reproductive aging (19). Neither the initial reproductive cycle frequency nor the age-related increase in cycle length differed between the Pohn 2–5 mice and the standard laboratory mice (1MLS1 and B6-ref groups) (Supplemental Figure 1A, see page 1198). Propagation of the Pohn stock did not influence these biomarkers of reproductive aging (Supplemental Figure 1B). In the PohnB6F1 hybrid, the interlitter interval was shorter compared to both parental groups (Pohn 9–10 and B6-rb) (Supplemental Figure 1C), indicating that heterosis increases litter frequency. The initial interlitter interval was similar for the heterogeneous comparison groups (1MLS0 and 3MLS0) and the PohnB6F1 mice, although the age-related increase was slower for the PohnB6F1 group than for the 3MLS0 group (Supplemental Figure 1D). These results demonstrate that the longer reproductive life spans of Pohn and PohnB6F1 breeders are not due to longer reproductive cycles, and that expression of a biomarker of reproductive aging, reproductive cycle lengthening, may be delayed in PohnB6F1 mice compared to some other heterogeneous groups.

View larger version (18K): [in this window] [in a new window]   >Supplemental Figure 1. Age-specific interlitter interval. For each breeding pair, we calculated the mean interval between births within each 150-day period. Data shown are the mean ± standard error of the mean (SEM) of interlitter intervals among all breeding pairs that were fertile during each 150-day period. To simplify the display, not all error bars are shown. Statistical analyses were performed on log-transformed data to eliminate the correlation of means with standard deviations. A, The interlitter interval was comparable for young Pohn 2–5 breeding pairs and young 1MLS1 breeding pairs. The effect of age on the interlitter interval (p <.0001, analysis of variance [ANOVA]) was the same for Pohn 2–5 and 1MLS1 breeding pairs (no interaction of stock with age, ANOVA). Breeding pairs are the same as in Figure 3A. Historical data from C57BL/6J breeding pairs (B6-ref) are provided as a reference. B, Neither the initial interlitter interval nor the effect of age (p <.0001, ANOVA) changed over generations from Pohn 2–5 to Pohn 9–10 (no main effect or interaction of generation with age, ANOVA). Breeding pairs are the same as in Figure 3B. C, Separate ANOVAs were performed for the comparison of PohnB6F1 breeders to each parental stock because the C57BL/6J-rb parental stock (B6-rb) stopped breeding earlier than the Pohn 9–10 parental stock. The interlitter interval was shorter for the PohnB6F1 breeders than for either parental stock (p <.0001, B6-rb stock; p =.02, Pohn 9–10 stock; ANOVA). There were no interactions with age. Breeding pairs are the same as in Figure 4A. D, Separate ANOVAs were performed for the comparisons of PohnB6F1 breeders to each heterogeneous stock (1MLS0 and 3MLS0) because the 3MLS0 stock stopped breeding earlier than the 1MLS0 stock. Neither the initial interlitter interval nor the effect of age differed between the PohnB6F1 breeders and the 1MLS0 breeders (no main effect or interaction of stock with age, ANOVA). However, the interlitter interval increased more slowly for the PohnB6F1 breeders compared to the 3MLS0 breeders (p =.03, interaction of age with stock, ANOVA)

View larger version (19K): [in this window] [in a new window]   Figure 3. Age-specific litter size: total pups born per litter per breeding pair within 50-day periods. If a breeding pair produced more than one litter in a 50-day period, the mean of the litter sizes was used as the value for the pair. A, Effect of age on litter size differed between Pohn 2–5 and 1MLS1 breeders (interaction of age with stock, p <.0001, analysis of variance [ANOVA]). Age-specific litter size was greater for 1MLS1 breeders, compared to Pohn females, from 101 to 250 days (Fisher's protected least significant difference [PLSD] test). Age-specific litter size was greater for Pohn breeders at 401–450 days (Fisher's PLSD). Only Pohn breeders were fertile after 550 days (mean litter size = 3.0, 5 females, 10 litters). Within each group at each time period, standard errors were 0.2–0.6 pups per litter. Historical data from C57BL/6J-ref breeding pairs are provided as a reference. B, Effect of age on litter size was large (p <.0001, ANOVA), but did not change from generations 2 to 5 (Pohn 2–5) to generations 9 to 10 (Pohn 9–10) (no interaction of generation with the age, ANOVA). Within each group at each time period, standard errors were 0.3–1.1 pups per litter

Age-Specific Litter Size
For Pohn 2–5 females compared to 1MLS1 control females, mean litter size was 34% smaller up to 250 days of age. But from 301 to 550 days, mean litter size for Pohn 2–5 females was 40% greater (Figure 3A). After 550 days, no 1MLS1 females were fertile; in contrast, five Pohn 2–5 females remained fertile and produced 10 additional litters with a mean of 3.0 pups per litter. For Pohn 9–10 females, even though reproductive life span was shorter than for Pohn 2–5 females, age-specific litter size was not affected significantly (Figure 3B).

For the PohnB6F1 breeders, litter size was comparable to, or slightly larger than, the litter size for either parental stock up to 250 days, and was always greater after that (Figure 4A). Thus, for females of this cross, initial litter size was not affected by heterosis; however, the ability to maintain litter size after 250 days of age was dramatically enhanced.

View larger version (22K): [in this window] [in a new window]   Figure 4. Age-specific litter size for PohnB6F1 mice. A, Litter size is maintained in PohnB6F1 females at the age when it begins to decrease in parental stocks (interaction of age with stock, p <.0001, analysis of variance [ANOVA]). Beginning with the 251–300 day interval, litter size was greater for the PohnB6F1 females than for either parental stock (Fisher's protected least significant difference [PLSD] test). Within each group at each time period, standard errors were 0.3–1.1 pups per litter. B, Effect of age on litter size differed for PohnB6F1 breeders compared to breeders of two highly heterogeneous stocks, 1MLS0 and 3MLS0 (interaction of age with stock, p <.0001, ANOVA). Up to 250 days, litters were 35%–45% smaller for PohnB6F1 females than for 3MLS0 breeders (Fisher's PLSD); however, after 250 days, litter size declined much more rapidly for 3MLS0 breeders than for PohnB6F1 breeders. After 400 days, PohnB6F1 breeders consistently had larger litters than 3MLS0 breeders (Fisher's PLSD). The pattern was similar for the comparison of PohnB6F1 to 1MLS0 breeders, although the litter size of 1MLS0 breeders during the first 250 days was intermediate, between that of PohnB6F1 and 3MLS0 breeders. Within each group at each time period, standard errors were 0.3–0.8 pups per litter

Age of First Decline in Litter Size
The age for the earliest significant reduction from the peak litter size proved to be a consistent biomarker for reproductive aging. The peak litter size occurred at different time periods for the various groups, from 51 to 250 days. The earliest significant decline from the peak litter size within a group occurred at the 251–300 day interval for the five control groups, a pattern also seen in the Pohn 9–10 group (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 5. The age period of the first significant decline from the peak litter size for each group. Litter size declined with age for all stocks (p <.0001, except C57BL/6J-ref, for which p =.02, analysis of variance [ANOVA]). Peak litter size occurred at different time periods, between 51 and 250 days, for the various groups. The criterion for a significant reduction from the peak litter size was p .05 for the protected least significant difference (PLSD) test. Details for each group are in Figures 3 and 4

Ovarian Oocyte Numbers
To investigate the physiological basis for the altered reproductive life history in Pohn mice, we determined ovarian oocyte numbers at 100, 200, and 400 days of age in groups of virgin females (Table 4). Pohn 9–10 females had more oocytes than B6 females throughout adulthood. Between 100 and 400 days, Pohn females lost 69% of their oocytes, whereas B6 females lost 87%; however, the difference in rate of oocyte loss was not statistically significant (i.e., no significant interaction of stock with age).

We used 1MLS7 females, as genetically heterogeneous controls, at 400 days of age. Surprisingly, although Pohn 9–10 females at this age had only half as many oocytes as the 1MLS7 females, their fertility rates were comparable (Pohn 9–10, 45%; 1MLS7, 40%). At ages when Pohn 9–10 and B6 females had the same number of oocytes as the 400-day-old 1MLS7 females (200 days for Pohn 9–10 females, 100 days for B6 females), fertility rates for both groups were 100%—much higher than the 40% for the 1MLS7 females. These results indicate that reproductive life span in 1MLS7 females is limited by something other than oocyte depletion, and that Pohn females, like B6 females, are less affected by such extraovarian determinants of reproductive senescence.

Total Life Span
Total life spans did not differ between parous Pohn 2–5 and 1MLS1 females (707 ± 36 days [mean ± SEM], n = 19 vs 694 ± 34 days, n = 22, respectively). Life spans of these continuously bred Pohn females were 12% shorter than those of sibling virgin Pohn females (812 ± 34 days, n = 45); life spans of continuously bred 1MLS1 females were 15% shorter than those of sibling virgin 1MLS1 females (813 days, n = 87). This finding is consistent with our general experience (our unpublished data) that continuous breeding shortens female mouse life spans.

For group-housed virgin Pohn 2–5 males, life spans were 897 ± 37 days (n = 42). The sex difference for Pohn mice was marginal (p =.09, Mantel–Cox log-rank test). To determine if life span might be affected by genetic variation among the founding Pohn breeding pairs, we compared life spans for the 15–19 virgin offspring of each of the five founding breeding pairs (i.e., the five families at Pohn generation 2). Because we found no significant effect of sex on life span, and no interaction of sex with family, we combined data from males and females within each family. Pohn mouse families exhibited significant variation in life span (Table 5). We could not attribute the variance in life span among the families to their descent from any of the three founding grandfathers. We could not further study the genetic basis for this family difference because, to minimize inbreeding, we cross-bred offspring.

	DISCUSSION

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Reproductive Life History
Fecundity is measured as the total number of pups produced by a breeding pair. It is affected by reproductive life span, total litter number, litter size, reproductive cycle length, and the age at which litter size declines. We analyzed these components of fecundity for the initial and subsequent generations of Pohn mice (Pohn 2–5 and Pohn 9–10) and for an out-crossed generation (PohnB6F1).

Pohn 2–5 versus controls.-- Although the overall fecundity of the Pohn 2–5 group was similar to that of our 1MLS1 heterogeneous control group (Table 2), the components of fecundity differed. In particular, the Pohn 2–5 breeders had a longer reproductive life span (Figure 1A), primarily because they produced more litters (Table 2), not because reproductive cycle length was altered (Supplemental Figure 1A). Also, litter size differed between the groups in an age-dependent manner (Figure 3A). Whereas the Pohn 2–5 breeders had smaller litters than the 1MLS1 breeders up to about 250 days of age, the age-related reduction in litter size occurred 100 days later for Pohn 2–5 breeders. In fact, older Pohn 2–5 breeding pairs had larger litters than age-matched 1MLS1 breeding pairs. Thus, components of reproductive senescence were delayed in Pohn 2–5 mice, relative to 1MLS1 mice, as indicated by two biomarkers—reproductive life span and the age at which litter size first decreases. This relative delay in reproductive senescence offset the effects of an initially smaller litter size in Pohn 2–5 mice to result in an overall fecundity equal to that of the heterogeneous 1MLS1 control group.

Pohn 2–5 versus Pohn 9–10.-- Reproductive capacity diminished in the Pohn stock by the ninth and tenth generations (Pohn 9–10), even though we minimized inbreeding by avoiding brother–sister and first cousin matings. The effect was due primarily to shortened reproductive life span (Figure 1B, Table 2). In addition, although litter sizes were generally comparable (Figure 3B), the age-related decline in litter size began earlier in the Pohn 9–10 group than in the Pohn 2–5 group (Figure 5). Reproductive cycle length was unaffected by propagation of the stock (Supplemental Figure 1B). Our results are consistent with the common observation (E. Eicher, L. Washburn 2005, personal communication) that fecundity diminishes as wild mice are initially propagated in captivity. Our findings suggest that this loss of fecundity might occur partly through the advancement of reproductive senescence, possibly indirectly, through an affect on a maturational determinant of reproductive potential (18).

As a stock is propagated, a common means by which a phenotype can be altered involves the loss of alleles, typically due to inadvertent selection and genetic drift. The resulting diminished heterosis can affect phenotypic expression through at least three mechanisms: (i) the loss of an allele that directs the expression of a specific phenotype; (ii) the emergence of expression of a deleterious recessive allele that becomes fixed when the wild-type allele is lost; and (iii) diminished "genetic buffering" (20), as when each allele optimizes function for a different set of environmental conditions. The altered phenotype can be restored by out-crossing if mechanisms ii or iii are involved, because most unrelated strains will carry the wild-type allele (mechanism ii), or because newly introduced alleles can restore genetic buffering (mechanism iii). In the present study, when we out-crossed the Pohn stock to the B6 strain, we observed that a number of reproductive phenotypes were affected.

PohnB6F1 versus parental stocks and heterogeneous crosses.-- The reproductive life spans for PohnB6F1 hybrid females are the longest we have seen for mice not diet restricted: 4 of 10 females reproduced at ages > 700 days; one weaned litters of two and three pups born at 753 and 777 days of age. This reproductive longevity was greater than for either of the parental stocks, which had maximum reproductive life spans of 629 days (Pohn 9–10) and 452 days (B6-rb), and was much greater than for either of two highly heterogeneous controls that we had available for comparison, 615 days (1MLS0) and 467 days (3MLS0). In fact, the maximum reproductive life span that we found in the literature for non-diet-restricted F1 hybrid mice was 648 days for an AB6F1 female, and she failed to wean this last litter (11).

The increased reproductive life span for the PohnB6F1 breeders, relative to either parental stock, resulted entirely from an increased number of litters produced (Table 3) and not from an increased length of reproductive cycles (Supplemental Figure 1C). In fact, out-crossing decreased the length of reproductive cycles. Litter size was comparable among the young adults for the PohnB6F1 mice and the parental groups; however, the age-related decline in litter size occurred much later for the PohnB6F1 hybrids (Figure 5). Thus, compared to the parental groups, the PohnB6F1 mice expressed differences in the same two biomarkers that distinguished the Pohn 2–5 breeders from their control group—reproductive life span and the age at which litter size first decreases. The values of these two biomarkers for the parental B6 and Pohn 9–10 groups were similar to each other, and much lower than in the PohnB6F1 hybrid, indicating that the mode of inheritance for reproductive senescence in this cross is characterized by heterosis. Surprisingly, in this cross, litter size for young adults did not exhibit heterosis; in fact, initial litter size was no greater than it was for the parental stocks, and smaller than for the heterogeneous 1MLS0 and 3MLS0 crosses. The greater overall fecundity in PohnB6F1 breeders, relative to their parental stocks, resulted primarily from their delayed reproductive senescence. Thus, in the parental stocks, a genetic potential to produce hybrids with extremely long reproductive life spans has not been lost during propagation or inbreeding.

Genetic diversity and heterosis alone do not appear to be sufficient to account for the increased fecundity of the PohnB6F1 hybrids; neither of the highly heterogeneous and genetically diverse crosses we used for comparison (1MLS0 or 3MLS0) produced breeders with a comparable delay in their age-related decrease in litter size or a comparably long reproductive life span. Furthermore, the age-related increase in reproductive cycle length was delayed in PohnB6F1 mice compared to the heterogeneous mice of the control crosses. Thus, unique alleles were provided by the PohnB6F1 parental stocks to substantially delay reproductive senescence. The similarity of this delay in PohnB6F1 mice to that in the early generations of the Pohn stock (rather than to the B6 strain) suggests that the Pohn stock was the principle genetic contributor to the delayed reproductive senescence in the PohnB6F1 mice.

Oocyte Number
The roles of ovarian and extraovarian (including neuroendocrine) aging in determining the age of reproductive cessation is genetically determined in mice and can be evaluated by heterochronic ovarian transplants. For example, in CBA females, which have fewer ovarian follicles as young adults than most other strains (21,22), ovarian aging determines the relatively early cessation of reproductive cycles (21), whereas, in C57BL/6NNia females, extraovarian factors are primary (23). In C57BL/6J females, both ovarian and extraovarian aging interact to determine the timing of reproductive senescence (24).

Studies of ovarian aging have focused on atresia, the progressive age-dependent depletion of ovarian oocytes (reproductive cycles cease when a threshold oocyte number is reached) (21). To begin an investigation of the physiological and cellular determinants of prolonged female reproductive life span in Pohn mice, we determined ovarian oocyte numbers. Although ovaries of the 400-day-old Pohn females contained less than half as many oocytes as the age-matched 1MLS7 females (Table 5), their fertility rates were comparable. In fact, at the age of 200 days, when Pohn females have the same number of ovarian oocytes as 400-day-old 1MLS7 females, 100% of the Pohn females were fertile compared to 40% of the 1MLS7 females. Therefore, something other than oocyte follicle number limits reproductive life span in the 1ML stock. Our results demonstrate that reproductive life span in Pohn females is not restricted by the extraovarian limitations that govern reproductive life span in 1ML females.

We also compared oocyte numbers in Pohn and B6 females of various ages. Pohn females had more oocytes than did B6 females. This difference was particularly evident at later ages (Table 5). Thus, to sustain fertility to advanced ages, Pohn females not only retain greater oocyte stores than standard inbred mice such as B6, they also avoid the age-related causes of infertility that are not dependent on oocyte number, such as those that limited reproductive life span in 1MLS7 mice.

This analysis of fecundity in the Pohn stock demonstrates its potential value as a genetic model for studies of both ovarian and extraovarian mechanisms of reproductive senescence.

Body Weight
Body weight may be a key factor mediating tradeoffs among litter size and other life history traits, including reproductive life span. Roberts (25) demonstrated that mouse stocks selected for low body weight produced fewer pups per litter in their first litters, but produced more litters, resulting in a longer reproductive life span, than stocks selected for heavier body weight from the same founder populations. These results indicate that some genes that regulate body weight may also mediate a tradeoff between reproductive life span and litter size that parallels differences between Pohn and standard laboratory mice. Pohn mice are very small compared to standard laboratory strains of mice. Body weight (mean ± SEM) at 6 months for female virgins in a separate study was 16.1 ± 2.2 g for Pohn and 20.7 ± 3.4 g for PohnB6F1. Both groups were smaller than age-matched B6 females (24.2 ± 3.4 g). Body weight is inversely associated with another senescence-related life history trait, life span [(9,26,27), discussed in (28)]. Therefore, it may be possible to rapidly find genetic loci that mediate the evolution of delayed female reproductive senescence by first identifying genetic loci that determine body size in crosses of standard laboratory mice with wild-derived mice, such as the Pohn mice, and then testing effects of the identified body size locus on reproductive life history traits.

Other Aspects of Senescence
The total life span of nonbreeding Pohn mice is comparable to that of our standard 1MLS1 control mice and B6 mice. Miller and colleagues (9) also reported that the life span for another group of Pohnpei-derived mice, the PoG0 mice, was similar to that of their control stock of laboratory mice, the 4-way cross "DC" stock, and similar to the life span we report for Pohn mice. However, we observed "family" differences in life span among the progeny of the five primary breeding pairs we used to establish the Pohn stock (Table 5). Progeny of the longest-lived of these five families had a mean life span of 998 days, with the longest-lived 10% (i.e., the two oldest) surviving to 1434 days. These are very long life spans for stocks of Mus musculus, approaching the 1008-day mean life span of the wild-derived "Id" mice reported by Miller and coworkers (9). These results indicate that alleles with important effects on overall life span are segregating within the Pohn stock.

Other aspects of delayed senescence are expressed in Pohn mice. Tail skin fibroblasts from Pohn mice, compared to tail skin fibroblasts from B6 mice, have a greater in vitro proliferative ability, a decreased expression of senescence markers, and an increased resistance to oxidative stress (13), demonstrating that cellular aging, as defined by in vitro replicative potential, is slower in cells from Pohn mice. Thus, the expression of multiple aspects of senescence may be altered in Pohn mice. This is consistent with predictions of evolutionary theory that conditions of a stable natural environment and low predation, such as those that exist on some tropical islands such as Pohnpei, diminish ecological mortality and may enhance selective pressure for delayed senescence (4,5).

Conclusion
Our studies highlight the potential of the Pohn stock and the PohnB6F1 hybrid as unique resources for the allelic diversity needed to identify genes that regulate female reproductive longevity and senescence—and perhaps other life history traits—in mammals. The Pohn stock is readily available, and PohnB6F1 hybrids can be easily produced.

	Acknowledgments

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This work was supported by grants RR-14455, AG-025007, and AG025707 (DEH); Maine Biomed Res Infrastructure/NCRR/2 P20 RR016463 and The Goldwater Fellowship Foundation (YB); and The Jackson Laboratory cancer core grant HL63230.

We are grateful for the expert assistance of K. Davis, L. Bottinelli, J. M. Currer, and C. M. Astle. We extend special thanks to Dr. C. Peterson (College of the Atlantic) for his valuable comments.

Yaniv Brandvain is now with the Department of Biology, Indiana University, Bloomington.

Simon Klebanov is now with the Obesity Research Center, St. Luke's-Roosevelt Hospital Center, New York, New York.

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received December 20, 2006

Accepted July 9, 2007

	References

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