We hypothesized that a polycystic ovary syndrome (PCOS) background associated with obese-prone genotype, coupled with preconditioning by caloric restriction, would confer a survival benefit in genetically prepubertal obese/PCOS (O/PCOS)-prone rats faced with an unpredictable challenge of food shortage. Female, juvenile JCR:LA-cp rats, O/PCOS- and lean-prone, were exposed to 1.5 h of daily meals and 22.5 h of voluntary wheel-running, a procedure that leads to activity anorexia (AA). One week before the AA challenge (AAC), O/PCOS-prone rats were freely fed (O/PCOS-FF) or pair fed (O/PCOS-FR) to lean-prone, free-feeding animals (Lean-FF). O/PCOS-FR and lean-prone, food-restricted (Lean-FR) groups were matched on relative average caloric intake. Animals were removed from protocol at 75% of initial body weight (starvation criterion) or after 14 days (survival criterion). The AAC induced weight loss in all rats, but there were significant effects of both genotype and feeding history on weight loss (lean-prone rats exhibited a higher rate of weight loss than O/PCOS-prone; P < 0.001), and rats with prior caloric restriction retained more weight than those free fed previously (90.68 ± 0.59% vs. 85.47 ± 0.46%; P < 0.001). The daily rate of running was higher in lean-prone rats compared with O/PCOS-prone. This difference in running rate correlated with differences in mean days of survival. All O/PCOS-FR rats survived at day 14. O/PCOS-FF rats survived longer (10.00 ± 0.97 days) than Lean-FR (6.17 ± 1.58 days) and Lean-FF (4.33 ± 0.42 days) rats (P < 0.05). Thus preconditioning by caloric restriction induces a substantial survival advantage, beyond genotype alone, in prepubertal O/PCOS-prone rats.
- JCR:LA-cp rat
- obese- and PCOS-prone genotype
- food restriction
over the last few decades, there has been a worldwide increase in pediatric obesity in both developed and developing countries (23, 32). Excess adiposity at a young age is linked to immediate and long-term health risks, including increased risk of cardiovascular disease and type 2 diabetes (10, 22) and increased middle-age mortality and morbidity, regardless of adult weight status (8, 27). At the same time, polycystic ovary syndrome (PCOS), one of the most common endocrine metabolic disorders in women of reproductive age, is increasing dramatically in Western societies (7). PCOS is a complex and heterogeneous disorder, characterized by hyperandrogenism, oligo-/anovulation, and polycystic ovaries, resulting in menstrual dysfunction and reproductive difficulty, as well as metabolic dysfunction, such as insulin resistance (5). Importantly, PCOS has a unique relationship with the obesity that is increasingly prevalent. The current literature indicates that >50% of women with PCOS are overweight or obese, and obesity significantly exacerbates some features of PCOS, such as hyperandrogenism, insulin resistance, infertility, and cardiovascular diseases (45). As in the case of obesity, the aetiology of the PCOS remains uncertain.
Interpopulation differences in prevalence and phenotypic expression of PCOS lend support to the notion that PCOS has a genetic component (12, 13). Furthermore, familial linkage studies (3, 48) and evidence that PCOS is more common in monozygotic twins than in dizygotic twins (43), together with its high worldwide prevalence, suggest an evolutionary contribution to the disease. In fact, Neel's (28) adaptive “thrifty genotype” hypothesis of obesity has been extended to the fitness benefits of PCOS (14, 16). PCOS-prone women, who have an increased risk of obesity and anovulation during times of food abundance, would have a selective advantage toward reproduction during periods of food shortage and famine, when non-PCOS-prone women become anovulatory (4, 14). Under this evolutionary scenario, PCOS can thus be seen as a “fertility storage condition,” which favors human reproductive success, allowing the human species to maintain fertility during adverse environmental circumstances, such as famine. Taken together, these observations suggest that obesity and PCOS are not the result of defective physiology but are natural, adaptive responses to food environmental changes, such as overnutrition and lack of physical activity.
A major problem for living organisms is to maintain energy balance when faced with environmental challenges, such as food depletion or famine (24). Such challenges activate a complex interplay of stress—metabolic and reproductive processes that initiate and maintain food-related behavior and homeostasis (1, 42). These processes, however, have limitations—a fact demonstrated when rodents are placed on time-limited, daily meals (1.5 h) and provided with unlimited access to running wheels (23.5 h). Under these conditions, animals reduce food intake, lose body weight, and escalate wheel-running, essentially becoming decompensated. The excessive physical activity further suppresses food consumption, body weight plummets, and animals die of this vicious feedback cycle [self-starvation or activity anorexia (AA) (9, 30, 34)], an animal analog of human anorexia nervosa. Our ongoing research is based on the JCR:LA-cp rats incorporating the autosomal-recessive cp gene [a Tyr763Stop mutation for the leptin receptor (19)]. With the use of the animal model of AA, our group has demonstrated recently that male, juvenile, obese-prone (cp/cp) JCR:LA-cp rats, especially those with a history of food restriction, ran more and survived longer in the AA challenge (AAC) relative to lean-prone (+/?) rats. This supports the notion that genotype and food environment contribute to survival during unpredictable periods of food shortage (29). However, less is known about the survival benefit of a PCOS background associated with the obese-prone (cp/cp) genotype in suboptimal nutritional environments.
In the present study, we imposed a 10-day period of food restriction or a period of free access to food on obese/PCOS (O-PCOS)-prone (cp/cp) and lean-prone (+/?) female rats, allowing us to isolate the separate and combined effects of genotype and feeding history on survival in the AAC. We used nonovulating, juvenile JCR:LA-cp female rats as an experimental model, as our group has demonstrated that female JCR rats reliably produce the spectrum and heterogeneity of traits that closely reflect the PCOS phenotype in women. That is, the cp/cp genotype of the JCR:LA-cp strain at 12 wk of age spontaneously presents hyperandrogenemia, oligo-ovulation, a decreased number of corpora lutea, and an increased number of total follicles, in particular, atretic and cystic follicles (38, 39).
We hypothesized that juvenile (nonestrous) cp/cp female rats would survive longer than lean-prone (+/?) in the AAC, based on the additive effects of genetic proneness for obesity and proneness for PCOS; we also predicted that prior food restriction of juvenile cp/cp female rats would give them an additional advantage over young cp/cp female rats with free access to food before the challenge. To test these hypotheses, we measured days ofsurvival in the AAC, as well as a number of behavioral, metabolic, and reproductive hormonal parameters.
MATERIALS AND METHODS
Twenty-four female JCR:LA-cp rats [12 O/PCOS-prone cp/cp and 12 non-PCOS, lean-prone (+/?)], 35-40 days of age, were obtained from the established breeding colony at the University of Alberta (35). Rats, in groups of 12, were housed individually in clear, polycarbonate cages (47 cm × 27 cm × 20 cm), with sterile wood-chip bedding in a temperature (22 ± 2°C)- and humidity-controlled environment, and maintained on a 12-h light/dark cycle (lights off 0700–1900). Throughout the experiment, animals had free access to water and were fed as outlined in the feeding schedule below. The care and use of animals were in accordance with Guidelines of the Canadian Council of Animal Care and subject to prior review and approval by the Animal Care and Use Committee, Health Sciences, of the University of Alberta.
Apparatus and materials for AAC.
Twelve Wahmann running wheels (1.1 m circumference) with metal side cages (25 cm × 15.5 cm × 12.5 cm) were used. A computer recorded the number of wheel turns in 1-min intervals, as described previously (25). Feeding was conducted in home cages or in feeding cages with the same materials and dimensions as the home cages but without bedding. Animals were fed standard laboratory chow (LabDiet 5010 rodent diet; PMI Nutrition International, Brentwood, MO; metabolizable energy = 3.42 kcal/g). An electronic scale (Sartoris Model TE4101; Sartorius AG, Göttingen, Germany) was used to measure food and body weight to the nearest gram. Rats were given a fixed amount of food (50 g) at the beginning of the feeding period, and the amount of chow remaining after the meal was subtracted from this fixed amount to provide a measured food intake (g). Subsequently, food intake was converted to calories [food (g) × 3.42 kcal/g], yielding a measure of daily caloric consumption.
One day after arrival, rats were acclimatized over 10 days to the feeding schedules (free access to food vs. food restricted), with food intake and body weights recorded daily. O/PCOS-prone rats were matched for body weights and randomly assigned to free-feeding (O/PCOS-FF) or food-restricted (O/PCOS-FR) groups (six rats/group). Food restriction consisted of pair-feeding the O/PCOS rats the daily mean amount of food consumed by the lean free-feeding (Lean-FF) group on the previous day. To allow for comparisons by genotype and feeding conditions, a group of lean-prone, food-restricted (Lean-FR) rats was included in the experimental design. The daily food intake of Lean-FR rats was a percentage of the food consumption of Lean-FF animals; we calculated this percentage from the difference in daily food intake between O/PCOS-FF and O/PCOS-FR groups [Lean-FR intake = Lean-FF intake × (O/PCOS-FF intake − O/PCOS-FR intake)/O-PCOS-FF intake × 100]. Thus O/PCOS-FR and Lean-FR groups were matched on relative average degree of food restriction. Blood was taken by tail-bleeding on day 7 of the acclimatization period for baseline biochemical parameters after an overnight fast, and the rats were then allowed to recover for 2 days. On day 10 and subsequent days, rats were weighed and transferred to feeding cages (home cages without bedding) at 0700 with 1.5 h free access to food, followed by 22.5 h of access to running wheels (AAC). Rats were removed from the protocol when body weight reached 75% of initial weight (starvation criterion) or after 14 days of the AAC (survival criterion). During the AAC, body weight, food intake, and wheel turns were measured daily.
When animals met the criterion for starvation or survival, they were removed from their home cages and anesthetized with isoflorane. Blood was taken by cardiac puncture. Liver and total white adipose tissue (WAT) pads (retroperitoneal + periovarian + subcutaneous) were dissected and weighed. The blood was collected in 10 ml polyethylene tubes containing EDTA and stored on ice until centrifugation. Plasma samples were stored at −80°C until analysis.
Plasma biochemical analysis.
Plasma corticosterone was measured using Milliplex multianalyte profiling (Millipore, Billerica, MA). Total testosterone, free testosterone, and sex hormone-binding globulin (SHBG) levels were assessed by an ELISA kit for rats (Cusabio Biotech kit, Hubei Province, China).
Data are presented as mean ± SE. Food intake, body weight, wheel turns, and days lasted in the AAC were analyzed by mixed-effects ANOVAs with genotype (O/PCOS-prone vs. lean-prone) and feeding history (free access vs. food restriction) as between-subjects factors and days (acclimatization or AAC) as the within-subjects term. We used paired t-tests as well as one-way and two-way repeated-measure ANOVAs to further elucidate the within-subject and mixed effects. The interaction of genotype and feeding history was explored by one-way ANOVAs on the four experimental groups, followed by post hoc comparisons of the means using a Tukey procedure. One-way ANOVAs were conducted on the four treatment groups to analyze plasma biochemical measures, followed by pair-wise contrasts and multiple comparisons of means. Planned contrasts were used when the difference by feeding history could be predicted within each genotype (lean-prone or O/PCOS-prone). For interactions having days, Greenhouse-Geisser correction was used if Mauchly's test of sphericity was significant (46). Correlations among total distance run, number of days lasted, body weight loss, and caloric intake during the AAC were also calculated. Alpha was set at 0.05 for all analyses. For all pair-wise contrasts and multiple comparisons of means tests, the least-significance alpha level for the several contrasts is given in the text. Due to lack of variability in days lasted in the AAC for the O/PCOS-FR group (all animals survived 14 days), the nonparametric Kruskal-Walis and Mann-Whitney U-tests (40) were used to assess differences among the four experimental groups. We also conducted a survival analysis without the O/PCOS-FR group (all animals were right censored).
Body weight and survival.
We conducted an analysis of body weight over the 10 days of acclimatization by genotype and feeding schedule. There were significant main effects on body weight of genotype (P < 0.001) and food schedule (P < 0.001). O/PCOS-prone rats had higher body weight than lean-prone animals, and free-fed rats had greater body weight than food-restricted groups. There was also significant interaction of genotype-by-days (P < 0.001) and feeding schedule-by-days (P < 0.001). O/PCOS-prone rats increased body weight over days more than lean-prone rats; free-feeding rats increased body weight over days, whereas food-restricted rats did not.
Daily body weight (g) over the period is shown in Fig. 1, together with retained body weight during the AAC (as a percentage of initial weight; Fig. 1). Body weight declined more for lean-prone rats than O/PCOS-prone rats over the AAC (Fig. 1). There were significant genotype and genotype-by-days effects, as well as a reliable effect of feeding history on absolute body weight (rats with a history of free-feeding = 144.78 ± 9.31 g; rats with prior food restriction = 132.61 ± 7.86 g; P = 0.001). The lower body weight of food-restricted rats compared with free-feeding animals reflects their low initial body weight before exposure to the AAC.
Over the first 3 days, when all rats remained in protocol, there was a significant main effect of feeding history on weight retention (prior free-fed rats retained 85.47 ± 0.46%; prior food-restricted animals retained 90.68 ± 0.59%; P < 0.001; Fig. 1). There was also a significant interaction of genotype-by-days on weight retention (P < 0.001). Subsequent analysis of linear trend by genotype showed that lean-prone rats declined in weight faster than O/PCOS-prone (P < 0.001). Multiple regression analysis using initial body weight (last day of acclimatization) and retained body weight (percent) on day 1 of AAC to predict days lasted in the AAC were performed. Both predictors had significant effects on survival (P < 0.001), with a coefficient of determination = 0.68. Higher initial body weight and higher retained weight on day 1 of AAC were correlated with increased survival.
All O/PCOS-FR rats survived in the AAC at day 14 (Fig. 2). A nonparametric, independent samples test showed significant differences in days lasted among the four experimental groups (P = 0.001). Follow-up, two-group tests indicated that O/PCOS-FR differed significantly from O/PCOS-FF (P = 0.002), but Lean-FR rats did not differ significantly from the Lean-FF group. O/PCOS-FF rats survived 10.00 ± 0.97 days, Lean-FR survived 6.17 ± 1.58 days, and Lean-FF survived 4.00 ± 0.37 days in the AAC.
Kaplan-Meier survival analysis for three of the four experimental groups, omitting the O-PCOS-FR group, as all rats were censored (no variability to estimate error), indicated a significant difference in the survival distributions by condition [χ2 = 7.07; degree of freedom (df) = 2; P = 0.02; Fig. 2]. There was no significant difference in the survival distributions for the two lean-prone groups (χ2 = 0.60; df = 1; P = 0.4). It is notable, however, that one of the six rats in the Lean-FR group survived to day 14 of the AAC, whereas none of the Lean-FF rats lasted more than 5 days.
For the first 3 days of the AAC, when all rats remained in the protocol, there was only a significant genotype-by-days interaction on wheel-running (P = 0.003). Lean-prone rats increased their running substantially from days 2 to 3 of the AAC compared with O/PCOS animals (Fig. 3).
There was no statistical difference among the groups in total distance run over the first 3 days: Lean-FF, 8,505 ± 1,811 m; Lean-FR, 7,696 ± 838 m; O/PCOS-FF, 5,309 ± 522 m; O/PCOS-FR, 5,633 ± 754 m (P = 0.11; Fig. 4). All of the O/PCOS-prone rats remained in the protocol until day 8 of the AAC. O/PCOS-FF and O/PCOS-FR rats' wheel-running showed a significant increase in wheel-running over days 4–8 (P = 0.001), but there were no significant main or interaction effects (Fig. 4). The O/PCOS-FR rats continued to run at about the same pace until day 14 (survival criterion). Correlations among total distance run for each rat and body weight loss, caloric consumption, and number of days lasted showed that running distance negatively correlated with both the number of days lasted (r = −0.462; P = 0.023) and the weight loss (r = −0.438; P = 0.032) but not with the food intake (P = 0.248).
As expected, caloric consumption was different among groups over the 10-day acclimatization period (Fig. 5). All main and interaction effects were significant; in particular, we focused on the days-by-feeding-history interaction on caloric consumption (P < 0.001). Over the 10 days of acclimatization, free-fed rats consumed more calories than food-restricted animals. During this period, Lean-FR and O/PCOS-FR rats did not differ significantly in daily caloric intake as a percentage of intake of their free-feeding controls. Lean-FR rats, as a percentage of the Lean-FF, were 66.32 ± 2.48; the mean percentage intake of O/PCOS-FR rats was 64.51 ± 3.01 of O/PCOS-FF intake. Thus the two food-restricted groups were equated for relative caloric intake before the AAC.
Throughout the AAC, animals with prior food restriction ate more over the first 3 days compared with those with a history of free-feeding, yielding a significant days-by-feeding-history interaction (P < 0.001). From days 1 to 3, animals with a history of free-feeding increased their daily caloric intake, whereas those with prior caloric restriction maintained their daily caloric intake at the same level (Fig. 5). All of the O/PCOS-prone rats that remained in the protocol until day 8 of the AAC did not differ significantly in their daily caloric intake by feeding history.
Plasma biochemical parameters and body composition.
Biochemical parameters are shown in Table 1. Baseline plasma corticosterone levels revealed a significant effect of feeding history (P = 0.021). Rats raised on free access to food showed a lower mean corticosterone level than prior food-restricted animals (672.14 ± 68.33 vs. 833.56 ± 67.13 nmol/l). Corticosterone levels on the day of removal from the AAC showed heterogeneity of variance. After natural log transformation to obtain equality of variance, there was a significant effect of genotype (P = 0.002), with lean-prone rats having a higher mean corticosterone level than O/PCOS-prone animals (1,260.47 ± 252.17 vs. 570.02 ± 64.26 nmol/l).
To examine the plasma corticosterone level changes, we used a repeated-measure ANOVA with genotype, feeding history, and time of measure (before and after the AAC). After transformation of the corticosterone values to natural logs establishing homogeneity of variance, there was a significant interaction of genotype by the before/after period (P = 0.023). From baseline to after the AAC, plasma corticosterone levels increased in lean-prone rats and decreased in the O/PCOS-prone animals. Planned contrasts on the difference in plasma corticosterone levels from baseline to removal, an index of the hypothalamic-pituitary-adrenal (HPA) axis reactivity, revealed a significant difference between O/PCOS-FF and O/PCOS-FR groups (P = 0.026). This indicated a lower stress response in the O/PCOS-FR rats; however, a similar comparison between the lean-prone groups did not show a significant effect (Table 1).
Plasma total testosterone levels before and after the AAC indicated significant genotype (P = 0.001) and feeding-history (P = 0.018) effects but no significant interaction with time of measurement (before/after). Within the O/PCOS-prone rats, there was a significant difference between rats with a history of free-feeding and those with prior food restriction (P = 0.005), whereas there was no significant difference by feeding history for lean-prone animals. AAC reduced total testosterone in lean-prone rats (P = 0.048), as well as in O/PCOS-prone animals (P = 0.001). On removal from protocol, plasma-free testosterone and SHBG concentration did not differ significantly among groups (Table 1).
On removal from the AAC, WAT in the Lean-FF and Lean-FR groups was depleted completely (Table 1). Also, there was no significant difference in WAT between O/PCOS-FF and O/PCOS-FR, even though prior food-restricted rats survived longer and ran farther. Unlike WAT, lean mass was not significantly different among the four groups following the AAC (Table 1).
Our results show for the first time that prepubertal, female O/PCOS-prone rats gain a survival advantage over their lean-prone congenics when confronted with a challenge of time-limited feeding and an opportunity for food-related activity (wheel-running). On average, O/PCOS-prone rats raised on free access to food lasted 2.4 times longer than Lean-FF rats under the AAC, replicating our recent findings in obese-prone, male JCR rats (29). Age-matched JCR female rats, prone to obesity and PCOS, survived longer in the AAC than male obese-prone rats, demonstrating an additional survival benefit of the PCOS genotype. The survival advantage of these female rats supports the evolutionary “thrifty gene” hypothesis of obesity (28) and its extension to PCOS (14). The longer survival of female O/PCOS-prone rats compared with their lean-prone congenics relates to higher baseline energy reserves, greater caloric intake, and less energy expenditure by physical activity in the AAC. These differences reflect the absence of a functional leptin receptor in O/PCOS-prone rats. Leptin acts through hypothalamic centers, predominantly the arcuate nucleus, to decrease food intake and increase energy expenditure, leading to lower fat mass (33), suggesting that functional leptin receptors are detrimental to surviving the AAC. When leptin signaling is defective, as in the O/PCOS-prone rats, the energy homeostasis pathway involving feeding-related peptides, such as neuropeptide Y, is impaired. This impairment results in increased appetite, decreased energy expenditure, and greater fat accumulation (36)—effects that are advantageous for survival in the AAC.
On removal from the AAC, fat mass was depleted completely in lean-prone animals, whereas O/PCOS-prone rats still had WAT stores. Given this sufficient fat reserve in O/PCOS-FF rats, it is unexpected that all of them starved, indicating that available fat stores of O/PCOS-prone animals are not the key element for surviving the AAC.
Our findings for HPA axis activity are more informative about the underlying mechanism for surviving the AAC. The after–before difference in plasma corticosterone levels, an index of HPA axis reactivity, was 2.3 times lower in O/PCOS-prone rats (+40.59 nmol/l) than lean-prone animals (+94.09 nmol/l), indicating an inverse relationship between stress and survival (5, 29). Thus O/PCOS-prone, female rats have high-energy reserves, low-energy expenditure, and reduced HPA axis-reactivity factors, favoring prolonged survival in the AAC. These same factors, working in opposite directions, lead to the rapid demise of the lean-prone animals.
In the wild, the longer survival of prepubertal O/PCOS-prone rats would confer increased reproductive success but only if the female rats became fertile after an extensive search for food during a famine. A detriment to reproduction in O/PCOS-prone rats is the absence of the leptin receptor, as functional leptin is intimately involved in the hypothalamic-pituitary-gonadal axis (18). Thus adult-obese and infertile mice (ob/ob), lacking a functional leptin receptor, did not become fertile following moderate caloric restriction (26). Currently, there are no animal studies of reproduction of prepubertal, PCOS-prone rodents following more severe food restriction combined with physical activity. In humans, however, caloric restriction and exercise have been shown to restore fertility of women with PCOS (17). One possibility is that prepubertal O/PCOS-prone rodents also become fertile following the AAC or when maintained on caloric restriction with exercise throughout the early developmental period.
Our findings for reproductive function showed that AAC reduced plasma total testosterone levels in both genotypes, indicating a suppressive effect of AAC on gonadal function, as reported previously by Pirke et al. (31). Free testosterone and SHBG levels, indicators for bioavailable testosterone, showed no difference among the four experimental groups after the AAC. Other reproductive function indices, such as estrus cyclicity and ovarian histology, could be more informative about PCOS at the young age of the animals in this study. On the other hand, the AAC may have eliminated all indices of PCOS.
The present study is the first demonstration that prior caloric restriction in young O/PCOS-prone rats contributes to survival. In our previous study of male obese-prone rats (5a), approximately two-thirds of the juvenile animals with a history of food deprivation survived the AAC; all of the O/PCOS-FR female rats survived in the current study. This indicates that female rats with the PCOS genotype, when combined with obese proneness, benefit more than male obese-prone rats from preconditioning by caloric restriction.
To isolate the effects of the preconditioning, independent of genotype, it is informative to compare survival by feeding history within each genotype. Results showed that O/PCOS-FR rats survived about 1.5 times longer than O/PCOS-FF animals. In contrast, Lean-FR female rats and Lean-FF showed no reliable difference in survival. One of the six rats in the Lean-FR group survived to day 14, whereas all Lean-FF female rats starved within 3–5 days of AAC. One possibility is that prepubertal, female JCR rats gain a survival advantage from preconditioning by caloric restriction, but the survival is reduced substantially by having a lean-prone genotype.
To understand how preconditioning by caloric restriction improves survival of O/PCOS-prone rats, we note our findings for HPA axis reactivity (6). Change in plasma corticosterone levels was least for O/PCOS-FR rats, suggesting that physiological stress in these animals is reduced by pre-exposure to caloric restriction (34). In addition to low HPA axis reactivity, caloric restriction is known to reduce oxidative stress at the cellular level, especially in obese-prone rodents (11, 21, 41, 47). This process involves a reduction of mitochondrial electron flow and proton leaks that attenuate cell damage caused by reactive oxygen species (15, 20). Thus the longer survival of O/PCOS-FR animals could relate to their low HPA axis reactivity and reduced oxidative stress.
Overall,the current findings show that the obese/PCOS genotype allows animals to survive unpredictable challenges of food restriction and food-seeking activity. Preconditioning to caloric restriction enhanced this survival advantage significantly. Thus the adaptive role of the PCOS genotype may explain its high prevalence in countries faced with sudden and unpredictable climatic changes related to periods of feast and famine (37). A limitation of our study is that all obese-prone, female JCR rats spontaneously develop the PCOS phenotype in adulthood (38, 39). Thus we were unable to arrange an obese, non-PCOS control group, which would have allowed us to isolate the unique effects of PCOS proneness on survival. To strengthen the evidence for the adaptive effects of the PCOS genotype, subsequent research should identify or engineer obese-prone animals that are either phenotypic for obesity and PCOS or phenotypic only for obesity.
Funding for this research was provided by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to W. D. Pierce and C. D. Heth. Support for the research of S. D. Proctor was provided by the Alberta Livestock Industry Development Fund and NSERC.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: A.D. and W.D.P. conception and design of research; A.D. performed experiments; A.D. and W.D.P. analyzed data; A.D. and W.D.P. interpreted results of experiments; A.D. prepared figures; A.D. drafted manuscript; A.D., D.F.V., C.D.H., J.C.R., S.D.P., and W.D.P. edited and revised manuscript; A.D., D.F.V., C.D.H., J.C.R., S.D.P., and W.D.P. approved final version of manuscript.
- Copyright © 2013 the American Physiological Society