publications | Calen Patrick Ryan, PhD

2024

Accelerated biological aging six decades after prenatal famine exposure

Mengling Cheng, Dalton Conley, Tom Kuipers, and 11 more authors

Proceedings of the National Academy of Sciences, Jun 2024

Publisher: Proceedings of the National Academy of Sciences

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To test the hypothesis that early-life adversity accelerates the pace of biological aging, we analyzed data from the Dutch Hunger Winter Families Study (DHWFS, N = 951). DHWFS is a natural-experiment birth-cohort study of survivors of in-utero exposure to famine conditions caused by the German occupation of the Western Netherlands in Winter 1944 to 1945, matched controls, and their siblings. We conducted DNA methylation analysis of blood samples collected when the survivors were aged 58 to quantify biological aging using the DunedinPACE, GrimAge, and PhenoAge epigenetic clocks. Famine survivors had faster DunedinPACE, as compared with controls. This effect was strongest among women. Results were similar for GrimAge, although effect-sizes were smaller. We observed no differences in PhenoAge between survivors and controls. Famine effects were not accounted for by blood-cell composition and were similar for individuals exposed early and later in gestation. Findings suggest in-utero undernutrition may accelerate biological aging in later life.
Effect of long-term caloric restriction on telomere length in healthy adults: CALERIE™ 2 trial analysis

Waylon J. Hastings, Qiaofeng Ye, Sarah E. Wolf, and 11 more authors

Aging Cell, Jun 2024

_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/acel.14149

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Caloric restriction (CR) modifies lifespan and aging biology in animal models. The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE™) 2 trial tested translation of these findings to humans. CALERIE™ randomized healthy, nonobese men and premenopausal women (age 21–50y; BMI 22.0–27.9 kg/m2), to 25% CR or ad-libitum (AL) control (2:1) for 2 years. Prior analyses of CALERIE™ participants’ blood chemistries, immunology, and epigenetic data suggest the 2-year CR intervention slowed biological aging. Here, we extend these analyses to test effects of CR on telomere length (TL) attrition. TL was quantified in blood samples collected at baseline, 12-, and 24-months by quantitative PCR (absolute TL; aTL) and a published DNA-methylation algorithm (DNAmTL). Intent-to-treat analysis found no significant differences in TL attrition across the first year, although there were trends toward increased attrition in the CR group for both aTL and DNAmTL measurements. When accounting for adherence heterogeneity with an Effect-of-Treatment-on-the-Treated analysis, greater CR dose was associated with increased DNAmTL attrition during the baseline to 12-month weight-loss period. By contrast, both CR group status and increased CR were associated with reduced aTL attrition over the month 12 to month 24 weight maintenance period. No differences were observed when considering TL change across the study duration from baseline to 24-months, leaving it unclear whether CR-related effects reflect long-term detriments to telomere fidelity, a hormesis-like adaptation to decreased energy availability, or measurement error and insufficient statistical power. Unraveling these trends will be a focus of future CALERIE™ analyses and trials.
Pregnancy is linked to faster epigenetic aging in young women

Calen P. Ryan, Nanette R. Lee, Delia B. Carba, and 6 more authors

Proceedings of the National Academy of Sciences, Apr 2024

Publisher: Proceedings of the National Academy of Sciences

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A central prediction of evolutionary theory is that energy invested into reproduction comes at the expense of somatic maintenance and repair, accelerating biological aging. Supporting this prediction are findings that high fertility among women predicts shorter lifespan and poorer health later in life. However, biological aging is thought to begin before age-related health declines, limiting the applicability of morbidity and mortality for studying the aging process earlier in life. Here, we examine the relationship between reproductive history and biological aging in a sample of young (20 to 22yo) men and women from the Cebu Longitudinal Health and Nutrition Survey, located in the Philippines (n = 1,735). We quantify biological aging using six measures, collectively known as epigenetic clocks, reflecting various facets of cellular aging, health, and mortality risk. In a subset of women, we test whether longitudinal changes in gravidity between young and early–middle adulthood (25 to 31yo) are associated with changes in epigenetic aging during that time. Cross-sectionally, gravidity was associated with all six measures of accelerated epigenetic aging in women (n = 825). Furthermore, longitudinal increases in gravidity were linked to accelerated epigenetic aging in two epigenetic clocks (n = 331). In contrast, the number of pregnancies a man reported fathering was not associated with epigenetic aging among same-aged cohort men (n = 910). These effects were robust to socioecological, environmental, and immunological factors, consistent with the hypothesis that pregnancy accelerates biological aging and that these effects can be detected in young women in a high-fertility context.
A blood biomarker of the pace of aging is associated with brain structure: replication across three cohorts

Ethan T. Whitman, Calen P. Ryan, Wickliffe C. Abraham, and 17 more authors

Neurobiology of Aging, Apr 2024

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Biological aging is the correlated decline of multi-organ system integrity central to the etiology of many age-related diseases. A novel epigenetic measure of biological aging, DunedinPACE, is associated with cognitive dysfunction, incident dementia, and mortality. Here, we tested for associations between DunedinPACE and structural MRI phenotypes in three datasets spanning midlife to advanced age: the Dunedin Study (age=45 years), the Framingham Heart Study Offspring Cohort (mean age=63 years), and the Alzheimer’s Disease Neuroimaging Initiative (mean age=75 years). We also tested four additional epigenetic measures of aging: the Horvath clock, the Hannum clock, PhenoAge, and GrimAge. Across all datasets (total N observations=3380; total N individuals=2322), faster DunedinPACE was associated with lower total brain volume, lower hippocampal volume, greater burden of white matter microlesions, and thinner cortex. Across all measures, DunedinPACE and GrimAge had the strongest and most consistent associations with brain phenotypes. Our findings suggest that single timepoint measures of multi-organ decline such as DunedinPACE could be useful for gauging nervous system health.
Liver-derived plasminogen mediates muscle stem cell expansion during caloric restriction through the plasminogen receptor Plg-RKT

Akshay Bareja, David E. Lee, Tricia Ho, and 21 more authors

Cell Reports, Mar 2024

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An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.
DNA methylation-based estimators of telomere length show low correspondence with paternal age at conception and other measures of external validity of telomere length

Dan T. A. Eisenberg, Calen P. Ryan, Nanette R. Lee, and 6 more authors

GeroScience, Mar 2024

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In humans, DNA methylation (DNAm) based estimators of telomere length (TL) have been shown to better predict TL-associated variables (e.g., age, sex, and mortality) than TL itself. The biological significance of DNAm-based estimators of TL (DNAmTL) is unclear. In vitro DNAmTL shortens with cell replications, even when telomerase is maintaining TL. Telomerase is typically suppressed in humans, except in testes. Accordingly, sperm TL increases with age, and offspring with greater paternal age at conception (PAC) have longer TL. Thus, we expect that PAC associations with DNAmTL can shed light on whether in vivo cell replications in the presence of high telomerase activity (production of sperm) shorten DNAmTL or if PAC-lengthened TL causes lengthened DNAmTL. In a pre-registered analysis, using data from 1733 blood samples from the Philippines, we examined the association between paternal age at conception (PAC) and offspring DNAmTL. We did not find an association between PAC and DNAmTL but found a positive association of paternal grandfather’s age at father’s conception predicting grandchild’s DNAmTL. In post hoc analyses, we examined how DNAmTL versus qPCR-measured TL (qPCR-TL) correlated with measures typically associated with TL. Contrary to previous findings, on almost all measures of external validity (correlations with parental TLs, southern blot TL, and age), qPCR-TL outperformed DNAmTL. The “kilobase” units of DNAm-based estimators of TL showed considerable deviations from southern blot-derived kilobase measures. Our findings suggest that DNAmTL is not a reliable index of inherited aspects of TL and underscores uncertainty about the biological meaning of DNAmTL.
Educational mobility, pace of aging, and lifespan among participants in the Framingham Heart Study

Gloria H. J. Graf, Allison E. Aiello, Avshalom Caspi, and 7 more authors

JAMA Network Open, Mar 2024

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People who complete more education live longer lives with better health. New evidence suggests that these benefits operate through a slowed pace of biological aging. If so, measurements of the pace of biological aging could offer intermediate end points for studies of how interventions to promote education will affect healthy longevity.To test the hypothesis that upward educational mobility is associated with a slower pace of biological aging and increased longevity.This prospective cohort study analyzed data from 3 generations of participants in the Framingham Heart Study: (1) the original cohort, enrolled beginning in 1948; (2) the Offspring cohort, enrolled beginning in 1971; and (3) the Gen3 cohort, enrolled beginning in 2002. A 3-generation database was constructed to quantify intergenerational educational mobility. Mobility data were linked with blood DNA-methylation data collected from the Offspring cohort in 2005 to 2008 (n = 1652) and the Gen3 cohort in 2009 to 2011 (n = 1449). Follow-up is ongoing. Data analysis was conducted from June 2022 to November 2023 using data obtained from the National Institutes of Health database of Genotypes and Phenotypes (dbGaP).Educational mobility was measured by comparing participants’ educational outcomes with those of their parents.The pace of biological aging was measured from whole-blood DNA-methylation data using the DunedinPACE epigenetic clock. For comparison purposes, the analysis was repeated using 4 other epigenetic clocks. Survival follow-up was conducted through 2019.This study analyzed data from 3101 participants from the Framingham Heart Study; 1652 were in the Offspring cohort (mean [SD] age, 65.57 [9.22] years; 764 [46.2%] male) and 1449 were in the Gen3 cohort (mean [SD] age, 45.38 [7.83] years; 691 [47.7%] male). Participants who were upwardly mobile in educational terms tended to have slower pace of aging in later life (r = −0.18 [95% CI, −0.23 to −0.13]; P < .001). This pattern of association was similar across generations and held in within-family sibling comparisons. There were 402 Offspring cohort participants who died over the follow-up period. Upward educational mobility was associated with lower mortality risk (hazard ratio, 0.89 [95% CI, 0.81 to 0.98]; P = .01). Slower pace of aging accounted for approximately half of this association.This cohort study’s findings support the hypothesis that interventions to promote educational attainment may slow the pace of biological aging and promote longevity. Epigenetic clocks have potential as near-term outcome measures of intervention effects on healthy aging. Experimental evidence is needed to confirm findings.
Epigenetic clock work ticks forward

Calen P. Ryan, and Daniel W. Belsky

Nature Aging, Jan 2024

Publisher: Nature Publishing Group

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Ying et al. integrate Mendelian randomization into epigenetic clock making and pioneer a strategy to develop aging biomarkers with stronger causal ties to healthspan. They distinguish signs of aging-related molecular damage from responses to it that might signal resilience.
Diet, Pace of Biological Aging, and Risk of Dementia in the Framingham Heart Study

Aline Thomas, Calen P. Ryan, Avshalom Caspi, and 6 more authors

Annals of Neurology, Feb 2024

_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1002/ana.26900

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Objective People who eat healthier diets are less likely to develop dementia, but the biological mechanism of this protection is not well understood. We tested the hypothesis that healthy diet protects against dementia because it slows the pace of biological aging. Methods We analyzed Framingham Offspring Cohort data. We included participants ≥60 years-old, free of dementia and having dietary, epigenetic, and follow-up data. We assessed healthy diet as long-term adherence to the Mediterranean-Dash Intervention for Neurodegenerative Delay diet (MIND, over 4 visits spanning 1991–2008). We measured the pace of aging from blood DNA methylation data collected in 2005–2008 using the DunedinPACE epigenetic clock. Incident dementia and mortality were defined using study records compiled from 2005 to 2008 visit through 2018. Results Of n = 1,644 included participants (mean age 69.6, 54% female), n = 140 developed dementia and n = 471 died over 14 years of follow-up. Greater MIND score was associated with slower DunedinPACE and reduced risks for dementia and mortality. Slower DunedinPACE was associated with reduced risks for dementia and mortality. In mediation analysis, slower DunedinPACE accounted for 27% of the diet-dementia association and 57% of the diet-mortality association. Interpretation Findings suggest that slower pace of aging mediates part of the relationship of healthy diet with reduced dementia risk. Monitoring pace of aging may inform dementia prevention. However, a large fraction of the diet-dementia association remains unexplained and may reflect direct connections between diet and brain aging that do not overlap other organ systems. Investigation of brain-specific mechanisms in well-designed mediation studies is warranted. ANN NEUROL 2024

2023

Effect of long-term caloric restriction on DNA methylation measures of biological aging in healthy adults from the CALERIE trial

R. Waziry, Calen P. Ryan, D. L. Corcoran, and 19 more authors

Nature Aging, Feb 2023

Publisher: Nature Publishing Group

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The geroscience hypothesis proposes that therapy to slow or reverse molecular changes that occur with aging can delay or prevent multiple chronic diseases and extend healthy lifespan1–3. Caloric restriction (CR), defined as lessening caloric intake without depriving essential nutrients4, results in changes in molecular processes that have been associated with aging, including DNA methylation (DNAm)5–7, and is established to increase healthy lifespan in multiple species8,9. Here we report the results of a post hoc analysis of the influence of CR on DNAm measures of aging in blood samples from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, a randomized controlled trial in which n = 220 adults without obesity were randomized to 25% CR or ad libitum control diet for 2 yr (ref. 10). We found that CALERIE intervention slowed the pace of aging, as measured by the DunedinPACE DNAm algorithm, but did not lead to significant changes in biological age estimates measured by various DNAm clocks including PhenoAge and GrimAge. Treatment effect sizes were small. Nevertheless, modest slowing of the pace of aging can have profound effects on population health11–13. The finding that CR modified DunedinPACE in a randomized controlled trial supports the geroscience hypothesis, building on evidence from small and uncontrolled studies14–16 and contrasting with reports that biological aging may not be modifiable17. Ultimately, a conclusive test of the geroscience hypothesis will require trials with long-term follow-up to establish effects of intervention on primary healthy-aging endpoints, including incidence of chronic disease and mortality18–20.
Association between infectious exposures in infancy and epigenetic age acceleration in young adulthood in metropolitan Cebu, Philippines

Thomas W. McDade, Calen P. Ryan, Linda S. Adair, and 7 more authors

American Journal of Human Biology, Nov 2023

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Objectives: The drivers of human life expectancy gains over the past 200 years are not well-established, with a potential role for historical reductions in infectious disease. We investigate whether infectious exposures in infancy predict biological aging using DNA methylation-based markers that forecast patterns of morbidity and mortality later in life.
Reply to Nwanaji-Enwerema, 2023, “Exposome and low birthweight selection bias considerations in early life infections and epigenetic age relationships”

Thomas W. McDade, Calen P. Ryan, Linda S. Adair, and 7 more authors

American Journal of Human Biology, Nov 2023

_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1002/ajhb.23977

2022

Immune cell type and DNA methylation vary with reproductive status in women: possible pathways for costs of reproduction

Calen P. Ryan, Meaghan J Jones, Rachel D Edgar, and 4 more authors

Evolution, Medicine, and Public Health, Jan 2022

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Background: Consistent with evolutionarily theorized costs of reproduction (CoR), reproductive history in women is associated with life expectancy and susceptibility to certain cancers, autoimmune disor- ders and metabolic disease. Immunological changes originating during reproduction may help explain some of these relationships. Methodology: To explore the potential role of the immune system in female CoR, we characterized leukocyte composition and regulatory processes using DNA methylation (DNAm) in a cross-sectional cohort of young (20–22 years old) women differing in reproductive status. Results: Compared to nulliparity, pregnancy was characterized by differential methylation at 828 sites, 96}% of which were hypomethylated and enriched for genes associated with T-cell activation, innate im- munity, pre-eclampsia and neoplasia. Breastfeeding was associated with differential methylation at 1107 sites (71}% hypermethylated), enriched for genes involved in metabolism, immune self- recognition and neurogenesis. There were no significant differences in DNAm between nulliparous and parous women. However, compared to nullipara, pregnant women had lower proportions of B, CD4T, CD8T and natural killer (NK) cells, and higher proportions of granulocytes and monocytes. Monocyte counts were lower and NK counts higher among breastfeeding women, and remained so among parous women. Implications: Our findings point to widespread differences in DNAm during pregnancy and lactation. These effects appear largely tran- sient, but may accumulate with gravidity become detectable as women age. Nulliparous and parous women differed in leukocyte com- position, consistent with more persistent effects of reproduction on cell type. These findings support transient (leukocyte DNAm) and persistent (cell composition) changes associated with reproduction in women, illuminating potential pathways contributing to CoR.
Birth weight and maternal energy status during pregnancy as predictors of epigenetic age acceleration in young adults from metropolitan Cebu, Philippines

Christopher W. Kuzawa, Calen P. Ryan, Linda S. Adair, and 7 more authors

Epigenetics, May 2022

Publisher: Taylor & Francis _eprint: https://doi.org/10.1080/15592294.2022.2070105

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Epigenetic clocks quantify regular changes in DNA methylation that occur with age, or in relation to biomarkers of ageing, and are strong predictors of morbidity and mortality. Here, we assess whether measures of fetal nutrition and growth that predict adult chronic disease also predict accelerated biological ageing in young adulthood using a suite of commonly used epigenetic clocks. Data come from the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a long-running cohort followed since birth in metropolitan Cebu, Philippines. Past work has shown that birth weight (BW) and the mother’s arm fat during pregnancy (a measure of pregnancy energy status) relate inversely to health outcomes in the CLHNS but primarily in males. Genome-wide DNA methylation was assessed in whole blood using the Infinium EPIC array. Participants included males (n=895) and females (n=803) measured in 2005 (20.8–22.5 years). Clocks included the Hannum and Horvath clocks trained on chronological age, the DNAmPhenoAge and DNAmGrimAge clocks trained on clinical biomarkers, the Dunedin pace of ageing (DunedinPACE) clock trained on longitudinal changes in ageing biomarkers, and the DNAmTL clock trained on leukocyte telomere length. In males, lower BW predicted advanced biological ageing using the Hannum, DNAmPhenoAge, DunedinPoAm, and DNAmTL clocks. In contrast, BW did not predict any clock in female participants. Participants’ mothers’ pregnancy arm fat only predicted DNAmTL in males. These findings suggest that epigenetic clocks are a useful tool for gauging long-term outcomes predicted by fetal growth, and add to existing evidence in the CLHNS for sex differences in these relationships.
Climatic and biotic influences on the distributions of Calliphora augur and Calliphora dubia (Diptera: Calliphoridae)

Aidan P Johnson, Calen P Ryan, and James F Wallman

Austral Entomology, May 2022

_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1111/aen.12617

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Calliphora augur (Fabricius) and Calliphora dubia (Macquart) are two widespread and endemic Australian blowflies of applied importance. In order to better understand the biology of these species, this study used historical, field and laboratory data to determine and interpret their distributions. Locality records from insect collections and the literature were used to determine the known distributions of each species. The resultant maps were partially verified with field trapping data for each species. Calliphora augur was found to dominate in the east of Australia, while C. dubia dominates in the west. Comparative laboratory and field experiments were also used to derive parameters defining the distributions of these two closely related species, particularly in relation to temperature and moisture. Calliphora dubia had a greater tolerance for dry stress and high temperatures than C. augur, although maggots of both species displayed similar developmental temperature preferences. No difference was seen between species in the impact of low temperatures on the development of maggots through to the pupal stage. The greater ability of C. dubia to tolerate adverse environmental conditions is most likely linked to the shared evolutionary history of both species, in which increased aridity in central Australia is proposed to have triggered speciation by separating ancestral populations: C. dubia evolved in the hot, dry conditions in the west of Australia, while C. augur evolved in the cooler, moist environment of the east. Improved understanding of the influences on the distributions of these common Australian blowflies will assist in the further study of their application to agriculture and forensic science.
Maternal epigenetic clocks measured during pregnancy do not predict gestational age at delivery or offspring birth outcomes: a replication study in metropolitan Cebu, Philippines

Calen P. Ryan, Raviraj J. Rege, Nanette R. Lee, and 6 more authors

Clinical Epigenetics, Dec 2022

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Adverse birth outcomes, such as early gestational age and low birth weight, can have lasting effects on morbidity and mortality, with impacts that persist into adulthood. Identifying the maternal factors that contribute to adverse birth outcomes in the next generation is thus a priority. Epigenetic clocks, which have emerged as powerful tools for quantifying biological aging and various dimensions of physiological dysregulation, hold promise for clarifying relationships between maternal biology and infant health, including the maternal factors or states that predict birth outcomes. Nevertheless, studies exploring the relationship between maternal epigenetic age and birth outcomes remain few. Here, we attempt to replicate a series of analyses previously reported in a US-based sample, using a larger similarly aged sample (n = 296) of participants of a long-running study in the Philippines. New pregnancies were identified prospectively, dried blood spot samples were collected during the third trimester, and information was obtained on gestational age at delivery and offspring weight after birth. Genome-wide DNA methylation was assessed with the Infinium EPIC array. Using a suite of 15 epigenetic clocks, we only found one significant relationship: advanced age on the epigenetic clock trained on leptin predicted a significantly earlier gestational age at delivery (β = − 0.15, p = 0.009). Of the other 29 relationships tested predicting gestational age and offspring birth weight, none were statistically significant. In this sample of Filipino women, epigenetic clocks capturing multiple dimensions of biology and health do not predict birth outcomes in offspring.

2021

The temporary cost of dominance

Calen P. Ryan, and Christopher W Kuzawa

eLife, Apr 2021

Publisher: eLife Sciences Publications, Ltd

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In a population of wild baboons, a new way to assess biological age reveals a surprising effect of social hierarchy.

2020

Germline epigenetic inheritance: Challenges and opportunities for linking human paternal experience with offspring biology and health

Calen P. Ryan, and Christopher W. Kuzawa

Evolutionary Anthropology: Issues, News, and Reviews, Apr 2020

_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1002/evan.21828

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Recently, novel experimental approaches and molecular techniques have demonstrated that a male’s experiences can be transmitted through his germline via epigenetic processes. These findings suggest that paternal exposures influence phenotypic variation in unexposed progeny–a proposal that runs counter to canonical ideas about inheritance developed during the 20th century. Nevertheless, support for paternal germline epigenetic inheritance (GEI) in nonhuman mammals continues to grow and the mechanisms underlying this phenomenon are becoming clearer. To what extent do similar processes operate in humans, and if so, what are their implications for understanding human phenotypic variation, health, and evolution? Here, we review evidence for GEI in human and nonhuman mammals and evaluate these findings in relation to historical conceptions of heredity. Drawing on epidemiological data, reproductive biology, and molecular embryology, we outline developments and opportunities for the study of GEI in human populations, emphasizing the challenges that researchers in this area still face.
Evolutionary life history theory as an organising framework for cohort studies: insights from the Cebu Longitudinal Health and Nutrition Survey

Christopher W. Kuzawa, Linda Adair, Sonny A. Bechayda, and 12 more authors

Annals of Human Biology, Feb 2020

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By tracking a group of individuals through time, cohort studies provide fundamental insights into the developmental time course and causes of health and disease. Evolutionary life history theory seeks to explain patterns of growth, development, reproduction and senescence, and inspires a range of hypotheses that are testable using the longitudinal data from cohort studies. Here we review two decades of life history theory-motivated work conducted in collaboration with the Cebu Longitudinal Health and Nutrition Survey (CLHNS), a birth cohort study that enrolled more than 3000 pregnant women in the Philippines in 1983 and has since followed these women, their offspring and grandoffspring. This work has provided evidence that reproduction carries “costs” to cellular maintenance functions, potentially speeding senescence, and revealed an unusual form of genetic plasticity in which the length of telomeres inherited across generations is influenced by reproductive timing in paternal ancestors. Men in Cebu experience hormonal and behavioural changes in conjunction with changes in relationship and fatherhood status that are consistent with predictions based upon other species that practice bi-parental care. The theoretical expectation that early life cues of mortality or environmental unpredictability will motivate a “fast” life history strategy are confirmed for behavioural components of reproductive decision making, but not for maturational tempo, while our work points to a broader capacity for early life developmental calibration of systems like immunity, reproductive biology and metabolism. Our CLHNS findings illustrate the power of life history theory as an integrative, lifecourse framework to guide longitudinal studies of human populations.
“Epigenetic clocks”: Theory and applications in human biology

Calen P. Ryan

American Journal of Human Biology, Feb 2020

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All humans age, but how we age—and how fast—differs considerably from person to person. This deviation between apparent age and chronological age is often referred to as “biological age” (BA) and until recently robust tools for studying BA have been scarce. “Epigenetic clocks” are starting to change this. Epigenetic clocks use predictable changes in the epigenome, usually DNA methylation, to estimate chronological age with unprecedented accuracy. More importantly, deviations between epigenetic age and chronological age predict a broad range of health outcomes and mortality risks better than chronological age alone. Thus, epigenetic clocks appear to capture fundamental molecular processes tied to BA and can serve as powerful tools for studying health, development, and aging across the lifespan. In this article, I review epigenetic clocks, especially as they relate to key theoretical and applied issues in human biology. I first provide an overview of how epigenetic clocks are constructed and what we know about them. I then discuss emerging applications of particular relevance to human biologists—those related to reproduction, life-history, stress, and the environment. I conclude with an overview of the methods necessary for implementing epigenetic clocks, including considerations of study design, sample collection, and technical considerations for processing and interpreting epigenetic clocks. The goal of this review is to highlight some of the ways that epigenetic clocks can inform questions in human biology, and vice versa, and to provide human biologists with the foundational knowledge necessary to successfully incorporate epigenetic clocks into their research.
Parity predicts biological age acceleration in post-menopausal, but not pre-menopausal, women

Talia N. Shirazi, Waylon J. Hastings, Asher Y. Rosinger, and 1 more author

Scientific Reports, Nov 2020

Number: 1 Publisher: Nature Publishing Group

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Understanding factors contributing to variation in ‘biological age’ is essential to understanding variation in susceptibility to disease and functional decline. One factor that could accelerate biological aging in women is reproduction. Pregnancy is characterized by extensive, energetically-costly changes across numerous physiological systems. These ‘costs of reproduction’ may accumulate with each pregnancy, accelerating biological aging. Despite evidence for costs of reproduction using molecular and demographic measures, it is unknown whether parity is linked to commonly-used clinical measures of biological aging. We use data collected between 1999 and 2010 from the National Health and Nutrition Examination Survey (n = 4418) to test whether parity (number of live births) predicted four previously-validated composite measures of biological age and system integrity: Levine Method, homeostatic dysregulation, Klemera–Doubal method biological age, and allostatic load. Parity exhibited a U-shaped relationship with accelerated biological aging when controlling for chronological age, lifestyle, health-related, and demographic factors in post-menopausal, but not pre-menopausal, women, with biological age acceleration being lowest among post-menopausal women reporting between three and four live births. Our findings suggest a link between reproductive function and physiological dysregulation, and allude to possible compensatory mechanisms that buffer the effects of reproductive function on physiological dysregulation during a woman’s reproductive lifespan. Future work should continue to investigate links between parity, menopausal status, and biological age using targeted physiological measures and longitudinal studies.

2019

Tandem repeat disorders

Calen P. Ryan

Evolution, Medicine, and Public Health, Jan 2019

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Tandem repeat disorders (TRDs) are a family of neuropathological disorders linked to the accumulation of short-tandem repeats (STRs; repeating DNA sequences 2–6
Genome-wide analysis of DNA methylation in relation to socioeconomic status during development and early adulthood

Thomas W. McDade, Calen P. Ryan, Meaghan J. Jones, and 5 more authors

American Journal of Physical Anthropology, Feb 2019

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Objectives: Socioeconomic status (SES) is a powerful determinant of health, but the underlying biological mechanisms are poorly understood. This study investigates whether levels of DNA methylation at CpG sites across the genome are associated with SES in a cohort of young adults in the Philippines. Methods: DNA methylation was assayed with the Illumina HumanMethylation450 Bead Chip, in leukocytes from 489 participants in the Cebu Longitudinal Health and Nutrition Survey (mean age = 20.9 years). SES was measured in infancy/childhood and adulthood, and was based on composite measures of income, assets, and education. Genome-wide analysis of variable probes identified CpG sites significantly associated with SES after adjustment for multiple comparisons. Functional enrichment analysis was used to identify biological pathways associated with these sites. Results: A total of 2,546 CpG sites, across 1,537 annotated genes, were differentially methylated in association with SES. In comparison with high SES, low SES was associated with increased methylation at 1,777 sites, and decreased methylation at 769 sites. Functional enrichment analysis identified over-representation of biological pathways related to immune function, skeletal development, and development of the nervous system. Conclusions: Socioeconomic status predicts DNA methylation at a large number of CpG sites across the genome. The scope of these associations is commensurate with the wide range of biological systems and health outcomes that are shaped by SES, and these findings suggest that DNA methylation may play an important role.

2018

Reproduction predicts shorter telomeres and epigenetic age acceleration among young adult women

Calen P. Ryan, M. Geoffrey Hayes, Nanette R. Lee, and 5 more authors

Scientific Reports, Dec 2018

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Evolutionary theory predicts that reproduction entails costs that detract from somatic maintenance, accelerating biological aging. Despite support from studies in human and non-human animals, mechanisms linking ‘costs of reproduction’ (CoR) to aging are poorly understood. Human pregnancy is characterized by major alterations in metabolic regulation, oxidative stress, and immune cell proliferation. We hypothesized that these adaptations could accelerate blood-derived cellular aging. To test this hypothesis, we examined gravidity in relation to telomere length (TL, n = 821) and DNA- methylation age (DNAmAge, n = 397) in a cohort of young (20–22 year-old) Filipino women. Age- corrected TL and accelerated DNAmAge both predict age-related morbidity and mortality, and provide markers of mitotic and non-mitotic cellular aging, respectively. Consistent with theoretical predictions, TL decreased (p = 0.031) and DNAmAge increased (p = 0.007) with gravidity, a relationship that was not contingent upon resource availability. Neither biomarker was associated with subsequent fertility (both p \textgreater 0.3), broadly consistent with a causal effect of gravidity on cellular aging. Our findings provide evidence that reproduction in women carries costs in the form of accelerated aging through two independent cellular pathways.

2017

The role of testosterone in coordinating male life history strategies: The moderating effects of the androgen receptor CAG repeat polymorphism

Lee T. Gettler, Calen P. Ryan, Dan T. A. Eisenberg, and 5 more authors

Hormones and Behavior, Jan 2017

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Partnered fathers often have lower testosterone than single non-parents, which is theorized to relate to elevated testosterone (T) facilitating competitive behaviors and lower T contributing to nurturing. Cultural- and individual-factors moderate the expression of such psychobiological profiles. Less is known about genetic variation’s role in individual psychobiological responses to partnering and fathering, particularly as related to T. We examined the exon 1 CAG (polyglutamine) repeat (CAGn) within the androgen receptor (AR) gene. AR CAGn shapes T’s effects after it binds to AR by affecting AR transcriptional activity. Thus, this polymorphism is a strong candidate to influence individual-level profiles of “androgenicity.” While males with a highly androgenic profile are expected to engage in a more competitive-oriented life history strategy, low androgenic men are at increased risk of depression, which could lead to similar outcomes for certain familial dynamics, such as marriage stability and parenting. Here, in a large longitudinal study of Filipino men (n = 683), we found that men who had high androgenicity (elevated T and shorter CAGn) or low androgenicity (lower T and longer CAGn) showed elevated likelihood of relationship instability over the 4.5-year study period and were also more likely be relatively uninvolved with childcare as fathers. We did not find that CAGn moderated men’s T responses to the fatherhood transition. In total, our results provide evidence for invested fathering and relationship stability at intermediate levels of androgenicity and help inform our understanding of variation in male reproductive strategies and the individual hormonal and genetic differences that underlie it.
Androgen receptor CAG repeat polymorphism and hypothalamic-pituitary-gonadal function in Filipino young adult males

Calen P. Ryan, Thomas W. McDade, Lee T. Gettler, and 4 more authors

American Journal of Human Biology, Jan 2017

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Objectives Testosterone (T), the primary androgenic hormone in males, is stimulated through pulsatile secretion of LH and regulated through negative feedback inhibition at the hypothalamus and pituitary. The hypothalamic-pituitary-gonadal (HPG) axis also controls sperm production through the secretion of follicle-stimulating hormone (FSH). Negative feedback in the HPG axis is achieved in part through the binding of T to the androgen receptor (AR), which contains a highly variable trinucleotide repeat polymorphism (AR-CAGn). The number of repeats in the AR-CAGn inversely correlates with transcriptional activity of the AR. Thus, we predicted longer AR-CAGn to be associated with higher T, LH, and FSH levels. Methods We examined the relationship between AR-CAGn and total plasma T, LH, and FSH, as well as “bioavailable” morning (AM-T) and evening (PM-T) testosterone in 722 young (21.5 ± 0.5 years) Filipino males. Results There was no relationship between AR-CAGn and total T, AM-T, or LH (P \textgreater .25 for all). We did observe a marginally non-significant (P = .066) correlation between AR-CAGn and PM-T in the predicted direction, and a negative correlation between AR-CAGn and FSH (P = .005). Conclusions Our results both support and differ from previous findings in this area, and study parameters that differ between our study and others, such as participant age, sample time, and the role of other hormones should be considered when interpreting our findings. While our data point to a modest effect of AR-CAGn on HPG regulation at best, the AR-CAGn may still affect somatic traits by regulating androgenic activity at peripheral tissues.
Androgen receptor polyglutamine repeat length (AR-CAGn) modulates the effect of testosterone on androgen-associated somatic traits in Filipino young adult men

Calen P. Ryan, Alexander V. Georgiev, Thomas W. McDade, and 6 more authors

American Journal of Physical Anthropology, Mar 2017

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Objectives The androgen receptor (AR) mediates expression of androgen-associated somatic traits such as muscle mass and strength. Within the human AR is a highly variable glutamine short-tandem repeat (AR-CAGn), and CAG repeat number has been inversely correlated to AR transcriptional activity in vitro. However, evidence for an attenuating effect of long AR-CAGn on androgen-associated somatic traits has been inconsistent in human populations. One possible explanation for this lack of consistency is that the effect of AR-CAGn on AR bioactivity in target tissues likely varies in relation to circulating androgen levels. Materials and Methods We tested whether relationships between AR-CAGn and several androgen-associated somatic traits (waist circumference, lean mass, arm muscle area, and grip strength) were modified by salivary (waking and pre-bed) and circulating (total) testosterone (T) levels in young adult males living in metropolitan Cebu, Philippines (n = 675). Results When men’s waking T was low, they had a reduction in three out of four androgen-associated somatic traits with lengthening AR-CAGn (p \textless .1), consistent with in vitro research. However, when waking T was high, we observed the opposite effect—lengthening AR-CAGn was associated with an increase in these same somatic traits. Discussion Our finding that longer AR-CAGn predicts greater androgen-associated trait expression among high-T men runs counter to in vitro work, but is generally consistent with the few prior studies to evaluate similar interactions in human populations. Collectively, these results raise questions about the applicability of findings derived from in vitro AR-CAGn studies to the receptor’s role in maintaining androgen-associated somatic traits in human populations.
Second-to-fourth digit ratio (2D:4D) is unrelated to measures of somatic reproductive effort among young men from Cebu, the Philippines: GEORGIEV ET AL.

Alexander V. Georgiev, Calen P. Ryan, Lee T. Gettler, and 2 more authors

American Journal of Physical Anthropology, Mar 2017

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Objectives: A low second-to-fourth (2D:4D) digit ratio, a retrospective marker of high prenatal androgens, predicts increased investment in costly sexually dimorphic traits in men in some stud- ies, although results are mixed. Here we test the hypothesis that the association of low 2D:4D ratios with increased muscularity and decreased adiposity depends on current testosterone (T) lev- els, such that digit ratio will be a particularly strong predictor of outcomes among men exhibiting a mating-effort-oriented endocrinological profile (high T). We also test the association between 2D:4D and somatic traits independently of T. Materials and methods: We related 2D:4D digit ratios, and their interaction with T, to handgrip strength, lean mass, arm muscle area, and skinfold thickness in a sample of young, childess men (20–22 y) from Cebu, Philippines (N 5 623). Results: Digit ratio did not significantly predict men’s T-dependent somatic traits. Interactions between 2D:4D and morning T, similarly, did not predict male muscularity or adiposity. Although two of the interactions were significant or marginally significant (p \textless .1), after adjusting for multiple testing the evidence in support of our hypothesis was weak. Discussion: We found no evidence that 2D:4D predicted measures of somatic reproductive effort in this sample of young men from Cebu, who as a group could be considered mostly mating- oriented. These relationships were also not contingent upon, or stronger, when considering the moderating effect of concurrent T levels. In this sample, 2D:4D was therefore either a poor proxy of prenatal androgen exposure or prenatal androgens had limited influence on adult somatic outcomes.
Like mother, like daughter: heritability of female Richardson’s ground squirrel Urocitellus richardsonii cortisol stress responses

Kevin R. Bairos-Novak, Calen P. Ryan, Angela R. Freeman, and 2 more authors

Current Zoology, Mar 2017

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Activation of the hypothalamic–pituitary–adrenal (HPA) axis liberates glucocorticoids, which pro- vides an acute indication of an individual’s response to stressors. The heritability of the stress re- sponse in wild mammals, however, remains poorly documented. We quantified the cortisol stress response of female Richardson’s ground squirrels (RGSs) to handling and physical restraint, test- ing for: (1) the effects of individual age, time of day, and sample latency; (2) repeatability within in- dividuals; (3) narrow-sense heritability; and (4) differences among individuals owing to potential genetic and/or environmental effects. We detected a positive linear relationship between baseline plasma cortisol (BL-cortisol) concentration and stress-induced plasma cortisol (SI-cortisol) concen- tration that defined each individual’s cortisol stress response. BL-cortisol, SI-cortisol, and stress re- sponse did not differ according to the time the sample was taken, or by subject age. Cortisol stress response was highly repeatable within individuals, had a mother–offspring heritability of h2 1⁄4 0.40 6 0.24 (mean 6 SE), full-sibling heritability of h2FS 1⁄4 0:37 6 0:71, and half-sibling heritabil- ity of h2HS 1⁄4 0:75 6 1:41. Stress responses of sibling groups, immediate-family groups, and squir- rels within a given area did not differ, whereas those of individuals from more distantly related matrilines did. Our results highlight the natural variability in HPA axis reactivity among individuals by quantifying both BL- and SI-cortisol levels, demonstrate partial heritability of the stress re- sponse that is not attributable to environmental variation, and suggest that at least part of an indi- vidual’s stress response can be accounted for by differences in matrilineal history.
Social and physical environments early in development predict DNA methylation of inflammatory genes in young adulthood

Thomas W. McDade, Calen P. Ryan, Meaghan J. Jones, and 7 more authors

Proceedings of the National Academy of Sciences, Jul 2017

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Chronic inflammation contributes to a wide range of human diseases, and environments in infancy and childhood are important determinants of inflammatory phenotypes. The underlying biological mechanisms connecting early environments with the regulation of inflammation in adulthood are not known, but epigenetic processes are plausible candidates. We tested the hypothesis that patterns of DNA methylation (DNAm) in inflammatory genes in young adulthood would be predicted by early life nutritional, microbial, and psychosocial exposures previously associated with levels of inflammation. Data come from a population-based longitudinal birth cohort study in metropolitan Cebu, the Philippines, and DNAm was characterized in whole blood samples from 494 participants (age 20–22 y). Analyses focused on probes in 114 target genes involved in the regulation of inflammation, and we identified 10 sites across nine genes where the level of DNAm was significantly predicted by the following variables: household socioeconomic status in childhood, extended absence of a parent in childhood, exposure to animal feces in infancy, birth in the dry season, or duration of exclusive breastfeeding. To evaluate the biological significance of these sites, we tested for associations with a panel of inflammatory biomarkers measured in plasma obtained at the same age as DNAm assessment. Three sites predicted elevated inflammation, and one site predicted lower inflammation, consistent with the interpretation that levels of DNAm at these sites are functionally relevant. This pattern of results points toward DNAm as a potentially important biological mechanism through which developmental environments shape inflammatory phenotypes across the life course.
Is early postnatal growth velocity, a proxy of minipubertal androgen action, related to adult second-to-fourth digit (2D:4D) ratios in men? A test in Cebu, Philippines

Alexander V. Georgiev, Calen P. Ryan, Judith B. Borja, and 1 more author

American Journal of Human Biology, Jul 2017

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Objectives: The ratio of the length of the second to the fourth digit (2D:4D) of the hand is often used as an index of prenatal androgen exposure but it might also be affected by androgens during “minipuberty,” a period of temporarily high testosterone (T) production in the first few months of life. To examine this, we tested the prediction that men with lower 2D:4D ratios had greater weight growth velocities during the first months of life—a metric recently shown to correlate with concurrent T levels. Methods: We related early growth data to 2D:4D ratios of both hands measured in 756 men (25–26 years) from Cebu, The Philippines. Results: Birth-to-fourth-month (B4M) weight gain velocity (a proxy of early postnatal androgen action) was not associated with adult 2D:4D ratios of either hand, when the latter was measured continuously. When comparing men with more male-typical digit ratios (\textless1.0) to those with more female-typical ratios (≥ 1.0), the group of men with more male-typical ratios had greater B4M weight velocity, but this was only the case for the left hand. Conclusions: We found modest evidence that adult digit ratios relate to an anthropometric correlate of androgen exposure during minipuberty. Definitive assessment of the role of postnatal T in shaping digit ratios will require direct measures of perinatal T related to longitudinally assessed digit ratios.

2016

Hsp90 and physiological stress are linked to autonomous transposon mobility and heritable genetic change in nematodes

Calen P. Ryan, Jeremy C. Brownlie, and Steve Whyard

Genome Biology and Evolution, Jul 2016

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Transposable elements (TEs) have been recognized as potentially powerful drivers of genomic evolutionary change, but factors affecting their mobility and regulation remain poorly understood. Chaperones such as Hsp90 buffer environmental perturbations by regulating protein conformation, but are also part of the PIWI-interacting RNA pathway, which regulates genomic instability arising from mobile TEs in the germline. Stress-induced mutagenesis from TE movement could thus arise from functional trade-offs in the dual roles of Hsp90. We examined the functional constraints of Hsp90 and its role as a regulator of TE mobility by exposing nematodes (Caenorhabditis elegans and Caenorhabditis briggsae) to environmental stress, with and without RNAi-induced silencing of Hsp90. TE excision frequency increased with environmental stress inten- sity at multiple loci in several strains of each species. These effects were compounded by RNAi-induced knockdown of Hsp90. Mutation frequencies at the unc-22 marker gene in the offspring of animals exposed to environmental stress and Hsp90 RNAi mirrored excision frequency in response to these treatments. Our results support a role for Hsp90 in the suppression of TE mobility, and demonstrate that that the Hsp90 regulatory pathway can be overwhelmed with moderate environmental stress. By compromising genomic stability in germline cells, environmentally induced mutations arising from TE mobility and insertion can have permanent and heritable effects on both the phenotype and the genotype of subsequent generations.

2015

Uncoupling clutch size, prolactin, and luteinizing hormone using experimental egg removal

Calen P. Ryan, Alistair Dawson, Peter J. Sharp, and 1 more author

General and Comparative Endocrinology, Mar 2015

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Clutch size is a key avian fitness and life history trait. A physiological model for clutch size determination (CSD), involving an anti-gonadal effect of prolactin (PRL) via suppression of luteinizing hormone (LH), was proposed over 20 years ago, but has received scant experimental attention since. The few studies looking at a PRL-based mechanistic hypothesis for CSD have been equivocal, but recent experiments utilizing a pharmacological agent to manipulate PRL in the zebra finch (Taeniopygia guttata) found no support for a role of this hormone in clutch size determination. Here, we take a complementary approach by manipulating clutch size through egg removal, examining co-variation in PRL and LH between two breeding attempts, as well as through experimentally-extended laying. Clutch size increased for egg removal females, but not controls, but this was not correlated with changes in PRL or LH. There were also no differences in PRL between egg removal females and controls, nor did PRL levels during early, mid- or late-laying of supra-normal clutches predict clutch size. By uncoupling PRL, LH and clutch size in our study, several key predictions of the PRL-based mechanistic model for CSD were not supported. However, a positive correlation between PRL levels late in laying and days relative to the last egg (clutch completion) provides an alternative explanation for the equivocal results surrounding the conventional PRL-based physiological model for CSD. We suggest that females coordinate PRL-mediated incubation onset with clutch completion to minimize hatching asynchrony and sibling hierarchy, a behavior that is amplified in females laying larger clutches.

2014

Shyness–Boldness, but not Exploration, Predicts Glucocorticoid Stress Response in Richardson’s Ground Squirrels (Urocitellus richardsonii)

Dawson Clary, Lindsay J. Skyner, Calen P. Ryan, and 3 more authors

Ethology, Nov 2014

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The relationship between stress and personality has often been studied using captive animals in a laboratory context, yet less often in wild populations. Wild populations, however, may reveal aspects of the personality–stress relationship that laboratory-based studies cannot. Here, we assessed the personality and stress hormone response of adult females within a free-living population of Richardson’s ground squirrels (Urocitellus richardsonii). Personality was assessed by quantifying individual responses to a novel object, and physiological stress was measured from faecal glucocorticoid metabolites. Principal component and principal component regression analyses were performed to determine whether the behavioural and endocrine measures were related. Based on these analyses, shyness–boldness was found to best predict glucocorticoid levels, in that individuals expressing the greatest vigilance in response to the novel object also had the highest measured concentrations of faecal glucocorticoids. Exploration, however, was independent of measured glucocorticoid levels, consistent with a multidimensional interpretation of non-human animal personality.
Maternal Gestational Cortisol and Testosterone Are Associated with Trade-Offs in Offspring Sex and Number in a Free-Living Rodent (Urocitellus richardsonii)

Calen P. Ryan, W. Gary Anderson, Charlene N. Berkvens, and 1 more author

PLoS ONE, Oct 2014

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The adaptive manipulation of offspring sex and number has been of considerable interest to ecologists and evolutionary biologists. The physiological mechanisms that translate maternal condition and environmental cues into adaptive responses in offspring sex and number, however, remain obscure. In mammals, research into the mechanisms responsible for adaptive sex allocation has focused on two major endocrine axes: the hypothalamic pituitary adrenal (HPA) axis and glucocorticoids, and the hypothalamic pituitary gonadal (HPG) axis and sex steroids, particularly testosterone. While stress-induced activation of the HPA axis provides an intuitive model for sex ratio and litter size adjustment, plasma glucocorticoids exist in both bound and free fractions, and may be acting indirectly, for example by affecting plasma glucose levels. Furthermore, in female mammals, activation of the HPA axis stimulates the secretion of adrenal testosterone in addition to glucocorticoids (GCs). To begin to untangle these physiological mechanisms influencing offspring sex and number, we simultaneously examined fecal glucocorticoid metabolites, free and bound plasma cortisol, free testosterone, and plasma glucose concentration during both gestation and lactation in a free-living rodent (Urocitellus richardsonii). We also collected data on offspring sex and litter size from focal females and from a larger study population. Consistent with previous work in this population, we found evidence for a trade-off between offspring sex and number, as well as positive and negative correlations between glucocorticoids and sex ratio and litter size, respectively, during gestation (but not lactation). We also observed a negative relationship between testosterone and litter size during gestation (but not lactation), but no effect of glucose on either sex ratio or litter size. Our findings highlight the importance of binding proteins, cross-talk between endocrine systems, and temporal windows in the regulation of trade-offs in offspring sex and number.
Validation of a radioimmunoassay-based fecal corticosteroid assay for Richardson’s ground squirrels Urocitellus richardsonii and behavioural correlates of stress

J.F. Hare, C.P. Ryan, C. Enright, and 4 more authors

Current Zoology, Oct 2014
Circulating breeding and pre-breeding prolactin and LH are not associated with clutch size in the zebra finch (Taeniopygia guttata)

Calen P. Ryan, Alistair Dawson, Peter J. Sharp, and 2 more authors

General and Comparative Endocrinology, Jun 2014

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Clutch size is a fundamental predictor of avian fitness, widely-studied from evolutionary and ecological perspectives, but surprisingly little is known about the physiological mechanisms regulating clutch size variation. The only formal mechanistic hypothesis for avian clutch-size determination predicts an anti- gonadal effect of circulating prolactin (PRL) via the inhibition of luteinizing hormone (LH), and has become widely-accepted despite little experimental support. Here we investigated the relationship between pre-breeding and breeding plasma PRL and LH and clutch-size in captive-breeding female zebra finches (Taeniopygia guttata). Using a repeated-measures design, we followed individual females from pre-breeding, through multiple breeding attempts, and attempted to decrease PRL using the D2-receptor agonist, bromocriptine. Clutch size was independent of variation in pre-breeding PRL or LH, although pre- breeding LH was negatively correlated with the time between pairing and the onset of laying. Clutch size was independent of variation in plasma PRL on all days of egg-laying. Bromocriptine treatment had no effect on plasma PRL, but in this breeding attempt clutch size was also independent of plasma PRL. Finally, we found no evidence for an inverse relationship between plasma PRL and LH levels, as predicted if PRL had inhibitory effects via LH. Thus, our data fail to provide any support for the involvement of cir- culating PRL in clutch size determination. These findings suggest that alternative models for hormonal control of avian clutch size need to be considered, perhaps involving downstream regulation of plasma PRL at the level of the ovary, or other hormones that have not been considered to date.

2013

Androgen receptor polyglutamine repeat number: models of selection and disease susceptibility

Calen P. Ryan, and Bernard J. Crespi

Evolutionary Applications, Jun 2013

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Variation in polyglutamine repeat number in the androgen receptor (AR CAGn) is negatively correlated with the transcription of androgen-responsive genes and is associated with susceptibility to an extensive list of human disease. Only a small portion of the heritability for many of these diseases is explained by conventional SNP-based genome-wide association studies, and the forces shaping AR CAGn among humans remains largely unexplored. Here, we propose evolutionary models for understanding selection at the AR CAG locus, namely balancing selection, sexual conflict, accumulation-selection, and antagonistic pleiotropy. We evaluate these models by examining AR CAGn-linked susceptibility to eight extensively studied diseases representing the diverse physiological roles of androgens, and consider the costs of these diseases by their frequency and fitness effects. Five diseases could contribute to the distribution of AR CAGn observed among contemporary human populations. With support for disease susceptibilities associated with long and short AR CAGn, balancing selection provides a useful model for studying selection at this locus. Gender-specific differences AR CAGn health effects also support this locus as a candidate for sexual conflict over repeat number. Accompanied by the accumulation of AR CAGn in humans, these models help explain the distribution of repeat number in contemporary human populations.
Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: Implications for pest mosquito control

Aditi D. Singh, Sylvia Wong, Calen P. Ryan, and 1 more author

Journal of Insect Science, Jun 2013

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RNA interference has already proven itself to be a highly versatile molecular biology tool for un- derstanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsR- NA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only in- duced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited.

2012

Stress-induced sex ratios in ground squirrels: support for a mechanistic hypothesis

Calen P. Ryan, W. Gary Anderson, Laura E. Gardiner, and 1 more author

Behavioral Ecology, Feb 2012

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Systematic deviations in sex ratio may be adaptive in the face of the prevailing environmental or physiological cues experienced by the mother; yet some theoretical and mechanistic hypotheses remain at odds and are rarely examined noninvasively under natural conditions. Conventional interpretations of the Trivers and Willard (Trivers RL, Willard DE. 1973. Natural selection of parental ability to vary the sex ratio of offspring. Science 179:90–92) hypothesis (TWH) predict that higher stress should be associated with female-biased litters; yet, several mechanistic hypotheses predict the opposite. We tested the predictions of the TWH and 2 stress-related mechanistic hypotheses in a free-living polygynandrous sciurid, Richardson’s ground squirrel, Urocitellus richardsonii. We examined the relationship between litter sex ratio and indicators of maternal condition and investment. These included litter size, juvenile mass at emergence, as well as maternal age, changes in maternal body mass, and maternal fecal glucocorticoid (cortisol and corticosterone) levels during gestation, lactation, and post-weaning. Males born of small litters were significantly heavier at emergence than males from larger litters, which were female biased, whereas females showed no significant change in mass with litter size. Mothers with higher fecal cortisol levels during the gestation period (but not during lactation or post-weaning) were more likely to produce male-biased litters, whereas females producing larger litters showed significantly higher cortisol levels during lactation (but not gestation or post-weaning) than mothers producing smaller litters. Our results provide support for both the proximate glucose-mediated mechanistic model of Cameron (Cameron EZ. 2004. Facultative adjustment of mammalian sex ratios in support of the Trivers-Willard hypothesis: evidence for a mechanism. Proc R Soc Ser B Biol Sci. 271:1723–1728) and the broad-sense TWH, where social and life-history characteristics play an integral role.

2010

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish, the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate Raja eglanteria

W Gary Anderson, Patricia J Dasiewicz, Suadi Liban, and 4 more authors

Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, Apr 2010

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The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca(2+) (19.67+/-3.65mM) and Mg(2+) (43.99+/-5.11mM) were high in L. erinacea and Mg(2+) was high (130.0+/-39.8mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO(3)(-) values were low (8.19+/-2.42 and 8.63+/-1.48mM) in both skates but very high in C. plagiosum (73.3+/-16.3mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0+/-8.1 to 0mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.