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Age at menarche, a sentinel index of pubertal maturation, was examined in relation to early family relationships (conflict, cohesion) and polymorphic variation in the gene encoding estrogen receptor-α (ESR1) in a midlife sample of 455 European American women. Consistent with prior literature, women who reported being raised in families characterized by close interpersonal relationships and little conflict tended to reach menarche at a later age than participants reared in families lacking cohesion and prone to discord. Moreover, this association was moderated by ESR1 variation, such that quality of the family environment covaried positively with menarcheal age among participants homozygous for minor alleles of the two ESR1 polymorphisms studied here (rs9304799, rs2234693), but not among women of other ESR1 genotypes. In addition, a) family relationship variables were unrelated to ESR1 variation, and b) genotype-dependent effects of childhood environment on age at menarche could not be accounted for by personality traits elsewhere shown to explain heritable variation in reported family conflict and cohesion. These findings are consistent with theories of differential susceptibility to environmental influence, as well as the more specific hypothesis (by Belsky) that girls differ genetically in their sensitivity to rearing effects on pubertal maturation.
Early pubertal development in girls portends heightened risk for a number of adverse health outcomes. These include all-cause mortality and deaths attributable to cardiovascular disease and cancer, incident coronary heart disease, breast cancer, uterine fibroid tumors, and in adolescence, early emergence of risk factors for heart disease, such as increased blood pressure, progressive weight gain, insulin insensitivity, and an atherogenic lipid profile (e.g., Adair & Gordon-Larsen, 2001; Bernstein, 2002; Cooper, Ephross, Weinberg, Baird, Whelan & Sandler, 1999; Golub, Collman, Foster, Kimel, Rajpert-De Meyts, Reiter et al., 2008; Hsieh, Trichopoulos, Katsouyanni & Yuasa, 1990; Jacobsen, Oda, Knutsen & Fraser, 2009; Lakshman, Forouhi, Sharp, Luben, Bingham et al., 2009; Marshall, Spiegelman, Goldman, Manson, Colditz, Barbieri et al., 1998; Remsberg, Demeratch, Schubert, Chumlea, Sun & Siervogel, 2005). Problems of psychological adjustment are also common among girls experiencing early puberty, as evidenced by low self-esteem and depressive symptomatology (Ge, Conger & Elder, 1996; Graber, Lewinsohn, Seeley & Brooks-Gunn, 1997; Graber, Seeley, Brooks-Gunn & Lewinsohn, 2004; Hayward, Killen, Wislon, Hammer, Litt, Kraemer et al., 1997; Kaltiala-Heino, Marttunen, Rantanen & Rimpela, 2003; Stice, Presnell & Bearman, 2001), body image concerns and eating disorders (Attie & Brooks-Gunn, 1989; Kaltiala-Heino, Rimpel, Risannen & Rantanen, 2001; Janna, Kaltiala-Heino, Kovisto & Rantanen, 2003), affiliation with deviant peers, truancy, and experimentation with drugs and alcohol (e.g., Caspi & Moffitt, 1991; Deardorff, Gonzales, Christopher, Roosa & Millsap, 2005; Kim, Smith & Palermiti, 1997; Mezzich, Tarter, Giancola, Lu. Kirisci & Parks, 1997; Romans, Martin, Gendall & Herbison, 2003; Silbereisen, Petersen, Albrecht & Kracke, 1989; Stice et al., 2001). Compared to later developing girls, those who mature earliest begin dating at a younger age, have earlier sexual intercourse and more sexual partners, date older boys, and report earlier first cohabitation -- behaviors that also contribute to risk for sexually transmitted diseases, early pregnancy, and teenage childbearing (e.g., Deardorff et al., 2005; Dunbar, Sheeder, Lezotte, Dabelea & Stevens-Simon, 2008; Hoier, 2003; Jorm, Christensen, Rodgers, Jacomb & Easteal, 2004; Kim & Smith, 1999; Kim et al., 1997; Lam, Shi, Ho, Stewart & Fan, 2002; Mezzich et al., 1997; Miller, Norton, Curtis, Hill, Schvaneveldt & Young, 1997; Phinney & Jensen, 1998).
These sequelae of early puberty underscore the public health significance of pubertal timing and, hence, of understanding its determinants. In addition to well-established biological antecedents, such as nutritional status, body weight and adiposity (Ellis, 2004; Ellis & Essex, 2007; Lassek & Gaulin, 2007; Lipson, 2001; Sloboda, Hart, Doherty, Pennell & Hickey, 2007), psychosocial factors, and specifically, aspects of the family environment, have emerged as significant predictors of pubertal development. In an influential paper published nearly two decades ago, Belsky, Steinberg and Draper (1991) drew on life history models to posit an evolutionary theory of socialization having implications for reproductive maturation. These authors hypothesized a certain plasticity of pubertal timing that is molded by early cues to future prospects, particularly prospects germane to reproduction. They suggested that qualities of the rearing environment not only shape expectations of future material resources, but also girls' expectations for experiencing enduring and supportive marital and family relationships as adults. These, in turn, were thought to somehow modulate pubertal timing (possibly through changes in body weight and associated neuroendocrine mechanisms), such that investment in prepubertal growth and development is curtailed in favor of accelerated progression to reproductive maturity when, in the face of early adversity, girls internalize family life and marriage as unpredictable, fragile, and fractious. Subsequent research largely confirmed the model's novel predictions regarding pubertal development, and this work has been the subject of comprehensive empirical and theoretical review by Ellis (2004, 2005; also Ellis, Shirtcliff, Boyce, Deardoff & Essex, this issue).
It is now well established that pubertal development tends to occur earlier in girls who are raised without a father than among those reared in traditional two-parent families, and this association may be strongest when the father's absence is first experienced in early childhood or persists over an extended number of years (Hoier, 2003; Ellis & Garber, 2000; Maestripieri, Roney, DeBias, Durante & Spaepen, 2004; Moffitt, Caspi, Belsky & Silva, 1992; Quinlan, 2003; Romans et al., 2003; Surbey, 1990; Wierson, Long & Forehand, 1993). In addition, and largely independent of father-absence, family environments prone to discord, harsh parenting, or lacking in close interpersonal relations are associated with girls' earlier pubertal development (Belsky et al, 2007; Ellis & Garber, 2000; Ellis, McFayden-Ketchum, Dodge, Petit & Bates, 1999; Graber, Brooks-Gunn & Warren, 1995; Kim & Smith, 1998; Kim et al., 1997; Moffitt et al., 1992; Quinlan, 2003; Romans et al., 2003; Steinberg, 1988; Tither & Ellis, 2008), whereas girls mature more slowly when reared in affectionate, cohesive families (Ellis et al., 1999; Ellis & Essex, 2007). These relations are documented in both short- and long-term longitudinal investigations, in retrospective studies, and among studies employing diverse indices of family environment. Nonetheless, effect sizes are typically small, and in investigations employing multiple family measures, pubertal timing may be predicted inconsistently across indicators.
Although the conceptual framework articulated by Belsky et al (1991), Ellis (2004), and others (e.g., Chisholm, 1999) implies a causal relation between aspects of the family environment and pubertal development, genetic factors afford alternative interpretations. For instance, behavioral correlates of early puberty, such as sexual precocity and adjustment problems, might presage later marital and family difficulties among women who reach reproductive competence at a young age. If variation in pubertal timing is heritably transmitted over generations, early pubertal maturation in daughters could reflect shared genetic influences that occasioned the accelerated reproductive development of their mothers and then, indirectly, the adversities of family environment to which the daughters were later exposed (i.e., gene-environment correlation) (Moffitt et al., 1992; Surbey, 1990). Indeed, genetic factors are known to play an important role in reproductive development, and a sentinel indicator of pubertal maturation in females, age at menarche, correlates moderately across generations (e.g., Brooks-Gunn & Warren, 1988; Damon, Damon, Reed & Valadian, 1962; Graber et al., 1995; Kaprio, Rimpela, Winter, Viken, Rimpela & Rose, 1995; Towne, Czerwinski, Demerath, Blangero, Roche & Siervogel, 2004), with an estimated heritability of about 50% (e.g., Snieder, MacGregor & Spector, 1998; Doughty & Rodgers, 2000; Rowe, 2000; Kirk, Blomberg, Duffy, Heat, Owens & Martin, 2001; Towne et al., 2005). Nonetheless, the few studies reporting data on mothers' pubertal development suggest that absence of the father and qualities of the family environment predict girls' menarcheal ages even after adjusting for correlated variation in maternal age at menarche (Campbell & Udry, 1995; Ellis & Essex, 2007; Graber et al., 1995; Surbey, 1990).
A second genetic possibility is suggested by accumulating evidence of gene-environment interaction, in which DNA sequence variation (polymorphisms) of biologically relevant genes have been found to moderate environmental influences on such prominent behavioral phenotypes as childhood temperament, early cognitive abilities, juvenile and adult antisocial behavior, and depression (Caspi, McClay, Moffitt, Mill, Martin, Craig et al., 2002; Caspi, Sugden, Moffitt, Taylor, Craig, Harrington et al., 2003; Caspi, Williams, Kim-Cohen, Craig, Milne, Poulton et al., 2007; Cicchetti, Rogosch & Sturge-Apple, 2007; Moffitt, Caspi Rutter, 2005; Scheese, Voelker, Rothbart & Posner, 2007; Taylor & Kim-Cohen, 2007). Analogously, differences in family environments could conceivably affect the pace of pubertal maturation more strongly in some girls than others owing to heritable variation in susceptibility to rearing influences, a possibility anticipated in the seminal paper of Belsky et al. (1991, p. 630). In fact, the developmental theory prompting this Special Section of Development and Psychopathology, referred to as "differential susceptibility" (Belsky, 1997; Belsky & Pluess, 2009) or "biological sensitivity to context" (Boyce & Ellis, 2005), is premised on the observation that individuals differ in their responsiveness to varying environmental conditions. With respect to reproductive maturation, for instance, Belsky et al (2007) found maternal sensitivity and maternal harsh control in childhood to predict pubertal onset in opposite directions among children exhibiting, as infants, high versus low negative emotionality. In addition, Ellis, Shirtcliff et al (this issue) have shown the tempo of pubertal development to covary with quality of parent-child relationships more strongly among adolescents who exhibit heightened cardiac-autonomic and adrenocortical reactions to laboratory stressors (high biological sensitivity to context) than among less reactive counterparts. Because it is likely that individual differences in susceptibility to rearing influences are at least partly heritable (just as infant temperament and stress-related physiologic reactivity –putative markers of susceptibility – are known to be heritable [Manuck & McCaffery, in press; Saudino, 2005]), investigation is warranted into sources of genetic variation that might moderate family environmental effects on pubertal timing. Although according well with the models of differential susceptibility, as well as existing studies of gene x environment interactions, the genetic modulation of psychosocial influences on pubertal development has not been examined previously to our knowledge or in relation to specific gene polymorphisms.
Unfortunately, little is known yet regarding sources of molecular variation that may underlie differences in pubertal timing. Physiologically, onset of menstruation (menarche) occurs with advancing maturation of the hypothalamic-pituitary-ovarian axis, following a gradual increase in pulsatile release of the pituitary-derived gonadotropins (follicle-stimulating hormone, luteinizing hormone) and heightened exposure of tissues to the gonadal steroid, estrogen (Di Vall & Radovick, 2008,Di Vall & Radovick, 2009). Because the biological actions of estrogen are largely mediated through activation of intracellular estrogen receptors, variation in estrogen receptor genes could contribute to heritable influences on pubertal development. Recently, common polymorphisms of the gene encoding estrogen receptor-α (ESR1) have been associated with menarcheal ages of adolescent girls in Northwestern Greece (Stavrou, Zois, Ioannidis & Tsatsoulis, 2003) and, in interaction with other genetic variation, among Chinese (pre-menopausal) and Spanish (post-menopausal) women (Long, Xu, Zhao, Liu. Shen, Lin et al., 2005; Mendoza, Moron, Quereda, Vazquez, Rivero, Martinez-Astorquiza et al., 2008; Xu, Long, Li, & Deng, 2005). In four other investigations, however, ESR1 variation was unrelated to age at menarche (Boot, van der Sluis, de Munick Keizer-Schrama, van Meurs, Krenning, Pols et al., 2004; Gorai, Tanaka, Inada, Morinaga, Uchiyama, Kikuchi et al., 2003; Mitchell, Farin, Stapleton, Tsai, Tao, Smith-DiJulio & Woods, 2008; Weel, Uitterlinden, Westendorp, Burger, Schuit, Hofman et al., 1999). These inconsistencies could stem from an absence of true association; methodological differences among studies, including limited statistical power; unexamined gene-gene interaction (epistasis); or an interaction of ESR1 variation with unmeasured environmental moderators (gene-environment interaction).
In sum, while pubertal maturation among girls may be accelerated or retarded by characteristics of family relationships prevailing in childhood (father absence, conflict, cohesion), it remains unclear whether such associations reflect direct psychosocial effects, correlated genetic influences on pubertal timing, or possibly, an interaction of family environment with relevant genetic variation. In this regard, the purpose of the present study was to further evaluate associations of menarcheal age with family relationship variables and polymorphisms of ESR1, as seen in a study cohort of 455 middle-aged women. Specifically, we examined: a) whether age at menarche and ESR1 polymorphisms covaried with reported qualities of participants' early rearing environments; and b) in exploratory analysis, whether any association of menarcheal age with these family variables may have differed in magnitude as a function of allelic variation in ESR1.
Study data were derived from a sample of 550 non-Hispanic Caucasian women who had participated in the University of Pittsburgh Adult Health and Behavior (AHAB) project between 2001 and 2005. The AHAB project provides a registry of behavioral and biological measurements on midlife community volunteers recruited via mass-mail solicitation from communities of southwestern Pennsylvania (principally Allegheny County). Registry data include sociodemographic measurements; indices of personality and temperament; psychiatric history and symptomatology; aspects of social and cognitive functioning (e.g, social networks, cognitive abilities); health-impairing attributes of habit and lifestyle; physiological measurements germane to cardiovascular, autonomic, metabolic, immune, and central nervous system functioning; and DNA extracted for the study of genetic variation associated with registry phenotypes (Cf., Bleil, Gianaros, Jennings, Flory & Manuck, 2008; Halder, Muldoon, Ferrell & Manuck, 2007; Halder, Marsland, Cheong, Muldoon, Ferrell & Manuck, 2010; Hall, Muldoon, Jennings, Buyssee, Flory & Manuck, 2008; Manuck, Phillips, Gianaros, Flory & Muldoon, 2010). AHAB participants were 30–54 years of age, with no clinical history of atherosclerotic cardiovascular disease, chronic kidney or liver disease, cancer treatment within the preceding year, or major neurologic disorders, schizophrenia, or other psychotic illness. Other AHAB study exclusions included pregnancy and use of insulin, glucorticoid, antiarrhythmic, psychotropic or prescription weight-loss medications. Data collection occurred over multiple laboratory sessions, and informed consent was obtained in accordance with approved guidelines of the University of Pittsburgh Institutional Review Board.
Although the AHAB registry includes measurements on a small proportion of African American women, study analyses were limited to the larger Caucasian cohort to mitigate confounding by sample heterogeneity and race/ethnicity differences in allele frequencies of ESR1 polymorphisms (e.g., Kardia, Chu & Sowers, 2006). Unfortunately, separate analysis of African American women alone was not feasible here, as the number of completed data protocols that also met other family criteria listed below was too small (62) to detect genetic associations of plausible effect size. In addition, our measure of early family environment (described below) did not segment childhood by participant age or with respect to any alteration in family (parental) composition that may have happened during childhood, but instead, asked participants only to report globally on family relationships while "growing up." This instrument was therefore insensitive to variation or change in family constellation since, in these instances, the family unit referenced in self-report is ambiguous, or at least indeterminate. For this reason, as well as to avoid confounding any association of menarcheal age and reported family relationships with differences in family structure (e.g., father absence), we restricted our analyses to women who had been raised by their two biological parents through at least the first 14 years of life. This resulted in exclusion of 73 participants who had only ever lived with a single parent (8), were adopted or raised by a non-biological parent (20), or whose parents had divorced (45). An additional seven participants were excluded for missing data on one or more non-genetic study variables to yield a working sample of 470 women.
Genomic DNA was isolated from peripheral white blood cells using the PureGene kit (Gentra Systems, Minneaopolis, MN). The two single nucleotide polymorphisms (SNPs) of ESR1 examined in prior research on pubertal timing, rs9340799 and rs2234693, were genotyped in this investigation. These SNPs also figure prominently in genetic studies of other estrogen-dependent conditions, such as osteoporosis, breast cancer, and atherosclerotic cardiovascular disease (e.g., Boyapati, Shu, Ruan, Cai, Smith, Wen et al., 2005; Cai, Shu, Dai, Wen, Cheng, Gao & Zheng, 2003; Kunnas, Laippala, Penttilla, Lehtimaki & Karhunen, 2000; Lehtimaki, Kunnas, Mattila, Perola, Penttila, Koivula et al., 2002; Modugno, Zmuda, Potter, Cai, Ziv, Cummings et al., 2005; Onland-Moret, van Gils, Roest, Grobbee,& Peters, 2005; Schuitt, Oei, Witteman, van Kessel, van Meurs, Nijhuis et al., 2004; Shearman, Cupples, Demissie, Peter, Schmid, Karas et al., 2003; Shin, Kang, Nishio, Lee, Park, Kim et a., 2003; Wedren, Lovmar, Humphreys, Magnusson, Milhus, Syvanen et al., 2004), where they are often labeled for the restriction enzymes by which they are recognized (XbaI and PvuII) or identified by their respective locations at 351 and 398 base pairs 5′ to exon 2 of the ESR1 gene. The two ESR1 polymorphisms were genotyped by standard restriction enzyme digestion, with resolution on 2% agarose gels and ethidium bromide staining (Castagnoli, Maestri, Bernardi, & Del Senno, 1987).
Four hundred sixty-one participants (98%) were genotyped successfully for rs9340799 and 457 (97%) for rs2234693. Only women for whom genotypes of both SNPs were available were included in statistical analyses, leaving a final study sample of 455 participants (Table 1). Frequencies of the rs9340799 A and G alleles were 62 and 38 percent, respectively, and of the rs2234693 T and C alleles, 53 and 47 percent. In the absence of selection pressures, nonrandom mating, migration, or other disturbing influences, the distribution of genotypes at a given locus should remain constant over generations, as defined by the Hardy-Weinberg Equilibrium (for a two-allele locus, the expected distribution of genotypes is p2 + 2pq + q2, where p and q denote the frequencies of the two alleles). The distribution of ESR1 genotypes in our sample did not depart from equilibrium (rs9340799: AA: 173; AG: 215; GG: 67; rs2234693: TT: 128; TC: 204; CC: 103; χ12 's < 0.10, ns). The two SNPs were also in strong linkage disequilibrium (D' = 0.98, r2 = 0.67), a measure of nonrandom association among alleles of more than one polymorphism, and is consistent with the close proximity of these loci.
Because genetic data derived from individuals of common race/ethnicity may still exhibit stratification, we tested for possible genetic substructure in this sample. Fifteen additional, genome-spanning SNPs (rs1022106, rs1335995, rs1439564, rs1502812, rs1860300, rs548146, rs705388, rs715994, rs720517, rs722743, rs730899, rs734204, rs9059966, rs1328994, and rs1485405) were genotyped for analysis using the program STRUCTURE (Falush, Stephens & Pritchard, 2003; Pritchard, Stephens & Donnelly, 2000). A model with admixture, uncorrelated allele frequencies, individual alpha parameters, and independent Fst for all subpopulations was run separately assuming 1, 2, or 3 subpopulations. For each model, we used a burn-in of 40,000 simulations, followed by 80,000 repetitions, and compared the likelihoods of models fitting the data. Evidence of stratification could be inferred if the likelihood of data fitting a model with ≥2 subpopulations was greater than that of a model with 1 population. However, no evidence of genetic substructure was detected (log probabilities for k = 1, 2, and 3 subpopulations were −6675.7, −6740.1 and −6741.9, respectively), and therefore, no further adjustments were made for stratification.
The first menstrual period is a highly salient event in girls' lives and a well-recollected developmental milestone. Studies of the reliability of retrospectively reported menarcheal age yield remarkably similar correlations with contemporaneously recorded age at menarche, whether assessed over recall intervals of short and long duration (4–5 years: r's = 0.81–0.83; 17 years: r = 0.75; 19 years: r = 0.78; 29 years: r = 0.79) (Bergsten-Brucefors, 1976; Damon, Damon, Reed & Valadian, 1969; Koprowski, Coates & Bernstein, 2001; Livson & McNeill, 1969; Must, Phillips, Naumora, Blum, Harris, Dawson-Hughes & Rand, 2002). In the AHAB project, women were asked to report their age at first menstruation as a component of a medical history interview administered by project nurses. Reported age at menarche averaged 12.6 ±1.4 years, with a mean recall interval of 32.4 ±6.7 years. The average menarcheal age recorded here was similar to the 12.8 years reported in the National Health Examination Survey (NHES) for white girls studied in 1963–1970, whose years of birth would have overlapped, but slightly preceded, the average of the present sample (Anderson, Dallal, & Must, 2003). Also, the cumulative proportion of AHAB participants attaining menarche by ages 12 (47%), 13 (78%), 14 (92%), and 15 (96%) years closely match corresponding NHES values at the same ages (43%, 73%, 91% and 98%, respectively).
Degree of conflict and interpersonal warmth in participants' early family environments were indexed by the Conflict and Cohesion subscales of the widely-used Family Environment Scale (FES) (Moos and Moos, 1981), as modified by Plomin, McClearn, Pedersen, Nesselroade & Bergeman (1988) to assess self-reported childhood rearing environments among adults. Scale modifications included conversion of the FES response format from true-false to 5-point Likert scales (anchored by end-ratings of "strongly disagree" and "strongly agree") and shortening each scale from an original 9 items to the 5 items having the strongest factor loadings for the scale. Conflict scale items reference competition, criticism, expressed anger, and fighting within the family, whereas Cohesion scale items reference agreeable relationships among family members, mutual assistance, attention and support, and shared family spirit. The five items of each scale were averaged for each subject and the resulting score distributions standardized. In the current sample, internal reliabilities (α) of the Conflict and Cohesion scales were 0.86 and 0.89, respectively, and the two scales correlated inversely (r = −0.66). For primary analyses, an overall measure of Quality of Family Environment (QFE) was calculated by reverse scoring the Conflict scale (so that higher scores indicated low family conflict), then averaging the two scales and standardizing the sample distribution of QFE scores. The Conflict and Cohesion scales were also examined separately.
Measures of both experienced and perceived environmental exposures often reflect nontrivial genetic influences (Kendler & Baker, 2007) and, with respect to FES scales, minor heritable variation has been found on retrospective reports of both family Conflict and Cohesion (Plomin et al., 1988). The latter findings are consistent with other biometric studies using the same or analogous scales (Deater-Deckard, Fulker & Plomin, 1999; Jacobson & Rowe, 1999; Jang, Vernon, Liveley, Stein & Wolf, 2001; Krueger, Markon & Bouchard, 2003; Plomin, McClearn, Pedersen, Nellelroade & Bergman, 1989). Recently, Krueger et al (2003) reported on the heritability of a composite measure of "perceived cohesion versus conflict in the family environment," as derived from multiple environmental scales (on which FES Cohesion scores loaded positively and FES Conflict scores loaded negatively) in the Minnesota Study of Twins Reared Apart (MISTRA). The 16% of phenotypic variance attributable to heritable variation in that study could be fully explained by genetic covariance with two higher-order dimensions of personality, Negative Emotionality and Constraint, as measured by the Multidimensional Personality Questionnaire (MPQ) (Tellegen, 1982). Scores on an abbreviated version of the MPQ, the 155-item MPQ-Brief Form (MPQ-BF) (Patrick, Curtin & Tellegen, 2002), were also available in the AHAB registry. To evaluate whether any association of menarcheal age with FES-assessed family environment might be accounted for by correlated variation in these personality dimensions, MPQ-BF traits of Constraint and Negative and Positive Emotionality were included in the present study. MPQ-BF scales have good internal consistency, and the three broad traits examined here correlate strongly (r's = 0.94–.98) with their corresponding MPQ factors.
Participants had a mean age of 45.1 ±6.6 years and were well-educated on the whole, averaging 15.9 ±2.9 years of schooling. By level of educational attainment, 38% lacked a college diploma, 36% held a Bachelors degree, and 26% had completed graduate or professional training. Consistent with secular trends, women reported their parents having somewhat fewer years of schooling than themselves (highest parental education: M = 13.9 ±3.2 years). At the time of study participation, 73% of women were married and an equivalent proportion (76%) was employed full or part-time. As shown in Table 2, participant age and personal and parental education were variously related to self-reported early family environment. Our overall measure of family environment (QFE) correlated negatively with age and participant education, whereas Conflict scores separately correlated negatively with age, and family Cohesion scores covaried positively with both personal and parental education. However, these sample characteristics were independent of menarcheal age and, when tested for association with ESR1 polymophisms by one-way analysis of variance (ANOVA) (Table 2), were unrelated to ESR1 variation.
Table 2 shows a modest, but significant, inverse association between QFE and menarcheal age (r = 0.10). Consistent with prior literature, women who reported growing up in families characterized by less conflict and greater cohesion experienced menarche at a later age than those raised in families with more conflict and less cohesion. Age at menarche also correlated negatively with Conflict scores alone (r = −0.10) and positively with Cohesion scores (r = 0.09, p = 0.05).
Also summarized in Table 2 are simple effects of ESR1 polymorphisms on QFE and family Conflict and Cohesion scales separately, none of which proved significant. As a result, we next tested whether polymorphic variation in ESR1 predicted age at menarche or moderated the association of menarcheal age with QFE. We entered participants' QFE scores in the first step of hierarchical linear regression, followed by ESR1 genotype (Step 2), and finally, the multiplicative interaction of QFE and genotype (Step 3). Effects of genotype were tested on an additive model of genetic influence and analyses run separately for each ESR1 polymorphism (coded −1, 0, 1 for genotypes AA, AG and GG of rs9340799 and TT, TC, and CC of rs2236693). Subsequent analyses produced virtually identical results when including demographic factors (age, personal and highest parental education) as covariates, so that for simplicity these variables are not considered in analyses summarized below.
Regression outcomes are presented in Table 3. Consistent with the bivariate correlations reported above, age at menarche was predicted modestly by QFE (b = .149, se = .067, t = 2.21, p = 0.028), but was unrelated to genotype of rs9340799. However, the association of menarcheal age with QFE varied significantly by genotype, as indicated in the interaction term of the final regression model (b = .259, se = .096, t = 2.71, p = 0.007). As illustrated in Figure 1, QFE scores, plotted to one standard deviation above and below the sample mean, covaried with menarcheal age more strongly in women homozygous for the rs9340799 G allele than among those of other genotypes. Bivariate correlations within the three genotypes of this polymorphism (Table 4) show approximately 9% of the variance in menarcheal age explained by QFE scores among GG homozygous women (r67 = .304, p = 0.012). The corresponding correlation among women carrying one G allele (AG heterozygotes) only approached significance (r215 = .123), and no association at all obtained among participants homozygous for the alternate A allele (r173 = −.034). Correlations calculated for the separate Conflict and Cohesion scales (also listed in Table 4) showed menarcheal age to covary positively with family cohesion (r67 = .319, p = 0.009) and negatively, albeit marginally, with family conflict (r67 = −.234, p = 0.057) among GG homozygotes. In contrast, age at menarche was unrelated to either dimension of family functioning in participants of heterozygous genotype and those homozygous for the A allele. These genotype-dependent associations were corroborated in parallel regression analyses (Table 5), where genotype of rs9340799 predicted menarcheal age significantly in interaction with reported family Cohesion (b = .294, se = 0.097, t = 3.02, p = 0.003), but did so only weakly in interaction with family Conflict (b = −.179, se = 0.094, t = −1.90, p = 0.058). When the two family predictors and their corresponding interaction terms were entered together in a futher regression analysis, moreover, the interaction of rs9340799 genotype and Cohesion remained significant (b = .303, se = 0.124, t = 2.36, p = 0.019), suggesting that this relationship is independent of correlated variation in family Conflict.1
Interestingly, our second ESR1 polymorphism showed a similar pattern of within-genotype correlation between QFE and age at menarche (genotype: CC: r = −.218, p= 0.026; CT: r = −.053, ns; TT: r = −.044, ns), even though the corresponding interaction term in regression was not significant for this polymorphism (b = 0.134, se = 0.094, t = 1.44, p = 0.152) (Table 3). Like rs9304799, menarcheal age correlated positively with Cohesion scores alone (r = 0.218, p = 0.027) and inversely (and again weakly) with reported family Conflict (r = −0.192, p = 0.051) among women homozygous for the less common rs2234693 C allele. Among participants of TT or TC genotype, menarcheal age was unrelated to either Cohesion or Conflict scores. In parallel regression models (Table 5), rs2234693 genotype predicted age at menarche in interaction with family Cohesion (b = .194, se = 0.097, t = 2.01, p= 0.045), but not family Conflict (b = −0.063, se = 0.091, t= −0.69, p = 0.492). And like rs9340799, further analysis showed genotype of rs2234693 to predict menarcheal age in interaction with family cohesion even in a model including both family predictors and their respective interaction terms (genotype × cohesion: b = .276, se = 0.130, t = 2.13, p = 0.034).
Earlier we cited evidence that heritable variation in a composite measure of family "Cohesion vs. Conflict" (which incorporated the FES scales used here) could be accounted for by correlated variation in two MPQ-assessed dimensions of personality, Negative Emotionality and Constraint (Krueger et al., 2003). As shown in Table 1, Negative Emotionality correlated positively and Constraint negatively with both QFE and Conflict scale scores in the current study, whereas scores on the Cohesion scale covaried inversely with Negative Emotionality and positively with both Constraint and Positive Emotionality. Despite these associations, none of the personality dimensions correlated significantly with age at menarche. In addition, substituting trait measures for the family environment scales in regression analyses predicting menarcheal age revealed no significant interactions between genotypes of the two ESR1 polymorphisms and the three personality dimensions. And finally, entering the trait measures as covariates on re-analysis of family QFE, Conflict and Cohesion scores did not alter the previously reported interactions of these variables with ESR1 variation. Thus, QFE continued to predict age at menarche in interaction with rs9340799 genotype (b = 0.259, se = 0.096, t = 2.69, p = 0.007), as did Cohesion scores alone (b = 0.291, se = 0.097, t = 2.99, p = 0.003), and family Cohesion continued to predict menarcheal age in interaction with genotypes of rs2234693 (b = 0.189, se = 0.097, t = 1.96, p = 0.051).
In this study of 455 midlife women reared to adolescence in stable, two-parent families, participants who described their families as cohesive and lacking in conflict tended to reach menarche at a later age than women who reported being reared in families prone to discord and lacking cohesion. This association is consistent with previous studies, both retrospective and longitudinal, in which analogous dimensions of family relationship similarly predicted daughters' pubertal timing (Moffitt et al., 1992; Romans et al., 2003; Graber et al., 1995; Ellis & Garber, 2000; Ellis et al., 1999; Kim et al., 1997; Kim & Smith, 1998; Steinberg, 1988; Wierson et al., 1993). In addition, the association of early family environment with age at menarche in our study was moderated by polymorphic variation in ESR1, a gene of hypothesized association with reproductive maturation. Thus, Quality of the Family Environment (assessed here by composite of the Conflict and Cohesion scales of the FES) covaried positively with menarcheal age among women homozygous for the minor alleles of rs9304799 (GG genotype) and rs2234693 (CC), but not among women of other ESR1 genotypes. Moreover, this genotype-dependent association between family environment and age at menarche was principally attributable to variation in family cohesion. The main effect of family relationships on menarcheal age here is consistent with prior literature; its modulation by ESR1 variation is also consistent with predictions from differential susceptibility and accords with other research showing heritable "person variables," such as infant negative emotionality and stress reactivity, to moderate environmental influences on pubertal timing (Belsky et al., 2007; Ellis, Shirtcliff, et al., this issue).
In girls, the onset of menses defines a sentinel event in the sequence of physiological and anatomic changes initiating puberty. Because these changes are occasioned by heightened exposure of reproductive and other tissues to the ovarian hormone, estrogen, it is plausible that variation in genes encoding estrogen receptors affect the pacing of pubertal development. When activated by estrogen, estrogen receptors (of which there are two, ER-α and ER-β) migrate to the cell nucleus, where they exert transcriptional control of estrogen-dependent genes by binding to estrogen response elements within gene regulatory sequences. In this way, estrogen (like other steroid hormones) can promote or repress the expression of genes specifying an array of cellular proteins. Genetic variation that alters ER activity could therefore modulate a range of estrogen-sensitive phenotypes, including pubertal maturation. At present, though, it is unclear how the ESR1 polymorphisms genotyped here might influence girls' reproductive development. In fact, the absence of allele-dependent effects on menarcheal age among all study participants (i.e., the absence of a genetic "main effect"), albeit in agreement with all but one previous study, suggests a more complex etiology (Boot et al., 2004; Gorai et al., 2003; Hong et al., 2005; Long et al., 2005; Mendoza et al., 2008; Mitchell et al., 2008; Stavrou et al., 2002; Weel et al., 1999). And given the present findings, elucidating underlying biological mechanisms would also require understanding how ESR1 variation might variably influence pubertal development as a function of the quality of family relationships.
One possible mechanism could involve effects on pubertal development related to variation in body weight, which may be associated with differing family environments. For instance, certain ESR1 genotypes might promote a proportionately greater weight gain among girls who respond to family adversity by overeating, thus advancing menarcheal age in these genetically-susceptible girls via the well-established relationship between adiposity and age at menarche. Unfortunately, we do not have data on participants' childhood weight to address this possibility, but it may be noted that controlling for weight in several prior studies did not attenuate associations of family variables with pubertal timing (Campbell & Udry, 1995; Ellis & Essex, 2007; Graber et al., 1995; Moffitt et al., 1992).
In any case, little is yet known about the functionality of ESR1 polymorphisms. Because rs9340799 and rs2234693 are both located in a non-coding region of the gene (intron 1), they do not produce ER proteins of differing structure (unless, hypothetically, through alternative splicing of the primary mRNA transcript), but might be in linkage disequilibrium with functional variation elsewhere in ESR1. Introns are also known occasionally to affect rates of gene transcription, and it is noteworthy that the C allele of rs2234693 forms part of a binding site for a transcription factor, B-myb, that has been shown to enhance transcriptional efficiency in an in vitro assay system (Herrington, Howard, Brosnihan, McDonnell, Li, Hawkins et al., 2002). In addition, B-myb is itself activated by estrogen, which might further augment ESR1 expression to influence cellular activity in pathways underlying hormone-sensitive interactions (Santen et al., 1998). It is conceivable, for instance, that variation in ER-α signaling in certain hypothalamic nuclei (viz., arcuate, anteroventral periventricular nucleus) modulates reawakening of the gonadotropin releasing hormone (GnRH) pulse generator in puberty. Such modulation might act on the pubertal decline in estrogen-dependent inhibition of luteinizing hormone (and hence, GnRH) or contribute more directly to activation of GnRH neurons. Both processes are regulated critically by the protein, kisspeptin, and the expression of kisspeptin is induced by gonadal steroids, as mediated by ER-α (Smith, Clifton & Steiner, 2006). Until it is demonstrated that the molecular variation of rs2234693 actually alters receptor availability in tissues expressing ER-α, however, the in vivo functionality of this polymorphism remains uncertain.
Whatever the biological mechanisms, is the magnitude of association between early family environment and menarcheal age observed here developmentally meaningful? Quality of Family Environment (QFE) scores correlated positively, but only modestly, with pubertal timing across all women (r =0.10), and indeed, the spread of QFE defined by a departure of one standard deviation above and below the sample mean was associated with a difference of only about 3.5 months in participants' age at menarche. Yet the difference in estimated menarcheal age over the same range of QFE variation among women of an "environmentally sensitive" ESR1 genotype, such as those homozygous for the G allele of rs9340799 (as depicted in Figure 1), was fully a year. Interestingly, adult ovarian function is also established more slowly among those who reach menarche latest. Longitudinal observations show girls reaching menarche at ages <12, 12–12.9 and ≥13 years to experience 50 percent ovulatory menstrual cycles at, respectively, 1, 3 and 4.5 years following menarche (Apter and Vikho, 1983). Thus, developmental variation indexed to small differences in menarcheal age may be magnified on further progression to reproductive maturity, as in the transition from primarily anovulatory to reliably ovulatory cycling. Distal sequelae of early and late menarche may be linked also to the range of variation in menarcheal ages predicted here by the interaction of ESR1 variation and early family environment. A one-year delay in menarche reduces breast cancer risk by 10–20 percent (Bernstein, 2002), and in a recent prospective study of women with mean age at baseline similar to the present sample, a one-year delay in menarche lowered total, coronary, and stroke mortality risk, respectively, by 4.5, 6.0 and 8.6 percent over an average follow-up of 11.1 years (Jacobsen et al., 2009; see also Lakshman et al., 2009).
A previous report that paternal promiscuity, parental divorce, father absence, and menarcheal age were predicted by length variation in a trinucleotide (GGC) repeat polymorphism of the androgen receptor gene suggested that family instability and pubertal timing might be genetically correlated (Comings, Muhleman, Johnson & MacMurray, 2002; but see also Jorm et al., 2004) Here, in contrast, genetic variation in ESR1 was unrelated to family relationship variables, but instead moderated associations between early family environment and age at menarche. This observation is consistent with Belsky's hypothesis that girls differ genetically in their susceptibility to rearing effects on pubertal maturation (Belsky et al., 2000, 2005; Belsky, Bakermans-Kranenburg & IJzendoorn, 2007), as well as models of context-dependent development – differential susceptibility -- that are less explicit regarding genetic determinants of individuals' relative sensitivity to the environment (Boyce & Ellis, 2005; Ellis, Shirtcliff, et al., this issue). Interestingly, all strands of theorizing on the origins of differential susceptibility have invoked evolutionary considerations, speculating on the circumstances in which variation in the capacity to adjust development in response to environmental conditions (i.e., developmental plasticity) would tend to be favored or rejected by natural selection (Belsky et al., 1991; 2005; Boyce & Ellis, 2005; Ellis, Boyce, Belsky, Bakermans-Kranenburg & IJzendoorn, this issue; Ellis et al., 2006). When specific sources of genetic variation (gene polymorphisms) are found to underlie this variation (as the present findings might imply), an evolutionary account of differential susceptibility requires explaining how the implicated polymorphisms are perpetuated in a reproducing population. This is not a trivial consideration, as selection ordinarily acts to reduce genetic variation, not promote it, and if the latter, will maintain alternate alleles in their respective frequencies at a given locus over generations only if their bearers experience, on balance, equal reproductive fitness (the currency of selection).
Differential susceptibility, as defined by Belsky & Pluess (2009) and Ellis, Boyce, et al. (this issue), is present when regression lines reflecting associations between a phenotype and environmental factor cross over when plotted for different variants of a susceptibility marker (here, genotypes of a polymorphism), such that the line of greater slope (plasticity) predicts both higher and lower phenotypic values at opposite poles of an environmental gradient than an alternate variant (genotype). This is illustrated here in Figure 1, where QFE scores are not only associated more strongly with menarcheal age among women homozygous for the rs9304799 G allele, but also tend to predict both later and earlier age at menarche at higher and lower QFE, respectively, compared, for instance, to AA homozygotes. Life history variables, like pubertal maturation, that are related to reproductive timing can respond to natural selection if they affect lifetime reproductive outcomes, such as the number and quality of offspring (Chisholm & Burbank, 2001). Consider that early menarche presages a number of adverse sequelae, including problems of mood and conduct in adolescence, early metabolic changes that heighten risk for cardiovascular disease, and in adulthood, breast cancer, diseases of heart and vasculature, and premature mortality (e.g., Bernstein, 2002; Cooper et al., 1999; Ge et al., 1996; Golub et al., 2008; Hayward et al., 1997; Jacobsen et al., 2009; Kaltiala-Heino et al., 2003; Lakshman et al., 2009; Marshall et al., 1998; Remsberg et al., 2005; Stice et al., 2001). If, in addition, child-bearing risks and the other health-related consequences of early menarche are mirrored in a lower reproductive fitness (and oppositely for delayed menarche) – so that age at menarche predicts lifetime reproductive success – selection would, in the context of our findings, for instance, theoretically favor the G allele of rs9304799 among women raised in a positive family environment, but the A allele among those reared under less propitious family circumstance. That is, if a genotype conferring greater plasticity results in reproductive advantage at one end of the environmental gradient to which it is sensitive, and relative disadvantage at the other, net fitness over all environments (though more variable) might approximate that of an alternative genotype associated with lesser sensitivity to environmental variation. Because neither genotype is optimal in all environments, both will tend to be preserved (Stearns, 1992).
In contrast to the argument from competing advantage just cited, proponents of differential susceptibility models have not viewed early and later pubertal development as a predictor of lifetime reproductive success so much as potentiating different reproductive strategies that adapt individuals to the varied circumstances of their development. Thus, girls whose early life experiences encourage little expectation of a materially secure, stable, and emotionally supportive marital and family life in adulthood may trade off continued growth and development for accelerated sexual maturation, early childbearing, and a limited reliance on paternal investment in child care and parenting (Belsky et al., 1991; Chisholm, 1999; Ellis, 2004). Presumably, the higher risks of early sexual development and childbearing are offset by a potentially longer reproductive lifespan, or perhaps this "early and fast" reproductive strategy simply offers the best prospect against omens of an uncertain future. Conversely, girls experiencing stable, warm, and cohesive family relationships in early childhood may anticipate the same for themselves as adults and pursue a reproductive strategy entailing slower pubertal development and deferred childbearing, premised on expectations of a secure marital bond and high paternal investment in child rearing (Belsky et al., 1991; Ellis, 2004, 2005). This reasoning alone, however, does not explain how genetic variation underlying interindividual variability in responsiveness to environmental differences would be maintained in a population. If the function of developmental plasticity is to maximize the adaptive responses (reproductive strategies) of individuals born into family environments of differing qualities – good or bad – selection might be expected to favor genetic variation enabling such plasticity and, over time, supplant genotypes promoting a less flexible developmental phenotype.
However, if a capacity to adapt to differing environments carries potential costs, as well as benefits, or does so in certain circumstances, it is conceivable that phenotypes of both greater and lesser sensitivity to the environment might prove equally fit overall and, therefore, persist in ratios of prevalence defined by whatever circumstances condition cost and benefit. That sensitivity to environmental influences could entail costs is recognized explicitly in differential susceptibility models (see Ellis, Boyce et al., this issue). Belsky (1997, 2005) argues, for instance, that responsiveness to environmental differences should be favored where conditions of early life provide reliable cues to circumstances that are likely to prevail at maturity, but that absent such predictability, parents' reproductive fitness would be better served by conceiving offspring of varying, less flexible phenotypes matched to particular environmental conditions ("diversified bet-hedging"). By this reasoning, individual differences in sensitivity to environmental cues (and, by implication, their genetic etiologies) will be sustained in a population hosting environments of both predictability and unpredictability. In a second model, Boyce and Ellis (2005) propose that individuals vary in their "biological sensitivity to context" and, in children, have indexed this variability via autonomic and adrenocortical reactivity to standardized laboratory stressors (Boyce et al., 1995; Ellis, Shirtcliff, et al., this issue). The tempo of pubertal development among high stress-reactive children was found to be, respectively, delayed and accelerated in families of high- and low-quality parent-child relationships, relative to children exhibiting less stress reactivity (less biological sensitivity to context). Heightened reactivity may be seen as promoting developmental outcomes best adapted to rearing conditions of either advantage or adversity, but the same reacitivity to stress that supports conditional adaptation is also known to promote stress-related psychiatric and physical disorders, including pathologies associated with premature mortality (e.g., atherosclerotic cardiovascular disease) (Boyce & Ellis, 2005; Manuck, 1994; Manuck, Marsland, Kaplan & Williams, 1995). Thus, Ellis, Boyce, et al. (this issue) suggest that while fitness benefits may accrue from context-dependent biological sensitivity in the most positive and negative rearing environments, the majority of children, who are raised in normative family circumstances, may instead fare better if they are less sensitive to context.
These several propositions frame differential susceptibility in an evolutionary context and, in explaining how developmental phenotypes of more and less plasticity may co-exist through a balance of fitness benefits and costs, they also help explain how specific genetic variation associated with these differences might be retained in a reproducing population. Accounting for the persistence of a given gene polymorphism (such as ESR1 variation) via fitness effects of reproductive strategies that are entrained by differential susceptibility to varying childhood environments is still, though, only a theoretical possibility. Such speculation may be critiqued, if applied in the present context, for privileging a particular phenotype – pubertal timing – out of the myriad physiological events affected by estrogen signaling, so that any single instance of gene × environment interaction involving the estrogen receptor (or other molecule of similarly extensive biological action) is most likely a by-product of other regulatory processes (Stearns, 1992; Manuck, 2010). On the other hand, this objection is mitigated if timing of reproductive maturation predicts womens' later reproductive histories, as proposed in the foregoing evolutionary models.
To our knowledge, though, it is not known whether women's lifetime reproductive success is either correlated with age at menarche or, as a result of environmentally contingent (facultative) differences in reproductive strategy, equivalent in women who reached sexual maturity earlier versus later. The question, at least as an evolutionary proposition, may also prove difficult to adjudicate in modern societies, where contraception now frequently renders childbearing an active decision rather than a byproduct of sexual activity. And where menarche has been studied in relation to reproductive outcomes in non-industrialized societies, results may be confounded by dissimilar social and family structures and a proportionately greater impact of other environmental influences on pubertal development, such as variation in nutritional status, health, and body weight. Among the rural Kipsigis of Kenya, for instance, early maturing girls bear more living offspring over their reproductive lives and command a higher brideprice than girls who reach menarche later (Borgerhoff Mulder, 1988, 1989). Similarly, in the Pumé foragers of Venezuela, earlier age at first reproduction predicts higher completed fertility unless the mothers are exceptionally young at the birth of their first child (<14 years, when risks of infant mortality outweigh the benefits of early childbearing) (Kramer, 2008). Psychosocial influences on menarcheal age have not been modeled in these foraging populations, however, and owing to differences in family constellation, rearing practices, and cultural norms, might differ appreciably from those observed in study samples drawn from industrialized, Western societies.
Returning to the present findings, it may also be asked to what extent our observations really reflect an interaction of genetic and environmental variation. In this regard, it is noteworthy that personality traits previously shown to explain heritable variation in reported family conflict and cohesion, though correlated in expected directions with FES scales here, did not account for the interactive effects of ESR1 variation and family environment on age at menarche. This would seem to credit speculation that our findings reflect gene-environment interaction, even though our measures of early family environment are self-reported and retrospective. However, should any other heritable, but unmeasured, determinants of reported family relationships exist in this sample or when using these instruments, our measurement of the family "environment" would merit at least a partly genetic interpretation. Where genetic and non-genetic influences on ostensibly environmental measurements cannot be partitioned by study design (as may be achieved in certain twin and adoption studies), then, inferring gene-environment interaction remains ambiguous. As a result, genotype-dependent associations of early family environment with menarcheal age in the present study could conceivably entail interactive effects involving other genes (epistasis), strictly environmental factors (gene-environment interaction), or both.
Finally, we wish to point out several methodological and interpretive limitations of this study. Although a convenient starting point, for instance, retrospective report cannot substitute for prospective investigation, and reliance on adults' self-reported early environments is less informative than observational and contemporaneous family measurements. In addition, findings reported elsewhere suggest that father-daughter relationships predict pubertal timing uniquely or more strongly than mother-daughter relationships, that positive family relationships are more predictive than negative relationships, and that the frequency of father-daughter interactions (positive and negative) predicts later pubertal development (Ellis & Garber, 2000; Ellis et al., 1999; Moffitt et al., 1992). Unfortunately, the FES Conflict and Cohesion scales we administered do not index parent-specific behaviors or frequency of interactions and, while there was a slight indication that menarcheal age might covary more reliably with family cohesion than with conflict within "environmentally sensitive" ESR1 genotypes, common method variance may have rendered these scales too highly correlated (inversely) to detect differential effects. Our findings are also restricted to observations on non-Hispanic Caucasian women, so that their generalizability to other populations is unknown, and to limited variation within a single gene. And last, while menarche is a reliably measured event, progression through puberty encompasses other development milestones, including notably the appearance of secondary sexual characteristics. In sum, subsequent research would benefit from studies of longitudinal design that entail observational measurement of the family environment, broader participant sampling, greater assessment of relevant genetic variation, and inclusion of multiple indicators of pubertal timing. At present, though, our findings afford initial evidence that familial influences on the pace of pubertal maturation vary in magnitude among individuals and do so in relation to genetic differences plausibly implicated in pubertal development.
This research was supported, in part, by National Institutes of Health Grants PO1 HL040962 and RO1 HL065137
1As noted earlier, the present analyses included only women who were raised to adolescence by their two biological parents to prevent potential confounding of reported family environment with variation or change in family structure (e.g., parental divorce, father absence). This led to the exclusion of 73 AHAB participants, 69 of whom had been genotyped successfully for rs9340799. Regression analyses conducted on the full cohort (n = 524), though subject to the interpretive limitations identified above, were similar to those reported for the sample of 455 women raised in stable families by two biological parents. Thus, genotype predicted menarcheal age in interaction with both QFE (b = .229, se = 0.092, t = 2.50, p = 0.013) and family Cohesion scores (b = .256, se = 0.094, t = 2.74, p = 0.006), but not with family Conflict alone (b = .163, se = 0.090, t = 1.84, p = 0.071).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
My 12-year-old daughter wanted me to make her for Christmas a Star Trek Klingon bat'leth sword prop to hang up in her room. So I made one from plywood. It's really a very simple project. The basic steps were: measure, make and print a template, cut out with jigsaw, paint with metallic paint, wrap handles with leather cord.
- 4'x2' sheet of 1/4" plywood (I used cheap pre-sanded birch from Lowes; make sure it's decently flat, straightening if needed)
- glue (Titebond II)
- metallic silver paint (DecoArt)
- leather cord (or paracord or scrap leather)
- paper (for template)
- masking tape (for template)
- computer and printer
- jigsaw with blade for curves
- drill with hole saw (optional but makes inner holes easier)
- scissors
- paint brushes
- woodworking vise (or clamps)
- measuring tape
- ruler
- pencil
- cloth
- box-cutter
I also had a slip in the cutting process which required patching the wood. I ended up patching with JB Weld.
Confession: While this was a Christmas present, I only finished it by December 30.
Step 1: Measure
Bat'leths are sized by going down on one knee and measuring from the floor to the tip of the head. In our case, it was 45 inches.
Step 2: Template
I traced a bat'leth in Inkscape from a photo I found online, and resized it to the 45 inches that I needed.
Of course, I don't have a printer that can print something this size, so I overlaid a grid of 7.5"x10" rectangles, and printed out each rectangle as a separate page (shifting the drawing over in Inkscape as needed).
Here's the Inkscape svg file for my template. You'll want to resize the bat'leth to your dimensions, and then perhaps add more page-sized rectangles. To resize, load the template in Inkscape, click on the bat'leth itself, choose Object | Transform | Scale via the menu. Click on Clear. Select in (or a metric unit if you prefer) from the unit drop-down. Make sure Scale proportionally is checked. Put in your desired width. Press Apply. Then select the whole drawing (ctrl-a), and shift it around, printing one page at a time (you can see in Inkscape which area of the drawing will get printed), using the grid as a guide to make sure you're getting everything. (You may want to add more grid rectangles, too.)
Cut margins as needed and tape sheets together.
Cut out the template.
Tape to wood and trace with a pencil. (I then drew the lines where the tape was by hand.)
Step 3: Cut Outside
Cut the outer outline of the bat'leth with a jigsaw. Use a sharp and good quality blade (I used a Bosch T101AO) to give a smooth cut. I clamped the sheet to a work table, and cut, moving the sheet and re-clamping as needed. To avoid vibrations, don't cut too far from the table.
This was my first time cutting curves with the jigsaw, and it went very nicely, which I attribute to the good quality blade.
Step 4: Cut Inside
I used a 1.5" paddle bit to drill circular holes at the two ends of each of the areas, flush to the tracing, making sure that the bat'leth was clamped to a piece of sacrificial wood (to prevent a jagged edge on the bottom side), and then just joined up the holes with the jigsaw. If you are more skillful with a jigsaw, you could just drill a smaller hole and do the whole cut with the jigsaw.
(Unfortunately, the sacrificial lumber I used had a hole in it, which made the drill jump and one of the circles be really crooked. Luckily no one got hurt, and I ended up filling the defect in with JB Weld.)
Step 5: Sand, Prime and Sand
Sand carefully. I went to 320 grit.
Then I "primed". I'm cheap, so I just painted the whole thing with wood glue (Titebond II) diluted 1:1 with water. Then after that dried, I sanded it again. It was easy to make it very smooth at this point. I filled in some dents and small voids around the edge with undiluted wood glue.
Unfortunately my "priming" method didn't work that well with the acrylic paint I used later, which didn't adhere as well as I'd like. If I used spray paint, it might have adhered better, but I tend not to do well with spray paint.
Step 6: Paint, Paint, and Paint
I then painted several thin coats of DecoArt metallic silver. On the two sides, I used very long strokes with a foam brush, and I did the edges sometimes with a cheap small brush and sometimes a foam brush.
I didn't sand between coats except where the paint made drippy marks, because sanding would tend to remove the paint all the way down. (Not so great adhesion, I guess. Spray paint might have been better, but I don't have spray paint skills.)
Surprisingly, I used less than one of the 2 oz bottles.
I really like the DecoArt acrylic craft paints, and have been using them for about 10 years.
Step 7: Handles
I wrapped the handles in flat (about 1/8" wide) leather cord. Some rough calculations suggested I'd need about 35 feet of it so we bought a 50 foot spool.
More careful measurements suggest that a 5" wrapped handle requires 134 inches of leather cord. More for a larger bat'leth as the handles will be wider.
For each handle, mark off the 5" handle area, and put a generous serving of wood glue on the area (including the two edges).
Then do a tight spiral wrap, tucking the two ends of the cord under the spiral wrap. Work quickly so the glue doesn't set before you're finished so you can clean it.
Tucking in at the start is easy. At the end is a little harder. Make the last three loops loose, and pull the end through. Tighten the loops, and then cut the end. Shove some more glue in.
Wipe excess glue and clean the glue off the cord. (If you worked quickly, the glue wouldn't have set.)
Then clamp. I wrapped the handle with aluminum foil (so it wouldn't stick or get dirty) and put it in a woodworking vise for a couple of hours. Repeat.
Finally, I touched up a few areas of the paint which got scratched in the process or got some glue on them.

subset: commoncrawl


text:
Saturday, February 6, 2016
Foxhole Court & miscellaneous extra content
Lucie mentioned in a recent comment that she's seen some extra Andrew & Neil content floating around -- I think I forgot to mention when I abandoned this blog ((yet again)) that I've sort of migrated over to Tumblr? Not on purpose, mind -- I created a tumblr mostly so I could follow my sisters and a couple friends, and then was somehow spotted by a couple Foxhole Court readers.
Over the course of a couple months I managed to accumulate ... 130? 150? Asks about the Foxes' lives past/present/future, and with help from a friend it's in a somewhat organized list here:
((I really should go back and tidy it up one day, but.))
I ended up having to disable Asks temporarily so I could get out of Fox headspace and try to get back to other projects, but if anyone is bored or curious, there's some extra content for you.
Also, sorry I'm still a failure at updating anything anywhere, but I think ya'll've been around long enough to be used to it. Whoops. It's been a weird couple of months, but I don't have the right words or headspace to sort out a proper explanation. I'll be back when I sort things out, I hope!
11 comments:
- Olga Katri said...
Thank you!!! Been reading all day to catch up!!!
- February 7, 2016 at 10:48 AM
- Alondra Rowel said...
I have to say this again! But please consider writing a book 4 even if it takes time!
- February 18, 2016 at 4:10 AM
- yenifer santiago said...
Yes, Book 4! & You're amazing! Honestly even before I finished the first chapter in Book 1, I knew I was hooked on for the rest of the series and I loved them. Thank you!!!
- February 18, 2016 at 9:55 AM
- D. Michaels said...
I have just completed my initiation into the world of the foxes and exy. Wow. What an amazing read. One of the best I ever read. Unlike many others I don't care if you give up the world of exy, but please keep writing. You are an amazingly gifted and talented author. Please keep up the good work. I wouldn't even mind if you raise the price of your kindle books.
- June 10, 2016 at 11:38 AM
- Alyssa Skvarla said...
So I read these books non-stop and finished one book a day so I'm deep in my book hangover/recovery phase after finishing TKM because everything was amazing about the trilogy (especially your writing!!) I was wondering if there was any place I could either send a letter or email enclosing my love for you and All for the Games because I want to express how good these books were and how I haven't been obsessed with reading something this much for a long time.
- June 23, 2016 at 4:23 PM
- Patricia Robello said...
I just wanted to let u know that I love your series!! Thank u for hard work and dedication!! I'll miss them!!
- July 28, 2016 at 1:22 PM
- Patricia Robello said...
I just wanted to let u know that I love your series!! Thank u for hard work and dedication!! I'll miss them!!
- July 28, 2016 at 1:22 PM
- Alondra Rowel said...
Just bought and received the books on paperback! So excited! Book 4 please!! LOL
- August 6, 2016 at 1:48 AM
- Danny Aguilar said...
- This comment has been removed by the author.
- October 16, 2016 at 5:52 PM
- Lyz Loea said...
Dear Nora,
First off, I'd like to say that I love your books. I'm a student at the Nueva School in California and I was wondering if you would be willing to mentor me in a project–for mine I chose to write books. The project involves finding a mentor and I hoped you might be willing to be mine. I know that your schedule is busy and if you're unable to do this, I completely understand. In any case, thank you for considering this.
Best regards,
Lyz B-P
- November 2, 2016 at 6:07 PM
- Missy Jackson said...
First off, I want to thank you, the wonderfully amazing author and creator of a world I have quickly (over the two-week span it took me to discover, receive, and tear through your novels) adopted into my inner circle (being an avid reader, naturally there is one) of precious and favorite series. I cannot stress enough how much your work inspires me to strive to become the writer I so aspire to be. Your characters, animated and flawed and so darn lovely I have to stop, put the book down, and catch my breathe- I am as in love with them as with your style of writing. I have laughed, cried, and been stricken into utter silence in which I am forced to stop and stand still for a while to let myself catch back up with my mindset. You are so wonderfully talented, it hurts. Everything about this series will stay with me for a long time afterwards, if not forever.
From the bottom, top, and every vessel in my heart: thank you.
Yours sincerely.
- March 20, 2017 at 11:16 PM


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During the Stone Age, dressing for distress was all the rage, because cave persons knew tattered threads would make them less likely to be courted by a handsome triceratops. Dress for success with today's Groupon: for $20, you get $45 worth of designer lingerie at Change, with your choice of locations. Change's emporium of underthings stocks a Danish-designed selection of high-tech brassieres and luxurious lingerie. This vast bounty of dainty drawers and more are neatly folded into a variety of collections that run the gamut of function and form, from sportswear to romantic unmentionables for special occasions such as Poltergeist Awareness Day. The knowledgeable staff uses "on the spot" bra fittings to help customers enhance their natural beauty with the right bra, selected from 90 different sizes to hug every variety of curve. Like a well-oiled skeletal system, a perfectly fitted bra can make its wearer feel more beautiful and confident by giving a slimming effect, helping clothes fit better, and providing stalwart back support. Check out the curvaceous collections for all the strap-and-lace goods.
Change Lingerie was mentioned by She Does the City and Toronto Life, and its Toronto locations have a perfect five-star average from ChickAdvisor reviewers:
- no one will leave with an ill-fitted bra… and guess what – 80% of you are wearing the wrong size as I type (thanks, Oprah.) We've been in enough change rooms to know that breasts don't come in six different shapes and sizes – so why should the fittings? The staff at CHANGE take time to measure you properly – and considering they offer everything from an A to a J – you'll be sure to leave with a bra that will look and feel terrific. Many have even exclaimed "OMG…CHANGE lingerie changed my life!" Seriously. – She Does the City
- It's time for a change in your bra shopping routine, or so says the mantra behind this Scandinavian import that focuses on pairing every gal with the perfect undergarment. Women accustomed to enduring bras that look more like battle armour will be pleased to find a range of attractive pieces, such as a sexy black lace number…. Everyone gets a fitting with a perky but professional saleswoman, and then it's time to start sampling the merch. – Toronto Life
Change Lingerie
Every woman's body is different, which is why the European designers at Change Lingerie have worked to a selection that includes 108 varieties of colours and sizes. Whether lacy, sleek, patterned, or sporty, the designs include everything from supportive full-cup models to strapless bras that stay hidden under football shoulder pads.
Customer Reviews
West Vancouver
Park Royal Shopping Centre North
West Vancouver, British Columbia V7T 2W4
604-913-7792Get Directions
West Point Grey
103-4416 West 10th Ave.
Vancouver, British Columbia V6R 2H9
604-222-2287Get Directions
2815 West Broadway
Vancouver, British Columbia V6K 2G6
604-742-0557Get Directions


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Updated on the 1st of every month
In the realm of fashion, accessories can easily complement your outfit or define your personal style. Unlike clothing, accessories can look as good as new even if it's the third time you've worn it that week. Jewellery is also one of those things that you don't have to feel guilty splurging on - who wants 5 pairs of sweatshop made designer jeans when you can create 5 diverse outfits with just a peacock feathered choker. Whether you have a taste for earrings, necklaces, brooches/pins or cuffs, Montreal offers it all - from the generic runway styles to one-of-a-kind handmade finds.
Earrings have a way of completing an outfit (I feel naked if I don't have any on). Each time you wear a pair of earrings with a different outfit, you create a little bit of your own characteristic flare. Adorn a pair of red coral hoop earrings with your comfy jeans and a tee or wear it with that amazing dress that you never seem to have an occasion to put it on.
Tips for this season's earrings: the longer the better. Oblong (fancy word for long, skinny rectangle) is in style. Along with the rest of the 80's clothing craze, the earrings have caught on too. Look for stripes, neon and mod plastic pieces. For more exquisite taste, there are the dangling, feathered, and chandeliered styles. You can find these at standard trendy stores but better to hunt for one-of-a-kind's to avoid being a walking display of mass production. You'll also start seeing more long chain-like earrings - I've seen these decorated with skulls for the goth-chic and with sequins and crystals for the more punk-averse.
Necklaces come in every shape and form. To accentuate them as an accessory and not just your daily necessity, pile on the strands. Multi strands of beads, chains and pearls linked together are in 'la mode'. For a DIY version, wear a couple gold (or silver) chains together for the same effect, or gather beaded and chained necklaces and wear them together. A funky alternative are wrap necklaces. Worn with a plain coloured top or a plunging neckline, the two end strands of the accessory should dangle like icicles of gemstones or long feathers.
Other accessories making its way into the streets are bracelet cuffs and brooches & buttons. The first sighting of these cuffs began in the 80`s in the form of sweatbands paired with full Adidas jumpsuits. Now, they have evolved into the leather studded sort. More recently, the style of these cuffs have been ranging from lace, denim, covered in belt buckles to cuff wrist watches. People are fastening buttons to their blazers, shirt collars, bags, and sweaters. These buttons make a personal statement, such as: a devotion to a band, political cause, sexual orientation (my personal favourite read, "I Like Dudes") or simply serves as a purpose for accessorizing. Tip: there is a guy who sells these around McGill campus and tamtam's for very reasonable prices. Vintage brooches are good finds in any boutique, antique store or any thrift store (try value village). These can add sophistication to your look with a well-chosen cameo, diamond studded piece or unique find. Sexy flower pins also make any background material look striking - fasten one to your bag, purse or coat lapels.
To those of you already titillating your fashion senses with accessories, I say no more. For those who have yet to try glamming up an outfit with just a pair of earrings, venture out to the following suggested locations you never know, you may feel dressed with nothing but a pair of earrings on.
Lola and Emily
3475 St-Laurent Blvd.
3762 St-Laurent Blvd.
Bleu comme le ciel
2000 Peel
More generic stores, but decent buys if you search:
Aldo Accessories
Centre Eaton (St-Catherine)
Le Chateau
Centre Eaton (St-Catherine)
Alexis Nihon (Atwater)
Les Promenades de la Cathedrale (St-Catherine)
Latest Article :: Christina Aguilera
Archived Articles :: [ALL archived articles here]
Spring 2005 Fashion Preview
Crunk & Funk Fashion at Stereobar
Montreal Fashion Week
Stylin' 20 Below
Fashion Flashback 2003
To Chabanel for a Little Chanel
Improve Your Style with Accessories
The 80's are Back...And So are the 70's
The Urban Fashion Index


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Spock goes to the bridge to complete some later duties and work when he finds kirk watching the stars, he finds himself in an emotional state, and his humanity begins to break though the surface. Spock Looms over feeling all the heavy emotion trying to suppress it as best he can avoiding another explosion. He is brought to sickbay to meet and attempt mind meld with a strange new man found in the sick bay. (Spelling and grammar errors.) POV: Spock
pinkninjasquad Oct 26, 2016
Yeah... spelling errors are one thing but getting one of the main characters' name wrong...?
Haunt411 Oct 13, 2016
Trust me honey no one really knows what love is. What is love baby don't hurt me don't hurt me no more.
glitterasthetic Feb 04
great, now im thinking of a board with legs, pointed ears and black shiny hair
redspiderliles Feb 15, 2014
As a new trekkie, I'm finding that I'm loving this pair more and more!!! *fangirls into oblivion*
CrystalTheMineral Jan 24, 2014
I really really love this right now! please keep writing your amazing!!!!
PyroWaffle Jun 04, 2013
@JessicaShortyyRaiinb oh well you know, I provide the pervyness to the people who need it XD LOOL


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|Prep Time: 30 min
|Serves: 24
|Cooking Time: 60 min
|Yield: 6 qts
Nutritious soup, worth the extra work!
- 1 1/2 cups Lentil
- 4 medium Onion, Sweet
- 6 medium Zucchini (raw)
- 3 Leek
- 3 cups Kale
- 4 cups Carrot Juice
- 1 tbsp Table Blend Seasoning Blend
- 1 cup Raw Whole Cashews
- 8 oz Portobello Mushroom
- 6 cups Water
*VegiZest - from Dr. Fuhrman's website. May use Mrs. Dash instead.
*I used portobello and crimini mushrooms; may use shitake, and/oyster as well.
*Dr. Fuhrman recommends juicing 5 pounds of carrots to make the carrot juice; 2 bunches celery to make the celery juice. (May replace 2 cups of water with 2 cups celery juice. I made without celery juice.
Place beans/lentils/dried split peas and 4 cups water in a very large soup pot over low heat.
Add whole onions, whole zucchini, and whole leeks to the pot along with the chopped kale. (May use chopped collard greens or other greens). Add carrot juice, 2 cups water (or 2 cups celery juice), Mrs. Dash and 2 tbs. VegiZest (Dr. Fuhrman).
Simmer mixture until onions, zucchini, and leeks are soft, about 20 minutes. Remove the whole veggies from the pot, along with some of the soup liquid, being careful to leave the beans and some of the kale in the pot.
Using a blender or food processor, complete blend/puree the whole veggies. Add more soup liquid and the cashews to the mixture and blend thoroughly. (I had to blend in 2 different batches.)
Return the blended, creamy mixture back tot he pot. Add the mushrooms and simmer for 30 minutes or until beans are soft.
Serving size - 1 cup


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I'm keeping at it! My work life is far from zero waste but as far as I can tell, that can't be helped very much. That said, I've been doing quite well with my home life! So what's day 2 of the Zero Waste Nerd's challenge?
So today I did as I always did. I brought my stainless steel Starbucks tumbler along, and used it all day, as is the usual.
After work I went to Goodwill to see if I could get any deals for anything that'd be good to resell on my Poshmark and to take a look at scarves. Wednesdays are 20% student discount day, so I stocked up! Scarves are great for bento backgrounds(not that I've been doing much of that lately) and wrapping presents. Once I learned about the scarf thing, I loved the idea. I'm horrible at getting presents to wrap up the way I want them to, they look lumpy, the tape doesn't stick, etc. If you want to learn about how to wrap all manners of things in scarves, there's websites dedicated to Furoshiki.
A photo posted by Grace McCarter (@thebentobuff) on May 25, 2016 at 1:46pm PDT
The untagged scarves are more expensive, generally $3 or $5, I always forget which. I try to stick to the ones tagged at $1. I bought a hefty number of scarves, a Kate Spade wallet, and a shirt. To me it was a really nice haul. I'd looked in my suitcase last week and was dismayed at the lack of warm weather tops I have, so this is a great addition. The Vera Bradley scarf will likely go up on Poshmark. It's cute but I don't think it quite matches my aesthetic.
When I handed the cashier my reusable shopping bag, she put everything in a plastic bag before attempting to put that in the reusable bag. I'd spaced out and hadn't noticed until she said something. We had a bit of a chuckle over that. I haven't seen too many people using reusable shopping bags coming out of Goodwill in all the time I've been going there, so I'm assuming it's a force of habit for her to bag things in those plastic bags.
Afterwards, I had to go to Giant to buy some margarine. This time I bought the sticks of Earth Balance as opposed to the usual tub, meaning at least most of the packaging should be recyclable(the individual sticks are lined with a foil-like thing. I've already sent them an email asking what they are and if they're recyclable). I was shocked to see how much cheaper the sticks were per pound than the tubs! I should have snapped a photo but the tub was close to twice the price per pound as the sticks! That just goes and gives a big old nod to all the zero wasters that have been discussing just how much money we've been paying for packaging.
I have been using Mint for tracking my expenses all year. I'm excited to see how my expenses for this transition month, versus before and after are in terms of my bank statement.


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Budget Hotels in Laka
About Laka
Laka, the fields where we put up our tents, is worth a visit for its mysterious surroundings with enormous boulders and the most beautiful sunset you will ever see! The place is immensely beautiful with greenery and beautiful horses all around. But be aware of all the cattle dung you would have to deal with. There's no choice but to accept it as your ground, walking sitting and chilling on it!
How To Reach Laka
Book Laka Tour Package
0 Hotels found


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Helicopter Services in Uttarakhand during Char Dham Yatra
|Helicopter Service
|Packages Offered by Helicopter
|Base Stations
|Prabhatam Aviation
|Kedarnath, Badrinath, Do Dhams, Char Dham Yatra
|Delhi, Dehradun, Phata
|Timberline Helicharters
|Kedarnath, Badrinath, Do Dham, CharDham Yatra
|Dehradun, Harsil
In today's world, pilgrims no longer need to spend days of agonizing walking/ trekking in order to offer their prayers if their health does not allow them to, or if they are constrained for time. The helicopter service we operate to the holy temples in Uttrakhand helps them do it in an easy and hassle free way.
Dear Sir/Madam,
We are a Family of 4 and wish to go to Kedarnath Temple on 15th May from Phata , Guptkashi or any other such location from Helicopter and come back on the same day .Please let us Know the Availability and Charges for the same . Your Early Reply is Requested . My Mobile No. is XXXXXXXX in case you wish to contact me .


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Why is your current title important to you?
The Mr. Regiment title represents who I am as a person. To be honest, I didn't know much about the Regiment of the Black and Tans until I met Leo Iriarte and Esteban Bartholo, a couple that has been heavily involved in the leather community for many years. It was when Leo and Esteban introduced me to the organization that I became interested. Because of the Black and Tans' purpose and objective to "foster brotherhood and be of service to the community," I grew to love and respect its members. Through these men, I have learned to grow in my identity, as a Leatherman, and grow in my understanding of masculinity and sexuality. In addition to this, the title of Mr. Regiment is important because I can be myself: a proudly educated, passionate, sex-positive professional who seeks brotherhood and to be of service to others.
In addition to representing a group of men unified by a fetish for leather uniforms, Mr. Regiment also represents sex positivity and philanthropic support of a variety of communities. For example, I have seen previous title holders of Mr. Regiment who are dedicated to the leather community and provide financial support and care to others. In addition to this, these men have numerous sex parties and donate the money to the AIDS Lifecycle. Through these events, I witness these men express their sexuality with dignity and pride. This is who I am and who I want to be, and that is what Mr. Regiment represents to me.
What do the colors of the leather pride flag mean to you?
I smile at this question because I tend to use emoticons constantly – especially the heart emoji. That is what the leather pride flag means to me. It is a flag about love, acceptance, and pride in your sexuality through leather. I could Google the meaning of the colors and the history behind it and basically copy and paste, but I am not doing that. Instead, I will tell you a short story. About a year ago, I began a job as an Analyst for a bank holding company in Pasadena. When I got to the location on my first day, I drove around the parking lot looking for a space to park in. I found an open spot, and right next to me was a car with a Leather flag sticker. For about six months, I would always park next to the car hoping to see the driver. I never did, and I still haven't. However, every time I park next to the car with the Leather flag sticker, I feel comfort. I feel a sense of unity. I feel safe. I feel horny. I feel like we know each other, through the flag and the community to which we both belong.
If somebody contacted you about visiting LA, what would be on your must see/do list?
The first thing that came to mind was Elysium Park because I cruise there, and I have had some crazy hot sex there. But I also think about places like Slammer, the Zone, and Midtown Spa for some fun. Also, I would hope that this person would be come during the summer because there are tons of play parties, especially the Payaso pool parties and Stan's naked parties (splash and pig emoji).
But for others that may like to enjoy Los Angeles in a different way, I would recommend a visit to the ONE National Gay & Lesbian Archives near USC, the Tom of Finland house in Echo Park, and the bars of Los Angeles like the Eagle, the Bullet, Precinct, and Faultline.
What is your favorite movie and why?
My favorite movie is based on a true story set in the early 1900s called "The King of Devils Island" because there is a metaphor that is told throughout the movie. The metaphor is about a whale that is captured and is ultimately killed. The story goes that the whale fought and fought and fought all day, and didn't give up until its final breath. At the same time (in the movie), there is a teenage boy sent to a rehabilitation home on an Island where clergy are supposed to help those admitted. Instead, the clergy mistreat and abuse them. The relentless boy becomes a protector of another boy which is targeted by the clergy. As the story develops, the viewers find that the teenage boy does not give up and thus is like the whale in the story. This relentless boy is very much like me.
What is the funniest thing to happen to you during sex?
…I can't think of anything funny. I guess something that is close to being funny is that someone threw up on my dick, and I make them clean it, with their mouth. I said, "well it came out of you, so clean it up!" (Hey, some guys like that. Who am I to judge?)
What do you hope to accomplish in your title year?
I don't know yet. I will know when I get there, but I have some ideas. I would like to do some kind of Latino Tom of Finland art juxtaposition. I would like to continue the sex parties to raise money for various communities in need. I would also like to strengthen the understanding about the leather community in mainstream society by creating a documentary around title holders. And I am also inspired by Jeffry Erdman's activism/passion with the aging community. I don't know yet. I am very teachable, so with guidance and the passion I bring, I know I will accomplish what I set out to do.


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power-on self testAbbreviated POST, a diagnostic testing sequence run by a computer��s BIOS as the computer��s power is initially turned on. The POST will determine if the computer��s RAM, disk drives, peripheral devices and other hardware components are properly working. If the diagnostic determines that everything is in working order, the computer will continue to boot.
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After spending almost three months in prison, rapper P-Fla is set to thrill his fans in a concert dubbed welcome back P.
The party is organized by his close friends and will take place on 2nd march at Relax with us bar.
While speaking to Rwandashow.com, P-Fla requested fans to turn up in big number.
"It will be a good time for me to share my precious time with fans after spending three months in prison without performing for them," said P-Fla.
He added that people should come and see a new changed P-Fla because the jail time made him learn a lot.
The rapper was jailed last year following allegations that he is on drugs. He will be accompanied by girl friend Al poeta, for the show.


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Contact Us 4 weeks ago
TOYOTA PREVIA S/C AWD - $500 (Staunton)
Motor has a bad head gasket and i dont have the time to work on it. Has a new radiator and hoses. Selling for 500. Give me a text if interested: 5 4 0 3 2 4 5 4 5 1
4 weeks ago
Harrisonburg, VA
Contact Us
$3,300.00 1 month ago
Toyota Previa Van Camper AWD Runs Great! Extras - $3300 (Durango)
1991 Toyota Previa AWD (all wheel drive) 257,000 milesPower doors, windows and mirrorsAuto transmission, shifts perfect and cleanUpgraded to 15" rims Goodyear Viva 2 tires have 90% tread leftAC worksCruise controlNew stereo and speakersUpgraded windshield wipers to modern designRoof rack (I can sweeten the deal with 2 roof mount bike racks)2" towing hitchTinted windows (rear window and some spots
1 month ago
Western Slope, CO
$2,000.00 1 month ago
92 Toyota previa - $2000 (Augusta)
I have a 1992 Toyota previa van. These vans are very addictive and cool as can be. Very tough and almost indestructible it just goes and goes. Runs and drives excellent. Rebuilt JZE2 in-line four-cylinder with plenty of power and excellent on gas. These vans are rear wheel drive with disc brakes all the way around. Will spin the tires LOL. Very reliable and fun to drive. This one is fully loaded W
1 month ago
Augusta, GA
$2,200.00 2 months ago
1994 Toyota Previa - $2200 (Ellisville)
Up for sale is a 1994 Toyota Previa. Spacious and reliable van. Running condition with clean title. There's a leak in stering fluid a problem that can be easily fixed. 199k miles.
2 months ago
Hattiesburg, MS
Contact Us 5 months ago
1993 Toyota Previa Van
5 months ago
Las Cruces, NM
Contact Us
$1,600.00 6 months ago
It's 1993 Toyota previa with rebuilt engine . Tires are new,new break pads , new shocks and struts and radiator . It is a little ugly but engine is really good and it has 70k on it . I built a bench that turn in to bed and you can put your stuff under the bench . I drive from Portland to ukiah and have this vehicle for 3 months . I never had any problem . Odometer says it's 300k but its rebuilt en
6 months ago
Mendocino, CA
$3,500.00 6 months ago
1995 toyota previa LE supercharged minivan - $3500 (mattoon, il)
2wd supercharged, rare white color, spoiler, bra, pivoting captain chairs, leather interior which has recently been redone, looks great other than passenger seat with a few small tears which is the only surface that was not redone. These are great, reliable minivans, just read the online reviews on Edmunds, etc. Clean title and carfax, I have owned since april, 2015. I wanted another previa with n
6 months ago
Mattoon, IL
$2,000.00 6 months ago
1991 *Toyota Previa* Deluxe - $2000 (*Toyota Previa* Financing Options Available)
Lowest tax rate in the state! 1991 *Toyota Previa* DeluxeAvailable at: Anderson Toyota Scion ☎ Phone:(779) 208-4159 ext: 0095 Year: *1991* Make: *Toyota* Model: *Previa* Series *Deluxe* Exterior Color: Burgundy Interior Color: Engine: 2TZ-FE Doors: 0 Odometer: 157149 Stock #: B12642A3 VIN: JT3AC11R1M0002722**Toyota Previa**, CAL TODAY!!!! 157K MILES!!!! HARD TO FIND PREVIA!!!! CONVENIENCE P
6 months ago
Rockford, IL
$600.00 7 months ago
1996 Toyota Previa CHEAP Price! - $600 (santa margarita)
For Sale.. Running 1996 Toyota Previa. As is, for parts or repair. Currently non opted..Lots of new parts recently..Semi new radiator ( a year or two old) .battery,brakes,thermostat,water pump,rear shocks, plugs and more etc. Interior is good, minus the drivers seat wear. Starts fine.The van seemed, to want to over heat,I was close to home, so I parked it, and got something else. I haven't seen an
7 months ago
San Luis Obispo, CA
$1,550.00 8 months ago
1994 Toyota Previa DX!! - $1550 (Pacific Grove)
1994 Toyota Previa DX, RWD, golden/beige. Price reduced to sell.237700 miles, original owner, no accidents, clean title.Runs great -- smooth and strong. Issues: Vibration/hesitation at cold start. Coolant internal leak. Sometimes some white smoke from exhaust at start. Possible problem: cylinder head gasket.Cylinder head replaced 60000 miles back. Smog test just passed in September 2016.Registrati
8 months ago
Monterey, CA
$3,900.00 9 months ago
Toyota Previa 25 mpg 4 cylinder Best designed Japanese van EVER - $3900 (Eugene)
Up for grabs is my legendary Toyota Previa vanThe Japanese were definitely Taking mushrooms when they designed this van With a rear wheel mid engine powered van this thing handles like nothing I've ever driven it can drift side ways in the rains taking a off ramp going 80 mph without a sweat and you feel like your driving a ball or a cloud These vans get the best fuel mileage 22/28 miles per gallo
9 months ago
Eugene, OR
$1,450.00 10 months ago
TOYOTA PREVIA 92 - $1450 (Houston)
Toyota previa Years 1992 engine v6 good Transmission automatica run good $ 1,450.00Please call, or Ingles Español
10 months ago
Galveston, TX
$1,900.00 10 months ago
$1,900, 1991 Toyota Previa Used Car Lot OH
Showen Motors734 S. South St. (US 68 South) Wilmington, OH937-382-47991991 Toyota Previa DLX$1,900Year:1991Make:ToyotaModel:PreviaTrim:DLXStock #:6520VIN:JT3AC11R6M0001064Transmission:AutomaticExterior Color:WhiteInterior Type:Cloth Mileage:137950Primary Contact:Brian or BradPhone:937-382-4799Vehicle CommentsVisit Showen Motors online at www.showen4cars.com to see more pictures of this vehicle or
10 months ago
Cincinnati, OH
$900.00 10 months ago
Toyota Previa Van - $900 (Hickory)
Van runs good. Slight oil leak somewhere. Engine runs cool. Fluid levels stay full. AC does not work. Radio works, but CD player does not. Has been very reliable transportation. 1997 Toyota Previas were 5 star vans in their time. Vinyl is torn on driver's side door (see pic). Carpet and upholstery in very good shape. Never smoked in. Rear wiper (comes with vehicle) needs to be installed, and needs
10 months ago
Hickory, NC
$900.00 11 months ago
Toyota previa - $900
Hi Craigslist I have a Toyota previa for sale it has high miles and needs a oil change oil light just came on today its been snogged an tagged if your interested please call or text the number can be seen in the upper left hand corner click the reply box thank you.
11 months ago
Hanford, CA
$1,300.00 11 months ago
Toyota Previa - $1300 (Lansing)
Toyota Previa 93' rear wheel drive 2.4L, 4cyl, great van, $1300, will probably need break lines replaced and possibly gas lines, about 151k miles, (not original picture) I will post more pictures when i get a chance. van will need work has been sitting for some time and does not start.
11 months ago
Ithaca, NY
$1,500.00 11 months ago
Toyota Previa - $1500 (Lansing)
Toyota Previa 93' rear wheel drive 2.4L, 4cyl, great van, $1500/ obo, will probably need break lines replaced and possibly gas lines, about 151k miles, (not original picture) I will post more pictures when i get a chance. van will need work has been sitting for some time and does not start.
11 months ago
Ithaca, NY
$600.00 11 months ago
1992 Toyota Previa Van - $600 (Lompoc)
1992 Toyota Previa Van Tow company lien sale, Runs good,newer tires 437-b North 'R' street Lompoc,Ca M-F 8:00-12:00 1:00-5:00 Please park on 'R' street no calls. $600.00 OBO
11 months ago
Santa Maria, CA
$2,500.00 11 months ago
1991 Toyota Previa ALLTRAC 5-speed - $2500 (Skull Valley)
Good running Toyota Previa ALLTRAC with 5-speed manual transmission.Cosmetically it needs some TLC, like replace a broken headlight, and is ready for new shocks. Cost of that comes to about $600 total if done by a professional. Less if you can do it yourself with parts.Engine and transmission are good. Only has 212,000 miles. These cars easily go 500K and more.Has good tires, just had new oil chan
11 months ago
Prescott, AZ
$1,000.00 1 year ago
1992 Toyota Previa Van - $1000 (Lompoc)
1992 Toyota Previa Van Tow company lien sale, Runs good,newer tires 437-b North 'R' street Lompoc,Ca M-F 8:00-12:00 1:00-5:00 Please park on 'R' street no calls. $1000.00 OBO
1 year ago
Santa Maria, CA