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Endocrinology Vol. 147, No. 11 5132-5138
Copyright © 2006 by The Endocrine Society

A Quantitative Trait Locus on Chromosome 6 Regulates the Onset of Puberty in Mice

Brandon M. Nathan, Craig A. Hodges, Pamela J. Supelak, Lindsay C. Burrage, Joseph H. Nadeau and Mark R. Palmert

Division of Pediatric Endocrinology and Metabolism (B.M.N., M.R.P.), Rainbow Babies and Children’s Hospital, University Hospitals of Cleveland, and Departments of Pediatrics (B.M.N., C.A.H., P.J.S., M.R.P.) and Genetics (L.C.B., J.H.N., M.R.P.), Case School of Medicine, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Mark R. Palmert, M.D., Ph.D., Division of Pediatric Endocrinology and Metabolism, Rainbow Babies and Children’s Hospital, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: mark.palmert{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Puberty is a fundamental developmental process experienced by all reproductively competent adults, yet the specific factors that regulate variation in its timing remain elusive. Using a new approach to identifying these factors, we have performed a survey among a panel of chromosome substitution strains (for inbred strains C57BL/6J and A/J) followed by linkage analysis to map a quantitative trait locus (QTL) on the distal end of chromosome 6 that regulates pubertal timing (as assessed by vaginal opening) in mice. The location of the QTL was then refined to a region between marker D6MIT59 and the end of the chromosome by generating and phenotyping a panel of 12 congenic strains, each with a unique and overlapping homozygous segment of the A/J chromosome on an otherwise uniform C57BL/6J genomic background. Additional characterization of the QTL indicated that the effects of the responsible gene(s) are gender specific and inherited in a codominant manner without parent-of-origin effects. These findings represent an important advancement toward identification of novel factors that regulate maturation of the hypothalamic-pituitary-gonadal axis and determine the timing of puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PUBERTY IS A critical developmental process marking the transition into adulthood. Although the timing of the onset of puberty is influenced by environmental factors, it is increasingly recognized that at least 50% of the variation within the general population is genetically determined (1, 2, 3, 4). The genetic factor(s) that regulate variation in the maturation of the hypothalamic-pituitary-gonadal (HPG) axis remain largely unknown and are the focus of much ongoing investigation. Identification of these factors will be important to our understanding of pubertal development and will likely improve our understanding of the pathophysiology of many reproductive endocrine disorders, such as precocious and delayed pubertal development and hypogonadotropic hypogonadism.

The timing of puberty in mice is also genetically regulated (5, 6, 7). Although there are some species differences between mice and humans (3, 8, 9, 10), the presence of common reproductive endocrine pathways (11) makes the mouse an appropriate model for investigating the genetic regulation of pubertal timing. Genes and pathways shown to modulate the onset of puberty in mice would be high-likelihood candidates for regulating maturation of the HPG axis in humans.

To capitalize on the advantages of the mouse model, we have taken a new approach, the analysis of chromosome substitutions strains (CSS), to investigate the genetic regulation of pubertal timing. Evaluation of the timing of puberty in these strains, as assessed by vaginal opening (VO), revealed that chromosome 6 harbors a gene or genes that regulate the onset of puberty (12). We now report the use of linkage analysis coupled with the generation and evaluation of congenic mice to map and confirm a quantitative trait locus (QTL), identified through a whole-genome approach, that regulates the onset of puberty.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
A full panel of CSS has previously been generated for the inbred C57BL/6J [The Jackson Laboratory (Bar Harbor, ME), JR000664, referred to as B6] and A/J (JR000646) strains through a collaboration between the Department of Genetics at Case Western Reserve University and the Broad Institute (13). In the CSS panel, a single chromosome from a donor strain (A/J) has been substituted in a homozygous fashion for the corresponding chromosome in a host strain (B6), neatly partitioning the genome into 22 strains (19 autosomes, two sex chromosomes, and the mitochondria) that reside on a defined and uniform genetic background. These mice are available from The Jackson Laboratory.

For the current study, mice from the CSS with A/J chromosome 6 substituted into the B6 background (referred to as B6–6A) and from the progenitor B6 strain were studied and used to generate F2 and congenic mice. In addition, A/J mice were used in the analysis of ovarian follicle development and male pubertal timing. Because there was no evidence of parent-of-origin effects in our previous studies (12), B6 and B6–6A mice were mated in reciprocal [(B6–6A male x B6 female) and (B6 male x B6–6A female)] crosses to generate an F1 population that was heterozygous for A/J and B6 chromosome 6 on an otherwise uniform B6 genomic background. F1 mice were mated to generate F2 offspring that were then used in linkage analysis and generation of congenic strains.

Twelve congenic strains, each with a unique and overlapping homozygous segment of A/J chromosome 6 (see Fig. 3Go), were evaluated. To make these strains, F2 mice were genotyped to identify animals with the desired segments of B6 and A/J chromosome 6; these mice were then bred with mice from the B6 progenitor strain to generate mice heterozygous for the particular nonrecombinant region; finally, two heterozygous mice were identified and mated to generate homozygous congenic animals that were propagated for the current studies (Burrage, L. C., and J. H. Nadeau, unpublished).


Figure 3
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  		FIG. 3. Congenic panel. The genotype on chromosome 6 for each of the 12 congenic strains is represented by the horizontal bars. Shaded portions indicate a known homozygous B6 segment, unshaded portions represent a known homozygous A/J segment, and hatched regions depict an area where a crossover between A/J and B6 occurred. The QTL mapped from the F2 linkage analysis extends from dotted lines (60–75 cM). The proximal boundary of the refined QTL is shown by the dashed line.

 
Housing
All animals used in this study were cared for according to a Case Western Reserve University approved protocol and Institutional Animal Care and Use Committee (IACUC) guidelines. Mice from all strains were housed under uniform conditions in a barrier facility in individually vented standard polysulfone microisolator cages with corncob bedding and provided access to chow (Prolab Isopro RMH 3000; PMI Nutrition International, LLC, Brentwood, MO) [total isoflavone = 448 ppm (mg isoflavone per kg diet)] and sterile water ad libitum. Animals were maintained on a 12-h light, 12-h dark schedule (lights on at 0600 h) at a mean ambient temperature of 72 F.

Group breeding (two to three females with one male) was employed during generation of congenic strains. However, for all animals phenotyped for pubertal timing, a more controlled protocol was implemented, as previously described (12). Individual males were placed in a cage with two to three females for approximately 14–18 d. To minimize the exposure of pups to opposite-gender pheromonal and hormonal signals, each female breeder was then isolated in a clean cage containing a sterile cotton nestlet for the remainder of the pregnancy. These breeders were monitored daily, 7 d/wk, between 0800 and 1300 h, for birth, and the date of birth was designated as the day pups were observed. Pups were weaned at 20–21 d, and males and females were then housed separately. No more than five pups were housed per cage to ensure that access to food and water was unfettered. Assessment of multiple litters and periodic cage rotations were used to minimize the impact of environmental differences during phenotyping. In addition, to assess for any environmental or methodological changes that might have occurred, B6 and B6–6A animals were periodically bred and the timing of VO assessed throughout the course of the study. [Of note, between the F2 and congenic experiments, the mouse colony was relocated to a new facility within Case Western Reserve University. In this new environment, subtle differences in the age at VO were noted (see Results and Ref. 12), but the strain-specific differences in age of pubertal onset were unchanged.]

Genotyping
DNA from F2 mice was genotyped for the presence of A/J and/or B6 chromosomal material using 10 commercially available microsatellite repeat markers (MapPairs Mouse Markers from Invitrogen Corp., Carlsbad, CA) that spanned the length of chromosome 6 at approximately 8-cM intervals [marker 1, D6MIT138 (0.7 cM); marker 2, D6MIT159 (7.3 cM); marker 3, D6MIT274 (20.8 cM); marker 4, D6MIT384 (27.5 cM); marker 5, D6MIT391 (35.3 cM); marker 6, D6MIT36 (46 cM); marker 7, D6MIT287 (49 cM); marker 8, D6MIT254 (60.6 cM); marker 9, D6MIT59 (67 cM); and marker 10, D6MIT15 (74 cM)]. DNA was obtained from tail clippings or ear punches, digested with proteinase K (QIAGEN, Inc., Los Angeles, CA), and PCR were carried out at an annealing temperature of 60 C for 35 cycles. PCR products from amplification of F2, A/J, and B6 DNA were separated on 6% Protogel polyacrylamide gels (National Diagnostics, Atlanta, GA) and stained with ethidium bromide. Genotypes (homozygous for A/J or B6 or heterozygous) were then designated for all markers.

Phenotyping: pubertal onset and ovarian follicle size
Beginning on the day of weaning, female pups from the (F2) linkage analysis and all congenic strains were examined daily, 7 d/wk, between 0800 and 1300 h, and the age at VO and concurrent body weight were recorded. Examiners were blinded to the genotype of each congenic strain throughout the phenotyping. Pubertal timing was assessed in male A/J, B6, and B6–6A mice by determining the age and concurrent body weight at preputial separation.

To assess follicular maturation, ovaries from 26-d-old female A/J, B6, B6–6A, and congenic strain no. 2 (see Fig. 3Go) mice were dissected, fixed in formalin, embedded in paraffin, and cut into 5- to 8-µm sections. A single section from each ovary was selected, based on greatest ovarian width, and the diameter of all antral follicles was measured. Antral follicular diameter was assessed by averaging the length of two perpendicular lines drawn from each edge of the follicle (14). A minimum of eight ovaries from four mice from each strain was assessed. Follicles were visualized on a Leica DMLB microscope and measured using the program Micromeasure 3.3 (http://www.colostate.edu/Depts/Biology/MicroMeasure).

Data analysis
Linkage analysis was performed using the R-QTL software package designed for mapping QTL in experimental crosses (http://www.biostat.jhsph.edu/~kbroman/qtl/) (15). It is important to note that in linkage studies performed using a CSS, only the alleles from a single chromosome segregate during the experimental crosses. This results in a substantially lower threshold for statistical significance than is used in traditional linkage analyses during which alleles from all chromosomes segregate (13, 16). Thus, empirical significance thresholds were determined through analysis of 10,000 permutations of the F2 genotype and phenotype data; LOD scores of 2.24 and 4.21 were established as the cutoffs for statistical significance (P < 0.05 and P < 0.001, respectively).

Because it is possible that VO is not a normally distributed trait in all strains and because relatively small numbers of animals were assessed for other phenotypes, two-tailed, nonparametric tests for independent variables (Mann-Whitney U tests) were used for comparisons of VO timing, ovarian follicle size, and preputial separation among strains (Complete Statistical System: Statistica; StatSoft, Inc., Tulsa, OK). Significance was attributed to P < 0.05 for all tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 269 F2 animals were generated for linkage analysis. The mean age at VO among the F2 animals was 30.1 ± 2.3 d with the age at VO approximating a Gaussian distribution (Fig. 1Go).


Figure 1
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  		FIG. 1. Distribution of age of VO in F2 mice.

 
Linkage analysis for VO revealed a QTL with a peak LOD score of 2.9 at the distal end of chromosome 6, centered between 60 and 75 cM from the centromere, just proximal to marker 8, according to LOD-1 criteria (Fig. 2AGo). A second analysis, controlling for body weight as a covariate, altered the profile slightly but did not lead to a statistically significant change in the linkage results (Fig. 2BGo).


Figure 2
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  		FIG. 2. QTL for VO on chromosome 6. A, Results from linkage analysis for timing of VO (peak LOD, 2.9); B, results for timing of VO controlled for covariate of body weight at VO (peak LOD, 3.2). Dotted vertical lines demarcate LOD – 1 QTL localization on the chromosome. Dashed horizontal line displays P < 0.05 significance level calculated from permutation analysis. Note differences in scales on y-axis between figures.

 
To validate and refine the location of the QTL, a minimum of 15 mice from 12 different congenic strains were phenotyped. Of the 12 congenic strains, numbers 1–7 contained a homozygous A/J segment spanning all or part of the initially identified QTL, whereas numbers 8–12 were homozygous B6 throughout this region (Fig. 3Go). Data from the individual congenic strains are shown in Table 1Go. The strains that did not contain the QTL had consistently later timing of puberty than those initially designated as containing the QTL (P < 0.0001; Table 1Go and Fig. 4Go). The mean timing of puberty among the strains without the QTL approximated B6 mice, whereas those with the QTL had significantly earlier timing of VO, similar to B6–6A mice (B6 vs. congenics with QTL, P < 0.0001; B6–6A vs. congenics with QTL, P value not significant).


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  		TABLE 1. Timing of VO in congenic strains

 

Figure 4
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  		FIG. 4. Comparison of timing of VO in congenic mice. The cumulative percentage of mice with VO is displayed. + QTL refers to all congenic strains with A/J chromosomal material within the initially mapped QTL, whereas – QTL represents congenic strains without A/J segments in that region.

 
Inspection of Table 1Go and Fig. 3Go demonstrates that strain 1 had significantly earlier pubertal timing than the B6 progenitor strain (P < 0.001) and that the region of chromosome 6 distal to marker 9 [D6MIT59 (67 cM)] is shared among all strains with early VO. Thus, at least one responsible gene within the QTL is located distal to this marker. Moreover, the finding that strain 7 has timing of VO similar to B6 strongly suggests that the QTL should be refined to include only the region distal of maker 9. With this refined localization, the difference between strains with (numbers 1–6) and without (numbers 7–12) this critical region was even more robust [26.9 ± 2.3 d (n = 133) vs. 29.5 ± 1.9 d (n = 103); P < 0.0001]. No statistical difference was observed between the mice without the critical region and the B6 mice.

To provide evidence that the difference in VO reflected a change in the level of gonadotropin activity among strains, we assessed ovarian antral follicle diameter in A/J, B6, B6–6A, and congenic strain no. 2 (chosen as a representative congenic strain with A/J sequence throughout the initially mapped QTL; see Table 1Go and Fig. 3Go) mice. Antral follicle diameter was assessed because the development of these follicles is reflective of gonadotropin secretion and/or activity (17, 18, 19, 20, 21, 22); 26-d-old animals were used so that almost all B6 animals would be prepubertal by VO measurement (see Fig. 4Go).

Mice from AJ, B6–6A, and congenic strain no. 2 all had greater follicle diameters than B6 (Table 2Go), consistent with their earlier ages at VO. To verify that the data did not reflect an underlying difference in follicle size between strains (i.e. A/J having bigger follicles than B6 regardless of pubertal timing), we measured antral follicle size in mature, 6-wk-old A/J and B6 mice. In these adults, no significant difference in follicle size was observed (data not shown), indicating that the difference seen at 26 d stems from earlier maturation.


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  		TABLE 2. Ovarian follicle maturation

 
We next sought to test for parent-of-origin effects and to investigate the mode of inheritance for the responsible gene(s) within the QTL. F1 mice for the congenic no. 2 strain were generated by reciprocal crosses with B6 mice and phenotyped. The F1 animals had a mean VO time (28.1 ± 1.6 d; n = 55) that was intermediate compared with the congenic no. 2 strain (26.4 ± 1.9 d) and the B6 mice (30.0 ± 2.1 d) (P < 0.0001 vs. congenic no. 2; P < 0.0001 vs. B6), suggesting that the phenotypic effects of the gene(s) within the QTL are sensitive to gene dosage. No apparent parent-of-origin effect was observed, consistent with previous studies of B6–6A mice (12).

Finally, to investigate whether the allele(s) detected on chromosome 6 modulate the timing of puberty in males, preputial separation (the separation of the foreskin from the glans penis, an androgen-dependent indicator of pubertal maturation in male rodents) (23) was assessed. No statistically significant difference was seen in the timing of preputial separation among A/J (27.5 ± 1.1 d; n = 13), B6 (27.6 ± 1.1 d; n = 34), or B6–6A (27.2 ± 1.5 d; n = 15) mice. These data are consistent with previous investigations that showed no apparent difference in tempo of testicular growth between progenitor A/J and B6 animals (data not shown). Given these negative results, no additional analysis among the congenic strains was performed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used a genome-wide screen among CSS (12) followed by linkage analysis and evaluation of congenic strains to map a QTL that regulates the timing of puberty in mice. To our knowledge, this is the first such QTL, identified using a whole-genome approach, that has been confirmed in either an animal or human study.

Other investigations of genetic regulation of pubertal timing have been performed in animal models (3, 10) and in humans (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Important discoveries have been derived from identifying genes that underlie several human monogenic disorders (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48), but variation in these genes has not yet been shown to account for variation in pubertal timing within the general population. To investigate this variation further, biological candidates have been evaluated in association studies (24, 26, 27, 28, 31, 32, 33, 49, 50) with only a few genes (COMT, ERS1, and CYP17) having alleles identified that associate with age of menarche in more than one study. More recently, two human whole-genome analyses, using age of menarche as a surrogate for the onset of puberty, have been reported (50, 51). The combined results from both studies reveal only one region in one of the manuscripts with significant linkage (22q13) (50), but several regions with suggestive or possible linkage were also identified. It is not clear whether the lack of replication of significant linkage stems from genetic or environmental heterogeneity or statistical fluctuations.

The divergent results in human studies highlight the need for complementary investigations, using a variety of approaches, to identify and confirm QTLs that regulate variation in pubertal timing. Indeed, because the timing of puberty is regulated by a complex architecture of multiple genes and neuroendocrine networks (10), there are likely many QTL that affect the trait. Animal models, such as CSS, are important complements to human studies because environmental differences can be minimized and phenotypes can be assessed in multiple animals with identical genomes, greatly enhancing the ability to detect and confirm QTL (13, 16).

The distal end of mouse chromosome 6 is homologous to 12q11 and 12q12 in the human genome. Our data suggest that this region may harbor a gene or genes that are an important modulator of pubertal timing. The region of chromosome 6 bordered by the initial QTL contains approximately 22.8 megabases and 83 identified genes. From current knowledge, the most likely candidate in that region is the glutamate receptor, ionotropic, N-methyl-D-aspartate, subunit 2B (NR2B) (at 64.5 cM), a gene encoding an N-methyl-D-aspartate receptor subunit found in GnRH neurons (52, 53, 54, 55). However, the data from our congenic mice indicate that the refined QTL location is distal to 67 cM and that the responsible gene is, therefore, probably one that was not previously known to modulate the reproductive endocrine axis. There are only 21 genes in the refined region, and although comparative sequence data are not complete, a query of the Mouse Phenome Database (http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home, queried April 7, 2006) reveals that only five expressed sequences [the lymphoid-restricted membrane protein (Lrmp) gene, Riken cDNA 4930469P12, cancer susceptibility candidate 1 (Casc1), transmembrane 7 superfamily member 3 (Tm7sf3), and hypothetical protein LOC626177] in the refined region harbor nonsynonymous polymorphisms that differ between the A/J and B6 genomes. Thus, unless one of these five single-nucleotide polymorphisms is causal, the responsible gene(s) may well have important sequence differences in intronic or regulatory regions. These data underscore the importance of whole-genome approaches that do not require a priori assumptions about candidate genes or pathways and that may lead to the discovery of novel regulators.

It is important to note that our data do not argue against the presence of other genes or QTL that modulate variation in the timing of puberty. The most substantial limitation of our approach is that it can detect only regulators that have functionally significant sequence differences between the B6 and A/J genomes; an important gene with no such sequence variants would not be detected. In addition, although the various measures of puberty in mice (VO, organ weights, first estrus, ovulation, and first pregnancy) correlate well with each other, each trait is likely regulated by both common and independent factors (12). Thus, it is also unlikely that identifying genes that modulate VO will characterize fully the reproductive endocrine axis and identify all genes that regulate the onset of puberty. On the other hand, available data strongly suggest that a gene that regulates VO will be among the important determinants of reproductive maturation in mice.

Once the responsible gene(s) have been identified, the mechanism for the difference in pubertal timing generated by the A/J and B6 sequence variant(s) will be fully explored. However, we have initially tested the hypothesis that a gene or genes modulating gonadotropin secretion or action reside within the QTL. To begin to distinguish this possibility from estrogen responsiveness alone, we examined antral follicle diameter in ovaries of several strains. Antral follicle formation (mature follicles with a follicular fluid space, i.e. antrum) is dependent on gonadotropin secretion and functional gonadotropin receptors in the ovary (17, 18, 19, 20, 21, 22). Therefore, measurement of the first wave of developing follicles provides a reasonable, integrated measure of gonadotropin action. The larger antral follicles in A/J, B6–6A, and congenic mice suggest that these follicles have been exposed to gonadotropins longer than B6 follicles and provide early evidence that the responsible gene(s) may either modulate hypothalamic-pituitary function or alter the function of gonadotropin receptors in the ovary.

There are several noteworthy observations about the genetic regulation of VO by the QTL we have identified. First, the data suggest that the QTL may exert gender-specific effects. Although assessment of pubertal timing in male mice is more difficult and likely less reliable than in females, we have no evidence for differences in pubertal timing among male A/J, B6, or B6–6A mice. It is not surprising that we have detected a QTL that might exert gender-specific effects because the regulation of puberty in humans likely involves gender-specific and gender-independent modulators (56, 57). This will be an area of important future investigation.

Second, although environment clearly plays an important role in regulation of pubertal timing, alterations in body weight did not mediate the effects of our QTL. When the linkage analysis was reevaluated controlling for body weight at VO, no significant alterations in the results were observed. Moreover, the congenic strains with the earliest pubertal timing (numbers 1–5, Table 1Go) were younger and smaller at VO than those without the QTL. These mice were, therefore, not simply growing more quickly and reaching a similar weight to B6 animals at a younger age.

Finally, it is interesting that one of the congenic strains containing the QTL, congenic strain no. 6, displayed later pubertal timing than the other strains (numbers 1–5) that harbor the critical A/J sequence (Table 1Go; Fig. 3Go). These data may simply represent a phenotypic outlier, but other explanations should be considered. For example, a second QTL in a more proximal portion of chromosome 6 may interact with the QTL we have mapped and delay VO. Alternatively, a spontaneous mutation in the critical substituted A/J region may have occurred in strain no. 6 and negated the effect on VO. Lastly, although crossover interference (58) makes it unlikely, it is possible that a double crossover during generation of this congenic strain resulted in a small segment of undetected B6 material in the critical region. These explanations are worthy of future investigation because they may lead to insights about the gene(s) on chromosome 6 that modulate the timing of puberty.

In summary, our findings represent an important advancement toward identification of novel factors that regulate the maturation of the HPG axis. When identified, the responsible gene(s) and pathway(s) will be high-likelihood candidates for modulating variation in the timing of puberty among humans and ultimately may represent new therapeutic targets for disorders of reproductive maturation such as precocious puberty and hypogonadotropic hypogonadism.


   Acknowledgments
 
We thank Mr. Ramon Jin for his assistance with phenotyping of mice for VO.


   Footnotes
 
This work was funded by National Institutes of Health (NIH) Grants K23RR15544 and R01HD048960 to M.R.P. and RR12305 to J.H.N. B.M.N. was supported by Training Grant DK007319-27 and a grant from the Endocrine Fellows Foundation; C.A.H. was supported by NIH Training Grant DK07678; L.C.B. was supported by NIH Training Grant GM007250-30.

Disclosure statement: The authors have nothing to disclose.

First Published Online July 27, 2006

Abbreviations: CSS, Chromosome substitutions strains; HPG, hypothalamic-pituitary-gonadal; QTL, quantitative trait locus; VO, vaginal opening.

Received June 6, 2006.

Accepted for publication July 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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