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Department of Laboratory Medicine, University of California, San Francisco, California 94143-0134
Address all correspondence and requests for reprints to: Farid F. Chehab, Ph.D., Department of Laboratory Medicine, University of California, San Francisco, 505 Parnassus Avenue, San Francisco, California 94143-0134. E-mail: chehab{at}pangloss.ucsf.edu * This
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Generation of the intercross
The outcross consisted of inbred BALB/cJ lean females that were
mated to leptin-treated C57BL/6J ob/ob males. Treatment of
the ob/ob males with recombinant leptin was as previously
described (5). The F1 heterozygous mice were intercrossed
to generate an F2 progeny consisting of genetically
heterogeneous males and females segregating the ob mutation
in a Mendelian fashion. F2 mice were genotyped at the
ob locus for the R105X mutation as previously described (4).
None of the F2 ob/ob mice was treated with
leptin.
Fertility assay
At 8 weeks of age, each F2 ob/ob male was
housed with two 16- to 20-week-old reproductively competent lean
C57BL/6J females that had had one to two pregnancies each since 8 weeks
of age. Pregnant lean females were removed to individual cages and
replaced by another lean female such that the obese males had two
females continuously throughout the mating period. Pregnancies,
deliveries, and number of pups were constantly monitored during the
15-week mating period. If obese males did not induce pregnancies in
lean females within 4–6 weeks, two new females were substituted to the
first pair to reduce chances of infertility in lean females. Similarly,
ob/ob females were mated with two to three 16- to
20-week-old C57BL/6J stud males that had plugged lean females within
2–3 weeks before mating. Fertility was similarly assayed in males and
females from the congenic C57BL/6J ob/ob strain. All obese
males were killed at 23 weeks of age to collect blood and determine
testes weights.
Metabolic assays
ob/ob mice were fasted for 15–17 h at 15–17 and
19–21 weeks of age, during which time they had access only to water.
Blood was collected by tail bleeding and assayed immediately in
duplicate for whole blood glucose with blood glucose test strips and
the Encore Glucometer (Bayer Diagnostics, Elkhart, IN). Plasma glucose,
albumin, amylase, cholesterol, and triglycerides were determined at 23
weeks of age on a Hitachi 747 Clinical Chemistry Analyzer
(Hitachi Scientific Instruments, Inc., Mountain View, CA)
from 14 fertile and 18 sterile obese males from whom blood was
available. Plasma testosterone levels were determined in duplicate with
a coated tube RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX).
Genome scan
Microsatellite markers that are polymorphic between the C57BL/6J
and BALB/cJ mouse strains were selected from the mouse genome map. PCR
primers were purchased from Research Genetics, Inc.
(Huntsville, AL) or synthesized in-house on an ABI DNA synthesizer. All
markers were chosen such that their amplification products were 5–50
bp different between the two strains with an average difference of
8–10 bp. DNA was extracted from either mouse tail biopsies or spleen
recovered at 23 weeks of age by standard proteinase K/SDS method
followed by phenol-chloroform extraction and DNA precipitation.
Genotyping was performed by amplification of the PCR products for 35
cycles in standard conditions of 1.5 mM MgCl2,
100 µM deoxynucleoside triphosphates and
thermocycling at 95 C, 55–58 C, and 72 C. The resulting PCR products
were fractionated on 6% denaturing polyacrylamide gels and blotted
onto Hybond N+ (Amersham Pharmacia Biotech, Arlington
Heights, IL) nylon membranes by capillary transfer for at least 1
h. The Southern blots were then hybridized at 42 C from 1 h to
overnight to a biotinylated (CA)10 oligonucleotide probe in
6xSSC and 0.5% SDS. The blots were then washed twice at room
temperature in 2xSSC, 0.5% SDS for 5 min and finally in 0.5x SSC,
0.5% SDS at 42 C for 10 min. Detection of the hybridized probe was
performed via the avidin-biotin conjugate system using a streptavidin
horseradish peroxidase system. Briefly, the washed blot was incubated
with 0.1 M Na citrate, pH 5, for 5 min at room temperature
and then incubated for 15 min in the same buffer supplemented with
0.3 m g/ml streptavidin-peroxidase conjugate
(Boehringer-Mannheim, Indianapolis, IN). Excess conjugate
was washed off by incubation with two changes of 0.5xSSC, 0.5% SDS
for 5 min at room temperature. The hybrids were visualized in a final
incubation of the blot with 0.1 mg/ml of tetramethyl benzidine in 0.1
M Na citrate, pH 5.0. The reaction was stopped by
transferring the blot to distilled water. This entire procedure of PCR
and Southern blotting was necessary to increase confidence in
genotyping and eliminate the interference of spurious PCR products
associated with amplification of microsatellites. All double
cross-overs were retyped for confirmation of the genotype.
Quantitative trait loci (QTL) analysis
Genotyping data were entered in the computer program
MapManager/QTL vb21 that was downloaded from the Map Manager web site
(http://mcbio.med.buffalo.edu/mmQT.html). Initially, the linkage map
was constructed using 130 polymorphic microsatellite markers and then
extended to additional 23 markers for fine mapping around the
significant loci on chromosomes 1, 3, 5, and 14. Quantitative traits
were correlated with genotypes at a specific locus by a regression
analysis algorithm that measures associations using the likelihood
ratio statistic (LRS) suggested by Haley and Knott (14). LRS
values were converted to logarithm of the odds (LOD) scores
according to Lander and Kruglyack (15) essentially by dividing
them by 2(ln10)
4.6. LOD scores were also calculated by importing the
data from MapManager to the computer program QGene (a kind gift of Dr.
Clare Nelson). Linkage was also evaluated with the MapManager
permutation assay using 1000 permutated data sets (16) at each
locus, thus setting up internal linkage thresholds. The LRS thresholds
for number of pregnancies, testes weight (TW)/body weight (BW) ratio,
TW, testosterone, and BW at 8 weeks were, respectively, 15.6, 14.4,
15.9, 16.7 and 21.7. All markers were analyzed as an F2
intercross except for X chromosome markers, which were analyzed as a
backcross since there is only one chance of recombination for the X
chromosome in a F2 male progeny when the outcross is
between two inbred strains.
Statistics
Unless otherwise noted, data are expressed as means ±
SEM. Statistical analysis was performed with the Statistica
package obtained from Statsoft (Tulsa, OK). Student’s t
test and linear regression analysis were performed to evaluate,
respectively, differences between the fertile vs. sterile
groups and to evaluate the correlation of various traits
combined.
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Results |
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, A and B), thus demonstrating the severity and the highly penetrant
nature of the obesity phenotype in males and females. Highly
significant differences in BWs between mice on the C57BL/6J and F2
BALB/cJ-C57BL/6J backgrounds were found only in males at 23 weeks of
age with respective BWs of 57.8 ± 7.1 g vs.
71.6 ± 4.9 g (means and SD; P =
10-7; t test) indicating that an
adiposity-modifying effect is evident at a later age.
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) and the ages at which they copulated with the lean females
(Table 1). Thus, fertile F2
obese males induced their first and second pregnancies in lean females
at 10.8 ± 0.6 and 14.2 ± 1.4 weeks of age, respectively.
Furthermore, BW differences between fertile and nonfertile obese mice
failed to reveal any statistical significance (Fig. 1D), indicating
that excess BW was not an impediment to the onset of fertility that
resulted from the segregation of modifier genes promoting fertility
(mof). Furthermore, BWs of F2 obese mice at 6,
8, 10, 12, 16, and 23 weeks of age were normally distributed and
revealed age-specific variations (Fig. 2, A–F), suggesting that modifier genes for adiposity (moa)
are also expressed by the mixed genetic background and would thus be
inherited with mof genes. Although there was no correlation
between the number of pregnancies and BWs of the 34 F2
obese mice at any age, a correlation was found between the number of
pregnancies of the 14 fertile obese mice and their respective BWs at 12
weeks of age (r = 0.55, P = 0.04). Taken together,
these data suggest that moa genes segregate with
mof genes contributing to the variation in the fertility
phenotype observed in F2 fertile obese males.
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Metabolic and physiological indexes in F2 obese
males
To assess some of the metabolic parameters that could have
contributed to the fertility of F2 obese males, glucose,
cholesterol, triglycerides, amylase, albumin, and testosterone were
measured in F2 obese males from plasma collected at 23
weeks of age. In addition, fasted blood glucose was determined at
15–17 weeks and 19–21 weeks of age (Table 2). Although albumin, amylase, glucose,
and cholesterol levels were not statistically significant between
fertile and sterile groups, testosterone and triglycerides levels were
significantly more elevated in fertile obese males with respective
P values of less than 0.05 and 0.01. Interestingly,
triglyceride levels in fertile mice correlated with glucose levels at
23 weeks of age (r = 0.54; P < 0.002).
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; P < 0.05). We also examined whether
testes weight of F2 obese mice could be an indicator of
fertility as previous studies have shown that obese mice on the
C57BL/6J background exhibit a hypogonadism characterized by lower
testes weights than their lean counterparts (17, 18, 19). Paired testes
weights of F2 obese mice followed a Gaussian distribution
(Fig. 2G) but were not significantly different between fertile and
sterile groups (Table 2). Furthermore, F2 obese mice showed
a skewed ratio of TW vs. BW (TW/BW) as normal mice have
testes weights approximating 0.38% of their BW (20) or 3.8 mg/g BW. In
this study, TW/BW ratios were normally distributed (Fig. 2H) in the
F2 population with the F2 fertile obese mice
showing a higher ratio than F2 sterile obese mice (Table 2).
Chromosomal mapping of modifier loci
To identify the chromosomal localization of modifier loci
affecting fertility and adiposity, we constructed a linkage map to
assess the correlation of different fertility and obesity-related
quantitative traits with the genotypes of F2 obese males at
various loci throughout the genome. Thus, 153 microsatellite markers
that are polymorphic between C57BL/6J and BALB/cJ strains were selected
from the mouse genome map and spanned all except the Y chromosome.
These markers were used to type the 34 F2 genetically
heterogeneous obese males and the resulting genotypes entered in the
Map Manager/QTL vb21 computer program. The ensuing F2
linkage map showed the markers to have an average spacing of 10.4
± 0.5 centimorgans (cM). Since the number of pregnancies induced by
fertile F2 obese males during the 15-week mating period was
variable, we used the number of pregnancies as a fertility quantitative
trait. Other quantitative traits included BW, absolute TW, TWs divided
by BW (TW/BW), BW measured at 6, 8, 10, 12, 16, and 23 weeks of age,
and each of the metabolic measurements shown in Table 2. Regression
analysis of individual marker genotypes with the number of pregnancies
quantitative trait identified a major locus on chromosome 1 at the
D1MIT459 marker with a P value of 2.6 x
10-6 and an additive mode of inheritance. Interval mapping
on chromosome 1 with this quantitative trait returned a likelihood
ratio statistic (LRS) of 25.7 and a LOD score of 5.6 (Fig. 3A). These values vastly exceed the LOD
score of 3.4 proposed by Lander and Kruglyack (15) as threshold for
significant linkage. Furthermore, linkage to the D1MIT459 locus
accounted for 53% of the phenotypic fertility variation. Two other
loci that fell into the lower category of suggestive linkage were
D3MIT316 (P = 0.0043; LRS = 10.9, LOD = 2.4)
and D9MIT297 (P = 0.002, LRS = 12.4, LOD =
2.7).
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) showed that fertile mice inherited more
BALB/cJ alleles than nonfertile mice at both the D1MIT459 and D3MIT316
loci. Thus at both loci, F2 fertile obese mice had
inherited 59% of BALB/cJ alleles vs. 33% in sterile obese
(P = 0.005) mice, indicating that BALB/cJ modifier
genes at these two loci may account, at least partly, for the
differential fertility phenotype in the two groups. When fertility of
obese mice was expressed as a discontinuous pseudodichotomous binary
trait, rather than a continuous trait reflecting variation in the
number of pregnancies, the number of BALB/cJ alleles inherited by the
F2 obese population at the D1MIT459, D3MIT316 loci, or both
correlated significantly (P < 0.05) with the binary
fertility trait with regression coefficients of 0.40, 0.45, and 0.55,
respectively.
On the other hand, with the TW/BW quantitative trait,
D3MIT316, the marker previously identified with the number of
pregnancies trait, emerged as the most significant association
(P = 4.5 x 10-4; LRS = 15.6;
LOD = 3.4). Therefore, the mapping of two different
quantitative traits, TW/BW and number of pregnancies to the same region
of chromosome 3 (Fig. 3B) strongly strengthens this association, thus
identifying a second locus that influences the fertility of
F2 obese males. Furthermore, since plasma testosterone
levels were more elevated in fertile vs. nonfertile obese
males, we sought to determine whether a modifier locus is associated
with this quantitative trait. Thus, absolute testosterone levels in
F2 obese males correlated significantly with genotypes at
the D14MIT113 (LRS = 17.6; P = 3.6 x
10-4) and D14MIT203 (LRS = 15.7; P =
3.9 x 10-4) loci yielding respective LOD scores of
3.8 and 3.4 and recessive modes of inheritance (Fig. 3C).
To map BW modifier genes that could segregate with fertility, we
analyzed BWs at various ages as a quantitative trait and found that the
D5MIT271 locus is significantly associated with BW at 8 weeks of age
(LRS = 22.9; P = 1.2 x 10-5)
with a LOD score of 5.0 and a recessive mode of inheritance (Fig. 2D).
However, this linkage decreases at later ages with respective LOD
scores of 3.5, 2.5, 1.7, and 1.7 at 10, 12, 16, and 23 weeks of age,
respectively, suggesting that this association gradually decreases with
the development of morbid obesity. Previous linkage studies aimed at
localizing obesity modifier loci in mouse crosses involved lean mice
from different strains (21, 22, 23, 24); however, this study is the first to
report BW modifiers in an obese state. Presumably, the effects of
modifier genes on adiposity are more likely to be encountered in a
cross involving morbid obesity such as the ob/ob phenotype
due to the relative ease of detection of BW fluctuations and their
relative stability over time. In summary, four loci on chromosomes 1,
3, 5, and 14 were identified and appear to affect the onset and extent
of fertility in ob/ob mice on the mixed C57BL/6J-BALB/cJ
genetic background. Thus, the fertility phenotype in F2
obese mice would be conferred by a combination of these loci and
possibly by other loci yet to be uncovered.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Furthermore, while glucose levels were not significantly different between the two groups, they demonstrate that the transient hyperglycemia that is characteristic of young C57BL ob/ob mice and that recedes to normoglycemia and elevated insulin levels in older ob/ob mice (25) is similar in both fertile and sterile groups. Therefore, the diabetic state does not appear to affect the fertility of F2 obese males. The interesting correlation of triglyceride levels with glucose in fertile F2 obese mice suggests that perhaps the conversion of glucose to acetyl coenzyme A and its subsequent entry into the fatty acid synthesis pathway may be more efficient in fertile than sterile obese mice. Whether this is a contributing factor to fertility or a secondary effect remains to be determined.
Interestingly, the leptin-independent genetic rescue of sterility in F2 ob/ob mice in this cross was applicable only to obese males but not to obese females. The lack of fertility among females further reinforces the notion that leptin is essential for the initiation of reproduction in females and cannot be substituted, as in males, by the effects of modifier genes. This hypothesis is consistent with previous findings that show that leptin is a contributing factor to the initiation of female reproduction in normal mice (11, 12) and rats (13). Alternatively, it is possible that the sterility rescue of ob/ob females by this genetic approach necessitates the combination of additional modifier genes, thus decreasing the frequency of such an occurrence in a single animal as in the present F2 intercross. Instead, transfer of the ob mutation on a pure BALB/cJ background by 10 successive backcrosses followed by an F2 intercross will result in a congenic strain consisting of ob/ob mice homozygous for almost all BALB/cJ alleles. Assay of the resulting obese females for fertility will test the hypothesis whether additional BALB/cJ genes are needed for induction of fertility in obese females.
Finally, the uncovering of chromosomal regions spanning modifier genes that altogether rescue the sterility of morbidly obese mice provide an entrypoint to narrow down the regions of interest and subsequent characterization of candidate genes within such regions. Ultimate proof will emanate from an alteration in expression or DNA sequence of candidate genes between the C57BL/6J and BALB/cJ strains at the proposed locus. Alternatively, consomic strains segregating these particular loci will uncover the contribution of their effect on the observed phenotype. Eventually, the elucidation of these complex pathways will lead to a better understanding of obesity-associated reproductive disorders.
Received July 29, 1998.
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