Copyright 2001 by the Genetics Society of America
Genetic Architecture of Testis and Seminal Vesicle Weights in Mice Isabelle Le Roy,* Sylvie Tordjman,* Danie`le Migliore-Samour,* Herve´ Degrelle† and Pierre L. Roubertoux*,‡ *Ge´ne´tique, Neuroge´ne´tique, Comportement, UPR CNRS, 45071 Orle´ans Cedex, France, †Biochimie Endocrinienne, Centre Universitaire, 75006 Paris, France and ‡University of Orle´ans, France Manuscript received March 28, 2000 Accepted for publication January 24, 2001 ABSTRACT Comparisons across 13 inbred strains of laboratory mice for reproductive organ (paired seminal vesicles and paired testes) weights indicated a very marked contrast between the C57BL/6By and NZB/BINJ mice. Subsequently these strains were selected to perform a quantitative genetic analysis and full genome scan for seminal vesicle and testis weights. An F2 population was generated. The quantitative genetic analyses indicated that each was linked to several genes. Sixty-six short sequences for length polymorphism were used as markers in the wide genome scan strategy. For weight of paired testes, heritability was 82.3% of the total variance and five QTL contributed to 72.8% of the total variance. Three reached a highly significant threshold (⬎4.5) and were mapped on chromosome X (LOD score 9.11), chromosome 4 (LOD score 5.96), chromosome 10 (LOD score 5.81); two QTL were suggested: chromosome 13 (LOD score 3.10) and chromosome 18 (LOD score 2.80). Heritability for weight of seminal vesicles was 50.7%. One QTL was mapped on chromosome 4 (LOD score 9.21) and contributed to 24.2% of the total variance. The distance of this QTL to the centromere encompassed the distance of the QTL linked with testicular weight on chromosome 4, suggesting common genetic mechanisms as expected from correlations in the F2. Both testis and seminal vesicle weights were associated with a reduction in the NZB/BINJ when this strain carried the YNPAR from CBA/H whereas the YNPAR from NZB/BINJ in the CBA/H strain did not modify reproductive organ weights, indicating that the YNPAR interacts with the non-YNPAR genes. The effects generated by this chromosomal region were significant but small in size.
R
EPRODUCTIVE organ size (testes and seminal vesicles) occupies a place of special importance among morphological measures because of its direct implication in fertility. Reproductive organ sizes are markers of the timing of puberty (Argyropoulos and Shire 1989) and testicular weight is connected with total sperm count in mice (Krzanowska 1971; Hunt and Mittwoch 1987; Chubb 1992). A correlated response to selection also appeared for ovulation rates in lines of mice selected for testicular weight (Islam et al. 1976). Weight is also an excellent index of either the biochemical or the anatomical state of reproductive organs. The balance and quantity of phosphocreatine and creatine in the fluid secreted by the epithelial cells of seminal vesicles varies according to their developmental stage (Lee et al. 1991). Small testis size is often associated with low androgen concentration (McKinney and Desjardin 1973; Carlier et al. 1990; Franc¸ois et al. 1990) and reactivity to testosterone (Michard-Vanhee and Roubertoux 1990). Testis size may also provide an indirect evaluation of the functionality of connected systems as it was demonstrated for hypothalamic activities (Jean-Faucher et al. 1983). Previously reported as-
Corresponding author: Pierre L. Roubertoux, UPR CNRS 9074, Ge´ne´tique, Neuroge´ne´tique, Comportement, CNRS, 3 B rue de la Fe´rollerie, 45071 Orle´ans Cedex, France. E-mail:
[email protected] Genetics 158: 333–340 (May 2001)
sociations between the fine anatomical structure of reproductive organs and their weight (mice with small testes having a high percentage of abnormal tubules and of reduced Sertoli cells; Chubb 1992) suggest that this simple measure might be used as a marker for complex morphological events. Finally, correlations between reproductive organ sizes and neurobehavioral measures have been reported. Testis weight is higher in mice with the largest brain asymmetries and low direction of laterality assessed from a paw preference test (see Collins 1985, for a review). Mice initiating attack behavior against conspecific males or initiating successful mating behavior (McKinney and Desjardin 1973) have higher testicular weight. As reproductive organ weights are the sum of different physiological events, elucidating the putative genes implicated in weight should help to dissect the biological bases of these phenotypes and to understand the mechanisms behind the correlations that have been reported above. Moreover, identifying the genes linked with testis and seminal vesicle weights in mice should have the practical outcome of facilitating the discovery of corresponding human genes via the use of comparative maps and subsequently developing animal models for sterility. Although more than 70 genes are known to be at work in cell activities of testes and 12 for seminal vesicles (Mouse Genome Database 1999), their puta-
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tive involvement in reproductive organ weights remains to be demonstrated. Very few studies have been performed with seminal vesicle weight in mice, notwithstanding its potential medical interest for modeling prostate cancer. Weight could be related to the abnormal vesicle shape described by Shukri et al. (1988), who identified Svs, one of the corresponding genes on chromosome 7. The implication of the H-2 complex on chromosome 17 has also been suggested (Gregorova et al. 1977). More attempts have been made to elucidate genes linked with testicular weight. Although four genes are known to contribute to testis differentiation signaling in mice (tdy, tda1, tda2, tda3; Mouse Genome Database 1999), the genes underlying the variation in testis weight in mice remain unknown. Marked polymorphisms for weight have been reported in inbred strains of laboratory mice (Shire and Bartke 1972; Shukri and Shire 1989; Chubb 1992) and several analyses either using recombinant inbred strains (Argyropoulos and Shire 1989; Shukri and Shire 1989; Chubb 1992) or generating segregating generations between inbred strains (Hunt and Mittwoch 1987; Washburn and Eicher 1989) have confirmed that testis weight follows a polygenic transmission. Unfortunately, the few chromosomal linkages that were suggested now appear controversial. Probably due to its major implication in testis differentiation signaling, the specific part of the Y chromosome was considered as encompassing putative candidates for variation in testis weight, one of these candidates being tdy itself. Results from backcross or intercross designs fit with an involvement of the specific part of the Y chromosome interacting with non-Y genes (Hunt and Mittwoch 1987; Washburn and Eicher 1989) in testis weight. This Y-chromosome effect was interpreted as a possible contribution of tdy. However, a more recent study (Chubb 1992) has shown that three strains differing in testis size did not reveal any polymorphism for the Y chromosome checked with a Y-specific probe (pY2). This result, indicating no implication of the tdy gene in testis weight, also excluded the contribution of the other genes carried by the Y chromosome in this phenotype. This conclusion might be limited to the C57BL substrains that were used in Chubb’s article. Contrasting with other morphological measures that have widely used wide genome scan strategies, only one attempt was made to investigate other regions. With recombinant inbred mice derived from C57BL/6J and DBA/2J (B ⫻ D), Zidek et al. (1998) found only one highly significant quantitative trait locus (QTL) linked to testis weight that was labeled Twq1, close to the D13Mit3 marker, on chromosome 13. At the D13Mit3 locus, the alleles from C57BL/6J were, surprisingly, linked with high testicular weight and those from DBA/ 2J with low testicular weight, although C57BL/6J parental strain had lower testis weight than DBA/2J. With the C57BL/6J and DBA/2J strains having almost extreme
weights among the set of B ⫻ D recombinant inbred strains, few “minoring” alleles in DBA/2J and few “majoring” alleles in C57BL/6J would have been expected. Considering that Twq1 contributed to 75% of the genetic variance, little room would remain for other QTL, indicating an incrementation of testis weight associated with DBA/2J alleles. For these reasons, we performed a wide genome scan for paired testis and seminal vesicles weights in an F2 population derived from C57BL/6By (B) and NZB/ BINJ (N) strains. These two strains were selected among 13 inbred strains of laboratory mice because they showed large differences for both the measures. Moreover, B and N appeared suitable for the wide genomic scan or QTL mapping because they differed for 92% of the tested simple sequence length polymorphisms (SSLPs; Le Roy et al. 1998). We had derived a quartet of congenic strains for the specific part of the Y chromosome (YNPAR) from NZB/BINJ (N) and CBA/H (H). This quartet was used to test for the implication of this region in testis and seminal vesicle weights.
MATERIALS AND METHODS General rearing conditions: All the mice had been maintained in a pathogen specific free area of our animal facilities under brother ⫻ sister mating for several generations when the experiment began. Mice from parental strains, F1 and F2 generations, were raised contemporarily. They were reared under the following general conditions: cages, 42 ⫻ 20 ⫻ 18 cm; bedding, dust-free sawdust; food, IM UAR and tap water ad libitum; temperature, 23⬚ ⫾ 0.5⬚; photoperiod, 12:12 with lights on at 8 AM; weaning at 29 ⫾ 2 days. Pregnant mothers were isolated from the mating cages. Litters with fewer than seven pups were discarded and the others culled to seven pups to prevent possible litter size effect on reproductive organ weights. At weaning, each male was housed with one NMRI female. Reproductive organ weights: Male mice were killed after cervical dislocation. Testes were removed and excised of adhering tissues. To prevent the loss of secretory fluid, the base of each seminal vesicle was grasped with forceps before removing. Paired seminal vesicle and paired testis weights were recorded to the nearest 0.1 mg. Comparisons across 13 inbred strains of mice: Reproductive organs were weighed in male mice at 90 ⫾ 5 days of age. They belonged to strains of mice developed from identified breeders and currently used for our experiments on intermale aggression (Le Roy et al. 1999). We used 13 strains: A/J, BALB/cJBy, CAST/Ei, C57BL/6By, DBA/2J, and NZB/BINJ purchased from The Jackson Laboratory (Bar Harbor, ME); CBA/H, C57BL/6J, and XLII from CSEAL (Orle´ans, France); BA and CPB-K provided generously by Dr. Hans van Abeelen (Nijmegen, The Netherlands); and DBA/1Bg and C57BL/ 10Bg given by Dr. Stephen C. Maxson (Storrs, CT). Mean values were compared using a one-way ANOVA procedure (SAS Institute 1987). Nine male mice per strain were used. Crosses and components of the mean differences: Parental N and B strains and reciprocal F1’s were separately compared with a Student’s t-test. We used the four reciprocal F2’s: NB.NBF2’s and NB.BNF2’s vs. BN.NBF2’s and BN.BNF2’s to test for a maternal contribution on the one hand and
Testis and Seminal Vesicle Weights NB.NBF2’s and BN.NBF2’s vs. NB.BNF2’s and BN.BNF2’s to test for an effect of the specific part of the Y chromosome (YNPAR) on the other hand (Carlier et al. 1991). This design was analyzed with a two-way ANOVA with the origin of the mother and the origin of YNPAR as main factors. Sample sizes are indicated in Table 2. Components of the mean differences (Mather and Jinks 1971) were estimated using seven parameters: [m] (mean), [d] (additivity), [h] (dominance), [i] (interaction between homozygous loci), [j] (interaction between homozygous and heterozygous loci), [l] (interaction between heterozygous loci), and [om] (origin of the mother). Heritability in the broad sense was estimated as h2L ⫽ VG/(VG ⫹ VE) where VE ⫽ 1⁄4VN ⫹ 1⁄4VB6 ⫹ 1⁄2VF1 and VG ⫽ VF2 ⫺ VE. Mapping QTL: QTL linked with paired testis and seminal vesicle weights were investigated with a full genome scan in the F2 intercross population derived from N and B mice. We used 193 males (17 B, 16 N, 18 NBF1’s, 12 BNF1’s, 34 NB.NBF2’s, 28 NB.BNF2’s, 30 BN.NBF2’s, and 38 BN.BNF2’s) maintained under the general conditions described above until they were killed at 150 ⫾ 6 days old. Given that the correlations between body weight and testis or seminal vesicle weight were not significant in the F2’s (r ⫽ 0.09 and r ⫽ ⫺0.03), respectively, the absolute paired testis and seminal vesicles weights were used for subsequent analyses. Individual measures were transformed (square root transformation) to ensure homoscedasticity in the nonsegregating generations (untransformed values were reported in the tables). DNA scoring: Tails and spleens were collected and stored at ⫺80⬚ until DNA extraction. DNA was extracted from tails and amplified with the usual procedure (Sambrook et al. 1989): initial denaturation (94⬚ for 3 min and then 94⬚ for 30 sec during each of the 40 amplification cycles), annealing (1 min 15 sec at 42⬚ to 55⬚, according to the primers), extension (1 min 15 sec at 72⬚), and final extension step of 3 min. We used a 4% agarose gel (3% NuSieve, 1% Sigma type II agarose) stained with ethidium bromide for visualization. The three possible genotypes were read with an UVP PMW 20 computer system on a screen with 4.5 magnification. The first and last authors performed blind and independent genotyping. Discordant observations resulted in a second amplification. Full genome scan: Genotyping was performed individually with the DNA from the F2 male mice. Sixty-six SSLPs were selected as genetic markers: 5 (chromosomes 1 and 2), 4 (chromosomes 4, 5, 17, 19), and 3 on the others (average interval length 22.5 cM). Significant differences between the three genotypes N//N, N//B, and B//B were assessed with the Kruskal-Wallis test. The chromosomes where these differences reached a P ⬍ 0.05 threshold were selected for QTL mapping. Subsequent likelihood ratios and LOD score computations were calculated with the interval mapping method using MapQTL package (van Ooijen and Maliepaard 1996; MapQTLtm-version 3.0). The expected confidence interval is expressed as 530/N · v (Darvasi and Soller 1997), where 530 is a constant obtained from simulations, N is the number of informative meioses, and v is the proportion of variance explained by the QTL. We tested for epistatic effects between two QTL using a twoway ANOVA with the values of the three genotypes at the closest SSLPs to the peaks of the QTL and the two QTL as main sources of variation. Epistasis was deduced when an interaction occurred. Congenic strains for the nonpairing region of the Y chromosome: To test directly an effect of the nonpairing region of
335 TABLE 1
Paired testis and seminal vesicle weights (g, mean ⫾ SEM) from 13 inbred strains of laboratory mice (90 ⫾ 5 days of age)
Strains A/J CAST/Ei C57BL/10Bg C57BL/6By (B) C57BL/6J BA BALB/cJBy CBA/H (H) CPBK DBA/1Bg DBA/2J NZB/BINJ (N) XLII
Paired testis weight 0.132 0.074 0.139 0.191 0.165 0.181 0.200 0.127 0.140 0.171 0.230 0.287 0.131
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.070 0.007 0.002 0.007 0.010 0.009 0.010 0.028 0.009 0.005 0.008 0.015 0.020
Paired seminal vesicle weight 0.137 0.069 0.290 0.324 0.270 0.190 0.201 0.129 0.230 0.170 0.229 0.381 0.170
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.025 0.004 0.060 0.012 0.042 0.023 0.020 0.031 0.030 0.023 0.067 0.030 0.030
the Y chromosome (YNPAR) on reproductive organ weights, the paired testis and seminal vesicle weights were measured in a quartet of congenic strains for this chromosomal region that we had developed from NZB/BINJ (N) and CBA/H (H) (Roubertoux et al. 1994). Briefly, the N-YNPAR was substituted in the place of the H-YNPAR in the H strain to obtain its congenic H.N-YNPAR for this region of the Y chromosome and second, the H-YNPAR was substituted in the place of the N-YNPAR in the N strain to obtain its congenic N.H-YNPAR. The congenic N.H-YNPAR was developed with N as recipient strain and H as donor, the N females being sired by NHF1 males and the backcross females subsequently sired again with N. The same design was used to transfer the YNPAR from N onto H and to obtain the second congenic H.N-YNPAR. At every generation it was assumed that the congenic progeny had lost 50% of the alleles from the congenic donor. Thus, for the measure of reproductive organ weights, few residual allelic forms located throughout the genotype including the YPAR of the parental donor strain were expected in the congenic strains at 35 and 37 backcross generations for N.H-YNPAR and H.N-YNPAR, respectively. Mean values were compared using a two-way ANOVA procedure with the recipient strain (H vs. N) and the origin of the YNPAR (H-YNPAR vs. N-YNPAR) as main sources of variation.
RESULTS
Measurement of reproductive organ weights in 13 inbred strains: Table 1 presents means ⫾ SEM for the two measures. Strain differences appeared for paired testis weight (F ⫽ 6.99, P ⬍ 0.0001) and seminal vesicle weight (F ⫽ 3.84, P ⬍ 0.001). The N and B strains presented a significant (t ⫽ 4.27, P ⬍ 0.0001) and marked contrast for testis weight. The difference for seminal vesicle weight was not the largest across the 13 strains even if it remained significant (t ⫽ 3.31, P ⬍ 0.001). Components of the mean differences in populations derived from N and B strains: Reproductive organ weights were measured in new individuals from the N
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I. Le Roy et al. TABLE 2
Paired testis and seminal vesicle weights (g, mean ⫾ SEM) in C57BL/6By (B), NZB/BINJ (N) strains, and reciprocal F1’s and F2’s Generations and sample size
Testis weight
B (n ⫽ 17) N (n ⫽ 16) BNF1 (n ⫽ 12) NBF1 (n ⫽ 18) F2 (n ⫽ 130)
0.194 0.298 0.254 0.288 0.256
⫾ ⫾ ⫾ ⫾ ⫾
0.007 0.007 0.012 0.004 0.003
Seminal vesicle weight 0.327 0.408 0.448 0.438 0.448
⫾ ⫾ ⫾ ⫾ ⫾
0.012 0.020 0.027 0.019 0.022
and B strains. Sample sizes and values in parental strains and reciprocal F1’s and F2’s are shown in Table 2. Parental strains differed for the two measures (t ⫽ 4.75, P ⬍ 0.0001, and t ⫽ 3.63, P ⬍ 0.001, for testis and seminal vesicle weights, respectively), the reproductive organs being heavier in the N than in the B strain. No significant difference was shown (NB.NBF2’s and BN.NBF2’s did not differ from NB.BNF2’s and BN.BNF2’s) for the origin of the YNPAR in the F2’s. Heritabilities were 0.823 and 0.507 for testis and seminal vesicle weights, respectively, in this population. The difference between the two values may result from the lower contrast between seminal vesicle weights in B and N strains. The best fitting models (2 ⫽ 8.140; P ⬍ 0.10 for testicular weight and 2 ⫽ 3.015; P ⬍ 0.70 for seminal vesicle weight) for estimation of the components of the mean differences were selected according to Kerbusch et al. (1981; Table 3). They indicated a polygenic mode of inheritance for both testis and seminal vesicle weights because of a significant [l] interaction between heterozygous loci for the two measures. The parameters contributing to the mean differences differed for the two phenotypes since a dominance effect was shown for testis and not for seminal vesicle weight, suggesting partially different genetic bases. A contribution of the origin of the mothers appeared for testis weight and not for seminal vesicle weight. The NBF1 males had higher testis weight than BNF1 males (t ⫽ 4.75, P ⬍ 0.01). Although the testes were heavier TABLE 3 Components of the mean differences for paired testis and seminal vesicle weights in generations derived from C57BL/6By (B), NZB/BINJ (N) mice (150 ⫾ 6 days of age) Paired testis weight [m] ⫽ 0.123 ⫾ 0.004 [d] ⫽ 0.012 ⫾ 0.002 [h] ⫽ 0.020 ⫾ 0.005 [l] ⫽ 0.015 ⫾ 0.005 [om] ⫽ 0.012 ⫾ 0.003 NS, not significant.
Paired seminal vesicle weight [m] ⫽ 0.446 ⫾ 0.009 [d] ⫽ 0.042 ⫾ 0.011 NS [l] ⫽ 0.078 ⫾ 0.014 NS
in males from NB.NBF2 or NB.BNF2 mothers than those in males from BN.NBF2 or BN.BNF2 mothers, the difference failed to reach significance (P ⬍ 0.057). In F1’s and F2’s the greater weights were observed for the males from the N mothers or grandmothers. A global estimate for the effect of the origin of the mother was significant for testis weight (Table 3). The product-moment correlation coefficient between testis and seminal vesicle weights in the F2 population was r ⫽ 0.45, P ⬍ 0.001. Mapping QTL: The three genotypes N//N, N//B, and B//B differed for testis weight (Kruskal-Wallis test) at the P ⬍ 0.05 threshold for the following SSLPs: chromosome 4, D4Mit205a (K ⫽ 20.89, P ⬍ 0.0001), D4Mit12 (K ⫽ 16.94, P ⬍ 0.0005); chromosome 10, D10Mit20 (K ⫽ 6.94, P ⬍ 0.05); D10Mit14 (K ⫽ 14.57, P ⬍ 0.005); chromosome 13, D13Mit3 (K ⫽ 8.36, P ⬍ 0.01), D13Mit13 (K ⫽ 6.76, P ⬍ 0.05); chromosome 18, D18Mit17 (K ⫽ 9.83, P ⬍ 0.005); and chromosome X, DXMit25 (K ⫽ 8.87, P ⬍ 0.005), DXMit223 (K ⫽ 19.29, P ⬍ 0.0005). For seminal vesicle weight, differences between the genotypes N//N, N//B, and B//B appeared only for chromosome 4 at D4Mit205a (K ⫽ 35.21, P ⬍ 0.0001), D4Mit12 (K ⫽ 20.71, P ⬍ 0.0001). Hence, QTL mapping was performed with MapQTL for chromosomes 4, 10, 13, 18, and X (Figure 1). For paired testis weight, highly significant linkages were reached for the QTL found on chromosomes 4, 10, and X (LOD scores ⱖ 4.3; Lander and Kruglyak 1995) and suggested linkage on chromosomes 13 and 18 (LOD scores ⱖ 2.8). A highly significant linkage emerged also on chromosome 4 for paired seminal vesicle weight (Figure 1). The sum of the contributions of the QTL to the phenotypic variance was lower for each of the two measures (72.8% for testis weight and 24.2% for seminal vesicles weight) than the corresponding estimated total genetic variance (82.3 and 50.7% for testis and seminal vesicle weights, respectively). The values that were computed for the three genotypes at the SSLP closest to the peak of the QTL indicated that alleles from the N strain contributed to increased testis and seminal vesicles weights (Table 4). No deviation due to dominance was detected for the QTL linked with testis weight. This conclusion fits with inspection of Table 4 indicating that B//N genotype had midvalues between B//B and N//N but not with the results from biometrical analysis. As epistasis is suspected to induce false QTL detection, we performed square root raw data transformation to eliminate the interaction between homozygous loci that appeared in the analyses of the components of the means for the two measures (Table 3). The subsequent analysis showed that epistatic contribution disappeared [l] (did not reach the significance for testis or for seminal vesicle weights) when this transformation was performed. A new QTL analysis with the transformed individual values was carried out. The LOD scores and chromosomal positions of the QTL did not differ from those reported
Testis and Seminal Vesicle Weights
337
Figure 1.—LOD plot of paired testis and seminal vesicle weights as calculated by MapQTL (tm) version 3.0 package (van Ooijen and Maliepaard 1996). Genetic distances from centromere are indicated on the x-axis with SSLPs used as markers for maximum likelihood tests.
above. The absence of epistasis between the QTL that we detected for testis weight was confirmed by calculating the interactions between the SSLP closest to the peak of the QTL because none of these interactions reached the P ⬍ 0.05 threshold. Contribution of the YNPAR: No effect of the YNPAR was detected from the analysis of reciprocal F2’s, whereas it appeared in a quartet of congenic strains for the YNPAR (Table 5). The recipient strain (N vs. H) contributed to testis and seminal vesicle weights (F ⫽ 55.23, P ⬍ 0.0001 and F ⫽ 96.19, P ⬍ 0.0001, respectively). The YNPAR effect was present in interaction with the recipient
strain for seminal vesicle weight (F ⫽ 8.97, P ⬍ 0.0037) and testis weight (F ⫽ 7.18, P ⬍ 0.009). DISCUSSION
We reported large differences related to genetic variability in testis and seminal vesicle weights in a population of 13 inbred strains of mice. Genetic analyses were performed with different complementary approaches. Contribution of the YNPAR to paired testis and seminal vesicle weights: The YNPAR contributed to reproductive organ weights as shown by the results obtained from a
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I. Le Roy et al. TABLE 4 QTL for paired testis and seminal vesicle weights in reciprocal F2 mice derived from C57BL/6By (B) and NZB/BINJ (N) strains (150 ⫾ 6 days of age) Chromosomal positiona
LOD score
N//N
N//B
B//B
% of total variance
Testis weight Chromosome 4 D4Mit205a (48 cM ⫾ 11.62) Chromosome 10 D10Mit14 (57 cM ⫾ 17.13) Chromosome 13 D13Mit13 (13.4 cM ⫾ 15.10) Chromosome 18 D18Mit17 (20 cM ⫾ 22.16) Chromosome X DXMit25 (37.8 cM ⫾ 9.85)
5.96
0.285b
0.265b
0.246b
17.5
5.81
0.282
0.264
0.241
11.9
3.10
0.269
0.261
0.248
13.5
2.80
0.272
0.260
0.251
9.2
9.11
0.272
0.257
20.7
0.387
24.2
Seminal vesicle weight Chromosome 4 D4Mit205a (47.5 cM ⫾ 8.42)
9.21
0.540
0.438
a
SSLP closest to the peak of the QTL. Values in parentheses correspond to the distance from the centromere with the confidence interval. b Mean phenotypic values at the QTL for the three possible F2 genotypes.
quartet of congenic strains, for this chromosomal region, using H and N as parental strains. The YNPAR from the N strain that had heavier testes increased testicular weight in the H strain and the transfer of YNPAR from the H strain reduced testicular weight in N. For the seminal vesicle weight, the N strain was more reactive because the YNPAR from the H strain reduced this weight on the N background, whereas it had no significant effect on H. The sizes of the effects were small and contributed to only a small part of the difference observed between the parental strains, suggesting that non-YNPAR regions contribute to both testis and seminal vesicle weight. The non-YNPAR contribution to reproductive organ weights was investigated generating the two reciprocal F1’s and the four reciprocal F2’s between the N and B parental strains, these strains being among the most contrasted strains out of a sample of 13 inbred strains. QTL for paired testis weights: As in recent studies that had used backcross and intercross generations, we found a polygenic inheritance of testis weight, encour-
aging a wide genome scan to investigate QTL. In the present study, five QTL contributing to 72.8% of the total variance were detected for testis weight, the total genetic variance being 0.823. Three QTL involved in testis weight had the values corresponding to a highly significant level (LOD score ⱖ 4.3; Lander and Kruglyak 1995). The strong genetic contribution that appeared for the QTL mapped on the X chromosome is compatible with the previously published results from a biometrics analysis (Hunt and Mitwoch 1987) and with the observed difference between our reciprocal F1’s (Table 2). The other highly significant QTL mapped on chromosomes 4 and 10. We have also found two “suggested” QTL on chromosomes 13 and 18. The QTL mapped on chromosome 13 was at 13.4 ⫾ 15.10 cM from the peak of Twq1 (Zidek et al. 1998), which is a debated QTL, for the reasons presented above. It is, moreover, at the limit of the confidence interval of our suggested QTL. Zidek et al. (1998) had also reported a suggested difference be-
TABLE 5 Paired testis and seminal vesicle weights (g, mean ⫾ SEM) in congenic strains for the specific part of the Y chromosome derived from CBA/H (H) and NZB/BINJ (N) (90 ⫾ 5 days of age) Measure Testis weight Seminal vesicle weight
Parental strains H ⫽ 0.125 ⫾ 0.019 (n ⫽ 21) N ⫽ 0.294 ⫾ 0.012 (n ⫽ 21) H ⫽ 0.133 ⫾ 0.005 (n ⫽ 21) N ⫽ 0.377 ⫾ 0.007 (n ⫽ 21)
Congenic strains for YNPAR H.N-YNPAR N.H-YNPAR H.N-YNPAR N.H-YNPAR
⫽ ⫽ ⫽ ⫽
0.134 0.228 0.126 0.281
⫾ ⫾ ⫾ ⫾
0.001 0.002 0.006 0.007
(n (n (n (n
⫽ ⫽ ⫽ ⫽
16)a 20) 16) 20)
a H.N-YNPAR indicates H strain with a nonpairing region of the Y chromosome from N and N.H-YNPAR indicates N strain with a nonpairing region of the Y chromosome from H.
Testis and Seminal Vesicle Weights
tween the genotypes B//B and D//D for D18Mit19 at 2 cM from the centromere. The mice carrying D//D alleles had, as expected, heavier testes than those with B//B. However, even if the LOD score found by Zideck and the LOD score detected in our study were low, the confidence intervals of the two QTL overlapped. QTL for paired seminal vesicle weights: Heritability was lower for this organ (50.7%) than for testis weight, this difference resulting, probably, from the smaller contrast between the N and B6 strains. The biometrics analysis performed with the mice that were used for testicular weights indicated a polygenic inheritance because an epistatic component ([l], interaction between heterozygous loci) reached significance. For this reason, chromosomal regions linked to paired seminal vesicle weights were investigated. In the present analysis, a substantial QTL (24.2% of the total variance, half of the genetic variance) has been detected on chromosome 4. Its distance from the centromere (47.5 cM) did not differ from the distance obtained for testicular weight on chromosome 4. Thus testis and seminal vesicle weights could be linked to the same QTL as expected from correlations in the F2 population. Candidate genes: Investigation of candidate genes always implies uncertainty in F2’s or recombinant inbred strains because the confidence interval is large, due to the reduced number of informative meioses, and hence encompassing a high number of genes. However, given that the physiological bases of the reproductive organ weights are documented, they may pave the way to suggesting potential candidates. Briefly, the hypothalamicpituitary axis is related to testes via two pathways. The morphogenic effect of follicle-stimulating hormone (FSH) on testes is activated by the development of epithelial tissues in seminiferous tubules and proliferation of Sertoli cells. This process is perinatal because the Sertoli cells’ proliferation stops at about 12 days after birth (Kluin et al. 1984). Inhibin, testosterone, 17estradiol, and dihydrotestosterone ensure a feedbackloop from Sertoli cells to hypothalamic-pituitary axis. A second pathway starting from this axis via luteinizing hormone reaches Leydig cells with a feedback loop to the hypothalamic-pituitary axis by testosterone and 17estradiol. The implication of hypothalamic-pituitary axis on these pathways is modulated by several factors. Leptin reduces its sensitivity, modifying the fasting-induced inhibition of gonadotropin releasing hormone (Barash et al. 1996) acting on frequency and amplitude of pulses of FSH. The implication of leptin in reproductive organ weights has been directly observed because leptin injection increases epithelial heights, producing an augmentation of both testes and seminal vesicles in mice (Barash et al. 1996). Candidate genes implicated in these mechanisms and included in the confidence intervals of putative QTL were screened using the Mouse Genome Database (1999).
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For the QTL linked with testis and seminal vesicle weights, the leptin receptor gene (Lepr) may be a common candidate, as leptin is involved in reproductive organ weights. The Lepr gene is mapped on chromosome 4 at 46.7 cM from the centromere, this distance being included in the confidence interval of both these QTL (48 and 47.5 cM from the centromere, respectively). The confidence interval of the QTL mapped on the X chromosome (38.6 cM) encompasses the androgen receptor gene (Ar), which is mapped at 36 cM from the centromere on the X chromosome (Mouse Genome Database 1999). Ar gene is a member of the nuclear receptor superfamily that acts as a ligand-dependent tissue, specific transcription factor (Magelsdorf et al. 1995). It is activated by binding testosterone or dihydrotestosterone. A single point mutation in the N-terminal region of Ar results in a premature stop codon leading to the expression of a nonfunctional truncated form of Ar (Gaspar et al. 1991). Due to X-link, Tfm mice are genetically males with smaller testes that are consistently found in the inguinal region upon dissection (Hutson et al. 1994). During development, the fetal testes secrete testosterone and antimulerian hormones, which are essential to proper differentiation and growth of the male reproductive tract. During later sexual maturation, dihydrotestosterone, the more potent testosterone metabolite, results in virilization of external genitalia. The susceptibility to testicular teratomas depends on the Ter gene in the 129 strain. It was mapped on chromosome 18 near D18mit 62 (Asada et al. 1994; Sakurai et al. 1995), which is close to the peak of the QTL that we found on this chromosome. As the Ter gene reduces germ cells in many strains of mice and was mapped in crosses derived from males having small vs. normal-sized testes, it could be a candidate for the QTL that we mapped on chromosome 18. The contribution of glucocorticoid receptor 1 (Grl-1) remains open (Sakurai et al. 1995). Conclusions: The present QTL analysis led to describing five chromosomal regions implicated in testis weight and one implicated in seminal vesicle weight. The genetic contribution of the QTL linked to testis weight reached the heritability value, suggesting that a small number of QTL were undetected in the F2 population. The picture was different for seminal vesicle weight for which 26.5% of the genetic variance was uncovered by the QTL described herein. The contribution of the specific part of the Y chromosome to reproductive organ weights is not in contradiction with this conclusion. Several genes are linked to YNPAR and other genes such as Tdy could be implicated in this phenotype. This work was supported by the CNRS (UPR 9074), Ministry for Research and Technology (Paris V-Rene´ Descartes and University of Orle´ans), Re´gion Centre and Pre´fecture de la Re´gion Centre, and
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I. Le Roy et al.
Fondation pour la Recherche Me´dicale. UPR 9074 is affiliated with INSERM and the University of Orle´ans.
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