Printer Friendly

Effects of quantitative trait loci determining testicular weight in DDD/Sgn inbred mice are strongly influenced by circulating testosterone levels.

INTRODUCTION

Testis weight is a physiologically important quantitative trait because of its direct implication in male fertility, i.e., spermatogenic ability. Several lines of evidence suggest that sperm production rate depends on testis weight [1,2]. Besides spermatogenesis, testis weight might be associated with various physiological processes; e.g., mating and aggression behaviors [3]. Interestingly, when lines were selected for testis weight, a correlated response for ovulation rate in females emerged in mice [4] and golden hamsters [5]. This suggests that sex-limited phenotypes are mutually correlated probably by sharing common physiologic pathways [6].

Therefore, elucidating the genetic basis underlying testis weight will be help to reveal various physiologic mechanisms of reproductive processes in males as well as females.

Since testis weight varies widely among inbred mouse strains (pared testis weight ranged about 100 to 300 mg), several studies pursuing testis weight genes have been performed [3,7-10]. Since males of the inbred mouse DDD/Sgn strain are known to have extremely heavy testes among inbred mouse strains, we have previously performed quantitative trait loci (QTL) mapping analyses in two [F.sub.2] intercross populations using DDD mice; i.e., [F.sub.2] mice between DDD and DH/Sgn (DH) inbred mice [9] and [F.sub.2] mice between DDD and CBA/N (CBA) inbred mice [10]. In DDDxDH [F.sub.2] mice, we identified significant testis weight QTL on chromosome (Chr) 9 (Twdql) and suggestive QTL on Chrs 4, 5, 14, 17, and 18. In DDDxCBA [F.sub.2] mice, we identified significant QTL on Chrs 1 (Twdq2) and X (Twdq3) and suggestive QTL on Chrs 2, 8, 9, 11, 13, and 14. The localization of QTL differed substantially between two crosses, and if any, there were some overlapping QTL. This finding suggests that there would be additional, yet-to-be-identified genes, which contribute to the heavy testes of DDD mice. In the present study, as a third QTL mapping study aimed at genetically dissecting the heavy testes in DDD mice, we produced and analyzed [F.sub.2] intercross populations between DDD and C57BL/6J (B6) inbred mice.

In addition to heavy testes, DDD males are known to have very high blood testosterone level [11,12]. Indeed, while the average plasma testosterone level in B6 males was 0.49 [+ or -] 0.18 ng/mL, that in DDD males was 13.07 [+ or -] 3.12 ng/mL [12]. Testicular growth and development is strongly influenced by androgen [13,14]. Although both testis weight and plasma testosterone level are inherited traits, interrelationship between them is not fully established. We have previously performed QTL mapping for plasma testosterone level in [F.sub.2] male mice between DDD and B6 [12] but did not detect any significant QTL. Even though the genetic basis of plasma testosterone level in DDD mice is unclear, we hypothesize that high blood testosterone level influence extremely heavy testes in DDD mice. Therefore, in this study, we first performed QTL mapping for testis weight and then investigated the influence of plasma testosterone level on the effect of the testis weight QTL.

We further performed whole-exome sequencing analysis in DDD mice to identify genes underlying testis weight QTL. Since extremely heavy testes are a prominent phenotype of DDD mice among inbred mouse strains, it was expected that there would be candidate genes carrying DDD-specific variations.

MATERIALS AND METHODS

Animal ethics statement

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the NIAS (authorization number H25-001).

Mice

The inbred mouse strains DDD and B6 were maintained at the National Institute of Agrobiological Sciences (NIAS, Tsukuba, Japan). Reciprocal crosses between DDD and B6 strains produced DB ([female]DDDx[male]B6) [F.sub.1] and BD ([female]B6x[male]DDD) [F.sub.1] mice, both of which were intercrossed to produce DB [F.sub.2] (n = 150) and BD [F.sub.2] (n = 150) male mice. Testicular weight was determined in DDD, B6, and [F.sub.1] mice at the age of 13 to 14 weeks (90 [+ or -] 5 days after birth), and in [F.sub.2] mice at the age of 11 to 12 weeks (71 to 80 days after birth).

All mice were weaned at 4 weeks of age and four to five mice were housed together in each cage during the experiments. All mice were maintained in a specific pathogen-free facility with a regular light cycle, controlled temperature, and humidity. Food (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and water were freely available throughout the experimental period.

Testosterone and testis weight analyses

The plasma testosterone level was determined in parental, [F.sub.1] and [F.sub.2] mice, as previously described [12]. The paired testes were weighed on an electric balance to the nearest milligram. The weight of the paired testes is simply designated as "testis weight."

Genotyping

Microsatellite sequence length polymorphisms were identified by electrophoresis after polymerase chain reaction (PCR) amplification of genomic DNA. The PCR amplification was carried out using the Thermal Cycler Dice (TaKaRa Bio Inc., Shiga, Japan). The PCR products were separated on 10% polyacrylamide gel (Nacalai Tesque Inc., Kyoto, Japan) and were visualized by ethidium bromide (Nacalai Tesque, Japan) staining. In total, 117 microsatellite loci were genotyped. Their chromosomal positions were retrieved from Mouse Genome Informatics (MGI, http://www.jax.org). Because the chromosomal positions of six markers were unavailable, they were determined based on our own linkage map.

Quantitative trait loci analysis

Normality of testis weight was assessed using the Shapiro-Wilk W test (JMP13, SAS Institute Japan Inc., Tokyo, Japan). QTL mapping was performed using R/QTL version 1.38-4 [15,16]. Single-QTL scans were performed by computing at 1 cM intervals across the entire genome with or without using the lineage (i.e., the direction of the cross) effect as a covariate. First, we included the lineage effect as an additive covariate in the single-QTL scan, if the lineage effect had a strong effect on testis weight. Next, we included the lineage effect as an interactive covariate because the effect of QTL may vary with the covariate (i.e., QTLxcovariate interaction). Threshold logarithm of the odds (LOD) scores for significant (p<0.05) and suggestive (p<0.63) linkages were determined by performing 1,000 permutations [17,18]. After single-QTL scans, two-QTL scans were performed to identify pairwise interactions. In this case, we strictly adhered to a recommended threshold [15]. Finally, the covariates and the combined effects of all QTL, including those that were significant and suggestive, were assessed using multiple-QTL models [19].

Whole-exome sequence analysis

To identify nonsynonymous single-nucleotide variation (nsSNV) and/or insertion-deletion in the coding regions of candidate genes, whole-exome sequence analyses were performed. Genomic DNA was extracted from the tail of DDD mice using a genomic DNA purification kit (Wizard Genomic DNA Purification Kit, Promega KK, Tokyo, Japan) and was submitted to Filgen, Inc. (Nagoya, Aichi, Japan) for exome capture and sequencing. Sequence reads were mapped to the mouse reference genome (GRCm38, mm10). Read mapping and variant analyses were performed using CLC Genomics Workbench 7.0.4 and 8.5.1 (Filgen, Japan).

Alpha tubulin acetyltransferase 1 sequencing

Genomic DNA from inbred strains including DDD, B6, DH/Sgn, and CBA/N were extracted according the protocol described above. PCR was performed using Atat1-specific primers (F: 5'-ttcccgttcgatgtggat and R: 5'-gtaaataacgtgccggttgc). The PCR product was purified by PCR purification kit (Labo-Pass PCR, Hokkaido System Science., Ltd. Sapporo, Japan) and submitted to Hokkaido System Science for direct sequence with these primers.

Statistical analysis

Statistical analyses were performed using JMP 13 (SAS Institute, Japan). Testis weight was represented as the mean [+ or -] standard error of the mean (mg). Statistical differences between two groups were analyzed using Students or Welch's t-test. For statistical comparison among more than two groups, the Tukey-Kramer honest significant difference test was used. Statistical significance is defined when p values are less than 0.05.

RESULTS

Localization of testis weight quantitative trait loci

Testis weight in DDD males (n = 28) was significantly higher than that in B6 males (n = 16) (277.6 [+ or -] 3.7 mg vs 206.2 [+ or -] 5.3 mg, p<0.0001). A clear lineage effect was identified in [F.sub.1] mice; i.e., testis weight in BD [F.sub.1] males (n = 12) was significantly higher than that in DB [F.sub.1] males (n = 15) (301.5 [+ or -] 4.8 mg vs 279.7 [+ or -] 3.7 mg, p<0.002).

Figure 1 depicts a histogram showing the distribution of testis weight in 297 [F.sub.2] male mice (We failed to determine the testis weight in one BD [F.sub.2] and one DB [F.sub.2] mouse. In addition, we excluded one DB [F.sub.2] mouse from the analysis because the paired testis weight of this mouse was abnormally low; i.e., only 76.8 mg. Thus, we phenotyped 297 [F.sub.2] mice but genotyped 300 [F.sub.2] mice). The testis weight followed a normal distribution. The average testis weight was 255.8 [+ or -] 1.7 mg. Although the difference between BD [F.sub.2] and DB [F.sub.2] males was not statistically significant (257.0 [+ or -] 2.3 mg vs 254.6 [+ or -] 2.4 mg, p>0.4), we included the lineage effect as an additive covariate in the subsequent single-QTL scan.

A LOD score plot for testis weight in [F.sub.2] males is shown in Figure 2 (solid lines). As shown in Table 1, four significant QTL were identified on Chr 1@57.5 cM (Twdq2: this locus was coincidental with that identified in DDDxCBA [F.sub.2] mice [10]; accordingly, the same QTL symbol was assigned), Chr 8@41.5 cM (Twdq4), Chr 14@32.3 cM (Twdq5), and Chr 17@36.1 cM (Twdq6), along with three suggestive QTL on Chr 4@33.1 cM (Twdq7: QTL symbol was assigned to this suggestive QTL because a significant testis weight QTL has been mapped to this region [3]), Chr 7@2.0 cM, and Chr 10@ 61.5 cM (Twdq8: QTL symbol was assigned to this suggestive QTL because a significant testis weight QTL has been mapped to this region [3]). The DDD allele was associated with increased testis weight at all QTL except for the Twdq8 on Chr 10. We next performed QTL mapping by including the lineage effect as an interactive covariate, but found no significant QTLxcovariate interactions (in other words, there were no QTL whose effect differed significantly between BD [F.sub.2] and DB [F.sub.2] males). Two-QTL scans with R/QTL revealed a possible pairwise interaction between Chr 1 and Chr 8; however, the LOD score for interaction (lod.int) was substantially lower (2.58) than the recommended threshold value (i.e., 6.3). Multiple regression analysis indicated that the detected QTL explained 41.4% of the testis weight variation (Table 2).

In the above analysis, we found strong evidence for Twdq2 on Chr 1. Accordingly, we next performed QTL mapping by including the nearest marker (D1Mit102) for Twdq2 as an additive covariate. While the LOD score on Chr 1 shrank to 1.1, the LOD scores for the remaining significant QTL, particularly those for Twdq4 (Chr 8) and Twdq5 (Chr 14), were increased to 6.2 and 4.8, respectively. In contrast, the LOD score for Twdq6 (Chr 17) was changed subtly (LOD score 4.4) (data not shown). One additional suggestive QTL was identified on Chr 5 (Table 1).

We also performed a single-QTL scan by including body weight as an additive covariate. Since the suggestive QTL on Chr 7 was identified as significant (Table 1), we newly assigned the QTL symbol Twdq9 to this QTL. Clearly, the LOD score for Twdq6 (Chr 17) was substantially increased to 7.7 (Figure 2, dashed lines). One additional suggestive QTL was identified on Chr 18 (Table 1).

Influence of testosterone level on the localization of quantitative trait loci

Next, we analyzed testis weight by taking circulating testosterone level into consideration, because DDD male has extremely high plasma testosterone level. The [F.sub.2] mice were then divided into two groups according to the plasma testosterone level; i.e., [F.sub.2] mice with less than 0.49 ng/mL testosterone (an average testosterone level in B6 male) were designated as [F.sub.2]L ([F.sub.2] with "low" testosterone, n = 124, an average and a median testosterone level were 0.33 [+ or -] 0.01 ng/mL and 0.34 ng/mL, respectively) mice and [F.sub.2] mice with more than 0.49 ng/mL testosterone were as [F.sub.2]H ([F.sub.2] with "high" testosterone, n = 176, an average and a median testosterone level were 7.84 [+ or -] 0.78 ng/mL and 2.02 ng/mL, respectively) mice. The difference in testosterone level between [F.sub.2]L and [F.sub.2]H mice was statistically significant (p<0.0001) when judged by either parametric (Student's t-test) or non-parametric (Wilcoxon rank sum test) analysis. On the other hand, there was no significant difference in testis weight in [F.sub.2]L and [F.sub.2]H mice (256.1 [+ or -] 2.6 mg vs 255.6 [+ or -] 2.2 mg, p>0.8). Single-QTL scans were performed for [F.sub.2]L and [F.sub.2]H mice separately. In [F.sub.2]L mice, one significant QTL was identified Chr 1@52.5 cM, along with two suggestive QTL on Chr 10@64.5 cM and Chr 14@ 53.3 cM (Table 3; Figure 3). On the other hand, in [F.sub.2]H mice, three significant QTL were identified on Chr 1@68.5 cM, Chr 8@ 45.5 cM, and Chr 17@52.1 cM, along with one suggestive QTL on Chr 4@33.1 cM. Substantial difference in LOD scores between [F.sub.2]L and [F.sub.2]H mice was found for QTL on Chrs 1, 4, 8, and 17. Notably, the LOD score for QTL on Chr 17 in [F.sub.2]H mice was higher than that identified in all [F.sub.2] mice (Table 1). Although QTL on Chr 1 explained similar variance in [F.sub.2]L and [F.sub.2]H mice, QTL identified in [F.sub.2]H mice accounted for slightly more variance compared with those identified in [F.sub.2]L mice (Table 2). Allele effect of significant QTL was investigated in all [F.sub.2], [F.sub.2]L, and [F.sub.2]H mice (Figure 4). Except for Twdq5 on Chr 14 (Figure 4C), high testosterone level tended to be associated with increasing the effect of the DDD-derived allele and with decreasing the effect of the B6-derived allele. Testosterone level appeared not to influence the effect of heterozygotes.

Candidate gene identification for Twdq6 on Chr 17

By submitting the term "abnormal testis weight" as a query to the MGI database (Mammalian Phenotype Browser), we retrieved 25 genes that were localized within 95% confidence intervals (CIs) of four significant single-QTL on Chrs 1, 8, 14, and 17 (Table 4). In most cases, testis weight was reduced in the mutant mice of these genes; only 2 of 25 mutants (protein tyrosine phosphatase, receptor type, V [Ptprv] and TBC domain family, member 4 [Tbc1d4]) were associated with increased testis weight. Since DDD mice have extremely heavy testis relative to other inbred mice, we assumed that there are DDD-specific genetic variations. To identify such genetic variations, we performed whole-exome sequence analysis. We identified nsSNVs in eight genes, i.e., tudor domain containing 5 (Tdrd5), neuregulin 1 (Nrgl), nanos C2HC-type zinc finger 3 (Nanos3), synaptonemal complex central element protein 2 (Syce2), Tbc1d4, mutS homolog 5 (Msh5), Atatl, and follicle stimulating hormone receptor (Fshr), but none of them except for one that detected for Atatl were DDD-specific. p.Pro29Ser resulting from a c.85C>T substitution (Chr 17:35909951) in Atatl seems to be novel because no single nucleotide polymorphism (SNP) ID has been assigned, and because no SNP were known at this chromosomal location in other inbred mouse strains including 129S1/SvImJ, A/J, AKR/J, BTBR T+ Itp3tf/J, C3H/HeJ, C57BL/6J, CAST/EiJ, CBA/J, DBA/1J, DBA/2J, FVB/NJ, I/LnJ, KK/HIJ, MOLF/EiJ, NOD/ShiLtJ, NZB/BINJ, NZO/HILtJ, PWK/PhJ, RF/J, SPRET/ EiJ, and WSB/EiJ according to "Mouse SNP retrieval utility" by Mouse Phenome Database (Figure 5A). Furthermore, Pro29 was well conserved among mammalian species including human, rat, chimpanzee, rhesus macaque, cattle, and dog (Figure 5B).

DISCUSSION

This study identified nine QTL, of which five were significant and four were suggestive. The result was satisfactory because the number of QTL underlying testis weight was thought to be small [7,20]. Seven of the nine QTL, i.e., Twdq2 (Chr 1), Twdq7 (Chr 4), Twdq4 (Chr 8), Twdq5 (Chr 14), Twdq6 (Chr 17), and the suggestive QTL on Chrs 5 and 18, had also been identified in either of our preceding studies using DDD mice [9, 10]. Therefore, only Twdq9 (Chr 7) and Twdq8 (Chr 10) were novel in a series of QTL mapping studies using DDD mice. Based on the results of three studies, most of the QTL underlying high testis weight in DDD mice might have been identified. Notably, the DDD-derived allele was associated with higher testis weight at most QTL.

The lineage effect was observed in the [F.sub.1] populations, i.e., BD [F.sub.1] mice had significantly higher testis weight than DB [F.sub.1] mice. The lineage effect was also found in our previous studies; e.g., DHxDDD [F.sub.1] mice had significantly higher testis weight than that of the DDDxDH [F.sub.1] mice [9], and CBAxDDD [F.sub.1] mice had significantly higher testis weight than that of the DDDxCBA [F.sub.1] mice [10]. We attribute the lineage effect to the difference in the genes on the Chr Y because the [F.sub.1] mice carrying [Y.sup.DDD] had significantly higher testis weight than the reciprocal [F.sub.1] mice in all crosses. There is experimental evidence supporting the contribution of Chr Y to testis weight in mice [21]. Our studies in Y-consomic strains clearly showed the effect of Chr Y; Chr [Y.sup.DDD] produced significantly heavier testes than did Chr [Y.sup.B6] [9,10]. Further evidence was that DDD mice had significantly heavier testis than DDD-Chr [Y.sup.CBA] mice (296.0 [+ or -] 4.4 mg vs 252.2 [+ or -] 3.8 mg, p<0.0001), although the body weight did not significantly differ between the two strains (32.3 [+ or -] 0.4 g vs 31.8 [+ or -] 0.3 g, p>0.3) (unpublished data). It was suggested that the native YDDD was indispensable to sustain high testis weight in DDD mice. The effect of Chr Y appeared to be independent of the effect of autosomes.

To control the effect of body weight, we analyzed testis weight by including body weight as an additive covariate. Accordingly, we re-analyzed previously published testis weight data in the DDDxDH [F.sub.2] mice [9] and DDDxCBA [F.sub.2] mice [10] by including body weight as an additive covariate. As a result, two of four suggestive QTL identified in DDDxDH [F.sub.2] mice, i.e., QTL on Chrs 14 and 17, were identified as significant QTL (the maximum LOD scores for these QTL were 4.8 and 4.7, respectively). The result suggests that these QTL may have an indirect effect on testis weight, acting through the body weight [15]. The QTL on Chr17 identified in DDDx DH [F.sub.2] mice might be allelic to that identified in this study because of considerable increase of LOD score after the inclusion of body weight as an additive covariate. In contrast, it was uncertain whether the QTL on Chr 14 identified in the two crosses were allelic, because the LOD score for Twdq5 identified in the present study was not substantially changed after the inclusion of body weight as an additive covariate.

Results of separate [F.sub.2] analyses (i.e., [F.sub.2]L and [F.sub.2]H

mice) strongly suggested that we should take the endocrinological background of the mice into consideration when assessing the effect of genes on testis weight. As it was particularly evident for the QTL on Chr 17, "high" testosterone level altered the mode of inheritance of QTL allele. In other words, "high" testosterone level tended to be associated with increasing the effect of the DDD-derived allele and with decreasing the effect of the B6-derived allele. We considered the possible mechanism for this phenomenon to be that, testosterone level might change the magnitude of expression level of genes underlying QTL. Indeed, testosterone is known to regulate gene expression levels [22,23]. Thus, high testosterone might be associated with up-regulation of the DDD-derived genes and was associated with down-regulation of the B6-derived genes. Otherwise, high testosterone might sensitize the cells or cellular receptors, on which the genes underlying QTL act. In this context, we were interested in the genetic basis of plasma testosterone level. Like other blood components, testosterone level is suggested to be genetically determined. Serum testosterone level is inherited in an autosomal dominant mode in pig breeds Meishan and Landrace [22], and significant QTL was identified in White DurocxChinese Erhualian resource population [24]. However, the mode of inheritance of circulating testosterone level was ambiguous in mice, and we could not identify any significant QTL [12]. In part, this result is associated with the extensive variation in the plasma testos terone levels in DDD mice. Since the episodic testosterone secretion is known in mice [25,26], variable testosterone level even in an inbred mouse strain might not be surprising. We should be cautious about species difference when interpreting the experimental results regarding the blood testosterone levels.

By searching MGI database, we found 25 candidate genes that potentially influence testis weight within 95% CIs for four significant single-QTL on Chrs 1, 8, 14, and 17 (Table 4). Interestingly, testis weight was decreased in most mutants of the candidate genes. Dysfunctional gene mutations tend to be associated with lower testis weight; thus, the higher testis weight of DDD mice might be a consequence of altered gene functions. We performed whole-exome sequencing in DDD mice on the assumption that the higher testis weight of DDD mice is caused by coding-region variants, which are specific to the DDD strain. The analysis identified nsSNV that differed between DDD and B6 mice in 8 of 25 candidate genes: Tdrd5, Nrgl, Nanos3, Syce2, Tbc1d4, Msh5, Atatl, and Fshr (Table 4). However, most of nsSNV identified for these genes were not DDD strain-specific. For example, although there were six nsSNVs in Tdrd5, all were also found in many other inbred mouse strains. In particular, two of six nsSNV in Tdrd5 resulting in Thr396Ala and Ile111Met were also identified in NZB/BINJ strain, which have extremely heavy testes like DDD [3]. However, no significant testis weight QTL was identified on Chr 1 in C57BL/6ByJ x NZB/BINJ [F.sub.2] mice, suggesting that these were unlikely to be causative of Twdq2. Similarly, two nsSNVs in Tbc1d4 resulting in Ile834Val and Arg659Gly were identified in NZB/BINJ, but no significant testis weight QTL was identified on Chr 14 in abovementioned [F.sub.2] mice [3]; therefore, these were unlikely to be causative of Twdq5. Although the nsSNVs in Fshr are plausible candidates underlying Twdq6, these nsSNVs were also found in other inbred mouse strains including a strain with very light testes such as CAST/EiJ.

One nsSNV, Pro29Ser, identified in Atat1 appears to be novel, given that no SNP ID has yet been assigned. Accordingly, we searched for this nsSNV among the various inbred mouse strains with consequent that this nsSNV was not shared by any other strains. We then investigated Atat1 sequence in other mammalian species and found that Pro29 was well conserved. Thus, we concluded that Ser29 was a mutation occurred specifically in DDD mice. A targeted disruption mutation in Atat1 resulted in reduction of testis weight in mice [27]. Therefore, Pro29Ser was unlikely to be associated with loss or hypofunction of this gene, if this gene is a causative of Twdq6. Although further-in-depth in vivo studies are necessary for validating the function of this mutation, this was an important finding to understand the molecular basis of mechanisms underlying high testis weight in mice. Taken together, results of the present study provide insights into genetic and endocrinological mechanisms determining testis weight in mice.

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. https://doi.org/10.5713/ajas.18.0783

Submitted Oct 17, 2018; Revised Feb 18, 2019;

Accepted Mar 6, 2019

ACKNOWLEDGMENTS

We would like to thank Editage (www.editage.jp) for English language editing. This study was supported by the NIAS Strategic Research Fund.

REFERENCES

[1.] Harcourt AH, Harvey PH, Larson SG, Short RV Testis weight, body weight and breeding system in primates. Nature 1981; 293:55-7. https://doi.org/10.1038/293055a0

[2.] Leader-Williams N. Age-related changes in the testicular and antler cycles of reindeer, Rangifer tarandus. J Reprod Fertil 1979;57:117-26. https://doi.org/10.1530/jrf0.0570117

[3.] Le Roy I, Tordjman S, Migliore-Samour D, Degrelle H, Roubertoux PL. Genetic architecture of testis and seminal vesicle weights in mice. Genetics 2001;158:333-40.

[4.] Mafizul Islam ABM, Hill WG, Land RB. Ovulation rate of lines of mice selected for testis weight. Genet Res 1976;27: 23-32. https://doi.org/10.1017/S0016672300016207

[5.] Furukawa T, Awata T, Satoh M. Selection for testis size and correlated responses of female reproductive traits in golden hamsters. Anim Sci J 2013;84:382-8. https://doi.org/10.1111/asj.12015

[6.] Land RB. The expression of female sex-limited characters in the male. Nature 1973;241:208-9. https://doi.org/10.1038/241208a0

[7.] Rocha JL, Eisen EJ, Van Vleck LD, Pomp D. A large-sample QTL study in mice: II. Body composition. Mamm Genome 2004;15:100-13. https://doi.org/10.1007/s00335-003-2308-6

[8.] L'Hote D, Serres C, Laissue P, et al. Centimorgan-range one-step mapping of fertility traits using interspecific recombinant congenic mice. Genetics 2007;176:1907-21. https://doi.org/10.1534/genetics.107.072157

[9.] Suto J. Genetic dissection of testis weight in a mouse strain having an extremely large testis: major testis weight determinants are autosomal rather than Y-linked on the basis of comprehensive analyses in Y-chromosome consomic strains. Proc Jpn Acad Ser B Phys Biol Sci 2008;84:393-406. https:// doi.org/10.2183/pjab.84.393

[10.] Suto J. Genetic dissection of testis weight in mice: quantitative trait locus analysis using [F.sub.2] intercrosses between strains with extreme testis weight, and association study using Y-consomic strains. Mamm Genome 2011;22:648-60. https://doi.org/10.1007/s00335-011-9353-3

[11.] Goto N, Nakajima Y, Imamura K, Yoshida T. Influence of testosterone on hydronephrosis in the inbred mouse strain DDD. Lab Anim 1985;19:85-8. https://doi.org/10.1258/002367785780942651

[12.] Suto J, Kojima M. Quantitative trait loci that control body weight in DDD/Sgn and C57BL/6J inbred mice. Mamm Genome 2017;28:13-9. https://doi.org/10.1007/s00335-0169666-3

[13.] O'Shaughnessy PJ, Monteiro A, Abel M. Testicular development in mice lacking receptors for follicle stimulating hormone and androgen. PLoS One 2012;7:e35136. https://doi.org/10.1371/journal.pone.0035136

[14.] Koskenniemi JJ, Virtanen HE, Toppari J. Testicular growth and development in puberty. Curr Opin Endocrinol Diabetes Obes 2017;24:215-24. https://doi.org/10.1097/MED.0000000000000339

[15.] Broman KW, Sen S. A guide to QTL mapping with R/qtl. New York, USA: Springer; 2009.

[16.] Broman KW, Wu H, Sen S, Churchill GA. R/qtl: QTL mapping in experimental crosses. Bioinformatics 2003;19:889-90. https:// doi.org/10.1093/bioinformatics/btg112

[17.] Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics 1994;138:963-71.

[18.] Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995;11:241-7. https://doi.org/10.1038/ng1195-241

[19.] Sen S, Churchill GA. A statistical framework for quantitative trait mapping. Genetics 2001; 159:371-87.

[20.] Chubb C. Genes regulating testis size. Biol Reprod 1992;47: 29-36. https://doi.org/10.1095/biolreprod47.L29

[21.] Hayward P, Shire JGM. Y chromosome effect on adult testis size. Nature 1974;250:499-500. https://doi.org/10.1038/250499a0

[22.] Kojima M, Degawa M. Serum androgen level is determined by autosomal dominant inheritance and regulates sex-related CYP genes in pigs. Biochem Biophys Res Commun 2013;430: 833-8. https://doi.org/10.1016/j.bbrc.2012.11.060

[23.] Kojima M, Sekimoto M, Degawa M. Androgen-mediated down-regulation of CYP1A subfamily genes in the pig liver. J Endocrinol 2010;207:203-11. https://doi.org/10.1677/JOE-100160

[24.] Ren DR, Ren J, Xing YY, et al. A genome scan for quantitative trait loci affecting male reproductive traits in a White Duroc x Chinese Erhualian resource population. J Anim Sci 2009; 87:17-23. https://doi.org/10.2527/jas.2008-0923

[25.] Bartke A, Dalterio S. Evidence for episodic secretion of testosterone in laboratory mice. Steroids 1975;26:749-56. https:// doi.org/10.1016/0039-128X(75)90107-5

[26.] Klomberg KF, Garland, Jr T, Swallow JG, Carter PA. Dominance, plasma testosterone levels, and testis size in house mice artificially selected for high activity levels. Physiol Behav 2002;77: 27-38. https://doi.org/10.1016/S0031-9384(02)00767-9

[27.] Kalebic N, Sorrentino S, Perlas E, Bolasco G, Martinez C, Heppenstall PA. alphaTAT1 is the major alpha-tubulin acetyl-transferase in mice. Nat Commun 2013;4:1962. https://doi. org/10.1038/ncomms2962

Jun-ichi Suto (1), * and Misaki Kojima (2)

* Corresponding Author: Jun-ichi Suto Tel: +81-29-838-6052, Fax: +81-29-838-7106, E-mail: jsuto@affrc.go.jp

(1) Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8634, Japan

(2) Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-0901, Japan

ORCID

Jun-ichi Suto

https://orcid.org/0000-0003-0659-2257

Misaki Kojima

https://orcid.org/0000-0002-1921-8160

Caption: Figure 1. A histogram showing the distribution of testis weight in 297 [F.sub.2] mice (data from BD [[female]B6x[male]DDD] [F.sub.2] and DB [[female]DDDx[male]B6] [F.sub.2] mice are combined). The mean [+ or -] SEM is 255.8 [+ or -] 1.7 mg (The mean [+ or -] SEM testis weight in BD [F.sub.2] mice is 257.0 [+ or -] 2.3 mg and that in BD [F.sub.2] mice is 254.6 [+ or -] 2.4 mg. The difference was not statistically significant). The average testis weight of parental B6 and DDD strains are indicated by arrows. SEM, standard error of the mean.

Caption: Figure 2. Genome-wide LOD score plot for testis weight when the lineage effect was included as an additive covariate (solid lines) and when the body weight was included as an additive covariate (dashed lines). The x-axis represents chromosomal and microsatellite marker positions and the y-axis represents LOD scores. Horizontal broken lines indicate the genome-wide threshold LOD score for significant (p<0.05) and suggestive (p<0.63) linkage. Threshold LOD scores for significant and suggestive quantitative trait loci were 3.56 and 2.10 for autosomes and 2.73 and 1.48 for Chr X, respectively. LOD, logarithm of the odds.

Caption: Figure 3. Genome-wide LOD score plot of the QTL for testis weight when the [F.sub.2]H (solid lines) and [F.sub.2]L (dashed lines) mice were analyzed separately. The x-axis represents the chromosomal and microsatellite marker position, and the y-axis represents the LOD score. Horizontal broken lines indicate the genome-wide threshold LOD score for significant (p<0.05) and suggestive (p<0.63) linkage. Threshold LOD scores for significant and suggestive QTL were 3.37 and 2.08 for autosomes and 2.72 and 1.50 for Chr X, respectively. LOD, logarithm of the odds; QTL, quantitative trait loci.

Caption: Figure 4. Comparison of allele effect of significant QTL among all [F.sub.2], [F.sub.2]L, and [F.sub.2]H mice. The x-axis represents the genotype at the microsatellite marker nearest QTL, and the y-axis represents average testis weight. Mice with different superscripts denote the presence of significant difference. Statistical difference between mice with three genotypes within each [F.sub.2] group (i.e., [F.sub.2] all, [F.sub.2]L, and [F.sub.2]H) was identified by Tukey-Kramer honestly significant difference tests. Error bars indicate standard error of means. QTL, quantitative trait loci.

Caption: Figure 5. Atatl sequence analysis. (A) Sequencing chromatograms for Atatl from DDD/Sgn, C57BL/6J, DH/Sgn, and CBA/N mice showing C>T polymorphism. (B) Atatl sequence alignment from various species with the novel polymorphism in red. Atatl, alpha tubulin acetyltransferase 1.
Table 1. Significant and suggestive QTL identified by the
genome-wide scan of F2 mice

QTL1)           Chr   Peak cM   95% CI2)

Twdq2            1     57.5     49.5-72.5
Twdq7            4     33.1     8.1-61.1
                 7      2.0     2.0-20.2
Twdq4            8     41.5     14.5-50.1
Twdq8           10     61.5     35.5-66.7
Twdq5           14     32.3     16.3-57.3
Twdq6           17     36.1     17.1-60.7
By including
  D1Mit102 as
  an additive    5     41.6     17.8-72.8
  covariate
By including
  body weight
  as an
  additive
  covariate
Twdq9            7      2.0     2.0-16.0
                18     45.9     28.9-55.9

QTL1)           lod3)    Nearest     High
                         marker     strain4)

Twdq2           11.5    D1Mit102      DDD
Twdq7            3.3    D4Mit178      DDD
                 2.7    D7Mit178      DDD
Twdq4            5.6    D8Mit263      DDD
Twdq8            2.2    D10Mit180      B6
Twdq5            3.7    D14Mit64      DDD
Twdq6            4.3    D17Mit152     DDD
By including
  D1Mit102 as
  an additive    2.5    D5Mit113      DDD
  covariate
By including
  body weight
  as an
  additive
  covariate
Twdq9            3.5    D7Mit178      DDD
                 2.4    D18Mit188     DDD

QTL, quantitative trait loci; CI, confidence interval; LOD,
logarithm of the odds.

Lineage effect was included as an additive covariate.

1) QTL symbols were assigned if they were significant, or if
they were suggestive but identified as significant at least
once previously in different genetic crosses.

2) 95% CI was defined by a 1.5-LOD drop.

3) LOD scores for significant QTL are indicated in bold.

4) High strain-derived alleles were associated with higher
testis weight.

Table 2. Multiple regression analysis for testis weight

Mice            Chr (cM)     df (1)  Variance    F
                                     (%) (2)     value

All             Chr1@57.5     2       13.7     33.0
  [F.sub.2] (3) Chr4@33.1     2       2.0       4.8
                Chr7@2.0      2       3.1       7.4
                Chr8@41.5     2       4.9      11.8
                Chr10@61.5    2       1.8       4.4
                Chr14@32.3    2       5.6      13.5
                Chr17@37.1    2       4.5      10.7
                Total         14      41.4      --
[F.sub.2]L      Chr1@52.5     2       12.7     10.5
                Chr10@64.5    2       5.6       4.7
                Chr14@53.3    2       8.3       6.9
                Total         6       29.9      --
[F.sub.2]H      Chr1@68.5     2       12.0     16.4
                Chr4@33.1     2       2.7       3.7
                Chr8@45.5     2       5.8       8.0
                Chr17@52.1    2       9.0      12.4
                Total         8       39.8      --

(1) Degrees of freedom.

(2) Percentage of total [F.sub.2] phenotypic variance.

(3) Lineage effect was included as a covariate.

Table 3. Significant and suggestive QTL identified by the genome-
wide scan of separate [F.sub.2] mice

Mice         QTL (1)   Chr   Peak cM   95% CI (2)  LOD (3)

[F.sub.2]L   Twdq2      1     52.5     16.5-72.5    4.3
             Twdq8     10     64.5     45.5-66.7    2.4
             Twdq5     14     53.3     22.3-62.3    2.8
[F.sub.2]H   Twdq2      1     68.5     51.5-75.5    8.3
             Twdq7      4     33.1     15.1-52.1    3.3
             Twdq4      8     45.5     15.5-58.5    5.2
             Twdq6     17     52.1     25.1-60.7    5.1

QTL, quantitative trait loci; CI, confidence interval; LOD,
logarithm of the odds.

(1) QTL symbols were assigned if they were significant, or if
they were suggestive but identified as significant at least
once previously in different genetic crosses.

(2) 95% CI was defined by a 1.5-LOD drop.

(3) LOD scores for significant QTL are indicated in bold.

Table 4. Candidate genes, mutant phenotypes, and nsSNV identified
for candidate genes of significant QTL

Chr   Position   Gene      Testis          Location        Nucleotide
         cM                weight in                        change
                           mutants

1      57.91     Kiss1     Decrease            -               --
       57.94     Snrpe     Decrease            -               --
       58.24     Ptprv     Increase            -               --
       61.45     Aspm      Decrease            -               --
       67.71     Tdrd5     Decrease     Chr1:156262881     c.2594C>G
                                        Chr1:156263413     c.2495C>T
                                        Chr1:156267355     c.2177G>A
                                        Chr1:156270646     c.2080G>A
                                        Chr1:156285523     c.1186A>G
                                        Chr1:156301806      c.333A>G
       71.56     Slc19a2   Decrease           --               --
8      18.75     Nrg1      Decrease     Chr8: 31818081     c.2050T>A
       20.59     Tex15     Decrease           --               --
       23.89     Cnot7     Decrease           --               --
       26.87     Ing2      Decrease           --               --
       40.45     Nanos3    Decrease     Chr8: 84176519      c.13A>G
       41.25     Syce2     Decrease    Chr8: 848722541      c.13G>A
14     18.79     Tkt       Decrease           --               --
       25.36     Otx2      Decrease           --               --
       27.98     Bcl2l2    Decrease           --               --
       36.32     Piwil2    Decrease           --               --
       37.62     Fndc3a    Decrease           --               --
       50.09     Tbc1d4    Increase    Chr14: 101458822    c.2500A>G
                                       Chr14: 101477059    c.1975A>G
17     18.57     Msh5      Decrease    Chr17: 35028832     c.2411C>A
                                       Chr17: 35031264     c.1693A>G
       18.59     Tnf       Decrease           --               --
       18.75     Atat1     Decrease    Chr17: 35909951     c.85C>T
       25.86     Dazl      Decrease           --               --
        29.4     Safb      Decrease           --               --
       58.35     Lhcgr     Decrease           --               --
       58.76     Fshr      Decrease    Chr17: 88985240     c.2009C>A
                                       Chr17: 88985243     c.2006A>G
                                       Chr17: 88986290     c.959G>A

Chr   Position   Gene      Testis            Amino         db SNP ID
         cM                weight in         acid
                           mutants          change

1      57.91     Kiss1     Decrease           --               --
       57.94     Snrpe     Decrease           --               --
       58.24     Ptprv     Increase           --               --
       61.45     Aspm      Decrease           --               --
       67.71     Tdrd5     Decrease       p.Pro865Arg      rs49650703
                                          p.Ala832Val      rs13476193
                                          p.Gly726Glu      rs48483855
                                          p.Asp694Asn      rs30769841
                                          p.Thr396Ala      rs31654838
                                          p.Ile111Met      rs32356404
       71.56     Slc19a2   Decrease           --               --
8      18.75     Nrg1      Decrease       p.Leu684Ile      rs32559738
       20.59     Tex15     Decrease           --               --
       23.89     Cnot7     Decrease           --               --
       26.87     Ing2      Decrease           --               --
       40.45     Nanos3    Decrease        p.Asn5Asp       rs38027221
       41.25     Syce2     Decrease        p.Gly5Arg       rs32744209
14     18.79     Tkt       Decrease           --               --
       25.36     Otx2      Decrease           --               --
       27.98     Bcl2l2    Decrease           --               --
       36.32     Piwil2    Decrease           --               --
       37.62     Fndc3a    Decrease           --               --
       50.09     Tbc1d4    Increase       p.Ile834Val      rs48744612
                                          p.Arg659Gly      rs235588405
17     18.57     Msh5      Decrease       p.Thr804Lys      rs33561826
                                          p.Met565Val      rs50143709
       18.59     Tnf       Decrease           --               --
       18.75     Atat1     Decrease       p.Pro29Ser           --
       25.86     Dazl      Decrease           --               --
        29.4     Safb      Decrease           --               --
       58.35     Lhcgr     Decrease           --               --
       58.76     Fshr      Decrease       p.Pro670His      rs107828637
                                          p.Asn669Ser      rs108743480
                                          p.Ser320Asn      rs51040992

nsSNV, nonsynonymous single-nucleotide variation; QTL, quantitative
trait loci; SNP, single nucleotide polymorphism; Kiss1, KiSS-1
metastasis-suppressor; Snrpe, small nuclear ribonucleoprotein E;
Ptprv, protein tyrosine phosphatase, receptor type, V; Aspm,
abnormal spindle microtubule assembly; Tdrd5, tudor domain
containing 5; Slc19a2, solute carrier family 19 (thiamine
transporter), member 2; Nrg1, neuregulin 1; Tex15, testis expressed
gene 15; Cnot7, CCR4-NOT transcription complex, subunit 7; Ing2,
inhibitor of growth family, member 2; Nanos3, nanos C2HC-type zinc
finger 3; Syce2, synaptonemal complex central element protein 2;
Tkt, transketolase; Otx2, orthodenticle homeobox 2; Bcl2l2, BCL-like
2; Piwil2, piwi-like RNA-mediated gene silencing 2; Fndc3a,
fibronectin type III domain containing 3A; Tbc1d4, TBC1 domain
family, member 4; Msh5, mutS homolog 5; Tnf, tumor necrosis factor;
Atat1, alpha tubulin acetyltransferase 1; Dazl, deleted in
azoospermia-like; Safb, scaffold attachment factor B; Lhcgr,
luteinizing hormone/choriogonadotropin receptor; Fshr, follicle
stimulating hormone receptor.
COPYRIGHT 2019 Asian - Australasian Association of Animal Production Societies
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2019 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Suto, Jun-ichi; Kojima, Misaki
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9JAPA
Date:Dec 1, 2019
Words:6594
Previous Article:Analysis of allele-specific expression using RNA-seq of the Korean native pig and Landrace reciprocal cross.
Next Article:Application of single-step genomic evaluation using social genetic effect model for growth in pig.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters