Genomic analysis of the appearance of ovarian mast cells in neonatal MRL/MpJ mice

Abstract

In MRL/MpJ mice, ovarian mast cells (OMCs) are more abundant than in other mouse strains, and tend to distribute beneath the ovarian surface epithelium at birth. This study investigated the factors regulating the appearance of neonatal OMCs in progeny of the cross between MRL/MpJ and C57BL/6N strains. F1 neonates had less than half the number of OMCs than MRL/MpJ. Interestingly, MRLB6F1 had more neonatal OMCs than B6MRLF1, although they were distributed over comparable areas. Furthermore, in MRL/MpJ fetuses for which parturition was delayed until embryonic day 21.5, the number of OMCs was significantly higher than in age-matched controls at postnatal day 2. These results suggest that the number of OMCs was influenced by the environmental factors during pregnancy. Quantitative trait locus analysis using N2 backcross progeny revealed two significant loci on chromosome 8: D8Mit343-D8Mit312 for the number of OMCs and D8Mit86-D8Mit89 for their distribution, designated as mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively. Among MC migration-associated genes, ovarian expression of chemokine (C-C motif) ligand 17 at mcom1 locus was significantly higher in MRL/MpJ than in C57BL/6N, and positively correlated with the expression of OMC marker genes. These results indicate that the appearance of neonatal OMCs in MRL/MpJ is controlled by environmental factors and filial genetic factors, and that the abundance and distribution of OMCs are regulated by independent filial genetic elements.

Figure 1. Appearance of ovarian mast cells in MRL/MpJ and C57BL/6N mice, and F1 and F2 intercross mice at postnatal day 0.

(A–F) Toluidine blue-stained sections of ovaries in (A) MRL/MpJ, (B) C57BL/6N, (C) MRLB6F1, (D) reciprocal F1 (B6MRLF1), (E) MRLB6F2, and (F) reciprocal F2 (B6MRLF2). Bars: 100 µm. (G) Number of mast cells per total ovarian area (OMC density). (H) Number of mast cells facing ovarian surface epithelium per total ovarian area (SEMC density). (I) Ratio of SEMC density to OMC density (SEMC ratio). Data represent mean ± SEM (n = 10–20 per group). Significant differences were analysed using Scheffé’s method following the Kruskal-Wallis test. Statistical significance is indicated by different letters (P < 0.05).

Figure 2. Effect of delayed parturition on appearance of ovarian mast cells in MRL/MpJ and C57BL/6N mice.

(A and B) Toluidine blue-stained sections of MRL/MpJ mice ovaries at (A) embryonic day 21.5 with artificially delayed parturition, and (B) postnatal day 2. Bars: 100 µm. (C) Number of mast cells per total ovarian area (OMC density) in MRL/MpJ mice. (D) OMC density in C57BL/6N mice. (E) Ratio of number of mast cells facing ovarian surface epithelium per total ovarian area to OMC density (SEMC ratio) in MRL/MpJ mice. (F) SEMC ratio in C57BL/6N mice. (G) Plasma estradiol concentration at embryonic day 18.5. (H) Plasma progesterone concentration at embryonic day 18.5. Data represent mean ± SEM (n = 3–13 per group). Significant differences were analysed using the Mann-Whitney U test. *P < 0.05; ns, not significant.

Figure 3. Appearance of ovarian mast cells in BMM progeny at postnatal day 0.

(A) Distribution of number of mast cells per total ovarian area (OMC density). (B) Distribution of number of mast cells facing ovarian surface epithelium per total ovarian area (SEMC density). (C) Distribution of ratio of SEMC to OMC densities (SEMC ratio). (D) Correlation between OMC and SEMC densities (Pearson’s correlation test, n = 200). (E) Correlation between SEMC density and SEMC ratio (Pearson’s correlation test, n = 200). (F) Correlation between SEMC ratio and OMC density (Pearson’s correlation test, n = 200).

Figure 4. Quantitative trait loci linkage analysis for the appearance of ovarian mast cells.

(A) Interval mapping for number of mast cells per total ovarian area (OMC density). (B) Interval mapping on Chr 8 for OMC density. (C) Interval mapping for number of mast cells facing ovarian surface epithelium per total ovarian area (SEMC density). (D) Interval mapping on Chr 8 for SEMC density. (E) Interval mapping for ratio of OMC to SEMC densities (SEMC ratio). (F) Interval mapping on Chr 8 for SEMC ratio. (G) Allele effect of D8Mit312 on OMC density. (H) Allele effect of D8Mit312 on SEMC density. (I) Allele effect of D8Mit200 on SEMC density. (J) Allele effect of D8Mit89 on SEMC ratio. M/B indicates mice heterozygous with both a MRL/MpJ and C57BL/6N allele. M/M indicates mice homozygous for the MRL/MpJ allele. Data represent mean ± SEM. Significant differences were analysed using the Mann-Whitney U test. Statistical significance is indicated by different letters (P < 0.01).

Figure 5. Epistatic interactions regulating the appearance of ovarian mast cells.

(A) Genotypic interactions between D8Mit312 and D6Mit74 for the number of mast cells per total ovarian area (OMC density). (B) Genotypic interactions between D8Mit312 and D6Mit74 for the number of mast cells facing ovarian surface epithelium per total ovarian area (SEMC density) (C) Genotypic interactions between D8Mit248 and D19Mit33 for SEMC density. (D) Genotypic interactions between D8Mit89 and D19Mit91 for SEMC ratio. M/B indicates mice heterozygous with both a MRL/MpJ and C57BL/6N allele. M/M indicates mice homozygous for the MRL/MpJ allele. Data represent mean ± SEM.

Figure 6. Candidate genes regulating the appearance of neonatal ovarian mast cells.

(A) Quantitative real-time PCR analyses of candidate gene expression at postnatal day 0. Significant differences were analysed using the Mann-Whitney U test. **P < 0.01; ND, not detected. (B) Correlation between Tpsb2 and Ccl17 (Pearson’s correlation test, n = 9). (C Correlation between Tpsb2 and Mt4 (Pearson’s correlation test, n = 8).