Sylvain Bellier

Genetic, nutritional, and gut microbiota-derived factors have been proposed to play a role in the development of the whole intestine that is around 40% longer in PRM/Alf mice compared with other mouse strains. The PRM/Alf genotype explains 60% of this length difference. The remaining 40% are due to a maternal effect that could depend on the gut microbiota transmitted by the mother to their pups. Germ-free PRM/Alf mice and C3H/He mice were associated with a simplified human microbiota (SIHUMI) to study its impact on gut length. The small intestines of the SIHUMI-associated mice were 16.4% (PRM/Alf) and 9.7% (C3H/He) shorter than those of the corresponding germ-free counterparts. Temporal temperature gradient gel electrophoresis and quantitative real-time PCR revealed differences in microbiota composition between both SIHUMI-associated mouse strains. Anaerostipes caccae was one log lower in PRM/Alf mice than in C3H/He mice. Since polyamines and short-chain fatty acids (SCFAs) are important intestinal growth factors, their concentrations were explored. Cecal concentrations of putrescine, spermine, spermidine, and N-acetylspermine were 1.5-fold, 3.7-fold, 2.2-fold, and 1.4-fold higher, respectively, in the SIHUMI-C3H/He mice compared with the SIHUMI-PRM/Alf mice. In addition, cecal acetate, propionate, and butyrate concentrations in SIHUMI-C3H/He mice were 1.4-fold, 1.1-fold, and 2.1-fold higher, respectively, than in SIHUMI-PRM/Alf mice. These results indicate that polyamines and SCFAs did not promote gut lengthening in any of the two mouse strains. This suggests that as yet unknown factors provided by the SIHUMI prevented gut lengthening in the SIHUMI-associated mice compared with the germfree mice.

Background: Due to their high level of genotypic and phenotypic variability,Mus spretusstrains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci.Mus spretusdiverged fromMus musculusaround 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative ofMus spretusandMus musculusspecies, respectively.Results: The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/Lvs. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (β and α_s1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/PasCsn1s1(11),Csn2(7) andWap(8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/PasWapgene.Conclusion: SNP frequencies found in three milk protein-encoding genes betweenMus spretusandMus musculusis twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple α_s1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.

The PRM/Alf inbred mice exhibit a huge intestinal lengthening. Since milk contains bioactive factors implied in numerous biological processes, one hypothesis is that PRM/Alf milk contains intestinotrophic factors contributing to this remarkable phenotype. A comparison between the milk from PRM/Alf and C57BL/6J (as a control) strains could be helpful in the identification of such factors, including proteins. However, a complete description of the mouse milk major protein fraction is still missing. Hence we adapted a reliable technique to separate and identify the major mouse milk proteins. This approach was achieved through the protein study of milk from C57BL/6J and PWK/Pas strains representative of two Mus musculus subspecies, M. m. domesticus and M. m. musculus respectively. C57BL/6J milk samples were first skimmed and fractionated by reverse phase-HPLC (RP-HPLC). The protein content of each chromatographic peak was analysed by SDS-PAGE and identified by mass spectrometry. This methodological approach allowed characterization of nine major mouse milk proteins: alpha(s1), beta, gamma, epsilon and kappa-caseins, Whey Acidic Protein, lactoferrin, Serum Albumin, Fatty Acid Binding Protein, as well as an alpha(s1)-casein isoform. Then, RP-HPLC patterns of C57BL/6J milk proteins were compared with those obtained starting from the milk of PWK/Pas females. This comparison revealed a protein polymorphism for the alpha(s1)-casein.

The interstitial cells of Cajal (ICC) play an important role in coordinating intestinal motility, and structural alterations in ICC are found in several human digestive diseases. Mouse models with defects in ICC allow a better understanding of their functions. We investigated the pattern of intestinal motility and the distribution of ICC in the PRM/Alf inbred mouse strain, characterized by a selective intestinal lengthening. In PRM/Alf mice, the digestive transit time, evaluated by using thermophilic Bacillus subtilis spores, was normal, indicating accelerated transit. The contractility and slow-wave frequency, recorded on isolated segments from the proximal small intestine, were significantly increased. The number of ICC was also significantly higher along the small intestine and the colon. The concomitant increase of the contractility, the slow-wave frequency, and the number of ICC is consistent with the proposal of a role of ICC number increase in the higher intestinal transit speed. The PRM/Alf model should be useful to further investigate the roles of ICC in the control of digestive motility.