Signature of evolution of genes involved in sperm-oocyte interaction in Vertebrates.

<p>Genes (names are indicated on the top) are classified into three main functional groups (from left to right): sperm-zona pellucida binding, acrosome reaction, and sperm-oolemma fusion. Species are indicated in the tree (left). Blue square: duplication; purple square: positive selection; pink square: duplication + positive selection. Black square: pseudogene; Black dot: no event. Black circle: no calculation of positive selection. White square: no gene found.</p

BMP15 positive selection pressure among mammals.

<p>Phylogenetic tree of BMP15 from 24 mammals species reconstructed using TreeBeST and rooted by minimizing with the number of duplications and losses. Bootstrap values are given when nodes are strongly supported (>80%). Branches or species that have a significant LRT for positive selection calculation are indicated in bold (threshold of <i>q</i>= 5% of false positives). The scale represents the substitution rate. </p

Rate of gene appearance, gene duplication, pseudogenization, loss and intensity of positive selection in Teleostei, Aves and Eutheria. a.

<p>Rate of events per million years (Myr) per lineage for the set of genes studied. This includes up to 69 genes, but in most cases, the number of relevant genes is lower because some types of events cannot happen in some taxa (losses or duplications cannot happen if a gene is primitively absent, and gains cannot occur in a clade if the gene appeared before the base of a given clade), and we have incorporated that factor into our calculations to obtain comparable rates across taxa. The geological age of lineages is used in the rate calculation through the phylogenetic diversity of the clades. <b>b</b> and <b>c.</b> Proportion of genes under positive selection in Teleostei, Aves and Eutheria for genes belonging to the third step, the sperm-oolemma fusion (B), and for all genes (C). Three types of confidence intervals are shown: 78%, 90% and 97% representing testing at 0.05, 0.01 and 0.001 probability thresholds, respectively. Statistically significant comparisons, for which confidence intervals do not overlap, are indicated.</p

Functional analysis of amino acids under positive selection in human BMP15.

<p>A, Human BMP15 full sequence showing prodomain, cleavage site (underlined) and mature peptide (framed sequence); known human variants for BMP15 found in POI patients (framed grey highligthed); and identified amino acids under positive selection (framed colored highlighted): BMP15^F146, BMP15^L189, BMP15^Y235. B, <i>In </i><i>vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/- 100ng recombinant human BMP15 (mock +/- BMP15) or wild type human BMP15 expressing vector (WT) or the different BMP15 variants vectors obtained by directed-mutagenesis of residues under positive selection. Results are expressed as the mean (±SD) of 4 independent experiments. Differences between means were analyzed by one-way ANOVA by comparing each condition to WT (*, p<0.05; **, p<0;01; ***, p<0.001).</p

Recombinant BMP15 inhibition of progesterone secretion.

<p>Granulosa cells from small antral (1–3 mm in diameter) ovine follicles were cultured for 96 h in serum-free conditions. Cultures were performed in the absence (control) or in the presence of increasing dose (10, 50, 200ng/ml) of wild type recombinant human BMP15^wt or BMP15^Y235C mutant. Each treatment was tested in triplicate in each of 5 independent experiments. The results represent the amount of progesterone secreted by 50 000 cells between 48 h and 96 h of culture (mean ±SEM). Dose effect was analyzed by one-way ANOVA by comparing each dose to control (**, p<0.01; ***, p<0.001). Mutation effect was analyzed by Student t-test within each dose ($, p<0.05).</p

BMP15 evolution among TGFß/BMP family members.

<p>Phylogenetic tree of thirteen members of the TGFβ/BMP superfamily expressed by the mammalian ovary reconstructed using maximum likelihood. Bootstrap values are given when nodes are strongly supported (>80%). The scale represents the substitution rate.</p

Phylogenetic tree of protein sequence of chicken GLUT12 and GLUT12 from other vertebrates (see Table 2).

<p>The tree was constructed as described in Materials and Methods using PhyleasProg analysis web server (Phylogenetic Analysis Programs v2.7).</p

Effects of insulin on <i>SLC2A12</i> gene expression in skeletal muscles.

<p>mRNA levels of <i>SLC2A12</i> were measured by Real-Time PCR in <i>Pectoralis major</i> and leg muscles of fed chicken controls (Fed group) and fed chickens injected with an anti-insulin antibody (Ins immuno-neutalized group) (N = 7). Cytochrome b (Cytb) and β-actin were used as housekeeping genes.Values represent means and standard errors. Data were subjected to analysis of variance (ANOVA) to detect significant intergroup differences (Fed vs Ins immuno-neutralized). The means were compared by Fisher’s least significant difference test. P<0.05 was considered statistically significant.</p

Tissue expression pattern of GLUT12 in the chicken.

<p>(A) Tissues from fed chickens were analysed for <i>SLC12A12</i> expression by Real-Time PCR (N = 6). Values represent means and standard errors. Data were subjected to analysis of variance (ANOVA) to detect significant intergroup differences. Statistical model was tissue comparison. The means were compared by Fisher’s least significant difference test. P<0.05 was considered statistically significant. (B) Tissues from fed chickens were analysed by Western blotting using an anti-GLUT12 antibody for protein distribution analyses (N = 5). In grey: skeletal muscles (<i>Pectoralis major</i> and leg muscle). Vinculin was used as a protein loading control. (C) Protein distribution was analysed from <i>Pectoralis major</i> and the leg muscles, collected at 19 days during embryogenesis, hatch and 5 days post-hatch. (N = 7).</p

Phylogenetic tree of multiple alignments of all known chicken GLUT protein sequences.

<p>The analysis of chicken GLUTs was performed on the Phylogeny.fr platform (<a href="http://www.phylogeny.fr/" target="_blank">http://www.phylogeny.fr</a>; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139517#pone.0139517.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139517#pone.0139517.ref021" target="_blank">21</a>]) using Ensembl protein IDs. The tree was constructed as described in Materials and Methods. Reliability of internal branches was assessed using the aLRT test. The scale represents the substitution rate.</p