Ovarian histological structure and expression of leptin receptors in ovaries of IUGR piglet treated with either saline (IUGR<i>Sal</i>) or leptin (IUGR<i>Lep</i>).

<p>(A) Germ cell populations in piglet ovaries at d21. a: oogonia (oog) within germ cell nests or cysts, b: oocytes in meiotic prophase (proph) within germ cell nests or cysts, c: oocytes in dictyate stage (cysts) grouped within germ cell cysts, d: oocytes enclosed in primordial follicles (foll prim), e: oocytes enclosed in primary follicles (foll I), f: oocyte enclosed in a secondary follicle. (foll II). The arrows indicate examples of germ cells. Bar = 20 µm, g: Percentages of different germ cell types in IUGR<i>Sal</i> and IUGR<i>Lep</i> piglet ovaries. (B) Immunostaining for leptin receptors in piglet ovaries at d21. Immunostaining of piglet ovarian sections using Ob-R antibody, without (a) and with (b)displacement by the peptide used as immunogen for Ob-R antibody preparation; o = oocyte. Bar = 20 µm. (C) Detection of leptin receptors by Western Blot using Ob-R antibody in piglet IUGR<i>Sal</i> and IUGR<i>Lep</i> ovaries at d21 and in a spermatozoa lysate as a positive control. Six Ob-R isoforms were observed, including the long form (Ob-Rb at 120 kDa) and the first short form (Ob-Ra at 90 kDa).</p

Histological structure and enzymatic activity of the pancreas of IUGR<i>Sal</i> and IUGR<i>Lep</i> piglets at d21.

<p>(A) Microscopic analysis and histological measurements in pancreas sampled from IUGR animals treated either with saline or leptin. Arrowheads highlight cells undergoing apoptosis. Bars = 10 µm. (B) Total protein content and enzymatic activities in pancreas sampled from IUGR animals treated either with saline or leptin. BW: Body weight, IU: International Unit<b>.</b> Values represent the mean ± SEM, (n = 6 per group).*: p<0.05; **: p<0.01, for leptin effect in IUGR piglet.</p

FOXO3A expression in ovaries of IUGR piglet treated with either saline (IUGR<i>Sal</i>) or leptin (IUGR<i>Lep</i>).

<p>(A) Detection of FOXO3A by Western Blot using FOXO3A antibody in piglet IUGR<i>Sal</i> and IUGR<i>Lep</i> ovaries at d21. (B) Immunostaining of piglet ovarian sections using FOXO3A antibody (a, b and c) showing a diffuse cytoplasmic staining in the oocytes of primary follicles (a), a strong perinuclear (arrows) and diffuse cytoplasmic staining in the oocytes of primordial follicles (b) and a strong perinuclear staining in the oocytes of secondary follicles (c), Bar = 20 µm. Graph in (d) shows FOXO3A staining grade in germ cells; four grades of staining were defined, corresponding to numbers of stained germ cells <5 (grade 0), 5–25 (grade 1), 25–50 (grade 2), and >50 (grade 3) per microscopic field (objective ×40). Graph in (e) shows the numbers of oocytes with a strong perinuclear staining. Data in panels d and e represent results of germ cell counting in the sub-epithelial and in the deep zones of ovarian cortex of IUGR<i>Sal</i> (n = 6) and IUGR<i>Lep</i> (n = 8) piglet ovaries, each zone corresponding to a tissue area of 0.62 mm2 analyzed per animal. Values represent the mean ± SEM, *: p<0.05; **: p<0.01, for leptin effect in IUGR piglet.</p

Morphometric parameters and organ weights at weaning (d21) of IUGR piglets treated with either saline (IUGR<i>Sal</i>) or leptin (IUGR<i>Lep</i>).

<p>Values represent the mean ± SEM, (n = 20 per group),</p>*<p>p<0.05;</p>**<p>p<0.01, for leptin effect in IUGR piglets.</p

Analysis of the histological structure of secondary immune structures in IUGR<i>Sal</i> and IUGR<i>Lep</i> piglets at d21.

<p>(A) Histological structure of spleen and immunodetection of CD79+ cells in IUGR<i>Sal</i> and IUGR<i>Lep</i> piglets at d21. Left panels: Microscopic observation of white pulpe (W) in spleen samples processed with a hemalun-Eosin-Safran staining. Bars = 50 µm. Central arteriolae are indicated by arrows. Primary follicles were easily identified in IUGR<i>Lep</i> (b) and delineated with the small circle but were not visible in IUGR<i>Sal</i> (a). The inset in (b) illustrates cells undergoing intense mitotic activity (arrowhead) found in this area. Right panels: Immunolabelling for CD79 in the white pulp of IUGR piglets treated either with saline (c) or leptin (d) postnatally. (B) Histomorphometrical analysis of Peyer’s patches in IUGR<i>Sal</i> and IUGR<i>Lep</i> piglets at d21. The areas of the whole Peyer’s Patches (PP) and the B follicle were measured and the ratio corresponding to the B follicle area reported to the whole Peyer’s patche surface was determined. Values represent the mean ± SEM (n = 6 per group), *: p<0.05; **: p<0.01, for leptin effect in IUGR piglets.</p

Growth and organ development of IUGR piglets treated with either saline (IUGR<i>Sal</i>) or leptin (IUGRL<i>ep</i>).

<p>(A) Morphometric parameters and organ weights at weaning (d21). (B) Evolution of body weight gain from birth to d21. BW: Body weight. Values represent the mean ± SEM, (n = 20 per group), *: p<0.05; **: p<0.01, for leptin effect in IUGR piglets.</p

B4GALNT2 transferase activity revealed by DBA lectin after in vitro overexpression of <i>B4GALNT2</i> in ovine granulosa cells.

<p>Primary ovine granulosa cells from <i>+/+</i> small antral follicles were transiently transfected with either the pCDNA-hB4GALNT2 expressing the human form of B4GALNT2 or the empty pCDNA3.1 vector. Twenty-four hours after transfection, cells were stained with biotinylated-DBA lectin (500 ng/ml). Arrows indicated DBA positive staining only in B4GALNT2 transfected cells. Cells were counterstained with hematoxylin. A black bar indicates the microscopy magnification scale.</p

Polymorphisms within the minimal <i>FecL</i> locus and allele sharing.

<p>Polymorphism positions are given relative to OAR11 v3.1 and <i>FecL</i> locus (Genbank:KC352617). Polymorphism type, single nucleotide polymorphism (SNP), microsatellite (Ms) or insertion/deletion (ins/del) referred to <i>+/+</i> vs. <i>L/L</i> sequence. Allele sharing indicated number of (<i>+</i>) chromosomes carrying the (<i>L</i>) allele relative to the number of (<i>+</i>) chromosomes tested.</p>a<p>SNP corresponding to the interval borders and therefore excluded from the minimal locus.</p>b<p>Allele segregating as the <i>FecL^L</i> mutation.</p>c<p>Ms marker genotyped by fluorescent SSCP, no information on the dinucleotide repeat variation.</p><p>NA, non-available coordinate on OARv3.1.</p

Quantitative PCR primer sequences and their efficiency.

<p>Quantitative PCR primer sequences and their efficiency.</p

Western immunoblotting analysis of B4GALNT2 transferase activity in Lacaune sheep granulosa cells and follicular fluids.

<p>Granulosa cell protein extracts (50 µg) and follicular fluids (200 µg) from <i>+/+</i> and <i>L/L</i> large antral follicles were precipitated (P) by agarose-DBA lectin or sepharose-protein A-KM694 monoclonal antibody. The resulting purified glycoproteins were separated on SDS-PAGE, transferred on nitrocellulose membrane and revealed after blotting (B) using biotinylated-DBA lectin or KM694 monoclonal antibody.</p