Mitogenomes of Polar Bodies and Corresponding Oocytes

<div><p>The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in <i>MT-ND5</i>, 2 in <i>MT-RNR2</i>, and 1 each in <i>MT-ATP8</i>, <i>MT-ND4</i>, <i>MT-CYTB</i>. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of ‘normality’.</p></div

Distribution of the 9 detected mutations in the 3 studied triads.

<p>wt: wild type.</p><p>Nd: not detected.</p

Amplification of the entire mitochondrial genome from polar body obtained by the amplification of 46 overlapping fragments using MitoAll kit.

<p>Amplification of the entire mitochondrial genome from polar body obtained by the amplification of 46 overlapping fragments using MitoAll kit.</p

Representative images of colon whole mounts in <i>En/En</i> and <i>en/en</i> rabbit myenteric plexuses.

<p><b>A</b>) E<i>n/En</i> myenteric ganglia (arrows) with human neuronal protein immunoreactive (HuC/D-IR) neurons. <b>B</b>) <i>En/En</i> myenteric ganglia (arrows) with nitric oxide synthase immunoreactive (nNOS-IR) neurons, (arrowheads) nNOS-IR nerve bundles. <b>C</b>) <i>en/en</i> myenteric ganglia (arrows) with HuC/D-IR neurons. <b>D</b>) e<i>n/en</i> myenteric ganglia (arrows) with nNOS-IR neurons, (arrowheads) nNOS-IR nerve bundles arranged within primary, secondary and tertiary nerve strands. In <i>En/En</i> (<b>A</b> and <b>B</b>) the ganglia and nerve bundles are less dense, have a lower number of HuC/D-IR and nNOS-IR neurons compared to <i>en/en</i> rabbits (<b>C</b> and <b>D</b>). <b>E</b>) and <b>F</b>): differences between <i>En/En</i> vs <i>en/en</i> SP-IR neurons (arrow), ganglia and nerve bundles (arrowhead) in the morphology of myenteric plexus in the ascending colon. In <i>En/En</i> (<b>E</b>) ganglia (arrow) are small and have a lower number of substance P immunoreactive (SP-IR) neurons than <i>en/en</i> rabbits (<b>F</b>). <b>A–D </b><i>scale bars</i> 200 µm; <b>E–F </b><i>scale bars</i> 100 µm.</p

Representative images of rabbit colonic mucosa in <i>En/En</i> vs <i>en/en</i>.

<p><b>A</b>) <i>En/En</i>; <b>B</b>) <i>en/en</i>. The arrow indicate c-kit immunoreactivity in the <i>tunica muscularis</i> of the ascending colon in an <i>en/en</i> rabbit.</p

F1 and backcrossed families obtained from Checkered Giant parental animals.

<p>Black squares and circles  =  rabbits with <i>en/en</i> genotype at the <i>English spotting</i> locus; spotted squares and circles  =  rabbits with <i>En/en</i> genotype; white squares and circles  =  rabbits with <i>En/En</i> genotype. ID of the animals is reported in blue or red (for those used as parental animals). The genotype of the g.93948587T>C SNP is reported for all animals. Rabbits that are circled in green have been used to collect specimens for anatomo-histochemical, gene expression analyses and resequencing (rabbits with n. 1–9). Rabbits that are circled in orange have been inspected after slaughtering. Animals with underlined ID showed hard feces in the intestine.</p

The <i>KIT</i> Gene Is Associated with the <i>English Spotting</i> Coat Color Locus and Congenital Megacolon in Checkered Giant Rabbits (<i>Oryctolagus cuniculus</i>)

<div><p>The <i>English spotting</i> coat color locus in rabbits, also known as <i>Dominant white spotting</i> locus, is determined by an incompletely dominant allele (<i>En</i>). Rabbits homozygous for the recessive wild-type allele (<i>en</i>/<i>en</i>) are self-colored, heterozygous <i>En/en</i> rabbits are normally spotted, and homozygous <i>En/En</i> animals are almost completely white. Compared to vital <i>en</i>/<i>en</i> and <i>En/en</i> rabbits, <i>En/En</i> animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the <i>KIT</i> gene as a candidate for the <i>English spotting</i> locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of <i>En/En</i> rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (<i>En/en</i>) with nine Checkered Giant (<i>En/en</i>) and two <i>en/en</i> does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the <i>KIT</i> gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the <i>English spotting</i> coat color phenotype (<i>θ</i> = 0.00 LOD  = 75.56). <i>KIT</i> gene expression in cecum and colon specimens of <i>En/En</i> (pathological) rabbits was 5–10% of that of <i>en/en</i> (control) rabbits. <i>En/En</i> rabbits showed reduced and altered c-kit immunolabelled ICC compared to <i>en/en</i> controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (<i>P</i><0.05) and substance P (<i>P</i><0.01) immunoreactive neurons in <i>En/En</i> vs. <i>en/en</i>. Electron microscopy analysis showed neuronal and ICC abnormalities in <i>En/En</i> tissues. The <i>En/En</i> rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.</p></div

Mean density of immunoreactive myenteric neurons in <i>En/En</i> vs <i>en/en</i> rabbits.

<p>Different immunoreactive neurons are reported: human neuronal protein (HuC/D) (<b>A</b>: <i>En/En</i> vs <i>en/en</i>, <i>P</i><0.05), nitric oxide synthase (nNOS) (<b>B</b>) and substance P (SP) (<b>C</b>: <i>En/En</i> vs <i>en/en</i>, <i>P</i><0.01) myenteric labeled neurons. Results are expressed as mean ± standard error of the mean.</p

Electron microscopy of Interstitial cells of Cajal (ICC).

<p><i>en/en</i> (<b>A</b> and <b>B</b>) control and <i>En/En</i> (<b>C</b> and <b>D</b>) pathological animals. <b>A</b>) ascending colon: an intramuscular ICC close to a nerve bundle (NB) and smooth muscle cells (SMC). Arrows indicate cell-to-cell contacts between ICC and SMC. x12,500. <b>B</b>) ascending colon: ICC at the submucosal border of the circular muscle layer showing in the cytoplasm several cisternae of the smooth endoplasmic reticulum and filaments, and caveolae along the plasma membrane; on the upper side, a nerve bundle and, on the lower side, the smooth muscle cells (SMC). x12,500. <b>C</b>) ascending colon: an intramuscular ICC with swollen mitochondria and extremely dilated cisternae of rough endoplasmic reticulum; on the upper side, a blood capillary (BC); on the right side, a nerve bundle (NB). The arrowhead indicates a nerve ending near the ICC. x15.000. <b>D</b>) descending colon: several intramuscular ICC with large intracytoplasmatic vacuoles; the arrows indicate cell-to-cell contacts between ICC and SMC. x7,500.</p

Breeds and rabbits genotyped for the <i>KIT</i> g.93948587T>C single nucleotide polymorphism.

1<p>The number of rabbits with the different genotypes is reported.</p