Expression of genes involved in meiosis.

<p>Expression levels of the <i>STRA8</i>, <i>DMC1</i> and <i>NANOS2</i> genes were determined in male and female gonads. Values are given as percentages after normalisation to the highest value (100%). Note that at 120 and 180 d<i>pp</i>, only male samples were collected.</p

Histological features from the onset of gonad development until early puberty.

<p>Paraffin sections of ovaries (2A) and testes (2B) were stained as described in Materials and Methods at 28 d<i>pc</i>, 4, 7, 14, 18 and 28 d<i>pp</i>, 2 months (ovary, 60 d<i>pp</i>), 4 (120 d<i>pp</i>) or 6 months <i>post partum</i> (testis, 180 d<i>pp</i>). Images magnified 2.5× (testes) or 3× (ovaries) are presented for each stage. Arrows point some nuclei harbouring characteristic figures: Sertoli cells (S), granulosa cells (G), germ cells (GC), stages of meiosis (PL = preleptoten; L = leptoten; Z = zygoten; P = pachyten; D = diploten), R = round spermatid, E = elongated spermatid, CN = condensed nuclei. The blue star labels proliferating germ cells (2Bc).</p

Expression of germinal-specific marker genes.

<p>Expression levels of the <i>OCT4</i> and <i>VASA</i> genes were determined in testes and ovaries. Values are given as percentages after normalisation to the highest value (100%). A2, A3 and B2, B3: immunofluorescence detection of OCT4 and VASA proteins in paraffin sections of gonads. Immunofluorescence pictures are given with each DAPI counterstaining. The antibody against OCT4 stains the nuclei of germ cells in 28 d<i>pc</i> testis (A2) or ovary (A3). Using the antibody against VASA, a cytoplasmic fluorescence is visible in germ cells in 18 d<i>pp</i> ovaries (B2) and 150 d<i>pp</i> testes. Red arrows point to the specifically labelled nuclei. Horizontal bars represent 100 µm (A2 and A3) or 200 µm (B2 and B3).</p

Schematic representation of gene expression patterns and main histological observations.

<p>Continuous lines represent a significant gene expression in ovaries (pink) or testes (blue) extracts. Dotted lines indicate that expression is significant but at a lower level. Thin black lines are drawn when no samples were assayed.</p

Expression of genes involved in ovary differentiation.

<p>Expression levels of the <i>FOXL2</i>, <i>WNT4</i>, <i>RSPO1</i>, <i>CYP19A1</i> and <i>BMP-15</i> genes were determined in males (black bars) and females (grey bars). Values are given as percentages after normalisation to the highest value (100%). The horizontal scale represents the age of animals in d<i>pc</i> and d<i>pp</i>. ad = adults, 2-year old rabbit. Note that at 120 and 180 d<i>pp</i>, only male samples were collected. <b>A2, B2</b>: immunofluorescence detection of FOXL2 (A2) and RSPO1 (B2) on paraffin sections of gonads. The antibody against FOXL2 specifically stains the nuclei of granulosa cells (female gonad at 18 d<i>pp</i>). The antibody against RSPO1 specifically stains the cytoplasm of germ cells (female gonad at 4 d<i>pp</i>).</p

Schematic representation of the principal features of gonad differentiation in the rabbit species.

<p>Schematic representation of the principal features of gonad differentiation in the rabbit species.</p

Gonad Differentiation in the Rabbit: Evidence of Species-Specific Features

<div><p>The rabbit is an attractive species for the study of gonad differentiation because of its 31-day long gestation, the timing of female meiosis around birth and the 15-day delay between gonadal switch and the onset of meiosis in the female. The expression of a series of genes was thus determined by qPCR during foetal life until adulthood, completed by a histological analysis and whenever possible by an immunohistological one. Interesting gene expression profiles were recorded. Firstly, the peak of <i>SRY</i> gene expression that is observed in early differentiated XY gonads in numerous mammals was also seen in the rabbit, but this expression was maintained at a high level until the end of puberty. Secondly, a peak of aromatase gene expression was observed at two-thirds of the gestation in XX gonads as in many other species except in the mouse. Thirdly, the expression of <i>STRA8</i> and <i>DMC1</i> genes (which are known to be specifically expressed in germ cells during meiosis) was enhanced in XX gonads around birth but also slightly and significantly in XY gonads at the same time, even though no meiosis occurs in XY gonad at this stage. This was probably a consequence of the synchronous strong <i>NANOS2</i> gene expression in XY gonad. In conclusion, our data highlighted some rabbit-specific findings with respect to the gonad differentiation process.</p></div

Expression of genes involved in testis differentiation.

<p>Expression levels of the <i>SRY</i>, <i>DMRT1</i>, <i>SOX9</i> and <i>AMH</i> genes were determined in males (black bars) and females (grey bars) by quantitative RT-PCR, as described in the Materials and Methods. The levels of expression are given as percentages after normalisation to the highest level (100%). Values are means +/− sem of expression levels determined on gonads from at least 3 animals per stage. The horizontal scale indicates the age of the rabbit in d<i>pc</i> (days <i>post coitum</i>) and d<i>pp</i> (days <i>post partum</i>). ad = adults, 2-year old rabbit. Note that at 120 and 180 d<i>pp</i>, only testes were collected and analysed. <b>C2, D2</b>: immunofluorescence detection of SOX9 (C2) and AMH (D2) on paraffin sections of gonads. The antibody against SOX9 specifically stains the nuclei of Sertoli cells (male gonad at 28 d<i>pp</i>). The white arrows label some of the Sertoli cells. The antibody against AMH stains the cytoplasm of Sertoli cells (gonads at 20 d<i>pc</i>).</p

Indirect estimation of the level of “CAA” to “UAA” editing.

<p><b>A:</b> schematic representation of the rabbit <i>APOB</i> mRNA from the AUG translation initiation codon until the STOP codon. At the 2177^th codon, the “C” residue is edited in a “U” residue. Using reverse transcribed RNA as template, the LApob48F/LApoB48R set of primers amplifies a 455 bp long amplicon encompassing the 2177^th codon. When using the APOBR4 primer as sequencing primer, the chromatogram shows the antisense sequence. <b>B:</b> detail of a characteristic chromatogram showing how the heights of the peaks were measured at the level of the 2177^th codon. Here, the “A” residue was the major one (a1), and the “G” the minor one (g1). Consequently, a large majority of DNA strands in this mixture encompassed the edited TAA (STOP) codon at position 2177. (a2) and (g2) are measured as references. <b>C:</b> standard equations obtained by plotting the a1/a2 and g1/g2 ratios against the amount of “A” or “G” containing DNA 455 bp fragment in the sequenced sample. Amounts are given as percentage of “A” or “G” containing DNA.</p

Analysis of rIFABP-hapobec1 transgenic and double transgenic rabbits.

<p><b>10A: Structure of the recombinant gene to express the human APOBEC1 cDNA in the intestine of transgenic rabbits.</b> The rIFABP-hAPOBEC1 construct encompassed two copies of the chicken ß-<i>GLOBIN</i> gene fragment 5′HS4 (gene expression insulator element, dotted box), the promoter of the rat intestinal fatty acid binding protein gene (r<i>IFABP</i>; grey box), the rabbit (rb) ß-<i>GLOBIN</i> second intron (black boxes and thick line), the human <i>APOBEC1</i> cDNA (white box) produced by PCR amplification from reverse transcribed RNA of HT29 cells (derived from a human colon tumor that have retained the ability to express the <i>APOBEC1</i> gene), and the human growth hormone polyadenylation sequences (box with vertical bars). The horizontal black arrow points the position of the transcription start site. ATG =  translation initiation site of <i>hAPOBEC1</i> cDNA. Numbers and small horizontal arrows represent the sets of primers. All studied transgenic animals were PCR positive for the sets 1–4. <b>10 B: gene expression.</b> The levels of human <i>APOBEC1</i> mRNA (left panel) and shRNA (middle panel) were measured in RNAs prepared from duodenum cells in two rIFABP-hapobec1 lines (L01 and L02) and in double transgenic animals (shL21+L01; shL21+L02; shL27+L01). Values are given in females (F) and males (M) after normalization to the level of reference gene expression determined simultaneously in each sample: Let7c miRNA in the case of shRNA, and <i>RPL19, YHWAZ, HPRT</i> in the case of human <i>APOBEC1</i>. In double transgenic animals, males and females were not distinguished, considering the small number of animals in these groups. The number of animals in each group is indicated in brackets. Values are given with the standard error of the mean (sem). The mean level of shRNA in shL21 and shL27 as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106655#pone-0106655-g002" target="_blank">Figure 2</a> is indicated with a horizontal line. The level of expression of the human <i>APOBEC1</i> transgene measured in the liver is given in L02. In L01, this level was not significantly detected. The level of rabbit <i>APOBEC1</i> mRNA (right panel) was measured in intestinal RNAs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106655#pone-0106655-g003" target="_blank">Figure 3</a>. The level found in wild type rabbits and in lines shL21 and shL27 is indicated with a horizontal line. <b>10 C: APOBEC1 dependent editing in intestine and liver and Plasma concentration of APOB48 in rabbits expressing the human APOBEC1 gene.</b> The estimation of editing was made as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106655#pone-0106655-g005" target="_blank">Figure 5</a>. Plasma concentration of APOB48 was performed as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106655#pone-0106655-g006" target="_blank">Figure 6</a> in 3 transgenic rabbits from line L02. Wild type animals are the same than those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106655#pone-0106655-g006" target="_blank">Figure 6</a>.</p