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 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

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

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

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

And act into two distinct cellular types during goat ovarian differentiation-0

EST), two human transcripts and the human gene. Genbank accession numbers of the different sequences are given on the left. A bovine-specific 5' non-coding exon 1 is depicted in red. The goat-specific part of exon 1 is depicted in pink. In goat, the second ATG (ATG) is not conserved in human (ACG). Sp = Signal peptide; Fu = Furin domain; Tsp = Thrombospondin domain; Nls = Nuclear localization signal; AG = conserved acceptor spicing site. b) A Neighbor-Joining tree was constructed with 28 DNA sequences from the four genes belonging to seven mammalian species, plus the putative goat ORF (the corresponding goat sequence is depicted in bold). Confidence values (higher than 50%) after bootstrap test are shown at each node. Genbank accession numbers are given in the methods section.<p><b>Copyright information:</b></p><p>Taken from "and act into two distinct cellular types during goat ovarian differentiation"</p><p>http://www.biomedcentral.com/1471-213X/8/36</p><p>BMC Developmental Biology 2008;8():36-36.</p><p>Published online 2 Apr 2008</p><p>PMCID:PMC2329615.</p><p></p

Manhattan plot of genome-wide TDT analysis for sex-reversal phenotype.

<p>For each marker the nominal significance (log(1/p)) is indicated. The dotted and the solid lines correspond to the genome-wide suggestive and significant thresholds respectively. </p