Gonad differentiation in the rabbit: evidence of species-specific features

Abstract

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 SRY 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 STRA8 and DMC1 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 NANOS2 gene expression in XY gonad. In conclusion, our data highlighted some rabbit-specific findings with respect to the gonad differentiation process.

Figure 1. Schematic representation of the principal features of gonad differentiation in the rabbit species.

Figure 2. Histological features from the onset of gonad development until early puberty.

Paraffin sections of ovaries (2A) and testes (2B) were stained as described in Materials and Methods at 28 dpc, 4, 7, 14, 18 and 28 dpp, 2 months (ovary, 60 dpp), 4 (120 dpp) or 6 months post partum (testis, 180 dpp). 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).

Figure 3. Expression of genes involved in testis differentiation.

Expression levels of the SRY, DMRT1, SOX9 and AMH 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 dpc (days post coitum) and dpp (days post partum). ad = adults, 2-year old rabbit. Note that at 120 and 180 dpp, only testes were collected and analysed. C2, D2: 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 dpp). The white arrows label some of the Sertoli cells. The antibody against AMH stains the cytoplasm of Sertoli cells (gonads at 20 dpc).

Figure 4. Expression of genes involved in ovary differentiation.

Expression levels of the FOXL2, WNT4, RSPO1, CYP19A1 and BMP-15 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 dpc and dpp. ad = adults, 2-year old rabbit. Note that at 120 and 180 dpp, only male samples were collected. A2, B2: 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 dpp). The antibody against RSPO1 specifically stains the cytoplasm of germ cells (female gonad at 4 dpp).

Figure 5. Expression of germinal-specific marker genes.

Expression levels of the OCT4 and VASA 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 dpc testis (A2) or ovary (A3). Using the antibody against VASA, a cytoplasmic fluorescence is visible in germ cells in 18 dpp ovaries (B2) and 150 dpp testes. Red arrows point to the specifically labelled nuclei. Horizontal bars represent 100 µm (A2 and A3) or 200 µm (B2 and B3).

Figure 6. Expression of genes involved in meiosis.

Expression levels of the STRA8, DMC1 and NANOS2 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 dpp, only male samples were collected.

Figure 7. Schematic representation of gene expression patterns and main histological observations.

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.