SRY, DMRT1, and POU5F1 location during early gonadal development.

(A) Key stages of gonadal development in rabbits with 31 days of gestation. Germ cells are first detected at 9 days post-coïtum (dpc), before the genital ridge formation, which occurs between 10 and 12 dpc. In XY gonads, testicular cords begin forming at 16 dpc, and germ cells enter meiosis a few months after birth. In XX gonads, the ovigerous cords appear at 20 dpc, and meiosis begins around birth. Location of SRY, DMRT1, and POU5F1 by in situ hybridization (RNAscope technology) on XY and XX control gonads at (B) 12 dpc or (C) 13 dpc. Dotted line: developing genital crests. Yellow arrowheads: coelomic epithelial cells expressing DMRT1. Scale bar = 50 μm.

Somatic markers location during testis differentiation.

Location of SRY by in situ hybridization (RNAscope technology), DMRT1, and SOX9 by immunohistochemistry on XY control testes from 14 to 18 dpc. The dotted line at 15 dpc: territory with cells expressing SRY and DMRT1 but not SOX9. Yellow arrowheads: tunica albuginea in formation. Scale bar = 50 μm.

Somatic markers location and expression during ovarian differentiation.

(A) Immunostaining of DMRT1 on XX control gonads from 14 to 18 dpc. (B) RT-qPCR analyses of RSPO1, DMRT1, and FOXL2 expression from 16 to 20 dpc in control gonads of both sexes (n=3-5). (C) RSPO1 in situ hybridization (RNAscope technology), immunostaining of DMRT1 and FOXL2 on 20 dpc control ovaries. Scale bar = 50 μm.

Ovarian-like morphology and transcriptomic signature of XY DMRT1-/- gonads at 20 dpc.

(A) Hematoxylin and eosin staining of gonads sections from control and DMRT1-/- 20 dpc rabbits. The enlarged area shows the characteristic ovarian surface epithelium found on XY DMRT1-/- gonads. Scale bar = 50 μm. Heatmap representation of (B) 3460 deregulated genes (adjusted p-value <0.05 and |log2FC|>1) or (C) 27 selected genes between XY control, XY DMRT1-/-, XX DMRT1-/-, and XX control at 20 dpc.

Somatic markers expression and location on control and DMRT1-/- gonads at 20 dpc.

RT-qPCR analyses of (A) testicular-related differentiation genes (SOX9, AMH, DHH, and SRY) or (B) ovarian-related differentiation genes (FOXL2, CYP19A1, and RSPO1) in XY control, XY DMRT1-/-, XX DMRT1-/- and XX control gonads (n=4-5) at 20 dpc. Statistical analyses were performed using the non-parametric Kruskal-Wallis test, followed by a pairwise permutation test: * p-value < 0.05; ns: non-significant. (C) Immunostaining of DMRT1, SOX9, and FOXL2 on XY control, XY DMRT1-/-, XX DMRT1-/-, and XX control gonad sections at 20 dpc. Scale bar = 50 μm.

Evolution of gonadal morphogenesis in XY and XX DMRT1-/- rabbits.

Hematoxylin and eosin staining of gonad sections from XY and XX DMRT1-/- gonads and XX control ovaries at 3 days post-partum (dpp), 18 dpp, and in adulthood (4-9 months). The enlargements for the first two panels correspond to the nuclei pointed by an arrow. PL: preleptotene stage; L: leptotene stage; Z: zygotene stage; D: diplotene stage; F: ovarian follicle; CL: luteal cells. Scale bar = 50 μm.