Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

Abstract

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Figure 1. XY^DSD phenotype of cloned dog produced by SCNT.

(a) Karyotyping analysis of the fibroblast of a sex reversed cloned dog (78, XY); (b) Agarose gel electrophoresis of SRY-specific PCR product; (c) External genitalia of a cloned male (XY genotype, XX phenotype) showing complete gonadal dysgenesis (left) and its gonad (right); (d–f) Hematoxylin/eosin (H&E) stained gonad and epididymic sections from adult XY^DSD cloned dog. (d) Uterine horn. (e) Uterine tube. (f) Ovary. CT, cortical tubule; EG, endometrial gland; EM, endometrium; F, fold; FS, follicle-like structure; IG, interstitial gland; ML, muscular layer; MM, myometrium; S, serosa; V, vessel. Scale bars, 50 μm (d–f), 100 μm (1), 200 μm (2 and 3).

Figure 2. Genome wide DNA methylation patterns of donor and XY^DSD cloned dogs.

(a) Hierarchical clustering of genome-wide CpG methylation in donor and XY^DSD cloned dogs using PVclust software. (b) Methylation levels (y-axis) of 100-bp intervals around genic regions (x-axis).

Figure 3. Methylation status of SRY gene in donor/XY^wt and XY^DSD cloned dogs in E34 fetal gonads.

(a) Methylation level (y-axis) across SRY gene (x-axis) of skin fibroblast in the donor and XY^DSD cloned dogs, data generated from WGBS; (b) Promoter methylation status of SRY gene at E34 gonad in the XY^wt and XY^DSD cloned dogs, using Bisulfite Sequencing PCR (BSP). Each BSP product was subcloned. Ten clones were subjected to nucleotide sequence analysis. Open and filled circles indicate unmethylated and methylated status, respectively.

Figure 4. Gene expression of sex-determining genes in E34 fetal gonads of XY^wt, XX^wt, and XY^DSD cloned dogs.

mRNA expression of SRY (a) and other (c) testis or ovarian determining factors were examined by QPCR. Significance of expression changes is indicated (one-way ANOVA). (b) Immunohistochemical analysis of SRY, VASA, SOX9, AMH and DMRT1 in E34 fetal gonads. The cells were counterstained with hematoxylin. Scale bars in = 150 μm; in inset = 40 μm.

Figure 5. SRY methylation and gene expression of sex-determining genes in 1 year postnatal fetal gonads of XY^wt, XX^wt, and XY^DSD cloned dogs.

(a) Promoter methylation status of SRY gene at 1 year postnatal gonad in the XY^wt and XY^DSD cloned dogs, using Bisulfite Sequencing PCR (BSP). (c) mRNA Expression of testis or ovarian determining factors were examined by QPCR. Significance of expression changes is indicated (one-way ANOVA). (b) Immunohistochemical analysis of SRY, SOX9, AMH, AMHRII and Inhibitinα. The cells were counterstained with hematoxylin. Scale bars in = 150 μm.