Introduction

Sexual development in mammals depends on a complex network of regulatory genes controlled by the presence or absence of the testis-determining gene SRY (sex-determining region on the Y chromosome) [1]. The protein encoded by SRY up-regulates the expression of transcription factor SOX9. The activation and maintenance of SOX9 expression triggers other genes (e.g. SOX8) required for testis and male differentiation, whereas SOX9 itself actively suppresses the expression of genes involved in ovarian development. In the absence of SRY, the alternative, female pathway is activated, resulting in ovary formation [2]. Derangements in these processes cause malformations of internal and external genitalia, varying from sexual ambiguity to complete sex reversal. The sex reversal syndrome in humans and other mammals is a congenital abnormality, characterized by inconsistency between chromosomal and gonadal sex [3]. In humans, sex reversal is a rare disorder of sterile XX males or XY females, with an incidence of one in 20,000-25,000 males [4]. XX sex reversals occur in different mammals, including European roe deer (Capreolus capreolus) [5]. Phenotypes can range from females carrying antlers to true hermaphrodites, although pronounced sexual dimorphism is usually evident [5]. C. capreolus has a high population density in Europe [6] and is the commonest cloven-hoofed game in Germany [7]; nonetheless, encountering sexual defects in natural populations appears to be extremely rare. Here, we studied a roe deer specimen with an intersex phenotype, resulting from incomplete male-determination. In order to explore the genetic basis of this defect, we sequenced the genome of roe deer.

Results

Fortuitously, we had access to a bagged, one year-old C. capreolus with a distinct intersex phenotype (Figure 1). It had cranial outgrowths typical of a buck and a rear phenotype characteristic of a doe. Close inspection revealed no vaginal opening, abdominal testes and a retro-posed pizzle. In contrast to a healthy male C. capreolus, there was no evidence of the single copy amelogenin (AMEL) gene [8] in the Y chromosome-specific region, indicating a female sex chromosome status, in spite of male external genitalia.

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Figure 1. European roe deer (C. capreolus) specimen with intersexual appearance.

(A) Habitus of the one-year old deer with short unbranched antlers (arrows) and a displaced penis opening in a brush appearing from the distance as a Schürze (arrowhead, characteristic tuft of hair in females normally located just above the vaginal entry). (B) Closer view of the back of the investigated deer, including anus and retro-posed penis with urethral opening (arrowhead). (C) Inguinal testicles under the abdominal skin (arrows). (D) Situs of the reproductive tract including the left and right small testicles (arrows) which had not descended into a scrotum; the right epididymis is also discernible.

https://doi.org/10.1371/journal.pone.0073734.g001

We then proceeded to explore the genetic basis of this defect by investigating ten genes [sex-determining region Y gene (SRY), SRY-related HMG-box gene 3 (SOX3), SRY-related HMG box gene 9 (SOX9), SRY-related HMG box gene 10 (SOX10), R-spondin 1 (RSPO1), forkhead transcription factor FOXL2 (FOXL2), double-sex- and MAB3-related transcription factor 1 (DMRT1), fibroblast growth factor 9 (FGF9), Wilms tumor gene 1 (WT1), androgen receptor (AR)] which are all known, in humans and mice, to be involved in sex determination or SRY-negative XX sex reversal, if mutated [3]. Since no sequence data are presently available for roe deer, we sequenced genomic DNA from a healthy male European roe deer using Illumina technology to produce 920 million paired-end reads (21-fold coverage). We assembled contigs from these reads, identified the aforementioned ten full-length genes of roe deer by comparison with homologous genes in the bovine genome (90-95% similarity), and independently sequenced each of these genes in the intersex, male and female control deer to verify the sequences in assembled contigs. We identified 42 sequence variations in SOX3, SOX9, SOX10, RSPO1, FOXL2, DMRT1, FGF9, WT1 and AR (Table 1) but none in SRY. Most of these alterations (n=25) are located intronically or in untranslated regions, and do not involve splice sites or regulatory regions. All variations in exonic regions are silent, except for two, which were also present in healthy roe deer controls, thus excluding them as a causal link to intersexuality in the present roe deer case. Two sequence variations identified in the X-chromosomally-located genes AR and SOX3 were detected in a heterozygous state in the intersex deer. Together with the lack of AMEL and SRY gene sequences, these data indicate the sole presence of two X chromosomes, representing the female sex chromosome status.

Since in humans, female-to-male sex reversal can be due to a translocation of Y chromosome sequences to an X chromosome encompassing SRY [9], we analyzed this gene. Yet, no SRY-specific fragment was detectable by PCR in the intersex deer, in contrast to XY male controls. Since the copy number of some genes have been shown to interfere with normal sexual differentiation [10], we investigated the dosages for SOX3, SOX9, SOX10, RSPO1, DMRT1, WT1, RSPO1, FOXL2 and AR using selected PCR amplicons for initial duplication deletion screening via quantitative real-time PCR analysis. While the copy numbers were identical in the intersex as compared with male and female control deer, the SOX9 gene showed a triple dosage compared with the controls. In order to exclude the possible involvement of a pseudogene, in-depth analyses of all three individual exons, both introns and the 5’- and 3’-untranslated regions were performed for the intersex as well as for one female and one male control deer. The analysis revealed the presence of three copies of the SOX9 gene in the intersex roe deer, in contrast to controls. Also the dosage of the 5’- and 3’-untranslated regions of SOX9 was increased by 3-fold, but further 5’-upstream and 3’-downstream regions showed normal, double dosage in the intersex individual (Figure 2). Duplication breakpoints can be suspected > 1.5 kb downstream of SOX9 and in a 24 bp region 890 bp upstream of the gene, including the entire coding region of SOX9 and the promoter (compared with the highly homologous sequence of SOX9 promoters in humans and mouse [11]). This information suggests that the extra copy of the SOX9 gene is functionally active, although long-range PCR analyses did not reveal the exact location or orientation of the extra copy of SOX9.

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Figure 2. Analysis of copy number variation by quantitative real-time PCR for the SOX9 gene, including exons, introns, 5’ and 3’ untranslated regions in the intersex roe deer as compared with a healthy female control.

In the intersex, the entire SOX9 gene and regions 5’ and 3’ of the gene is duplicated (red bars) as compared with the female control showing normal gene dosage for all regions investigated (grey bars).

https://doi.org/10.1371/journal.pone.0073734.g002

Discussion

Here, we report on a specimen of C. capreolus with a distinct intersex phenotype. It had cranial outgrowths typical of a buck, and a rear phenotype characteristic of a doe. Close inspection revealed no vaginal opening, abdominal testes and a retro-posed pizzle. Our results of molecular genetic testing demonstrate a case of SRY-negative XX sex reversal in the investigated intersex roe deer. Similar cases of SRY-negative XX sex reversal have been reported previously for dogs, goats, horses and pigs [12–15] but rarely in wild mammals, such as roe deer [5]. A number of genes other than SRY (e.g. WT1) have been reported as underlying causes of mammalian sex reversal in humans and mouse models [10], but the genetic causes have usually remained elusive. Several genes known to be involved in sex determination or SRY-negative XX sex reversal (SOX3, SOX10, RSPO1, DMRT1, WT1, RSPO1 and FOXL2) were inferred not to be linked to the phenotype in the present intersex roe deer by sequencing analyses and CNV analyses except of SOX9. For this gene, we identified a 3-fold increased gene dosage in this deer.

SOX9 is a direct downstream target of SRY and plays a critical role in male sexual differentiation [16]. SOX9 mutations can lead to haplo-insufficiency, being responsible for campomelic dysplasia, a skeletal malformation syndrome often associated with XY sex reversal in humans [17]. In contrast, duplication of SOX9 has been implicated in XX sex reversal [18–20]; its ectopic expression in mice induces testis formation in XX gonads [21]. Together with this information, our findings indicate that the extra copy of SOX9 is sufficient to initiate testis differentiation in the absence of SRY, inferring the intersex phenotype in the present roe deer. In this case, the link between phenotype and genotype serves as an excellent example of how a deep sequencing-based approach can gain insights into naturally occurring genetic defects in wild animals, which also has broad implications for understanding complex sex disorders.

Materials and Methods

Supporting Information