Clinical and gonadal transcriptome analysis of 38,XX disorder of sex development pigs†.

Pigs serve as a robust animal model for the study of human diseases, notably in the context of disorders of sex development (DSD). This study aims to investigate the phenotypic characteristics and molecular mechanisms underlying the reproductive and developmental abnormalities of 38,XX ovotestis-DSD (OT-DSD) and 38,XX testis-DSD (T-DSD) in pigs. Clinical and transcriptome sequencing analyses were performed on DSD and normal female pigs. Cytogenetic and SRY analyses confirmed that OT/T-DSD pigs exhibited a 38,XX karyotype and lacked the SRY gene. The DSD pigs had higher levels of follicle-stimulating hormone, luteinizing hormone, and progesterone, but lower testosterone levels when compared with normal male pigs. The reproductive organs of OT/T-DSD pigs exhibit abnormal development, displaying both male and female characteristics, with an absence of germ cells in the seminiferous tubules. Sex determination and development-related differentially expressed genes shared between DSD pigs were identified in the gonads, including WT1, DKK1, CTNNB1, WTN9B, SHOC, PTPN11, NRG1, and NXK3-1. DKK1 is proposed as a candidate gene for investigating the regulatory mechanisms underlying gonadal phenotypic differences between OT-DSD and T-DSD pigs. Consequently, our findings provide insights into the molecular pathogenesis of DSD pigs and present an animal model for studying into DSD in humans.

Pathological characteristics of SRY-negative 38,XX-DSD pigs: A family case report.

Disorders of sex development (DSD) are congenital conditions characterized by atypical development of chromosomes, gonads, or anatomical sex. XX-DSD pigs disrupt the production of high-quality breeding pigs and impede the advancement of the pig industry. However, the etiology of XX-DSD pigs remains unclear. Systematic reports on the genetic and pathological characteristics of prepubescent XX-DSD pigs in familial contexts are sparse. This study aimed to investigate the genetic and pathological features of one-month-old XX-DSD pigs within a familial context and to provide phenotypic information to elucidate the pathogenic mechanisms of XX-DSD pigs. The findings revealed that inbreeding within the XX-DSD family may contribute to the pathogenesis of XX-DSD pigs. All XX-DSD pigs in the family had a chromosomal sex of female and were male pseudohermaphrodites. The degree of masculinization of the reproductive organs varied among XX-DSD pigs, demonstrating phenotypic heterogeneity. HE staining showed that the testes of prepubescent XX-DSD pigs contained vesicles in the seminiferous tubules, with or without vestigial germ cells. Ultrastructural analyses indicated that sertoli cells, leydig cells and germ cells in the testes of XX-DSD pigs exhibited pathological damage, confirming impaired testicular function. Immunofluorescence staining revealed high expression of SRY-box transcription factor 9 (SOX9) in XX-DSD pig testicular tissues, while forkhead box L2 (FOXL2) was minimally expressed. Disordered secretion of reproductive hormones in prepubescent XX-DSD pigs indicated abnormal hypothalamic-pituitary-gonadal axis (HPGA) function. This study elucidates the genetic and pathological characteristics of prepubescent XX-DSD pigs in familial case, providing valuable insights for further exploration of the pathogenic mechanisms underlying XX-DSD.

Whole-genome de novo sequencing reveals genomic variants associated with differences of sex development in SRY negative pigs.

BACKGROUND: Differences of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. In more than 50% of human DSD cases, a molecular diagnosis is not available. In intensively farmed pig populations, the incidence of XX DSD pigs is relatively high, leading to economic losses for pig breeders. Interestingly, in the majority of 38, XX DSD pigs, gonads still develop into testis-like structures or ovotestes despite the absence of the testis-determining gene (SRY). However, the current understanding of the molecular background of XX DSD pigs remains limited. METHODS: Anatomical and histological characteristics of XX DSD pigs were analysed using necropsy and HE staining. We employed whole-genome sequencing (WGS) with 10× Genomics technology and used de novo assembly methodology to study normal female and XX DSD pigs. Finally, the identified variants were validated in 32 XX DSD pigs, and the expression levels of the candidate variants in the gonads of XX DSD pigs were further examined. RESULTS: XX DSD pigs are characterised by the intersex reproductive organs and the absence of germ cells in the seminiferous tubules of the gonads. We identified 4,950 single-nucleotide polymorphisms (SNPs) from non-synonymous mutations in XX DSD pigs. Cohort validation results highlighted two specific SNPs, "c.218T > C" in the "Interferon-induced transmembrane protein 1 gene (IFITM1)" and "c.1043C > G" in the "Newborn ovary homeobox gene (NOBOX)", which were found exclusively in XX DSD pigs. Moreover, we verified 14 candidate structural variants (SVs) from 1,474 SVs, identifying a 70 bp deletion fragment in intron 5 of the WW domain-containing oxidoreductase gene (WWOX) in 62.5% of XX DSD pigs. The expression levels of these three candidate genes in the gonads of XX DSD pigs were significantly different from those of normal female pigs. CONCLUSION: The nucleotide changes of IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment of the WWOX were the most dominant variants among XX DSD pigs. This study provides a theoretical basis for better understanding the molecular background of XX DSD pigs. DSD are conditions affecting development of the gonads or genitalia. These disorders can happen in many different types of animals, including pigs, goats, dogs, and people. In people, DSD happens in about 0.02-0.13% of births, and in pigs, the rate is between 0.08% and 0.75%. Pigs have a common type of DSD where the animal has female chromosomes (38, XX) but no SRY gene, which is usually found on the Y chromosome in males. XX DSD pigs may look like both males and females on the outside and have testis-like or ovotestis (a mix of ovary and testis) gonads inside. XX DSD pigs often lead to not being able to have piglets, slower growth, lower chance of survival, and poorer meat quality. Here, we used a method called whole-genome de novo sequencing to look for variants in the DNA of XX DSD pigs. We then checked these differences in a larger group of pigs. Our results reveal the nucleotide changes in IFITM1 (c.218T > C), NOBOX (c.1043 C > G), and a 70 bp deletion fragment in intron 5 of the WWOX, all linked to XX DSD pigs. The expression levels of these three genes were also different in the gonads of XX DSD pigs compared to normal female pigs. These variants are expected to serve as valuable molecular markers for XX DSD pigs. Because pigs are a lot like humans in their genes, physiology, and body structure, this research could help us learn more about what causes DSD in people.