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The normal course of DNA methylation depends on the correct functioning of the DNA methylation machinery, which include DNA methyltransferase enzymes (DNMTs) and methyl-CpG-binding domain proteins (MBDs). So far, little is known about the activity of these components during adipogenesis in the pig. The aim of this study was to analyze the expression of ten genes (DNMT1, DNMT3a, DNMT3b, MBD1, MBD2, MBD3, MBD4, MeCP2, UHRF1, and CBX5) during in vitro differentiation into adipocytes of porcine mesenchymal stem cells derived from adipose (AD-MSC) and from bone marrow tissue (BM-MSC). We found that, in undifferentiated cells, the global methylation level was higher in BM-MSC than in AD-MSC, but had similar levels in adipocytes. The transcript level of the DNMT1 gene increased at the beginning of adipogenesis and then decreased, while DNMT3a and DNMT3b transcripts increased during differentiation. All the examined MBD genes show similar expression patterns within the studied system (AD-MSC and BM-MSC). The transcript abundance of UHRF1 and CBX5 decreased in both systems. The changes in the expression patterns of these genes points to the dynamic nature of DNA methylation during porcine adipogenesis.

Freemartinism is a disorder of sex development (DSD) caused by the formation of placental anastomoses between fetuses of different sex. It leads to development of an abnormal reproductive tract in the female fetus, including the presence of testes or ovotestes. In pig production, such conditions can affect both reproductive performance and meat quality, through undesired boar taint. A common approach to diagnosing freemartinism is to detect leukocyte chimerism (XX/XY). In the study described here, we performed a complex evaluation of 28 DSD pigs with ambiguous external genitalia (enlarged clitoris, presence of scrotum, etc.). Sex chromosomes were cytogenetically identified by Giemsa staining, as well as with fluorescence in situ hybridization (FISH), while Y-linked (ZFY) and X-linked (ZFX) genes were detected by PCR-RFLP. Histological analysis of the gonads was performed on eosin–hematoxylin-stained microscope slides. Chromosome studies revealed that, of 28 DSD animals, 20 were freemartins (38,XX/38,XY), 2 had a female karyotype (38,XX), and 6 had a male karyotype (38,XY). Analysis of breeding data showed that the mean litter size in which DSD animals occurred was about 17. Histology of the gonads could be performed for 8 DSD pigs, including 6 freemartins and 2 XX DSD. In all these cases, testicular tissue without signs of spermatogenesis was seen. In XX DSD pigs, a copy number variation (CNV) polymorphism downstream from SOX9, tentatively associated with this type of DSD, was detected by FISH. On the other hand, the background of the XY DSD remains unknown. Since cytogenetic analysis is labor-intensive and time-consuming, we evaluated the usefulness of the droplet digital PCR (ddPCR) approach to diagnosing XX/XY leukocyte chimerism. Estimation of X and Y chromosome copy numbers was based on the quantitative detection of AMELX and AMELY genes. Different sex chromosome sets (XX, XY and XX/XY) were unequivocally identified by ddPCR. In conclusion, we suggest that freemartinism, beside testicular or ovotesticular XX DSD, is an emerging problem in highly prolific pigs and that detection of XX/XY leukocyte chimerism can be performed using a fast and reliable ddPCR approach.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

Testicular XX disorder of sex development (XX DSD, SRY -negative), causing sterility, is quite frequently diagnosed in pig populations, however, its molecular background is still unknown. This disorder may affect carcass quality (boar taint) due to the presence of testicular tissue in animals with ambiguous female external genitalia. A chromosome fragment encompassing the SOX9 gene was previously considered as a candidate region in functional and association studies. We analyzed distribution of polymorphic variants in 5’UTR and in 3’-flanking regions of the SOX9 gene in a cohort of 12 XX DSD pigs and a panel of 116 normal females. Altogether, 8 previously known polymorphic sites were analyzed and no significant association under Bonferroni correction (P>0.0063) between DSD phenotype and identified SNPs was found. The region harboring the SOX9 gene was also searched for the presence of copy number variation (CNV) by fluorescence in situ hybridization technique (FISH), using a set of 7 BAC probes. A potential CNV region, manifested by size variation (large and small) of fluorescence signals, produced by a single BAC probe hybridizing downstream (approx. 500 kb) of the SOX9 gene, was observed in 4 DSD cases. We conclude, that a new CNV polymorphism in the region harboring SOX9 gene can be considered as a promising marker for the DSD phenotype.

The miR-30 gene family includes potential regulators of adipogenesis. We searched for polymorphism in this gene family in 4 pig breeds: Duroc, Pietrain, Polish Landrace (PL), Polish Large White (PLW) and a synthetic line (L990). Altogether 5 single nucleotide polymorphisms (SNPs) in 3 genes were found, including one already known SNP rs340704946 within the pre-miR-30d genomic sequence. An association of the rs340704946 with intramuscular fat (IMF) content, abdominal fat accumulation and daily body mass gain in L990 was found. Moreover, the AG genotype was associated with an increased content of monounsaturated fatty acids (MUFA) in subcutaneous (P=0.027) and visceral fat (P=0.007) tissues when compared to the GG genotype. Analysis of PLW pigs revealed an association of the rs340704946 with transcript level of the ME1 gene in longissimus dorsi (LD) muscle (P=0.002), as well as the ME1 protein product in the LD muscle (P

Disorders of sex development (DSD) are rarely reported in cats, but this does not mean these occurrences are an insignificant reproductive and health problem in this species. The DSD condition affects reproduction and can be associated with an increased risk of gonadal tumorigenesis. In this review, an overview of findings since 2012 are presented that focus on cytogenetic and molecular genetic studies of cats with abnormal external genitalia. Results from advanced cytogenetic analysis of sex chromosomes indicate there is a range of abnormalities, including aneuploidies, structural rearrangements and freemartinism, which manifests as leukocyte XX/XY chimerism. The molecular abnormalities that result in feline monogenic and multifactorial DSD (such as hypospadias and cryptorchidism) are very few. There are only two mutations of genes (CYP11B1 and TAC3) which are known to be responsible for syndromes associated with abnormal sexual development. Several candidate genes (SRY, AR, SRD5A2, MAMLD1, DHH, HSD3B2, and HSD17B3) have also been examined, but no associations were identified between these polymorphisms and DSD phenotypes. The findings in developing the present review indicate sex chromosome abnormalities are quite common causes of feline DSD. The study of the molecular disorders that lead to the development of DSD in cats with normal XX or XY sex chromosome complements is still in its infancy, and further research is needed into this topic. It can be anticipated that the use of next generation sequencing technologies to study the genetic disorders that result in the DSD condition in cats will lead to an increase the detection of several causative mutations.