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The Effects of Peruvian maca (Lepidium meyenii) Root Extract on In Vitro Cultured Porcine Fibroblasts and Adipocytes
Peruvian maca (Lepidium meyenii) is a plant known for its nutritional and medicinal properties whose use as a supplement in animal diets has attracted much interest. We studied the effects of powdered maca root extract on the growth potential of in vitro cultured porcine cells prior to its use as an additive in animal nutrition. Fibroblast cell viability (MTT), cell proliferation (BrdU), and apoptosis level (TUNEL) were measured for a range of extract doses (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, and 10 mg/mL). Transcript levels of CCND1, MCM2, and PCNA genes as molecular markers of cell proliferation were also determined. Next, the effects of maca extract at 2 and 5 mg/mL on in vitro induced adipogenesis were evaluated over eight days of differentiation. The transcript levels of three adipocyte marker genes (CEBPA, PPARG, and FABPB4) were measured at days 0, 4, and 8 of adipose differentiation, and lipid droplet accumulation (BODIPY staining) was also noted. No cytotoxic effect was detected on fibroblast cell viability, and the inhibitory concentration (IC50) value was determined to be IC50 > 10 mg/mL. Doses of maca extract above 3 mg/mL decreased cell proliferation. The transcript level decreased in concentrations above 5 for the MCM2 and PCNA genes. For the CCND1 gene, the transcript level decreased when the greatest maca dose was used. In the in vitro adipogenesis experiment, it was found that the rate of lipid droplet formation increased on day 4 of differentiation for both doses, while decreased lipid droplet formation was observed on day 8 for 5 mg/mL of maca extract. Significant changes were seen in the mRNA level for CEBPA and PPARG on days 4 and 8, while the transcript of FABP4 increased only on day 8 at 2 mg/mL dose. It can be concluded that the addition of Peruvian maca in small doses (<3 mg/mL) has no negative effect on porcine fibroblast growth or proliferation, while 2 mg/mL of maca extract enhances adipocyte differentiation.
Altered Transcript Levels of MMP13 and VIT Genes in the Muscle and Connective Tissue of Pigs with Umbilical Hernia
Umbilical hernia (UH) and inguinal hernia (IH) are among the most common defects in pigs, affecting their welfare and resulting in economic losses. In this study, we aimed to verify the association of previously reported differences in transcript levels of the ACAN, COL6A5, MMP13, and VIT genes with the occurrence of UH and IH. We examined mRNA levels in muscle and connective tissue from 68 animals—34 affected by UH and 34 controls. In a second cohort, we examined inguinal channel samples from 46 pigs (in four groups). We determined DNA methylation levels in muscle tissue for the UH and control animals. The transcript level of MMP13 changed in the UH cases, being upregulated and downregulated in muscle and connective tissue, respectively, and the VIT gene also showed an increased muscular mRNA level. The transcript of the ACAN gene significantly decreased in old pigs with IH. We further observed an increased DNA methylation level for one CpG site within the MMP13 gene in UH individuals. We conclude that these alterations in gene mRNA levels in the UH animals depend on the tissue and can sometimes be a consequence of, not a cause of, the affected phenotype.
Prevalence of the SOD1, PRCD and SLC2A9 gene mutations responsible for degenerative myelopathy, progressive rod-cone degeneration, and hyperuricosuria in Polish population of Labrador Retriever dogs
Polymorphism of the HMGA2 gene in Polish children and adolescents with short stature and diverse growth hormone secretion
SOX9 gene variants in 27 French Bulldogs with disorder of sex development (XX, SRY-negative): identification of first case of skeletal abnormalities associated with SOX9 triplication
Genetic and epigenetic markers in the METTL21C gene associated with umbilical hernia in pigs
Abstract Background Hernias, particularly umbilical hernias (UH), are prevalent anatomical anomalies in swine, leading to significant welfare issues and economic losses. Besides environmental factors also genetic components contribute to the development of UHs, though the exact mechanisms remain unclear. This study employed a multiple approaches integrating RNA-seq, DNA methylation analysis, Sanger sequencing, droplet digital PCR and western blot analysis to investigate the genetic and epigenetic underpinnings of UH in pigs. Muscle tissue from affected and control pigs was examined to identify differentially expressed genes (DEGs) and associated pathways. Results We found 59 significant DEGs, including SIM1 , PITX1 , HOXA7 , METTL21C , PVALB , ALX1 , EYA2 , and TBX1 . Interestingly, RNA-seq analysis of METTL21C revealed its significant upregulation in UH-affected pigs. This was corroborated by epigenetic analysis, which identified hypomethylation at four CpG sites in the METTL21C within potential regulatory region, aligning with increased mRNA levels. Furthermore, Sanger sequencing uncovered an SNP (rs330073569) in the METTL21C regulatory region, which was significantly associated with UH condition. This SNP can potentially alter transcription factor binding leading to enhanced METTL21C transcription, and putatively contributing to the gene’s increased expression in UH pigs. Conclusions This study highlights potential genetic and epigenetic factors in UH etiology. The most significant result suggests that METTL21C plays an important role in the development of UH. This finding makes this gene a promising candidate for further research aimed at better characterizing umbilical hernia in pigs and at potentially eliminating undesirable variants from the gene pool.
A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism
Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid ( n = 122) and control ( n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3′UTR (16:23716202G>A) in KATA6 , responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism ( P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3′UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.
GH1 gene polymorphism in Polish children and adolescents with short stature - reanalysis based on diverse growth hormone secretion
Wykorzystanie analizy transkryptomicznej do identyfikacji genów związanych z występowaniem przepuklin pępkowych świń
XX/XY Chimerism in Internal Genitalia of a Virilized Heifer
Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.
Elevated serum concentration of anti‐Mullerian hormone and its association with SNP variants in the AMH gene in a tortoiseshell tomcat with a disorder of sex development (38,XX; SRY-negative)
AbstractTesticular disorders of sex development (DSD) in cats with XX sex chromosomes and the absence of the SRY gene are rare congenital abnormalities. A Maine Coon tomcat with a normal penis, gonads in the scrotum, low serum testosterone concentration, and an elevated level of anti‐Müllerian hormone (AMH) was subjected to genetic analyses due to an unusual tortoiseshell coat color for males. Primary studies revealed the presence of XX sex chromosomes, the lack of SRY and the presence of two copies of the candidate SOX9. The DSD tomcat and its parents were analyzed using whole genome sequencing. Candidate SNPs in AMH, ORC1, DOCK8, PRKAR1A, and TMEM186 genes, as well as a known intronic 5‐kb deletion in X‐linked ARHGAP36 gene, which is responsible for orange coat, were identified. Potentially pathogenic homozygous genotypes were observed in all candidate genes; however, only in AMH and ORC1 were these genotypes rare in a control cohort. Further studies were focused on two SNPs located in the 5′‐and 3′‐untranslated regions (UTRs) of AMH. It has been experimentally demonstrated that only a short AMH transcript is present in feline testes. In silico analysis revealed that the SNP located in the 3′UTR of AMH occurs within a sequence that partially matches the canonical binding site for human miR‐5571‐5p. This microRNA is expressed in mammalian testes, which we confirmed in feline testicular tissue. We concluded that SNP in the 3′UTR of AMH is associated with elevated expression of the encoded hormone; however, it is not the cause of the testicular DSD phenotype in the studied Maine Coon tomcat.
First report on the presence of a common pathogenic variant in the PKD1 gene, causing polycystic kidney disease, in a series of related Sphynx cats
Lack of causative mutation in the AMH and AMHR2 genes in a cat (38,XY) with persistent Mullerian duct syndrome (PMDS)
AbstractA 1‐year‐old European shorthair male cat with a normally developed penis was subjected to genetic, endocrinological and histological studies due to unilateral cryptorchidism. The blood testosterone level was typical for males, while the level of anti‐Mullerian hormone (AMH) was very low. Surgical removal of internal reproductive organs was followed by a histological study, which revealed inactive testicles with neoplastic changes and derivatives of Mullerian ducts. Cytogenetic analysis showed a normal XY sex chromosome complement and molecular analysis confirmed the presence of Y‐linked genes (SRY and ZFY). Although the level of AMH was low, two normal copies of the AMH gene were found using droplet digital PCR (ddPCR). Analysis of the coding sequences of two candidate genes (AMH and AMHR2) for persistent Mullerian duct syndrome (PMDS) in the affected cat and in control male cats (n = 24) was performed using the Sanger sequencing method. In the affected cat, homozygosity was found for three novel missense variants in Exon 1 (one SNP) and Exon 5 (two SNPs) of AMH, but the same homozygous genotypes were also observed in one and two control cats, respectively, whose sex development was not examined. Three known synonymous variants with homozygous status were found in AMHR2. We conclude that the DNA variants identified in AMH and AMHR2 are not responsible for PMDS in the affected cat.
