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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.
Droplet digital PCR quantification of selected microRNAs in raw mastitic cow’s milk from the west of Poland
Abstract Introduction MicroRNAs (miRNAs), a class of noncoding small RNAs, have been recognised as potential biomarkers of mammary gland conditions, including bovine mastitis diagnosis. The aim of this study was to quantify selected miRNAs in the milk of mastitic cows. Material and Methods Milk samples (n = 90) were collected from healthy and mastitic dairy cows originating from local dairy cattle farms located in the west of Poland. MicroRNAs of the miR-21a, miR-92a, miR-146a and miR-383 species were quantified using the highly sensitive droplet digital PCR method. Direct measurement of somatic cell count (SCC) was performed using a cell counter. Cows were divided into three groups: those with an SCC below 200,000/mL were designated Low (n = 25), those with an SCC between 200,000 and 999,999 were Medium (n = 34), and those with an SCC of 1,000,000 or higher were High (n = 31). Microbiological analyses were performed using standard culture testing. Results The level of miR-383 was very low and this miRNA was excluded from analysis. The miR-92a was used to normalise miR-21a and miR-146a expression levels. The obtained results of expression of miR-21a and miR-146a correlated with somatic cell number (R = 0.53 and 0.79, respectively). Conclusion These results show that ddPCR is a useful method for quantifying miRNAs in raw cow milk. It seems that miR-146a is a promising marker for bovine mastitis, although further studies are needed to select a panel of miRNAs that can be used in mastitis monitoring in Poland.
Droplet Digital PCR Quantification of Selected Intracellular and Extracellular microRNAs Reveals Changes in Their Expression Pattern during Porcine In Vitro Adipogenesis
Extracellular miRNAs have attracted considerable interest because of their role in intercellular communication, as well as because of their potential use as diagnostic and prognostic biomarkers for many diseases. It has been shown that miRNAs secreted by adipose tissue can contribute to the pathophysiology of obesity. Detailed knowledge of the expression of intracellular and extracellular microRNAs in adipocytes is thus urgently required. The system of in vitro differentiation of mesenchymal stem cells (MSCs) into adipocytes offers a good model for such an analysis. The aim of this study was to quantify eight intracellular and extracellular miRNAs (miR-21a, miR-26b, miR-30a, miR-92a, miR-146a, miR-148a, miR-199, and miR-383a) during porcine in vitro adipogenesis using droplet digital PCR (ddPCR), a highly sensitive method. It was found that only some miRNAs associated with the inflammatory process (miR-21a, miR-92a) were highly expressed in differentiated adipocytes and were also secreted by cells. All miRNAs associated with adipocyte differentiation were highly abundant in both the studied cells and in the cell culture medium. Those miRNAs showed a characteristic expression profile with upregulation during differentiation.
Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs
The role of the CEBPB gene in porcine adipogenesis: a study using CRISPR/Cas9-edited mesenchymal stem cells
Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for gene editing and the regulation of gene expression. It enables the introduction of targeted mutations, thereby facilitating functional studies of specific genes in various cellular processes. In this study, we aimed to generate a deletion in the promoter region of the CEBPB gene, which encodes a transcription factor involved in adipogenesis, and to evaluate the impact of this modification on the adipogenic differentiation potential of porcine mesenchymal stem cells (MSCs). A 575-bp deletion was introduced in the target region, resulting in the generation of both homozygous and heterozygous mutant cells. Adipogenic differentiation was assessed by quantifying transcript levels of adipocyte marker genes ( GATA2 , CEBPA , PPARG , and FABP4 ) at days 0, 4, 6, 8, and 10 of the differentiation process. Disruption of CEBPB expression led to the downregulation of these adipogenic markers, indicating impaired adipocyte differentiation. Additionally, to assess the proliferative capacity of the edited cells, the expression levels of proliferation-associated genes ( CCND1 , MCM2 , and PCNA ) were measured. A reduction in their transcript levels was observed in both homozygous and heterozygous mutant cells. These findings indicate that both homozygous and heterozygous deletions in the CEBPB promoter completely block adipogenesis and alter MSC proliferation, highlighting the pivotal role of CEBPB not only in adipogenic differentiation but also in the regulation of cell proliferation in porcine mesenchymal stem cells. These results provide new insights into the molecular mechanisms underlying adipose tissue development and have implications for pig breeding strategies aimed at optimizing carcass composition, as well as for biomedical research focused on adipose tissue biology.
Zastosowanie technologii CRISPR-Cas9 do wyjaśnienia czy zmiany architektury jądra interfazowego w trakcie adipogenezy są przyczyną czy skutkiem aktywności transkrypcyjnej genów
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
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.
Cytogenetic and molecular insight into the genetic background of disorders of sex development in seventeen cats
AbstractThe genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XXSRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.
Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing
Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5′-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.
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.
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.
A case of non-mosaic X trisomy (65,XXX) in a Thoroughbred mare confirmed by cytogenetic and molecular analysis
Polymelie bei einem weiblichen Kalb – ein Fallbericht mit operativer Entfernung
ZusammenfassungDas hier vorgestellte weibliche Kalb der Rasse Deutsch Holstein Farbrichtung Schwarzbunt wurde mit einer kompletten zusätzlichen Vordergliedmaße geboren. Der Ansatzpunkt der Vordergliedmaße lag zwischen den Schulterblättern. Man spricht somit von einer Notomelie, eine Form der Polymelie. Am 15. Lebenstag des Kalbes wurde die zusätzliche Gliedmaße operativ entfernt. Die Operation und Wundheilung verliefen gut. Die zytogenetischen Untersuchungen ergaben keine chromosomalen Anomalien. Die genetischen Untersuchungen zeigten keinen Hinweis auf eine Chimäre, die die Ursache der Anomalie sein könnte. Antigene des Schmallenberg Virus konnten nicht nachgewiesen werden, ein Antikörpertiter gegen das Virus lag bei der Geburt und am 15. Lebenstag vor.
