The Role And Mechanisms Of LSD1 In Porcine Early Embryonic Development

Early embryonic development is a complex and precise biochemical process that involves the regulation of a large number of gene transcriptions and functions,along with complex epigenetic regulation.Recent research has highlighted the crucial involvement of histone modifying enzymes in the regulation of histone modification,chromatin structure,and gene transcription during early embryonic development.Specifically,the histone demethylase Lysine specific demethylase(LSD1)has been shown to catalyze the demethylation of mono-and di-methylated H3K4me1/2 or H3K9me1/2,as well as other nonhistone substrates.LSD1 plays an important role in the regulation of fundamental biological processes like gene transcription.However,further investigation into the role and mechanism of LSD1 in the early embryonic development of pigs is necessary.This data is anticipated to serve as a valuable reference for enhancing the efficiency and quality,reducing embryo mortality and loss of early embryonic development.To elucidate the potential function of LSD1 in the regulation of porcine early embryonic development.This study utilized porcine early embryos as an experimental model.The findings suggest that LSD1 plays a crucial role in porcine early embryonic development,as demonstrated by inhibiting LSD1 expression with the LSD1 inhibitor GSK-LSD1.Transcriptomic sequencing was conducted following LSD1 inhibition in porcine early embryos,and functional enrichment analysis was performed on the resulting differential genes.The analysis revealed that LSD1 primarily influences early porcine embryonic development through autophagy.Functional verification experiments,including RT-q PCR,immunofluorescence,and Western blotting,were conducted.The study further elucidated the molecular mechanism by which m TOR mediates cell autophagy to regulate porcine early embryonic development.The specific results are as follows:1.LSD1 was localized in the nucleus and expressed during all stages of early porcine embryo.2.Inhibition of LSD1 resulted in decreased cleavage rate,blastocyst rate,and diameter of PA parthenogenetic activation and in vitro fertilization embryos.It also inhibited the m RNA and protein expression levels of pluripotency-related genes OCT4,SOX2,KLF4,and NANOG.3.Inhibition of LSD1 resulted in increased levels of H3K4me1/2 and H3K9me1/2methylation modifications in porcine early embryos.4.Inhibition of LSD1 resulted in DNA damage and cell apoptosis,as evidenced by elevated expression levels of the DNA damage marker protein γH2AX,DNA damage response genes(ATM and ATR),and decreased expression levels of DNA double-strand break repair pathway-related genes(BRCA1,53BP1,XRCC6,and PRKDC).Additionally,inhibition of LSD1 up-regulated the ratio of TUNEL-positive cells and the expression of apoptosis-related proteins Caspase3,Caspase8,and Caspase9,while down-regulating the expression of BCL2/BAX,ultimately inducing DNA damage and apoptosis in porcine early embryos.5.Differentially expressed genes were identified through bioinformatics analysis in early porcine embryos following LSD1 inhibition.A total of 2,992 genes showed differential expression,with 1,347 being up-regulated and 1,645 down-regulated.These genes were predominantly enriched in pathways related to ribosomes,phagosomelysosome fusion,autophagy,apoptosis,and the m TOR signaling pathway.6.Inhibition of LSD1 resulted in heightened cell autophagy in early pig embryos,leading to decreased levels of p-m TOR/m TOR and increased levels of p-ULK1/ULK1.Additionally,there was an upregulation of autophagy-related proteins LC3B-II/LC3 BI and p62,along with increased expression of autophagy-related genes such as ATG5,ATG7,ATG16,and ATG101.7.The m TOR activator inhibited the expression of autophagy-related proteins LC3B-II/LC3B-I and p62,as well as autophagy-related genes(ATG7 and ATG101).It also up-regulated the expression of BCL2/BAX,down-regulated the expression of Caspase8,and subsequently restored the early embryonic development rate that had decreased due to LSD1 inhibition.8.Inhibition of LSD1-induced selective autophagy primarily triggers mitophagy,resulting in reduced mitochondrial membrane potential,ATP levels,and increased ROS levels,ultimately leading to mitochondrial dysfunction.This study elucidated the expression pattern of LSD1 and its regulatory mechanism on autophagy through m TOR during early porcine embryonic development.These findings enhance our comprehension of LSD1’s role and the regulatory mechanisms involved in porcine early embryonic development,offering a theoretical foundation for enhancing the efficiency of early embryonic development in pigs.