| Spermatogonial stem cells(SSCs),also known as male germline stem cells(mGSCs),are the only germ cells in male with self-renewal and differentiation function.SSCs are the basis for the continuous production of sperm to maintain male fertility even though they only account for 0.03% of the total male germ cells.Dairy goat is an important economic livestock in China,which provides abundant dairy and meat products.However,its development and application are limited by the low fertility.Understanding the selfrenewal,proliferation and differentiation of dairy goat SSCs is important for preservinghigh quality germplasm resources and acquiring excellent strains by gene editing technology.LIN28 A is the only mammalian protein with both cold-shock and CCHC zinc finger structures.It regulates gene expression at the transcriptional or post-transcriptional level.But little is known about LIN28 A in livestock SSCs.In this study,we found that LIN28 A is widely expressed in different tissues of dairy goat.It is specifically expressed in the cytoplasm of SSCs and is a biomarker of dairy goat mGSCs.LIN28 A promoted proliferation and maintained the self-renewal of GmGSCs-I-SB in vivo and in vitro.We revealed the role of LIN28 A in NANOG transcriptional regulation via DNA epigenetic modifications to maintain the stemness of GmGSC.The results are shown in detail as following:1.LIN28 A is highly conserved among different species.In this study,we successfully cloned LIN28 A CDS of dairy goat and preliminarily explored its sequence,structure and expression profile.The LIN28 A amino acid sequences of Capra hircus,Bos caurus,Gallus gallus,Homo sapiens,Mus musculus,Ovis aries and Sus scrofa have similar hydrophilic and hydrophobic distributions,and the similarity of sequences is as high as 95.01%.Dairy goat’s LIN28 A protein also has the cold-shock structure and CCHC zinc finger structure.The expression of LIN28 A is highest in liver tissues followed by pancreatic tissues but lower in testis.The expression of LIN28 A is highest in 3 months old testicular tissue and decreases as testis developed.Immunofluorescence staining showed that LIN28 A mainly expressed in the cytoplasm of SSCs and slightly expressed in the nucleus,suggesting that LIN28 A could be a biomarker of dairy goat mGSCs.2.We successfully got GmGSCs-I-SB-Lin28 a cell line which stably overexpresses LIN28 A.LIN28A enhanced the viability and proliferation of GmGSCs-I-SB cell lines without inducing apoptosis.The expression level of PCNA in the GmGSCs-I-SB-Lin28 a was remarkably increased compared with that of the control cell line.Moreover,the percentage of BrdU-positive cells in LIN28A-overexpressing cell line was also significantly higher than that of the control group.Overexpressing LIN28 A up-regulated the expression of pluripotent related genes OCT4,SOX2 and self-renewal related genes PLZF,ETV5 and GFRα1.These results indicated that LIN28 A plays an important role in the proliferation,self-renewal and stemness maintenance of dairy goat mGSCs.3.To evaluate whether LIN28 A promotes the self-renewal and proliferation of dairy goat mGSCs in vivo,GmGSCs-I-SB-Lin28 a and GmGSCs-I-SB-GFP cells were transplanted into seminiferous tubules of busulfan-treated infertile mice.They had similar testicular size and weight.But testis transplanted with GmGSCs-I-SB-Lin28 a cells has more VASA-and PCNA-positive germ cells compared with control which indicated that LIN28 A promotes the self-renewal and proliferation of dairy goat mGSCs in vivo.However,there were no sperm in the epididymis which indicates the GmGSCs-I-SB cells could not complete the differentiation process.In addition,we found that LIN28 A via activating the mTOR/S6 signaling pathway to promote the proliferation of dairy goat mGSCs.4.Dairy goat has two NANOG genes.The NANOG gene located in chromosome 15 has only one exon and its promoter is highly methylated with no promoter activity.The one located on chromosome 5 with four exons is a functional gene as it has a lower methylation level in GC enriched promoter region and a strong promoter activity.5.The-1167+201NANOG-chr5 with the strongest promoter activity has two possible LIN28 A binding sites(-783 and-210).Promoter truncation experiments showed that LIN28 A equally enhanced the promoter activity of-573→+201 region and-1167→+201 region,suggesting that LIN28 A mainly promoted NANOG expression by binding to-210.6.Overexpressing LIN28 A does not perturb the histone(H3K4ME1/2/3)and DNA methylation level of GmGSCs-I-SB but demethylates some special DNA loci.The GmGSCs-I-SB-Lin28 a cell line showed lower DNA methylation level of NANOG promoter located on chromosome 5.LIN28 A and TET1 are co-localized in dairy goat mGSCs and the Co-IP experiment confirmes the interaction between them.Mechanistically,LIN28 A specifically binds to NANOG promoter and recruits TET1 for epigenetic DNA methylation modifications to activate the expression of NANOG,which is independent on the LIN28A/Let-7g negative feedback axis.In summary,we explored the expression patterns of LIN28 A in male dairy goat and uncovered the effects of LIN28 A on proliferation and self-renewal of mGSCs.We revealed that LIN28 A recruits TET1 to NANOG promoter for epigenetic DNA methylation modifications to activate the expression of NANOG.This study provides a reliable scientific basis for studying proliferation,self-renewal and pluripotency mechanism of goat and a new scheme for the preservation and breeding of dairy goat germplasm resources and obtaining high-quality dairy goat strains through gene editing technology. |
