Identification Of Important Genes Involved In The Development Of Uterus-Vagina Junction Epithelial Fold (Containing Sperm Storage Tubules) In White Leghorns Hens

Uterus-vagina junction(UVJ)is an important part of the oviduct of female birds.The epithelium is formed to the epithelial fold,which contains tubular sperm storage tube s(SST).The UVJ epithelial fold(containing SST)is closely related to the reproduction of birds.The morphological differences formed during the development of UVJ epithelial fold(containing SST)are considered to be an important source of individual differences in hen sperm storage traits.However,there is a lack of study on the development of UVJ epithelial fold(containing SST)in birds,especially in layers.Therefore,this study explored the process of UVJ epithelial fold development and identified the important genes related to UVJ epithelial fold(containing SST)development.Through morphological anatomical measurement,histological section and statistical analysis,it was found that there were significant individual differences in the morphogenesis of UVJ epithelial fold(containing SST);the morphogenesis development level of UVJ epithelial fold was moderately correlated with other parts of reproductive system(oviduct weight,oviduct length,the largest follicle diameter),and the Pearson correlation coefficient was between 0.35 and 0.60;There was no significant difference in the area and height of UVJ epithelial fold between the onset of laying period and the peaking laying period in white leghorn population(random population),whereas,there was a significant difference in the area and height of UVJ epithelial fold between the high and low sperm storage individuals.According to the morphological characteristics,the development of UVJ epithelial fold(containing SST)was divided into four stages: T1,T2,T3 and T4,and the corresponding stage classification criteria were formulated.In order to better understand the mechanism of developmental regulation in UVJ epithelial folds,a total of 19 individuals(individual differences were controlled)were used to construct m RNA library and sequencing by bulk RNA-seq.After data analysis,the original morphological periods(T1 ～ T4)were combined into three periods: S1(n =5),S2(n = 10),S3(n = 4).Then enrichment analyses(such as Gene Set Enrichment Analysis,Panther classification and etc.)were used to identify candidate genes from S2 vs.S1 and S3 vs.S2 respectively,and in doing so,we identified the cytoskeleton(representative genes: KIF5 C,CAP2,DSP,DNAH5,PDLIM3,SGCZ etc.),cell polarity &planar cell polarity(representative genes: WNT11,SFRP1,MLLT3,etc.),cell adhesion(representative genes: CDH2,CDH6,ITGB6,ITGB8,etc.),cell junction(representative genes: MCAM,PCDH8,SPON2,SLIT2,etc.),cell migration regulation(representative genes: CDC42,RHOV),regulation of proliferation and differentiation(representative genes: BMP4,FGF family)and so on and so forth.Two important pathways were also identified: TGF-β(including BMP4,BMPR2,SMAD9,TGFBR2,etc.),and Wnt pathway(including WIF1,WISP1,WNT7 A,WNT9A,etc.).Among which,several key potential genes,such as BMP4,CDC42,CDH2,WIF1,NOV,CA family,FGF family were screened out for further study.Then,a UVJ epithelium fold(containing SST)sample at S2 stage was used to identify genes by single cell transcriptome sequencing(scRNA-seq).After quality control and data analysis,the cells of UVJ epithelial fold(containing SST)were divided into 17 subgroups.After subgroup identification,it was found that there were 5 ciliated epithelial subgroups,3 non ciliated epithelial subgroups(including SST subgroup),1 epithelial progenitor cell subgroup,1 fibroblast subgroup and several other subgroups at S2 stage.The differentially expressed genes in important subpopulations included CA4,CA8,FUT9,DNAH5 and etc.The expression of PCNA,MKI67 and MCM-2 in the epithelial subgroup was higher than that in the non-epithelial subgroup,which suggested the proliferation difference was considered to be an important factor in the formation of UVJ epithelial fold.The expression levels of BMP4,CDC42,CDH2,WIF1,NOV and other important candidate genes in different subgroups were compared,and these genes were suggested to have played different roles in different subgroups.Finally,the histological section confirmed the morphogenesis of SST belonged to budding;the immunohistochemical detection of PCNA showed that the proliferation of UVJ and SST epithelium belonged to global proliferation,and the proliferation of UVJ epithelium was higher than that of non-epithelial cells,which is an important reason for UVJ epithelial fold formation.Immunohistochemistry of other important candidate genes suggested that CDC42 gene was mainly related to cilia and participates in the regulation or maintenance of cell polarity;WIF1 mainly played a role in inhibiting Wnt signaling pathway ligands;CDH2 was mainly involved in cell adhesion molecules in the development of SST cells;BMP4 and NOV were related to the proliferation of epithelial cells and fibroblasts respectively.Through the culturing of UVJ primary epithelium(containing SST)by making use of BMP4 protein and pathway inhibitor LDN193189,it was showed that BMP4 and its downstream genes SMAD,ALK2 and ALK3 had different regulatory effects on cell proliferation in different periods of epithelium,and BMP4 protein also had regulatory effects on DF-1 cell(Chicken embryo fibroblasts)proliferation;the overexpression of NOV gene in DF-1 cell showed no effect on the proliferation of fibroblasts,but it inhibited the migration of fibroblasts.The above studies have revealed several developmental and regulatory factors in the formation of UVJ epithelial folds(containing SST).