Mechanisms Of 1α,25（OH）₂VD₃ Regulating The Follicular Development And Egg Production Performance In Layers

Vitamin D₃（VD₃）is an important nutrient known for its classical role in maintaining bone homeostasis and regulating calcium and phosphorus metabolism.1α,25（OH）₂VD₃（VitD₃）,a steroid compound,is the active form of VD₃ that really functions in the organism.VitD₃ is affects a variety of cellular functions including cell proliferation,apoptosis,and inflammatory,and the female reproductive system has recently been found to be an important target organ for VitD₃,and VD₃ deficiency is associated with various female reproductive disorders.VitD₃ act through Vitamin D receptor（VDR）,and the expression of VDR in female reproductive system is progressively expanded.Therefore,VitD₃ has an important modulatory role in female reproduction.In vitro studies demonstrated VitD₃ has positive effects on follicular development and steroid synthesis,but the mechanisms of VitD₃ function on follicular development and reproductive performance in poultry have yet to be revealed.The aim of this study was to investigate the mechanism by which VitD₃ affects follicular development and delays ovarian decline in Hyland Brown hens.In this study,we first verified the direct regulation of VitD₃on follicle development in young layers and follicle cells,based on which we used aged hens as model animals further investigated the effect of VitD₃ on ovarian recession and reproductive performance,and built an in vitro follicle culture model to reveal the mechanism of VitD₃’s function on ovarian regression in aged hens.The analyses of transcriptomic,targeted metabolomics,and proteomics data obtained from three biological levels to clarify the effects of VitD₃ on follicle development and egg production performance in laying hens,and the regulation of in vitro active substances to promote follicular development in hens,we attempted to give a theoretical basis for improving follicular development and egg production performance of hens.The main results are as follows:1.VitD₃ improved the development of follicles in young layers.Our study investigated the effects of VitD₃ on follicle development and steroid hormone biosynthesis in young layers.VitD₃ supplementation promoted follicle development,increasing the number of small yellow follicles（SYFs）and large yellow follicles（LYFs）and the thickness of the granulosa layer（GL）of SYFs.Transcriptome analysis revealed that VitD₃ supplementation altered gene expression in the ovarian steroidogenesis,cholesterol metabolism,and glycerolipid metabolism signaling pathways.Steroid hormone-targeted metabolomics profiling identified 20 steroid hormones altered by VitD₃ treatment,with 5 being significantly different among the groups.VitD₃ treatment altered the gene expression related to steroid metabolism and the production of testosterone,estradiol,and progesterone in the SYFs of young layers.2.VitD₃ regulated the proliferation,apoptosis,and steroid synthesis of phGC and ph TC.We isolated and cultured granulosa cells from pre-hierarchical follicles（phGCs）and theca cells from prehierarchical follicles（ph TCs）,and the effects of different dosages of VitD₃ on cell proliferation,apoptosis,cell cycle and steroid synthesis-related genes and E2,P4 contents in culture supernatants were determined.It was found that VitD₃ increased cell proliferation,promoted cell cycle progression,regulated the transcription of cell-cycle-related genes,and inhibited the apoptosis of phGCs and ph TCs.In addition,the steroid hormone biosynthesis-related genes,estradiol（E2）and progesterone（P4）concentrations,and Vitamin D receptor（VDR）expression level was significantly altered by VitD₃.3.VitD₃ delayed ovarian decline in aged hens.VitD₃ treatment significantly increased the follicle number and the GL thickness in aged hen ovary.The KEGG analysis of RNA-seq revealed that VitD₃supplementation regulated the expressions of genes involved in the cell cycle,DNA replication,extracellular matrix（ECM）-receptor interaction,steroid hormone biosynthesis,TGF-β,and MAPK signaling pathways.Additionally,the proteomics analysis showed that these differentially abundant proteins（DAPs）among three groups focused on proteins related to the regulation of follicular development such as the PI3/AKT signaling pathway,ECM-receptor interaction,focal adhesion,and ribosome pathways and the cellular components was also affected by the high dose of VitD₃.Mechanistically,VitD₃ promotes proliferation and anti-apoptosis in PHFs and activates both the PI3K/AKT and MAPK/ERK pathways.4.VitD₃ activates PI3K/AKT and MAPK/ERK pathways in vitro cultured follicles to delay ovarian recession.In order to study the mechanism of VitD₃ on follicle development in aged hens,an in vitro SYF culture model was constructed and used it to further validate the mentioned mechanism of action of VitD₃.Our results showed that VitD₃ promoted the synthesis of steroids in vitro cultured follicles,promoted cell proliferation and inhibited apoptosis;consistent with our in vivo results,it promoted follicle development by activating the PI3K/AKT and MAPK/ERK signaling pathways.5.VitD₃ enhanced the reproductive performance of aged hens.Aged hens（72 wk）were used as model animals to study ovarian recession and this study evaluated the effects of different VitD₃ doses on productive,egg quality and reproductive performance decline in aged hens.The results showed that VitD₃supplementation in aged hens significantly increased production rate and shell strength and improved the microstructure of eggshells.Besides,it significantly increased the serum levels of FSH,LH,P4 and Preg,and inhibited the decline of ovaries and oviducts contributing to the morphological structure and secretory capacity of the uterine part.Conclusion:Our findings demonstrate that VitD₃ can promote follicular cell proliferation,inhibit apoptosis and increase steroid hormone synthesis,and activate PI3K/AKT and MAPK/ERK signaling pathways to delay ovarian regression in aged hens and improve reproductive performance.These results could provide a basis for understanding that exogenous active substances modulate follicle development and ovarian recession in laying hens.