| Gonadotropin-releasing hormone（GnRH）,synthesized by hypothalamic neurons,acts through the pituitary portal system on GnRH receptors（GnRHR）on the surface of anterior pituitary gonadotropin cells to regulate gonadotropin synthesis and secretion.Follicle stimulating hormone（FSH）,which is synthesized and secreted by pituitary gonadotropin cells,plays a key role in reproduction as an intermediate factor in the hypothalamic-pituitary-gonadal axis（HPGA）,regulating mammalian gonadal development and hormone secretion,follicular development,gametogenesis and other physiological activities.Meanwhile,FSH has also been shown to play important regulatory functions in skeletogenesis,lipid metabolism and cholesterol synthesis.Currently,FSH and its preparations have been widely used in the fields of reproductive regulation in livestock and assisted reproduction in humans.In cells,GnRH signaling molecules can regulate the synthesis and secretion of FSH through various signaling pathways such as MAPK signaling pathway and Ca2+/Ca MKII signaling pathway,and its regulatory mechanism is very complex.Among them,Ca MKII,as an important intracellular signaling molecule,is involved in the regulation of various pituitary hormone synthesis and secretion.However,the molecular mechanism by which GnRH regulates the synthesis and secretion of FSH through Ca MKII is not clear.Previous findings suggest that non-coding RNA mediates the regulatory process of GnRH signaling molecules on pituitary gland,which is closely related to the synthesis of FSH.However,it is not clear whether pituitary-specific expression of non-coding RNAs is involved in Ca MKII regulation of FSH synthesis and secretion.In order to clarify the changes of m RNA,mi RNA and protein expression in the pituitary gland after GnRH stimulation and to resolve the molecular mechanism of GnRH regulation of FSH synthesis and secretion.In this study,proteomic,phosphorylated proteomic and transcriptomic correlation analyses were performed in the GnRH-treated rat adenohypophysis.By bioinformatics analysis,the differential expression profiles of adenopituitary proteins and phosphorylated proteins after GnRH treatment were established,and the protein functions were clustered.Combined with TMT quantitative proteomics,HE staining,and immunofluorescence assay,the differential expression and cellular localization of CAMK2D and CREB1 proteins were verified.The regulatory effects of CAMK2D on CREB1 and FSH were verified by transfection of the corresponding phosphorylation site mutant plasmids.The targeting and regulation of mi R-23b-3p with lnc RNA-m23b and CAMK2D was verified using a dual luciferase reporter assay.The experiments resolved the molecular mechanism of GnRH regulating FSH synthesis and secretion through CAMK2D,and the results were as follows:1.81 differentially expressed proteins were identified in the GnRH-treated rat pituitary gland,of which 28 were up-regulated and 53 were down-regulated.2.The differential expression profile of phosphorylated proteins in rat pituitary gland after GnRH treatment was successfully constructed,and a total of 621 differentially expressed phosphorylated proteins and 1000 phosphorylation modification sites with significant differences were identified.3.GnRH treatment increased the protein expression and phosphorylation modification level of CAMK2D,increased the phosphorylation modification level of CREB1 protein,and enhanced the co-localization signal between CAMK2D and CREB1 in the adenohypophysis.4.GnRH promotes the phosphorylation of CREB1 S133 site by phosphorylating the T287 site of CAMK2D,which in turn promotes the synthesis and secretion of FSH.5.385 differentially expressed m RNAs were identified after GnRH treatment of rat adenohypophysis,of which 180 m RNAs were significantly up-regulated and 205 m RNAs were significantly down-regulated.Twenty differentially expressed mi RNAs were identified,of which 13mi RNAs were significantly up-regulated and 7 mi RNAs were significantly down-regulated.6.GnRH promotes the upregulation of lnc RNA-m23b expression in the adenohypophysis.Highly expressed lnc RNA-m23b competitively binds mi R-23b-3p and promotes CAMK2D expression,which in turn regulates the synthesis and secretion of FSH.In this study,we investigated the effects of GnRH on m RNA,mi RNA,protein and phosphorylated protein expression in the adenohypophysis,and revealed the molecular mechanisms by which GnRH promotes FSH synthesis and secretion through the lnc RNA-m23b/mi R-23b-3p/CAMK2D/CREB1 pathway.The results of the study complemented the epigenetic level to improve the regulatory network of GnRH on the synthesis and secretion of FSH in the pituitary gland,and also provided a theoretical basis for the application of reproductive hormones in assisted reproduction and the development of related hormone preparations. |
