| In vertebrate,progestin is a steroid hormone essential for the regulation of reproductive processes.In most teleost,the major progestin derived from follicular layers of a follicle is 17a,20β-dihydroxy-4-pregnen-3-one(DHP).Studies have revealed that DHP is the naturally occuring maturation-inducing steroid,which triggers meiosis resumption of oocyte via a non-genomic action.It is reported that DHP also mediates the LH-initaited ovulation via a genomic action;however,the molecular mechanism underlying this genomic action is poorly understood.Progestin exerts its effect by binding to its receptors.The classical nuclear progestin receptor(nPR/Pgr)is able to bind DHP,then serves as a transcription factor or coactivator to regulate the expressions of downstream target genes.Studies in mammals have demonstrated that PGR is indispensable for ovulation;but downstream signaling molecules and pathways involved in ovulation have not been clearly resolved,and therefore require further explorations.To date,due to the lack of available genetic models,the role of Pgr in ovulation in non-mammalian species remains unclear.In this study,zebrafish(Danio rerio)was used as a model to study the molecular functions of DHP and Pgr in the regulation of ovulation using various techniques including TALENs,histology,RNA-seq,in vitro oocyte culture,Real-time quantitative PCR(qPCR)and Western blot analyses.Some of the key findings are briefly summarized below.1.Three lines of Pgr knockout fish(pgr-/-)that harbor three different frameshifted mutations were established using two different pairs of TALENs targeted two different sites of first exon of pgr.DNA sequence and Western blot analyses confirmed the loss of Pgr in female pgr-/-fish which exhibits infertility.Further,histological analysis showed that pgr-/-follicles(follicular cell enclosed oocytes)matured normally but failed to ovulate.For the first time,we demonstrated that Pgr is essential for ovulation in fish,but not required for final oocyte maturation in a non-mammalian model.2.High-throughput RNA-seq analysis revealed 3567 genes significantly differentially expressed(DEGs)between wt and pgr-/-fish(fold change ≥ 2,P≤0.05).Among those,1543 gene transcripts were significantly more expressed,while 2024 genes were significantly less expressed,in wt than those in pgr-/-.Principle component analysis showed that pgr-/-samples were clustered together and distant away from wt samples on PC1 axis.Intriguingly,enrichment analysis indicated that the genes with higher expression in wt were involved in multiple ovulatory pathways and processes such as inflammatory response,angiogenesis,cytokine production,chemotaxis,cell migration and cytoskeleton reorganization.In contrast,the genes with lower expression were mainly involved in cell growth and proliferation processes,such as DNA replication,DNA repair,DNA methylation,RNA processing,telomere maintenance,catabolic processes,and nuclear and cell division.These results suggest the Pgr-controlled cell fate transition in preovulatory follicular cells is essential for ovulation.3.Based on transcriptomic analyses,six metalloproteinase genes that are potentially regulated by Pgr were screened out,including A Disintegrin And Metalloproteinase domain 8b(adam8b),A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif 1(adamts1),adamts8a,adamts9,matrix metalloproteinase 2(mmp2)and mmp9.The expression patterns of these genes during folliculogenesis were analyzed by qPCR.The result showed that expressions of adam8b,adamtsl,adamts8a,mmp2 and mmp9 were found in all stages of follicles,but no significant change in the expressions of these genes was found between immature and mature follicles.In contrast,the expression of adamts9 in follicles was extremely low before oocyte maturation(stage Ⅰ-Ⅳa)but increased drastically prior to ovulation(stage Ⅳb)in wt or pgr-/-.Increase of adamts9 in wt was about ten times of that in pg-/-.For stage IV fully grown follicles,follicular layers and denuded oocytes were collected at different time points within a day,and gene expressions were determined for six representative metalloproteinase genes.In oocyte,adam8b,adamts1,mmp2 and mmp9 were expressed stably,but expression of adamts8a and adamts9 were extremely low and sometimes even undetected.In follicular cells,expressions of adam8b,adamtsl,adamts9 and mmp9 increased dramatically following oocyte maturation(stage IVb);however,expressions of adam8b,adamts9 and mmp9 were found to be significantly reduced in pgr-/-.The transcript and protein of pgr exhibited a transient increase at 1-2 hours prior to the completion of oocyte maturation(stage IVa).These results suggest that during oocyte maturation of stage IV follicles,the transient increase of Pgr precedes those of adam8b,adamtsl,adamts9 and mmp9,indicative of these genes belonging to Pgr-regulated target genes and their possible involvements in follicular rupture.The expression of adamts9 is specific to follicular cells,and increases sharply following maturation,indicating a special role of this gene in ovulation.4.Considering the special expression pattern of adamts9 during folliculogenesis and its specificity to follicular cells,in vitro follicle cultrue was conducted to investigate the relationship between the expression of adamts9 and DHP or Pgr.The results showed that,DHP induced oocyte maturation(significant elevation in GVBD)and the expression of adamts9 in stage IV follicles in wt in a dose and time dependent manner.DHP(100 nM)induced oocyte maturation,but did not affect the expression of adamts9 in pgr-/-follicles.Western blot analysis showed that the protein level of Pgr was significantly reduced by DHP following oocyte maturation in vitro,similar to those in naturally mature follicles in vivo;both high dose of RU486(10 μM)or testosterone(T,500 nM)induced oocyte maturation,but did not affect the expression of adamts9 as well as the protein level of Pgr in wt follicles.These results demonstrated that DHP induces the expression of adamts9 via Pgr during oocyte maturation.However,the expression of adamts9 induced by DHP was much lower than that in naturally mature follicles,which indicates other endocrine factors may be involved.In fish,human chorionic gonadotropin(hCG)is able to induce oocyte maturation and ovulation via its cognate Lh receptor(Lhcgr).In vivo experiment showed that the expression of lhcgr significantly increased following oocyte maturation,similar to that of adamts9.In vitro,the effect of hCG on oocyte maturation was far slower than that of DHP,and hCG alone had no effect on the expression of adamts9.Interestinly,when follicles were co-treated with hCG(10 or 50 IU/mL)and DHP(100 nM),expression of adamts9 was not only induced in pgr-/-,but also further elevated in wt in a hCG dose-dependent manner.Co-treatment of RU486(10 uM)with hCG(50 IU/mL)resulted in significant increases of adamts9 and oocyte maturation in wt and pgr-/-follicles,similar to the result caused by co-treatment of DHP with hCG;Co-treatment of T(500 nM)with hCG(50 IU/mL)reproduced these observations,suggesting that oocyte maturation is likely a prerequisite for the expression of adamts9 to become directly induced by hCG.These results suggest DHP initiates the expression of adamts9 before oocyte maturation(stage IVa),while following maturation(stage IVb),Lh signaling further potentiates the expression of adamts9.Taken together,the research presented in this study demonstrated for the first time,Pgr is an essential regulator for ovulation in zebrafish by generation and characterization of Pgr knockout lines.Using this Pgr mutant and RNA-seq,Pgr was shown to be essential for ovulation by controlling cell fate transition in preovulatory follicular cells.Based on transcriptomic analyses,the preovulatory expression patterns of candidate metalloproteinase genes were further explored,among which adamts9 is specifically expressed in follicular cells and rapidly increases following oocyte maturation.In vitro experiments suggest that,during oocyte maturation,DHP-Pgr and LH-Lhcgr signalings jointly upregulate the expression ofadamts9.This cooperative action of DHP and LH may be important for the maximal expression of adamts9 required for ovulation. |
PDF Full Text Request