Studies On The Expression And Regulation Of Nuclear Progesterone Receptor ?pgr? And Prostaglandin F_2? Receptor ?Ptgfr? In The Brain Of The Mudskipper Boleophthalmus Pectinirostris

In most fishes,17a,20?-dihydroxy-4-pregnen-3-one(DHP)and PGF2a play important roles in oocyte maturation and ovulation,and their mechanism of action has been studied in depth.Recent studies have shown that these two hormones also participate in the regulation of reproductive behavior of female fish,but the mechanism has not been elucidated.Our previous studies showed that the mudskipper Boleophthalmus pectinirostris presented semilunar oviposition rhythm during the reproductive season,and the blood level of 17a-OHP(precursor of DHP)also presented semilunar rhythm in which the peak coincided with the oviposition period.Based on recent research progress,we hypothesized that during oocyte maturation and ovulation of sexually mature female mudskipper,DHP and PGF2a synthesized by the ovaries may feed back to the brain tissue and induce the reproductive behavior of females.Based on this assumption,we use Pgr and Ptgfr,receptors for DHP and PGF2a as research object in this paper.Using molecular biotechnologies,we first cloned the CDS sequences of Pgr and Ptgfr.We also studied the tissue distribution patterns of pgr and ptgfr in females,and the expression trend of pgr and ptgfr in the semimonthly cycle of reproductive season.as well as the regulation of pgr and ptgfr in brain tissue of B.pectinirostris by DHP.The main results are presented as follows:1.Cloning,functional trait analysis and tissue expression of pgr in mudskipper.The complete CDS of pgr is 1968bp,encoding a protein with 655 amino acids.Phylogenetic tree analysis showed that the Pgr of the mudskipper and the Pgr of other fish formed a branch,independent of PGRs of other species such as mammals,birds,reptiles and amphibians.Real-time quantitative PCR(qPCR)analysis showed that pgr was expressed in many tissues of female mudskippers.Among peripheral tissues,the expression of pgr in ovary was the highest.In brain tissues,pgr expression was highest in pituitary,followed by that in diencephalon.pcDNA3,1+ expression vector was used to construct the expression plasmid of pgr gene in mudskipper,which was transfected with expression plasmid of plasmid promoter pGL3-MMTV-Lu into HEK293T cell line for expression.The results showed that under the action of hormones DHP,P4,and 17a-OHP,Pgr could transactivate MMTV promoter,and that their activation effect was dependent on the concentrations of these three hormones,among which the semi-maximum effect concentration(EC50)of DHP was the lowest.2.The expression of ptgfr in different tissues of mudskipper.According to the published gene sequence of ptgfr in B.pectinirostris,ptgfr expression in many tissues of the female B.pectinirostris was detected by fluorescence real-time quantitative PCR.The expression of ptgfr in skin was the highest in peripheral tissues,ptgfr expression was highest in the medulla oblongata,followed by the diencephalon,in brain tissue.During a complete lunar cycle in the reproductive season(May 19,2015 to June 18,2015),brain tissues of six female mudskippers were collected at 3 days' intervals(body length 11.1-14.1 cm,body weight 17.1-21.7 g).qPCR results showed that the expression level of ptgfr presented a rhythm close to the half-moon cycle.3.Regulation of pgr and ptgfr in the diencephalon and fshb,lhb,pgr in pituitary by DHP of mudskippers.(1)in June and September,2017,January,April and June,2018,brain tissues of adult female mudskippers(body length 9.2-13.0 cm,body weight 18.0-24.1 g)were collected after intraperitoneal injection of DHP(50?g/fish).qPCR results showed that DHP significantly unregulated ptgfr expression in the diencephalon in June and September,but had no significant effect on pgr expression.DHP had no effect on pgr and ptgfr expression in January and April.(2)during the reproductive season(April to June,2018),DHP(50 ?g/fish)was intraperitoneally injected into the sexually mature female mudskippers(body length 9.5-13.0 cm,body weight 18.1-25.9 g).The results showed that the expressions of fshb,lhb and pgr in pituitary did not significantly effect compared with the controlgroup.(3)during the reproductive season(July 13,2018),the diencephalon of female mudskippers(body length 11.0-13.1 cm,body weigh 22.1-27.8 g)were cultured with 100 nM DHP for 3 hours in vitro.qPCR analysis showed that the expressions of pgr and ptgfr in the female mudskippers were significantly increased than the control group.To sum up,this study has confirmed that DHP is the most suitable ligand for Pgr in mudskippers.pgr and ptgfr were highly expressed in the diencephalon part of the brain tissue of the mudskippers.During the reproductive season,ptgfr was expressed in the diencephalon of sexually mature females mudskippers in a semilunar cycle.Further studies have observed that DHP injection in vivo can significantly promote the expression of ptgfr in the diencephalon only during the reproductive season.In vitro,DHP significantly promoted the expression of pgr and ptgfr in the diencephalon.These results suggest that DHP may up-regulate the expression of ptgfr gene through pgr.