Expression Regulation And Function Of Coagulation Factors 3a And 5 During Ovulation In Zebrafish

The ovary is a solid organ rich in blood vessels.The microvascular network around the follicle changes constantly during follicular development,ovulation and luteinization,especially during ovulation,where the ovum ejects the follicle,the blood flow slows down,the blood clots,and the blood vessel breaks.A recent study in our laboratory found that female zebrafish from a progesterone nuclear receptor knockout line(pgr-/-)had a phenotype of non-ovulation of mature eggs,and further transcriptomics showed that the expression levels of coagulation factors f3a and f5 in the follicular layer cells of mature follicles of this line were much lower than those of wild type.Although increased expression of coagulation factors f3 and f5 during ovulation has been reported in animal models such as humans and mice,little is known about the function of these two factors during ovulation.Therefore,this paper takes zebrafish as the research object and uses modern molecular biology techniques to systematically study the expression,regulation,and function of coagulation factors f3a and f5 in the ovulation process of zebrafish at both in vivo and in vitro levels.The main research results are as follows:1.We first compared the expression of f3a and f5 in follicles at different developmental stages.The qPCR results showed that the expression levels of f3a in follicles at stage Ⅰ,stage Ⅱ,stage Ⅲ,stage Ⅳa were significantly higher than those of f5,but the expression levels of both were stable.The expression levels of f3a and f5 were significantly increased when the follicles developed into stage Ⅳb(after completion of oocyte maturation but prior to ovulation).Since zebrafish have a daily spawning cycle and ovulate one hour before the light is turned on at 8 AM,we selected four time points at 12:00,20:00,5:00,and 7:00 to compare the expression levels of f3a and f5 in the stage Ⅳ follicles.The qPCR results showed that the expression levels of f3a and f5 were low at 12:00 and 20:00,and slightly increased at 5:00,and the follicles in the ovary were all in the stage IVa.At 07:00,a large number of the stage Ⅳb follicle appeared in the ovaries,and the expression of f3a and f5 increased sharply.The results of the Whole-mount in situ hybridization also showed that there were no positive signals of f3a and f5 cRNA probes in the follicular layer of stage Ⅳa follicle,but there were obvious positive signals of f3a and f5 cRNA probes on the surface of stage IVb follicles.Finally,RT-PCR was used to locate the expression sites of f3a and f5 in the stage Ⅳb follicles,and it was found that they were only expressed in the follicular layers,but not in the oocyte.2.To further study the molecular mechanism of regulating f3a and f5 expression in follicles,we first studied the effects of hCG and DHP,which regulate the ovulation of zebrafish,on the regulation of f3a and f5.The results showed that hCG(50 IU/mL)only induced the expression of f5 in the stage Ⅳ follicles after 3 h exposure in vitro.After DHP(100 nM)exposure for 3 h,the expression levels of f3a and f5 were significantly increased,and the up-regulation effect of DHP on f5 was significantly stronger than that of hCG.The dose-time effect experiment further showed that DHP had dose-dependent and time-dependent effects on the expression of f3a and f5.The expression of f3a and f5 in the stage IVa follicle reached the maximum under the culture condition of DHP exposure at 100 nM for 2 h.By DHP(100 nM)exposure to follicles at different developmental stages,f3a was found to be significantly upregulated only in stage IVa follicle after 3 h,and f5 has significantly upregulated in both stages Ⅲ and stage IVa follicle,but the late one much stronger.Subsequently,we observed that 3a and f5 expression levels in the stage Ⅳb follicles of pgr-/-were significantly lower than those of wild-type(wt).Moreover,DHP could not up-regulate the expression levels of f3a and f5 in the stage Ⅳb follicles of pgr-/-female.By cloning the core promoter sequences of zebrafish f3a and f5 and combining them with transactivation assays,it was proved that DHP could activate the core promoters of f3a and f5 through Pgr in HEK293T cells.These results fully indicate that DHP can up-regulate the expression of coagulation factors f3a and f5 in the stage Ⅳa follicle by Pgr.3.Based on confirming that Pgr is a key transcription factor regulating f3a and f5,we constructed transgenic lines to trace the spatiotemporal expression of Pgr in follicles.We first cloned the 2.8-kb sequence of the core promoter of pgr in zebrafish,and then successfully constructed a transgenic zebrafish Tg(fliI:eGFP)line using the Tol2 transposon system.RT-PCR results showed that the expression patterns of pgr and egfp mRNA in the transgenic line were consistent in all organs,and both of them were expressed in the brain,pituitary gland,gonad and kidney,but not in the heart and liver.In the pituitary gland,we found that GFP signals were mainly expressed on the proximal pars distalis(PPD)region of the pituitary gland,in which most cells expressing GFP signal were clustered,but also some independent cells were expressing GFP signal.GFP is widely and uniformly expressed in the kidney.In order to more accurately determine the expression position of the GFP signal in the gonad,we crossed the Tg(gsdf:nfsB-mCherry)transgenic zebrafish line constructed in our laboratory with Tg(fliI:eGFP).To obtain the Tg(pgr:eGFP/gsdf:nfsB-mCherry)transgenic zebrafish line,in the testis,GFP signal was expressed in the stromal cells between the spermatogenic tubules,mCherry signal was expressed in the Sertoli cells,and no fluorescence signal was observed in the germ cells.In the ovary,GFP signaling is expressed throughout the follicular layer.However,both GFP and mCherry signals were observed in the granulosa cell layer adjacent to the oocyte.However,in the theca cell layer,only a few cells were observed to express GFP signal,and the signal intensity was significantly stronger than that in the granulosa cell layer.Intriguingly,confocal microscopy of the follicle surface revealed that cells expressing GFP signal in the theca cell layer were distributed next to vascular-like structures.4.To explore the distribution relationship between cells expressing Pgr in theca cells and the vascular network on the follicle surface,we hybridized Tg(fliI:eGFP)line and Tg(fli1:DsRed)transgenic zebrafish line with vascular endothelial cells specifically expressing DsRed fluorescent protein,then obtain the Tg(pgr:eGFP/fliI:DsRed)transgenic zebrafish line.Confocal microscopy was used to observe the follicle surface,and it was found that the cells expressing strong GFP signal in the theca cells were distributed close to the vessels expressing DsRed signal,and most of them were concentrated in the branches of the vessels.By observing the ovulation process of zebrafish in vitro,we found that a small pore appeared in the local area of the follicular layer at the early stage of ovulation.With the enlargement of the pore,oocytes were finally released from the enlarged pore,and then the residual follicular layer gradually shrank back and remained in place.During this process,there are broken blood vessels at the point of ovulation.By looking at the number of blood cells in the follicle surface vessels found that the blood cells are less before ovulation(at 6:45),and a large number of blood cells accumulated in the vessel during ovulation(at 7:00).The blood vessels became deep red after ovulation and remain in a frozen state,which means blood coagulation appeared during ovulation.In order to verify the correlation between the coagulation in the blood vessels on the surface of follicles and ovulation,the zebrafish ovulation model was constructed to explore the effects of EDTA and heparin sodium on ovulation rate.The results showed that both EDTA and heparin sodium could significantly inhibit ovulation rate,and the effect of EDTA was stronger than that of heparin sodium.These results suggest that near ovulation,DHP can up-regulate the expression levels of f3a and f5 through Pgr in thecal cells,and then act on adjacent blood vessels to induce coagulation,thus promoting ovulation.In conclusion,this study systematically elucidates the physiological mechanisms,namely,DHP regulates the expression of coagulation factors f3a and f5 through Pgr during ovulation in zebrafish,causing the blood vessels on the surface of follicles to coagulate,and then regulating ovulation in zebrafish.The regulation of f5 mediated by Pgr is reported for the first time.The results of this research not only enrich the basic knowledge of fish reproductive biology but also contribute to a more comprehensive understanding of the function of coagulation factors in the process of ovulation invertebrates.