Functions Of Calcium Signalling In Mediating Follicular Development In Laying Ducks And Its Mechanism

In the past years,it is widely accepted that calcium plays an important role in maintaining eggshell and bone formation in laying birds.However,more evidence shows that there is a close relationship between the level of calcium in the diet and the number of laid eggs.It is found that dietary calcium deficiency has a significant negative impact on the egg production rate,which is related to the ovulation number of mature follicle.We speculate that,dietary calcium probably also plays important role in the regulation of follilce development and maturation.Up to date,however,there are few reports on this issue in laying ducks.The biological regulation function of calcium in mediating follicle maturation still needs to be further confirmed,and the related mechanism needs to be studied further.Therefore,this study firstly was to find out the differential key signal proteins in granulosa cells cells among different developmental stage of follicles through DIA proteomics,and then screened the differential genes related to follicle development between the peak-and late-laying ducks.And finally,the effects of calcium channel agonist on the reproductive functions were studied and its molecular mechanism were also studied in vitro.The biological function of calcium in poultry provides a new theoretical basis.The results of this study were shown as follows:1.Differential signal proteins screening in granulosa cells from developmental follicle in laying ducksIn order to identify the differential key protein signals during follicular development of laying ducks,granulosa cells were isolated from small white follicles（diameter 1-3 mm）,large white follicles（diameter 3-6 mm）,small yellow follicles（diameter 6-8 mm）and smallest hierarchical follicles（F4）of 180 d-old laying ducks,and were subjected to DIA analysis.The most significant 10 pathways during the follicular development included metabolism,secondary metabolite synthesis pathways,endocytosis,adhesion,cytoskeleton regulation,MAPK signaling pathways,RNA transport,tight junctions and calcium signaling pathway.GO enrichment analysis indicated that the biological processes of differentially expressed proteins mainly included biological adhesion,reproduction,and immune processes;cell components included cell connections,outer cell matrix,and cell membrane;molecular functions included antioxidant activity,transport activity,catalytic activity and signal molecule transduction activity.Trend analysis of differential proteins showed that 310 proteins showed a stable expression and then F4 significantly decreased,284 protein expression decreased linearly with follicle development,223 proteins increased linearly with follicle development,and 202 proteins were significantly increased only in F4 follicular granulosa cells.With regard to calcium signaling,it was demonstrated that expression abundance of 6proteins were increased with development of follicle,including calcium permeable stress-gated cation channel 1,calcium/calmodulin-dependent protein kinase type II subunit gamma isoform X1;the expression abundance of 12 proteins were decreased with follicle development,including 45 k Da calcium-binding protein and calcium-binding mitochondrial carrier protein.The results indicate that the signaling pathway related to calcium,MAPK,AMH change significantly during the follicle development in ducks,which may play an important role in the development of follicles.2.Differential screening of calcium signal-related genes in granulosa cells from growing follicles between peak-and late-laying ducksIn order to screen the signal related to calcium during delayed follicle development by employing late-laying ducks,plasma and granulosa cells were collected from 180-day（peak）and 700-day（late）-old laying ducks,RNA-seqing were then conducted after RNA extraction from granulosa cells.Compared with peak-laying ducks,the number of dominant follicles（diameter>8 mm）and 1-3 mm follicles were significantly decreased（P<0.05）.Both the plasma concentration of calcium and ovarian calcium content in late-laying ducks were significantly lower than peak-laying ducks（P<0.05）.The ovarian calcium content in late-laying ducks was significantly lower than peak-laying ducks（P<0.05）.GO enrichment pathway results showed that the differential genes related to biological processes included cell development,cell signaling,adhesion,reproduction and other biological processes;cell components were mainly classified into cell membrane,outer cell matrix,and cell connection,etc.;molecular functions mainly include nucleic acid binding and catalytic activity.KEGG enrichment pathway analysis showed that the signal pathways are important pathways for performance differences,including follicular extracellular matrix,basement membrane,collagen,and cell connections.A total of 8 protein-encoding DEGs involved in calcium transport in granulosa cells from 1-3 mm follicle of young and old duck have been significantly changed,of which 6 DEGs were significantly up-regulated,including Ca²⁺-ATPase（ATP2B1,ATP2C2）,calcium-binding protein calbindin（CALB1）,two DEGs（calcium channel protein CACNG3,cycle dependent kinase A2CDK5A2）,etc.The ovarian m RNA expression of Gpx1and Mn-SOD were significantly down-regulated in late-laying ducks,while the m RNA expression of Caspase 3,ATF6 and FAS were increased in late-laying ducks.The results of this study demonstrate that in late-laying ducks,antioxidant enzymes were down-regulated in the ovary,imbalance in calcium homeostasis are caused and atretic signal were enhanced,suggesting that changes in calcium signal and decreased antioxidant capacity may be important factors in inducing follicular atresia in laying birds.3.The effects of capsaicin on the ovarian calcim signaling and follicular development in laying ducksIn order to evaluate the calcium function in mediating follicle development,this study was conducted to examine the effects of dietary supplementation with calcium channel agonist capsaicin（CAP）on follicular development and ovarian antioxidant capacity in laying ducks.Three hundred and seventy eight 58-week-old laying ducks were randomly divided into 3treatments,with 6 replicate of 21 laying ducks.Ducks fed a corn-soybean basal diet served as controls.The other two groups of ducks were fed the same diet,with supplementation of 150mg CAP/kg,but pair-fed or ad libitum fed.The experiment lasted 8 weeks.The results showed that,when ducks were ad libitum fed,dietary supplementation of CAP increased the feed intake of laying ducks（P<0.001）,egg production and egg weight,but reduced feed conversion ratio（P<0.001）.The relative weight of large yellow follicles from the two CAP-fed ducks was significantly higher than that from control（P=0.01）.The relative weight of small yellow follicles of CAP ad libitum fed ducks was significantly higher than that of the other two groups（P<0.01）.Compared with control,the number of dominant follicles in laying ducks tended to be increased by supplementing CAP under ad libitum fed condition（P=0.06）.The ovarian m RNA expressions of TRPV4,ATP2A2,ITPR1,Ca M and CDK1 in the CAP ad libitum fed ducks were significantly higher than those of the other two groups（P<0.05）.Compared with control,CAP supplementation significantly increased plasma Gpx activity（P<0.01）,and reduced the ovarian MDA content in laying duck（P<0.01）.The results indicate that dietary supplementation of CAP could increase feed intake and improve egg production performance probably by activating calcium signaling pathways and improving redox status.4.Mechanism of calcium in affecting granulosa cell functionIn order to explore the potential mechanism by which calcium affects follicle development,in vitro trial was conducted on granulosa cells.It was found here that the cellular viability of granulosa cells were decreased linearly（P<0.01）with increasing concentrations of EGTA or verapamil.The cellular viability of granulosa cells from 3-6 mm follicles were gradually increased and then decreased with increased medium A23187 concentration.The results of present study indicate that calcium promotes proliferation in granulosa cells.Further study showed that c AMP production in granulosa cells is linearly increased with increasing A23187 but linearly decreased with increasing EGTA or verapamil,indicating that the cellular production of c AMP is activated by calcium.Both verapamil and EGTA increased the cellular m RNA expression of PDE1A（P<0.01）,indicating that calcium participates in the production of c AMP in granulosa cells probably through modulating PDE1A.Furthermore,EGTA significantly reduced the tight junction in granule cells（P<0.05）.m RNA expression of GJC1or GJA1 were decreased by EGTA or verapamil（P<0.01）,whereas,addition of 0.5 and 2.0μM dc AMP based on EGTA significantly increased the m RNA expression of GJA1 or GJC1（P<0.05 or P<0.0001）.The results showed that calcium affected signal cellular communication through c AMP,therefore exerting biological function in follicle development.In conclusion,the calcium signal of granulosa cells changes significantly during follicular development in peak-laying ducks.The level of calcium in the ovaries of late-laying ducks decreased and the activation of follicular atresia signals were also enhanced.Dietary supplementation of calcium channel agonist capsaicin could activate calcium signals in the ovary and promoted follicular development,therefore improving egg production performance,and its mechanism may be related to the production of cAMP in granule cells.