Preliminary Study Of Effect Of Notch Signaling On The Ovarian Follicular Development In The Mammals

The Notch signaling pathway is an evolutionarily conserved mechanism that is involved in the regulation of many biological functions,such as apoptosis,cell proliferation,differentiation and lineage decisions during embryonic development,and homeostasis of adult self-renewing organs.The Notch gene was first described in the Drosophila melanogaster,in which haploinsufficiency resulted in"notches"at the wing margin.The Notch family possess Notch ligands,receptors,downstream molecules and regulators.The ligands and receptors are all transmembrane proteins,and Notch signaling is activated upon cell-to-cell contact as a result of interactions between Notch receptors and their ligands.mammals possess five Notch ligands,including 3 Delta-like ligands(Dll1,3,4) and two ligands related to Drosophila Serrate(Jagged1 and 2).Four Notch receptors are Notch1,2,3,4 respectively.At present.Almost all of the studies about the effect of Notch signaling pathway on the ovarian follicular development are focusing on the Drosophila.In the mammalian ovary,it is found that the Notch receptors 2,3 were expressed in the granulosa cells,and Jagged1 is expressed in the oocytes.The ovarian blood vessels also express Notch receptors1,4 and ligand Jagged1.However,we know nothing about these molecular functions on the mammalian ovarian follicular development.Transcriptional factor RBP-J(Recombination signal Binding Protein-J,RBP-J) is the main effector of Notch signaling in the nucleus.In the conditional RBP-J knockout mouse,we occasionally find the follicular number decreasing, percentage of medulla increasing,and no mature follicles and corpus luteum forming.The RBP-J knockout in other organs and systems may have impact on the ovarian follicular development,because the RBP-J encoding gene is extensively deleted in the mouse.The inductive period is relatively longer than the cycle of follicular development,and this is not benefit to our study.In order to avoid these advantages,we have used the ovary organ culture system,and added the inhibitor of Notch signaling pathway to study the effect of Notch signaling on the preantral follicular development.The results are as following:(1)We have successfully constructed the postnatal day 4 rat ovary culture system.PND4 rat ovary were dissected from freshly killed rat pups.Whole ovary were placed on floating filters in 0.5ml Dulbecco Modified Eagle medium(DMEM)-Ham F-12 medium(vol/vol 1:1).In the eight days culture periods,change the medium every two days.After the culture,the ovary was fixed ,embedded in paraffin,sectioned, stained with Hematoxylin and eosin.We can watch the clear histological structure, integrated follicles.The follicles developed obviously comparing to the postnatal day 4 follicles.These all mean that the ovary was cultured successfully.(2)In order to estimate the whole ovary follicle numbers,The fixed,paraffin- embedded ovary was serially sectioned.The starting section is seclected randomly from the first five sections,and then every fifth section was choosed as samples.The whole follicle number was calculated by the application of correction factor 5.After calculating the different stage follicle number in the whole ovary Using this mothod,we can get the conclusion that the preantral follicular development is inhibiting after interrupting the Notch pathway.(3)In the sections,we also find that the experimental group has more apoptotic cells than the control groups.These apoptotic cells comprised not only granulos cells but also oocytes according their morphology and structure.The Tunel assay reach the same point.(4)We have found some oocyte cluster-like structure,and there are more than ten oocytes in this structure in the experimental group.There are rare oocyte cluster-like structure with as much as three oocytes.(5)The estradiol concentration in the culture medium was determined using Radio- Immuno Assay,and there is no significant difference between the groups.(6)We have detected the expression of Notch associated molecules in the rat ovary using RT-PCR for the purpose of analyzing implication of these molecules in the follicular development in the mammals.The ligands and receptors can be expressed in the ovary except Delta-like 3.Althouth the articles about Notch receptor and ligans Jagged1and Jagged2 have beed published,there is no paper which say the Delta-like 1 and Delta-like 4 expression in the mammalian ovary.In the Notch down stream molecules HES1,3,5,Only the Hes1 can be found in the.But the Hes1 can't be checked using Western blot.We have amplified ovary granulosa cell specific promoter,and cloned it upstream of the Cre encoding sequence after determineding it's activity and specificity.In summary,we can conclude that the Notch signaling pathway is implicated in the follicular development in the ovary of the mammals. But the molecular mechanism remain to be elucidated.