Effects Of Estrogen And TGF-?1 On Development Of Chicken Primordial And Growing Follicles

In the avian ovaries,there are many follicles with different diameters simultaneously.The ovarian's follicle is the basic functional unit in the females and the development of follicles is complex process with precise regulation.The follicle consists of oocyte and follicular cells including granulosa and theca cells.There is a dual directional regulation mechanism between the follicles and follicular cells,so the system in the follicles is very complex.During the development process of follicles,most of the follicles undergo atresia and failed to reach ovulation.Egg yield is the key index to evaluate poultry varieties and individual quality.So it was significant to study the follicles in different development periods.In this experiment,we studied the dynamic change of follicle amount in the chicken ovary and establishment the culture models of ovaries and follicles in vitro.The ovary and follicles were treated with E2 and/or TGF-?1 then studied the regulation effect and mechanism in the hen ovaries to provide theoretical basis for the study of poultry reproduction performance.1.Morphology of the development follicles in the chicken ovariesThe process of avian follicle development is continuous and quickly development.It was benefited to study the morphology of chicken ovaries in the period of hatching,1-4 wk and 1-4 month.We observed the morphology of ovaries and the results showed that the germ cell cyst began to breakdown after hatching 3-4d and then formed the primordial follicles.Then the primordial follicles transition to growing follicles.The morphology of ovaries in the chickens of 1-4 wk showed that the volume of ovaries enlarged and a lot of follicles were observed in the cortex.In the 4 month ovaries,there are some follicles bulged the surface of ovaries.Meanwhile,the morphology of follicles is changed.The granulosa cells transition from flat to cubic and the layers of granulosa cells increased from one layer to more layers.We also counted the follicle number in the ovaries of 1-4 wk and the follicle distribution followed by the diameter in the ovaries of 1-4 month.The results showed that the follicle number of the chicken in 4 wk is 4-fold of 1 wk.In the ovaries of 1 month,the diameter of the largest follicle is nearly 200 ?m,and the largest follicle is almost 1000?m in the ovaries of 4 month hen.There is an interesting phenomenon that some follicles have more than one oocyte in the ovaries.The multioocyte follicles divided into three classes:1)symmetric follicle,the same volume oocytes in the follicle;2)asymmetric follicle,one oocytes is smaller and the other oocyte is bigger in the follicle;3)fused follicle,is formed during follicle development.We cultured the two individual follicles for 4 days and the follicles fused to form one follicle.In conclusion,the early development of chicken ovaries is mainly the dynamic change of follicle amount,the growth and development of oocyte and follicular cells.2.Effect of estrogen on chicken primordial follicle development and activationThe building of primordial follicle pool is important for follicle selection and ovulation,also significant for maintain the reproduction for female animals.The building process of primordial follicle pool is regulated by endocrine/autocrine/paracrine factors.Steroid hormones and their receptors play pivotal roles in avian reproduction and development.E2 is an essential mediator in ovarian development.In this study,Day 3(3d)chickens were injected with E2(0.025,0.25 or 2.5 mg/kg body weight)for 4 days to assess the effects of estrogen on.avian follicle development.Morphological analysis showed that treatment of E2 at 2.5 mg/kg increased the number of growing follicles by 28.5%and activated follicles by 8.9%(P<0.05),compared with the control group.Treatment of E2 also increased the size of three kinds of follicles and oocytes,respectively.However,this stimulating effect of E2 was inhibited by combined treatment with the estrogen receptor antagonist tamoxifen,resulting in decreased number of the growing follicles by 14.3%(P<0.05)and the activated follicles by 5.8%,respectively.Immunohistochemical staining of PCNA revealed that the ratios of the PCNA-positive granulosa and interstitial cells were increased by 14.0%and 21.5%,respectively.Western blot analysis of PCNA confirmed this stimulating effect of E2(P<0.001).Expression of ERa mRNA was elevated by administration of E2 in vivo and in vitro.Furthermore,E-and N-cadherin displayed increased expression after E2 treatment in vivo and in vitro.In conclusion,E2 can promote chicken primordial follicle development and activation involving its increased receptor and cadherin production at the early stage of ovarian development.3.Regulation of TGF-?1 in the maintenance and activation of primordial follicle in the chicken ovaryThe TGF-?1 superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions.TGF-?1 and its receptors are highly expressed in ovary cells in many species.However,whether TGF-?1 can regulate the development of germ cell cyst and primordial follicle in avian remain unknown.In this study,5d chickens were injected with TGF-?1(2.5,12.5 and 62.5 ?g/kg or PBS)for 3d to assess the effects of TGF-?1 on avian follicle development.Morphological analysis showed that treatment with TGF-?1 at 12.5 ?g/kg increased the number of germ cell cyst and reduced the number of primordial and growing follicle when compared with the control group.The size of oocyetes and follicles have decreased after treatment with TGF-?1.Immunohistochemical staining of PCNA revealed that the ratios of the PCNA-positive somatic and granulosa cells were decreased by 16.2%and 2.48%,respectively.Western blot analysis of PCNA confirmed this negative effect of TGF-?1.Furthermore,we detected the apoptosis cells by TUNEL assay and the results shown that higher percentage positive cells in TGF-?1 treatment group compared with the control group.What's more,we cultured the 5d chicken ovaries for 3d in vitro.Morphological analysis showed that treatment of TGF-?1 at 10 ng/mL also increased the number of germ cell cyst and reduced the number of primordial and growing follicle,compared with the control group.However,the negative effect of TGF-?1 on early ovary development was rescued by treatment of SD208,resulting in increaed the gene expression of steroidogenic enzymes and cell cycle-regulating proteins compared with the TGF-?1 group.In conclusion,TGF-?1 could maintain the primordial follicle development and activation involving reduced cell proliferation and relative gene expression at the early stage of ovarian development.4.Isolation and culture of chicken growing follicles in 2-and 3-dimensional modelsThe isolation and culture of ovarian follicles is essential for the studies of follicular development and function.In mammalians,oocytes and different size follicles have been successfully isolated and cultured in vitro.In avian,because the size of growing follicles is much larger than mammalians so that it proven to be more challenging.The isolation and culture of ovarian follicles is essential for the studies of follicular development and function.In this study,the growing follicles from domestic hens were isolated using enzymatic and mechanical methods and then investigated for the optimized conditions for culture.Assessments of viability and hormonal responsiveness were also considered.A larger percentage of healthy follicles was achieved by mechanical separation than enzymatic dissociation(83.5%vs.54.7%by collagenase I or 63.4%by trypsin),despite a lower recovery yield for the former(126.0 vs.275.7 by collagenase I or 261.0 by trypsin)from each ovary.All of the mechanically isolated follicles(800 ?m)survived when cultured in the 3-dimensional(3D)system for 7d whereas only 92.6%of the follicles survived in the 2-dimensional(2D)group.Follicles cultured in the 3D system also had a higher cell proliferation rates but lower apoptotic rates as assessed by BrdU incorporation and TUNEL assays.Ultrastructural examination showed that the granulosa cells in the 3D group were organized tightly with adjacent layers in contrast to the loose attachment in the 2D system group.After treatment with FSH in the 3D culture for 3d,the mechanically isolated follicles(800 ?m)displayed elevated mRNA expression of steroidogenic enzymes,cytokines and cell cycle-regulating proteins.The 3D culture model established in this study thus provides a useful tool for in vitro culture using growing follicles in a large diameter to study the mechanisms of growing follicle development in the avian species.5.Regulation of E2 and TGF-?1 on the follicle development in vitroThere are many studies on primordial germ cells and hierarchical follicle,but less for the growing follicles.The process of follicle development is regulated by many kinds of cytokines and steroids.In the study,we detected the levels of steroidal hormone(progesterone,testosterone and estrogen)in the isolated follicles(400,800 and 1600 ?m).The results showed that the level of P4 is increased by the follicle size,the level of T is decreased by the follicle size,and the level of E2 has no significant difference between the three classes of follicles.The cultured follicles in 3D model were treatment with different doses E2(0,10,100,1000 ng/mL)for 5d.The results of BrdU labeled the proliferation cells showed that the dose of E2(100 ng/mL)could increase the proliferation cells and inhibited the apoptosis cells significantly.After treatment with E2,the gene expression of steroids receptors(ERa,FSHR and LHR)and steroidogenic enzyme(Cyp19al)has increased in different degrees.ICI182780,is the specific inhibitor of ERca.After treatment with the inhibitor,the facilitated effect of E2 decreased.And the number of proliferation cells decreased the gene expression of ERa and FSHR also decreased.Treatment the follicle with E2 and TGF-?1(100 ng/mL),the number of proliferation cells were increased by synergistic effect.SD208,is the specific inhibitor of TGF?R1.Treatment the follicles with TGF-?1 and SD208,the gene expression of Nr5a1and Cypl9al have decreased in the SD208 treatment group,the gene expression of the cell cycle-regulate proteins also decreased after treatment with SD208.In conclusion,E2 and TGF-?1 could regulate the development of follicles by increased cell proliferation and relative gene expression.Combined treatment of E2 and TGF-?1 would regulate the development of the avian follicles by a synergistic effect.In conclusion,we investigated the morphological change of chicken ovaries and showed that the development of avian ovaries is mainly based on the dynamic changes of the follicles pool.We investigated the regulation effect of E2 and TGF-?1 on formation and activation of primordial follicles by building a chicken ovary culture model in vitro.We revealed that combined mechanical isolation method with 3-dimensional culture method is the proper ways to study the development of large growing follicles in vitro after comparing the several methods of isolation follicles and culture follicles.By combining E2 and TGF-?1 treatment with follicles could promote the growth and development of follicles by synergistic effect.These results could be contributed greatly to studies of the development of poultry follicles and improvement of poultry reproductive performance.