| The characteristic of follicle development of laying hens is the well-organized follicle hierarchy,it is important to ensuring the follicle hierarchy.In the ovary of hens,one follicle is selected from small yellow follicles pools with a diameter of about 6-8 mm to the preovulatory follicle group,which is called follicle selection.Follicle selection is a key step in determining follicle development and ovulation.The process of follicle selection is closely related to the functional differentiation of the granulosa cell layer.The transition from undifferentiated state to differentiated state of granulosa cells is directly related to follicle selection.The development of ovarian follicle cells is controlled by a variety of hormones and cytokines,including follicle-stimulating hormone(FSH)and insulin-like growth factor 1(IGF1).The response of granulosa cells to FSH is considered to be a key marker of follicular selection,and IGF1 plays a key role in the proliferation and differentiation of granulosa cells.More and more studies show that FSH and IGF1 may play important roles in regulating the proliferation,apoptosis and progesterone synthesis of granulosa cells,but the basic mechanism is still unclear.In order to study the mechanism of FSH and IGF1 influencing the function of granulosa cells,this experiment optimized the granulosa cell culture system of chicken small yellow granulosa cells,screened out the optimal concentration of FSH and IGF1 for treating granulosa cells,studied the effect of FSH and IGF1 on the proliferation、apoptosis and the progesterone synthesis of granulosa cells during chicken follicle selection.It provides a theoretical basis for the regulatory role of FSH and IGF1 in chicken follicle selection.1.The granulosa cells of chicken small yellow follicles have poor adherence capacity and are prone to death in vitro.The culture conditions were optimized by pre-plating extracellular matrix,serum source,and serum concentration.Finally,pre-plated rat tail collagen was used as the extracellular matrix,and 5% chicken serum was added to the culture medium as an optimal culture system.In that culuture system,the granulosa cells of the chicken could guarantee its proliferation characteristics after passaged.2.In order to determine the optimal concentration of FSH and IGF1 for treating granulosa cells,we added different concentrations of FSH(0,50,100 ng / m L)and IGF1(0,100,200 ng / m L)to treat granulosa cells for 24 h.The proliferation of granulosa cells were detected by Ed U.The results showed that 200 ng / m L IGF1 could significantly increase the proliferation of granulosa cells,but FSH had no significant effect.Finally,50 ng / m L FSH and 200 ng / m L IGF1 were selected as the optimal concentrations for treating granulosa cells.3.In order to study the function of FSH and IGF1 in the granulosa cells of chicken small yellow follicle,s 50 ng / m L FSH and 200 ng / m L IGF1 were added to the culuture medium.The expression of m RNA related to proliferation、apoptosis and progesterone synthesis was detected by q PCR.The results showed that IGF1 promoted granulosa cells proliferation,FSH and IGF1 inhibited granulosa cells apoptosis and inhibited the expression of progesterone-related genes.In summary,IGF1 can promote granulosa cells proliferation.FSH and IGF1 can inhibit granulosa cells apoptosis and the expression of genes related to progesterone synthesis.The results of this study reveal the effect of FSH and IGF1 on the function of granulosa cells in small yellow follicles,clarify their role in follicle selection,and provide a theoretical basis for the regulation of follicle development and follicle selection. |
