| The development and maturation of the follicles and hierarchically ovulation are controlled by a series of hormones and growth factors. A lot of studies show that epidermal growth factor (EGF) is involved in the growth regulation of the primordial follicles and follicular development and maturation of the chicken, human and rat. Goose is a seasonal reproduction poultry which has a low egg production, at present, there is no report whether the growth and development of follicles was regulated by the hormones and growth factors or not in goose. In this study, on the basis of the histomorphology of goose follicle development, it is depth to explore the mechanism of EGF participation in FSH mediated goose granulosa cell proliferation.With the purpose of investigate the morphological changes of the follicles, the morphological changes were studied by the tissue morphology in the development of the follicles. Compared with the early stage of production, the ovary weight of laying geese increased rapidly, containing many follicles stayed at different developmental stages. There are 5～6 ovulation follicles in the ovary of the female goose, divided by hierarchical follicular diameter (according to the volume descending named F1, F2... F5 or F6) and the prehierarchical follicles (SWF, LWF, SYF, LYF). In the broody period, many follicles were atretic, the follicles stopped the growth basically, the atrophy and degradation were observed in the ovaries. Transmission Electron microscopy observation showed that the follicle border of the broody goose was not clear, and lose the integrity of the structure. In laying period, the thickness of granulosa layer was 22.18 μm in the LYF follicles, when the follicles were developed to the hierarchical stage, the thickness became thinner and was only 12.53 μm in Fl follicles. In the hierarchical stage, the theca layer became thicker slowly, the thickness was 40.61 μm in the SWF follicles. In the hierarchical stage, the theca layer became thicker fast, the thickness was 308.30 μm in F4 follicles,7.7 times as the thickness of the SWF follicles. The FSH, LH, P4, E2 and EGF levels were measured by enzyme-linked immunosorbent assay (ELISA), there were two peeks of the concentration of FSH in the follicles development (59.24 mlU/mg in SYF and 46.06 mlU/mg in F2), the concentration of LH had the same trend, but the peeks were observed at LYF and F4. While E2 and P4 showed a down trend in the follicle development process and the concentration in LWF and SWF follicles was higher significantly than that in the other follicles (P<0.05). The concentration of EGF was higher significantly in SYF and LYF than the other follicles, up to 68.65 pg/mg and 46.73 pg/mg (P<0.05), the expression levels of EGF and its receptor EGFR gene were highest in SYF follicles, the expression levels were 10.17 and 5.87 times of that in F1, and showed a trend of firstly increase and then decrease. In conclusion, the morphological structure, the concentration of the hormones and EGF had changed significantly in the development of the follicles in goose, the SYF were the key stage of the changes.To further analysis the regulation function of epidermal growth factor and FSH in the follicle development and granulosa cell proliferation, the prehierarchical follicles were cultured suspensively in vitro, the effect was compared with different concentrations of EGF (0 ng/mL,1 ng/mL,10 ng/mL and 100 ng/mL) and FSH (0 mIU/mL,25 mIU/mL,50 mIU/mL and 100 mlU/mL) treatment, the results showed that the follicles were treated with EGF and FSH alone or combined for 24 h, the thickness of granulosa layer and theca layer were significantly increased, and with the treatment of EGF combined FSH for 24 hours, the thickness of granulosa layer and theca layer were increased for 4.16 μm and 8.36 μm (P<0.05). The SYF follicles were selected to isolate and culture the granulosa cells, it was found that EGF and FSH could promote the proliferation of granulosa cell. The effect of granulosa cell proliferation is significant after the treatment with EGF (10 ng/mL) and FSH (50 mIU/mL) (P<0.05). Besides, the expression of anti-apoptotic Bcl-2 gene was increased for 12 times (P<0.05), and the expression of pro-apoptotic Box gene was decreased for 3 times (P<0.05). The EGF could promote the granulosa cell proliferation, and this effect could be enhanced in the case of the presence of FSH.In order to investigate the effect of EGF on the key gene Smad4 gene which was involved in the apoptosis of granulosa cells, goose Smad4 gene was cloned and obtain 1656 bp fragment (genbank:KU363745), a spliced variant was found which was 1536 bp named Smad4-b (genbank:KU363746), and found the fifth exon was missing. The RT-qPCR results showed that two alternatively spliced variants were expressed in the hypothalamus, pituitary, ovaries, oviduct and other reproductive tissue, but mainly on Smad4-a (wild-type), and the abundance of tissues was:oviduct> ovary> pituitary> hypothalamus. After EGF treatment, the expression level of Smad4 gene was significantly down-regulated in granulosa cells. The granulosa cells were treated with EGF, the expression level of Smad4-a was down-regulated.To investigate the effect of miRNA in the ovaries of laying and broody goose, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using solexa deep sequencing and bioinformatics to determine differential expression of the miRNAs. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary,127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs was validated using real-time quantitative PCR. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the differentially expressed miRNAs were involved in ovarian function, including hormone secretion, reproduction processes and so on. After adding EGF and FSH into treated granulosa cells to detect expression of related miRNA, the expression levels of miR-26b, miR-27a, miR-27b were up-regulated significant (P<0.05), the expression levels of miR-22, miR-27c. miR-34 were down-regulated. The results of bioinformatic prediction showed that Smad4 was one tai’get gene of miR-26b. and it was opposite with the expression level of miR-26b, so it was speculated that EGF was involved in the proliferation of goose granulosa cells may depend on the miR-26b targeted to Smad4 gene. These results indicate that the key miRNAs played a certain role in the EGF-mediated process of the granulosa cell proliferation.In summary, the morphological changes of the follicles were investigated in this study, the concentration of hormones and growth factor were detected, the prehierarchical follicles were cultured suspensively in vitro, the granulosa cells were isolated and cultured, and investigate the effect of EGF on the key gene Smad4 gene which was involved in the apoptosis of granulosa cells. These results are useful to the comprehensively understand molecular mechanism of EGF in goose reproductive regulation, provide technical support for improvement of the fertility of goose and strengthen goose industry in future. |
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