The Role Of Smad9 Gene In The Establishment Of Goose Hierarchy Follicles

The low egg production of geese is a bottleneck restricting the development of modern goose industry in China.It is of great commercial value to develop goose breeds or strains with high egg production performance.In addition to the genetic,environmental and nutritional factors,the level of egg production in poultry is more important depending on the growth and development level of follicles in the ovary.The process of avian follicle development and maturation is a priority,and it is a complex physiological and biochemical process regulated by multiple factors inside and outside.Therefore,it is of great significance to carry out research on the regulatory mechanism of goose follicle development.In the previous experiments,our lab for the first time discovered that the high expression of the transcription factor Smad9（also known as Smad8）,downstream of the BMP/Smad signaling pathway during the early stage of hierarchy follicles development of Wanjiang White Goose（50-60 eggs per year,two-line hybrid goose of Wanxi White Goose and Sichuan White Goose）.However,Smad9 gene becomes silence during follicular maturation.It is suggested that Smad9 may be involved in the development of goose ovary and follicles.Therefore,it is assumed that the low-yielding geese,such as Wanxi White Goose（20-25 eggs per year）,may be caused by the high expression of Smad9 in the mature stage of follicles.If the hypothesis is true,we speculate that Smad9may play a key role in regulating follicular development and ovulation of goose.In order to verify the above hypotheses and conjecture,this study used the Wanxi White Goose as the research object.In vitro and in vivo experiments were conducted to investigate the localization and expression pattern of Smad9 in ovarian and hierarchical follicles.The effects of Smad9 expression on egg production,follicular hormone receptor and serum hormone levels,as well as hormones secretion,hormone receptor expression and cell proliferation of granulosa cells were studied by in vivo injection of Smad9inhibitor（LDN-193189）and in vitro Smad9 interference expression.On the basis of these studies,Chromatin Immunoprecipitation（ChIP）assays were used to elucidate the specific roles and molecular mechanisms of Smad9 in the development of follicular hierarchy and the regulation of ovulation in birds.The main research contents and results are as follows:1.In order to test whether the hypothesis and conjecture of the subject are valid.Firstly,the Wanjiang White Goose and the Wanxii White Goose,which are in different stages of growth and development,are selected as experimental subjects to obtain early ovary,prenatal ovary,laying ovary and hierarchical follicle samples.Smad9 mRNA expression in these samples was quantitatively detected and compared by qPCR.The results showed that:（1）Smad9 gene showed some expression differences in the progressive process of goose ovary development;（2）There was no significant difference in Smad9 gene expression between the two goose species at the early stage of ovary development and prenatal ovary,while the Smad9 mRNA expression of low-yielding Wanxi White Goose at the mature stage of ovary development and the hierarchical development of follicles was significantly higher than that of high-yielding Wanjiang White Goose（P<0.05）.The results of this experiment preliminarily indicate that the research hypothesis of the subject has a certain experimental basis,and it also shows that the follow-up discussion based on this hypothesis has strong scientific significance.2.Ovary and various hierarchical follicles were obtained during the egg production period of Wanxi White Goose.Histological observation,H&E staining,immunohistochemistry,qPCR and Western blot were used to study the localization and quantitative expression of Smad9.The results showed that:（1）The surface of goose ovary during laying period containing a large number of pre-hierarchy and pre-ovulatory follicles.The pre-hierarchy follicles included small white follicle（SWF）,large white follicle（LWF）and small yellow follicle（SYF）,pre-ovulatory follicles include F5,F4…F1（diameter from small to large）.The number of pre-hierarchy follicles is greater at each stage,whereas there is only one at each stage of pre-ovulation;（2）H&E staining showed that the thickness and density of granulosa cells in goose hierarchy follicles showed regular changes with follicular development;（3）Immunohistochemistry showed that Smad9protein is only expressed in the granulosa cell layer of the follicles in the goose ovary,no signal was detected in other parts of the ovary;（4）For the first time,quantitative study found that Smad9 showed differential expression in goose hierarchy follicles,and further Western blot results showed that Smad9 expression in hierarchy follicles was consistent at the level of transcription and translation.These results suggest that Smad9 may be involved in the establishment and maintenance of follicular hierarchy.3.The Smad9 gene inhibitor LDN-193189 was injected by wing vein to suppress Smad9 expression during the earlier stage of laying of the Wanxi White Goose.Changes in Smad9 and Smad1/5 expression in hierarchy follicles after injection of LDN-193189 were detected by qPCR and Western blot.The levels of E₂,P₄,FSH,LH and the expression of aromatase（P450 arom）in serum were detected by ELISA,and the total egg production of goose during the whole laying period was recorded.The results showed that:（1）After injection of LDN-193189 in vivo,Smad9 expression was significantly inhibited in follicles other than SYF（P<0.05）,especially in the follicular phase of F3 and F2 of pre-ovulatory follicles（P<0.01）;（2）There was no significant difference between the two groups in the Smad1/5 mRNA and FSHR mRNA expression.However,LHR mRNA expression in the pre-ovulatory follicles of the inhibitor group was significantly different from that in the control group（P<0.05）.It showed a gradual upward trend until the difference in F1follicles was extremely significant（P<0.01）;（3）31.8 eggs in inhibitor group were increased by 7.0 eggs compared with 24.8 eggs in the same period control group（saline group）,and the difference was significant（P<0.05）;（4）The results of ELISA showed that the concentration of E₂ and P450 arom in serum was significantly higher in the inhibitor group than in the control group（P<0.05）,while the concentration of P₄,FSH and LH in serum was not significantly different between the two groups.These results demonstrate that Smad9 inhibitor can regulate the expression of Smad9 in vivo,inhibition of Smad9expression may lead to follicular development-associated estrogen synthesis and ovulation-associated LHR transcriptional changes.Therefore,we believe that Smad9 gene is involved in the regulation of egg production in geese.These data also provided experimental basis for the initial hypothesis of this study that inhibiting the expression of Smad9 may improve the egg production of low-yielding Wanxi White Goose.However,it is unclear about the ways and means by which the Smad9 gene affects the expression of follicular development and ovulation-related factors.4.It is well known that the growth and development of follicles are closely related to the proliferation,differentiation and endocrine function of follicular granulosa cells.In order to elucidate the biological role of Smad9 gene in follicular development and egg production,we used the SYF follicular granulosa cells as the research object for primary culture and carried out in vitro studies.Six pairs of Smad9-siRNA interference sequences were designed based on the Smad9 gene sequence（XM₀13192591.1）of goose in GenBank.After screening,the primary cultured granulosa cells were transfected.Previous studies have shown that BMP-4 can induce the expression of Smad9,so in the same period,BMP-4,BMP-4/LDN-193189 and Smad9-siRNA were used to induce primary cultured granulosa cells,repectively.The results show that compared with the control group:（1）Smad9-siRNA group effectively silenced the expression of Smad9 in granular cells.The expression of FSHR,LHR and CYP19A1 mRNA in granulosa cells was significantly downregulated（P<0.05）,and the concentrations of E₂,P₄,FSH,LH and P450 arom in cell culture supernatants were significantly decreased（P<0.05）,and cell proliferation was also significantly reduced（P<0.05）.（2）BMP-4 significantly stimulated Smad1/5/9 expression in granulosa cells,but had no significant effect on hormone synthesis,hormone receptor expression and cell proliferation.（3）BMP-4/LDN-193189 group significantly blocked the expression of Smad9（P<0.05）,and the expression of LHR and CYP19A1 mRNA in granulosa cells was significantly increased（P<0.05）,and the concentration of E₂ and P450arom in cell supernatants was significantly increased（P<0.05）,and the cell proliferation ability was also significantly enhanced（P<0.05）.These results suggest that partial blockade of Smad9 expression by the inhibitor LDN-193189 is beneficial to the growth and function of granulosa cells.In addition,the results also suggest that Smad9 may have some dose-effect on the regulation of goose follicular development.5.In vivo,inhibition of Smad9 expression by LDN-193189 promoted egg production in geese.To elucidate this molecular mechanism,subcellular localization of Smad9expression in granulosa cells was performed by immunofluorescence in this study.Followed by a ChIP test analysis was carried out.The results showed that:（1）Smad9 is located in the cytoplasm of granulosa cells,while its active form p-Smad9（phosphorylated Smad9 protein）is located in the cytoplasm and nucleus;（2）ChIP analysis showed that p-Smad9 could bind to the promoter region of luteinizing hormone receptor（LHR）to achieve the regulation of LHR transcription.A large number of studies have shown that lutropin（LH）plays an important role in the growth,development,predominance and ovulation of follicles,and LH functions must be mediated by LHR on follicles,especially in the follicle predominance and ovulation process has an important significance;（3）In this study we also found that Smad9 regulates LHR transcription closely related to its expression level.Compared with the blank control group,the LHR transcriptional activity did not change significantly when Smad9 was overexpressed,and decreased significantly after Smad9 was inhibited by interference（P<0.05）,but increased significantly after Smad9 was blocked by the inhibitor（P<0.05）.These results indicate that the dose-effect found in our in vitro experiments is real.In this study,the differential expression information of Smad9 gene in hierarchical follicle of goose was obtained for the first time.The results of functional study on this gene suggest that:（1）Smad9 gene may regulate the hierarchical development and ovulation of goose follicle by affecting the proliferation and endocrine function of granulosa cells and the transcription of reproductive hormone receptor LHR;（2）Smad9 gene has a dose-effect in participating in this regulation process.Subsequent studies may regulate the hierarchical development and ovulation of goose follicle by regulating the expression of Smad9 gene in the egg-laying period,which provides a new strategy for improving the egg production of geese.In addition,Smad9 may be used as a candidate gene in the future to provide a theoretical basis for the selection and breeding of high-yield geese.