The Function Of STMN1,DENND1A In Ovarian Granulosa Cells

BACKGROUNDPolycystic ovary syndrome(PCOS)is one of the most complicated endocrine metabolic disorders in 6%to 8%of Asian women of reproductive age.The characteristic clinical features of PCOS include oligomenorrhea or amenorrhea,hyperandrogenism,polycystic ovarian morphology,and obesity.It accounts for 75%of anovulatory infertility.Besides,PCOS also causes risk of diabetes,cardiovascular disease and endometrial cancer,making PCOS a disease that beyond the range of gynaecology and obstetrics and affect the whole life and whole body of women.PCOS is with genetic heterogeneity and phenotypic complexity,making it a complex endocrinometabolic disorder and its pathogenesis remains obscure.At present,PCOS is considered to be caused by genetic factor and non-genetic factor(mainly environmental factor).In terms of genetic factor,our research center have conducted a series of studies,including study of differently expressed genes in case-control study and genome-wide association study(GWAS),and found some key genes and loci closely connected with PCOS.Although our results has been repeated by independent studies,many genes are firstly reported to be associated by PCOS and additional experimental evidence is needed to shed light on the mechanism of these genes within the occurrence and development of PCOS,which may promote the prevention,diagnose and treatment of PCOS.This study aims to investigate the possible role of DENND1A and STMN1 in female reproductive system.Chapter 1.The function of STMN1 in ovarian granulosa cells Part 1.The expression change of STMN1 in PCOS patients’granulosa cellsOBJECTIVE:STMN1 is a highly conserved gene that codes for cytoplasmic phosphoproteins.As a small regulatory protein,it integrates diverse intracellular signaling pathways involved in the control of cell proliferation,di□erentiation,and other activities.STMN1 also plays a potential role in the regulation of hormone secretion in rodent pituitary and insulinoma cell lines.Furthermore,STMN1 mediates nerve growth factor(NGF)-induced GCs apoptosis through tumor necrosis factor a(TNF-α).NGF promotes steroidogenesis by enhancing the expression of enzymes involved in progesterone,testosterone,and estradiol(E2)synthesis.These results indicates us that STMN1 may participate in regulating sexual hormone secretion and reproductive disease.Granulosa cells,as an important generator for estradiol,progesterone and many cytokines,paly an important role in regulation of menstrual cycle and the pregnancy process.Whether STMN1 is involved in the development of PCOS needs to be investigated.We try to make it clear that whether STMN1 expresses abnormally in PCOS patients’ granulosa cells.METHODS:We collected 38 samples of follicular fluid granulosa cells from PCOS patients accepting in vitro fertilization(IVF)treatment and 36 samples of control women,who were accepting IVF treatment because of oviduct factors or male factors.The total RNA was extracted and reverse-transcripted to cDNA,and quantitative real-time polymerase chain reaction,(RT-qPCR)method was conducted to detect the expression level of STAMN1.RESULTS:The mRNA levele of STMN1 in PCOS patients’ granulosa cells is 2 times higher than that of control patients,with statistical significance.CONCLUSIONS:The expression level of STMN1 in PCOS patients’ granulosa cell is higher than that of control patients,indicating that STMN1 may play a role in granulosa cells and may be involved in the pathophysiology of PCOS.Part 2.The role of STMN1 in mouse primary granulosa cells OBJECTIVE:The expression level of STMN1 in granulosa cells of PCOS patients is 2 times than that of control patients,indicating that STMN1 may play a role in the realization of GC’s function.But,at the present time,little is known about the potential role of STMN1 in ovarian function,such as follicular development,oocyte maturation,and ovarian steroidogenesis.In this experiment,we try to investigate whether STMN1 is involved in GCs’ steroidogenic process.METHODS:Firstly,in order to localize the expression site of STMN1 within ovary,ovaries of mouse and rhesus monkey are used in immunohistochemistry(IHC)assay.Further,immunofluorescence(IF)was conducted to localize STMN1 in subcellular level.Then,we up-regulated and down-regulated STMN1 in ovary GCs,and detected the hormone level change.RTCA real time cell proliferation monitor was used to detect the possible role of STMN1 on GC’s proliferation.If hormone level in the cell culture supernatant is changed,the genes encoding key enzymes involved in steroidogenic pathway would be detected both in transcriptional and translational level.Luciferase assay was also designed to check whether STMN1 could promote the transcriptional activity of promoter sequence of regulated genes.RESULTS:In this study,we demonstrated that STMN1 is predominantly expressed in GCs in ovaries of mice and rhesus monkey.Specifically,STMN1 is mostly expressed in cytoplasm of GCs,and slightly in nucleus.In mouse primary GCs,we found that STMN1 could affect progesterone production:knockdown of STMN1 decreased progesterone production,whereas overexpression of STMN1 stimulated progesterone production.The RTCA assay shows that knockdown of STMN1 has no significant effect on the proliferation of GCs,and overexpression of STMN1 could slightly inhibit GC’s proliferation.Obviously,the proliferation change caused by STMN1 couldn’t account for the progesterone change.We also checked the expression of key genes involved in progesterone production both in transcriptional level and translational level and found that when STMN1 was up-regulated,Star and Cypllal expression increased,and when STMN1 was down-regulated,Star(steroidogenic acute regulatory protein)and Cyp11a1(cytochrome P450 family 11 subfamily A member 1)expression decreased.Luciferase assay found that STMN1 could both promote the transcriptional activity of Star and Cyp11al promoter sequence in mouse primary GC,HEK293T,KGN and SVOG cells.Finally,we predicted several STMN1 binding site in Star transcription start site using bioinformatics method.And further Chromatin Immunoprecipitation(ChIP)shows that STMN1 could bind to Star-1(-1,196/-1,177),Star-3(+247/+269)and Star-4(+71/+741)directly.CONCLUSIONS:In this study,we found that STMN1 was predominantly expressed in GCs in ovary,and it could regulate the production of progesterone in mouse primary GCs.Subsequent pathway study indicated that STMN1 could enhanced the expression of Star,a crucial rate-limiting enzyme in progesterone production.We firstly found that STMN1 is involved in GC’s steroidogenesis.As is known,progesterone plays an important role in the menstrual cycle regulation,ovulation process,and maintenance of early pregnancy.Our study may help in the prognosis and treatment of related disease.Chapter 2.The Function of DENND1A in ovarian granulosa cellsPart 1.The localization study of DENND1A in ovaryOBJECTIVE:DENND1A has been firstly found to be associated with PCOS in GWAS study conducted by our research center.Three loci(rs10818854,rs2479106 and rs 10986105)were detected within DENND1A sequence.Further,three independent studies carried out in European also found that DENND1A is significantly associated with PCOS,making DENND1A a strong risk gene for PCOS.In 2014,some researcher found that DENND1A transcript 2(V2),a truncated transcript of DENNDIA,expression level is elevated in PCOS patients’ ovary theca cell,and forced overexpression of DENND1A.v2 in theca cells from control women could generate a phenotype similar to PCOS.However,there are no enough research on the ovarian function of DENND1A,even no detailed statement about the ovarian expression pattern of DENNDIA,and not to speak of the detailed mechanism study.So,we firstly try to locate DENND1A expression in both tissular and cellular level to define the ovarian expression pattern of DENNDIA,which may help clearing the future investigation direction.METHODS:Firstly,we detected the expression pattern of DENND1A in different tissues and organs using RT-PCR method.Then,we localized tissular DENND1A’s expression within 2-month-old mouse and human ovary using Immunohistochemistry(IHC)staining.We also localized cellular DENND1A’s expression in KGN cells using immunofluorescence(IF)method.RESULTS:DENND1A.V1 and V2 have different expression patterns.DENND1A.V1 is richly expressed in liver,testis,spleen,stomach,and intestine,and DENND1A.V2 is richly expressed in testis,ovary,muscle,and spleen.In ovary IHC staining,we found that DENND1A is predominantly expressed in GCs of ovary,and slightly expressed in theca cells and oocytes.Using IF in KGN cells,we discovered that DENND1A.V1 is mainly located in cytoplasm,and DENND1A is distributed both in cytoplasm and nucleus.CONCLUSIONS:DENND1A.V1 and V2 have different expression patterns.There is rich distribution of DENND1A in ovary,especially in granulosa cells.And in cellular level,the V1 and V2 have different expression sites:The V1 is located in cytoplasm of KGN cell,and V2 is located both in cytoplasm and nucleus of KGN cell.Part 2.The expression change of DENND1A in PCOS patients’granulosa cellsOBJECTIVE:The connection of DENND1A and PCOS are verificated by both GWAS study and genotype-phenotype association studies.DENND1A is abundantly expressed in ovarian granulosa cells.And granulosa cell’s dysfunction was reported in PCOS patients,such as steroidogenic dysregulation and abnormal proliferation state.Would DENND1A be involved in the granulosa cell’s dysfunction of PCOS?Since the realization of abnormal function relys on the quantitative,qualitative,temporal and spatial change of gene expression,we try to make it clear that whether there is DENND1A expression change in PCOS patients’ guanulosa cells.METHODS:We collected 109 samples of follicular fluid granulosa cells from PCOS patients accepting in vitro fertilization(IVF)treatment and 68 samples of control women,who were accepting IVF treatment because of oviduct factors or male factors.The total RNA was extracted and reverse transcripted to cDNA,and quantitative real-time polymerase chain reaction,(RT-qPCR)method was used to detect the expression level of DENND1A.V1 and V2.RESULTS:The expression of internal control gene GAPDH was set as 1.Results show that the expression level of DENND1A.V1 in PCOS patients’ granulosa cells and control granulosa cells are 0.00355 ± 0.00293(Mean ± SD)and 0.00241 ±0.00117 respectively,and the expression level of DENND1A.V2 in PCOS patients’granulosa cells and control patients’ granulosa cells are 0.00087 ± 0.00107 and 0.00050 ± 0.00042 respectively.It indicates us that the expression level of DENND1A in PCOS patients’ granulosa cells is elevated with statistical significance.CONCLUSIONS:The expression level of DENND1A in PCOS patients’granulosa cell is higher than that of control patients,indicating that DENNDIA may be involved in the occurrence and development of PCOS.Part 3.The role of DENND1 A in granulosa cellsOBJECTIVE:We have found that the PCOS risk gene DENND1A is abundantly expressed in granulosa cells of ovary,and the mRNA level of DENND1A is elevated in follicular fluid granulosa cells of PCOS patients.These results indicate that DENND1A may play an important role in granulosa cells.So far,there is no reported investigations about DENND1A in ovarian granulosa cells.And we try to explore its possible role in granulosa cells.METHODS:In our experiment,the KGN cell that could synthesize steroid hormone was used in function study.We down-regulate DENND1A by siRNA interfering and up-regulate DENND1A by conducting stable cell lines that overexpressing DENND1A.V1 and V2 respectively,and detect the possible steroid hormone level change in cell culture supernatant.At the same time,the proliferation change caused by DENND1A is detected.The expression change of key genes involved in steroid hormone synthesis is measured in both transcriptional and translational level.We designed the transgenic mice model that overexpressing Dennd1a in ovarian granulosa cells specifically by driving Dennd1a expression by Cyp19a1 promoter.We investigate the reproductive phenotypes of transgenic mice.RESULTS:In cell experiment,we found that stably transfected KGN cell lines expressing DENND1A.V1 and V2 could produce more E2 than that transfected with vector.When DENND1A.V1 and V2 were interfered by siRNA specifically,the E2 level in the culture supernatant decreased.Further pathway study shows that CYP19A1 is regulated by DENND1A.V1 and V2.When DENND1A.V1 and V2 was overexpressed,CYP19A1 expression increased in both mRNA and protein levels,and vise versa.The in vivo study shows that the transgenic mice have a lower fertility than the wild type mice.Follicles in ovaries of transgenic mice are activated excessively.But compared with wild type mice,less antral follicles are found in transgenic mice after PMSG stimulation.CONCLUSIONS:In ovarian granulosa cells,we found that DENND1A.V1 and V2 could promote the GC’s E2 synthesis by inducing the expression of CYP19A1,and enhance the proliferation ability of KGN cell.The Dennd1a transgenic mice’s follicles are activated excessively,but its development is disordered.