pcos is one of the most common reproductive endocrine disorders , which affects approximately 510% of premenopausal women and accounts for 75% of anovulatory infertility [ 1 , 2 ] . it is associated with elevated levels of circulating free testosterone , which is produced primarily in the ovary [ 3 , 4 ] . in women with pcos , ovarian theca cells are recognized as one of the primary sources of excessive androgen biosynthesis , which is associated with augmented expression of several steroidogenic enzymes , such as cytochrome p450c17 which has both 17-hydroxylase and c17 , 20 lyase activities [ 5 , 6 ] . in addition to p450c17 , the upregulated enzymes also include cytochrome p450 cholesterol side chain cleavage ( p450scc ) , encoded by the cyp11a gene . although some collective studies have identified important correlations between increased steroidogenic enzyme gene expression and increased androgen biosynthesis in pcos theca cells [ 7 , 8 ] , the exact molecular and cellular mechanisms remain unclear . our previous studies assessed the differential gene expression in human normal and polycystic ovaries using cdna microarray technology , which identified many genes expressed differentially in pcos . set ( patient  se translocation  ) was one of the altered genes , which was up - regulated in polycystic ovaries . the set protein , also known as taf-1 [ 1012 ] , i2pp2a , and inhat , belongs to a family of multitasking proteins involved in apoptosis , transcription , nucleosome assembly , and histone binding . we did not know what role(s ) set played in the regulation of ovarian steroidogenesis and whether set would make an attractive candidate for involvement in the hyperandrogenism of pcos .   in the present study , we firstly investigated the cellular localization of set protein in human ovaries and its different expression in the polycystic ovaries compared with human normal ovaries of adult women . it would provide us with preliminary knowledge in the regulation of androgen production in ovary and a fundamental mechanism in the pathophysiology of hyperandrogenism in pcos . ovarian tissues , samples of normal adult ovaries and polycystic ovaries , were collected during transsexual operation and surgical treatment with the signed consent form . pcos patients were recruited by the revised criteria of the european society of human reproduction and embryology / american society for reproductive medicine in 2003 : ( i ) oligomenorrhoea and/or anovulation ( eight or fewer menstrual cycles in a year or menstrual cycles more than 35 days in length ) ; ( ii ) clinical and/or biochemical signs of hyperandrogenism ; and ( iii ) polycystic ovaries on ultrasound ( presence of 12 or more follicles in each ovary measuring 29  mm in diameter and/or increased ovarian volume > 10  ml ) and exclusion of other aetiologies ( e.g. , congenital adrenal hyperplasia , androgen - secreting tumours , and cushing 's syndrome ) . the patients did not receive hormonal therapy or stopped it for at least 3 months before operation . controls were healthy women , with regular menstrual cycles and without family history of hirsutism . the ages of the pcos specimens group were matched to those of the control group . all samples were histologically examined and stored in liquid nitrogen for western blotting and paraffin - embedded for immunohistochemical staining . this study was approved by the institutional ethics committee of the first affiliated hospital of nanjing medical university . the detection of set protein was carried out by a two - step ihc procedure and the primary polyclonal antibody ( abcam , cambridge science park , cambridge , uk ) was diluted by 1  :  400 . immunohistochemical staining of negative controls was executed simultaneously , of which the primary antibody was the igg . the concentrations of protein extracted from the ovarian tissues were measured by the bradford method . equal amounts of protein were loaded and separated by 12% sds - page and then electroblotted onto polyvinylidene fluoride membrane ( bio - rad , hercules , ca ) . the membranes were incubated with polyclonal antibodies to set ( 1  :  1000 , santa cruz biotechnology , santa cruz , ca , usa ) . after washing with tbs three times , the membrane was incubated with hrp - conjugated secondary antibodies and then incubated with -tublin ( 1  :  2000 , abcam , cambridge science park , cambridge , uk ) . after washing with tbs three times , it was incubated with hrp - conjugated secondary antibodies ( 1  :  1000 ; beijing zhongshan biotechnology , china ) and examined by enhanced chemiluminescence ( amersham biosciences , uppsala , sweden ) . data were expressed as mean  sem from six independent experiments in two groups . in our study , all pcos patients , aged from 26 to 31 , had menstrual - cycle abnormalities ( amenorrhea or oligomenorrhea ) , polycystic ovaries , and hyperandrogenism ( presence of hirsutism and/or serum total t levels > 2.43 the control women , aged from 27 to 29 , were in good health . to determine the cellular localization of set protein in the human ovaries , set was expressed predominantly in theca cells and oocytes in both pcos ovarian tissues ( figures 1(b ) and 1(e ) ) and normal ovarian tissues ( figures 1(c ) and 1(f ) ) . no signal was detected in the negative control ( figures 1(a ) and 1(d ) ) . the different expression of set protein in polycystic ovaries and normal ovaries was detected by western blot . tubulin was used as internal control . as shown in figure 2 , expression of set protein in polycystic ovaries was triple times higher than that in normal ovaries ( p < 0.05 ) . it is well known that theca cells express p450scc , 3-hydroxysteroid dehydrogenase ( hsd3b2 ) , and p450c17 which has both 17-hydroxylase and c17 , 20 lyase activities . we found that set protein was predominantly expressed in theca cells and oocytes of human ovarian tissues ( figure 1 ) . this was in agreement with previous studies performed by zhang et al .   which showed that set was expressed in theca cells and oocytes of rats . since the primary function of theca cells is to produce androgen , set expression in theca cells indicates that set may be involved in androgen biosynthesis . it is a 39  kda phosphoprotein widely expressed in various tissues , especially in steroidogenic cells within the central nervous system , adrenal gland , and gonad [ 11 , 16 ] . as a transcriptional regulating factor , set not only exerts function by binding to the transcriptional coactivators cbp / p300 , but set - mediated promoter hypoacetylation is a prerequisite for coactivation of the estrogen - responsive ps2 gene by prmt1 . recent evidence suggested that set was essential for regulating both the promoter activity of cyp17 and the biological activity of p450c17 . in human nt2 neuronal precursor cells and ma-10 cells , set binds to rat p450c17 promoter at 418/399 and activates its basal transcription from a rat p450c17 luciferase reporter plasmid [ 15 , 18 ] . set has been identified as a potent inhibitor of the protein phosphatase 2a ( i2pp2a ) . set inhibited pp2a activity by binding to the catalytic subunit pp2ac through the amino terminal domain ( i2ntf ; aa 1175 ) and the carboxy terminal domain ( i2ctf ; aa 176277 ) . set can foster 17 , 20 lyase activity of p450c17 by inhibiting the dephosphorylation action of pp2a on p450c17 in nci - h295a cells . all these indicated that set regulated androgen synthesis in steroidogenic cells by regulation of both the transcriptional and posttranslational levels of p450c17 and cyp17 . in mouse eggs pp2a was needed for both continued metaphase arrest and successful exit from meiosis , which suggested that set may participate in oocyte maturation . however , the function of set protein in human ovary in regulating androgen production and oocyte development should be further studied . hyperandrogenism is the central defect in pcos patients , which is related to the increased expressions of steroidogenic enzymes and the increased androgen biosynthesis [ 23 , 24 ] . our previous study showed that set protein was differently expressed in polycystic ovaries by microarray assay . in this study , it is found that set protein was predominantly expressed in theca cells and oocytes of human ovarian tissues by immunohistochemistry ( figure 1 ) . and expression of set protein in polycystic ovaries was found to be triple times higher than that in normal ovaries by western blotting ( figure 2 ) . combined with the effect of set on cyp17/p450c17 , set may be involved in the regulation of androgen production in the theca cells , and the elevated set protein in polycystic ovaries could increase the level of cyp17 and the activity of p450c17 , which led partly to the increased production of androgen in theca cells . additional studies are required to examine whether and how set overexpression in theca cells contributes to hyperandrogenism of pcos . in summary , set protein expressed predominantly in theca cells , which suggested that set protein may play a role in modulating androgen production in follicular theca cells . the increased expression of set protein in polycystic ovarian tissues may be partly a potential mechanism in the pathophysiology of hyperandrogenism in pcos .
background . 
 we previously found that expression of set gene was up - regulated in polycystic ovaries by using microarray . 
 it suggested that set may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome ( pcos ) . in this study , expression and cellular localization of set protein 
 were investigated in human polycystic and normal ovaries . 
 method . 
 ovarian tissues , six normal ovaries and six polycystic ovaries , were collected during transsexual operation and surgical treatment with the signed consent form . 
 the cellular localization of set protein was observed by immunohistochemistry . 
 the expression levels of set protein were analyzed by western blot . 
 result . 
 set protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both pcos ovarian tissues and normal ovarian tissues . 
 the level of set protein expression in polycystic ovaries was triple higher than that in normal ovaries ( p < 0.05 ) . 
 conclusion . 
 set was overexpressed in polycystic ovaries more than that in normal ovaries . combined with its localization in theca cells , set 
 may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in pcos .