Dysfunction Of Rat Leydig Cells Induced By TGF-β1and Protective Effect Of Icariin

ObjectiveTransforming growth factor-β1（TGF-β1）, one of members belongs to TGF-β superfamily. In the testis, TGF-β1has been shown to be present at different stages ofdevelopment. Furthermore, TGF-β1can regulate testis development and spermatogenesisthrough autocrine and paracrine pathways. Previous studies had shown that TGF-β1down-regulated testosterone secretion of rat Leydig cells, inhibited Leydig cell sproliferation and affected the process of its differentiation. However, few studies havebeen done about the regulation of estradiol secretion of this cell as well asconnexin43-based gap junction intercellular communication （GJIC） between adjacent Leydig cells induced by TGF-β1. More and more researchers reported that Icarrin, as oneof the effective constituents of epimedium, a traditional C hinese herbal medicine, hasdefinite benififial effects on male reproductive system. The mechanisms may be related toimproving the secretion of testosterone or estradiol. But wehter it has protective effects onLeydig cell s function is not clear yet. Therefore, based on above evidences the aim of thisstudy is to figure out wether there is relationship between the alteration of estradiolsecretion and abnormal Cx43-based GJIC in primary cultured rat Leydig cells induced byTGF-β1. And furtherly to assay wether Icariin can protect Leydig cell from damaging byTGF-β1.Methods1．Isolation and purification of Leydig cells from3-month-old SD rats throughcontinuous Percoll density gradient centrifugation. After administration of differentconcentrations of TGF-β1to primarycultured Leydigcells, the effect of TGF-β1onbasaland hCG stimulated estradiol and testostorone secretions in vitro were evaluated.2．After administration of TGF-β1, immunocytochemical method was used to observethe expression of aromatase in Leydig cells; tritium release assay was used to investigatearomatase activity; Western blot and Real-time PCR were used to detect expression ofgene CYP19and data were measured by image analysis software.3．Observation of the location of connexin43in TGF-β1-treated Leydig cells byindirect immunofluorescence; Furthermore, the total protein and phosphorylated informswere detected by Western blotting. The function of Cx43-based GJIC between Leydigcells were investigated by FRAP techniques.4．Determined the optimal concentration of Icariin administrated in primary culturedLeydig cells by MTT. Observation of effect of Icariin on secretion of estradiol andP450arom activity induced by TGF-β1. Furthermore, FRAP experiment was used todetect the effect of Icariin on GJIC between Leydig cells treated by TGF-β1. Results1. When the purified Leydig cells were treated with different doses of TGF-β1invitro, the basal and hCG-stimulated estradiol and testosterone secretion were affected ina dose-dependent manner. Compared with control group, low concentration of TGF-β1（1ng/ml） inhibited basal and hCG-stimulated estradiol and testosterone secretionobviously（P＜0.01）. Although the inhibition of relatively high dose of TGF-β1（10ng/ml） didn t enhance with the dose increased, the result was still lower thancontrol group.2. Immunocytochemistry revealed that aromatase was localized in cytoplasm andpresented an attenuated trend in both dose and time-course TGF-β1treatment groups. Intritium release assay, we found that aromatase activity was decreased in TGF-β1-treatedcells at concentration of5ng/ml (2121.43±1.07foml mg protein^-1h^-1) compared withcontrol cells (53±0.71fmol mg protein^-1h^-1). When time-course was up to30h, TGF-β1induced a50%inhibition of aromatase activity. Besides, TGF-β1caused significantdown-regulation of both total protein and mRNA levels of CYP19in Leydig cells in atime-dependent manner.3. Indirect immunofluorescence results indicated that after TGF-β1-treated,expression of Cx43translocated from cell membrane to cytoplasm. Besides, there s noevident difference between control and TGF-β1-treated Leydig cells in total protein ofCx43. However, after administration of TGF-β1, phosphorylated Cx43（P2+P1）increased but there was no significant alteration in non-phosphorylated Cx43（P0）. Theeffect of TGF-β1on Leydig cells was observed byFRAP techniques. The results showedthat in the control groups, the fluorescence intensity was gradually recovered at differenttimes after bleaching. The mean fluorescence recovery rate of it was （81.25±1.25）%. Incontrast to the control groups, the fluorescence recovery of the bleached cells in theTGF-β1groups was not obvious in the same time. The mean fluorescence recovery ratesof them was （43.58±1.87）%. However, administration of estradiol can reverse thedown-regulation of GJIC by TGF-β1. 4. The proper concentration of Icariin is10μg/ml. After administration of Icariin, theamount of estradiol inhibited by TGF-β1was increased from0.22±0.04pg/ml to1.48±0.12pg/ml, and this trend was accompanied by up-regulated P450arom activity.Furthermore, Icariin could reversed the mean fluorescence recovery rates of Leydig cellsinduced by TGF-β1, from （24.71±1.87）%to （55.88±2.45）%.Conclusions1. We succeeded in isolation and purification Leydig cells of adult male rat. And aftertreatment with TGF-β1, the synthesis of estradiol and testosterone were significantlydecreased. TGF-β1might participate in the regulation of endocrine secretion of Leydigcells.2. TGF-β1could suppress expression of P450arom as well as down-regulate itsactivity in Leydig cells. This effect may depend on attenuated expression of its encodegene CYP19both in translation and transcription levels. The results above indicate thatthe reduction of estradiol by TGF-β1can be attributed to attenuation of aromataseactivity and expression of its encode gene CYP19.3. TGF-β1could significantly down-regulate gap junction channels accompanied byCx43hyperphosphorylation and translocation of Cx43from the gap junctions into thecytoplasm. Besides, estradiol could attenuate inhibitory effect of TGF-β1on GJIC.Therefore, estradiol may be involved in protecting of GJIC between Leydig cells.4. Icariin could interrupt the inhibition of TGF-β1onsecretionof estradiolof Leydigcells. This effect can be largely attributed to enhancing the P450arom activity. Besides,Icariin could reverse the effect of down-regulation of TGF-β1on GJIC between Leydigcells.