| Objectives1.3T3-L1 adipocytes were cultured in vitro. Differences among the effects of clenbuterol (CLB), zilpaterol (ZIL), and ractopamine (RAC) on the expression of peroxisome proliferators activated receptor γ (PPARγ) and adiponectin (ADP) in adipocytes were investigated, which was aiming to investigate the potential association of β2AR agonists on adipocyte endocrine function and related reproductive system diseases.2. Transwell system was established to examine the effects of adipocyte coculturing on the expression of INSL3 and 3β-HSD in Leydig cells, which was used to verify the molecular pathogenesis of obesity on male infertility.3. Transwell system was established to examine the effects of adipocyte coculturing and CLB on the expression of INSL3 and 3β-HSD in Leydig cells, which was used to verify the pathogenesis of β2AR agonists-associated male reproduction disturbance with adipocyte endocrine disorders.Materials and methods1. Materials1.1 3T3-L1 adipocytes3T3-L1 pre-adipocytes were purchased from Shanghai Cell Bank, Chinese Academy of Science, and cultured in adipogenic medium. After induction,3T3-L1 adipocytes were used for experiments.1.2 TM3 Leydig cellsTM3 Leydig cells were presented by Professor Yan-hong Yu from Jinan University. After passage, TM3 Leydig cells were used for experiments.2. Grouping and administration2.1 Grouping of 3T3-L1 adipocytesAdipocytes were cultured with different doses of CLB, ZIL and RAC, respectively, and the adipocytes were harvested after 12h and 24h to detect cell viability and the expression of PPARy and ADP. The grouping is shown as Table 1.2.2 Coculture experimentsTM3 Leydig cells were cultured in vitro with addition of 5μM CLB, and transwell system was used to do coculture of TM3 Leydig cells and 3T3-L1 cell-derived matured adipocyte, coculture of TM3 Leydig cells and 3T3-L1 cell-derived matured adipocyte with addition of 5μM CLB, respectively. TM3 Leydig cells cultured alone were normal control.3. Methods3.1 3T3-L1 adipocytes culture and differentiation3T3-L1 pre-adipocytes were maintained in DMEM supplemented with 10% fetal calf serum (FBS), humidified atmosphere with 5% CO2 at 37℃. The medium was replaced every 2d. Cell differentiation began 48h after confluence and took 2d in differentiation medium containing 0.5mM IBMX, 1μM dexamethasone, and 10μg/ml insulin. The cells were further incubated in basal medium containing 10μg/ml insulin alone for 2d. Then, the basal medium was changed every 2d until greater than 90% of cells had accumulated multiple lipid droplets on the eighth day and adipocyte differentiation was achieved. Lipid content was measured by Oil Red O staining.3.2 TM3 Leydig cells cultureTM3 Leydig cells were maintained in DMEM/F12 supplemented with 10% horse serum, humidified atmosphere with 5% CO2 at 37℃. The medium was replaced every 2d. The cells could be subcultured until cell fusion was more than 80%.3.3 Establishment of cell coculture systemA model of co-cultured system of 3T3-L1 adipocytes and TM3 Leydig cells in vitro was built by transwell. Adipocytes were plated in 6-well plates. After induction of adipocytes, Leydig cells were plated in transwell chambers. After 48h of coculture, Leydig cells were collected for total RNA and protein extraction.3.4 Evaluation of 3T3-L1 adipocytes viabilityAfter CLB, ZIL and RAC exposure, respectively, cell viability was quantified by methyl thiazolyl tetrazolium assay (MTT).3.5 Evaluation of the expression of PPARy, ADP, INSL3 and 3β-HSDRT-PCR and Western blot were used to assess the mRNA and protein expression of PPARγ, ADP, INSL3 and 3β-HSD.4. Statistical analysisResults were expressed as mean ± standard deviation. Statistical analysis of the data was performed by SPSS 21.0. software using one-way ANOVA analysis followed by Tukey’s test. A value of.P<0.05 was considered statistically significant.Results1. Determination of mature adipocytesAfter induction, more than 90% of the cells had accumulated fat droplets as determined by Oil Red O staining.2. Viability of 3T3-L1 adipocytes treated with CLB, ZIL and RAC, respectivelyTreatments with CLB, ZIL and RAC, respectively resulted in depressed cell viability compared with untreated groups (P<0.05). With increasing doses of three kinds of β2AR agonists, viability of the cells showed more severe decrease (P<0.05).In 12h, the viability of 3T3-L1 adipocytes:groups B<B1<B2, but no difference among groups (P>0.05); groups C<C2 (P<0.05), groups C<C1 (P>0.05), groups C1<C2 (P>0.05); groups D<D2 (P<0.05), groups D1<D2 (P<0.05), groups D<D1 (P>0.05). In 24h, the viability of 3T3-L1 adipocytes:groups B<B1<B2, groups C<C1<C2, but no difference among groups (P>0.05); groups C<C2 (P<0.05), groups D<D1<D2 (P<0.05).3. Expression of PPARy and ADP in 3T3-L1 adipocytes treated with CLB, ZIL, RAC, respectively in vitroAfter CLB, ZIL and RAC exposure, the expression of PPARy and ADP could be down-regulated and the decrease was more severe with increasing doses (P<0.05).In 12h, the expression of PPARy:groups B<B1 (P<0.05), groups B<B2 (P<0.05); groups B1<B2, but no difference between groups (P>0.05); groups C<C1<C2 (P<0.05), groups D<D1<D2 (P<0.05). In 24h, the expression of PPARγ:groups C<C1<C2 (P<0.05), groups D<D1<D2 (P<0.05).In 12h, the expression of ADP:groups B<B1<B2, groups C<C1<C2, but no difference among groups (P>0.05), groups D<D1<D2 (P<0.05). In 24h, groups B<B2 (P<0.05), groups B<B1 (P<0.05), groups B1<B2 (P>0.05); groups C<C2 (P<0.05), groups C1<C2 (P<0.05), groups D<D2 (P<0.05), groups D1<D2 (P<0.05); groups C<C1 (P>0.05), groups D<D1 (P>0.05).4. Expression of INSL3 and 3β-HSD in Leydig cells in vitroCompared with normal control group, the expression of INSL3 was a little lower in Leydig cells with CLB (P>0.05), while the expression of 3β-HSD was a little higher (P>0.05). Compared with normal control group, the expression of INSL3 and 3β-HSD of co-cultured cells was down-regulated (P<0.05), and the decrease was more severe in co-cultured cells with CLB group (P<0.05).Conclusions1. After CLB, ZIL and RAC exposure, respectively, the viability of the cells showed more severe decrease, and the expression of PPARy and ADP could be down-regulated and the decrease was more severe with increasing doses, which were involved in adipocyte endocrine disorders and then might disturb related reproductive system events.2. CLB had the greatest effects on adipocytes, while the effects of ZIL on adipocytes was intermediate to CLB and RAC.3. Adipocyte-induced downregulation of INSL3 and 3β-HSD in Leydig cells was an important mechanism by which obesity caused male infertility. In the coculture of TM3 Leydig cells and 3T3-L1 adipocyte with addition of CLB, the decrease of INSL3 and 3β-HSD expression was more severe, which indicated that β2AR agonists-mediated adipocyte endocrine disorders could involve in pathogenesis of male reproductive diseases. |
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