Influence Of Rat Testicular Macrophages On Function And Morphology Of Leydig Cells In Vitro

Objective: The present study has investigated the influence of testicular or peritoneal macrophages with or without stimulated by a bacterial lipopolysaccharide (LPS) on Leydig cells in vitro by using the methods of cell culture, cell hemotixylin and eosin (H&E) staining, cell histochemistry technique, magnetic enzyme immunoassay of testosterone. We observed mainly the effect of different concentration of macrophage-conditioned media (MCM) on Leydig cells' basal testosterone production and morphology in different periods. Meanwhile, we also assayed the role of IL-1β-one of macrophage's cytokines. According to these, we expect to understand the interaction of testicular macrophages and Leydig cells deeperly on physical or pathologic status, and discuss the effect factors of maintenance and disorder of testicular spermatogenic microenvironment in order to provide reliable reference for diagnosing and treating immune infertility. Methods: Experiments were performed on healthy adult male Wistar rats, weighting about 200g. Testicular or peritoneal macrophages and Leydig cells were obtained from the rats, studied as follows: 1) For preparation ofMCM, testicular or peritoneal macrophages were incubated in RPMI-1640 with or without LPS (50μg/ml) for 24h or 48h . Then eight kinds of MCM were got (LPS-TMCM-24h, LPS-PMCM-24h, LPS-TMCM-48h, LPS-PMCM-48h, TMCM-24h, PMCM-24h, TMCM-48h, PMCM-48h). 2) Every kind of MCM was added respectively to Leydig cells at different concentration (5%, 10%, 20%, 30%, 40% or 50%). After cultured for 24h, the culture medium was collected and testosterone in it was determined quantitatively by SerozymeⅠOperator made in Switzerland in order to get the most stimulating concentration. 3) According to this concentration, the testosterone of leydig cells' culture medium was assayed respectively after LPS-TMCM-24h was added for 1h, 6h, 12h, 24h, 48h or 72h so that the most stimulating time could be obtained. 4) As one of effect factors, human recombinant interleukin 1β(IL-1β) was added to Leydig cell culture and measure method was the same. 5) Leydig cells were counted with or without LPS-TMCM-24h for 24h, 48h, 72h, 96h or 120h. 6) Some Leydig cells were cultured on glass coverslips for 24h, added respectively to LPS-TMCM-24h, LPS-PMCM-24h, TMCM-24h or PMCM-24h,IL-1 and some as control group. Then they were H&E stained. 7) Leydig cells with or without LPS-TMCM-24h were performed with histochemistry staining of lactate dehydrogenase (LDH). Image analysissystem was used to measure the cellar positive granular light density. 8) All results were assayed by SPSS10.0 statistical analysis system.Results: 1) Cell preparation and purity evaluation: After cultured for 30min in RPMI-1640, testicular or peritoneal macrophages were allowed to attach to glass culture flask. The purity of testicular (more than 90%) or peritoneal macrophage (more than 95%) was assessed through histochemical staining for nonspecific esterase. Leydig cells were plated in 24-well culture plates and cultured for 24h, then most of them were attached. The typical Leydig cells were polygon or triangle, having steady microgranules which were lipid droplet in true. The purity of Leydig cell was more than 80% assessed by 3β-HSD histochemistry. 2) Determination of the most stimulating concentration: ①With the increasing of concentration of LPS-TMCM-24h added to Leydig cells, the testosterone production of Leydig cells was increased. When the concentration was 20%, testosterone production reached peak(0.412±0.052ng/ml),while in control group it was 0.201±0.039ng/ml. They were significant (P<0.01). ② LPS-PMCM-24h, TMCM-48h resulted in significant inhibition on testosterone production. When the concentration of LPS-TMCM-24h was 20%, 30%, 40% or 50% testosterone production was respective 0.270±0.076, 0.310±0.032, 0.285±0.058, 0.256±0.108ng/ml. Incontrast, in control group it was 0.479±0.048ng/ml(P<0.01)；When the concentration of TMCM-48h was 5%, 10%, 40% testosterone production was respec...