Recent research has also pointed to a putative role for CCR6/CCL20 axis in optimal germinal centre (GC) kinetics and B memory cell function [20, 21]
Recent research has also pointed to a putative role for CCR6/CCL20 axis in optimal germinal centre (GC) kinetics and B memory cell function [20, 21]. was used as unfavorable control. n?=?10 CP-640186 hydrochloride in each group. 40659_2018_161_MOESM3_ESM.pdf (360K) GUID:?0CD97216-FFE5-4C7F-8C4E-9F1488E8EFC1 Data TIAM1 Availability StatementNot applicable. Abstract Background Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly comprehended, especially in the process of spermatogenesis. Methods and results CCR6 was expressed in spermatogenic cell lines and its expression was shown in an age-dependent upregulation manner from puberty to adulthood in mouse testis. Immunostaining results confirmed the localization of CCR 6 in testis. Further chemotaxis assays exhibited that spermatogenic cells GC-1 and -2 exhibited a directional movement toward CCR6-specific ligand such as CCL20 or Sertoli cells in vitro. Conclusions The present findings indicate that CCR6 is usually involved in the chemotaxis of spermatogenic cells in vitro and promotes chemotaxis under non-inflammatory conditions during normal spermatogenesis. Electronic supplementary material The online version of this article (10.1186/s40659-018-0161-z) contains supplementary material, which is available to authorized users. was analyzed in the culture supernatant from TM4, 15P-1 cell line or primary Sertoli cells (SC) with an ELISA-based kit following manufacturers instructions. The culture supernatant was centrifuged and separated at 1000for 20?min. Transwells were used for the diluted standard, blank, and sample. The luminescent signal produced from TMB substrate was measured at 450?nm using a spectrophotometer (Thermo Fisher, Waltham, MA, USA). Each sample was tested in CP-640186 hydrochloride duplicate in two individual measurements. Chemotaxis assays Chemotaxis assays were performed using 24-well transwells (6.5?mm/diameter, 8?m/pore; Costar, Corning Inc., Corning, NY, USA) as previously described [13]. Briefly, GC-1 or ??2 (1??104/cells, 100?L) were added to 8?m-pore transwell inserts, coated with 25?g of growth factor-depleted Matrigel (BectonCDickinson Immunocytometry Systems, San Jose, CA, USA). The bottom chamber contained serum-free RPMI with or without CCL20 (50?ng/mL) or rDEFB1 (500?ng/mL) or TM4 or 15P-1 cell line or primary SC (1??104/cells, 100?L). For the purpose of blocking migration, each condition was prepared in a separate aliquot and incubated with anti-CCR6 antibody (ab78429 or SAB2702036). Normal rabbit IgG was used as unfavorable control. Migration was conducted at 37?C, 5% CO2 for 24?h. Migrated cells were collected from the lower compartment, centrifuged at 450(Additional CP-640186 hydrochloride file 1: Physique?S1). Therefore, the presence of CCR6 protein in testis and its expression in an age-dependent up-regulation manner may indicate the coincidence between the spermatogenesis and CCR6 expression in the testis from puberty to adulthood. Open in a separate window Fig.?1 Expression characteristics of CCR6 protein in spermatogenic cell lines and mouse testis. a Western blots showing the expression of CCR6 in spermatogenic GC-1 and GC-2 cell lines. -Tubulin was used as loading control. n?=?5 in each group. b Representative western blot results showing the age-dependent up-regulated expression of CCR6 protein in mouse testes from 3?days, 2, 4 and 8?weeks age groups (n?=?6 in each group). -Tubulin was used as loading control. c Statistical analysis of average optical density of western blotting bands in b. *p? ?0.05 compared with 8?weeks age group; **p? ?0.01 compared with 8?weeks age group; ***p? ?0.001 compared with 8?weeks age group Under normal physiological CP-640186 hydrochloride says, focal inflammatory cytokines can be found in the testicular interstitium, such as TNF- (Additional file 2: Physique?S2). However, no inflammatory infiltration exists in the seminiferous epithelium due to the integrity of blood-testis barrier (BTB). Then, immunostaining data revealed that CCR6-positive signals were detected in the spermatogenic cells, spermatids and testicular interstitial area of mouse and human testes (Fig.?2a, b), respectively. The co-localization of CCR6 and occludin, a key member of tight junction strands of blood-testis barrier (BTB), suggested that CCR6 signals were localized in the cell membrane, especially in the tight junction between Sertoli and germ cells (Fig.?2c). Then, this study attempted to determine the cellular origin of CCR6-specific ligand CCL20 in the seminiferous epithelium. The data from ELISA results confirmed that CCL20 was present in TM4, 15P-1 cell line and primary SC (Fig.?3), indicating that Sertoli cells may be the main cellular origin of CCL20. Open in a separate windows Fig.?2 Characteristics of CCR6 localization in normal testis. a Representative immunohistochemical images showing the expression of CCR6 in mouse and human testes (arrow). Nuclei were counterstained with hematoxylin. Enlarged images are shown in the inset;.