Effects of aging and macrophages on mice stem Leydig cell proliferation and differentiation in vitro

Abstract

Background: Testosterone plays a critical role in maintaining reproductive functions and well-beings of the males. Adult testicular Leydig cells (LCs) produce testosterone and are generated from stem Leydig cells (SLCs) during puberty through adulthood. In addition, macrophages are critical in the SLC regulatory niche for normal testicular function. Age-related reduction in serum testosterone contributes to a number of metabolic and quality-of-life changes in males, as well as age-related changes in immunological functions. How aging and testicular macrophages may affect SLC function is still unclear.

Methods: SLCs and macrophages were purified from adult and aged mice via FACS using CD51 as a marker protein. The sorted cells were first characterized and then co-cultured in vitro to examine how aging and macrophages may affect SLC proliferation and differentiation. To elucidate specific aging effects on both cell types, co-culture of sorted SLCs and macrophages were also carried out across two ages.

Results: CD51+ (weakly positive) and CD51++ (strongly positive) cells expressed typical SLC and macrophage markers, respectively. However, with aging, both cell types increased expression of multiple cytokine genes, such as IL-1b, IL-6 and IL-8. Moreover, old CD51+ SLCs reduced their proliferation and differentiation, with a more significant reduction in differentiation (2X) than proliferation (30%). Age matched CD51++ macrophages inhibited CD51+ SLC development, with a more significant reduction in old cells (60%) than young (40%). Crossed-age co-culture experiments indicated that the age of CD51+ SLCs plays a more significant role in determining age-related inhibitory effects. In LC lineage formation, CD51+ SLC had both reduced LC lineage markers and increased myoid cell lineage markers, suggesting an age-related lineage shift for SLCs.

Conclusion: The results suggest that aging affected both SLC function and their regulatory niche cell, macrophages.

Keywords: CD51; aging; macrophages; stem Leydig cells; testis; testosterone.

Figure 1

Co-localization of CD51 and macrophage marker F4/80. (A–C) Mounted seminiferous tubules were co-stained with CD51 and F4/80 antibodies. (D–F) Unsorted testicular cell suspension was co-stained with CD51 and F4/80 antibodies. (G–I) Macrophages labeled by Trypan blue phagocytosis (blue arrows) were positively-stained with the CD51 antibodies (red or white arrows). Scale bar: 50µm in length.

Figure 2

Fluorescence-activated cell sorting (FACS) of CD51-positive cells from young (Y) and old (O) testicular cell suspensions. (A) Testicular cell suspensions from Y or O testes without label (Y- and O-unlabeled) or with CD51-PE antibody labeled (Y- and O-CD51) were analyzed by PE and FITC channels. CD51-negative cells distributed below the diagonal line while CD51+ and CD51++ cells distributed above the diagonal line. (B, C) CD51-labeled Y (Yraw) and O (Oraw) testicular cells were separated into CD51 strongly positive (++) and weakly-positive (+) Y and O cells. Scale bar: 50µm in length.

Figure 3

Expression of testicular cell marker genes in isolated CD51-positive cells of the young and old testes. RNAs from whole testis (YT or OT) or CD51-negitive cells (Y- or O-) were used as controls. Cell types (marker genes) include: Leydig cell (Cyp17a1 and Scarb1), Smooth muscle cells (Acta2), vascular endothelial cells (Cd146 and Cd31), germ cells (Ddx4), macrophages (Adgre1and Cd115) and stem Leydig cells (Nes, Coup-tf2, Pdgfra). A few inflammatory cytokine genes (Tnfa, IL-6, IL-8, and IL-1b) are also shown. The data are expressed as mean ± SEM of cells from three individual experiments. *^,#Significantly different from age-matched YT/OT or Y-/O- controls (*) or from same cell type from young animals (^#) at P < 0.05 respectively.

Figure 4

Culture of CD51+ cells (SLCs) and CD51++ cells (macrophages) from young and old testes. YSLC1 and OSLC1: Newly isolated (day 1) young and old CD51+ SLCs were round with evenly distributed cytoplasm. YMF1 and OMF1: Newly isolated (day 1) young and old CD51++macrophages were not uniform in shape and had large numbers of granules in the cytoplasm. YSLC4 and OSLC4: Young and old SLC spread out after 4 days in culture with morphology of typical mesenchymal stem cells. Also, Y cells spread better than the O cells. YMF4 and OMF4: Young and old CD51++ macrophages did not spread as well as CD51+ SLCs after 4 days in culture. Thick scale bars: 50µm in length. Thin scale bars: 20µm in length.

Figure 5

Effect of aging on the proliferation and differentiation of CD51+ SLCs and CD51++ macrophages. (A, B) Young and old CD51+ SLCs were labeled with EdU for 6 hours in day 3 of the culture. (C) The rates of EdU+ CD51+ SLCs and CD51++ macrophages were quantified. (D) Testosterone production of CD51+ SLC and CD51++ macrophages after induction of differentiation. (E) Testosterone production by differentiated CD51+ SLCs on days 2 and 3 was expressed as percentage of day-4 cells. (F–I) Expression of HSD3B activity by CD51++ macrophages (F, H) or CD51+ SLC (G, I) 4 days after induction of differentiation. (F, G) Young cells. (H, I) old cells. (J–M) Expression of CYP17A1 proteins by young CD51+ SLC after 4 days in culture. (J) CD51+ SLC without primary CYP17A1 antibody. (K) CD51+ SLC before differentiation. (L, M) CD51+ SLC 4 days after induction of differentiation. ND: not detected. The data are expressed as mean ± SEM of three individual experiments. *^,#Significantly different from age-matched YSLC or OSLC controls (*) or from same cell type from young animals (^#) at P < 0.05 respectively. Black scale bars: 50µm in length. White scale bars: 20µm in length.

Figure 6

Effects of aging and macrophages on the proliferation of SLCs. (A) Dividing cells were labeled with EdU for 6 hours on day 3 in culture. YMF or OMF: Young or old CD51++ macrophages cultured alone. YSLC or OSLC Young or old CD51+ SLCs cultured alone. YMF/YSLC or OMF/OSLC: Young or old CD51++ macrophages were co-cultured with young or old CD51+ SLCs. (B) Quantification of EdU labeling indices of CD51+ SLCs cultured separately, CD51++ macrophages (MF) cultured separately, or co-culture of the two cell types (SLC/MF). (C) Quantification of the total cell numbers of cells cultured separately (SLC+MF) or co-cultured cells (SLC/MF). The data are expressed as mean ± SEM of three individual experiments. *^,#Significantly different from age-matched SLC (B) or SLC+MF (C) controls (*) or from same cell type from young animals (^#) at P < 0.05 respectively. Scale bars: 50µm in length.

Figure 7

Effect of aging on the interactions between CD51+ SLCs and CD51++ macrophages. (A, B) Testosterone production by separately cultured (CD51++ MF or CD51+ SLC) or co-cultured (SLC/MF) cells in the presence of differentiation inducing medium. (C, D) Percentage of testosterone production by separately cultured cells (YMF, YSLC, OMF or OSLC) or co-cultured cells within same ages (YSLC/YMF or OSLC/OMF) or across different ages (YSLC/OMF or OSLC/YMF) in the presence of differentiation inducing medium. The data are expressed as mean ± SEM of 3-7 individual experiments. (ND) not detected. *Significantly different from time-matched CD51+ SLC controls at P < 0.05 respectively.

Figure 8

Effects of aging and macrophages on the expressions of LC-, myoid cell- or macrophage-related genes. (A) LC-related genes. (B) Myoid cell-related genes. (C) Macrophage-related genes. (SLC+MF) RNA from the separately cultured cells combined after culture. (SLC/MF) RNA from co-cultured CD51+ SLCs and CD51++ macrophages. The data are expressed as mean ± SEM of three individual experiments. *^,#Significantly different from age-matched SLC+MF controls (*) or from same cell type from young animals (^#) at P < 0.05 respectively.