Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro

Abstract

Background: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG.

Methods: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated.

Results: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis.

Conclusions: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.

Keywords: Bioassay; LH; Leydig; Testosterone; cAMP; hCG.

Fig. 1

Comparison between LH- and hCG-induced cAMP production by dose–response experiment. Murine primary Leydig cells were stimulated by increasing doses of hCG and LH, in the presence of 500 μM IBMX. Total cAMP was measured after 3 h of incubation and cAMP levels were normalized as percentage of the maximal response. All the results are represented as means ± SEM in a logarithmic X-axis, then non-linear regressions were plotted. The EC₅₀ cAMP values were compared (LH EC₅₀ = 192 ± 53.96 pM; hCG EC₅₀ = 18.64 ± 10.14 pM; means ± SEM; Mann-Whitney’s U-test; P = 0.0286; n = 4)

Fig. 2

Evaluation of pErk1/2 and pCreb activation upon LH and hCG treatment. a Mouse Leydig cells were stimulated by increasing doses of LH and hCG. The phosphorylation of Erk1/2 and Creb was evaluated after 15 min by Western blotting (image representative of five independent experiments). b, c Densitometric analysis of pErk1/2 (b) and pCreb (c) signals. The values were normalized over total ERK and represented as means ± SEM, then statistically evaluated (§ = significant vs control; * = significant LH vs hCG; two-way ANOVA and Bonferroni post-tests; P < 0.05; n = 5)

Fig. 3

Stard1 gene expression analysis. The expression of Stard1 gene was evaluated in 12 h EC₈₀ LH- or hCG-stimulated mouse primary Leydig cells by real-time PCR. Unstimulated cells served as control. Each value was normalized over the Hprt gene expression and graphically represented as fold increase over unstimulated controls in relative units scale (means ± SEM). Gene expression levels were compared (§ = significant vs control; Mann-Whitney’s U-test; P < 0.05; n = 4)

Fig. 4

Comparison between LH- and hCG-induced testosterone production in murine primary Leydig cells. The steroid was measured in 24 h-stimulated cells by increasing LH and hCG doses, in the presence of 500 μM IBMX. a Total testosterone levels was measured by immuno-assay and represented as means ± SEM in ng/ml, then non-linear regressions were plotted. b Testosterone levels were normalized in percentage of the maximal response. EC₅₀ values were statistically compared (hCG EC₅₀ = 36.66 ± 12.25 pM; LH EC₅₀ = 50.08 ± 18.03 pM; means ± SEM; Mann-Whitney’s U-test; P = 1.00; n = 5)

Fig. 5

Analysis of LH/hCG-induced ERK and CREB phosphorylation in the presence/absence of specific inhibitors. Murine primary Leydig cells were stimulated 15 min by EC₈₀ LH or hCG, in the presence or in the absence of PKA and MEK inhibitors (H-89 and U0126, respectively). pERK and pCREB levels were evaluated by Western blotting (image representative of three independent experiments)

Fig. 6

Evaluation of testosterone production upon LH or hCG stimulation in the presence or absence of specific inhibitors, in mouse Leydig cells. The cells were stimulated 24 h by EC₈₀ LH and hCG, in the presence or absence of H-89 or U0126. Total testosterone levels were measured by immune-assay and represented as means ± SEM (§ = significant vs “unstimulated, no inhibitors”; * = significant LH/hCG vs corresponding LH/hCG samples without inhibitor; Mann-Whitney’s U-test; P < 0.05; n = 4)