17β-Estradiol and progesterone have independent and combined effects on C2C12 myotubes following electrical pulse...

17β-Estradiol (E2) and progesterone (P4) may influence exercise-induced muscle damage and repair during the menstrual cycle (MC), but their individual and combined effects remain unclear. This study investigated how hormones influence C2C12 myoblast proliferation, differentiation, and migration under basal conditions and after electrical pulse stimulation (EPS). Myoblasts were treated with E2 and P4 (alone or combined to mimic MC phases). Proliferation (MTT assay), migration (scratch assay), creatine kinase (CK), differentiation (immunohistochemistry), and protein expression (Western blot) were assessed. Elevated E2 (>100 pg/mL) and P4 (>1.5 ng/mL) reduced proliferation, as well as in the early follicular phase (EFP) and mid-luteal phase (P < 0.05). Migration decreased in EFP and late follicular phase (LFP) (P < 0.05), but not with isolated E2/P4 treatment. Differentiation was impaired across all MC phases (reduced myotube diameter; P < 0.05). Although greater E2 accelerated CK recovery post-EPS, the presence of P4 (MC phases) influenced this effect. E2 upregulated HSP70 and myogenin protein content, but P4 counteracted these benefits during specific MC phases. ERα increased in all conditions, suggesting a potential role in mediating the EPS response. In conclusion, E2 and P4 independently decrease myoblast activity, but their combined effects vary across the MC, with a potential role of ERα in this process. These findings highlight the complex interplay of sex hormones in the myogenic response to contractile stress.NEW & NOTEWORTHY 17β-Estradiol (E2) and progesterone (P4) may differentially regulate myoblast function, with high concentrations suppressing proliferation. Notably, E2 enhances muscle repair after electrical stimulation, but P4 may influence this response when combined with E2 to simulate menstrual cycle phases. The discovery that ERα increases across all hormone treatments suggests a key regulatory role in muscle contractile responses. These findings provide novel insights into how menstrual cycle hormones may influence exercise recovery and muscle adaptation.