Estradiol (E2) production, but not progesterone is induced by follicle stimulating hormone (FSH), whereas both were induced by dibutyryl-cAMP. KGN spheroids were incubated in media with testosterone (T) in the absence or presence of FSH (250 ng/ml) or varying concentrations of dibutyryl-cAMP (0–1 mM) for 72 h and levels of E2 (A, B) and progesterone (C, D) were measured by ELISA. FSH significantly induced E2 (A), but not progesterone (D) production. Dibutyryl-cAMP induced a dose-dependent increase in E2 (B) and progesterone (C) production. Representative images of hematoxylin and eosin-stained sections of spheroids treated with FSH (E) or dibutyryl-cAMP (F) showed that FSH did not alter the presence of pyknotic nuclei, but dibutyryl-cAMP at 1 mM increased the number of pyknotic nuclei as compared to the control. Scale bar = 50 µm. Data presented in mean ± SD overlayed with experimental values in dots, or as dot plots of all data points. Student’s t tests were used to examine the statistical difference between FSH treatments. p Value < .05 is denoted by *. One-way ANOVA with Tukey’s HSD for multiple comparisons was performed to examine the effects of dibutyryl-cAMP. Bars that do not share the same letters are significantly different (p value ≤ .05). Three independent experiments, n = 2–3 gels per experiment, each with 256 spheroids.