Sexual Dimorphism's impact on adipogenesis: A three-dimensional in vitro model treated with 17β-estradiol and testosterone

Abstract

Using a three-dimensional (3-D) in vitro culture model, we report the dose dependent effect of 17β-estradiol and testosterone on the adipogenic differentiation and maturation of human adipose derived stem cells (hASCs) obtained from female and male patients. Considering sexual dimorphism, we expected male and female adipocytes to respond differently to the sex steroids. Both male and female hASC spheroids were exposed to 100 nM and 500 nM of 17β-estradiol and testosterone either at the beginning of the adipogenic maturation (Phase I) to discourage intracellular triglyceride accumulation or exposed after adipogenic maturation (Phase II) to reduce the intracellular triglyceride accumulation. The results show that 17β-estradiol leads to a dose dependent reduction in intracellular triglyceride accumulation in female hASC spheroids compared to the both untreated and testosterone-treated cells. Affirming our hypothesis, 17β-estradiol prevented intracellular triglyceride accumulation during Phase I, while it stimulated lipolysis during Phase II. PPAR-γ and adiponectin gene expression also reduced upon 17β-estradiol treatment in female cells. Interestingly, 17β-estradiol and testosterone had only a modest effect on the male hASC spheroids. Collectively, our findings suggest that 17β-estradiol can prevent fat accumulation in adipocytes during early and late stages of maturation in females.

Keywords: 3-D spheroids; Adiponectin; Female adipocyte; Male adipocyte; PPAR-γ; triglyceride.

Figure 1.

Timeline of experiments (Created using Biorender.com)

Figure 2.

Bright field morphology of spheroids prepared using hASCs isolated from the female adipose tissue. Control = Adipogenic maturation medium containing no 17β-estradiol or testosterone. E = Adipogenic maturation medium containing either 100 nM or 500 nM 17β-estradiol. T = Adipogenic maturation medium containing either 100 nM or 500 nM testosterone. Scale bars = 100 μm.

Figure 3.

Biochemical characterization of spheroids formed from hASCs isolated from the female adipose tissue. Quantitative measurement of the spheroid sizes during (A) Phase I and (B) Phase II experiments (n > 50). DNA quantification during (C) Phase I and (D) Phase II experiments. Protein content during (E) Phase I and (F) Phase II experiments. Normalized triglyceride content during (G) Phase I and (H) Phase II experiments. Control = Adipogenic maturation medium containing no 17β-estradiol and testosterone. E = Adipogenic maturation medium containing either 100 nM or 500 nM 17β-estradiol. T = Adipogenic maturation medium containing either 100 nM or 500 nM testosterone. Error bars represent 95% confidence intervals. * p ≤ 0.05 against control on same day; # p ≤ 0.05 between E 100 and E 500 on the same day; $ ≤ 0.05 between T 100 and T 500 on the same day. The spheroid diameters and normalized triglyceride content showed statistically significant differences across days.

Figure 4.

Bright field morphology of spheroids prepared using hASCs isolated from the male adipose tissue. Control = Adipogenic maturation medium containing no 17β-estradiol or testosterone. E = Adipogenic maturation medium containing either 100 nM or 500 nM 17β-estradiol. T = Adipogenic maturation medium containing either 100 nM or 500 nM testosterone. Scale bars = 100 μm.

Figure 5.

Biochemical characterization of spheroids formed from hASCs isolated from the male adipose tissue. Quantitative measurement of the spheroid sizes during (A) Phase I and (B) Phase II experiments (n > 50). DNA quantification during (C) Phase I and (D) Phase II experiments. Protein content during (E) Phase I and (F) Phase II experiments. Normalized triglyceride content during (G) Phase I and (H) Phase II experiments. Control = Adipogenic maturation medium containing no17β-estradiol and testosterone. E = Adipogenic maturation medium containing either 100 nM or 500 nM 17β-estradiol. T = Adipogenic maturation medium containing either 100 nM or 500 nM testosterone. Error bars represent 95% confidence intervals. * p ≤ 0.05 against control on same day; # p ≤ 0.05 between E 100 and E 500 on the same day, $ ≤ 0.05 between T 100 and T 500 on the same day. The spheroid diameters and normalized triglyceride content showed statistically significant differences across days.

Figure 6.

PPAR-γ and adiponectin gene expression of spheroids formed from hASCs isolated from the female adipose tissue. The gene expression was measured after 21 days in Phase I and after 36 days in Phase II experiments. Error bars represent 95% confidence intervals. p ≤ 0.05 against control on same day.