Adipogenic differentiation is the process by which preadipocytes become mature adipocytes, cells that store energy and regulate metabolic homeostasis. During differentiation, neutral lipids that accumulate in adipocytes can be detected using stains and used as an index of cell differentiation. However, imaging tools for evaluating intracellular lipid droplets remain at their infancy. Nutrition, stress, or chemical exposure can dysregulate adipogenic differentiation and lipid metabolism. Therefore, the aims of this study were to develop an accurate, standardized approach to quantify lipid droplet size of mature adipocytes and a clustering approach to analyze the total lipid content per adipocyte. For the lipid droplet analysis, we used two approaches, the free online computer software of reference, ImageJ, and another free online computer software, CellProfiler. For ImageJ, we used an already developed macro designed to identify particles and quantify their area, and for CellProfiler, we developed a new analysis pipeline. Our results show that CellProfiler is able to accurately identify a greater number of lipid droplets compared to ImageJ. A clustering analysis is also possible using CellProfiler which allows for the quantification of total lipid content per individual adipocyte to provide insight into single-cell responsiveness to adipogenic stimuli. CellProfiler streamlines the lipid droplet phenotypic analysis of adipocytes compared to more traditional analysis methods. In conclusion, this novel image analysis tool can provide with a more precise evaluation of lipid droplet and adipogenesis dysregulation, a critical need in the understanding of metabolic disorders.

The endocrine disrupting chemical bisphenol A (BPA) increases adipose tissue mass in vivo and promotes adipogenesis in vitro. However, mechanisms explaining BPA’s obesogenic effect remain unknown. We investigated the effects of gestational BPA and its analogue, bisphenol S (BPS), exposure on the adipogenic differentiation ability of fetal preadipocytes and the role of endoplasmic reticulum stress in regulating this process. Pregnant sheep (n=7-8/group) mated to the same male were exposed to BPA or BPS from days 30 to 100 of gestation, and pregnancies terminated 20 days later. Adipose tissue was harvested and fetal preadipocytes isolated. Adipose tissue gene expression, adipocyte size, preadipocyte gene expression, adipogenic differentiation, and dynamic expression of genes involved in adipogenesis and endoplasmic reticulum stress were assessed. Gestational BPA enhanced adipogenic differentiation in female, but not male, preadipocytes. The unfolded protein response (UPR) pathway was upregulated in BPA-exposed female preadipocytes supportive of a higher endoplasmic reticulum stress. Increased expression of estradiol receptor 1 and glucocorticoid receptor in female preadipocytes suggests that this may be a potential cause behind the sex-specific effects observed upon BPA exposure. Gestational BPS affected adipogenic terminal differentiation gene expression in male preadipocytes, but not adipogenic differentiation potential. We demonstrate for the first time that gestational BPA exposure can modulate the differentiation ability of fetal preadipocytes. UPR upregulation in gestationally BPA-exposed female preadipocytes may contribute to the increased preadipocyte’s adipogenic ability. The marked sex-specific effect of BPA highlights higher susceptibility of females to bisphenol A and potentially, a higher risk to develop obesity in adulthood.