Evaluation of steroid hormone receptor protein expression in intact cells using flow cytometry

Abstract

Several methods are currently employed to evaluate expression of steroid hormone receptors in tissues and cells, including real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) and western blot assays.These methods require homogenization of cells, thereby preventing evaluation of individual cells or specific cell types in a given tissue sample. In addition, methods such as real-time RT-PCR assess mRNA levels, which may be subject to posttranslational modifications that prevent subsequent production of functional proteins. Flow cytometry is a fluorescence-based technique commonly used to evaluate expression of cell surface and intracellular proteins. This method is especially useful as it allows for single-cell analysis and can be utilized to determine the amount of receptor expressed by individual cells. Flow cytometry is commonly used to analyze immune cell activity and determine functionality based on changes in expression of cell surface molecules, as well as intracellular proteins (such as cytokines). Here, we describe a method to identify protein expression of steroid hormone receptors by rat leukocytes from different organs (spleen, liver and thymus) using flow cytometry. We examined expression of glucocorticoid receptor (GR), androgen receptor (AR) and progesterone receptor (PR) by cells at these sites and were able to demonstrate expression of receptors, as well as the intensity of expression of each receptor. This method is useful for rapid, high throughput measurement of steroid hormone receptors at the protein level in single, intact cells and would be valuable to determine which cells are more likely to respond to steroid hormone treatment.

Visualising Androgen Receptor Activity in Male and Female Mice

“…AR expression in the spleen is somewhat controversial, for instance immunohistochemical staining for AR in all mouse tissues by Takeda et al did not not show reactivity [58]. However, more recent studies have shown modest levels of AR expression and an important role for testosterone in the functioning of the immune system, for instance splenic B cells express AR in mice, as do leukocytes from both spleen and thymus in rats [59,60]. The AR activity seen by luciferase imaging is thus likely to be due to the immune cell contents of the spleen and indeed AR immunoreactivity is not uniform across the spleen tissue (Figure 7).…”

Impact of the Neuroendocrine System on Thymus and Bone Marrow Function

“…Moreover, PR KO mice have reduced evidence of the inhibitory effects of pregnancy hormones on T cell development. In addition, flow-cytometric data has shown the presence of PR on TECs [45] . Curiously there maybe an obscure link between PRs in the thymus and reproductive function: an early study demonstrated that athymic nude and thymec-tomized WT mice both display abnormally small uterus and ovaries, delayed puberty with abnormal estrous cycles and sterility in mature mice.…”

Effect of altrenogest‐treatment of mares in late gestation on adrenocortical function, blood count and plasma electrolytes in their foals

“…Although the low N/L ratio on the day of birth could be indicative for prematurity, it might also be due to an immunomodulatory effect of altrenogest. Progesterone receptors have been identified on rat leucocytes (Butts et al 2007) and immunosuppressive effects of progesterone have been suggested (Szekeres-Bartho et al 2005). On Day 2 after birth, N/L ratio and plasma cortisol levels were positively correlated, indicating that a gestagen-induced immunomodulation may have waned by this time.…”