Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function

Abstract

Progesterone and its interaction with nuclear progesterone receptors (PR) PR-A and PR-B play a critical role in the regulation of female reproductive function in all mammals. However, our knowledge of the regulation and possible cellular function of PR protein isoforms in the fallopian tube and uterus in vivo is still very limited. In the present study, we revealed that equine chorionic gonadotropin (eCG) treatment resulted in a time-dependent increase in expression of both isoforms, reaching a maximal level at 48 h in the fallopian tube. Regulation of PR-A protein expression paralleled that of PR-B protein expression. However, in the uterus PR-B protein levels increased and peaked earlier than PR-A protein levels after eCG treatment. With prolonged exposure to eCG, PR-B protein levels decreased, whereas PR-A protein levels continued to increase. Furthermore, subsequent treatment with human (h)CG decreased the levels of PR protein isoforms in both tissues in parallel with increased endogenous serum progesterone levels. To further elucidate whether progesterone regulates PR protein isoforms, we demonstrated that a time-dependent treatment with progesterone (P4) decreased the expression of PR protein isoforms in both tissues, whereas decreases in p27, cyclin D2, and proliferating cell nuclear antigen protein levels were observed only in the uterus. To define the potential PR-mediated effects on apoptosis, we demonstrated that the PR antagonist treatment increased the levels of PR protein isoforms, induced mitochondrial-associated apoptosis, and decreased in epidermal growth factor (EGF) and EGF receptor protein expression in both tissues. Interestingly, immunohistochemistry indicated that the induction of apoptosis by PR antagonists was predominant in the epithelium, whereas increase in PR protein expression was observed in stromal cells of both tissues. Taken together, these observations suggest that 1) the tissue-specific and hormonal regulation of PR isoform expression in mouse fallopian tube and uterus, where they are potentially involved in regulation of mitochondrial-mediated apoptosis depending on the cellular compartment; and 2) a possible interaction between functional PR protein and growth factor signaling may have a coordinated role for regulating apoptotic process in both tissues in vivo.

Dynamic regulation of estrogen receptor-α isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

“…To standardize the assay for measurement of ER proteins, we examined different starting protein concentrations for each sample. This study demonstrated the linearity and validity of ADU for all immunoreactive bands in Western blot analysis (55,57). All steps were carried out at room temperature unless otherwise stated.…”

“…The aim of experiment 2 was to examine whether exogenous estrogens regulated ER␣ isoform expression in mouse fallopian tubes; 26-day-old female mice, in which endogenous concentrations of E 2 were expected to be minimal (55,57), were given a single subcutaneous (sc) injection of 0. The aim of experiment 3 was to determine the effect of the steroidal ER antagonist ICI 182,780 (Faslodex; fulvestrant) (21) and progesterone (P 4) on the regulation of ER␣ isoform expression in the fallopian tube; 26-day-old female mice received either E 2 (0.5 g/g BW sc) or PPT (100 g/g BW sc) for 4 days and, in addition, an intraperitoneal injection of either 8.3 mg ICI 182,780 (Tocris Cookson)/ kg BW, 2.5 mg P 4/each animal in 100 l of sesame oil (vehicle), or vehicle alone for 1-2 days.…”

“…The protein content was determined using the BCA protein assay (Pierce). Western blot analyses were performed using standard procedures to evaluate the abundance and distribution of ER␣, ER␤, ␤-actin, IGF-I, IGF-II, and VEGF (55,57). For each time point or treatment, equivalent amounts of protein from at least five individual fallopian tube extracts were pooled; 30 g of protein were directly electrophoresed on either 4 -12% Bis-Tris gels or 10 -20% Tris-glycine gels (Novex, San Diego, CA).…”

Liver-derived IGF1 enhances the androgenic response in prostate

“…To standardize the assay for the measurement of AR proteins, we examined different starting protein concentrations for individual sample. This study demonstrated the linearity and validity of ADU for all immunoreactive bands in western blot analysis (Shao et al 2006). All steps were carried out at room temperature, unless otherwise stated.…”

“…The protein preparation of the selected VP lobes was essentially performed as described previously (Shao et al 2006(Shao et al , 2007. The protein content was determined using the BCA protein assay (Pierce, Rockford, IL, USA).…”