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.

To gain an insight into potential roles of AR in this tissue, we demonstrated that eCG treatment increased AR expression in a timedependent manner and subsequent treatment with hCG decreased AR expression in mouse fallopian tubes. This expression pattern was positively associated with 17␤-estradiol and testosterone levels in vivo. Immunohistochemical analysis of fallopian tube epithelial cells revealed that nuclear localization of AR increased in parallel with decreased AR in the cytoplasm following eCG treatment. Moreover, we found that treatment with flutamide upregulated AR expression in immature mice in association with a decrease in serum testosterone levels, whereas the same treatment resulted in downregulation of AR expression in gonadotropin-stimulated mice with concomitant decreases in serum 17␤-estradiol concentrations, suggesting that androgen differs from estrogen in the regulation of AR expression. Furthermore, we demonstrated that DES increased both AR protein expression and nuclear location over a 48-h time course. DHT had rapid effects, with induction of AR expression and translocation at 6 h after injection, but unlike DES it had prolonged efficacy. In addition, we provided direct in vivo evidence that nuclear protein interaction between AR and p21 Cip1 , a previously reported AR-regulated gene, was enhanced by gonadotropin stimulation. To our knowledge, this study provides the first demonstration to illustrate that estrogen as a principal regulator may contribute to regulate and activate AR in the fallopian tubes in vivo. androgen receptor; gonadotropins THE PHYSIOLOGICAL FUNCTIONS of androgen receptor (AR) in the male, in particular, regulating the development and function of reproduction, have been well characterized (5, 14, 25), but its regulation and biological roles in the female reproductive tissues still remains to be fully understood (15,20). The expression of AR protein has been investigated in human (2, 7, 17, 35, 60, 71) and rat (23, 44, 61, 65) ovaries and uteri, and a role for AR in the ovulatory process has been demonstrated. For example, hydroxyflutamide, a specific inhibitor of AR, suppresses ovulation rate in rats (8). Furthermore, female AR knockout mice (72) have longer estrous cycles and display reduced fertility with increased age due to lost follicle numbers (24, 58). These observations are consistent with studies in the testicular feminized mice, which have dysfunctional ARs in vivo (33). In addition, activated AR has been shown to modulate uterine growth in rats (48), and AR knockout mice lack exogenous gonadotropin-induced endometrial growth in uteri in vivo (24,58). These studies indicate a potential relationship between AR expression and normal female reproductive function.

Progesterone (P 4 ) regulates many aspects of physiological functions via two nuclear P 4 receptors (PR), PRA and PRB, which are members of a structurally related nuclear hormone receptor superfamily that includes glucocorticoid receptors (GR). The regulation and cellular distribution of PR protein isoforms have been extensively studied in reproductive tissues, but this is not the case in the lung. In the present study, reverse transcriptase (RT)-PCR, Western blotting, and immunolocalization supported the presence of PRA in the lung of female mice, with PRA protein levels significantly increased between postnatal day 7 and 12, declined at postnatal day 26, and minimal in adults when compared to postnatal day 2. The peak was temporally related to postnatal lung maturation in rodents. Immunoreactivity for PR was detected in the alveolar and bronchial epithelia. We then extended this study to examine, for the first time, the regulation of PRA protein expression in female mouse lung in vivo. Neither the increase in endogenous P 4 nor treatment with exogenous P 4 regulated PRA protein expression in female mouse lung. However, treatment of mice with the GR/PR antagonist RU 486, but not Org 31710 (a specific PR antagonist), significantly increased PRA protein expression in parallel to a decrease in GR protein expression. In addition, treatment with the synthetic glucocorticoid dexamethasone led to a decrease in PRA protein expression independent of endogenous P 4 levels. Furthermore, immunoprecipitation followed by Western blot analysis revealed that, under in vivo conditions, PRA physically interacted with GR in mouse lung. Confocal laser microscopy revealed that PRA and GR co-localized in the nuclei of alveolar epithelia cells, whereas nuclear PR and cytoplasmic GR were detected in bronchial epithelium. Taken together, our observations suggest that PRA may be an important physiological factor involved in postnatal lung development and that the regulation of PRA protein expression is not dependent on P 4 , but rather on functional glucocorticoid/GR signaling mediated by protein-protein interaction in the mouse lung.