Progesterone-mediated effects on gene expression and oocyte-cumulus complex transport in the mouse fallopian tube.

BACKGROUND: The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor are involved in regulating both tubal ciliary beating and muscular contractions, likely via both genomic and non-genomic actions. METHODS: To provide more details of the underlying mechanisms, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at 20 min, 2 h and 8 h post progesterone treatment using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice that were injected with one of the progesterone receptor antagonists, Org 31710 or CDB2194. RESULTS: Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, consistent with the proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were identified as up-regulated. Analyses using quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin1 and a down-regulation of its receptor Endothelin receptor A by progesterone. We also confirmed that treatment with progesterone receptor antagonists before ovulation accelerates the transport of the oocyte cumulus complex. CONCLUSIONS: This is the first study showing that progesterone regulates the expression of endothelin1 and endothelin receptor A in the fallopian tube. Together with previous studies of the effects of endothelin on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction and eventually gamete transport in the fallopian tube.

The classical progesterone receptor mediates the rapid reduction of fallopian tube ciliary beat frequency by progesterone.

BACKGROUND: The transport of gametes as well as the zygote is facilitated by motile cilia lining the inside of the fallopian tube. Progesterone reduces the ciliary beat frequency within 30 minutes in both cows and mice. This rapid reduction suggest the involvement of a non-genomic signaling mechanism, although it is not known which receptors that are involved. Here we investigated the possible involvement of the classical progesterone receptor in this process. METHOD: The ciliary beat frequency of mice fallopian tube was measured ex vivo using an inverted bright field microscope and a high speed camera. The effects of the agonists progesterone and promegestone and an antagonist, mifeprestone, were investigated in wildtype mice. The effect of progesterone was also investigated in mice lacking the classical progesterone receptor. RESULTS: Progesterone, as well as the more specific PR agonist promegestone, significantly reduced the CBF at concentrations of 10-100 nanomolar within 10-30 minutes. In the absence of progesterone, the PR antagonist mifeprestone had no effect on the ciliary beat frequency at a concentration of 1 micromolar. When ciliated cells were pre-incubated with 1 micromolar mifeprestone, addition of progesterone did not reduce the ciliary beat frequency. Accordingly, in ciliated cells from mice not expressing the classical progesterone receptor, exposure to 100 nanomolar progesterone did not reduce the ciliary beat frequency. CONCLUSIONS: This is the first study to provide comprehensive evidence that the classical progesterone receptor mediates the rapid reduction of the tubal ciliary beat frequency by progesterone.

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube.

BACKGROUND: The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations. METHODS: A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone. RESULTS: The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls. CONCLUSIONS: The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

S100A7 (Psoriasin), highly expressed in ductal carcinoma in situ (DCIS), is regulated by IFN-gamma in mammary epithelial cells.

BACKGROUND: The aim of the present work was to explore signal transduction pathways used in the regulation of S100A7 (psoriasin). Members of the S100 gene family participate in many important cellular functions. Psoriasin, S100A8 (calgranulin A) and S100A9 (calgranulin B) are expressed in ductal carcinoma in situ (DCIS), as well as in the hyperproliferative skin disease, psoriasis. In the latter condition, a disturbance in the STAT pathway has recently been reported. This pathway is implicated in the regulation of IFN-gamma, widely recognized as a key cytokine in psoriasis. IFN-gamma also exerts anti-tumor action in a number of tumor cell types, including breast cancer. We therefore examined the effect of IFN-gamma and STAT-signaling on the psoriasin expression. METHODS: We established a TAC2 mouse mammary epithelial cell line with tetracycline-inducible psoriasin expression (Tet-Off). Viability in cell culture was estimated using MTS assay. Protein and gene expression were evaluated by Western blotting and quantitative real-time PCR. Statistical analyses were assessed using a one-tailed, paired t-test. RESULTS: We report the downregulation of psoriasin by IFN-gamma in the MDA-MB-468 breast cancer cell line, as well as the downregulation of psoriasin induced by anoikis in cell lines derived from different epithelial tissues. In contrast, IFN-gamma had no suppressive effect on calgranulin A or calgranulin B. IFN-gamma is an important activator of the STAT1 pathway and we confirmed an active signaling pathway in the cell lines that responded to IFN-gamma treatment. In contrast, in the SUM190 breast carcinoma cell line, IFN-gamma did not suppress the expression of endogenous psoriasin. Moreover, a reduced phosphorylation of the STAT1 protein was observed. We showed that IFN-gamma treatment and the inhibition of the transcription factor NFkappaB had a synergistic effect on psoriasin levels. Finally, in TAC2 cells with tetracycline-induced psoriasin expression, we observed the increased viability of psoriasin-expressing cells after IFN-gamma treatment. CONCLUSION: Our data support the possibility that psoriasin expression is transcriptionally suppressed by IFN-gamma and that this effect is likely to be mediated by the activation of the STAT1 signaling pathway. The increased viability of psoriasin-expressing cells after IFN-gamma exposure suggests that psoriasin expression leads to the development of an apoptosis-resistant phenotype.