Comparison of the Effects of Inappropriate Meal Timing-Induced and Genetic Models of Circadian Clock Disruption on Uterine mRNA Expression Profiles.

BACKGROUND: Accumulating evidence reveals that inappropriate meal timing contributes to the development of lifestyle-related diseases. An underlying mechanism is thought to be the disruption of the intracellular circadian clock in various tissues based on observations in both systemic and tissue-specific clock gene-deficient mice. However, whether the effects of conditional clock gene knockout are comparable to those of inappropriate meal timing remains unclear. OBJECTIVES: This study aimed to compare the effects of a recently developed 28-h feeding cycle model with those of a core clock gene Bmal1 uterine conditional knockout (Bmal1 cKO) model on uterine mRNA expression profiles. METHODS: The models were generated by subjecting C57BL/6J mice to an 8-h/20-h feeding/fasting cycle for 2 wk and crossing Bmal1-floxed mice with PR-Cre mice. Microarray analyses were conducted using uterine samples obtained at the beginning of the dark and light periods. RESULTS: The analyses identified 516 and 346, significantly 4-fold and 2-fold, up- or downregulated genes in the 28-h feeding cycle and Bmal1 cKO groups, respectively, compared with each control group. Among these genes, only 7 (1.4%) and 63 (18.2%) were significantly up- or downregulated in the other model. Moreover, most (n = 44, 62.9%) of these genes were oppositely regulated. These findings were confirmed by gene set enrichment analyses. CONCLUSIONS: This study reveals that a 28-h feeding cycle and Bmal1 cKO differently affect gene expression profiles and highlights the need for considering this difference to assess the pathophysiology of diseases associated with inappropriate meal timing.

The Circadian Clock, Nutritional Signals and Reproduction: A Close Relationship.

The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period (Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 (Bmal1). By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock's circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review.

Uterine Deletion of Bmal1 Impairs Placental Vascularization and Induces Intrauterine Fetal Death in Mice.

Recently, it was demonstrated that the expression of BMAL1 was decreased in the endometrium of women suffering from recurrent spontaneous abortion. To investigate the pathological roles of uterine clock genes during pregnancy, we produced conditional deletion of uterine Bmal1 (cKO) mice and found that cKO mice could receive embryo implantation but not sustain pregnancy. Gene ontology analysis of microarray suggested that uterine NK (uNK) cell function was suppressed in cKO mice. Histological examination revealed the poor formation of maternal vascular spaces in the placenta. In contrast to WT mice, uNK cells in the spongiotrophoblast layer, where maternal uNK cells are directly in contact with fetal trophoblast, hardly expressed an immunosuppressive NK marker, CD161, in cKO mice. By progesterone supplementation, pregnancy could be sustained until the end of pregnancy in some cKO mice. Although this treatment did not improve the structural abnormalities of the placenta, it recruited CD161-positive NK cells into the spongiotrophoblast layer in cKO mice. These findings indicate that the uterine clock system may be critical for pregnancy maintenance after embryo implantation.

Androgen-responsive FOXP4 is a target for endometrial carcinoma.

Although low estrogen is considered to suppress uterine endometrial carcinoma, the most cases occur in the postmenopausal stage. After menopause, the production of androgen level also declines. Therefore, to resolve the above enigma, we hypothesize that the postmenopausal decline of androgen is a trigger of its progression. In the present study, to validate this hypothesis, we examine the pathological roles of androgen/AR by analyzing clinical data, culturing endometrioid cancer cell lines, and using murine models. Clinical data show that androgen receptor (AR) expression and serum dihydrotestosterone (DHT) are associated with lower disease-free survival (DFS). DHT suppresses malignant behaviors in AR-transfected human endometrial cancer cells (ECC). In ovariectomized Ptenff/PRcre/+ mice, DHT decreases the proliferation of spontaneously developed murine ECC. In AR-transfected human ECC and Ptenff/PRcre/+ mice, DHT suppresses FOXP4 expression. FOXP4-overexpressed human ECC increases, while FOXP4-knocked-down ECC shows decreased malignant behaviors. DHT/AR-mediated ECC suppression is restored by FOXP4 overexpression. The high FOXP4 expression is significantly correlated with low postoperative DFS. These findings indicate that the androgen/AR system suppresses the malignant activity of endometrial carcinoma and that downstream FOXP4 is another target molecule. These findings will also impact developments in clinical approaches to elderly health.