Tomoko Saito-Fujita

Combinatory approaches prevent preterm birth profoundly exacerbated by gene-environment interactions

There are currently more than 15 million preterm births each year. We propose that gene-environment interaction is a major contributor to preterm birth. To address this experimentally, we generated a mouse model with uterine deletion of Trp53, which exhibits approximately 50% incidence of spontaneous preterm birth due to premature decidual senescence with increased mTORC1 activity and COX2 signaling. Here we provide evidence that this predisposition provoked preterm birth in 100% of females exposed to a mild inflammatory insult with LPS, revealing the high significance of gene-environment interactions in preterm birth. More intriguingly, preterm birth was rescued in LPS-treated Trp53-deficient mice when they were treated with a combination of rapamycin (mTORC1 inhibitor) and progesterone (P4), without adverse effects on maternal or fetal health. These results provide evidence for the cooperative contributions of two sites of action (decidua and ovary) toward preterm birth. Moreover, a similar signature of decidual senescence with increased mTORC1 and COX2 signaling was observed in women undergoing preterm birth. Collectively, our findings show that superimposition of inflammation on genetic predisposition results in high incidence of preterm birth and suggest that combined treatment with low doses of rapamycin and P4 may help reduce the incidence of preterm birth in high-risk women.

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC‐1β

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post‐neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER‐resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild‐type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue‐specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator‐activated receptor coactivator (PGC)‐1β, and Syvn1 mutants showed upregulation of PGC‐1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS‐102 abolished the negative regulation of PGC‐1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post‐translational regulator of PGC‐1β and a potential therapeutic target in obesity treatment.

MicroRNA-200a Locally Attenuates Progesterone Signaling in the Cervix, Preventing Embryo Implantation

Although cervical pregnancy and placenta previa, in which the embryo and placenta embed in or adjacent to the cervix, are life-threatening complications that result in massive bleeding and poor pregnancy outcomes in women, the incidence of these aberrant conditions is uncommon. We hypothesized that a local molecular mechanism is normally in place to prevent embryo implantation in the cervix. The ovarian hormones progesterone (P(4)) and estrogen differentially direct differentiation and proliferation of endometrial cells, which confers the receptive state for implantation: P(4) dominance causes differentiation of the luminal epithelium but increases stromal cell proliferation in preparation of the uterus for implantation. In search for the cause of cervical nonresponsiveness to implantation, we found that the statuses of cell proliferation and differentiation between the uterus and cervix during early pregnancy are remarkably disparate under identical endocrine milieu in both mice and humans. We also found that cervical levels of progesterone receptor (PR) protein are low compared with uterine levels during this period, and the low PR protein levels are attributed to elevated levels of microRNA(miR)-200a in the cervix. These changes were associated with up-regulation of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (200α-HSD) and down-regulation of its transcriptional repressor signal transducer and activator of transcription 5 in the cervix. The results provide evidence that elevated levels of miR-200a lead to down-regulation of P(4)-PR signaling and up-regulation of (200α-HSD) in the cervix, rendering it nonresponsive to implantation. These findings may point toward not only the physiological but also the pathological basis of the cervical milieu in embryo implantation.

STAT3 accelerates uterine epithelial regeneration in a mouse model of decellularized uterine matrix transplantation

Although a close connection between uterine regeneration and successful pregnancy in both humans and mice has been consistently observed, its molecular basis remains unclear. We here established a mouse model of decellularized uterine matrix (DUM) transplantation. Resected mouse uteri were processed with SDS to make DUMs without any intact cells. DUMs were transplanted into the mouse uteri with artificially induced defects, and all the uterine layers were recovered at the DUM transplantation sites within a month. In the regenerated uteri, normal hormone responsiveness in early pregnancy was observed, suggesting the regeneration of functional uteri. Uterine epithelial cells rapidly migrated and formed a normal uterine epithelial layer within a week, indicating a robust epithelial-regenerating capacity. Stromal and myometrial regeneration occurred following epithelial regeneration. In ovariectomized mice, uterine regeneration of the DUM transplantation was similarly observed, suggesting that ovarian hormones are not essential for this regeneration process. Importantly, the regenerating epithelium around the DUM demonstrated heightened STAT3 phosphorylation and cell proliferation, which was suppressed in uteri of Stat3 conditional knockout mice. These data suggest a key role of STAT3 in the initial step of the uterine regeneration process. The DUM transplantation model is a powerful tool for uterine regeneration research.

F4/80+ Macrophages Contribute to Clearance of Senescent Cells in the Mouse Postpartum Uterus

Cellular senescence, defined as an irreversible cell cycle arrest, exacerbates the tissue microenvironment. Our previous study demonstrated that mouse uterine senescent cells were physiologically increased according to gestational days and that their abnormal accumulation was linked to the onset of preterm delivery. We hypothesized that there is a mechanism for removal of senescent cells after parturition to maintain uterine function. In the current study, we noted abundant uterine senescent cells and their gradual disappearance in wild-type postpartum mice. F4/80+ macrophages were present specifically around the area rich in senescent cells. Depletion of macrophages in the postpartum mice using anti-F4/80 antibody enlarged the area of senescent cells in the uterus. We also found excessive uterine senescent cells and decreased second pregnancy success rate in a preterm birth model using uterine p53-deleted mice. Furthermore, a decrease in F4/80+ cells and an increase in CD11b+ cells with a senescence-associated inflammatory microenvironment were observed in the p53-deleted uterus, suggesting that uterine p53 deficiency affects distribution of the macrophage subpopulation, interferes with senescence clearance, and promotes senescence-induced inflammation. These findings indicate that the macrophage is a key player in the clearance of uterine senescent cells to maintain postpartum uterine function.

How does Progesterone Support Embryo Implantation

Abstract: Pregnancy comprises multiple stages with complex interactions of molecules and cells, and previous studies have clarified that progesterone (P4) is a key player in pregnancy. Several animal experimental models have been established to address the detailed mechanisms of P4, and genetically engineered mouse models have especially helped us understand its function. P4 receptor (PR)-null female mice show no ovulation, while PR co-chaperone FKBP52-null mice exhibit implantation failure with normal ovulation. Moderate supplementation of P4 rescues implantation failure in FKBP52-deficient mice but does not restore the capability for pregnancy up to full term, resulting in embryo resorption. Supplementation of a large amount of P4, however, can rescue pregnancy and provide normal reproductive outcomes until parturition. Mouse studies by our groups, and others, have also shown that epigenetic regulation of uterine P4-PR signaling, P4-induced molecular crosstalk between the epithelium and stroma and uterine proliferation-differentiation switching are indispensable for successful implantation. Collectively, P4 orchestrates the whole process of pregnancy in spatiotemporal manners, eventually integrating them toward successful parturition. In this review article, we review the literature on the uterine functions of P4 in pregnancy, with a special focus on the knowledge gained about embryo implantation by studies utilizing mouse models.

Mdm2-p53-SF1 pathway in ovarian granulosa cells directs ovulation and fertilization by conditioning oocyte quality

Functions of tumor suppressor p53 and its negative regulator mouse double minute 2 homolog (Mdm2) in ovarian granulosa cells remain to be elucidated, and the current study aims at clarifying this issue. Mice with Mdm2 deficiency in ovarian granulosa cells [Mdm2‐loxP/progesterone receptor (Pgr)‐Cre mice] were infertile as a result of impairment of oocyte maturation, ovulation, and fertilization, and those with Mdm2/p53 double deletion in granulosa cells (Mdm2‐loxP/p53‐loxP/Pgr‐Cre mice) showed normal fertility, suggesting that p53 induction in the ovarian granulosa cells is detrimental to ovarian function by disturbing oocyte quality. Another model of Mdm2 deletion in ovarian granulosa cells (Mdm2‐loxP/anti‐Mullerian hormone type 2 receptor‐Cre mice) also showed subfertility as a result of the failure of ovulation and fertilization, indicating critical roles of ovarian Mdm2 in ovulation and fertilization. Mdm2‐p53 pathway in cumulus granulosa cells transcriptionally controlled an orphan nuclear receptor steroidogenic factor 1 (SF1), a key regulator of ovarian function. Importantly, MDM2 and SF1 levels in human cumulus granulosa cells were positively associated with the outcome of oocyte maturation and fertilization in patients undergoing infertility treatment. These findings suggest that the Mdm2‐p53‐SF1 axis in ovarian cumulus granulosa cells directs ovarian function by affecting their neighboring oocyte quality.—Haraguchi, H., Hirota, Y., Saito‐Fujita, T., Tanaka, T., Shimizu‐Hirota, R., Harada, M., Akaeda, S., Hiraoka, T., Matsuo, M., Matsumoto, L., Hirata, T., Koga, K., Wada‐Hiraike, O., Fujii, T., Osuga, Y. Mdm2‐p53‐SF1 pathway in ovarian granulosa cells directs ovulation and fertilization by conditioning oocyte quality. FASEB J. 33, 2610–2620 (2019). www.fasebj.org

HIF2α in the uterine stroma permits embryo invasion and luminal epithelium detachment

&NA; Although it has been reported that hypoxia inducible factor 2 &agr; (Hif2a), a major transcriptional factor inducible by low oxygen tension, is expressed in the mouse uterus during embryo implantation, its role in pregnancy outcomes remains unclear. This study aimed to clarify functions of uterine HIF using transgenic mouse models. Mice with deletion of Hif2a in the whole uterus (Hif2a‐uKO mice) showed infertility due to implantation failure. Supplementation with progesterone (P4) and leukemia inhibitory factor (LIF) restored decidual growth arrest and aberrant position of implantation sites in Hif2a‐uKO mice, respectively, but did not rescue pregnancy failure. Histological analyses in Hif2a‐uKO mice revealed persistence of the intact luminal epithelium, which blocked direct contact between stroma and embryo, inactivation of PI3K‐AKT pathway (embryonic survival signal), and failed embryo invasion. Mice with stromal deletion of Hif2a (Hif2a‐sKO mice) showed infertility with impaired embryo invasion and those with epithelial deletion of Hif2a (Hif2a‐eKO mice) showed normal fertility, suggesting the importance of stromal HIF2&agr; in embryo invasion. This was reflected in reduced expression of membrane type 2 metalloproteinase (MT2‐MMP), lysyl oxidase (LOX), VEGF, and adrenomedullin (ADM) in Hif2a‐uKO stroma at the attachment site, suggesting that stromal HIF2&agr; regulates these mediators to support blastocyst invasion. These findings provide new insight that stromal HIF2&agr; allows trophoblast invasion through detachment of the luminal epithelium and activation of an embryonic survival signal.