Possible Role of α1-Antitrypsin in Endometriosis-Like Grafts From a Mouse Model of Endometriosis.

Previous study indicated that bleeding into the peritoneum may accelerate inflammatory response in endometriosis-like grafts in mice. To identify changes in protein levels in the grafts from mice that underwent unilateral ovariectomy (uOVX), which causes bleeding from ovarian arteries and vein, the grafts were generated by injecting a suspension of human endometrial cells in BALB/c nude female mice, and protein profile changes were compared with non-uOVX control mice. The level of α1-antitrypsin (α1-AT) decreased in grafts from nude mice that underwent uOVX. The levels of phosphorylated Akt, mammalian target of rapamycin, S6K, regulatory factors for cell survival, and of phosphorylated nuclear factor κB, an inflammatory mediator, were higher in endometriosis-like grafts from the uOVX group than from the control. The grafts were mostly comprised of stromal cells. The bioactivity of α1-AT was assessed by investigating cytokine expression in protease-activated receptor (PAR) 1/2 agonists-stimulated stromal cells. The PARs promoted the expression of interleukin 8 (IL-8), but treatment with α1-AT blocked IL-8 expression dose dependently. Knocking down α1-AT expression increased the constitutive IL-6, IL-8, and cyclooxygenase 2 expression as well as PAR1 agonist-stimulated IL-6 expression. These findings support the notion that decreased α1-AT protein in the grafts constituted with human endometrial cells in mice may have exacerbated inflammation in endometriosis-like grafts, suggesting the possible involvement of α1-AT in the pathophysiology of endometriosis.

Androgens modulate the morphological characteristics of human endometrial stromal cells decidualized in vitro.

The activated androgen receptor (AR) in decidualizing human endometrial stromal cells (HESCs) regulates genes involved in cytoskeletal organization, cell motility, and cell cycle progression. Androgens also enhance the secretion of prolactin, a widely used marker of decidualized HESCs. The purpose of the present study was to investigate the direct effects of androgens on the ultrastructural changes associated with decidual transformation of HESCs. Primary HESC cultures were established and propagated, and confluent cultures were decidualized for 6 days with 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) and progesterone (P4) in the presence or absence of dihydrotestosterone (DHT). Phase-contrast image analysis demonstrated that DHT increases the shape index of decidualizing cells, which was reversed upon cotreatment with the AR antagonist flutamide. Electron microscopy demonstrated that DHT enhances many of the ultrastructural changes induced by 8-br-cAMP and P4 in HESCs. Decidualizing cells are characterized by an abundant cytoplasm, multiple cell surface projections and, unlike undifferentiated HESCs, form 2 or more cell layers. The DHT further stimulated cytoplasmic expansion, lipid droplet formation, the production of an abundant extracellular matrix, and gap junction formation in decidualized HESCs. The present study demonstrates that androgen signaling has an impact on the morphological and ultrastructural changes associated with the decidual process. Our findings show that androgens promote the development and expansion of cytoplasmic organelles and gap junctions in decidualizing HESCs. These results suggest that androgens in early pregnancy play an important role in promoting the cellular transformation associated with decidualization.

Effect of a dienogest for an experimental three-dimensional endometrial culture model for endometriosis.

The pathogenesis of endometriosis remains poorly understood at least in part because early stages of the disease process are difficult to investigate. Previous studies have proposed a three-dimensional fibrin matrix culture model to study human endometriosis. We examined the ultrastructural features of the endometriosis in this model and assessed the effect of a progestin on endometrial outgrowth and apoptosis in this culture system. Endometrial explants were placed in three-dimensional fibrin matrix culture and treated with and without various concentrations of the progestin dienogest. By the second week, endometrial gland-like formation was established in outgrowths both attached to and at a distance from the explants. These cells formed a combination of clumps and tubular monolayers surrounding a central cavity. Electron microscopy demonstrated that these cells are polarized with microvilli on the apical surface, desmosome-like structures, and basement membrane; features consistent with glandular epithelial cells. Outgrowth of endometrial stromal cells and glandular formation was impaired in response to dienogest in a dose-dependent manner. Our study shows that the human endometrial explants cultured in three-dimensional fibrin matrix establish outgrowths that ultrastructurally resemble ectopic endometrial implants. This model may provide insight into the cellular processes leading to endometriosis formation and enables screening of therapeutic compounds.

Androgen signaling in decidualizing human endometrial stromal cells enhances resistance to oxidative stress.

OBJECTIVE: To investigate the effect of androgens on the expression of genes involved in oxidative stress resistance in decidualized human endometrial stromal cells (HESCs). DESIGN: In vitro experiment. SETTING: University hospital. PATIENT(S): Premenopausal women undergoing hysterectomy for uterine fibroids. INTERVENTION(S): Human endometrial stromal cells isolated from hysterectomy specimens were decidualized with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) and P in the presence or absence of dihydrotestosterone (DHT) at various concentrations. Hydrogen peroxide was used as a source of reactive oxygen species. MAIN OUTCOME MEASURE(S): Prolactin secretion, apoptosis, FOXO1, and the free radical scavengers superoxide dismutase 2 (SOD2) and SOD1 protein expression. RESULT(S): Prolactin production was induced in HESCs in response to 8-br-cAMP and P. Dihydrotestosterone further enhanced the secretion of PRL in cells treated with 8-br-cAMP plus P. The effect of DHT was blocked by the antiandrogen flutamide. Dihydrotestosterone enhanced resistance to oxidative stress-induced apoptosis on decidualized HESCs. Moreover, DHT enhanced FOXO1 expression in parallel with increased SOD2 protein but not with SOD1. CONCLUSION(S): Androgens might play a critical role in the decidualization process at the time of embryo implantation and trophoblast invasion by promoting resistance to oxidative stress.

Human chorionic gonadotropin confers resistance to oxidative stress-induced apoptosis in decidualizing human endometrial stromal cells.

OBJECTIVE: To investigate the effect of hCG on the expression of genes involved in oxidative stress resistance observed in decidualizing human endometrial stromal cells (HESCs). DESIGN: In vitro experiment. SETTING: Saitama Medical University hospital. PATIENT(S): Premenopausal women undergoing hysterectomy for myoma uteri. INTERVENTION(S): HESCs from hysterectomy specimens were isolated and incubated with 8-bromo-cyclic adenosine monophosphate and medroxyprogesterone acetate in the presence or absence of recombinant hCG (rhCG) at various concentrations. Hydrogen peroxide was used as the source of reactive oxygen species (ROS). MAIN OUTCOME MEASURE(S): Apoptotic cells, FOXO1, superoxide dismutase 2 (SOD2), Bax, and Bcl-2 protein expression. RESULT(S): In a dose-dependent manner, rhCG conferred additional protection to decidualizing HESCs against oxidative stress-induced apoptosis. In parallel, rhCG augmented the expression of the forkhead transcription factor FOXO1 and its downstream target, the ROS scavenger SOD2. rhCG also inhibited the expression of the proapoptotic Bax protein and up-regulated antiapoptotic Bcl-2 levels in decidualizing HESCs exposed to ROS. CONCLUSION(S): The data suggest that hCG might improve the uterine environment upon implantation by suppressing apoptotic responses in the maternal decidua under oxidative stress.

Increased ovarian follicle atresia in obese Zucker rats is associated with enhanced expression of the forkhead transcription factor FOXO1.

It is well established that hyperinsulinemia, resulting from insulin resistance, plays a role in the pathophysiology of polycystic ovary syndrome (PCOS). The aim of this study was to investigate if ovarian follicular development and atresia are impaired in obese hyperinsulinemic (fa/fa) Zucker rats. To gain insight into the molecular mechanism of follicular atresia, we also examined the expression and localization of forkhead transcription factor FOXO1, a major regulator of cell fate decisions such as differentiation, cell-cycle arrest, and cell death. Serum insulin but not gonadotropin levels were significantly higher in obese (fa/fa) rats when compared to lean controls. Total ovarian follicle number and the percentage of atretic follicles were also significantly increased in obese (fa/fa) rats. Follicle atresia was associated with nuclear accumulation of FOXO1 transcription factor in TUNEL-positive granulosa cells. These results suggest a role for FOXO1 in granulosa cell apoptosis and increased ovarian follicle atresia associated with hyperinsulinemia.