The role of unfolded protein response in the pathogenesis of endometriosis: contribution of peritoneal fluid.

RESEARCH QUESTION: Endoplasmic reticulum stress (ERS) is caused by the accumulation of the misfolded or unfolded proteins in the endoplasmic reticulum and induces the unfolded protein response (UPR). Peritoneal fluid is important in the pathogenesis of endometriosis. In this study, the role of UPR associated with ERS in endometriosis, and peritoneal fluid, were investigated. DESIGN: Normal, eutopic and ectopic endometrium tissues were divided into menstrual cycle phases, and endometrial stromal cells (ESC) were treated with 10-20% concentration of control peritoneal fluid and peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min, and 24 and 48 h. The UPR signalling proteins were analysed immunohistochemically and immunocytochemically. Data were compared statistically. RESULTS: p-IRE1 was increased in ectopic glandular and stromal cells in the early proliferative phase compared with normal and eutopic endometrium. p-PERK increased in ectopic glandular and stromal cells in the late proliferative phase compared with normal endometrium. ATF6 was increased in ectopic glandular epithelium compared with normal endometrium in the proliferative phases, versus eutopic endometrium in the late secretory phase. p-IRE1 and p-PERK were increased in high concentrations of ESC treated with peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min compared with controls. In ESC treated with peritoneal fluid from women with endometriosis, p-IRE1 decreased at 24-48 h compared with 30 min. CONCLUSIONS: In endometriosis, UPR pathways are activated as highly dependent on cell type and phase. Also, p-PERK and p-IRE1 increased because of exposure to high-dose peritoneal fluid from women with endometriosis in stromal cells. Our findings provide a basis for further studies searching for a potential biomarker for the diagnosis of endometriosis.

Zika Virus-Infected Decidual Cells Elicit a Gestational Age-Dependent Innate Immune Response and Exaggerate Trophoblast Zika Permissiveness: Implication for Vertical Transmission.

Vertical transmission of the Zika virus (ZIKV) causes severe fetal defects, but the exact pathogenic mechanism is unclear. We identified up to a 10,480-fold higher expression of viral attachment factors AXL, GAS6, and PROS1 and a 3880-fold increase in ZIKV infectiousness/propagation in human term decidual stromal cells versus trophoblasts. Moreover, levels of viral attachment factors and ZIKV are significantly increased, whereas expression of innate immune response genes are significantly decreased, in human first trimester versus term decidual cells. ZIKV-infected decidual cell supernatants increased cytotrophoblasts infection up to 252-fold compared with directly infected cytotrophoblasts. Tizoxanide treatment efficiently inhibited Zika infection in both maternal and fetal cells. We conclude that ZIKV permissiveness, as well as innate immune responsiveness of human decidual cells, are gestational age dependent, and decidual cells augment ZIKV infection of primary human cytotrophoblast cultures, which are otherwise ZIKV resistant. Human decidual cells may act as reservoirs for trimester-dependent placental transmission of ZIKV, accounting for the higher Zika infection susceptibility and more severe fetal sequelae observed in early versus late pregnancy. Moreover, tizoxanide is a promising agent in preventing perinatal Zika transmission as well as other RNA viruses such as coronavirus.

Tumor necrosis factor alfa and interleukin 1 alfa induced phosphorylation and degradation of inhibitory kappa B alpha are regulated by estradiol in endometrial cells.

OBJECTIVE: When bound to the inhibitory kappa B (IкB) protein, the transcription factor nuclear factor kappa B (NF-кB) remains inactively in the cytoplasm. Activated NF-кB upregulates the gene expression of many chemokines including monocyte chemoattractant protein-1 and interleukin (IL)-8. We hypothesized that estrogen may regulate IкB phosphorylation and degradation thus influencing NF-кB-dependent gene expression. Regulation of chemokines by estrogen is different in uterine endometrial cells when compared to ectopic endometrial cells of endometriosis. MATERIALS AND METHODS: We investigated the in vivo expression of IкB in normal endometrium and in eutopic and ectopic endometrium of women with endometriosis. We then studied in cultured endometrial cells to assess the effects of estradiol on IкB and NF-кB function. RESULTS: Normal endometrium from mid-late proliferative phase revealed the strongest IкB immunoreactivity throughout the cycle (p<0.05). When compared to paired homologous eutopic endometrium, ectopic endometrium revealed significantly less immunoreactivity for IкB (p<0.05). Moreover, estradiol induced a decrease in tumor necrosis factor-and IL-1-induced IкB phosphorylation, and also decreased the levels of active-NF-кB (p<0.05). CONCLUSION: Our results support the conclusion that one pathway for estradiol-mediated NF-кB inhibition occurs through the down-regulation of IкB phosphorylation. We propose that the estradiol-induced regulation of IкB and consequent reduction in active-NF-кB may affect inflammatory responses in human endometrial cells.

hCG: Biological Functions and Clinical Applications.

Human chorionic gonadotropin (hCG) is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy. hCG can activate various signaling cascades including mothers against decapentaplegic homolog 2 (Smad2), protein kinase C (PKC), and/or protein kinase A (PKA) in several cells types by binding to luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or potentially by direct/indirect interaction with transforming growth factor beta receptor (TGFßR). The molecule displays specialized roles in promoting angiogenesis in the uterine endothelium, maintaining myometrial quiescence, as well as fostering immunomodulation at the maternal-fetal interface. It is a member of the glycoprotein hormone family that includes luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). The α-subunit of hCG displays homologies with TSH, LH, and FSH, whereas the ß subunit is 80-85% homologous to LH. The hCG molecule is produced by a variety of organs, exists in various forms, exerts vital biological functions, and has various clinical roles ranging from diagnosis and monitoring of pregnancy and pregnancy-related disorders to cancer surveillance. This review presents a detailed examination of hCG and its various clinical applications.

In vivo effects of curcumin and deferoxamine in experimental endometriosis.

BACKGROUND: Endometriosis is one of the most common chronic gynecological diseases. OBJECTIVES: The aim of the study was to examine the effects of curcumin and/or deferoxamine on cell proliferation in a rat model of endometriosis. MATERIAL AND METHODS: Thirty female 12-week-old albino Wistar rats, weighing 200-250 g, were used in this study. All the rats underwent ovariectomy and 0.1-mg ß-estradiol 17-valerate pellets were placed intraperitoneally. An experimental model of endometriosis was created in all the animals. To create the experimental model, an approximately 1-cm long section of the uterus was taken, primarily from the right horn of the uterus. Autologous fragments were then placed between the peritoneum and muscle. The animals were divided into 3 groups: Group A, treated only with the vehicle used for curcumin and deferoxamine; group B, treated with curcumin (100 mg/kg body weight); and group C, treated with deferoxamine + curcumin (100 mg/kg body weight). After biopsy samples were obtained, the sections were stained with hematoxylin and eosin. Immunostaining for cytokeratin-7 and proliferating cell nuclear antigen (PCNA) was performed. Blood iron levels were measured using a Perkin Elmer AAnalyst 800 Atomic Absorption Spectrophotometer. RESULTS: The endometrial implant size increased in Group A, but treatment with curcumin (p = 0.01) and deferoxamine + curcumin (p = 0.007) reduced the implant size. In ectopic endometrial epithelial cells, there were significant decreases in PCNA immunoreactivity between groups A and B (p = 0.044) and between groups A and C (p = 0.033). CONCLUSIONS: Treatment with curcumin alone and/or in combination with deferoxamine contributed to a reduction in implant size and cell proliferation in a rat endometriosis model. Iron-chelating agents may act in the same manner when used in women with endometriosis; however, further studies from different perspectives are still needed.

Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology.

The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition. Conversely, the contribution of impaired ER homeostasis by severe/prolong ER stress-mediated UPR signaling pathways to several reproductive tissue pathologies including endometriosis, cancers, recurrent pregnancy loss and pregnancy complications associated with pre-term birth have been reported. This review focuses on ER stress and UPR signaling mechanisms, and their potential roles in female and male reproductive physiopathology involving in menstrual cycle changes, gametogenesis, preimplantation embryo development, implantation and placentation, labor, endometriosis, pregnancy complications and preterm birth as well as reproductive system tumorigenesis.

The Abelson tyrosine kinase (c-Abl) expression on the mouse uterus and placenta during gestational period.

c-Abl is a protein tyrosine kinase which has very important roles in signal transduction, control of the cell cycle, cell motility, proliferation, and inhibition of apoptosis. We hypothesized that c-Abl may play an important role on uterine remodeling during pre-receptive, receptive and non-receptive endometrium. Our aim is to investigate the expression of c-Abl protein tyrosine kinase in uterine remodeling and placental development in mouse gestational stage. We performed c-Abl immunohistochemistry on mouse uterine tissue sections on days 1-9, 11, 13, and 15 of pregnancy. c-Abl was highly upregulated in the uterine luminal epithelium and other endometrial structures including glands and blood vessels in pre-receptive and receptive endometrium. Therefore these results demonstrate a role for c-Abl in uterine remodeling during decidualization, implantation, and placentation throughout gestation.

Increased c-Jun N-terminal kinase activation in human endometriotic endothelial cells.

Endometriosis is a common inflammatory gynecological disease characterized by the presence of endometrial tissue outside of the uterine cavity. The c-Jun N-terminal kinase (JNK) is a subfamily of the mitogen-activated protein kinases (MAPKs) involved in cellular processes ranging from cytokine expression to apoptosis, and is activated in response to inflammation and cellular stress. We hypothesized that inflammatory cytokines in the peritoneal microenvironment increase JNK MAPK activity in endometriotic endothelial cells, and that human endometrial endothelial cells (HEECs) may be involved in inflammatory pathogenesis of endometriosis. Thus, we evaluated the expression of the total- and phosphorylated-(phospho)-JNK in endometrial and endometriotic endothelial cells in vivo, and in HEECs treated with normal peritoneal fluid (NPF), endometriotic peritoneal fluid (EPF), and the inflammatory cytokines interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in vitro. Phospho-JNK immunoreactivity in HEECs in normal endometrium was significantly higher in the early proliferative and late secretory phases compared to other phases. Both eutopic and ectopic HEECs from the early secretory phase also revealed higher phospho-JNK immunoreactivity, compared to their respective cycle-matched normal HEECs. Moreover, HEECs treated with EPF showed significantly higher phospho-JNK levels compared to that in HEECs treated with NPF. In conclusion, our in vivo and in vitro findings suggest that increased phosphorylation of JNK in HEECs from women with endometriosis is likely due to high level of IL-1ß and TNF-α in peritoneal fluid; this in turn may up-regulate inflammatory cytokine expression and thus play a role in the pathogenesis of endometriosis.

Expression and role of interleukin-23 in human endometrium throughout the menstrual cycle and early pregnancy.

Interleukin-23 (IL-23) is a novel cytokine involved in the regulation of organ-specific immune responses. We hypothesized that expression of IL-23 in the human endometrium is menstrual cycle and pregnancy dependent, and is involved in endometrial immune regulation. IL-23 expression and regulation was investigated in the human endometrium and placenta in vivo using immunohistochemistry and in vitro using Western blot and cell viability analyses. IL-23 immunoreactivity in endometrial glandular cells was highest in the late proliferative and early secretory phases, as compared to other cycle phases and first trimester tissues. Endometrial stromal cells (ESC) showed weak IL-23 immunoreactivity without significant changes in intensity and distribution throughout the menstrual cycle. First trimester decidual cells revealed significantly stronger IL-23 staining compared to ESC from non-pregnant endometrium. Both villous cytotrophoblasts and syncytiotrophoblasts also showed positive IL-23 immunoreactivity, with a higher staining in syncytiotrophoblasts. In the trophoblastic cell line HRT8, IL-23 expression increased in a time-dependent manner, but was undetectable in stromal cells under all treatment conditions. ESC treated with recombinant IL-23 showed significantly decreased IL-8 secretion and cell viability. These results suggest a possible regulatory role for IL-23 in the menstrual cycle and in early pregnancy, although the extent and function of this role are yet to be determined.

Immune-endocrine interactions in endometriosis.

Endocrine and immune systems are among the most essential regulators of endometrial physiology, and immune-endocrine interactions are likely to be involved in the pathogenesis of endometriosis. Endometriosis is an inflammatory, estrogen-dependent disease defined by the presence of viable endometrial tissue outside the uterine cavity. Impaired immune response that results in inadequate removal of refluxed menstrual debris has been proposed as a possible causative factor in the development of endometriosis. Moreover, decrease in spontaneous apoptosis of endometrium is the other theory proposed for the development of endometriosis. Endometriotic tissues respond to sex steroids aberrantly and behave differently compared to endometrium in addition to their ability to produce local estrogen. The effects of estrogen on distinct intracellular signaling pathways including MAPK, PI3K/AKT and NF-kappa B may take a role in enhanced endometrial cell survival, altered immune response, and differential cytokine and chemokine expression in endometriosis. Better understanding of immune-endocrine interactions will set the stage for effective immune-targeted therapies not only for endometriosis but also for other endometrial diseases such as adenomyosis, recurrent reproductive failure and implantation-related infertility.

The role of growth factors and cytokines during implantation: endocrine and paracrine interactions.

Implantation, a critical step for establishing pregnancy, requires molecular and cellular events resulting in uterine growth and differentiation, blastocyst adhesion, invasion, and placental formation. Successful implantation requires a receptive endometrium, a normal and functional embryo at the blastocyst stage, and a synchronized dialogue between maternal and embryonic tissues. In addition to the well-characterized role of sex steroids, the complexity of embryo implantation and placentation is exemplified by the number of cytokines and growth factors with demonstrated roles in these processes. Disturbances in the normal expression and action of these cytokines result in an absolute or partial failure of implantation and abnormal placental formation in mice and human. Members of the gp130 cytokine family, interleukin-11 (IL-11) and leukemia inhibitory factor, the transforming growth factor beta superfamily, the colony-stimulating factors, and the IL-1 and IL-15 systems are crucial molecules for a successful implantation. Chemokines are also important, both in recruiting specific cohorts of leukocytes to the implantation site and in trophoblast trafficking and differentiation. This review provides discussion of the embryonic and uterine factors that are involved in the process of implantation in autocrine, paracrine, and/or juxtacrine manners at the hormonal, cellular, and molecular levels.

Expression and regulation of c-Jun N-terminal kinase (JNK) in endometrial cells in vivo and in vitro.

JNK(c-Jun N-terminal kinase) is one of the main types of mitogen-activated protein kinases. JNK modulates inflammation and apoptosis in response to stress. Our hypothesis is that temporal and spatial changes in JNK activity regulate inflammation in human endometrium and that fluctuation in estrogen and progesterone levels may play a role in JNK activation. Therefore, we aimed to determine total-(t-) and active-(phosphorylated, p-) JNK expression in endometrial tissues in vivo by immunohistochemistry, and in vitro by immunocytochemistry and Western blot analysis. Immunohistochemistry revealed moderate cytoplasmic and nuclear t-JNK immunoreactivity, and mostly nuclear p-JNK immunoreactivity throughout the menstrual cycle and early pregnancy. The highest p- and t-JNK immunoreactivity was detected in late secretory phase (P < 0.05). We observed that endometrial stromal cell (ESC)s showed a significant increase in p-JNK expression following 48 h of estrogen combined with progesterone (E(2) + P(4)) withdrawal from the culture conditions, compared to control and non-withdrawal groups (P < 0.05). Upon treatment with JNK inhibitor SP600125, we observed a significantly decreased interleukin (IL)-8 level (P < 0.05) in the presence and absence of E(2). These results demonstrate that JNK expression increases during the late secretory phase when the inflammatory response is highest. Inhibition of IL-8 expression by SP600125 suggests that JNK is involved in regulation of proinflammatory mediators of endometrium.

Effects of excess vitamin B6 intake on cerebral cortex neurons in rat: an ultrastructural study.

The aim of this study was to investigate whether excess of vitamin B6 leads to ultrastructural changes in cerebral cortex of forty-eight healthy albino rats which were included in the study. Saline solution was injected to to the control groups (CG-10, n = 12 for 10 days; CG-15, n = 12 for 15 days; CG-20, n=12 for 20 days). The three experimental groups (EG-10, n = 12; EG-15, n = 12; EG-20, n = 12) were treated with 5 mg/kg vitamin B6 daily for 10 days (EG-10), 15 days (EG-15) and 20 days (EG-20). Brain tissues were prepared by glutaraldehyde-osmium tetroxide double fixation for ultrastructural analysis. No significant changes were observed in the control groups. The ultrastructural analysis revealed that the numbers of damaged mitochondria, lipofuscin granules and vacuoles were significantly higher in all the experimental groups than in the control groups (p < 0.05). However, synaptic density was significantly decreased in the experimental groups as compared to the control groups (p < 0.05). The results suggest that the excess of vitamin B6 intake causes damage to the cerebral cortex due to cellular intoxication and decreased synaptic density. Thus, careful attention should be paid to the time and dose of vitamin B6 recommended for patients who are supplemented with this vitamin.

Immunohistochemical and ultrastructural changes in the renal cortex of cadmium-treated rats.

The aim of this study was to determine the cadmium-induced immunohistochemical and morphological changes in the renal cortex of adult male rats exposed to high doses of cadmium for 30 d. Animals used as controls received a standard diet and water ad libitum. The animals used for this study received 15 ppm CdCl2 in their drinking water for 1 mo. The mean arterial pressure (MAP), the mean blood Cd level, and the mean tissue Cd content were significantly higher when compared to controls (p < 0.01). Immunohistochemical studies demonstrated a weak labeling to type IV collagen and laminin, but a strong labeling to fibronectin in the renal cortex of the Cd-treated animals when compared to controls. The ultrastructural alterations found in Cd-treated rats were a diminution in the amount of filtration slits, increased fusion of foot processes in epithelial cells of the glomeruli, increase of lysosomal structures and pinocytic vesicles as well as large mitochondria in proximal tubule cells, and degenerated cells in distal tubules. Additionally, the glomerular basement membrane was slightly thickened. In conclusion, cadmium toxicity results in alterations in the renal extracellular matrix and tubular or glomerular cells, which could play an important role in renal dysfunction.

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles.

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus-oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an important regulatory molecule for the dominant follicle selection or atresia should be considered.

Estradiol suppresses vascular monocyte chemotactic protein-1 expression during early atherogenesis.

OBJECTIVE: This study was undertaken to determine whether estrogen down-regulates vascular monocyte chemotactic protein-1 expression during the development of atherosclerosis in vivo and to identify the cellular localization of monocyte chemotactic protein-1 expression under baseline conditions and in response to atherogenic stimuli. STUDY DESIGN: Female, homozygous low-density lipoprotein-receptor-deficient mice (n = 68) in a C57BL/6 background underwent ovariectomy, were implanted subcutaneously with 17beta-estradiol or placebo pellets, and were changed to a high cholesterol (1.25%) diet. Thereafter, four mice from each group were killed weekly for 8 weeks, and their aortae were frozen for immunohistochemical analysis. The lipid deposition was identified by Sudan black B staining. Monocyte chemotactic protein-1 expression was detected with a rabbit anti-mice monocyte chemotactic protein-1 antibody, and semiquantitative analysis was performed. RESULTS: Consistent with previous reports, estradiol resulted in diminished vascular lipid deposition (22% +/- 7% vs 15% +/- 6% at 8 weeks of gestation, P <.05). We found that the inhibition of lipid deposition in aortae of animals that were treated with estrogen is associated with a concomitant down-regulation of monocyte chemotactic protein-1 immunoreactivity in aortic endothelial and smooth muscle cells (P <.05). Serum total cholesterol concentrations did not differ between the two treatment groups, which suggests a direct effect of estradiol on the aorta. CONCLUSION: Our findings suggest that one of the mechanisms by which estrogen down-regulates atherogenesis is by the suppression of vascular monocyte chemotactic protein-1 expression, which leads to decreased macrophage recruitment to the arterial wall early in the process.