RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Establishment of an immortalized stromal cell line derived from human Endometriotic lesion.

BACKGROUND: Endometriosis is a benign gynecological disease with obviously feature of estrogen-dependence and inflammatory response. The applications of primary endometriotic stromal cells in research of endometriosis are restricted for short life span, dedifferentiation of hormone and cytokine responsiveness. The objective of this study was to establish and characterize immortalized human endometriotic stromal cells (ihESCs). METHODS: The endometriotic samples were from a patient with ovarian endometriosis and the primary endometriotic stromal cells were isolated from the endometriotic tissues. The primary cells were infected by lentivirus to establish telomerase reverse transcriptase (hTERT)-induced immortalized cells. Quantification of mRNA and proteins was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot. CCK-8 assay and EdU labeling assay were assigned to assess the growth of ihESCs. Karyotype assay was performed to detect the chromosomes of ihESCs. Colony formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities. RESULTS: ihESCs continuously overexpressed hTERT via infection of lentivirus and significant extended the life span reaching 31 passages. The morphology, proliferation and karyotype of ihESCs remained unchanged. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins and estrogen/progesterone receptors (ERs and PRs) were unaltered. Furthermore, the treatment of estrogen increased the proliferation and EMT of ihESCs. Lipopolysaccharides (LPS) and IL-1ß remarkably induced inflammatory response. The clonogenesis ability of ihESCs was consistent with primary cells, which were much lower than Ishikawa cells. In addition, nude mouse tumorigenicity assay demonstrated that ihESCs were unable to trigger tumor formation. CONCLUSION: This study established and characterized an immortalized endometriotic stromal cell line that exhibited longer life span and kept the cellular morphology and physiological function as the primary cells. The immortalized cells remained normal feedback to estrogen and inflammatory response. Moreover, the immortalized cells were not available with tumorigenic ability. Therefore, ihESCs would be serviceable as in vitro cell tool to investigate the pathogenesis of endometriosis.

Effect of interleukin-1ß and lipoxin A4 in human endometriotic stromal cells: Proteomic analysis.

AIM: Lipoxin A4 (LXA4 ) can function as an endogenous 'breaking signal' in inflammation and plays an important role in the progression of endometriosis. The proteome responses to interleukin-1ß (IL-1ß) or LXA4 in human endometriotic stromal cells (ESC) are not well understood. METHODS: In this study, primary ESC were cultured from ovarian endometriosis tissue. Three groups were established: the control group; the IL-1ß stimulation group; and the IL-1ß and LXA4 incubation group. Proteins were assessed on 2-D polyacrylamide gel electrophoresis (2D-PAGE), and differentially expressed protein spots were further identified on matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI-TOF-MS). Wound healing and transwell assays were performed to assess the migration and invasion of ESC after treatment. RESULTS: In total, 40 differentially expressed protein spots were identified successfully on MALDI-TOF-MS. The proteins identified were related to cell structure, metabolism, signal transduction, protein synthesis and membrane structure, processes that may be involved in the development of endometriosis. Vinculin and IL-4 were further analyzed on western blot and quantitative real-time polymerase chain reaction. Moreover, LXA4 could suppress the migration and invasion of ESC induced by IL-1ß. CONCLUSION: LXA4 may inhibit the progression of endometriosis partly by lowering or raising the effect of IL-1ß, mediated via some inflammation-related proteins (e.g. vinculin) and immune response-related protein (e.g. IL-4) in vitro.

Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Identification of a novel deletion mutation in DPY19L2 from an infertile patient with globozoospermia: a case report.

BACKGROUND: Male infertility is an increasing medical concern worldwide. In most cases, genetic factors are considered as the main cause of the disease. Globozoospermia (MIM102530) (also known as round-headed sperm) is a rare and severe malformed spermatospermia caused by acrosome deficiency or severe malformation. A subset of genetic mutations, such as DNAH6, SPATA16, DPY19L2, PICK1, and CCIN related to globozoospermia, have been reported in the past few years. The DPY19L2 mutation is commonly found in patients with globozoospermia. Herein, a 180-kbp homozygote deletion at 12q14.2 (g.63950001-64130000) was identified by copy number variation sequencing (CNVseq) in a patient with a globozoospermia, including the complete deletion of DPY19L2. CASE PRESENTATION: A 27-year-old patient at the First Affiliated Hospital of Xiamen University was diagnosed with infertility because, despite normal sexual activity for 4 years, his wife did not conceive. The patient was in good health with no obvious discomfort, no history of adverse chemical exposure, and no vices, such as smoking and drinking. The physical examination revealed normal genital development. However, semen tests showed a normal sperm count of 0% and the morphology was the round head. Sperm cytology showed that acrosomal enzyme was lower than normal. Reproductive hormones were in the normal range. B ultrasound did not show any abnormal seminal vesicle, prostate, bilateral testis, epididymis, and spermatic veins. The karyotype was normal, 46, XY, and no microdeletion of Y chromosome was detected. However, a homozygous deletion mutation was found in DPY19L2, which was further diagnosed as globozoospermia. CONCLUSIONS: The present study reported a male infertility patient who was diagnosed with globozoospermia. The analysis of gene mutations revealed that DPY19L2 had a homozygous mutation, which was the primary cause of globozoospermia.

High expression of ZEB1 in endometriosis and its role in 17ß-estradiol-induced epithelial-mesenchymal transition.

Endometriosis is an estrogen-dependent disease associated with pain and infertility. The objective of this study was to determine the expression of ZEB1 in endometriosis and its role in 17ß-estradiol (E2)-induced epithelial-mesenchymal transition (EMT). 25 patients with endometriosis and 16 endometriosis-free patients were recruited for the study. Tissue expression of EMT makers was investigated by immunohistochemistry, then the expression of ZEB1 was quantified by qRT-PCR, immunohistochemistry, and western blot. The proliferation, DNA replication, and migration and invasion in ZEB1 knockdown Ishikawa cells were further respectively performed by MTS, Edu, wound healing and transwell assays. Luciferase assay was used to measure the ZEB1 promoter activity. Our results show that protein levels of E-cadherin and Keratin 18 decreased in endometriotic tissues. Meanwhile the expressions of ZEB1, Vimentin, and N-cadherin were significantly increased in endometriotic tissues. Down-regulation of ZEB1 inhibited Ishikawa cells proliferation, migration, invasion and EMT. E2 promoted the expression of ZEB1 through the ER genomic pathway, which contributed to the EMT process. The -1401 bp - -1901 bp region in the ZEB1 promoter was the main target of the E2 activity. The present results suggest that a high expression of ZEB1 plays an important role in the pathogenesis of endometriosis, and it may serve as a potential therapeutic target for endometriosis.

Lipoxin A4 Suppresses Estrogen-Induced Epithelial-Mesenchymal Transition via ALXR-Dependent Manner in Endometriosis.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is essential for embryogenesis, fibrosis, and tumor metastasis. Aberrant EMT phenomenon has been reported in endometriotic tissues of patients with endometriosis (EM). In this study, we further investigated the molecular mechanism of which lipoxin A4 (LXA4) suppresses estrogen (E2)-induced EMT in EM. STUDY DESIGN: The EMT markers were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in eutopic endometrial epithelial cells (EECs) or investigated by immunohistochemistry and qRT-PCR in endometriotic lesion of EM mice. The invasion and migration under different treatments were assessed by transwell assays with or without Matrigel. The messenger RNA (mRNA) and activities of matrix metalloproteinase 2 (MMP-2) and MMP-9 were determined by qRT-PCR and gelatin zymography, respectively. Luciferase reporter assay was used to measure the activity of zinc finger E-box binding homeobox 1(ZEB1) promoter. The level of E2 in endometriotic tissues was assessed by enzyme-linked immunosorbent assay. RESULTS: In eutopic EECs, stimulatory effects of E2 on EMT progress, migration, and invasion were all diminished by LXA4. Lipoxin A4 reduced E2-induced ZEB1 promoter activity. Lipoxin A4 also attenuated the phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase induced by E2. Co-incubation with Boc-2 rather than DMF antagonized the influence of LXA4. Animal experiments showed that LXA4 inhibited the EMT progress, MMP expression, and proteinase activities of endometriotic lesion in an LXA4 receptor (ALXR) manner, which suppressed the progression of EM. ZEB1 mRNA expression was upregulated and well correlated with E2 level in human endometrium. CONCLUSION: Lipoxin A4 suppresses E2-induced EMT via ALXR-dependent manner in eutopic EECs, which reveals a novel biological effect of LXA4 in EM.

[Surveillance of schistosomiasis in national surveillance sites of Zhenjiang City, 2005-2010].

OBJECTIVE: To master the changes of schistosomiasis epidemic situation in national surveillance sites of Zhenjiang City, Jiangsu Province. METHODS: According to the scheme of the national schistosomiasis surveillance, the Shicheng Village of Yangzhong County and Sanzhou Village of Dantu District were selected as the national schistosomiasis surveillance sites, and from 2005 to 2010, the schistosomiasis morbidity and Oncomelania hupensis status were surveyed and the results were analyzed statistically. RESULTS: In 2010, in the Shicheng Village, the reduction rates of mean living snail density, infected snail density, area with infected snails, and positive blood tests in residents were 98.4%, 0, 0, 71.8% respectively, and in the Sanzhou Village, the reduction rates were 70.4%, 100%, 100% and 81.5%, respectively compared with those in 2005. No acute infections were found in the 2 villages during the period of 6 consecutive years. CONCLUSION: In the national surveillance sites of Zhenjiang City, the schistosomiasis morbidity has been effectively controlled. However, the areas with snails change little. Therefore, the comprehensive management of snail environment in the marshland should be strengthened in the future.

ZEB1 promotes epithelial-mesenchymal transition in cervical cancer metastasis.

OBJECTIVE: To investigate role of Zinc finger E-box binding homeobox 1 (ZEB1) in cervical cancer tissue (squamous cell carcinoma, SCC). DESIGN: Exploratory study. SETTING: University hospital. PATIENT(S): Sixty patients with SCC, including stage CINIII (n = 10), IB1 (n = 10), IB2 (n = 10), IIA1 (n = 10), IIA2 (n = 10), and IIB (n = 10) were studied. INTERVENTION(S): Caski cells were transfected with recombinant shZEB1 lentivirus or shCtrl lentivirus to generate stable ZEB1-knockdown Caski cells. MAIN OUTCOME MEASURE(S): ZEB1 expression was analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry in cervical cancer tissues. ZEB1 expression in Caski cells was down-regulated by short-hairpin RNA (shRNA) interference, and changes in ZEB1 expression corresponded with changes in the proliferation and migratory ability of Caski cells. RESULT(S): Quantitative real-time polymerase chain reaction and immunohistochemistry results revealed that ZEB1 expression and the ratio of Vimentin to E-cadherin were high in 27 of 50 SCC patients and correlated with advanced International Federation of Gynecology and Obstetrics stage, tumor size >4 cm, and parametrial invasion. However, the expression of ZEB1 in cervical cancer tissue was independent of age and SCC antigen level. Transfection of ZEB1 shRNA in Caski cells significantly decreased the messenger RNA and protein expression of ZEB1, parallel with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker Vimentin. Furthermore, the proliferation and migratory ability of Caski cells were significantly lower in the transfected group than in the nontransfected control group. CONCLUSION(S): Down-regulation of ZEB1 expression may protect the invasive front of the tumors from converting to a mesenchymal phenotype by reducing the proliferation and motility of cervical cancer cells, suggesting that ZEB1 might be a potential therapeutic target for SCC.

[Molluscicidal effect of 5% niclosamide ethanolamine granules against Oncomelania hupensis in a marshland field].

OBJECTIVE: To investigate the molluscicidal effect of 5% niclosamide ethanolamine granules against Oncomelania hupensis in a marshland field. METHODS: The 5% niclosamide ethanolamine granules were sprayed at a dose of 40 g/m2 on 3 snail-breeding marshlands in Yangzhong City of Jiangsu Province to assess its field molluscicidal actions, while 26% suspension concentrate of metaldehyde and niclosamide (MNSC); at a dose of 4 g/m2) and fresh water served as controls. RESULTS: After seven days spraying, 5% niclosamide ethanolamine granules resulted in a of snail mortal85.42%ity, while the mortality rates of snails were 82.35% and 2.86% in the MNSC and water control groups, respectively. CONCLUSION: 5% niclosamide ethanolamine granules exhibit a high molluscicidal activity, which is suitable to be used in the mashland.

Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17ß-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

OBJECTIVE: To study the role of lipoxin A4 (LXA4) in endometriosis. DESIGN: Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). SETTING: University hospital. PATIENT(S): Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). INTERVENTION(S): Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. MAIN OUTCOME MEASURE(S): Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. RESULT(S): The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERß increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERß in vivo. Moreover, administering LXA4 could augment ERß expression in ESCs and inhibit the 17ß-estradiol-induced phosphorylation of p38 MAPK very likely through ERß. CONCLUSION(S): Our findings indicate that LXA4 regulates ERß expression and inhibits 17ß-estradiol-induced phosphorylation of p38 MAPK, very likely through ERß in ESCs.

[Construction of platform of Schistosoma japonicum infection real-time monitoring and early warning along Yangtze River].

OBJECTIVE: To construct a platform for Schistosoma japonicum infection real-time monitoring and early warning in marshlands along the Yangtze River. METHODS: The data of the status of Oncomelania hupensis snails, schistosomiasis endemic situation, marshland environment status, and other basic information were collected and analyzed comprehensively in marshlands along the Yangtze River in Jiangsu Province, and a schistosomiasis endemic situation electronic map was established. Through the GPS and 3G technology, the short messaging service (SMS) alerts were sent to the fishermen when they entered the areas of different schistosomiasis endemic levels in real-time. RESULTS: The SMS alerts reminded the fishermen to adopt the suitable interventions to reduce or avoid the schistosome infection. In addition, the data of moving track of mobile phones of fishermen, and the examination and treatment of schistosomiasis at each monitoring point were collected and analyzed comprehensively and then systematic analysis reports of schistosomiasis situation prediction, early warning, related evaluation, etc. were created in real-time. CONCLUSION: The platform for Schistosoma japonicum infection real-time monitoring and early warning in marshlands along the Yangtze River has a certain application value for schistosomiasis prevention and control work of Jiangsu Province and even whole country.

[Survey of intestinal nematode infections in Yangzhong City from 2003 to 2011].

From 2003 to 2011, fecal specimens from 29 473 individuals were tested; of those, 52 were found to be infected with intestinal nematodes. The total infection rate was 0.18%, and the infection rates of Ascaris lumbricoides, hookworm, and Trichuris trichura were 0.08%, 0.01%, and 0.09%, respectively. Totally 10 954 children were surveyed for Enterobius vermicularis infection, and the infection rate was 0.29%. In conclusion, the prevalence of intestinal nematodes of population is low in Yangzhong City. However, we still need strengthen health education and regular monitoring.

[Malaria endemic and control in Yangzhong City from 1979 to 2010].

In Yangzhong City, the incidence of malaria decreased gradually from 883.48 per hundred thousand in 1979 to 0.36 per hundred thousand in 2010. The incidence was 0 from 2005 to 2009, but 1 local infected patient was found in 2010. The achievements of malaria control are significant, however, with the development of economy, the increase of mobile population, and the dimness of malaria control consciousness, the control strategy should be adjusted in order to eliminate malaria.

[Surveillance of schistosomiasis in a national surveillance site of Yangzhong City, 2005-2010].

The surveillance of schistosomiasis in Shicheng Village, a national schistosomiasis surveillance site in Yangzhong City from 2005 to 2010 showed that snail areas were 16.43 hm2, the occurrence rates of frames with living snails and mean densities of living snails exhibited a declining trend year by year, and the infected snails were only found in 2007. The sero-positive rates in residents were 1.81%, 0.98%, 0.29%, 0.28%, 0.47% and 0.51%, respectively from 2005 to 2010. However, no stool-examination -positive persons were detected. During the period, no acute infections occurred, and no advanced cases and infected animals were detected. It is indicated that schistosomiasis has been effectively controlled in the surveillance site. However, snails remain present, so comprehensive control and surveillance of infectious sources should be further strengthened.

[Surveillance of schistosomiasis in a national surveillance site of Yangzhong City, 2005-2009].

A longitudinal survey of schistosomiasis was carried out in Shicheng Village, Yangzhong City, Jiangsu Province, a national schistosomiasis surveillance site, from 2005 to 2009. The results showed that the infection situation of schistosomiasis in residents, and the status of snails, in general, remarkably decreased from 2005 to 2009. However, the snails still existed, so the comprehensive control in marshland and surveillance of infectious sources should be strengthened.