Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell–cell or cell–extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.
Dongmei He, Hong Zeng, Jingfei Chen, Lan Xiao, Yuhao Zhao and Nenghui Liu
Claire Stenhouse, Charis O Hogg and Cheryl J Ashworth
Integrins regulate adhesion at the foeto-maternal interface by interacting with secreted phosphoprotein 1 (SPP1) and fibronectin (FN). It is hypothesised that impaired foetal growth of ‘runt’ piglets is linked to altered integrin signalling at the foeto-maternal interface. Placental and endometrial samples associated with the lightest and closest to mean litter weight (CTMLW) (gestational day (GD18, 30, 45, 60 and 90), of both sex (GD30, 45, 60 and 90) (n = 5–8 litters/GD), Large White × Landrace conceptuses or foetuses were obtained. The mRNA expression of the integrin subunits (ITG) ITGA2, ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, ITGB8, SPP1 and FN was quantified by qPCR. Temporal changes in mRNA expression were observed, with different profiles in the two tissues. Endometrial ITGB1 (P ≤ 0.05, GD45) and SPP1 (P ≤ 0.05, all GD combined and GD60) expression was decreased in samples supplying the lightest compared to the CTMLW foetuses. Placentas supplying female foetuses had decreased expression of ITGB6 (GD45, P ≤ 0.05) and FN (GD90, P ≤ 0.05) compared to those supplying male foetuses. Endometrial samples supplying females had increased ITGB3 (P ≤ 0.05, GD60) and FN (P ≤ 0.05, GD30) expression and decreased SPP1 (P ≤ 0.05, GD60) expression compared to male foetuses. Correlations between mean within-gilt mRNA expression and percentage prenatal survival, number of live foetuses or conceptuses and percentage male foetuses were observed. This study has highlighted novel and dynamic associations between foetal size, sex and integrin subunit mRNA expression at the porcine foeto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these novel findings.
Jing Liu, Yang Wang, Peng Chen, Yue Ma, Shuo Wang, Ye Tian, Anna Wang and Danbo Wang
Previous lncRNA microarray screening found that the AC002454.1 gene was highly expressed in Endometriosis (EMS) and these expression levels were highly correlated with cyclin-dependent kinase-6 (CDK6). This study investigated the expression level and correlation between AC002454.1 and CDK6 in endometrium tissues and the influence of these changes in expression upon the biological behavior of eutopic endometrial cells. We confirmed AC002454.1 and CDK6 mRNA and protein were highly expressed in ectopic and eutopic endometrial tissue from patients with EMS and were clearly correlated. In vitro, both AC002454.1 and CDK6 positively regulated the proliferation, migration and invasion ability of eutopic endometrial cells and could promote the transformation of cells from G0/G1 phase to S phase. AC002454.1 and CDK6 may have synergistic effects, thereby affecting the biological behavior of endometrial cells, thus promotes the progression of EMS.
Jie Mei, Yuan Yan, Shi-Yuan Li, Wen-Jie Zhou, Qun Zhang, Ming-Qing Li and Hai-Xiang Sun
Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. The aim of this study was to determine the role of chemokine CXCL16 and its receptor CXCR6 in the decidualization during pregnancy. Here, the expression of CXCL16 was investigated in endometrial tissues, decidua and placenta in this study. Compared with endometrial tissue, protein expression of CXCL16 was significantly higher in tissues from the fertile control samples, especially in villus. Meanwhile, the primary trophoblast cells and decidual stromal cells (DSCs) secreted more CXCL16 and expressed higher CXCR6 compared to endometrial stromal cells (ESCs) in vitro. Stimulation with the inducer of decidualization (8-bromoadenosine 3′,5′-cyclic with medroxyprogesterone acetate, 8-Br-cAMP plus MPA) significantly upregulated the expression of CXCL16 and CXCR6 in ESCs in vitro. After treatment with exogenous recombinant human CXCL16 (rhCXCL16) or trophoblast-secreted CXLC16, decidualised ESCs showed a significant decidual response, mainly characterised by increased prolactin (PRL) secretion. Simultaneously, PI3K/PDK1/AKT/Cyclin D1 pathway in decidualised ESCs were activated by rhCXCL16, and AKT inhibitor GS 690693 abolished the PRL secretion of ESCs that was triggered by rhCXCL16. Finally, the impaired CXCL16/CXCR6 expression could be observed at the maternal–foetal interface from patients who have experienced spontaneous abortion. This study suggests that the CXCL16/CXCR6 axis contributes to the progression of ESC decidualization by activating PI3K/PDK1/AKT/Cyclin D1 pathway. It unveils a new paradigm at the maternal–foetal interface in which CXCL16 is an initiator for the molecular crosstalk that enhances decidualization of ESCs.
Shou-Bin Tang, Lei-lei Yang, Ting-ting Zhang, Qian Wang, Shen Yin, Shi-Ming Luo, Wei Shen, Zhao-Jia Ge and Qing-Yuan Sun
It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development, and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3); (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2, and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P<0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P<0.01). H3K9me2 and H3K27me3 were significantly increased in 4-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2, and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.
Belinda K M Lo, Sairah Sheikh and Suzannah A Williams
Follicle development requires complex and coordinated interactions between both the oocyte and its associated somatic cells. In ovarian dysfunction, follicle development may be abnormal due to defective somatic cell function; for example, premature ovarian insufficiency or malignancies. Replacing defective somatic cells, using the reaggregated ovary (RO) technique, may ‘rescue’ follicle development. ROs containing mature follicles have been generated when transplanted to a host mouse to develop. We have developed a RO culture technique and the aims were to determine how follicle development differed between transplanted and cultured ROs, and the influence of ovarian age (P2 vs P6). Mouse ROs were cultured for 14 days; P2 and P6 ovaries cultured as Controls. Follicle development was compared to ROs transplanted for 14 days and ovaries from P16 and P20 mice. ROs generated from either P2 or P6 exhibited similar follicle development in culture whereas in vivo follicle development was more advanced in P6 ROs. Follicles were more developed in cultured ROs than transplanted ROs. However, follicles in cultured ROs and ovaries had smaller oocytes with fewer theca and granulosa cells than in vivo counterparts. Our results demonstrate the fluidity of follicle development despite ovary dissociation and that environment is more important to basal lamina formation and theca cell development. Furthermore, follicle development within cultured ROs appears to be independent of oocyte nest breakdown and primordial follicle formation in source ovaries. Our results highlight the need for understanding follicle development in vitro, particularly in the development of the RO technique as a potential fertility treatment.
Song Li, Qi Fan, Yanqiu Xie, Haiyan Lin, Qi Qiu, Yihua Liang and Qingxue Zhang
In vitro activation of primordial follicles is becoming more essential in assisted reproductive technologies. Vasoactive intestinal peptide (VIP) is one of the members of the neurotrophin family which has demonstrated to have an impact on follicle development in recent years. This study aims to investigate the effect of VIP on the activation of primordial follicles in neonatal rat in an in vitro culture system and to determine the relevant molecular mechanism of their activation. Ovaries of 4-day-old rats were examined for the expression of VIP receptors and were cultured in mediums containing VIP with or without inhibitors of the ERK-mTOR signalling pathway. They were then collected for histological analysis or measurement of the molecular expression of this pathway. The receptors of VIP were found in granular cells and oocytes of primordial and early growing follicles in neonatal ovary. The ratio of growing follicle increased in the presence VIP at different concentrations, with the highest level of increase being observed in the 10-7mol/L VIP-treated group. The ratio of PCNA-positive granular cells was also increased, while that of the apoptotic oocytes were decreased, and protein analysis showed increased phosphorylation of ERK1/2, mTOR and rps6 in the VIP-treated group. However, the effect of VIP on the activation of primordial follicle became insignificant with the addition of MEK inhibitor (U0126), or mTORC1 inhibitor (rapamycin). This study indicated that VIP could activate neonatal rat primordial follicle through the ERK-mTOR signalling pathway, suggesting a strategy for in vitro primordial follicle recruitment.
Beverly G Reed, Samir N Babayev, Lucy X Chen, Bruce R Carr, R Ann Word and Patricia T Jimenez
MicroRNAs (miRs) are small molecules important for regulation of transcription and translation. The objective was to identify hormonally regulated miRs in human endometrial stromal cells and to determine the impact of the endocrine disruptor, bisphenol A (BPA), on those miRs. miR microarray analysis and multiple confirmatory cell preparations treated with 17β-estradiol (E2) and BPA altered miR-27b, let-7c, let-7e and miR-181b. Further, decidualization downregulated miR-27b. VEGFB and VEGFC were validated as targets of miR-27b. Identification of miR-27b target genes suggests that BPA and E2 downregulate miR-27b thereby leading to upregulation of genes important for vascularization and angiogenesis of the endometrium during the menstrual cycle and decidualization.
Caroline M Allen, Federica Lopes, Rod T Mitchell and Norah Spears
Chemotherapy treatment is a mainstay of anticancer regimens, significantly contributing to the recent increase in childhood cancer survival rates. Conventional cancer therapy targets not only malignant but also healthy cells resulting in side effects including infertility. For prepubertal boys, there are currently no fertility preservation strategies in use, although several potential methods are under investigation. Most of the current knowledge in relation to prepubertal gonadotoxicity has been deduced from adult studies; however, the prepubertal testis is relatively quiescent in comparison to the adult. This review provides an overview of research to date in humans and animals describing chemotherapy-induced prepubertal gonadotoxicity, focusing on direct gonadal damage. Testicular damage is dependent upon the agent, dosage, administration schedule and age/pubertal status at time of treatment. The chemotherapy agents investigated so far target the germ cell population activating apoptotic pathways and may also impair Sertoli cell function. Due to use of combined chemotherapy agents for patients, the impact of individual drugs is hard to define, however, use of in vivo and in vitro animal models can overcome this problem. Furthering our understanding of how chemotherapy agents target the prepubertal testis will provide clarity to patients on the gonadotoxicity of different drugs and aid in the development of cytoprotective agents.
Sathish Kumar, Geoffrey H Gordon, David H Abbott and Jay S Mishra
Adequate maternal vascular adaptations and blood supply to the uterus and placenta are crucial for optimal oxygen and nutrient transport to growing fetuses of eutherian mammals, including humans. Multiple factors contribute to hemodynamics and structuring of placental vasculature essential for term pregnancy with minimal complications. In women, failure to achieve or sustain favorable pregnancy progression is, not surprisingly, associated with high incidence of antenatal complications, including preeclampsia, a hypertensive disorder of pregnancy. While the pathogenesis of preeclampsia in women remains unknown, a role for androgens is emerging. The relationship between androgens and maternal cardiovascular and placental function deserves particular consideration because testosterone levels in the circulation of preeclamptic women are elevated approximately two- to three-fold and are positively correlated with vascular dysfunction. Preeclampsia is also associated with elevated placental androgen receptor (AR) gene expression. Studies in animal models mimicking the pattern and level of increase of adult female testosterone levels to those found in preeclamptic pregnancies, replicate key features of preeclampsia, including gestational hypertension, endothelial dysfunction, exaggerated vasoconstriction to angiotensin II, reduced spiral artery remodeling, placental hypoxia, decreased nutrient transport and fetal growth restriction. Taken together, these data strongly implicate AR-mediated testosterone action as an important pathway contributing to clinical manifestation of preeclampsia. This review critically addresses this hypothesis, taking into consideration both clinical and preclinical data.