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MiR199a was found to be differentially expressed in rat uteri between the prereceptive and receptive phase via microRNA (miRNA) microarray analysis in our previous study. However, the role of miR199a in rat embryo implantation remained unknown. In the study, northern blot results showed that the expression levels of miR199a were higher on gestation days 5 and 6 (g.d.5–6) in rat uteri than on g.d.3–4 and g.d.7–8. In situ localization of miR199a in rat uteri showed that miR199a was mainly localized in the stroma or decidua. The expression of miR199a was not significantly different in the uteri of pseudopregnant rats and evidently increased in the uteri of rats subjected to activation of delayed implantation and experimentally induced decidualization. Treatment with 17β-estradiol or both 17β-estradiol and progesterone significantly diminished miR199a levels. Gain of function of miR199a in endometrial stromal cells isolated from rat uteri inhibited cell proliferation and promoted cell apoptosis. Loss of function of miR199a displayed opposite roles on cell proliferation and apoptosis. Further investigation uncovered a significant inverse association between the expression of miR199a and growth factor receptor-bound protein 10 (Grb10), an imprinted gene, and miR199a could bind to the 3′UTR of Grb10 to inhibit Grb10 translation. In addition, in vivo analysis found that the immunostaining of GRB10 was attenuated in the stroma or decidua from g.d.4 to 6, contrary to the enhancement of miR199a. Collectively, upregulation of miR199a in rat uterus during the receptive phase is regulated by blastocyst activation and uterine decidualization. Enforced miR199a expression suppresses cell proliferation partially through targeting Grb10.

Although the relationship between polymorphisms in microRNAs (miRNAs) and recurrent pregnancy loss (RPL) has been studied, there is very little data available in the literature. In the present study, we scanned 55 potentially functional polymorphisms in the miRNA coding region in Chinese women with unexplained RPL (URPL; no. 2011-10). The rs6505162 C>A in the MIR423 coding region was found to be significantly associated with the occurrence of human URPL. The rare A allele contributed to an increase in the expression of mature MIR423. C to A substitution in the polymorphism rs6505162 in pre-MIR423 repressed cell proliferation and migratory capacity. Further investigations showed that MIR 423 could inversely regulate the expression of proliferation-associated 2 group 4 (PA2G4) by binding the 3′-UTR of PA2G4. Dual-luciferase assay indicated that the A allele in the polymorphism rs6505162 could more effectively suppress the expression of PA2G4 than the C allele could. Collectively, the present data suggest that rs6505162 C>A in pre-MIR423 may contribute to the genetic predisposition to RPL by disrupting the production of mature MIR42 3 and its target gene, which consequently interferes with MIR423 functioning.

Abstract

Emx2 deletion impairs the growth and maintenance of the genital ridge. However, its role in subsequent germ cell differentiation during embryonic stages is unknown. Using a tamoxifen-inducible Cre-loxP mouse model (Emx2 ^flox/flox, Cre-ER ^TM, hereafter called as Emx2 knockdown), we showed that germ cell differentiation was impaired in Emx2-knockdown testes. Representative characteristics of male germ cell differentiation, including a reduced ability to form embryonic germ (EG) cell colonies in vitro, down-regulation of pluripotency markers and G1/G0 arrest, did not occur in Emx2-knockdown testes. Furthermore, FGF9 and NODAL signalling occurred at abnormally high levels in Emx2-knockdown testes. Both blocking FGF9 signalling with SU5402 and inhibiting NODAL signalling with SB431542 allowed germ cells from Emx2-knockdown testes to differentiate in vitro. Therefore, EMX2 in somatic cells is required to trigger germ cell differentiation in XY foetuses, posterior to its previously reported role in the growth and maintenance of the genital ridge.

Small extracellular vesicles (sEVs) are important mediators of cell-to-cell communication involved in the successful establishment of a pregnancy. Human decidual stromal cells play a key role in regulating trophoblast invasion. Nevertheless, the regulatory functions of decidual stromal cells-derived sEVs in human trophoblast cells are still unclear. In this study, primary human decidual stromal cells were isolated, and immortalized human endometrial stromal cell line (HESCs) were decidualized into human decidual stromal cells (HDSCs) using hormonal cocktail containing medroxy progesterone 17-acetate (MPA), estrogen and cAMP analog. HDSC-sEVs were isolated from both primary human decidual stromal cells and immortal HDSCs, respectively, and identified by transmission electron microscopy and western blotting. EV uptake assay indicated that HDSC-sEVs could be uptaken by trophoblast cells. HDSC-sEVs could increase the invasiveness and the expression level of N-cadherin of trophoblast cells with elevated phosphorylation of SMAD2 and SMAD3 in the cells. Silencing of N-cadherin could block cell invasion induced by HDSC-sEVs, while knockdown of SMAD2 and SMAD3 could inhibit the upregulation of N-cadherin in trophoblast cells. Taken together, our results suggested a regulatory effect of HDSC-sEVs in the invasion of trophoblast cells, and HDSC-sEVs may be important mediators of trophoblasts during embryo implantation and placentation.