The transcriptomic and proteomic effects of ectopic overexpression of miR-30d in human endometrial epithelial cells.

miR-30d is known to be up-regulated during the acquisition of receptivity in the endometrium. In order to determine the transcriptomic and proteomic changes which occur after transient overexpression of miR-30d in primary endometrial epithelial cells, in vitro cultured human endometrial epithelial cells (hEECs) were studied experimentally. Two different miRNAs (scramble versus mimic; n = 15) were transiently transfected into primary hEECs from four different patients and were evaluated for mRNA and protein expression using Agilent's gene expression microarray and iTRAQ analysis techniques, respectively. A set of differentially expressed mRNAs were validated by qPCR and several differentially expressed proteins were validated by western blot. Finally, methylation differential immunoprecipitation (MeDIP) was used to validate the epigenetic changes in the H19 gene. The results showed that transient transfection with miR-30d miRNA induced the differential mRNA-expression of 176 genes (75 up-regulated and 101 down-regulated). Several of them have been associated with reproductive and endocrine system disorders, tissue development, and are implicated in epithelial cell proliferation. Also, the down-regulation of some genes such as H19 and N-methyltransferase (NNMT) may suggest that epigenetic alterations are induced. Furthermore, upstream effects of genes regulated by the estrogen receptor alpha 1 (ESR1) transcription factor have been predicted. Proteomic analysis identified 2290 proteins, of which 108 were differentially expressed (47 up-regulated and 61 down-regulated). Among these differentially expressed proteins DNA methyl transferase (DNMT)1 was found to be up-regulated; this protein participates in the maintenance of DNA methylation, supporting an epigenetic role for miR-30d. Finally MeDIP showed an increase in methylation in the H19 DMR region. In conclusion transient in vitro overexpression of the receptivity-up-regulated miRNA miR-30d in hEECs seems to activate genes which are associated with hormonal response and the epigenetic status of these cells.

Complete method to obtain, culture, and transfer mouse blastocysts nonsurgically to study implantation and development.

OBJECTIVE: To illustrate an efficient, complete, step-by-step protocol for studying implantation in mice. DESIGN: Video presentation of an animal model for research in reproductive biology. ANIMAL(S): Mouse (Mus musculus). INTERVENTION(S): A nonsurgical embryo transfer system very similar to that used for human embryo transfer. MAIN OUTCOME MEASURE(S): The protocols with recipient and donor mice are performed in parallel in the same week. For the donor mice: the first step is ovarian stimulation, followed by ovulation induction and mating; finally, the mice are sacrificed, and the embryos are collected and cultured. For recipient mice: first estrous synchrony is induced, followed by mating with a vasectomized male, visualization of the vaginal plug, and nonsurgical transfer of the embryos. Finally (optionally), the implantation sites can be visualized on day 7.5 of development. (All animal experiments were performed with the approval of the institutional review board.) RESULT(S): Implantation is an essential step in human reproduction although, because of technical and ethics considerations, still relatively little is known about human implantation and early development. Conversely, mouse models are well established and can be used for preliminary experiments. However, there are various bottlenecks in the procedure for obtaining and transferring murine embryos, which makes experimentation with this model more difficult. These difficulties include pseudopregnancy, ovarian hyperstimulation, and embryo collection, culture, and transfer. We have proposed a complete, efficient method for obtaining, culturing, and transferring mouse blastocysts that can be easily applied in research. Potential applications include testing new media components that do not affect preimplantation but do affect implantation and early development. The embryo transfer method proposed here has been demonstrated to achieve embryo implantation easier and faster than, and in approximately similar rates as other traditional surgery methods. CONCLUSION(S): This workflow is the first set of complete step-by-step instructions available that incorporate advances such as nonsurgical mouse embryo transfer. This will facilitate research into different reproduction events such as embryo development, embryo implantation, or contraception.