Expression of E-cadherin, leukemia inhibitory factor and progesterone receptor in mouse blastocysts after ovarian stimulation

The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors [progesterone receptor], cytokines [leukemia inhibitory factors [LIF]], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor [PgR] genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice [6- 8 weeks old] were injected with 7.5 IU pregnant mare serum gonadotropin [PMSG]. Their blastocysts [approximately n= 120] were flushed out after 3.5 days, following administration of human chorionic gonadotropin [hCG]. The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]. Data were analyzed by the student's t-test [one sample t-test]. Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts [p<0.05]. Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate

Designing and development of a silencer vector based on second generation of RNA interference [RNAi] technique to study downregulation of steroid receptor RNA activator [SRA]

RNA interference [RNAi] is the most potent technique for gene silencing in eukaryotic cellular system at transcriptomic level. Genetic disorders and cancers are important targets for therapeutic development of this technique. In order to bypass the temporary dpwnregulation by siRNA, a new generation of shRNA named shRNAmir has developed. Silencing construct with structure similar to microRNA [shRNAmir], mimics a natural microRNA pathway inside the cell. Steroid receptor RNA activator [SRA] is one of the regulators of steroid receptor like ER. Prostate, uterus and breast tissue express a low level of SRA, there is an increase of expression during their tumorgenesis. So SRA may participate in tumorgenesis or proliferation of tumors. We used RNAi technique to silence expression of SRA. The SRA silencer was designed and constructed by Soe-PCR, then cloned into an expression vector pEGFPCl. Human breast cancer [MCF7] cells were transfected with silencer plasmid then the changes in the SRA expression estimated by Real-Time PCR at 24, 72 hours and after l0days. The results showed about 60% decrease in relative expression of SRA gene, after 72 hours and 10 days, which shows that shRNAmir-SRA could successfully knockdown the expression of target gene. It seems that the designed shRNAmir may be a suitable tool for a variety of applications because it could stably knockdown the expression of target gene