MicroRNA Let-7a and dicer are important in the activation and implantation of delayed implanting mouse embryos.

STUDY QUESTION: Does Let-7a have a functional role in modulating dicer expression to activate dormant mouse blastocysts for implantation? SUMMARY ANSWER: Let-7a post-transcriptionally regulates dicer expression altering microRNA expression to affect the implantation competency of the activated blastocysts. WHAT IS KNOWN ALREADY: The Let-7a microRNA is up-regulated during blastocyst dormancy and its forced-expression suppresses embryo implantation in vitro and in vivo. Dicer is a Let-7 target, which processes pre-microRNA to mature microRNA. STUDY DESIGN, SIZE, DURATION: The effects on the expression of Let-7a and dicer in dormant blastocysts during the first 12 h after estradiol-induced activation, and the relationship between Let-7a and dicer in preimplantation embryos were determined. The effects on the microRNA expression and embryo implantation in vivo in dicer-knockdown mouse 5-8 cell embryos and dormant blastocysts at 1 h post estradiol activation were also studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICR female mice at 6 weeks of age were ovariectomized on Day 4 of pregnancy to generate the delayed implantation model. Mouse 5-8 cell embryos and/or dormant blastocysts at 1 h after estradiol injection were electroporated with dicer siRNA and Let-7a precursor or Let-7a inhibitor. At 48 h post electroporation, the Let-7a expression, dicer transcripts and proteins in the embryos were determined using qPCR and immunostaining/western blotting, respectively. All experiments were repeated at least three times. MAIN RESULTS AND THE ROLE OF CHANCE: Estradiol injection down-regulated Let-7a and up-regulated dicer in the dormant blastocysts during the first 12 h post-activation. Dicer knockdown at 1 h post-activation of blastocysts suppressed EGFR expression, attenuated EGF binding and compromised implantation of the transferred embryos. Let-7a transcriptionally regulated dicer by binding to the 3'-UTR of dicer in trophoblast cells. Dicer knockdown in blastocysts suppressed mature Let-7a expression and compromised implantation. LIMITATIONS, REASONS FOR CAUTION: Gain- and loss-of-function approaches were used by analyzing transient expressions of transfected microRNA modulators or genes. The consequence of the Let-7a-dicer interaction on pregnancy remains to be determined. The study used the mouse as a model and the applicability of the observed phenomena in humans warrants further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the Let-7a-dicer interaction leads to differential microRNA expression in dormant blastocysts after estradiol activation. Because the expression pattern of Let-7a in human blastocysts is similar to that in mouse blastocysts, our observation that the Let-7a-dicer interaction has a role in regulating the implantation potential of the mouse blastocysts could be applicable to humans. STUDY FUNDING/COMPETING INTEREST(S): This project is supported partly by a research grant from the Research Grant Council to W.S.B.Y. The authors have no competing interests to declare.

Involvement of microRNA lethal-7a in the regulation of embryo implantation in mice.

MicroRNAs interact with multiple mRNAs resulting in their degradation and/or translational repression. This report used the delayed implantation model to determine the role of miRNAs in blastocysts. Dormant blastocysts in delayed implanting mice were activated by estradiol. Differential expression of 45 out of 238 miRNAs examined was found between the dormant and the activated blastocysts. Five of the nine members of the microRNA lethal-7 (let-7) family were down-regulated after activation. Human blastocysts also had a low expression of let-7 family. Forced-expression of a family member, let-7a in mouse blastocysts decreased the number of implantation sites (let-7a: 1.1±0.4; control: 3.8±0.4) in vivo, and reduced the percentages of blastocyst that attached (let-7a: 42.0±8.3%; control: 79.0±5.1%) and spreaded (let-7a: 33.5±2.9%; control: 67.3±3.8%) on fibronectin in vitro. Integrin-ß3, a known implantation-related molecule, was demonstrated to be a target of let-7a by 3'-untranslated region reporter assay in cervical cancer cells HeLa, and Western blotting in mouse blastocysts. The inhibitory effect of forced-expression of let-7a on blastocyst attachment and outgrowth was partially nullified in vitro and in vivo by forced-expression of integrin-ß3. This study provides the first direct evidence that let-7a is involved in regulating the implantation process partly via modulation of the expression of integrin-ß3.

Up-regulation of endocrine gland-derived vascular endothelial growth factor but not vascular endothelial growth factor in human ectopic endometriotic tissue.

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF), endocrine gland-derived VEGF (EG-VEGF/PK1), and its receptors (PKR1 and PKR2) in eutopic and ectopic endometrial tissues. DESIGN: A case-control study. SETTING: University reproduction unit. PATIENT(S): Infertile women undergoing diagnostic laparoscopy for tubal patency. INTERVENTION(S): Endometrial and endometriotic tissue sampling from women with and without endometriosis. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction (PCR) analysis of genes in eutopic and ectopic endometrial tissues. The EG-VEGF protein was studied by immunohistochemistry. RESULT(S): In normal endometrium, EG-VEGF messenger RNA (mRNA) expression was 50-fold higher in the secretory than in the proliferative phase, but that of PKR1 was 6-fold higher in the latter than in the former. The PKR2 transcript was detected in the proliferative but not the secretory endometrium. In patients with endometriosis, eutopic endometrial PKR2 transcript level was 4-fold higher in the proliferative than in the secretory phase. No differences in EG-VEGF or PKR1 were found in proliferative versus secretory endometrium in these patients. There were no significant differences in the expression of EG-VEGF in eutopic endometrium of normal women and in those with endometriosis. In the paired laser-captured microdissected eutopic endometrial and ectopic endometriotic samples, a significantly higher EG-VEGF, but not VEGF, transcript level was detected in the ectopic when compared with eutopic samples; whereas the expressions of PKR1 and PKR2 were barely detectable. The H-scoring confirmed that the stroma of endometriotic samples had a significantly higher EG-VEGF protein expression than that in the paired eutopic endometrium. CONCLUSION(S): High levels of EG-VEGF expression may play an important role in angiogenesis in endometriotic tissues.

Oviductal microsomal epoxide hydrolase (EPHX1) reduces reactive oxygen species (ROS) level and enhances preimplantation mouse embryo development.

Somatic cell-embryo coculture enhances embryo development in vitro by producing embryotrophic factor(s) and/or removing harmful substances from the culture environment. Yet, the underlying molecular mechanisms on how somatic cells remove the toxicants from the culture medium remain largely unknown. By using suppression subtractive hybridization, we identified a number of mouse oviductal genes that were up-regulated when developing preimplantation embryos were present in the oviduct. Epoxide hydrolase 1, microsomal (Ephx1 previously known as mEH) was one of these genes. EPHX1 detoxifies genotoxic compounds and participates in the removal of reactive oxygen species (ROS). The transcript of Ephx1 increases in the oviductal epithelium at the estrus stage and in Day 3 of pregnancy as well as in the uterus of ovariectomized mice injected with estrogen or progesterone. Human oviductal epithelial cells OE-E6/E7 express EPHX1 and improve mouse embryo development in vitro. Addition of an EPHX1 inhibitor, cyclohexene oxide (CHO) or 1,1,1-trichloropropene 2,3-oxide (TCPO), to the culture medium increased intracellular and extracellular ROS levels of OE-E6/E7 cells and suppressed the beneficial effect of the cells on embryo development; CHO and TCPO at these concentrations had no adverse effect on OE-E6/E7 growth and embryo development in vitro. Taken together, EPHX1 in oviductal cells may enhance the development of cocultured embryos by protecting them from oxidative stress. Our result supports the notion that somatic cell coculture may enhance embryo development via removal of deleterious substances in the culture medium.

Regulation of complement-3 protein expression in human and mouse oviducts.

The human oviduct derived embryotrophic factor-3 (ETF-3) contains complement protein-3 (C3) and its derivates. Although C3 is not embryotrophic, it is converted into the embryotrophic derivative, iC3b in the presence of embryos and oviductal cells. The regulation of C3 production in the oviduct is not known. The objectives of this study were to investigate the effects of presence of preimplantation embryos and hormones on C3 expression in the oviducts in vitro and in vivo. The expression of C3 in the oviduct of pregnant mice was compared to that of pseudo-pregnant mice. The hormonal action on C3 expression was studied in the ovariectomized mouse oviducts and human oviductal epithelial (OE) cells. The results showed that the level of C3 mRNA in the mouse oviduct was high on Day 1 and Day 2, but decreased to a minimum on Day 4 of pregnancy, whereas that of pseudo-pregnancy remained relatively stable within the same period. The protein levels of C3 and iC3b specific fragments, alpha-115 and alpha-40, respectively in the mouse oviductal luminal fluid were highest on Day 3 of pregnancy, when the embryos were expected to be most sensitive to the embryotrophic activity of ETF-3. Estrogen elevated C3 expression in the ovariectomized mouse oviduct and the OE cells. Progesterone suppressed estrogen-induced C3 expression in the mouse oviduct, but had no effect on OE cells. In conclusion, the presence of embryo and steroid hormones regulate the synthesis and secretion of oviductal C3.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis.

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.