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Identification of embryo implantation-related proteins Mitra Arianmanesh Embryo implantation is a complex process involving an active dialogue between the endometrium and embryo. Tightly controlled communication between the hypothalamus, pituitary, corpus luteum (CL), endometrium and embryo is essential for implantation. Unravelling molecules involved in embryo implantation is essential since implantation failure is one of the main causes of female infertility. Therefore, identification of molecular events during embryo implantation may result in enhancing implantation rates in both natural and assisted reproductive cycles, improving contraceptive design and reducing the rate of multiple pregnancies following embryo transfer in IVF cycles. Thus, in this study, sheep was used as an animal model in order to study endometrial, corpus luteal and plasma proteome changes during embryo implantation and early pregnancy. Endometrium, CLs and plasma were harvested from cyclic ewes on days 12 and 16 of the oestrous cycle (n=4 ewes/group) and from pregnant ewes on days 12, 16 and 20 of pregnancy (n=4 ewes/group). Furthermore, ovine endometrium were collected from pregnant and non-pregnant horns on days 16 (n=4) and 20 (n=4) of pregnancy to compare endometrial protein profiles of the gravid horn (in the presence of the conceptus) with the non-gravid horn (in the absence of the conceptus) in response to the conceptus to elucidate local embryo-endometrial signalling. 2DE gel, LC-MS/MS, Western blot, IHC and qRT-PCR were employed to quantify implantation processes. This study has identified proteins in the CL and endometrium with involvement in biological pathways that are fundamental for embryo implantation and gestation. In addition, it was found that the implanting embryo is capable of regulating the expression of endometrial proteins to establish an ideal environment for its implantation and establishment of pregnancy. These findings provide an addition to the field and a solid base for targeted studies to improve our understanding of implantation and its regulation.

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"The Aurora Kinase proteins are a family of serine/threonine kinases that have been shown to play fundamental roles in controlling M phase progression in somatic cells. Aurora Kinase A protein is known to be vital for proper spindle assembly and therefore, chromosome segregation. Previous reports have shown that Aurora Kinase B is vital for proper completion of karyokinesis and cytokinesis in somatic cells. The role of Aurora Kinase C in somatic cells has been found to be less clear; however it appears to play an important role in spermatogenesis. Little is known about the role of these Aurora Kinase proteins mouse oocytes during oogenesis, and even less is known about them in embryos during early development. The objective of these studies was to characterize the presence, localization, and function of Aurora Kinase B and Aurora Kinase C protein and mRNA in mouse oocytes and early embryos. Oocytes and embryos were collected from hormone stimulated CF-1 mice and cultured for varying amounts of time. Cumulus denuded oocytes were either fixed for immunofluorescence microscopy studies, lysed for analysis of mRNA levels through the use of reverse transcription PCR (rtPCR) and quantitative rtPCR (q-rtPCR), lysed for protein analysis employing Western blotting, treated with Aurora Kinase protein inhibitor drugs, or microinjected with a siRNA pool targeting Aurora Kinase B. Samples were processed for immunofluorescence analysis using markers of spindle morphology (tubulins), Aurora Kinase B, Aurora Kinase C, and Aurora Kinase B activity (phospho Histone H3). Analysis of relative levels of Aurora Kinase B and Aurora Kinase C mRNA were assessed by rtPCR and q-rtPCR methods. Western blotting was performed on oocytes and early embryos to quantitate Aurora Kinase B and C protein levels. Aurora Kinase inhibitors, Hesperadin and ZM447439, were added to culture medium with mouse oocytes to determine the effects of the loss of Aurora Kinase activity. siRNAs were used to inhibit Aurora Kinase B mRNA in early embryos to ascertain the effect of functional loss of this transcript on embryo development. Marked differences were observed in the localization of Aurora Kinase B when unfertilized oocytes or pre-zygotic genome activation (ZGA) embryos were compared to post-ZGA samples. There was no evidence of Aurora Kinase B protein localized to the mitotic spindle or resultant midbody in oocytes and blastomeres of early embryos. Western blotting results supported this data. Embryos fixed post-ZGA demonstrated Aurora Kinase B localization at midbodies between dividing cells, as was found in mouse embryonic fibroblast control cells. Aurora Kinase C protein was not demonstrable in mouse oocytes, embryos, or control cells using immunocytochemistry or Western techniques. In contrast, Aurora Kinase B and Aurora Kinase C mRNAs were both found to be present in mouse oocytes and early embryos. q-rtPCR data further supported this finding for Aurora Kinase B and revealed that the mRNA level of this transcript is relatively constant until ZGA at which point a decrease relative to the earlier stages was observed. Transcript levels recovered post-ZGA and were comparable to the pre-ZGA levels. Functional inhibition of the Aurora Kinase family through the use of Hesperadin or ZM447439 demonstrated the importance of these proteins for proper microtubule and spindle organization, as these drugs disrupted both karyokinesis and cytokinesis in mouse oocytes and blastomeres of early embryos. Aurora Kinase B targeting siRNA also established a role for Aurora Kinase mRNA in embryos at the 2-cell stage based on the disruption of the cell cycle that was observed in treated embryos. Given earlier reports showing the vital role of the Aurora Kinase proteins in proliferating somatic cells, knowledge of the expression and localization of these proteins in oocytes and early embryos is vital for the understanding of cell cycle control during oogenesis and early embryogenesis. Our data indicate that Aurora Kinase B mRNA may also play a role in early embryogenesis, demonstrating a need for analysis of transcript as well as protein. Our results, as well as outcomes of future experiments suggested by our work, may provide significant insight into cell cycle regulation differences between somatic and embryonic cells. These differences may have a profound impact upon manipulated embryos including those reconstructed through somatic cell nuclear transfer. "

The ability to create transgenic livestock is a tremendous benefit in scientific research for many disciplines including functional genomics, pharmaceutical synthesis and development of enhanced production animals. Transgenes can either be stably or transiently expressed to alter gene function and obtain a specifically engineered phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and skill associated with both methods, a preliminary investigation exploring alternative delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel and that, although bovine zygotes are not susceptible to chemical transfection, the trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA (Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1 Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts transfected at a ratio of 9:6 with GeneJammer® truly experience gene knock down after siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P < 0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than the transfected blastocysts (P < 0.05). From these data, it can be concluded that although siRNA injection may be capable of knocking down gene expression for the first 7 days of embryonic development, it does not persist to the hatching stage; however, blastocysts transfected at day 7 do express altered gene expression in the trophectoderm which can continue through embryonic hatching events.

Mouse tumor susceptibility gene, tsg101, was discovered by Dr. Stanley Cohen at Stanford University in 1996. Subsequent studies have revealed multiple functions of tsg101. It participates in MDM2/p53 regulatory circuit, protein trafficking and regulation of cellular proliferation and differentiation. Knockout mouse experiment has demonstrated an essential role of tsg101 in early embryonic development, since tsg101 deficiency results in malformation of mesoderm, hence embryonic lethality. To further scrutinize the role of tsg101 in mouse tissues during the stages of organogenesis, we analyzed tsg101 protein steady state expression profile in the developing embryo. The data indicated that tsg101 was ubiquitously and diffusedly expressed in all early embryonic tissues, suggesting a role of tsg101 in rapid dividing early embryonic cells. Along the developmental path, tsg101 expressions became more specific in the epithelial cells of lung, intestine, kidney, thyroid, thymus and adrenal gland. This is concordant with previous report of tsg101 function in regulation of epithelial cell differentiation. In addition, tsg101 expression was found in pyramidal and stellate cells in cerebral cortex, substantia nigra cells of thalamus and Purkinje cells in cerebellum of adult mouse. Whether tsg101 participates in regulating the trafficking of neurotransmitters in these neuronal cells awaits further investigation. Surprisingly, we found congruous expression profile of thyroglobulin in these neuronal cells, except in stellate cells. Besides, the expression of TTF-1 was found in the VI layer of cerebral cortex and neuronal fiber in thalamus. Both thyroglobulin and TTF-1 are thyroid specific proteins, which are important for the development of brain tissues. Further investigation is urged to clarify whether these two proteins really present in these brain tissues and their functional role in these neuronal cells.