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Dawit Tesfaye, Samuel Gebremedhn, Dessie Salilew-Wondim, Tsige Hailay, Michael Hoelker, Christine Grosse-Brinkhaus, and Karl Schellander

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

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Michael Hoelker, Franka Rings, Qamaruddin Lund, Nasser Ghanem, Chirawath Phatsara, Josef Griese, Karl Schellander, and Dawit Tesfaye

The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

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Helmut Torner, Nasser Ghanem, Christina Ambros, Michael Hölker, Wolfgang Tomek, Chirawath Phatsara, Hannelore Alm, Marc-André Sirard, Wilhelm Kanitz, Karl Schellander, and Dawit Tesfaye

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus–oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB (colourless cytoplasm, high G6PDH activity) and BCB+ (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB+ and BCB oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB+ (33.1±3.1%) and BCB (12.1±1.5%) oocytes. Moreover, BCB+ oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP) and protein biosynthesis (RPS274A and mRNA for elongation factor 1α, EF1A). BCB oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.

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Korakot Nganvongpanit, Heike Müller, Franca Rings, Michael Hoelker, Danyel Jennen, Ernst Tholen, Vitezslav Havlicek, Urban Besenfelder, Karl Schellander, and Dawit Tesfaye

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

Free access

Salilew-Wondim Dessie, Franca Rings, Michael Hölker, Markus Gilles, Danyel Jennen, Ernst Tholen, Vitezslav Havlicek, Urban Besenfelder, Vladimir L Sukhorukov, Ulrich Zimmermann, Joerg M Endter, Marc-André Sirard, Karl Schellander, and Dawit Tesfaye

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB+) and without (PB) first polar body and zygotes were subjected to DEP at 4 MHz and 450 μm electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB+ and PB oocytes was significantly (P < 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P < 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.