Federica Ariu

Expression pattern of zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in ovine oocytes and in vitro-produced preimplantation embryos

The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription-polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal-embryonic transition. The transcription of the four genes did not resume with activation of the genome.

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38 °C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P<0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P<0.05) and TE (P<0.01) cell number and a lower percentage of picnotic nuclei (P<0.05) compared with the control group. Significantly lower abundance for BAX (P<0.01) and OCT4 (P<0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.

Prooxidant Effects of Verbascoside, a Bioactive Compound from Olive Oil Mill Wastewater, on In Vitro Developmental Potential of Ovine Prepubertal Oocytes and Bioenergetic/Oxidative Stress Parameters of Fresh and Vitrified Oocytes

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

Expression of maternally derived KHDC3, NLRP5, OOEP and TLE6 is associated with oocyte developmental competence in the ovine species

BackgroundThe sub-cortical maternal complex (SCMC), located in the subcortex of mouse oocytes and preimplantation embryos, is composed of at least four proteins encoded by maternal effect genes: OOEP, NLRP5/MATER, TLE6 and KHDC3/FILIA. The SCMC assembles during oocyte growth and was seen to be essential for murine zygote progression beyond the first embryonic cell divisions; although roles in chromatin reprogramming and embryonic genome activation were hypothesized, the full range of functions of the complex in preimplantation development remains largely unknown.ResultsHere we report the expression of the SCMC genes in ovine oocytes and pre-implantation embryos, describing for the first time its expression in a large mammalian species.We report sheep-specific patterns of expression and a relationship with the oocyte developmental potential in terms of delayed degradation of maternal SCMC transcripts in pre-implantation embryos derived from developmentally incompetent oocytes.In addition, by determining OOEP full length cDNA by Rapid Amplification of cDNA Ends (RACE) we identified two different transcript variants (OOEP1 and OOEP2), both expressed in oocytes and early embryos, but with different somatic tissue distributions.In silico translation showed that 140 aminoacid peptide OOEP1 shares an identity with orthologous proteins ranging from 95% with the bovine to 45% with mouse. Conversely, OOEP2 contains a premature termination codon, thus representing an alternative noncoding transcript and supporting the existence of aberrant splicing during ovine oogenesis.ConclusionsThese findings confirm the existence of the SCMC in sheep and its key role for the oocyte developmental potential, deepening our understanding on the molecular differences underlying cytoplasmic vs nuclear maturation of the oocytes.Describing differences and overlaps in transcriptome composition between model organisms advance our comprehension of the diversity/uniformity between mammalian species during early embryonic development and provide information on genes that play important regulatory roles in fertility in nonmurine models, including the human.

Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization

Parthenogenetic activation of the mammalian oocyte constitutes an essential step to a number of oocyte- or embryo-related technologies. Mammalian parthenotes are useful tools for studying the roles of paternal and maternal genomes in early mammalian development and are considered potential candidates for an ethical source of embryonic stem cells. We investigated the in vitro developmental competence of pre-implantation ovine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) together with the expression of a panel of fourteen genes at different times of development. IVF and PA embryos showed similar developmental competence. No differences in gene expression were observed between PA and IVF two cell-stage embryos, while PA morulae showed a significantly higher expression of IGF2. At the blastocyst stage, parthenotes exhibited up-regulation of TP-1, CDC2, and IGF2 transcripts and significantly lower levels of AQP3, ATP1A1, H2A.Z, hsp90beta, and OCT4, while NANOG, BAX, CCNB1, CDH1, GAPDH, and IGF2R displayed similar expression patterns in the two groups. Our study indicates that oocyte parthenogenetic activation does not impair in vitro pre-implantation development to the blastocyst stage, but affects the gene expression status of the embryo after the activation of its own genome.

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.

Amniotic fluid l-ergothioneine concentrations in pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos

L-ergothioneine levels were measured in amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos. Amniotic fluids were collected between 60 and 65 and 80-85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC)method with fluorescence detection. L-Ergothioneine concentrations ranged between 0.23 and 9.36 μmol/L and were significantly higher in pregnancy obtained by the transfer of vitrified/thawed in-vitro produced embryos. Conversely, no significant changes in amniotic fluid L-ergothioneine concentrations were observed according to the stages of pregnancy considered in this study. These findings suggest that L-ergothioneine concentrations, are not affected as much by the gestational age, but rather by the method used to induce the pregnancy. On the whole, the measurement of L-ergothioneine in amniotic fluid could serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy.

The effect of okadaic acid on meiotic maturation of canine oocytes of different size

The present study was conducted to determine the effect of okadic acid (OA), a potent inhibitor of seronine/treonine 1 and 2A phosphatase, on meiotic resumption and progression in canine oocytes with different diameters. Cumulus-oocyte complexes were collected from ovaries of bitches at different oestrous phases. In Experiment 1, to determine the optimal concentration of OA (0.5 or 2 μM), the oocytes were pre-incubated for 1, 3, and 20 h in TCM 199 supplemented with 20% SCE and thereafter cultured in the same medium without OA. In Experiment 2, the selected oocytes were divided into three groups according to their diameter: <110 μm, 110-120 μm, >120 μm, and pre-incubated in OA 0.5 μM for 1 h. Oocytes were cultured in vitro as previously described. After 72 h of IVM, in Experiment 1, significantly more oocytes reached MII stage with 0.5 μM for 1 h (30.8% P<0.001%) for oocytes cultured in other OA condition and in control group. In Experiment 2, OA induced a significantly higher incidence of MII oocytes in the 110-120 μm and >120 μm groups (P<0.001) compared to control group, but a significantly higher proportion of the oocytes>120 μm pre-incubated with OA progressed to MII (51.3% P<0.001). In contrast, smaller oocytes (<110) did not develop to MII stage with or without OA. In conclusion, treatment of canine oocytes with 0.5 μM for 1 h, improves meiotic maturation. The culture of fully grown (>120 μm) oocytes with OA at the onset of in vitro maturation can result in a higher frequency of meiotic maturation.