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In vitro maturation induces glycoconjugate changes in equine cumuls-oocyte complexes
No full textIn vitro matured oocytes (IVM) suffer some inadequacies when compared with in vivo matured ones1. Inadequate IVM can yield under- or overmature oocytes, which will not undergo normal fertilization and embryo development2. Glycoconjugates play a key role in oocyte maturation, and in oocyte-sperm interactions leading to fertilization 3,4, thus the knowledge of oligosaccharide pattern of equine COCs could provide useful information about the comparison between immature and matured COCs. Cumulus enclosed oocytes from abbattoir ovaries were fixed in Bouin’s solution and embedded in paraffin wax either before or after IVM. Sections were stained with 13 lectin (MAL II, SNA, PNA, DBA, RCA120, SBA, HPA, Con A, WGA, GSA I-B4, GSA II, UEA I, LTA). The radiata zone of immature COCs reacted with all used lectins, whereas matured COCs stained with MAL II, SNA, HPA, SBA, and Con A. The zona pellucida of both COCs types bound MAL II, SNA, SBA, and Con A, whereas immature COCs reacted also with RCA120, WGA, and matured ones stained with UEA I. The ooplasm of both types of COCs reacted with HPA, Con A, GSA II, UEA I and LTA, whereas immature oocytes bound also SNA, SBA, WGA, GSA I-B4. These results indicate that IVM modifies glycoprotein pattern of equine COCs and prompted us to undergo further studies to investigate the role of the modified oligosaccharides in oocyte viability, capacity to undergo fertilization and normal embryonic development.
References
1. Deleuze S et al Theriogenology. 2009, 72:203-9.
2. Hinrichs K & Di Giorgio LM J Reprod Fertil Suppl 1991, 44:369–74.
3. Dell A et al Biochim Biophys Acta 1999, 1473:196-205.
4. Clark GF & Dell A. J Biol Chem 2006, 281:13853-6
Holding in meiosis inhibitors-free medium does not affect oocyte oxidative status and development.
No full textAttività mitocondriale e stress ossidativo in ovociti maturati in vivo o in vitro in un modello animale con schema intra-soggetto
No full textEffects of in vitro opioidergic stimulation on proliferative and differentiative abilities of canine umbilical cord matrix mesenchymal stem cells
No full textOpioid receptors (ORs) are G protein-coupled receptors. Other than antinociception, they have been recently shown to be involved in the crucial switch phase between cell proliferation and differentiation, from which the stem cell fate depend. We detected mu-OR subtype 1 (MOR-1) and kappa-OR subtype 1 (KOR-1) expression in canine umbilical cord matrix (UCM) mesenchymal stem cells (MSCs). MOR expression decreased with passage numbers, whereas KOR was expressed at constant levels throughout passages. Both ORs type were functionally active, since DAMGO and U69593, MOR- and KOR- selective agonists respectively, and CTAP and nor-BNI, MOR- and KOR- selective antagonists respectively, were significantly able to modulate cell proliferation. Both opioid agonists, when used at the concentration of 1μM, inhibited cell proliferation of canine UCM-MSCs. Inhibitory effect on cell proliferation was also observed after CTAP treatment, whereas no effect was noticed after nor-BNI treatment. By specific stain and morphology analysis, no differences were observed in the neurogenic differentiation potency of UCM-MSCs in both treatments and control conditions. Collectively our data suggest that, in canine UCM-MSC, opioids modulate cell proliferation, but further studies are needed to evaluate whether opioid modulation may play a role in directing these cells to neurogenic lineages
Bioenergy/oxidative status of equine oocytes held in the absence of meiosis inhibitors before in vitro maturation.
No full textEffects of gestational age on proliferative and differentiation potency of mesenchymal stem cells isolated from canine amnion and umbilical cord matrix
No full textABSTRACT - Amniotic membrane (AM) and umbilical cord matrix (UCM)
mesenchymal stem cells (MSCs) have been isolated and characterized in humans and
large animal models. In order to distinguish which cells retain the best features for different purposes, the effects of gestational age on proliferation and differentiation potency of canine AM-MSCs and UCM-MSCs was analyzed. Samples were recovered after elective ovariohysterectomy from bitches in early (35 to 40 days) and late (45 to 55 days) fetal stage of pregnancy. The proliferation study and the molecular analysis of
embryonic, mesenchymal and hematopoietic markers were performed. Cell neurogenic and osteogenic differentiation were followed. No differences were noticed when
comparing data obtained from cells isolated at different gestational ages. Doubling times, cell viability and Oct-4, CD29 and CD44 stemness markers expression were similar in cell isolated from bitches in early or late pregnancy. In both gestational ages, morphological features of neuronal and osteogenic differentiation were observed which
need to be confirmed by molecular analysis. In conclusion, our data indicate the possibility to isolate MSCs from canine fetuses at early and late gestational ages with the same proliferative and differentiative capabilities
ATP content and SOD activity in single oocytes before and after in vitro maturation
No full textThe developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Analyses of energy and redox status parameters are emerging technologies useful for further oocyte quality characterisation. Mitochondrial (mt) activity is a necessary feature involved in cytoplasmic maturation, and the primary function of mitochondria is adenosine triphosphate (ATP) production. Mitochondria distribution pattern and ATP content are important parameters in the evaluation of oocyte metabolic activity, particularly activities driving microtubules dynamics leading to chromosomes segregation. Superoxide dismutase (SOD), a first-line antioxidant enzyme, has also been hypothesised as being associated to oocyte quality. The aim of the present study was to analyse ATP content and SOD activity in single equine oocytes examined before and after in vitro
maturation. Cumulus–oocyte complexes surrounded by a compact cumulus oophorus were recovered from the ovaries of slaughtered mares and analysed before or after in vitro maturation (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent evaluation of signs of meiotic maturation, and only those oocytes showing cumulus expansion, regular ooplasmic size (4160 mm in diameter) and morphology, and 1st polar body extrusion were selected for analysis. Adenosine triphosphate intracellular levels were analysed by luciferin-luciferase bioluminescent reaction (ATPlite, PerkinElmer, Monza, Italy). Quantification of SOD activity was performed by spectro- photometrical assay with WST1 and by polyacrylamide native gel and nitro blue tetrazolium reduction method. Intracellular ATP levels were influenced by meiotic stage in that oocytes at the germinal vesicle stage (GV, n 1⁄4 15) showed 1.25 ` 0.8 pmol cell1, whereas metaphase II (MII) oocytes (n1⁄415) showed significantly higher levels (2.29`1.69 pmolcell1; Po0.05). This is in line with our previous observations on mt distribution pattern analysed by Mitotracker Orange CMTM Ros staining and confocal microscopy (Ambruosi et al. 2009). In vitro-matured MII oocytes showed significantly higher rates of perinuclear mt distribution pattern, indicating mt aggregation around meiotic metaphase spindle, compared with GV oocytes (3/12, 25% v. 0/13, 0% in GV oocytes; Po0.05). Superoxide dismutase spectrophotometrical activity was 0.72 ` 0.55 U mg1 prot in GV oocytes (n 1⁄4 4) and 2.33 ` 0.33 U mg1 prot in MII oocytes (n 1⁄4 2; P o 0.001). In native gel SOD activity was 16 285.05 arbitrary densitometric units (ADU) in a GV oocyte and 22 501.35 ADU in a MII oocyte. To our knowledge, this is the first study reporting intracellular SOD activity in single oocytes in mammals. Moreover, this is the first study reporting ATP content in single equine oocytes. Observed quantitative differences seem to be related to meiotic stage
Effects of DEHP exposure on energy/oxidative parameters of the cumulus-oocyte complexes in the mare
No full textABSTRACT - The aim of the present study was to analyze the effects of in vitro
exposure to Di-(2-ethylhexyl) phthalate (DEHP), an industrial plasticizer, on cumulus-oocyte maturation and energy/oxidative status in the horse. After in vitro maturation (IVM) in presence of 0.12, 12 and 1200 μM DEHP, cumulus cells (CCs) were removed and evaluated for apoptosis and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase-II
stage; MII) oocytes were further evaluated for cytoplasmic energy/oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 μM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 μM), DEHP induced apoptosis
(P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial (mt) distribution patterns, apparent energy status, intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with the antioxidant N-Acetyl-Cysteine
reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without improving oocyte maturation. In conclusion, in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy/oxidative parameters in
matured oocytes
MITOCONDRI E SPECIE REATTIVE DELL’OSSIGENO (ROS) IN OVOCITI DI PECORE ADULTE SUPEROVULATE E MATURATI IN VIVO O IN VITRO. MITOCHONDRIA AND REACTIVE OXYGEN SPECIES (ROS) IN OOCYTES FROM SUPEROVULATED ADULT EWES AND MATURED IN VIVO OR IN VITRO
No full textDifferential Expression and Localization of Glycosidic Residues in In Vitro and In Vivo Matured Cumulus-Oocyte Complexes in Equine and Porcine Species
Full text linkGlycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and bN-acetylgalactosamine (GalNAc)-termi- nating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models
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