Differential Expression and Localization of Glycosidic Residues in In Vitro and In Vivo Matured Cumulus-Oocyte Complexes in Equine and Porcine Species

Glycoprotein 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

Pro-oxidant 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

Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential

BACKGROUND: Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression. METHODS: Experiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls. RESULTS: EH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls. CONCLUSIONS: Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential

In vitro maturation induces glycoconjugate changes in equine cumuls-oocyte complexes

In 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

Effects of DEHP exposure on energy/oxidative parameters of the cumulus-oocyte complexes in the mare

ABSTRACT - 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

Effects of in vitro opioidergic stimulation on proliferative and differentiative abilities of canine umbilical cord matrix mesenchymal stem cells

Opioid 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

Effects of gestational age on proliferative and differentiation potency of mesenchymal stem cells isolated from canine amnion and umbilical cord matrix

ABSTRACT - 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