Nuclear Maturation Of Bovine Oocytes Research Articles

Cytokine-Supplemented Maturation Medium Enhances Cytoplasmic and Nuclear Maturation in Bovine Oocytes.

Bovine in vitro oocyte maturation (IVM) is an easy way to obtain oocytes for subsequent assisted reproductive techniques but is inefficient compared to in vivo maturation. Supplementation of three cytokines, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), or FLI, has increased oocyte maturation and embryo development in multiple species, but studies have not explored the oocyte differences caused by FLI IVM supplementation. This study aimed to assess important nuclear and cytoplasmic maturation events in high-quality oocytes. FLI-supplemented oocytes had a decreased GV (3.0% vs. 13.7%, p < 0.01) and increased telophase I incidence (34.6% vs. 17.6%, p < 0.05) after IVM, increased normal meiotic spindles (68.8% vs. 50.0%, p < 0.001), and an increased nuclear maturation rate (75.1% vs. 66.8%, p < 0.001). Moreover, in metaphase II oocytes, the percentage of FLI-treated oocytes with a diffuse mitochondrial distribution was higher (87.7% vs. 77.5%, p < 0.05) and with a cortical mitochondrial distribution was lower (11.6% vs. 17.4%, p < 0.05). Additionally, FLI-supplemented oocytes had more pattern I cortical granules (21.3% vs. 14.4%, p < 0.05). These data suggest that FLI supplementation in bovine in vitro maturation medium coordinates nuclear and cytoplasmic maturation to produce higher-quality oocytes.

Glutamine, proline, and isoleucine support maturation and fertilisation of bovine oocytes

Successful in-vitro production of bovine embryos relies on meiotic maturation of oocytes in vitro (IVM) before they can be fertilised. High levels of IVM are currently achieved using a complex medium that contains all 20 common amino acids, namely TCM199, but can also be achieved using a simple inorganic salt solution containing non-essential amino acids, proline, and glutamine. Further simplification of the amino acid content of medium used for IVM could lead to a more defined medium that provides reproducible IVM. The aim of this study was, therefore, to determine the minimal amino acid requirements for bovine oocyte nuclear maturation, as measured by progression to metaphase II (MII) of meiosis. Supplementation of a simple medium composed of inorganic salts (M1 medium) with multiple amino-acid combinations showed that M1 containing glutamine, proline, and isoleucine resulted in nuclear maturation comparable to that of TCM199 (57.4 ± 3.4% vs 67% ± 1.7%, respectively) but was reduced when cystine (Cys2) to that seen with M1 alone (38.0 ± 2.2%). Viability of oocytes matured in this simplified medium was equal to those matured in TCM199 since the same proportion of zygotes with 2 pronuclei were observed following fertilisation in medium containing no amino acids (33.9 ± 6.5% vs 33.3 ± 3.6%, respectively). Addition of glutamine, proline and isoleucine to fertilisation medium also increased the proportion of zygotes but did not increase blastocyst development rates. Thus, a defined medium containing only glutamine, proline and isoleucine is sufficient for oocyte maturation and successful fertilisation.

Effect of Sericin Supplementation in Collection Medium on Bovine Oocyte Nuclear Maturation

Sericin is a water-soluble globular protein derived from silkworm Bombyx mori and has the competency as an antioxidant. This study was conducted to examine the effect of sericin supplementation in the collection medium on bovine oocyte nuclear maturation. Sericin with different concentration (0 (control), 0.1 %, 0.5 %, and 1%) was added to collection medium and maturated for 24 hour at 38.5 °C in 5% of CO2 air. Matured oocytes were stained with acetic-orcein and determined the oocyte nuclear stage under a stereomicroscope. After in vitro maturation, 74-87% of oocytes were reached nuclear maturation (metaphase II). The maturation rates of oocytes were significantly higher at 0.1% group (87.7%) (P<0.05) compared to other groups. There was no significant differences were observed between control group (74.6%), 0.5% group (79.4%), and 1% group (78.3%) (P>0.05). These findings showed that supplementation of 0.1% sericin in the collection medium improved the nuclear maturation of bovine oocytes.

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes.

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P &gt; 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P &gt; 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

Maturação nuclear in vitro de ovócitos bovinos selecionados pelo método azul cresil brilhante

Objetivou-se investigar a competência in vitro da maturação nuclear de ovócitos selecionados pela técnica do corante Azul Cresil Brilhante (ACB), após manutenção em meio Talp-Hepes. Complexos cumulus ovócito (CCOs) foram obtidos de ovários de matadouro e distribuídos aleatoriamente em 6 tratamentos. Quanto percentual de ovócitos que apresentaram configuração nuclear de MI (Metáfise I) ao término da MIV, notou-se que T3 e T5 apresentaram-se superiores ao T1, T2 e T4 (p&lt;0,05). Quanto à capacidade dos ovócitos de completarem a MIV, expresso pela competência em atingirem MII (Metáfise II), observou-se que os tratamentos T1, T2, T4 apresentaram maior percentual em MII que T3 e T5 (p&lt;0,05). Dos ovócitos submetidos a MIV, T1 apresentou menor percentual de degenerados que T2, T3, T4, T5 (p&lt;0,05). Concluiu-se que a capacidade de coloração de ovócitos por ACB não alterou após manutenção dos CCOs por 5 horas em Talp-Hepes e que o corante ACB mostrou-se eficaz na seleção de ovócitos mais competentes após manutenção de CCOs por 5h em Talp-Hepes.

Effect of season on follicular population, quality and nuclear maturation of bovine oocytes under tropical conditions

The aim was to determine the effect of season of the year and the presence of a corpus luteum (CL) on follicular population (FP) and the quality of the oocytes, and of season on nuclear maturation of the bovine oocytes under tropical conditions. Three seasons were evaluated: hot-dry (March–June), hot-humid (July–October) and fresh-humid (November–February). In a first study, 1112 bovine ovaries were obtained from a local slaughterhouse. Follicles were classified as small (≤4mm), middle (4.1–8mm) and large (≥8.1mm); and the maximum diameter of the follicle (MDF) and CL (MDCL) were also recorded. The oocytes were collected by aspiration and classified as viable (grade I and II) and damaged (grade III and IV). In the second study, 2261 viable oocytes were matured in vitro, and then fixed and stained with Lacmoid to classify the stage of development as mature (metaphase II), immature or degenerate. Data were analyzed using analysis of variance and chi-square procedures. The largest FP of large follicles (0.67), MDF (1.18mm), MDCL (1.87mm), and the highest proportion of viable oocytes (34.19%) were obtained during the hot-humid season (P<0.05). The ovaries without CL had the greatest FP (10.34) with more viable oocytes (24.44%). The highest proportion of mature oocytes (76.92%) was also obtained in the hot-humid season. In conclusion, season influenced FP, MDF, MDCL, and the quality and nuclear maturation of oocytes. The presence of a CL in the ovary resulted in a decrease of FP and viability of oocytes.

Effect of lipopolysaccharide on developmental competence of oocytes

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation resulting in low fertility. The aim of this study was to determine the effect of LPS on the developmental competence of bovine oocytes in vitro. LPS perturbed the nuclear maturation of bovine oocytes by inhibiting meiotic progression. Although LPS did not affect the copy number of mitochondrial DNA, it decreased mitochondrial membrane potential in matured oocytes. LPS inhibited mitochondrial redistribution throughout the cytoplasm. Oocytes matured under LPS treatment showed decreased development to the blastocyst stage. Moreover, the trophoblast cell number of blastocysts was significantly lower when the oocytes were matured in the presence of LPS. Our findings suggest that LPS might impair the nuclear and cytoplasmic maturation of oocytes and obstruct subsequent embryonic development in dairy cows.

Effect of kit ligand on natriuretic peptide precursor C and oocyte maturation in cattle

In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.

Folikül Büyüklüğü ve Folikül Uyarıcı Hormon Konsantrasyonunun Sığır Oositlerinin İn Vitro Nükleer Olgunlaşması Üzerine Etkisi

The aim of the study was to investigate the effect of follicle size and follicle stimulating hormone (FSH) concentration on nuclear maturation of bovine oocytes in vitro. Follicles on bovine ovary were classified into 3 groups according to the diameter; small (

A simple and high-throughput method to assess maturation status of bovine oocytes: Comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4′,6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.

Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation

Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P4) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P4 induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P4 receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P4 actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P4-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P4 in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P4, providing further support to the AngII-P4 sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P4 and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation.

Protein kinase implication in prolactin signaling in bovine oocyte-cumulus complexes

The mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles are poorly understood. In this work, participation of tyrosine kinases and protein kinase C in mediation of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic and functional state of surrounding cumulus cells in vitro has been investigated. It was found that a tyrosine kinase inhibitor genistein suppresses the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas a protein kinase C inhibitor calpostin C does not affect the hormonal effect. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time, the retarding action of prolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. These results show that the stimulating influence of prolactin on oocyte nuclear maturation accompanied by cumulus expansion is achieved with participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation both of tyrosine kinases and protein kinase C.

Effect of maternal age on the developmental competence and progression of nuclear maturation in bovine oocytes

Progression of meiotic division in oocytes and early embryonic development are affected by oocytes quality. In most mammals, oocyte quality declines with increase in maternal age. The main aim of the present study is to investigate the effect of maternal age on developmental competence, progression of meiotic division, and associated kinetics of maturation promoting factor (MPF) activity in bovine oocytes. Oocytes were collected from the ovaries of young and old cows (here after referred to as young cow oocytes and old cow oocytes, respectively). When old cow oocytes were matured and fertilized in vitro, the rate of abnormal fertilization was greater than that in young cow oocytes. Moreover, progression of nuclear maturation and activation of MPF during oocyte maturation (or inactivation of MPF and formation of pronucleus after insemination) were faster in old cow oocytes than in young cow oocytes. Relative expression of cyclin B, cyclin-dependent kinase 1 and MAD2 transcripts in either immature or mature oocytes did not differ between the two groups. When cumulus cells (CC) were removed and denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two age groups. Moreover gap junctions between oocytes and CC disappeared more rapidly during maturation of old cow oocytes than of young cow oocytes. These results suggest that the fertilization ability of old cow oocytes is low and that premature progression of meiotic division in these oocytes is partly due to impaired oocyte-CC gap junctions communication.

Effect of melatonin on in vitro maturation of bovine oocytes

To investigate the effect of different concentrations of melatonin on bovine oocytes in vitro maturation, varying concentrations of melatonin (0, 0.01, 1, 100 iM), were included in the the maturation medium. Slaughterhouse derived oocytes were subjected to standard in vitro maturation procedures in high oxygen tension. After culture for 24 h, over 80% of COCs had full cumulus cells expansion. As melatonin concentration increased, the degree of cumulus cells expansion did not changed significantly. Oocytes incubated in 0.01 and 1 iM melatonin-containing media for 24 h, result in 73.11 and 70.68% maturation rate, respectively, which were no different from the control (72.24%). The maturation rate decreased (P < 0.05) significantly when melatonin concentrations was increased from 0.01 to 1 and 100 iM (73.11 vs 70.68% and 65.24% respectively). After culture in melatonin-containing media for 24 h, 100% oocytes achieved GVBD stage in all groups. There were no significant differences among groups at GVBD stage. But oocytes that remained at metaphase-I stage were significant (P < 0.05) different with 100 iM in compare to other groups (21.32 Vs 17.67, 15.68, 16.53). In conclusion in this experiment, melatonin cannot improve cumulus cell expansion and nuclear maturation of bovine oocytes. When concentrations is high, melatonin may affect bovine oocytes meiotic maturation at metaphase-1 stage, but it is improbable melatonin be toxic for bovine oocytes.

355 INFLUENCE OF NITRIC OXIDE AND CYCLIC GMP SIGNALING PATHWAY ON THE IN VITRO MATURATION OF BOVINE OOCYTES: PRELIMINARY RESULTS

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthase enzyme (NOS) and has been shown to be involved in oocyte maturation. NO is known to act through the guanylate cyclase (GC) signaling pathway, stimulating the production of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG). The objective of the present study was to investigate the involvement of NO and GC/cGMP/PKG pathway on the IVM of bovine oocytes. Slaughterhouse ovaries were transported to the laboratory and oocytes were aspirated from 2 to 8 mm follicles. Oocytes were submitted to IVM (TCM-199+10% fetal calf serum + hormones) for 24 h (38.5°C and 5% CO2 in air) and were assessed for nuclear maturation by acetic-orcein (1%) staining. Maturation rates were analyzed by ANOVA. Five replicates were performed with 20 oocytes per group per replicate. When the oocytes were matured with the NO donor [(0, 10-9, 10-8 and 10-7M S-nitroso-N-acteyl-D,L- penicillamine (SNAP)] germinal vesicle break down (GVBD) rates after 7 h in IVM were 36, 31, 42, and 24%, respectively (P &gt; 0.05). Maturation rates after 24 h IVM ranged from 80 to 85% (P &gt; 0.05). The inhibition of GC [(0, 0.1, 10, and 100 μM 1, H-[1, 2, 4]oxadiazole[4, 3-a]quinoxalon-1-one (ODQ)] and PKG (0, 1, 10, and 100 μM KT5823) did not affect (P &gt; 0.05) the ability of oocytes to form the first polar body (average of 83 and 88%, respectively). When the cGMP-analogue (0, 1, 2, and 4 mM 8-Bromo-cGMP) and the GC-stimulator (0, 5, 10, and 50 μM Protoporphyrin IX) were used during IVM, maturation rates were over 85% in all groups (P &gt; 0.05). To confirm the lack of effect of the inhibitors, another evaluation with higher concentrations of inhibitors in semi-defined IVM medium (TCM-199 + 0.04% BSA) was carried out. Maturation rates were 70 to 75% (P &gt; 0.05) with ODQ and 57 to 76% (P &gt; 0.05) with KT5823. The evaluation with the GC stimulator and the cGMP analogue in semi-defined medium is currently underway. In conclusion, under the conditions studied, the GC/cGMP/PKG signaling pathway is not involved in the nuclear maturation of bovine oocytes. Supported by FAPESP, Brazil.

Retinoic acid effects on nuclear maturation of bovine oocytes in vitro

In the present study, the effect of all-trans retinoic acid (t-RA) administration during in vitro maturation (IVM) on bovine oocytes maturation was determined. Concentrations of t-RA (RA; 0, 0.25, 0.5 and 1 μM) and 0.1% ethanol (vehicle) were included in the maturation medium. Ovaries collected from the local abattoir were transported to the laboratory in in 0.9% NaCl with 100 IU/ml penicillin and 100 in vitro maturation (IVM) on bovine oocytes maturation was determined. Concentrations of t-RA (RA; 0, 0.25, 0.5 and 1 μM) and 0.1% ethanol (vehicle) were included in the maturation medium. Ovaries collected from the local abattoir were transported to the laboratory in in 0.9% NaCl with 100 IU/ml penicillin and 100 in vitro maturation (IVM) on bovine oocytes maturation was determined. Concentrations of t-RA (RA; 0, 0.25, 0.5 and 1 μM) and 0.1% ethanol (vehicle) were included in the maturation medium. Ovaries collected from the local abattoir were transported to the laboratory in in 0.9% NaCl with 100 IU/ml penicillin and 100 g/ml streptomycin at 30 - 35°C within 1-2 h after collection. The oocytes of antral follicles, 2 to 8 mm in diameter, were recovered by aspiration. After preliminary evaluation, the oocytes were selected and washed four times in HEPES-TCM 199 supplemented with 2% FBS, 0.2 mM sodium pyruvate, 100 IU/ml penicillin and 100 g/ml streptomycin. Then 10 cumulus-oocyte complexes (COCs) were subjected to each droplet of maturation medium and incubated at 38.5°C, 5% CO 2 and 95% humidity for 24 h. Maturation medium was bicarbonate-buffered TCM199 supplemented with 10% FBS, 0.2 mM sodium pyruvate, 5 μg/ml bovine FSH, 0.01 IU/ml bovine LH, 100 IU/ml penicillin and 100 g/ml streptomycin. Results show different concentrations of t-RA have no effect on cumulus expansion. The rate of oocytes developing to the MII stage compared to control, vehicle, and 0.25 μM groups was significantly increased with 1 μM t-RA treatment (p

Effects of Serum, Gonadotropins, Epidermal Growth Factor and Estradiol 17-Beta on Cumulus Expansion and Nuclear Maturation of Bovine Oocytes

Effects of Serum, Gonadotropins, Epidermal Growth Factor and Estradiol 17-Beta on Cumulus Expansion and Nuclear Maturation of Bovine Oocytes

Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by prostaglandins E2 and F2α

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 μM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P&lt;0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 μM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P&lt;0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE2, PGF2α or AngII was present in the co-culture system with follicular cells (PGE2 77.4%, PGF2α 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE2 or PGF2α produced by follicular cells.

ATP-dependent Chromatin Remodeling Factors Regulate Nuclear and Epigenetic Maturation of Bovine Oocytes.

Chromatin remodeling involves in dynamic transition of chromatin structure and includes alterations for the DNA methylation, histone modification, histone composition and chromatin conformation through the action of ATP-dependent chromatin remodeling enzymes. During oocyte maturation, transition of chromatin to chromosome occurs via a series of processes including germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and metaphase II. However, dynamics of chromatin remodeling factors in the progress is unclear. This study was performed to understand how ATP-dependent chromatin remodeling factors (ACRFs) act during the oocyte maturation. To inhibit the activity of ACRFs, oocytes were first treated with 20 U/ml apyrase, an enzyme for ATP hydrolysis. Oocytes that were obtained at 1 h, 9 h, 24 h and 36 h after maturation, respectively, were immunostained with various antibodies specific for ACRFs and epigenetic markers such as Brg-1, BAF-170, Mi-2, hSNF2H, 5-MeC, AcH3K9, AcH4K5, mono-, di- and tri-methyl H3K9. After apyrase treatment, the localization and the distribution of ACRFs such as Brg-1, BAF-170, Mi-2 and hSNF2H showed normal patterns in chromatin or metaphase chromosomes of maturing oocytes, but the timing of their chromatin remodeling was retarded. In contrast to normal oocytes, Mi-2 didn't disappear at 1 h after maturation in the nucleus of apyrase-treated oocytes. These results demonstrate that suppression of the activity of ACRFs give rise to delayed chromatin remodeling in the nuclear maturation. The distribution patterns of epigenetic markers, including DNA methylation of 5-MeC, histone acetylation of H3K9 and H4K5, and histone mono-, di-, tri-methylation of H3K9, were temporally consistent with the timing of chromatin remodeling in apyrase-treated oocytes. Thus, nuclear maturation of bovine oocytes was delayed by inhibition of ACRFs. Developmental competence of apyrase-treated oocytes fertilized in vitro was significantly decreased compared with that of normal oocytes. Our findings suggest that ACRFs control timely nuclear maturation by chromatin remodeling, thereby leading to oocyte maturation and early embryonic development.