Bovine COCs Research Articles

The effect of cadmium on the bovine in vitro oocyte maturation and early embryo development

Common pollutants such as heavy metals and cadmium is among those with high environmental concerns. In vivo studies had shown that cadmium (Cd) causes oocyte degeneration and embryo mortality, and lowers pregnancy rates in mammals. However, there is limited information available about direct effects of Cd on oocyte maturation and/or embryo development. This study was aimed to investigate if Cd has any effect on the oocyte maturation and/or embryo development in vitro. Bovine COCs were collected from the slaughter house and cultured for 24 h in serum-free media only (Controls) or supplemented with 0.2, 2.0 and 20.0 μM CdCl2. At 24 h cumulus cell expansion was assessed in all COCs. COCs were either denuded and stained for determination of nuclear maturation or fertilized for assessment of subsequent embryo development. Cd at the lowest concentration (0.2 μM) did not affect any of the parameters studied. However, at higher concentrations (2.0 and 20.0 μM) it significantly (P < 0.05) reduced the percentage of fully-expanded COCs and significantly (P < 0.05) increased the percentage of partially and/or non-expanded COCs compared to controls and 0.2 μM. Cadmium at higher concentrations (2.0 and 20.0 μM) also significantly (P < 0.01) reduced the percentage of oocytes reaching metaphase II stage compared to controls and 0.2 μM. Post-fertilization cleavage rate in presumptive zygotes and blastocyst development significantly (P < 0.05) reduced 0.2, 2.0 and 20.0 μM CdCl2 compared to the controls (0.0 μM). In conclusion, these results suggest that Cd had direct detrimental effects on the bovine oocyte maturation and its developmental competence.

The influence of morphology, follicle size and Bcl-2 and Bax transcripts on the developmental competence of bovine oocytes.

This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1-3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6-9 mm (95.6; 41.1%), but similar (p > 0.05) to 3-6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1-3 mm (Bax; 1.0 and Bcl-2; 1.0), 3-6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6-9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.

Tribbles expression in cumulus cells is related to oocyte maturation and fatty acid metabolism.

BackgroundIn mammals, the Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that are involved in multiple biological processes including cell proliferation and fatty acid (FA) metabolism. Our recent studies highlighted the importance of FA metabolism in cumulus cells (CC) during oocyte maturation in vertebrates and reported a higher TRIB1 expression in CC surrounding in vivo mature oocytes as compared to immature ooocytes in mice and cows. The objective was to investigate Tribbles expression patterns in bovine CC during in vitro maturation (IVM) and to examine their roles in the cumulus-oocyte complex.MethodsTribbles gene expression was analyzed in bovine and murine CC using quantitative RT-PCR. Proteins were detected using Western blot and intracellular localization was assessed by immunofluorescence. Bovine COCs were treated with etomoxir, an inhibitor of FA oxidation (FAO) which blocks CPT1 activity, during 6 h and 18 h IVM. Oocyte meiotic stage was assessed and expression of Tribbles and lipid metabolism genes was quantified in CC.Results and discussionTRIB1 and TRIB3 were more strongly expressed whereas TRIB2 was less expressed in CC surrounding the oocytes from preovulatory follicles than in CC of immature ones. In CC, Tribbles were located in the cytoplasm and nucleus; in mitotic cells TRIB2 and TRIB3 were detected in the spindle. In the oocyte, Tribbles proteins were detected in the ooplasm; also TRIB2 and TRIB3 were more accumulated in the germinal vesicle. In bovine CC, expression of TRIB1 and TRIB3 was transiently increased at a time preceding oocyte meiosis resumption in vitro. Treatment with etomoxir (150 μM) during IVM resulted in a significant reduction of oocyte maturation rate and decreased MAPK3/1 phosphorylation in the oocytes. In CC, 18 h IVM of etomoxir treatment significantly increased expression of TRIB1, TRIB3, CPTA1 (enzyme regulating FA entry in mitochondria for FAO) and CD36 (thrombospondin receptor involved in FA transport). Under the same conditions, expression of TRIB2 and ACACA (Acetyl coenzyme A carboxylase involved in FA synthesis) decreased in CC.All considered, Tribbles family members may be involved in cell proliferation and in FAO signaling in CC and participate in oocyte meiotic resumption regulation.

Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM

We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9h of maturation did not affect cleavage rate, but improved blastocyst yield (P<0.05) on Day 8 (51 and 61%, respectively; P<0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9h compared with no incubation. The addition of 175 ng mL(-1) GDF-9 or 10%v/v BMP-15 from partially purified transfected 293H cell supernatant for 24h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P<0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.

The influence of cumulus cells during in vitro fertilization of buffalo ( Bubalus bubalis) denuded oocytes that have undergone vitrification

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M–0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control. An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01). In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.

329 EXPRESSION AND REGULATION OF GENOMIC AND NON-GENOMIC PROGESTERONE RECEPTORS IN BOVINE CUMULUS - OOCYTE COMPLEXES DURING MATURATION IN VITRO

The steroid hormone, progesterone (P4) plays an essential role in the regulation of female reproductive events. It acts by binding to specific receptors classified as genomic (nPR-A, nPR-B) and non-genomic (mPRα, mPRβ, PGRMC1, PGRMC2) progesterone receptors (PRs). The aim of the present work was to identify and characterize the PRs that are expressed in bovine COCs during IVM. Immature COCs collected from the ovaries of slaughtered animals and either allocated to analysis at the immature, germinal vesicle stage (GV) or processed for IVM and collected at the metaphase II stage (MII) of meiosis. Groups of up to 50 COCs were placed in 500 mL of maturation medium (TCM-199) supplemented with 10% (v/v) FCS and 10 ng mL-1 epidermal growth factor without or with FSH (100 ng mL-1), LH (100 ng mL-1), or P4 (10, 100, 500 ng m-1), in 4-well dishes and cultured at 39°C for 24 h in a humidified atmosphere containing 5% CO2. GV and MII COCs were stripped of their cumulus cells and 100 oocytes and 10 μg of protein from cumulus cells were resolved by SDS-PAGE using specific antibodies against the different PRs. In addition, the pattern of PR expression was characterized in wholemount preparations of GV and MII COCs by immunocytochemistry using the same antibodies. nPR-A, mPRα, and mPRβ were expressed in both bovine oocytes and their surrounding cumulus cells, while expression of nPR-B, PGRMC1, and PGRMC2 was detected solely in the cumulus cells. Cumulus cell expression of nPR-A and nPR-B, mPRα, and mPRβ increased following IVM, while PGRMC1 and PGRMC2 expression decreased. In contrast to the expression profile of the cumulus cells, nPR-A, mPRα, and mPRβ expression was downregulated following IVM in the corresponding oocytes. Supplementation of the IVM media with FSH or LH resulted in an increase in the expression of nPR-A and nPR-B in cumulus cells and a decrease of nPR-A in oocytes. In contrast, P4 supplementation (500 ng mL-1) during IVM resulted in increased cumulus cell nPR-B expression and decreased nPR-A, mPRα, and mPRβ expression in oocytes. Oocyte mPRβ expression was also downregulated by LH and FSH. PGRMC1 and PGRMC2 were upregulated by 500 and 10 ng mL-1 of P4 respectively. In conclusion, the dynamic changes observed in the protein expression of genomic and non-genomic P4 receptors in bovine COCs after IVM and after treatment with LH, FSH, and P4 suggest that some or all of these receptors may be involved in oocyte maturation in cattle. Supported by Science Foundation Ireland (07/SRC/B1156). IMAparicio was supported by a postdoctoral contract (POS07011) from Consejeria de Economia, Comercio e Innovacion, Junta de Extremadura, Spain.

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

360 EMBRYONIC DEVELOPMENT AND STEROIDOGENESIS OF BOVINE CUMULUS - OOCYTE COMPLEXES MATURED IN α-MEM MEDIUM SUPPLEMENTED WITH PVA OR PVP-40

The influence of the culture medium and its supplements on IVM and steroid secretion by bovine COCs was examined. Immature COCs were matured for 24 h in a-MEM supplemented with IGF-1 (10 ng mL-1), insulin (100 ng mL-1) and 0.1% polyvinyl alcohol (PVA; Sigma Chemical Co., St. Louis, MO, USA) or 0.1% polyvinylpyrrolidone-40 (PVP; molecular weight of 40 000; Sigma). Neither FSH nor LH was used in either treatment. The control group consisted of COCs matured in TCM supplemented with FSH (20 μg mL-1) and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Steroid production was measured in culture medium after IVM and cumulus cells (CC) aromatase activity was estimated by estradiol (E2) production and by the determination of the ratio of E2 to testosterone (T). Cleavage, blastocyst, and hatching rates were evaluated 168-224 h post-insemination. Hormone determination data were analyzed using the GraphPad 5.0 for Windows software (GraphPad Software, San Diego, CA, USA). The means and standard deviations were first calculated for all variables, followed by the Kolmogorov-Smirnov normality test. Since the values were not normally distributed, the treatments were compared by the Kruskal-Wallis test. Blastocyst formation, cleavage, and hatching rates were analyzed by the chi-square test. The level of significance was set at P &lt; 0.05 in all analyses. High aromatase activity (E2:T ratio &gt; 1.0) was detected in the culture medium of both chemically defined IVM systems. E2 concentrations were 22.86 ng mL-1 and 22.46 ng mL-1 for PVA and PVP-40, respectively, and 0.27 ng mL-1 (P &lt; 0.001) for the control group. Progesterone secretion was lower in a-MEM + PVP-40 medium. Testosterone was not secreted by COCs matured in control medium. There was a significantly higher cleavage rate in the control group, but no differences (P &gt; 0.05) in blastocyst (48.92%, 49.56%, and 44.21%) or hatching (38.46%, 41.96%, and 40.78%) rates between the PVA, PVP, and control groups, respectively. Our results show that CC of COCs cultured in serum-supplemented medium show decreased aromatase activity. Also, the addition of IGF-I/insulin and PVA or PVP-40 to IVM medium had no significant effect on the rates of oocyte maturation and embryonic development when compared with results obtained in medium supplemented with estrous cow serum and FSH. Supported by FAPESP/FAEPA-HCFMRP/USP.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles &lt;8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&amp;D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P &lt; 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P &lt; 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P &lt; 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P &lt; 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 � 25 v. 109 � 43 (P &lt; 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP- N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5 mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal–zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-α- d-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.

300 GLUCOSAMINE SUPPLEMENTATION DURING IN VITRO MATURATION LEADS TO PERTURBED DEVELOPMENTAL CAPACITY OF BOVINE CUMULUS OOCYTE COMPLEXES

Glucose is a primary energy substrate required for successful in vitro oocyte maturation (IVM). However, most maturation media contain more glucose than that seen in follicular fluid (2.3 mM vs. 5.6 mM in TCM199). Glucosamine (Glc) as an alternative substrate for extracellular matrix during cumulus expansion reduced glucose uptake by bovine cumulus oocyte complexes (COCs, Sutton-McDowall et al. 2004 Reproduction 128, 313–319). As this could enable a reduction in glucose concentrations to physiological levels in IVM medium of COCs, the aim of this study was to investigate the influence of Glc supplementation on oocyte developmental capacity. Bovine COCs were matured in synthetic follicular fluid medium (SFFM, a defined medium based on the composition of follicular fluid, plus 5.6 mM glucose, FSH, hCG and BSA, Sutton-McDowall et al. 2004 Reprod. Fertil. Dev. 16 sup, 204) ± 5 mM Glc. After 24 h, either nuclear maturation (rep = 8, n = 160) or blastocyst development 8 days post-fertilization (rep = 5, n = 400) was determined. Data was arcsine transformed and analyzed by ANOVA, followed by Tukey's test. While the presence of Glc did not affect the completion of nuclear maturation and early cleavage, +Glc led to severely perturbed blastocyst development (−Glc, 32.5 ± 1.9% vs. +Glc, 4.7 ± 3.9%, P &lt; 0.001). Glc supplementation in somatic cells is well-known to down-regulate the phosphatidylinositol-3-kinase (PI3K) signalling pathway, reducing protein synthesis and other cell survival mechanisms. Therefore, oocyte protein synthesis (measured by [2,3,4,5,6-3H] phenylalanine incorporation, rep = 5, n = 200) and embryo development (rep = 6, n = 720) following IVM in SFFM ± Glc ± EGF (a PI3K pathway stimulator) was determined. Glc supplementation led to a 40% decrease in protein synthesis compared to −Glc, while the combination of +Glc + EGF significantly increased protein synthesis by 60%. However, IVM + EGF + Glc did not improve blastocyst rates (main effect: −Glc 41.6 ± 6.6% vs. +Glc, 6.6 ± 1.7%, P &lt; 0.001). Additionally, COCs were also cultured in SFFM ± 50 μM LY294002 (a specific PI3K inhibitor) and nuclear maturation (rep = 5, n = 200) or blastocyst development 8 days post-fertilization (rep = 4, n = 200) was determined. Despite the presence of LY294002 leading to 43% less COCs completing nuclear maturation (P &lt; 0.001), blastocyst development was not affected (mean = 38.8 ± 3.2%). These results demonstrate that Glc supplementation during IVM has no effect on nuclear maturation or early development but is detrimental to oocyte developmental capacity by severely perturbing blastocyst development. However, the diminished developmental capacity appears to be independent of the well-characterized Glc down-regulation of the PI3K signalling pathway. This work was supported by the Australian Research Council (SPIRT, C00107702) and Cook Australia Pty Ltd.

327THE EFFECTS OF GLUCOSE AND GLUCOSAMINE ON CUMULUS EXPANSION AND NUCLEAR MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES

Glucose is the primary energy substrate consumed by bovine COCs during in vitro maturation (IVM), with most accounted for by glycolysis (L-lactate production). However, antral follicular fluid (FF) contains less than half the glucose of standard IVM media (TCM199=5.6mM, FF=2.3mM). We have previously demonstrated that from 20 to 24h of IVM, a significant proportion of the glucose utilized is directed into pathways other than lactate production (Sutton et al., 2003 Reproduction 126, 27–34). We hypothesize that glucose is utilized for cumulus matrix synthesis. The aim of this study was to determine the influence of glucosamine (an intermediate for matrix components) on FSH-stimulated glucose uptake and cumulus expansion. The influence of different glucose concentrations and glucosamine on nuclear maturation was also investigated. Bovine COCs were collected from abattoir-derived ovaries. In Exp. 1, individual COCs (n=60, 3 replicates) were cultured in 10-μL drops of TCM199 (plus pyruvate, hCG and BSA, containing 5.6mM glucose), ±FSH (0.1IUmL−1) and ±glucosamine (5mM). After 20h, COCs were transferred to fresh media and cultured a further 4h. Cumulus expansion and glucose/L-lactate levels in spent medium from 0–4-h and 20–24-h culture periods were measured. In Experiment 2, COCs (n=300, 6 replicates) were cultured in groups of 10 in 100μL of Bovine FF medium (a defined medium based on the composition of bovine antral FF, also containing amino acids, FSH, hCG and BSA) ±glucosamine (5mM) in 2.3 or 5.6mM glucose, or in conventional TCM199 IVM media (as above). Nuclear maturation was assessed at 24 and 30h using orcein staining. Treatment differences were determined using two-way ANOVA. The influence of FSH and glucosamine (Exp. 1) on the measured parameters was evident at 20–24h, with FSH increasing diameter, glucose uptake and L-lactate production (P&amp;lt;0.05). Although glucosamine alone did not influence diameter or glucose/L-lactate concentrations, glucosamine plus FSH led to a decrease in glucose uptake compared to FSH-stimulation alone (P&amp;lt;0.05). The proportion of oocytes at MII (Exp. 2) was significantly lower when COCs were cultured in low glucose (main effect, 24h: 2.3mM=38% v. 5.6mM=64%; P&amp;lt;0.005). The presence of glucosamine tended to stimulate meiotic maturation (main effect, 24h: 0mM=45% v. 5mM=59%; P=0.1). MII frequency in TCM199 controls at 24h was 68%. These experiments support the hypothesis that synthesis of cumulus matrix is a major pathway for glucose metabolism, especially in the absence of glucosamine. Furthermore, oocytes matured in media based on a physiological concentration of glucose (2.3mM), have delayed meiosis compared to oocytes cultured in higher glucose (5.6mM). Thus, glucose has multiple functions, involving matrix formation and meiosis regulation during bovine IVM. Supplementation of medium with glucosamine appears to partly reduce the dependency of COCs on glucose. Supported by Australian Research Council and COOK Australia.

128CHANGES WITHIN OXYGEN ENVIRONMENT DURING IVF AND IVC IMPROVE BOVINE EMBRYONIC DEVELOPMENT IN VITRO

Oxygen concentration during both IVF and IVC affects embryonic development. A low O2 atmosphere during IVC has been reported to be beneficial for embryos in culture without somatic cells. Similarly, a reduction in spermatozoa (sp) concentration can influence the oxygenation grade of fertilization medium, and O2 requirements can vary according to the embryonic stage of development. This experiment investigated whether prolonged contact with sperm cells interacts with effects of oxygen tension during the first days of culture. Slaughterhouse bovine COCs matured with Vero-cells in TCM199 with FCS and EGF were co-incubated (CI) with swim-up separated sp. COCs with attached sp were either removed after 2h and placed in IVF medium without sp up to completion of 18h (sp-restricted CI), or COCs and sp were left together for 18h (sp-prolonged CI). Zygotes were cultured in serum-free, B2 medium conditioned with Vero cells (Marquant-Le Guienne et al., 1999 Theriogenology 51, 386), modified as described by Gomez and Diez (2000 Anim. Reprod. Sci. 58, 23–37). According to IVF procedure (sp-restricted or sp-prolonged), embryo culture was made either in 20% O2 from Day 0 up to Day 3 and in 5% O2 later on, or in 5% O2 from Day 0 up to Day 8, in a 2×2 factorial design. IVF medium was monitored for Na+, K+, Ca2+, pH, O2, CO2 , HCO3− and lactate (i-STAT analyzer; 5 replicates -R-) at 0h, 2h and 18h without cells (controls), at 2h in CI and at 18h both in sp-restricted and sp-prolonged CI. Embryo development was analyzed by CATMOD for effects, and all data were processed by GLM and Duncan test and expressed as LSM±SE. The presence of cells decreased pO2 (P&amp;lt;0.01) in IVF medium at 2h of CI (154±2.3 without cells v. 144±2.3 with cells). There were differences in pO2 at 18h (P&amp;lt;0.05) between sp-prolonged (140.8±2.3) and sp-restricted CI (148.6±2.3). At 18h in CI there was K+ depletion (P&amp;lt;0.05), while Na+, Ca2+, pH and pCO2 remained invariable throughout. Lactate production increased (P&amp;lt;0.05) in CI at 2h and, together with HCO3− (P&amp;lt;0.01), at 18h in sp-prolonged CI. Increasing O2 up to 20% during the first 3 days of culture improved total and medium to late (M+L; early blastocysts excluded) blastocyst rates upon sp-prolonged CI, which is comparable to sp-restricted CI under both % O2 conditions during embryo culture. Under 5% CO2 throughout culture, higher expansion rates occurred in sp-restricted CI, suggesting that this system could improve embryonic viability. Metabolic effects can be inferred from differences in pO2 , lactate and HCO3− , and research will explore the role of oxygen free radicals in these experimental conditions. Grant support: Eureka 2573; CDTI, PROFIT, and FICYT.