Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (> 18 mm) follicles. RNeasy Micro Kit (Qiagen(A (R))) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo-and in vitro-derived embryos in cattle.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Analysis of the impact of rainfall assimilation during LBA atmospheric mesoscale missions in Southwest Amazon

The assimilation of satellite estimated precipitation data can be used as an efficient tool to improve the analysis of rainfall generated by numerical models of weather forecast. The system of data assimilation used in this study is cumulus parameterization inversion based on the Kuo scheme. Reanalysis were performed using the field experiment data of the LBA Project (WETAMC and DRYtoWET-AMC), where it was possible to verify an improvement in the simulations results, since the data assimilation corrects the position and the intensity of rainfall in the numerical model. (C) 2012 Elsevier B.V. All rights reserved.

Fog- and cloud-induced aerosol modification observed by the Aerosol Robotic Network (AERONET)

Large fine mode-dominated aerosols (submicron radius) in size distributions retrieved from the Aerosol Robotic Network (AERONET) have been observed after fog or low-altitude cloud dissipation events. These column-integrated size distributions have been obtained at several sites in many regions of the world, typically after evaporation of low-altitude cloud such as stratocumulus or fog. Retrievals with cloud-processed aerosol are sometimes bimodal in the accumulation mode with the larger-size mode often similar to 0.4-0.5 mu m radius (volume distribution); the smaller mode, typically similar to 0.12 to similar to 0.20 mu m, may be interstitial aerosol that were not modified by incorporation in droplets and/or aerosol that are less hygroscopic in nature. Bimodal accumulation mode size distributions have often been observed from in situ measurements of aerosols that have interacted with clouds, and AERONET size distribution retrievals made after dissipation of cloud or fog are in good agreement with particle sizes measured by in situ techniques for cloud-processed aerosols. Aerosols of this type and large size range (in lower concentrations) may also be formed by cloud processing in partly cloudy conditions and may contribute to the "shoulder" of larger-size particles in the accumulation mode retrievals, especially in regions where sulfate and other soluble aerosol are a significant component of the total aerosol composition. Observed trends of increasing aerosol optical depth (AOD) as fine mode radius increased suggests higher AOD in the near-cloud environment and higher overall AOD than typically obtained from remote sensing owing to bias toward sampling at low cloud fraction.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemProA (R) medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Our data show that Ca2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

Photobiological effect of low-level laser irradiation in bovine embryo production system

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism