Oocyte Transport Research Articles

Estradiol increases IP3 by a nongenomic mechanism in the smooth muscle cells from the rat oviduct.

Estradiol (E2) accelerates egg transport by a nongenomic action, requiring activation of estrogen receptor (ER) and successive cAMP and IP3 production in the rat oviduct. Furthermore, E2 increases IP3 production in primary cultures of oviductal smooth muscle cells. As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E2 in the oviduct, herein we determined the mechanism by which E2 increases IP3 in these cells. Inhibition of protein synthesis by Actinomycin D did not affect the E2-induced IP3 increase, although this was blocked by the ER antagonist ICI182780 and the inhibitor of phospholipase C (PLC) ET-18-OCH3. Immunoelectron microscopy for ESR1 or ESR2 showed that these receptors were associated with the plasma membrane, indicating compatible localization with E2 nongenomic actions in the smooth muscle cells. Furthermore, ESR1 but not ESR2 agonist mimicked the effect of E2 on the IP3 level. Finally, E2 stimulated the activity of a protein associated with the contractile tone, calcium/calmodulin-dependent protein kinase II (CaMKII), in the smooth muscle cells. We conclude that E2 increases IP3 by a nongenomic action operated by ESR1 and that involves the activation of PLC in the smooth muscle cells of the rat oviduct. This E2 effect is associated with CaMKII activation in the smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity necessary to accelerate oviductal egg transport.

On-chip 3D rotation of oocyte based on a vibration-induced local whirling flow

We propose a novel on-chip 3D cell rotation method based on a vibration-induced flow. When circular vibration is applied to a microchip with micropillar patterns, a highly localized whirling flow is induced around the micropillars. The direction and velocity of this flow can be controlled by changing the direction and amplitude of the applied vibration. Furthermore, this flow can be induced on an open chip structure. In this study, we adopted a microchip with three micropillars arranged in a triangular configuration and an xyz piezoelectric actuator to apply the circular vibration. At the centre of the micropillars, the interference of the vibration-induced flows originating from the individual micropillars induces rotational flow. Consequently, a biological cell placed at this centre rotates under the influence of the flow. Under three-plane circular vibrations in the xy, xz or yz plane, the cell can rotate in both the focal and vertical planes of the microscope. Applying this 3D cell rotation method, we measured the rotational speeds of mouse oocytes in the focal and vertical planes as 63.7 ± 4.0° s−1 and 3.5 ± 2.1° s−1, respectively. Furthermore, we demonstrated the transportation and rotation of the mouse oocytes and re-positioned their nuclei into a position observable by microscope. Three-dimensional cell observation and manipulation, required during cellular surgery, rely on proper orientation of the target cell. Conventional cell rotation techniques using microscopic hand-like devices demand sophisticated equipment and highly skilled operators. Additionally, existing on-chip approaches exploiting electric, magnetic and optical forces involve intricate structures that are complex to fabricate. To simplify these systems, Takeshi Hayakawa and co-workers from Nagoya University, Japan, have developed a microfluidic chip that controls cell rotation through vibration. The chip consists of a glass surface decorated with three small pillars in a triangular arrangement. To achieve stable motion, the researchers apply in-plane and vertical circular vibrations, creating local vortexes around the pillars that cause a cell placed at their centre to rotate. This simple, high-performance method is expected to play a central role in future biotechnology research.

1P1-O09 振動誘起流れを用いたオープンチップでの細胞操作

We present a novel cell manipulation method based on vibration-induced flow in open-chip environment. By applying circular vibration to a chip having micropillars, local flow is induced around the micropillars. Therefore, by patterning micropillar array on the chip and applying the circular vibration, we can achieve cell transportation along the array in the open chip environment. We theoretically analyzed this phenomenon with a single micropillar, and experimentally evaluated behaviors of manipulated microbeads with two micropillars. We confirmed there are different manipulation modes according to the pitch of micropillars. We designed the micropillar array for transportation of mouse oocytes based on these evaluations. We succeeded in transportation of oocytes with up to 35.6 μm/s and confirmed that the transportation velocity can be changed by changing the frequency and amplitude of the applied vibration.

Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives.

Cryopreservation techniques for mammalian oocytes and embryos have rapidly progressed during the past two decades, emphasizing their importance in various assisted reproductive technologies. Pregnancies and live births resulting from cryopreserved oocytes and embryos of several species including humans have provided proof of principle and led to the adoption of cryopreservation as an integral part of clinical in vitro fertilization. Considerable progress has been achieved in the development and application of the cryopreservation of mammalian oocytes and embryos, including preservation of the reproductive potential of patients who may become infertile, establishment of cryopreserved oocyte banks, and transport of oocytes and embryos internationally. However, the success rates are still far lower than those obtained with fresh oocytes and embryos, and there are still obstacles that need to be overcome. In this review, we address the major obstacles in the development of effective cryopreservation techniques. Such knowledge may help to eliminate these hurdles by revealing which aspects need improvement. Furthermore, this information may encourage further research by cryobiologists and increase the practical use of cryopreservation as a major part of assisted reproductive technologies for both humans and animal species.

Cloning, localization and characterization of a Zn transporter (Zip10) from fly, dog, and human (893.39)

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63Zn, with varying pHs and ion concentrations. 63Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping).Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl‐ (with HCO3舉) or all Na+ (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral).Conclusions: Our results indicated that all three Zip10 clones transport Zn2+. Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption.Grant Funding Source: Supported by DK83007, DK92408

Uptake of Betaine into Mouse Cumulus-Oocyte Complexes via the SLC7A6 Isoform of y+L Transporter1

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

VALIDAÇÃO DE UM PROGRAMA DE PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS COM TRANSPORTE DE OÓCITOS E DE EMBRIÕES POR LONGAS DISTÂNCIAS

This study aimed to evaluate the program of in vitro embryo production (IVEP) feasibility whose cattle oocyte maturation and embryo development occurred partly during the transport, fertilization (IVF) was performed with conventional or sexed semen and the embryos were transferred at different periods of development. Therefore, oocytes aspirated in the state of Bahia were transported to a commercial laboratory in São Paulo city, where the steps of in vitro production continued, the IVF was performed with sperm derived from conventional semen (CONV group) or sexed group (SEX group). On the sixth day of cultivation, the embryos produced were sent to Bahia and transferred to recipients at different periods of development (6.5, 7.0 and 7.5 days). The females had a mean of 46.18 ± 32.7 oocytes recovered, and 30.74 ± 24.3 viable oocytes by aspiration. The rates of embryos produced and pregnancy were 32.85% and 33.12%, respectively. The type of semen did not affect neither the number of cleaved oocytes nor the pregnancy rates, however, it affected the number of embryos produced, and the age of the embryo interfered in the pregnancy rates. The results demonstrated that the transport of oocytes and embryos was effective in cattle IVEP program, thus, it can be used in large scale.KEYWORDS: Bos indicus; sexed semen; transfer of embryos.

Serum and Glucocorticoid-Inducible Kinase1 Increases Plasma Membrane wt-CFTR in Human Airway Epithelial Cells by Inhibiting Its Endocytic Retrieval

BackgroundChloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells.Methods and ResultsWe report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR).ConclusionThis study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane.

Chapter 18 - Analysis of RNA Transport in Xenopus Oocytes and Mammalian Cells

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.

Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice

We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High-affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two-electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and (33) P-labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild-type plants were observed under both high-Pi and low-Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double-knockdown plants grown under both high- and low-Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake.

Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse

Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. In vitro experiment. Oocytes were recovered by transvaginal ultrasound-guided follicular aspiration either from dominant follicles 24 h after deslorelin administration (dominant stimulated follicle [DSF]), or from subordinate (immature) follicles at the same time. To mimic transport, DSF oocytes were incubated overnight under differing conditions before ICSI; immature oocytes were placed in varying conditions overnight before in vitro maturation, followed by ICSI. The rate of blastocyst production was compared among treatments. Blastocysts were produced in all groups. Dominant stimulated follicle oocytes held in sealed tubes in pre-equilibrated control maturation medium maintained at 37°C yielded blastocyst development equal to that obtained for control incubated oocytes (70%). Dominant stimulated follicle oocytes held similarly in a warm passive device yielded poor blastocyst development (10%). Immature oocytes held for one or 2 nights in modified M199 medium, or for one night in commercial embryo holding solution, in air at room temperature, yielded 35-37% blastocyst development per injected oocyte. A commercially available medium can be used for shipping immature oocytes at room temperature with good resulting blastocyst rates. Better blastocyst rates per oocyte are obtained from DSF oocytes; however, these require maintenance at 37°C and as they are already maturing at the time of collection, are more sensitive to delays. This new, practical information supporting transport of both immature and DSF oocytes for ICSI may allow wider use of this procedure.

A live birth from vitrified-warmed oocytes in a Philadelphia chromosome-positive acute lymphoid leukemia patient 5 years following allogenic bone marrow transplantation and after a magnitude 9.0 earthquake in Japan.

To report a live birth from vitrified-warmed oocytes for a Philadelphia chromosome-positive acute lymphoid leukemia (Ph-ALL) patient. A 20-year-old single woman with Ph-ALL requested oocyte cryopreservation at a private fertility clinic using assisted reproduction technology (ART). In cases of leukemia, there is a very short time before chemotherapy, follwed shortly by total body irradiation (TBI), and although she had already received the chemotherapy, ten oocytes were vitrified and stored for 59 months before warming. Soon after the oocyte cryopreservation, she received TBI and bone marrow transplant (BMT). During the storage, a magnitude 9.0 earthquake occurred making oocyte transport necessary. The embryo transfer was planned in a hormone replacement cycle, and intracytoplasmic sperm injection (ICSI) was performed on the vitrified-warmed oocytes. On day 3, two embryos were transferred. The patient became pregnant and delivered a healthy girl after ICSI using vitrified-warmed oocytes. Oocyte cryopreservation is the best option for fertility preservation of young single women with leukemia. Oncologists and gynecologists who conduct ART should cooperate to improve the quality of life of cancer patients.

ABCB1 single nucleotide polymorphisms (1236C>T, 2677G>T, and 3435C>T) do not affect transport activity of human P-glycoprotein

P-glycoprotein (P-gp) is a multidrug efflux transporter that has a defined role in the absorption and disposition of drugs. Many studies have investigated the potential influence of ABCB1 polymorphisms on the disposition of its substrates. However, there remains significant controversy regarding the role of these polymorphisms. Our aim was to generate a P-gp expression system for single nucleotide polymorphisms (SNPs) and the reference sequence to assess the functional significance of these variants on transport. P-gp reference, a P-gp ATPase deficient mutant (G534D) and a triple SNP variant of P-gp (1236C>T, 2677G>T, and 3435C>T) were expressed in Xenopus laevis oocytes and used to assess the influence of these SNPs on transport of digoxin and imatinib. The inhibition of P-gp-mediated transport in Caco-2 cells and oocytes was also assessed. No effect of the triple SNP variant of P-gp on molecular transport of digoxin or imatinib was observed. The rank order of inhibition of P-gp in Caco-2 cells and ABCB1-injected oocytes was tariquidar>elacridar>PSC-833>laniquidar>cyclosporine>verapamil>dipyridamole. These data suggest there is no functional consequence of these SNPs for molecular transport of model substrates or inhibition by model inhibitors for P-gp. Transporter-injected oocytes may be a useful tool for probing the mechanism for unexplained drug-drug interactions or to characterize therapeutic transport inhibitors.

Directional Transport Is Mediated by a Dynein-Dependent Step in an RNA Localization Pathway

Author SummaryLike traffic on highways, molecular cargos are transported within cells on tracks that are collectively referred to as cytoskeletal networks. RNA molecules are one such cargo, and in many species, the localization of RNAs in egg cells or oocytes is essential for establishing the first asymmetries that are necessary for proper embryo development. RNAs can be actively transported by molecular motors that move cargos along the cytoskeletal tracks, but how such motors are capable of directing cargos to specific destinations within the cell is not yet known. Here we show that two motors, dynein and kinesin—known to carry out transport in opposite directions—are both directly involved in RNA localization in frog oocytes. To understand how these motors can promote directional cargo transport, we developed a system to monitor RNA transport in live oocytes. We find that the motor acting first in the pathway, dynein, is responsible for unidirectional transport. Bidirectional transport, mediated by kinesin, occurs subsequently on cytoskeletal tracks of opposing polarity near the RNA's final destination. Our results suggest a new model for directional transport comprising an initial directional cue that dominates over a later nondirectional step, acting to refine the ultimate cargo distribution.

Trans-Resveratrol and ε-viniferin decrease glucose absorption in porcine jejunum and ileum in vitro

trans-Resveratrol and ε-viniferin are used as dietary supplements. They are reported to be supportive in preventing arteriosclerosis and diabetes and a previous study could demonstrate an inhibitory potential on sodium-dependent glucose transport (SGLT1) in oocytes und mouse intestinal everted rings (Schulze et al., 2012, Genes Nutr. 6, S61). The in vitro effects of trans-resveratrol and ε-viniferin on intestinal glucose uptake in the porcine small intestines (Sus Scrofa) have not yet been evaluated. It was hypothesized that trans-resveratrol/ε-viniferin may have an adverse effect on porcine intestinal sodium-dependent glucose uptake. The effects on electrogenic small intestinal glucose absorption and sodium-dependent 3H-glucose uptake in brush border membrane vesicles (BBMV) were evaluated. Pieces of mucosa were mounted into Ussing chambers and were incubated with either trans-resveratrol (0.3mmol/L), ε-viniferin (0.3mmol/L), or ethanol. Sodium-dependent glucose absorption into BBMV was measured. 3H-glucose uptake studies were performed using the same concentrations of the respective substances. SGLT1-mediated glucose absorption was approximately 3-fold higher in ileum compared to jejunum. After preincubation with trans-resveratrol and ε-viniferin, glucose-induced increases of short-circuit currents were significantly decreased. BBMV-studies revealed comparable results and glucose uptake was also significantly decreased. As the glucose transport/uptake was decreased after preincubation with either trans-resveratrol or ε-viniferin this active transport mechanism was directly influenced by inhibiting the SGLT1 transport system.

A novel inhibitory mechanism of nitrogen-containing bisphosphonate on the activity of Cl− extrusion in osteoclasts

Nitrogen-containing bisphosphonates have been well known to be inhibited farnesyl diphosphate synthase (FDPS), an enzyme in mevalonic acid metabolism, resulting in disturbance in polymerization of cytoskeleton structure in bone resorption and promotion of apoptosis in mature osteoclasts. Although bisphosphonates have been reported to activate ion transporters in native epithelium and Xenopus oocytes, little is known whether bisphosphonates affect acid hydrochronic acid extrusion in osteoclasts during bone resorption. The aim of this study was to determine the role of bisphosphonates on inhibition of hydrochronic acid extrusion in osteoclasts. Effects of zoledronic acid, a nitrogen-containing bisphosphonate, on the Cl(-) current activated by extracellular acidification were examined in two types of osteoclasts derived from RAW264.7 cells and mouse bone marrow macrophages (BMMs). Extracellular acidification induced outwardly rectifying Cl(-) currents in mouse osteoclasts. Zoledronic acid dose-dependently inhibited the acid-activated Cl(-) current. The non-nitrogen bisphosphonate etidronic acid had no effect on the acid-activated Cl(-) current. Tetracycline-induced FDPS silencing caused a significant decrease in the Cl(-) current. The inhibitor of geranylgeranyl transferase suppressed the Cl(-) current. By contrast, the inhibitory action of zoledronic acid was rescued by addition of geranylgeranyl acid, a derivative of mevalonic acid. The activity of acid-activated Cl(-) currents was dependent on expression of ClC-7 during osteoclastogenesis. These results suggest that nitrogen-containing bisphosphonates suppress the activity of osteoclastic acid-activated Cl(-) currents through FDPS inhibition, suggesting the inhibition of Cl(-) extrusion activity.

Transporte de oócitos in vitro através do oviduto de búfalas e vacas de corte cruzadas

The present study was conducted to verify if the elevation of plasma concentrations of estradiol during superovulatory treatments affects the oocyte transport in buffalo females, as well as if the inferior quality of buffalo oocytes and/or some functional difference on the oviduct of these animals is responsible for the low embryo recovery rate in superovulated uffaloes when compared to cows subjected to the same treatment. Oviducts of 10 buffaloes and 15 of cows, treated to induce a single ovulation were used. The oviducts were placed on Petri dishes and received the following treatments: 5 buffalo oocytes with no E2 (G-BufBuf and G-BovBuf), 5 bovine oocytes with no E2 (G-BufBov and G-BovBov), 5 buffalo oocytes with E2 (G-BufE2Buf and G-BovE2Buf) and 5 bovine oocytes with E2 (G-BufE2Bov and G-BovE2Bov; factorial 2x2x2). Oocytes were incubated for 24h. Subsequently, oviducts were washed and oocytes were recovered and counted. Since no interactions were found between E2 treatment, oviducts and oocytes species, main effects were analyzed separately. Recovery rate and number of oocytes was higher on cattle compared to buffaloes (35.0+8.6% and 1.4+0.3 vs. 10.0±4.6% and 0.5±0.2, respectively; p<0.05); no effect of E2 treatment was observed on recovery rate and number of oocytes (29.8±9.0% and 1.3±0.4 vs. 16.9±6.1% and 0.7±0.2, respectively; p>0.05); the number of buffaloes and bovine oocytes recovered were similar (1.4±0.4 and 0.6±0.2, respectively; p>0.05). Oocytes recovery rate showed a trend (P=0.07) to be higher when buffalo oocytes were implanted when compared to bovine oocytes (35.2±9.2% vs. 12.9±5.4%). Present results suggest that oocyte transport by the oviduct of buffaloes and bovine was not dependent on oocytes species or E2 supplementation to the culture medium.

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

228 IN VITRO EMBRYO PRODUCTION AFTER EXPOSURE OF BOVINE OOCYTES TO DIFFERENT TRANSPORTATION MEDIA AND PERIODS: PRELIMINARY RESULTS

This study was designed to evaluate the effect of different oocyte transportation media and time. Immature oocytes were recovered from slaughterhouse bovine ovaries. Oocytes (n = 492) of quality grades I to III were randomly allotted to one of the following transportation media based on TCM-199, either buffered with HEPES (control) or buffered with NaHCO3 and added with FSH/LH (maturing). Both media were supplemented with pyruvate, penicillin G (10 000 IU), streptomycin (0.05 mg mL–1), and 10% fetal bovine serum (FBS). In control medium, the oocytes were kept for 1 or 8 h at 37°C, and thereafter were transferred to maturing medium until the maturation period (24 h) was completed, under controlled atmosphere (5% CO2) and temperature, in an incubator. The maturing oocytes were distributed in two types of equipment developed for oocyte and embryo transportation, with and without 5% CO2, and kept for 1, 8, or 24 h before maturation or fecundation (IVF) procedures. The oocytes kept for 24 h in the transportation device were placed directly for IVF. All procedures used for in vitro maturation, IVF, and in vitro culture were the same as those adopted for commercial in vitro embryo production at Biotran LTDA (Alfenas, Minas Gerais, Brazil). The cleavage rate was evaluated on Day 3 post-insemination, and the blastocyst production was evaluated on Day 7. The statistical model included the main effects of treatment (control and maturing with or without 5% CO2), time, replicate, and the interaction of media × time. Data (3 replicates) were analysed by ANOVA and differences were identified by Tukey’s test. The time before in vitro maturation at the incubator negatively (P < 0.007) affected cleavage rates (76.4 ± 16.9 v. 58.8 ± 13.2 and 52.2 ± 18.5%, respectively, for 1, 8, and 24 h). However, treatment had no effects (P = 0.3) on cleavage (66.9 ± 15.0, 57.8 ± 19.7, and 67.7 ± 19.8% for the control and maturation with and without 5% CO2, respectively). Similarly, blastocyst production rates differed (P < 0.04) between 1 h (33.4 ± 14.7) and 24 h (19.3 ± 17.0%), whereas blastocyst production at 8 h did not show significant effects (19.3 ± 17.0). Although it was not significant (P > 0.05), only 14.1% of the oocytes kept in medium 2 with a controlled atmosphere for 24 h became embryos. This difference was probably related to the trend in treatment effect (P = 0.06) and the lower rate of embryo production (22.2 ± 13.6%) in this treatment compared with the control (33.8 ± 18.1%) and maturation without 5% CO2 (29.8 ± 17.3%). These results showed that 24 h of transportation is detrimental for the oocyte development potential and that the buffered medium used in this study with 5% CO2 did not efficiently maintain embryo production. Supported by CNPq, CAPES, FAPEMIG, and Biotran LTDA.

In vitro embryo production in sheep: Pregnancy after long periods of oocyte and embryo transport

To evaluate the feasibility of long distance transport of ovine oocytes and in vitro produced embryos. Santa Ines pluriparous sheep (n=5) were synchronized with intravaginal devices containing 0.3mg progesterone (CIDR®, Pfizer, Netherlands) for 10 days. Thirty-six hours prior to progesterone device removal and follicular aspiration, 0.04mg d-cloprostenol sodium (Ciosin®, Coopers, Brasil), 200 IU FSH (Folltropin®, Bioniche, Canada) and 300IU eCG (Novormon®, Syntex, Argentina) were administered intramuscularly. Oocyte retrieval was performed via mid-ventral laparotomy, under general anesthesia. The oocytes were evaluated and classified under a stereomicroscope and placed in cryovials with TCM 199, aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and stored in a transport incubator at 38.5°C. Oocytes were then transported via the road for 14h to the laboratory, where the oocytes then completed more than 10h of IVM. In vitro fertilization (IVF) was performed with frozen sperm from a single ram. On Day 3 the embryos were placed in cryovials containing SOFovine (In Vitro Brasil, Sao Paulo, Brazil), aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and placed into the same portable incubator. The embryos were returned to the farm under the same transport conditions (km and h). Upon arrival at the farm, all embryos were evaluated and transferred into recipient ewes 4 days after the initial oocyte retrieval. Embryos were transferred in sets of 3 to 4, into the apex of the uterine horn, ipsilateral to the corpus luteum, by means of mid-ventral laparotomy, under general anesthesia. Pregnancy was diagnosed using transrectal ultrasonography 30 days after embryo transfer. On average 9.8±5.7 oocytes (49 oocytes/5 donors) were obtained per donor, and 25 grade I morulae were transferred into 7 recipients. One pregnancy (14.3%) was confirmed resulting in a healthy lamb. These results indicate the feasibility of in vitro embryo production in sheep after maturation and embryonic development following transport over long distances.