Mycoplasma hyopneumoniae vaccination at or shortly before weaning under field conditions: a randomised efficacy trial.

This study assessed the efficacy of two different Mycoplasma hyopneumoniae vaccination programmes in relation to the time of weaning. Eight hundred and twenty-eight piglets were randomly divided into three groups: group V1 was vaccinated three days before weaning, group V2 at weaning (21 days of age) and group NV was left non-vaccinated. Vaccinations were performed using Ingelvac MycoFLEX. After the nursery period, 306 pigs were allocated to fattening unit (F1) and 501 pigs to a second unit (F2). Efficacy was evaluated using performance parameters and pneumonia lesions at slaughter. Statistically significant differences were obtained in F2 where group V1 had a higher average daily weight gain compared to groups V2 and NV for the entire study period (17 and 18 g/day, respectively) and the fattening period (26 and 36 g/day, respectively) (P<0.05). Considering respiratory disease scores for both fattening units, group V1 was the only group where coughing severity did not increase significantly between placement and the end of the fattening period (P>0.05). Between groups, there were no statistically significant differences for the average lung lesion scores (V1=3.44; V2=4.61; NV=4.55, P>0.05) and the prevalence of pneumonia (V1=35.0 per cent; V2=38.0 per cent; NV=41.4 per cent, P>0.05). Overall, vaccination against M hyopneumoniae before weaning provided numerically better performance than vaccination at weaning, but did not reach statistical significance. An influenza outbreak in F1 and the presence of coexisting mixed respiratory infections in both F1 and F2 could have possibly influenced the performance of both vaccinated groups across all measured parameters.

Impact of particulate matter and ammonia on average daily weight gain, mortality and lung lesions in pigs.

The present study investigated the simultaneous influence of particulate matter (PM10) and ammonia (NH3) on performance, lung lesions and the presence of Mycoplasma hyopneumoniae (M. hyopneumoniae) in finishing pigs. A pig herd experiencing clinical problems of M. hyopneumoniae infections was selected. In total, 1095 finishing pigs of two replicates in eight compartments each were investigated during the entire finishing period (FP). Indoor PM10 and NH3 were measured at regular intervals during the FP with two Grimm spectrometers and two Graywolf Particle Counters (PM10) and an Innova photoacoustic gas monitor (NH3). Average daily weight gain (ADG) and mortality were calculated and associated with PM10 and NH3 during the FP. Nasal swabs (10 pigs/compartment) were collected one week prior to slaughter to detect DNA of M. hyopneumoniae with nested PCR (nPCR). The prevalence and extent of pneumonia lesions, and prevalence of fissures and pleurisy were examined at slaughter (29 weeks). The results from the nasal swabs and lung lesions were associated with PM10 and NH3 during the FP and the second half of the FP. In the univariable model, increasing PM10 concentrations resulted in a higher odds of pneumonia lesions (second half of the FP: OR=8.72; P=0.015), more severe pneumonia lesions (FP: P=0.04, second half of the FP: P=0.009), a higher odds of pleurisy lesions (FP: OR=20.91; P<0.001 and second half of the FP: OR=40.85; P<0.001) and a higher number of nPCR positive nasal samples (FP: OR=328.00; P=0.01 and second half of the FP: OR=185.49; P=0.02). Increasing NH3 concentrations in the univariable model resulted in a higher odds of pleurisy lesions (FP: OR=21.54; P=0.003) and a higher number of nPCR positive nasal samples (FP: OR=70.39; P=0.049; second half of the FP: OR=8275.05; P=0.01). In the multivariable model, an increasing PM10 concentration resulted in a higher odds of pleurisy lesions (FP: OR=8.85; P=0.049). These findings indicate that the respiratory health of finishing pigs was significantly affected by PM10.

Efficacy of vaccination against Actinobacillus pleuropneumoniae in two Belgian farrow-to-finish pig herds with a history of chronic pleurisy.

The efficacy of an Actinobacillus pleuropneumoniae subunit vaccine based on ApxIA, ApxIIA, ApxIIIA and OMP-2 (Porcilis App, MSD) was investigated in two farrow-to-finish pig herds (A and B) affected by chronic pleurisy. In total, 1161 pigs were included. At three weeks of age, the pigs were randomly allocated to non-vaccinated control (NV; n=580) and vaccinated (V; n=581) groups. At 6 and 10 weeks of age, pigs were injected with Porcilis-APP (V group) or adjuvant (NV group). At slaughter (26 weeks), pleurisy and pneumonia lesions were assessed. All pigs were weighed individually at 6 and 26 weeks of age, and average daily weight gain (ADG; g/pig/day) was calculated. Mortality and days of additional treatment (DAT) were registered during the whole experiment. Data were analysed using binary logistic regression or analysis of variance for proportions or continuous variables, respectively. The prevalence of pleurisy and pneumonia was (NV-A=19.3, V-A=7.9, (P=0.000); NV-B=17.9, V-B=0.7, (P=0.000)) and (NV-A=42.4, V-A=21.2, (P=0.000); NV-B=46.7, V-B=19.0, (P=0.000)), respectively. The ADG was NV-A=632±157, V-A=647±91, (P=0.162); NV-B=660±115, V-B=670±82, (P=0.232). The mortality during the experiment was NV-A=5.7, V-A=1.8, (P=0.015); NV-B=2.3, V-B=1.0, (P=0.170) per cent. The DAT was: NV-A=15.04±1.41, V-A=14.95±0.67, (P=0.010); NV-B=21.68±2.43, V-B=16.99±0.62, (P=0.000). The present study showed a significant reduction of the prevalence of pleurisy and pneumonia, and antimicrobial use in V pigs from both herds, and in mortality in V pigs from one herd.

Lenalidomide in relapsed refractory myeloma patients: impact of previous response to bortezomib and thalidomide on treatment efficacy. Results of a medical need program in Belgium.

The prognosis of multiple myeloma patients has significantly improved since the introduction of the novel agents thalidomide, bortezomib and lenalidomide. We report the data of a medical need programme with lenalidomide plus dexamethasone, conducted in Belgium between August 2007 and March 2008, and including 98 relapsed refractory multiple myeloma patients. In addition to chemotherapy and steroids, all patients had received prior treatment with bortezomib, and 84% of them had been exposed to thalidomide. In 52 patients response data could be retrieved by post-hoc analysis. A partial remission or better was achieved in 52% (49% partial and 3% complete response) of patients, despite a median of 5 previous anti-myeloma treatment lines. Responses were rapid while the majority of patients received lenalidomide with once weekly (also called low-dose) dexamethasone. Treatment with lenalidomide plus dexamethasone did prolong overall survival by nearly half a year in this population with end-stage myeloma. Overall response and quality of response were independent of previous response to thalidomide and bortezomib, although the time to progression tended to be shorter in thalidomide- and bortezomib-refractory patients. It can be concluded that lenalidomide plus dexamethasone is an effective and safe treatment regimen in highly refractory multiple myeloma patients, and that these responses are irrespective of previous exposure or sensitivity to thalidomide and bortezomib.

Giant liposarcoma of the stomach involving the mediastinum.

A case of a liposarcoma of the stomach in a 27-year old woman is described. Initially the patient consulted with epigastric pain. MRI showed a giant tumour of the stomach wall, invading the surrounding organs, as well as the mediastinal region. After surgical 'en-bloc' resection of the tumour, histopathologic examination yielded a diagnosis of pleiomorphic liposarcoma. Because of the bad prognosis of this histologic type, the patient received adjuvant chemotherapy: a combination of doxorubicin and ifosfamide (MAI). Nine months after surgery, she represented with a relapse of the tumour that had become inoperable. Palliative chemotherapy was started with the intent to prolong the young patient's life. However 6 months later, the patient died of the recurrent disease. Although liposarcoma is a very common soft tissue sarcoma, it is rarely seen in the stomach. The standard therapy is surgical excision. Over the last years, adjuvant therapy became more accepted. Drugs of choice are doxorubicin and ifosfamide, although the benefits of this therapy are still largely unknown and doubtful.

ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004.

The sixth report of the ESHRE PGD Consortium is presented, relating to cycles collected for the calendar year 2003 and follow-up of the pregnancies and babies born up to October 2004. Since the beginning of the data collections, there has been a steady rise in the number of cycles, pregnancies and babies reported. For this report, 50 centres participated, reporting on 2984 cycles, 501 pregnancies and 373 babies born. Five hundred and twenty-nine cycles were reported for chromosomal abnormalities, 516 cycles were reported for monogenic diseases, 137 cycles were reported for sexing for X-linked diseases, 1722 cycles were reported for preimplantation genetic screening (PGS) and 80 cycles were reported for social sexing. Data VI is compared to the cumulative data for data collections I-V.

Which patients with recurrent implantation failure after IVF benefit from PGD for aneuploidy screening?

Patients with recurrent IVF failure are defined as patients who are younger than 37 years and who had at least three consecutive unsuccessful IVF/intracytoplasmic sperm injection (ICSI) cycles with good quality embryos. These patients might be predisposed to chromosome errors in their embryos and therefore might benefit from preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). This technique is, however, expensive and some normal embryos might be lost due to the error rate. The aim of this retrospective study was to define those patients who would benefit most from it. One hundred and twenty-one first PGD-AS cycles for recurrent IVF failure were analysed. The aneuploidy rate, 'no embryo transfer' rate, live birth rate per embryo transfer and implantation rate were respectively 48.3, 22.3, 29.7 and 19.5%. A multivariate logistic regression analysis gave us a predictive model demonstrating that to have a 90% probability of having an embryo transfer after PGD-AS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes and six embryos for biopsy. This study suggests that most patients with recurrent IVF failure may benefit from PGD-AS. Future studies, however, should more strictly define this heterogeneous group of patients, so that comparison is easier.

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA.

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

ESHRE PGD Consortium data collection IV: May-December 2001.

The ESHRE PGD Consortium was formed in 1997 to survey the practice of preimplantation genetic diagnosis (PGD). Since then, three reports have been published giving an overview on PGD from an ever-increasing number of centres and reporting on an increasing number of PGD cycles and pregnancies and babies born after PGD. After these initial influential publications, important shortcomings were identified primarily on the method of data collection, i.e. with Excel spreadsheets, and in the timing of the collection (cycles were collected in a different time frame from pregnancies and babies, making the follow-up of cycles very difficult). This is why the Steering Committee has made a major investment in developing and implementing a new database in FileMaker Pro 6. It was also decided that cycles would be collected from one calendar year, as well as the pregnancies and babies ensuing from that particular calendar year. This gave us the opportunity to take a closer look at the data collected earlier, and to attempt to improve their quality. This is a report on the corrected data from the first three data collections (I-III) as well as the result of the last data collection (IV) that was completely carried out using the new database.

Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men.

BACKGROUND: The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis (not sufficient to spill over into the ejaculate) may benefit from the technique. Previous reports suggest a higher pregnancy rate after ICSI treatment in patients with obstructive azoospermia (OA) compared to their non-obstructive azoospermia (NOA) counterparts, which could be due to a higher aneuploidy frequency in the embryos of the latter group. We therefore conducted a prospective cohort study to compare the aneuploidy frequency of the screened embryos between the two groups. METHODS: From May 2001 until September 2003, we offered couples with an OA or NOA partner ICSI in combination with preimplantation genetic diagnosis for aneuploidy screening. RESULTS: No difference in age (30.6 and 33.5 years) or stimulation parameters was observed between the two groups; 53 and 60% of the embryos were abnormal in the NOA and OA groups respectively (P = not significant). CONCLUSIONS: The aneuploidy frequency in embryos obtained from NOA as well as OA men is similar and very high, despite the young age of their female partners.

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells.

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.

PGD in 47,XXY Klinefelter's syndrome patients.

The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis, may benefit from the ICSI technique in order to father a child. As ICSI use has become more common, centres have introduced infertility treatment for Klinefelter patients. To date, 34 healthy children have been born using ICSI without PGD, and the conception of one 47,XXY fetus has been reported. In view of the possible risk of an increased gonosome number in the spermatozoa of Klinefelter patients, a safer approach--offering these couples ICSI combined with PGD--has been used, and has resulted in the birth of three healthy children. Couples in which the male suffered from Klinefelter's syndrome were first treated in 1995; these patients were offered ICSI + PGD using FISH technology, notably to enumerate the X and Y chromosomes. ICSI + PGD was performed in 32 cycles of 20 couples with spermatozoa originating from a fresh ejaculate (n = 1), testicular biopsy (n = 21) or frozen-thawed testicular biopsy (n = 10). Normal fertilization occurred in 56.0 +/- 22.4% of the successfully injected oocytes. On day 3 of development, 119 embryos from 29 cycles were of sufficient quality to undergo biopsy and subsequent PGD; a positive result was obtained in 113 embryos. Embryos were available for transfer in 26 cycles, with a mean of 1.6 +/- 0.6 embryos per transfer. Eight pregnancies were obtained, and five resulted in a delivery. A total of 113 embryos from couples with Klinefelter's syndrome was compared with 578 embryos from control couples with X-linked disease where PGD was used to determine gender. A significant fall occurred in the rate of normal embryos for couples with Klinefelter's syndrome (54.0%) compared with controls (77.2%). Moreover, a significantly increased risk of abnormalities was observed for sex chromosomes and autosomes; for each autosome separately, this reached significance level for chromosomes 18 and 21 only. Hence, a cautious approach is warranted in advising couples with non-mosaic Klinefelter's syndrome. Moreover, the use of ICSI + PGD or prenatal diagnosis should be carefully considered.

Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

Cloning and functional analysis of chicory root fructan1-exohydrolase I (1-FEH I): a vacuolar enzyme derivedfrom a cell-wall invertase ancestor? Mass fingerprint of the 1-FEH I enzyme.

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.

Cloning, developmental, and tissue-specific expression of sucrose:sucrose 1-fructosyl transferase from Taraxacum officinale. Fructan localization in roots.

Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels.

Efficacy and safety of vancomycin constant-rate infusion in the treatment of chronic Gram-positive bone and joint infections.

OBJECTIVE: To evaluate the efficacy and safety of vancomycin constant-rate infusion over 24 h in the treatment of Gram-positive bone infections, METHODS: Vancomycin (40 mg/kg/day) was administered without a loading dose to 15 patients (12 male, three female) aged 23--90 years, weighing 46--85 kg, with postoperative chronic bone and joint infections. The 24-h dose was adjusted to maintain plasma levels between 25 and 35 mg/L. Mean duration of therapy was 6.2 months (4--8.5) via a portable infusion pump. Sites of infection included hip and femur (10), tibia (three), patella (one) and vertebrae (one). Sequestrectomy (two), removal of material (7/8 prosthetic hips, 1/5 metal implants) and debridement (two) were performed at the beginning of the treatment. Involved bacteria included Staphylococcus aureus (eight, six methicillin resistant), S. epidermidis (four methicillin-resistant), Enterococcus faecalis (one), Enterococcus avium (one) and Streptococcus bovis (one). RESULTS: MIC of vancomycin ranged from 1 to 4 mg/L. The mean vancomycin bone concentration when available was 67.7plus minus38.9 microg/L. Based on a mean post-treatment follow-up of 14plus minus4 months (6--20.6), cure was achieved in 10 patients (66.6%). Failures were related to the inability to remove the infected prosthesis (one) or implants (three) and to the persistence of a deep wound abcess (one). Adverse events included pruritus (four cases), tinnitus (two), mild transient elevation of creatinine level (three) and transient neutropenia (two). Vancomycin was maintained in all the patients. CONCLUSIONS: Prolonged treatment with vancomycin constant-rate infusion is effective and safe for treatment of Gram-positive chronic bone and joint infections, providing that complete surgical débridement and prosthetic material removal are performed.

Taurine acts as an osmolyte in human and mouse oocytes and embryos.

Taurine transport in mouse embryos has been shown to be osmotically regulated. We studied release of taurine from mouse and human oocytes and embryos when exposed to conditions that created osmotic imbalances, either by incubation in anisosmotic media or by inhibition of Na(+)-K(+)-ATPase with ouabain. Furthermore, we studied the effect of cleavage in mouse embryos on release of taurine. When human oocytes that remained unfertilized after in vitro fertilization, human embryos (2- to 8-cell), and mouse 2-cell embryos were loaded with [3H]taurine and subsequently incubated for 4 h in hyposmotic media (200 and 240 mOsm/kg), they showed significantly lower radioactivity as compared to those incubated in media of 280, 320, and 360 mOsm/kg and higher radioactivity of the incubation media. Incubation with 1.5 mM ouabain resulted in decreased radioactivity of mouse embryos and increased radioactivity of incubation medium. When mouse 2-cell embryos were cultured for 24 h after loading with [3H]taurine, radioactivity of embryos that cleaved to the 4-cell stage was significantly lower than that of uncleaved embryos. This finding is in accordance with the theory that cell division induces cell volume-regulatory mechanisms. In contrast, when 1-cell embryos were cultured for 24 h, radioactivity of embryos developing to the 2-cell stage was significantly higher than that of uncleaved embryos. These data support the theory that taurine is released by embryos when they have to adjust their cell volume because of either extracellularly induced or intracellularly occurring osmotic imbalances. When culture is performed without taurine, the resultant taurine depletion of embryos may be disadvantageous, either because the embryo has to rely more on its inorganic osmolytes for volume regulation or because taurine can no longer provide its other protective functions.

Modulation of embryonic Na(+)-K(+)-ATPase activity and mouse preimplantation development in vitro in media containing high concentrations of potassium.

The effect of various potassium concentrations (ranging from 1.4 mM to 30 mM K+) in modified Tyrode's medium on the culture of mouse zygotes obtained after in vitro fertilization to the blastocyst stage was examined. A clear dose-dependent negative effect of increasing K+ concentrations on the preimplantation embryonic development in vitro was found. We have previously shown that significantly more two-cell embryos reach the blastocyst stage when cultured during the second day postinsemination in medium supplemented with taurine. Because taurine, an amino acid that abounds in the reproductive tract, has been reported to inhibit the enzyme Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase), we used two other conditions known to inhibit the Na(+)-K(+)-ATPase to study their effect on mouse embryo development. Culturing embryos during a short period (the second day postinsemination) in low extracellular K+ concentrations (1.4 mM) or in medium supplemented with ouabain (50 microM) showed positive effects similar to those of culturing in medium with taurine (10 mM). This beneficial effect of ouabain was found in various K+ concentrations tested, including the high concentrations present in the oviduct. Although the effects of low K+ and taurine can possibly be ascribed to their other cellular effects, the effect of ouabain shows that inhibition of the Na(+)-K(+)-ATPase during the two-cell stage in the mouse is beneficial for further embryonic development to the blastocyst stage.

Temporal effects of ouabain on in-vitro development of mouse zygotes.

Ouabain is a specific inhibitor of Na(+)-K(+)-ATPase, an enzyme which controls the intracellular Na+ and K+ levels. In this study, in-vitro fertilized zygotes from a hybrid mouse strain were used to examine the temporal effects of 50 microM ouabain on embryonic development in vitro during the preimplantation period. A higher incidence of blastocyst formation at the end of the culture period was found when embryos were cultured in the presence of ouabain from 22 to 46 h post-insemination, or any other period that included this time period. When zygotes from randomly bred mice were used, inhibition of Na(+)-K(+)-ATPase with ouabain clearly promoted development through the 2-cell block in vitro. As Na(+)-K(+)-ATPase is the most important regulator of intracellular electrolyte concentrations in mammalian cells, these results suggest that an ionic imbalance exists in embryos cultured in conventional media which can be positively influenced by inhibiting this enzyme.