Culture of pre-implantation Chinese hamster (Cricetulus griseus) embryos in vitro

Pre-implantation embryos from Chinese hamsters (Cricetulus griseus) were cultivated under completely defined conditions. The embryos were placed in drops of chemically defined medium under liquid paraffin and cultured in an atmosphere of 10% CO2 in air. By this method, development will proceed in vitro from the two-cell stage up to the blastula within 72 h. It is possible to stop the cultivation at different stages of development, to fix the embryos and analyse the chromosomes. The method described in detail seems to be appropriate for examination of the induction of genetic defects during the first days of embryogenesis.     

Current and future methodology for monitoring sleep

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.     

Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin-1 system during embryonic development

In the study reported here, we localized at the protein level the major components of the interleukin (IL)-1 system in the human embryo, and we investigated the endometrial factors influencing their secretion during embryonic development. To localize these components, we performed immunohistochemical experiments in 44 oocytes and 78 embryos. The following primary antibodies were used: monoclonal mouse anti-human IL-1 receptor type I (IL-1R tl), monoclonal mouse anti-human IL-1 beta, and polyclonal rabbit anti-human IL-1 receptor antagonist (IL-1ra). For embryo culture, human embryos at different developmental stages were cultured in 100-microliters drops of Ham's F-10 medium + 4 mg/ml BSA (n = 33), in 100-microliters drops of Menezo B2 culture medium (n = 18), or in wells with 1 ml of Menezo B2 culture medium (n = 8). For embryo coculture, endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) were isolated from human secretory endometrium and cultured until confluence in 75% Dulbecco's Modified Eagle's Medium and 25% MCDB-105 containing antibiotics and supplemented with 10% charcoal-Dextran-treated fetal bovine serum. Individual human embryos were cocultured with experimental EEC and ESC (n = 23 and n = 4, respectively) for 5 days in 600-microliters drops of Menezo B2 medium, and conditioned medium was removed every 24 h. Human embryos were also cultured with EEC-conditioned medium (n = 9). IL-1 alpha, IL-1 beta, and IL-1ra levels were determined by ELISA in the 24-h culture- or coculture-conditioned media. Immunostaining confirmed the presence of IL-1 beta, IL-1ra, and IL-1R tl in oocytes and embryos in all stages analyzed, with no statistical differences. IL-1 alpha, IL-1 beta, and IL-1ra were absent in conditioned media of cultured embryos and embryos cocultured with ESC. However, when human embryos were cocultured with EEC or with EEC-conditioned medium alone, two different populations of embryos were observed: IL-1 producers (57% and 56%) and IL-1 nonproducers (43% and 44%, respectively). Finally, the IL-1 profile of a single human embryo cocultured with maternal EEC which successfully implanted and developed is presented, this pattern being similar to that described in the IL-1 producer population. These results demonstrate the presence of the IL-1 system in the human embryo. However, the selective release of IL-1 only when embryos were cocultured with EEC or EEC-conditioned medium indicates an obligatory role of the endometrium in the regulation of the embryonic IL-1 system. Furthermore, the differential embryonic production of IL-1 may be related to the implantation capability of the embryos.     

Molecular markers of acute upper airway inflammation in workers exposed to fuel-oil ash

In-vitro fertilization (IVF) embryos are selected for transfer on the basis of morphology and rate of development. However, when a number of embryos have similar characteristics, the selection of the best embryos is left to chance. Recently, we proposed a simple, novel method to overcome this problem, based on pre-selection of embryos cleaving early to the two-cell stage. In this study we have adopted the same method to choose embryos fertilized after intracytoplasmic sperm injection (ICSI). Fertilized embryos that had cleaved to the two-cell stage by 27 h post-injection were designated as 'early cleavage' embryos, while those that had not yet reached the two-cell stage were designated as 'no early cleavage'. In all cases, the early cleavage embryos were transferred when available. Early cleavage was observed in 54 (61.4%) of the 88 cycles assessed. There were significantly (P = 0.04) more clinical pregnancies in the early cleavage group, 14/54 (25.9%), compared with the no early cleavage group 2/34 (3.2%). No differences between the groups were found when comparing key parameters (age, stimulation protocol and semen characteristics) of the couples. Using the ICSI technique, we have shown that early cleavage to the two-cell stage is not influenced by the timing of fertilization, and is more likely due to intrinsic factors within the oocyte or embryo that promote embryo cleavage after fertilization.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.     

Clinical course of human immunodeficiency virus type 1 associated pulmonary tuberculosis during short-course antituberculosis therapy

PURPOSE: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. METHODS: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. RESULTS: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. CONCLUSIONS: Matrigel may be a deleterious addition to embryo culture or coculture systems.     

Aggressive behavior of psychiatric patients among geriatric population

The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In Experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following: 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P < 0.01) in MK-1 + BSA (74.4%), MK-1 + FCS (56.1%), and M-199 + FCS (51.4%) than in MK-1 + HS (94.7%). A greater (P < 0.01) percentage of blastocysts was obtained in MK-1 + HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P < 0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulae cultured in MK-1 + BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1 + HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET.     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.     

Smoking cessation in pregnancy. Intervention among heavy smokers

OBJECTIVE: Human recombinant leukemia inhibitory factor (rLIF) has been shown to stimulate hatching of murine and ovine embryos in vitro. The temporal and dose-dependent effects of murine rLIF (mrLIF) and human rLIF (hrLIF) on embryo development in two different mouse strains were investigated in this work. METHODS: Two-cell embryos were recovered from the fallopian tubes of superovulated/mated females and cultured in Krebs medium plus bovine serum albumin in microdroplets under oil. RESULTS: In the B6CBF1 strain, mrLIF significantly stimulated blastocyst formation and decreased embryo fragmentation/degeneration when added simultaneously at the initiation of culture or 24 hours thereafter. Human rLIF also had a positive effect on development. In the CD1 strain (lower fecundity), mrLIF dose-dependent effects were observed, with enhanced developmental stimulation achieved with higher doses. CONCLUSIONS: These findings confirm that hrLIF stimulates mouse embryo development in vitro and that different mouse strains show distinct responses to the cytokine. In addition, mrLIF enhances blastocyst formation and decreases embryo fragmentation when added to the embryo culture as early as the two-cell stage.     

Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9-16-cell stage embryos and blastocysts to 0 M; 10(-7) M ; 10(-6) M, and 10(-5) M H2O2 in pyruvate-free mSOF was evaluated. Developmental rates of the H2O2-treated zygotes to the 5-8-cell or blastocyst stages and survival of H2O2-treated blastocysts were reduced in a dose-dependent manner whereas the 9-16-cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide-stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10(-5) M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9-16-cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium.     

Variation in porcine zona pellucida morphology following pronase treatment

The effects of cell stage and day of embryo collection on the pronase-induced zona pellucida morphology in pig embryos were investigated. Three hundred and seventy-two two- to eight-cell embryos were treated for 3.0 min in 5.0% pronase in Whitten's Medium. Responses in zona pellucida morphology observed after pronase treatment were: (1) absent, (2) stretched and (3) intact. Each pig was coded as to the type of zona pellucida morphology its embryos possessed after enzyme treatment according to the following scale: (1)--only intact, (2)--intact and stretched, (3)--only stretched, (4)--stretched and absent and (5)--only absent. Also, the number of embryos of a particular cell stage was expressed as a fraction of the total number of embryos collected from each pig. The incidence of intact zonae pellucidae was greatest (P less than .05) among two- to three-cell embryos and the incidence of absent zonae pellucidae was greatest (P less than .05) among six-to eight-cell embryos. A significant correlation (r = .79) was observed between day of embryo collection and coded zona pellucida morphology. The results suggest that the pronase-induced zona pellucida morphology observed is dependent on cell stage and day of embryo collection.     

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61% in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts.     

Bilirubin helps to overcome the two-cell block in mouse oocyte cultures

PURPOSE: In vitro fertilization and culture of mouse oocytes, under normal atmospheric oxygen tension, subjects them to severe oxidative stress. Oocytes from some strains of mice lack the natural protective mechanism that guards them against this oxidative stress and fail to develop beyond the two-cell stage. METHODS: We could overcome the toxic effects of oxygen metabolites by adding 0.2-0.4 mg/dl bilirubin in a lactate-pyruvate culture medium defined by Whitten (1971). Six- to 8-week-old ICR (Institute of Cancer Research) female mice were super ovulated by intra peritoneal injection of 5 IU PMSG (pregnant mare serum gonadotropin) followed by 10 IU hCG 48 h later. The oocytes were collected from the distended fallopian tubes and inseminated with 1-2 million sperm from 3-4-month-old ICR male mice. The eggs were scored at 24, 48, and 72 h after the hCG injection. CONCLUSIONS: With 0.4 mg/dl bilirubin supplement, by the end of 72 h, 82% of the eggs progressed from the two-cell stage to the four-cell stage. Routine inclusion of bilirubin can improve embryo development in vitro.     

Functional analysis of a rat sodium channel carrying a mutation for insect knock-down resistance (kdr) to pyrethroids

As a model for establishing an optimized medium for human in vitro fertilization (IVF), modified human tubal fluid (HTF) media containing amino acids at concentrations found in human serum and follicular fluid were prepared, and the effect of the media on development of random-bred (ICR) and F1 hybrid (CBF1) mice embryos was studied. The total concentrations of amino acids found in serum and follicular fluid were about one-third to one-half the concentrations present in two conventional media used in human IVF: Ham's F-10 and Eagle's minimal essential medium (MEM). When ICR mouse embryos were cultured in the HTF medium containing 21 amino acids at concentrations found in follicular fluid, the number of embryos developing to morulae at 72 h and to blastocysts at 96 h increased in comparison with those cultured in HTF medium. When HTF containing amino acids at concentrations found in serum was used, only induced morula formation at 72 h was enhanced. The number of hatching blastocysts at 96 h also increased when CBF1 mouse embryos were cultured with HTF supplemented with amino acids at concentrations found in follicular fluid. When ICR mouse embryos were cultured in modified HTF media containing concentrations of amino acids found in Ham's F-10 and MEM that contained higher concentrations of glutamine, embryo development was inhibited. The amount of ammonium produced during incubation for 3 days was significantly less when embryos were cultured in media containing concentrations of amino acids found in follicular fluid compared with when Ham's F-10 or MEM was the culture medium. Ammonium is produced by the breakdown of glutamine in the culture medium during incubation with or without embryos. These results suggest that the concentrations of amino acids found in follicular fluid are more effective and safer for embryo culture than those in other media currently in use.     

Carbon monoxide inhibits hypoxic pulmonary vasoconstriction in rats by a cGMP-independent mechanism

Hypoxia activates erythropoietin-producing cells, chemoreceptor cells of the carotid body and pulmonary artery smooth muscle cells (PSMC) with a comparable arterial PO2 threshold of some 70 mmHg. The inhibition by CO of the hypoxic responses in the two former cell types has led to the proposal that a haemoprotein is involved in the detection of the PO2 levels. Here, we report the effect of CO on the hypoxic pulmonary vasoconstriction (HPV). Pulmonary arterial pressure (PAP) was measured in an in situ, blood-perfused lung preparation. PAP in normoxia (20% O2, 5% CO2) was 15.2+/-1.8 mmHg, and hypoxia (2% O2, 5% CO2) produced a DeltaPAP of 6.3+/-0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the DeltaPAP by 88.3+/-2.7% and 78.2+/-6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the DeltaPAP produced by angiotensin II. The effect of CO was studied after inhibition of the NO-cyclic guanosine monophosphate (cGMP) cascade with N-methyl-l-arginine (5.10(-5) M) or methylene blue (1.4.10(-4) M). It was found that both inhibitors more than doubled the hypoxic DeltaPAP without altering the effectiveness of CO to inhibit the HPV. In in vitro experiments we verified the inhibition of guanylate cyclase by measuring the levels of cGMP in segments of the pulmonary artery. Cyclic GMP levels were 1.4+/-0.2 (normoxia), 2.5+/-0.3 (hypoxia) and 3.3+/-0.5 pmole/mg tissue (hypoxia plus 8% CO); sodium nitroprusside increased normoxic cGMP levels about fourfold. Methylene blue reduced cGMP levels to less than 10% in all cases, and abolished the differences among normoxic, hypoxic and hypoxic plus CO groups. It is concluded that CO inhibits HPV by a NO-cGMP independent mechanism and it is proposed that a haemoprotein could be involved in O2-sensing in PSMC.     

N-acetyl-cysteine causes a late re-specification of the anteroposterior axis in the Xenopus embryo

N-acetyl cysteine is an agent which has been shown to interrupt signal transduction processes linking a wide range of stimuli to the activation of NF-kappa B in mammalian cells. We have investigated its effect on the early development of Xenopus embryos by injecting it into blastulae, using concentrations comparable to those effective on cultured cells. High concentrations at the late blastula or early gastrula stage suppress posterior and enhance anterior development, yielding embryos with enlarged cement glands and otherwise consisting of little except head in extreme cases. Reducing the amount of N-acetyl cysteine injected leads to progressively more posterior structures developing. Injection into one- or two-cell embryos gives similar phenotypes, but of reduced severity and the cement gland is not so enlarged. Explants of animal cap cells taken several hours after injection develop to give large amounts of cement gland material. We have examined the expression of a number of genes in the anteriorised embryos. Posterior markers and Xsna are reduced. Noggin and Goosecoid mRNA are up-regulated through the gastrula and persist at these levels until at least the late neurula stage, whereas in controls Noggin is much lower and Goosecoid is absent at these stages. The most anteriorised phenotype may be a consequence of this changed expression.     

The NMDA antagonist MK-801 blocks the extinction of Pavlovian fear conditioning

We have evaluated the safety of intracytoplasmic sperm injection(ICSI) procedures by using bovine zygotes. Bovine zygotes were injected with a small amount (2-3 pl) of either medium alone or medium containing polyvinylpyrrolidone (PVP) (sham-ICSI, without spermatozoon) using the same procedure as ICSI, and the subsequent in-vitro embryonic development and embryo quality (number of cells/blastocyst) were examined. Control zygotes which had not been injected were similarly evaluated after in-vitro development. The sham-ICSI of either medium alone or medium containing PVP into bovine zygotes had no harmful effects on the rate of normal fertilization and on the rate of development to hatched blastocyst stage compared with those of controls (P > 0.05). In addition, no harmful effects were observed in the number of cells per blastocyst (embryo quality). The results suggest, for the first time, that the ICSI procedures currently used for animal and human ICSI are neither detrimental to embryonic development nor detrimental to embryo quality.     

Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol

Embryos were collected on Days 5 and 6 after breeding to investigate the effectiveness of ethylene glycol (ETG) and glycerol (GLY) as cryoprotectants of sheep morulae and blastocysts and to determine their optimum stage of development for cryopreservation. Only excellent (grade 1) and good (grade 2) embryos (196 morulae and 188 blastocysts) were incubated in increasing concentrations of GLY or ETG and submitted to a slow-freezing and quick-thawing procedure. Both cryoprotectants were removed using 0.25 M sucrose solution, and then embryos were cultured or transferred to determine their viability. Freezing medium containing ETG yielded higher in vitro survival rates (P < 0.01) than medium containing GLY (64.6% vs 16.0%); the difference between cryoprotectants was greater when morulae were used (57.9% vs 4.2%, P < 0.005) as compared with blastocysts (70.4% vs 21.5%, P < 0.05). There was a strong interaction between type of cryoprotectant and embryo stage (P < 0.005). After transfer of morphologically viable embryos, the in vivo development rate of embryos frozen with ETG was also higher than that of embryos frozen with GLY (45.5% vs 27.7%, P < 0.05). There were no significant differences in the number of lambs born among procedures and embryo stage, though the lowest lambing rate was obtained with morulae frozen with GLY (21.4%). Similar lambing rates were produced when blastocysts were frozen with either GLY or ETG (36.6% vs 43.0%). The best embryo survival after thawing was observed when blastocysts were frozen with ETG as cryoprotectant.