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PubMed2003

Placental villitis of unclear etiology during ovum donor in vitro fertilization pregnancy.

OBJECTIVE: Preliminary observations by a single pathologist at our institution revealed a 75% incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies. Because the incidence of villitis of unexplained etiology in the general population is approximately 10%, we conducted a controlled study to compare the incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies to that in in vitro fertilization pregnancies that do not use donated ova. STUDY DESIGN: Placental specimens of ovum donor in vitro fertilization pregnancies were matched randomly with pregnancies that resulted from both fresh and frozen/thawed native oocyte in vitro fertilization from March 5, 1995, to October 10, 2001, and examined in a blinded fashion by a single pathologist (D. J. R.) for villitis of unexplained cause. The incidence of villitis of unexplained etiology was analyzed in 27 patients who underwent ovum donor in vitro fertilization versus 37 patients who underwent native oocyte in vitro fertilization. RESULTS: Villitis of unexplained cause occurred in 22.2% of ovum donor in vitro fertilization pregnancies, 10.8% of native oocyte in vitro fertilization pregnancies (fresh and frozen/thawed combined), and 14.3% of frozen/thawed cycles (P=.21). CONCLUSION: Although the incidence was not statistically different than in in vitro fertilization that used native maternal oocytes, there was a 2-fold increase in villitis of unexplained cause in the ovum donor in vitro fertilization placentas, which suggests that immune-related disorders may be increased in ovum donor pregnancies.

Adult↗
PubMed1989

Relation of in vitro drug activity to clinical response in a prospective trial for advanced germ cell testicular tumors.

An in vitro assay, which evaluates the effect of drugs on labelled DNA precursor incorporation into fresh specimens after 3 h of in vitro treatment, was used for a prospective study. Drugs were used as monochemotherapy or in different combinations according to their in vitro activities for salvage or palliation treatment of 16 patients with germ cell testicular tumors, for a total of 21 correlations between in vitro and clinical results. In 11 instances, drugs active in vitro were used; in 10 instances, because no drug was active in vitro, patients were treated with inactive drugs. There was a significant correlation between in vitro and clinical activity (p = 0.02), with a very high true-negative rate (90%) and a lower true-positive rate (54%). No patient treated with drugs inactive in vitro survived 2 years, whereas freedom from progression and overall survival for patients treated with drugs active in vitro were 27.3 and 36.4%, respectively (active vs. inactive, p = 0.007). The overall response in the group of patients treated prospectively was comparable to that observed in a group of 9 patients treated in the same period of time with drugs of unknown in vitro activity (33 vs. 11% objective and long-term responses, respectively, p = n.s.) notwithstanding the high percentage of patients treated with drugs inactive in vitro in the former group.

Antineoplastic Agents↗
PubMed2000

Periodic tension development in the membrane of the in vitro contractile vacuole of Paramecium multimicronucleatum: modification by bisection, fusion and suction.

The contractile vacuole of the freshwater protozoan Paramecium multimicronucleatum is a membrane-bound exocytotic vesicle that expels excess cytosolic water. The in vitro contractile vacuole isolated from P. multimicronucleatum along with a small amount of cytosol and confined under mineral oil showed periodic rounding and slackening at fairly regular intervals. Activity lasted for over 30 min at room temperature (24-27 degrees C). The rounding of the in vitro contractile vacuole corresponded to the increased membrane tension of the in vivo contractile vacuole that occurs immediately before fluid expulsion. Unlike the in vivo contractile vacuole, the in vitro contractile vacuole did not expel fluid, since it lacked a mechanism to form a pore. The subsequent slackening of the in vitro contractile vacuole corresponded to the fluid-filling phase of the in vivo contractile vacuole that occurs at decreased membrane tension. Fluid filling occurred in the in vitro contractile vacuole only when it was isolated together with its radial arms. In vitro membrane-bound vesicles obtained by 'bisecting' (although the two parts were not always identical in size) an in vitro contractile vacuole established their own independent rounding-slackening cycles. In vitro contractile vacuole vesicles could fuse again when the vesicles slackened. The fused vesicle then showed a rounding-slackening cycle with a period closer to that of the vesicle that exhibited the shorter cycle period. An additional rounding phase of the in vitro contractile vacuole could be induced by applying suction to a portion of its membrane with a micropipette when the contractile vacuole was in its slackened phase. This suggests that maximum tension development in the contractile vacuole membrane can be triggered when tension is increased in any part of the contractile vacuole membrane. The time from the start of an extra rounding phase to the next spontaneous rounding and for subsequent rounding-slackening cycles was nearly the same as that before the extra rounding phase. This implies that there is no master pacemaker to control the rounding-slackening cycle in the contractile vacuole membrane. Severed radial arms also became vesiculated and, like contractile vacuole membranes, these in vitro vesicles showed independent rounding-slackening cycles and vesicle-vesicle fusions. Thus, membrane derived from the radial arm seems to be identical in its tension-developing properties with the contractile vacuole membrane. ATP was found to be required for contractile vacuole rounding but inhibitors of actin or tubulin polymerization, such as cytochalasin B and Nocodazole, had no effect on the in vitro contractile vacuole's rounding-slackening cycle.

Actins↗
PubMedDate not supplied

Kinetic analysis of tolbutamide-sulfonamide interaction in rabbits based on clearance concept. Prediction of species difference from in vitro plasma protein binding and metabolism.

The interaction between tolbutamide (TB) and sulfonamide (SA) in rabbits was quantitatively investigated by both in vivo and in vitro experiments, and the mechanisms of species difference between rabbits and rats were analyzed by comparing the two total body clearances (CLtot) obtained from in vivo and in vitro studies. The sulfonamides used were sulfaphenazole (SP) and sulfadimethoxine (SDM). In vivo CLtot of TB was changed little by SA in rabbits, which was contrary to the phenomenon seen in rats, i.e. CLtot was markedly decreased by SA in rats (Sugita et al., Biochem. Pharmacol., 30, 3347, 1981). The total body clearance defined as CL in vitro tot.pred was predicted for TB in the presence and absence of SA by the equation: CL in vitro tot.pred congruent to fB CL in vitro int, where fB is the blood-free fraction and CL in vitro int is the hepatic intrinsic clearance of unbound drug obtained from in vitro experiments using liver microsomal fraction. A comparatively good agreement was observed between the two CLtot obtained from in vivo and in vitro data. The analysis in rabbits based on these in vitro experiments showed that the small gross changes of in vivo CLtot induced by SA were due to the compensative changes of two factors, i.e. fB and CL in vitro int which in the presence of SA were approximately 200 and 50% of the control, respectively. However, in rats, the marked decrease of in vivo CLtot induced by SA was explained by the greater degree of the decrease in CL in vitro int than that of the increase in fB.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Unresponsiveness in hapten-specific cytotoxic lymphocytes. III. Influence of H-2 and non-H-2 gene loci on the in vitro trinitrophenyl-specific CTL response following either acute or chronic in vivo treatment with trinitrobenzene sulfonic acid.

The regulation of the in vitro generation of cytotoxic T lymphocytes (CTLs) directed against hapten-modified syngeneic cells has been investigated. The results indicate that acute intravenous pretreatment with water-soluble hapten, trinitrobenzene sulfonic acid (TNBS), can either positively or negatively affect the in vitro generation of trinitrophenyl (TNP)-specific CTLs. In general, mice bearing the H-2d haplotype are most likely to develop a reduced in vitro response pattern following a single acute in vivo TNBS treatment, wheras mice bearing the H-2k or H-2b haplotypes display either unchanged or augmented in vitro response patterns. We have shown that, in addition to the influences of H-2 genes, non-H-2 genes can also influence the in vitro hapten-specific CTL response following intravenous pretreatment with water-soluble hapten. Further, in two (H-2k X H-2d) F1 combinations between an H-2k strain displaying an unchanged in vitro response pattern following acute in vivo TNBS treatment and an H-2d strain displaying a reduced in vitro response pattern following similar treatment, it was observed that a single in vivo TNBS pretreatment did not induce the unresponsive state when F1-TNP stimulator cells were used. These results suggest that the mechanisms responsible for the reduced in vitro response pattern are not dominant within the F1 environment. However, when TNP-modified parental stimulators are used, a split-response pattern is observed in cells from TNBS-treated F1 mice which reflect the response patterns of the respective parents. These latter results again emphasize the influence of gene loci on the in vitro response patterns following acute TNBS treatment. In contrast to the significant influence of H-2 and non-H-2 genes on the in vitro TNP-specific response following acute in vivo TNBS treatment, these genes do not appear to significantly influence the in vitro TNP-specific response pattern following chronic TNBS treatment. Chronic TNBS treatment renders all strains tested specifically unresponsive.

Animals↗
PubMed1991

Characterization of developmental arrest in early bovine embryos cultured in vitro.

The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19/45) of in vivo controls developed normally; none (0/55; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2/48) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33/33 (100%) of blocked embryos responded positively to fluorescein diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11/41, 27% vs 22/41, 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death.

Journal Article↗
PubMed1996

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Animal Testing Alternatives↗
PubMed2005

In vitro models for the blood-brain barrier.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Animals↗
PubMed1985

In vitro drug testing using hemopoietic cells: goals and limitations.

In vitro drug sensitivity is one of many biologic variables which may predict in vivo drug response. Even if in vitro assays provide relevant data, for some tumors, variable levels of stem-cell origin, differentiation, tumor heterogeneity, or self renewal may be more important than cytotoxicity to proliferating cells. Although ANLL has been used here frequently as a model, it may not be the most appropriate tumor for study. Unlike many cancers, in ANLL, primary drug resistance is unusual, and in relapse, secondary drug resistance is usually incomplete. It has been suggested that in vitro drug sensitivity predicts remissions for patients who do not die of infection or remain aplastic during induction therapy. However, for the majority of patients, this argument acknowledges the overriding importance of biologic variables other than in vitro drug cytotoxicity. For rapidly growing tumors, such as Burkitt's lymphoma, rapid emergence of drug resistance related to disease burden may be the most important response determinant. Perhaps in other tumors, in vitro drug sensitivity will be an independent variable of overriding importance. To determine the role of in vitro drug testing, trials examining in vitro drug sensitivity must meet stringent criteria. The assays should use well-defined and reproducible cultures and drug exposures. The trials must be large enough, contain homogenously treated patients, and use carefully defined response and survival endpoints. Decision rules derived from such trials must be further tested by prospective evaluation. Investigators conducting these trials must be prepared to search for important in vitro results reflecting tumor biology and to analyze in vitro drug sensitivity as only one continuous variable determining in vivo responses. Such trials will be difficult to conduct and expensive. In the final analysis, in vitro assays may find their most important roles as preclinical drug screens and models for in vitro drug resistance. Further insights into molecular genetics of malignant transformation and drug resistance may make such assays obsolete, but for the present, they provide important insights into tumor variability and mechanisms of drug response.

Animals↗
PubMed2004

Comparison of maturation, fertilization, development, and gene expression of mouse oocytes grown in vitro and in vivo.

PURPOSE: To investigate the difference of in vitro and in vivo grown oocytes, we compared maturation, fertilization, development, and maternal gene expression from both in vitro and in vivo grown mouse oocytes. METHODS: The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After culture, maturation, fertilization, and developmental rates were assessed. RT-PCR (reverse transcription-polymerase chain reaction) was performed to examine the expression of beta-actin, GDF-9, and IGF-II in matured oocytes. RESULTS: No difference in the nuclear maturation was detected between in vitro and in vivo grown oocytes, but the mean oocyte diameter of the in vitro group was smaller than that of the in vivo group. The fertilization rate was significantly lower in the in vitro group than in the in vivo group (p < 0.05). The capacities of in vitro grown oocyte to cleave and develop to blastocysts were significantly lower than those of the in vivo grown oocytes (p < 0.001). Moreover, blastocyst of in vitro group had fewer total cells than those of in vivo group (p < 0.05). In regards to the expression of genes in mature oocytes, growth differentiation factor-9 (GDF-9) expression was similar between the two groups, but beta-actin was significantly reduced in the in vitro group compared to the in vivo group. Particularly, the expression of insulin-like growth factor II (IGF-II) was not found in the in vitro grown oocytes. CONCLUSIONS: These results showed that in vitro grown oocytes did not have the same developmental capacity as in vivo grown oocytes. We assume that the aberrant expression of maternal-derived genes in the in vitro grown oocytes may cause the poor embryo viability.

Actins↗
PubMed1991

In vitro growth pattern and differentiation predict for progression of myelodysplastic syndromes to acute nonlymphocytic leukaemia.

In 153 consecutive patients with myelodysplastic syndrome (MDS) the prognostic value of FAB-classification, cytogenetics, Bournemouth score, a history of previous radio- or chemotherapy and in vitro bone marrow growth were retrospectively analysed, for both acute nonlymphocytic leukaemia (ANLL) development and survival. Thirty-eight of the 153 patients (25%) showed progression to ANLL, 63 (41%) died during the myelodysplastic phase due to infection or bleeding and three (2%) received allogeneic bone marrow transplantation (BMT). Univariate analysis showed that the FAB-classification, in vitro growth pattern and differentiation, and cytogenetics had a predictive value for ANLL development and survival. The Bournemouth score was predictive only for survival. Most predictive for the development of ANLL were in vitro growth pattern and maturation. Patients with normal in vitro growth progressed to ANLL in 6% of the cases, in patients with hypoplastic or leukaemic growth 32.5% developed ANLL (P less than 0.0001). The ANLL incidence in patients with normally differentiated in vitro colonies was 14.5%, compared with a 52% incidence in cases showing no in vitro cell maturation (P = 0.001). The combination of growth pattern and differentiation revealed an ANLL incidence of 4.2% in cases of normal growth and differentiation, and 60.4% if the in vitro growth and/or differentiation was abnormal (P = 0.006). In vitro maturation was the only parameter predictive for ANLL development in multivariate analysis. From our data it is concluded that the predictive value of in vitro bone marrow culturing in patients with MDS can be increased by including in vitro maturation as a distinct parameter. The in vitro prognostic data can be important in selecting MDS patients for intensive chemotherapy or BMT.

Bone Marrow↗
PubMed2002

Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica).

In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitrol 1-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.

Amino Acid Sequence↗
PubMed2002

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.

Animals↗
PubMed2000

Births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro.

In Experiment 1, cleavage frequency and in vitro development of domestic cat embryos produced after in vitro maturation of oocytes obtained from ovaries after ovariohysterectomy (in vitro) with that of oocytes retrieved from follicle-stimulating hormone-treated donors at 24 h after administration of luteinizing hormone (in vivo) and fertilization by intracytoplasmic sperm injection (ICSI) or IVF were compared. In each group presumptive zygotes were assessed for cleavage on IVC Days 1 and 4 and for development to blastocysts on IVC Day 7. In vitro matured oocytes had lower frequencies of meiotic maturation (59.2% v. 66.5%), cleavage at Day 1 (41.4% v. 64.9%) and development to the morula stage at Day 4 (65.8% v. 87.9%) than did in vivo matured oocytes, after ICSI and IVF. Development to the blastocyst stage was lower in in vitro matured oocytes (19.0%) than in vivo matured oocytes (29.5%) after ICSI. In Experiment 2, we evaluated the capacity of sperm injected oocytes without a visible polar body to undergo cleavage and in vitro development. More in vivo matured than in vitro matured oocytes underwent cleavage at Day 1 (46.6% v. 12.6%) and developed to the morula stage by Day 4 (66.7% v. 46.1%), but no blastocysts were obtained at Day 7 in either group. In Experiment 3, we evaluated the in vivo viability of domestic cat embryos derived from ICSI of in vitro matured oocytes. Morula stage embryos were transferred to 18 domestic cat recipients either on Day 4 or 5 after oocyte recovery. A total of 3 domestic cat recipients were pregnant after transfer to recipients on Day 5. Two pregnant cats delivered two normal and healthy live male kittens on Day 68 of gestation and the remaining cat delivered a male kitten on Day 62 that died during the last two days of gestation. These results demonstrate that: (1) inadequate cytoplasmic maturation of in vitro matured domestic cat oocytes is the main cause of deficient oocyte activation; (2) the injection of oocytes without a visible polar body is a useful technique to evaluate oocyte cytoplasmic maturation; and (3) blastocysts obtained after ICSI of in vitro matured oocytes are viable and not a result of parthenogenesis.

Animals↗
PubMed2002

Development of skeletal muscle and expression of candidate genes in bovine fetuses from embryos produced in vivo or in vitro.

The objectives of this study were to determine the effects of in vitro embryo production on histological development and gene expression in the skeletal muscle of bovine fetuses during late gestation. Blastocysts produced in vivo were obtained from superovulated Holstein cows. Blastocysts produced in vitro were obtained from oocytes of Holstein cows that were matured and fertilized in vitro. Single blastocysts were transferred into heifers at a synchronized estrous and fetuses were recovered at Day 222 of gestation (n = 12 each for in vivo and in vitro). Samples of semitendinosus muscle were obtained for histological analysis and assessment of gene expression. Individual muscle sections were stained for the assessment of primary muscle fibers, secondary muscle fibers, or total muscle fibers. Semiquantitative reverse transcription-polymerase chain reaction assays were performed for 5 different candidate genes. The ratio of secondary-to-primary fiber number was greater in fetuses from embryos produced in vitro compared with fetuses from embryos produced in vivo. Similarly, the ratio of secondary-to-primary fiber volume density tended to be greater in fetuses from embryos produced in vitro. The proportional volume of tissue present between myofibrils was greater in fetuses from embryos produced in vitro. The expression of mRNA for myostatin was decreased in skeletal muscle of fetuses in the in vitro group compared with controls. The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase tended to be increased in skeletal muscle of fetuses in the in vitro treatment group. There was no effect of treatment on the expression of mRNAs for myf-5, myoD, or myogenin. In conclusion, in vitro production of embryos resulted in fetuses with altered development of skeletal muscle fibers. Myostatin was identified as the candidate gene whose expression may contribute to the observed changes in muscle development of these fetuses.

Animals↗
PubMed2002

Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro.

Successful joint resurfacing by tissue-engineered cartilage has been limited, in part, by an inability to secure the implant to bone. To overcome this, we have developed the methodology to form a cartilage implant in vitro consisting of a layer of cartilagenous tissue overlying a porous, biodegradable calcium polyphosphate (CPP) substrate. As bone will grow into the CPP after implantation, it will result in anchorage of the cartilage. In this study, the cartilagenous tissue formed in vitro after 8 weeks in culture was characterized and compared to native articular cartilage. Light microscopic examination of histological sections showed that there was a continuous layer of cartilagenous tissue on, and integrated with the subsurface of, the CPP substrate. The in vitro-formed tissue achieved a similar thickness to native articular cartilage (mean +/- SEM: in vitro = 0.94 +/- 0.03 mm; ex vivo = 1.03 +/- 0.01 mm). The cells in the in vitro-formed tissue synthesized large proteoglycans (Kav +/- SEM: in vitro = 0.27 +/- 0.01; ex vivo = 0.27 +/- 0.01) and type II collagen similar to the chondrocytes in the ex-vivo cartilage. The in vitro-formed tissue had a similar amount of proteoglycan (GAG microg/mg dry wt.: in vitro = 198 +/- 10; ex vivo = 201 +/- 13) but less collagen than the native cartilage (hydroxyproline microg/mg dry wt.: in vitro = 21 +/- 1; ex vivo = 70 +/- 8). The in vitro-formed tissue had only about 3% of the load-bearing capacity and stiffness of the native articular cartilage, determined from unconfined mechanical compression testing. Although low, this was within the range of properties reported by others for tissue-engineered cartilage. It is possible that the limited load-bearing capacity is the result of the low collagen content and further studies are required to identify the conditions that will increase collagen synthesis.

Animals↗
PubMed1996

Intrinsic tone and distensibility of in vitro and in situ cat pulmonary arteries.

This study was designed to determine the in vitro and in situ diameter vs pressure relationship of 200- to 1,200-microns diameter pulmonary arteries in the cat. Diameter vs pressure relationships of these arteries were obtained using two methods, microscopic observation of in vitro cannulated and pressurized arteries and X-ray angiography of in situ arteries. Both in vitro and in situ arteries were studied first under normal conditions and then after reducing tone with Ca(2+)-free solution (in vitro) or papaverine (in situ). In vitro arteries commonly increased their tone in response to elevated transmural pressure, and in some cases, the diameter actually decreased as pressure increased. This behavior was not observed in the in situ arteries. The major difference between in vitro and in situ arteries was that when the in vitro arteries were relaxed, the slope of the diameter vs pressure curves increased, whereas the slope was not altered significantly by relaxation of the in situ arteries. This difference is emphasized by the increased distensibility with relaxation of the in vitro arteries but the decreased distensibility with relaxation of the in situ arteries. The results of this study suggest that, at least in the cat, small pulmonary arteries possess a mechanism that is dormant in the in situ environment within the normal lung. However, the potential for pressure-induced constriction may be unmasked by changing the vessel history and/or environment. Extrapolating results obtained from in vitro pulmonary arteries to the in situ situation should therefore be done with caution. Studies directed at what factors contribute to differences in the responses of in vitro and in situ arteries might help in understanding pulmonary vascular pathophysiology.

Angiography↗
PubMed2005

Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals.

The lower sensitivity of in vitro cytotoxicity assays currently restricts their use as alternative to the fish acute toxicity assays for hazard assessment of chemicals in the aquatic environment. In vitro cytotoxic potencies mostly refer to nominal concentrations. The main objective of the present study was to investigate, whether a reduced availability of chemicals in vitro can account for the lower sensitivity of in vitro toxicity test systems. For this purpose, the bioavailable free fractions of the nominal cytotoxic concentrations (EC50) of chemicals determined with a cytotoxicity test system using Balb/c 3T3 cells and the corresponding free cytotoxic concentrations (ECu50) were calculated. The algorithm applied is based on a previously developed simple equilibrium distribution model for chemicals in cell cultures with serum-supplemented culture media. This model considers the distribution of chemicals between water, lipids and serum albumin. The algorithm requires the relative lipid volume of the test system, the octanol-water partition coefficient (K(ow)) and the in vitro albumin-bound fraction of the chemicals. The latter was determined from EC50-measurements in the presence of different albumin concentrations with the Balb/c 3T3 test system. Organic chemicals covering a wide range of cytotoxic potency (EC50: 0.16-527000 microM) and lipophilicity (logK(ow): -5.0-6.96) were selected, for which fish acute toxicity data (LC50-values) from at least one of the three fish species, medaka, rainbow trout and fathead minnow, respectively, were available. The availability of several chemicals was shown to be extensively reduced either by partitioning into lipids or by serum albumin binding, or due to both mechanisms. Reduction of bioavailability became more important with increasing cytotoxic potency. The sensitivity of the Balb/c 3T3 cytotoxicity assay and the correspondence between in vivo and in vitro toxic potencies were increased when the free cytotoxic concentrations instead of the nominal cytotoxic concentrations were used as measure of cytotoxic potency. The few remaining prominent differences between cytotoxic and acute toxic concentrations can be explained by a more specific mechanism of acute toxic action than basal cytotoxicity. It is concluded that the frequently observed low sensitivity of in vitro cytotoxicity test systems, compared to fish acute toxicity assays, at least in part, can be explained by differences in the availability of chemicals in vitro and in vivo. Moreover, neglecting these differences systematically causes a bias of the correlation between in vivo and in vitro toxic potencies of chemicals. Taking them into account, however, increases the predictivity of the in vitro assays.

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