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PubMed2002

In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon.

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.

Animals↗
PubMed1994

Interferon-alpha-mediated prevention of in vitro apoptosis of chronic lymphocytic leukemia B cells: role of bcl-2 and c-myc.

Chronic B lymphocytic leukemia cells (B-CLL), characterized by the accumulation in vivo of long-life span B cells, exhibit spontaneous programmed cell death or apoptosis when cultured in vitro. We show that interferon-alpha (IFN-alpha), although able to decrease in vivo the number of leukemic cells, protects chronic B lymphocytic leukemia cells from in vitro programmed cell death or apoptosis. This inhibition of spontaneous in vitro apoptosis of leukemic B cells was observed after 24-48 hr of culture with 100-1000 U of either Interferon-alpha 2a or 2b. The protective activity was observed in the majority of the patients tested (6 out of 8) independent of the amount of apoptosis observed. Furthermore, in contrast to IL-4, IFN-alpha did not up-regulate the expression of Bcl-2. This suggests that B-CLL cells can be prevented from undergoing apoptosis in vitro by at least two different mechanisms: one, triggered for instance by IL-4, is associated with Bcl-2 production and the second triggered by Interferon-alpha is Bcl-2 independent. To elucidate the pathways mobilized by Interferon-alpha we also studied the regulation of c-myc expression in our experimental system. We found that (i) induction of in vitro B-CLL apoptosis was not associated with up-regulation of c-myc, (ii) c-myc expression as assessed by mRNA and protein determinations was increased after in vitro or in vivo Interferon-alpha stimulation. Additional experiments using c-myc specific oligonucleotides demonstrated that when Interferon-alpha-mediated c-myc expression was decreased by 60%, the in vitro protective effect of Interferon-alpha was not modified. Thus our data show that in contrast to the situation in vivo, Interferon-alpha prevents spontaneous in vitro B-CLL cells apoptosis through a Bcl-2-independent pathway which is probably not related to c-myc up-regulation.

Aged↗
PubMed1997

Translation and replication properties of the human rhinovirus genome in vivo and in vitro.

The poor translation efficiency of genome-length human rhinovirus RNA in vitro using HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficient in vitro translation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particles in vitro. A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesis in vivo. In order to test this hypothesis and extend in vitro translation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed the in vitro synthesis of T7 transcripts containing the intact poliovirus type 1 (PV1) 5' noncoding region (5' NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14) polyprotein-coding region and 3'-terminal sequences. These chimeric RNAs translated efficiently in vitro and were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimera in vitro. The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirus in vivo are determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5' NCR may be a limitation in rabbit reticulocyte lysate-based in vitro translation extracts, it does not appear to be a major limiting determinant for growth of rhinovirus in vivo or replication in the HeLa cell-free extract.

Animals↗
PubMed1976

In vitro colony growth of acute myelogenous leukemia.

Colony formation in vitro by marrow cells from patients with untreated acute myelogenous leukemia (AML) and from patients in AML relapse is infrequent using the standard Robinson assay. A newly developed culture system has been described in which marrow from AML patients in these disease stages form leukemic cell colonies. In this in vitro system, phytohaemagglutinin is the essential stimulator for colony formation. The leukemic origin of the colonies has been proven by ultrastructural morphology and cytogenetics. It appears that colony formation by leukemic cells in this system is predominantly independent from the leukocyte factor which is the main stimulator in the Robinson assay for growing colonies of marrow cells from haematologically normal individuals. Bone marrow cells in untreated acute myelogenous leukemia (AML) demonstrate abnormal growth in vitro in the Robinson assay (Robinson et al., 1971; and Bull et al., 1973). Characteristically, there is a near total failure of colony formation; predominantly clusters are formed containing 20 cells or less (Bull et al., 1973; Greenberg et al., 1971; Moore et al., 1973 and 1974, and van Bekkum et al., in press). The absence of colonies has been shown to be due to a marked decrease of the normal myeloid precursor cell population in untreated AML. The small agregate formation of AML cells has been attributed to the suboptimal response of leukemic cells to the leukocyte stimulation factor. Because this poor proliferation in vitro might not represent the maximal in vitro and in vivo proliferation potential of the leukemic cells, we studied a number of modifications of the in vitro culture system. A number of factors were studied which may have some influence on cell proliferation in general, notably phytohaemagglutinin (PHA), which induces lymphocyte colonies in vitro (Rozenszajn et al., 1974), and endotoxin which has been demonstrated to increase the labelling index of leukemic cells in vivo (Golde et al.). In this paper an in vitro system is described in which marrow cells from untreated AML and AML in relapse were stimulated by phytohaemagglutinin (PHA) to form leukemic cell colonies in soft agar. These (similar) cells predominantly formed small aggregates (20 cells or less) in the presence of the normal leukocyte feeder layer alone. Moreover, in the course of the experiments, it appeared that by adding low concentrations of endotoxin to the cultures, the stimulating effect of PHA could be amplified.

Bone Marrow↗
PubMed1991

Effect of in vitro culture on the dynamics of uteroglobin distribution in rabbit blastocysts.

The purpose of this study was to investigate the localization and transport of uteroglobin in normal rabbit blastocysts (day 4-day 6 p.c.) and in those cultured for 6-48 h in vitro, using a specific radioimmunoassay and immunocytochemistry. The results of the radioimmunoassay showed that in day 4 p.c. blastocyst tissue (based on homogenate measurements) a significant decrease of the uteroglobin content started after only 6 h of culture in vitro. A significant concomitant rise of uteroglobin was observed in the culture medium after 12 h of in vitro culture. Using immunocytochemistry it was not possible to detect uteroglobin in any compartment of the non-cultured or in vitro cultured day 4 p.c. blastocysts. The efflux of uteroglobin down a concentration gradient was confirmed by the immunocytochemistry in non-cultured and in vitro cultured day 5 p.c. and day 6 p.c. blastocysts. Uteroglobin immunoreactions were mainly detected in non-cultured blastocysts (day 5 and 6 p.c.) in large vesicles of the trophoblast cells. In addition endocytotic vesicles at the inside of the apical membrane of trophoblast cells, some cell debris within the perivitelline space and the neozona were labelled. During in vitro culture of day 5 and 6 p.c. blastocysts, uteroglobin labelling in the coverings did not change. In non-cultured and cultured day 5 and 6 p.c. blastocysts neither the compartments of the embryoblast, the endoderm cells nor the blastocyst cavity showed any uteroglobin immunoreactions. After only 6 h of in vitro culture, uteroglobin immunoreactions were no longer found within the trophoblast cells. The reaction did not reappear during the course of in vitro culture up to 48 h, suggesting a complete lack of de novo synthesis of uteroglobin by blastocysts.

Animals↗
PubMed1981

Quantitative association between the in vitro human tumor stem cell assay and clinical response to cancer chemotherapy.

Objective methods have been developed to quantitate results of the in vitro human tumor stem cell assay, the degree of the association between the in vitro assay and clinical response as well as the likelihood of response. Methods considered to quantitate in vitro assay data included first-order kinetics of percent survival with drug concentration, minimal percent of tumor colony-forming unit survival at low drug concentrations, and area under the in vitro percent survival-drug concentration curve. Based upon experimental data, the percent tumor colony survival and the area under the curve (i.e., in vitro sensitivity indices) were concluded to better account than other methods for the commonly observed nonlinear shape of the in vitro curves. The two methods also yielded equivalent quantitative descriptions of the in vitro data. A logistic regression model was used for explicit quantitation of the relationship between the in vitro sensitivity index and predicted probability of clinical response. Very high association was observed between the predicted in vivo and actual clinical response for the cytotoxic drugs considered. Incorporation of other pharmacologic and patient prognostic factors into the quantitative methods is discussed and shown to improve their effectiveness.

Antineoplastic Agents↗
PubMed1988

Penbutolol: pharmacokinetics, effect on exercise tachycardia, and in vitro inhibition of radioligand binding.

The pharmacokinetics of penbutolol 40 mg, its reduction in exercise-induced tachycardia, and the in vitro inhibition of radioligand binding to beta-adrenoceptors by plasma have been investigated in 7 healthy volunteers. The peak penbutolol concentration of 285 ng/ml was observed 1.2 h after administration, and the maximum of 4'-OH-penbutolol of 4.76 ng/ml was found after 1.64 h. Penbutolol was detected for up to 48 h, and 4'-OH-penbutolol dropped below the limit of detection after about 10 h. The terminal plasma concentration of penbutolol declined with an average half-life of 19 h. The maximum reduction in exercise-induced tachycardia was 33 beats/min 2.6 h after taking penbutolol. There was still a significant reduction of about 7 beats/min after 48 h. This effect could be adequately explained by the concentration-time course of penbutolol in combination with Clark's model of the concentration-effect relationship. Antagonist activity in plasma caused 91% inhibition of radioligand binding in vitro to beta 2-adrenoceptors on rat reticulocyte membranes 1.6 h after intake of penbutolol. By 48 h after intake, radioligand binding was still significantly inhibited (23%). The in vitro inhibition of radioligand binding by plasma showed a linear correlation with the reduction in exercise-induced tachycardia for all phases of the workload. The time course of the reduction in heart rate was completely explained by the in vitro inhibition of radioligand binding. However, it was not possible to explain the in vitro inhibition of radioligand binding by the concentration-time course of penbutolol using a simple competition model, although both variables were based on the same sampling site. When the in vitro inhibition of radioligand binding was plotted against the penbutolol concentration at the same sampling times (with both variables transformed to multiples of the apparent inhibition constant) the discrepancy became even more apparent as time-related counterclockwise hysteresis. None of the known metabolites of penbutolol can explain the discrepancy between the penbutolol concentration and the inhibition of radioligand binding in vitro. It appears that an other active metabolite is formed, which contributes to the effect in vitro and in vivo and so can explain the observed discrepancy.

Adrenergic beta-Antagonists↗
PubMed1995

Impact of cephaloridine on glutathione and related enzymes: comparison of in vivo and in vitro rat models.

The aim of this study was to investigate the early effects of cephaloridine (CPH) on glutathione-dependent phase II detoxification in the rat proximal tubular cell and to find an in vitro alternative to the in vivo model. The in vivo study was conducted in three groups of rats which received CPH at doses of 250, 500 or 750 mg/kg per day for 3 days, while another group received 500 mg/kg as a single dose. For the in vitro study, rat renal proximal tubular cultured cells were exposed to CPH at concentrations of 0.3, 0.6, 1, 1.7 mM for 24, 48 and 72 h. Glutathione-dependent detoxification was evaluated in vivo and in vitro on the basis of total intracellular glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPX). Glutathione reductase (GRED) and GST mRNA levels were also determined. Results of in vivo and in vitro models were comparable in terms of the early increase of GSH, GST and GRED. This increase had a bell-shaped dose-response with a maximum at 500 mg/kg in vivo and 1 mM in vitro. Beyond these doses, GSH and its dependent enzyme levels decreased, associated with cytotoxicity in vitro and renal insufficiency in vivo. The increased GST activity was associated with an increased level of GST7 in vivo and a markedly increased level of GST1-2 in vitro. We concluded that the in vitro model can be used as an alternative to animal experimentation to study glutathione-dependent detoxication. Low cytotoxic doses of CPH induced an early increase of glutathione phase II-dependent detoxification enzymes.

Animals↗
PubMed2005

Suitability of a batch in vitro fermentation model using human faecal microbiota for prediction of conversion of flaxseed lignans to enterolactone with reference to an in vivo rat model.

BACKGROUND: In vitro fermentation models have been used widely for studies of shortchain fatty acid (SCFA) formation from carbohydrates, whereas the suitability of these methods for enterolactone (ENL) formation has received less attention. AIM: The aim was to study the suitability of an in vitro fermentation model for prediction of bioconversion of lignans to ENL, to compare the approach with that of an in vivo rat model and to study the SCFA formation in both models. METHODS: Predigested samples of rye bran (R), flaxseed meal (F) alone, or in combination with rye bran (R&F) and a faecal control were incubated in an in vitro fermentation model using human faecal microbiota. In the in vivo experiment rats consumed a non-fibre control diet (C) or diets supplemented either with rye bran (R), flaxseed meal (F) alone, or with their combination (R&F) for four weeks. Enterodiol (END), ENL and SCFA concentrations were measured from in vitro faecal fermentation samples and from the intestinal contents of rats. Plasma ENL concentrations from rats were also measured. RESULTS: The highest ENL production was found in vitro with the F supplement (areas under curve: 740 +/- 4, 7,500 +/- 400, 2,600 +/- 500 and 1,520 +/- 70 nmol x h for the R, F, R&F supplements and faecal control, respectively). In vivo, the concentration of ENL in caecal digesta from flaxseed meal was significantly (P < 0.05) enhanced by the presence of rye bran (medians 261, 407 and 24 nmol/g in the F, R&F and C groups, respectively). No correlation was found between the models regarding ENL production, possibly due to different responses to the presence of rye bran matrix, differences in microbiota or application of a batch in the in vitro fermentation model. Rye bran supplementation enhanced butyrate production both in vitro and in vivo. CONCLUSION: In vitro fermentation and the in vivo rat models responded differently to the presence of rye bran and no correlation with regard to the ENL formation from flaxseed lignans was observed.

4-Butyrolactone↗
PubMed1998

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.).

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the invitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277-1289; N. Sinha et al., 1993 Genes Dev. 7: 787-797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro.

Antibodies↗
PubMed2000

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.

Animals↗
PubMed1984

Covalent binding of benzene and its metabolites to DNA in rabbit bone marrow mitochondria in vitro.

Rabbit bone marrow mitochondria, stripped of their outer membrane (mitoplasts), have been shown to carry out the NADPH-dependent bioactivation of radiolabelled benzene in vitro to metabolites capable of covalently binding to mtDNA, thereby inhibiting transcription. The metabolites of benzene produced in bone marrow cells by the microsomal cytochrome P-450 are thought to be phenol, catechol, hydroquinone and p-benzoquinone (Andrews et al., Life Sci., 25 (1979) 567; Irons et al., Chem.-Biol. Interact., 30 (1980) 241). Incubation of mitoplasts from rabbit bone marrow cells in vitro with varying concentrations of the putative microsomal metabolites showed a concentration-dependent inhibition of RNA synthesis. The 50% inhibitory molar concentration (IC50) for each metabolite was determined to be: 1,2,4- benzenetriol , 6.3 X 10(-7); p-benzo-quinone, 2 X 10(-6); phenol, 2.5 X 10(-5); hydroquinone, 5 X 10(-5); catechol, 2 X 10(-3); benzene, 1.6 X 10(-2). DNA, isolated from rabbit bone marrow cell or rat liver mitoplasts prelabelled in DNA with [3H]dGTP and exposed to [14C]benzene in vitro, was enzymatically hydrolyzed to nucleosides which were chromatographed on a Sephadex LH-20 column to separate free nucleosides from nucleoside-adducts. The elution profiles indicated that rat liver mtDNA contained six guanine nucleoside-adducts and rabbit bone marrow cell mtDNA contained seven guanine nucleoside-adducts. Incubation of bone marrow mitoplasts in vitro in the presence of benzene and the hydroxyl radical scavenger, mannitol, resulted in the inhibition of formation of four of the guanosine-adducts. When [3H]dATP was substituted as the prelabelled precursor nucleotide, the LH-20 column profile indicated that two adenine nucleoside-adducts were also formed from benzene in vitro. Furthermore, a comparison of the Sephadex LH-20 column profiles of purine adducts derived from [14C]benzene- and [3H]dGMP-labelled mtDNA with profiles generated by individually incubating each of the putative unlabelled metabolites with bone marrow mitoplasts in vitro has indicated that p-benzoquinone, phenol, hydroquinone and 1,2,4- benzenetriol form adducts with guanine. One of the two adenosine-adducts may arise from hydroquinone; the compound forming the other adduct is unknown at the present time. Exposure of mitoplasts to catechol in vitro resulted in the formation of a guanine nucleoside-adduct that was present in rat liver mtDNA but absent from the DNA isolated from rabbit bone marrow cell mitoplasts exposed to [14C]benzene in vitro. This suggests that catechol is probably not a major metabolite of benzene formed in bone marrow cell mitochondria.

Animals↗
PubMed1993

In vitro cell aggregation and cell adhesion molecules in Crohn's disease.

BACKGROUND: Lack of a suitable model has hindered efforts to understand inflammation and granuloma formation in Crohn's disease. METHODS: Granulomalike aggregates of circulating mononuclear cells are produced in vitro by cultures of cells with polyacrylamide beads. To identify features of in vitro aggregates, which are similar to tissue granulomas of Crohn's disease, the gross morphology and immunohistological appearance of the aggregates produced with peripheral blood mononuclear cells from patients with Crohn's disease were analyzed, and the size of in vitro aggregates was correlated with clinical activity of the disease. Blocking antibodies were used to evaluate the role of cell-adhesion molecules in the formation of in vitro aggregates. RESULTS: The size of in vitro aggregates correlates very significantly with clinical activity (P < 0.001). In active Crohn's disease, in vitro aggregates show immunohistological features of hypersensitivity type granulomas. Blocking antibodies against leukocyte function associated antigen LFA-2 (CD2), LFA-3 (CD58), and Mac-1 (CD11b/CD18) inhibit in vitro aggregate formation. CONCLUSION: In vitro aggregates model in vivo granulomas in size and organization. Cell adhesion molecules like CD2, CD58, and CD11b/CD18 may be involved in granuloma-formation of Crohn's disease.

Antigens, CD↗
PubMed1992

Maturation and fertilization of porcine oocytes in vitro.

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.

Journal Article↗
PubMed1993

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes.

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.

Journal Article↗
PubMed1995

In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media.

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P < 0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P > 0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.

Journal Article↗
PubMed1986

Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo.

The inhibitory effects of monoclonal antibodies on the fertilization of mouse oocytes were evaluated in vitro and in vivo. Among the 40 sperm-specific monoclonal antibodies that had been examined, nine showed significant inhibition of the fertilization of mouse oocytes in vitro. MS 204 was shown to cause a high incidence of penetration of the zona pellucida of mouse eggs by multiple sperm, when the sperm concentration for insemination exceeds 1 X 10(5)/ml. This antibody prevented further penetration of the vitelline membrane by sperm. On the other hand, sperm penetration to zona was inhibited in the presence of MS 207. However, neither MS 204 nor MS 207 caused significant inhibition of penetration of zona-free mouse or hamster eggs by sperm. MS 204 and MS 207 were also found to inhibit the fertilization of mouse oocytes in vivo and embryo development in vitro, when superovulated mice were injected intraperitoneally with given doses of antibodies prior to the mating. Further in vitro culture of the recovered 2-cell oocytes revealed little or no further embryo development beyond two- to four-cell stages. In the controls, greater than 80% of the retrieved oocytes were fertilized and successively developed to the blastocyst stages in vitro when the ascites fluid from NS-1 cells was administered. Two of the monoclonal antibodies generated against human sperm antigens, HS 11 and HS 63, were shown to cross-react specifically with mouse sperm acrosomal antigens and also inhibited the fertilization of mouse oocytes in vitro and in vivo. The results of this study suggest that some monoclonal antibodies to sperm acrosomal antigens exhibit strongly inhibitory effects on the in vitro and in vivo fertilization of mouse oocytes as well as subsequent development of early embryos. As a comparative control, rabbit antisera against sperm-specific enzymes, lactate dehydrogenase-X and 3-phosphoglycerate kinase-2, showed little or no inhibition on the fertilization of mouse oocytes in vitro or in vivo or the subsequent embryo development.

Animals↗
PubMed2004

Effect of serial transfer of three strains of Paecilomyces fumosoroseus on growth in vitro, virulence, and host specificity.

Serial passage of entomopathogenic Hyphomycetes has been shown to alter virulence and host specificity. We evaluated virulence, host specificity, biomass production, conidiation, conidial germination, and a genetic fingerprint of 3 strains of Paecilomyces fumosoroseus after passage in vitro or in vivo in Diuraphis noxia or Plutella xylostella. Strain 4461 did not change in virulence toward D. noxia or P. xylostella after 30 passages in vitro nor after 15 passages in D. noxia. However, it lost virulence toward D. noxia after 15 passages in P. xylostella and did not regain virulence after 5 passages in D. noxia. Passage in D. noxia did result in a loss in conidiation for strain 4461, and passage in vitro resulted in a reduction in the speed of germination. Strain 4481 was the least variable and did not change in any of our tests. Strain 4491 did not change in virulence after passage in vitro nor after passage in D. noxia. It lost virulence toward D. noxia after passage in P. xylostella but regained virulence after re-passage in D. noxia. Mycelial dry weight and conidiation were both reduced after passage in vitro, but were increased after passage in D. noxia. These two traits did not change after passage in P. xylostella. Germination speed was reduced after in vitro passage of strain 4491. No change in banding pattern was observed for any strain using 14 primers for RAPD-PCR. These results demonstrate the intraspecific variability and phenotypic plasticity of strains of P. fumosoroseus. Stability of virulence after in vitro passage is clearly a desirable trait for a mass-produced biocontrol agent. However, a change in host specificity or productivity in vitro, as we observed for some strains, must be monitored and minimized.

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