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PubMed2000

Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients.

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O(2), 5% CO(2), and 90% N(2). In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 microM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 microM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 microM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm(2) vs. 0.71 mm(2) surface area; n = 27, P < 0.04).

Animals↗

PubMed2001

Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Adenosine Triphosphate↗

PubMed1992

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.

Adenoviridae↗

PubMed1996

In vitro fertilization of in vitro matured oocytes obtained from the follicles without hCG exposure for prevention of severe ovarian hyperstimulation syndrome: a case report.

We aspirated ovarian follicles of an infertile patient who encountered a great risk of ovarian hyperstimulation (OHSS) during her ovulation-induction cycle, which had been stimulated with gonadotropins, in order to avoid OHSS without interrupting the treatment. If this action were to yield a considerable number of immature oocytes, some of which were to achieve in vitro maturation and subsequent fertilization, this method could be an alternative to conventional ovulation-induction methods for patients who repeatedly present imminent signs of hyperstimulation. However, the number of oocytes collected from the patient was disappointingly low, even though a large number of follicles were thoroughly aspirated. One of these oocytes was matured in vitro in a medium containing 30% follicular fluid and granulosa cells of mature follicles, fertilized, cryopreserved, and transferred in a later artificial cycle; but pregnancy was not achieved. A low number of recovered oocytes from patients in this situation might be a factor that greatly limits the clinical applications of this method.

Adult↗

PubMed1994

The effect of exposure to chlorhexidine gluconate in vitro and in vivo on in vitro adhesion of Candida albicans to buccal epithelial cells from diabetic and non-diabetic subjects.

The effect of 0.2% chlorhexidine gluconate on in vitro adhesion of Candida albicans to buccal epithelial cells (BEC) was studied in 12 healthy subjects and 12 patients with diabetes mellitus. Exposure of BEC for one minute with 0.2% chlorhexidine gluconate in vitro, or by rinsing the mouth in vivo, resulted in a significant reduction in candidal adhesion to BEC in both diabetic and non-diabetic subjects and between both groups. In addition to the known fungicidal effect of chlorhexidine, it also reduces Candida albicans adhesion to oral mucosal cells, a factor of importance in the establishment of candidal infection.

Adult↗

PubMed1985

In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product.

Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.

Animals↗

PubMed1988

Ultraviolet microbeam irradiation of microtubules in vitro. The action spectrum for local depolymerization of marginal band microtubules in vitro matches that for reducing birefringence of chromosomal spindle fibres in vivo.

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which microtubules are locally depolymerized.

Animals↗

PubMed1996

Effects of cryopreservation of DNA synthesis in the inner cell mass of in vitro matured/in vitro fertilized bovine embryos frozen in various cryoprotectants.

The rate of DNA synthesis in the inner cell mass (ICM) of frozen-thawed bovine embryos was examined. Bovine blastocysts derived from in vitro matured/in vitro fertilized oocytes were frozen with 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.4 M glycerol plus 0.25 M sucrose (GL). Viable embryos, after thawing and culture beyond the blastocysts stage, were examined by immunocytochemical staining for detection of DNA synthesis by ICM cells. The numbers of bromodeoxyuridine-immunoreactive ICM cells of frozen-thawed embryos prepared with EG (10.7) and GL (11.5) were significantly lower than those of unfrozen embryos (17.3). The results suggest that the rates of proliferation of ICM cells of frozen-thawed bovine embryos tend to be lower than those of unfrozen embryos irrespective of the cryoprotectant used.

Analysis of Variance↗

PubMed1998

Effect of oxygen concentration in the gas atmosphere during in vitro insemination of bovine oocytes on the subsequent embryonic development in vitro.

In vitro matured bovine oocytes were co-incubated with sperm for 18 hr in a droplet of fertilization medium under a gas atmosphere of 5% CO2 with 5 or 20% O2. After removing the cumulus cells, they were fixed to examine their fertilization rate, or cultured for another 154 hr in a chemically defined medium under 5% O2 to determine their development to the blastocyst stage. There was no difference between the 5 and 20% O2 groups in the fertilization rate. However, the percentage of inseminated oocytes which developed to the blastocyst stage was higher when in vitro insemination was conducted under 5% O2 compared with that under 20% O2 (34.4 vs. 24.7%, P < 0.05).

Animals↗

PubMed2004

Tn5 as a molecular genetics tool: In vitro transposition and the coupling of in vitro technologies with in vivo transposition.

The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory. In this chapter we describe some of these tools with a focus on the Tn5 transposition system. Tn5 technologies are particularly useful because the Tn5 transposition system has simple requirements, is efficient, random in target recognition, and robust. In particular we will describe the use of in vitro Tn5 transposition in transposon tagging and in the generation of nested deletions. We will also describe a unique in vitro/in vivo technology in which Tn5 inserts can be generated in a wide spectrum of bacterial species through the electroporation of preformed tranposase-transposon DNA complexes.

Base Sequence↗

PubMed1992

Effects of pig follicular fluid on maturation of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro.

This study examines the effects of pig follicular fluid on the maturation of pig oocytes and on their subsequent fertilizing and developmental capacity in vitro. The addition of pig follicular fluid or its fractions obtained by ultrafiltration, gel filtration and ion-exchange chromatography to maturation medium significantly increased the rates of nuclear maturation, normal fertilization and normal cleavage of pig oocytes after fertilization in vitro: the rates of normal fertilization and cleavage were 2-4 times higher than those in the control medium. The efficacy of pig follicular fluid was lost after heating at 56 degrees C for 30 min, whereas no significant decrease in activity was observed after defatting. In addition, the effective component(s) was partially purified by ultrafiltration, gel filtration and ion-exchange chromatography: the activity was observed in the fraction (UF2; M(r) 10,000-20,000) obtained by ultrafiltration. Activity was found in the first fraction (G1) obtained by gel filtration of UF2. Among three fractions obtained by ion-exchange chromatography of G1, only the third fraction had the activity. The results indicate that pig follicular fluid contains an acidic substance(s) (M(r) 10,000-200,000) that promotes oocyte maturation.

Animals↗

PubMed1997

Support for the development of bovine embryos in vitro by secretions of bovine trophoblastic vesicles derived in vitro.

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.

Animals↗

PubMed1993

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

Animals↗

PubMed1976

Studies on the binding of 5,5-diphenyl-hydantoin to nucleic acids in vitro and to rat brain subcellular fractions in vitro and in vivo.

The binding of 5,5-diphenylhydantoin (DPH) to nucleic acids (bovine brain RNA, rat liver ribosomal and tRNA, Torula utilis RNA, and calf thymus and Escherichia coli DNA was studied using ultraviolet spectroscopy, gel chromatography and thermal transition profiles. Within the sensitivity of these methods, it was found that there is essentially little or no interaction between DPH and nucleic acids in vitro as has been reported previously. Little, if any evidence of DPH intercalation with DNA was noted during thermal transition studies. DPH does not interfere with DNA reassociation. Further studies into the nature of the in vivo subcellular distribution of 14C-DPH in rat brain revealed accumulation of the radioactivity primarily in the soluble fractions. The nuclear fraction and the microsomes, containing high DNA and RNA tissue ratios, demonstrated the greatest particulate association with radioactivity at 2 and 12 hours, respectively. This association with particulate fractions was not demonstrated after gel chromatography. These data do not support a hypothesis relating DPH binding to nucleic acids in vitro or in vivo to a possible mechanism of action of the drug.

Animals↗

PubMed1976

In vitro generation of suppressor cell activity: suppression of in vitro induction if cell-mediated cytotoxicity.

It was observed that when normal mouse spleen cells were cultured alone in vitro (precultured) for 3 to 7 days, these cells lost the ability to generate cell-mediated cytotoxicity (CML) during subsequent in vitro sensitization with allogeneic spleen cells, trinitrophenyl (TNP)-modified syngeneic spleen cells, or syngeneic tumor cells. These precultured cells, which were themselves unable to generate CML, were also shown in mixing experiments to suppress, actively, the generation of CML by freshly explanted spleen cells. Suppression occurred at the sensitization phase of CML, and not at the effector level; supernatants from suppressive precultured cells were not suppressive. Suppression was totally abrogated by the treatment of spleen cells with a T cell-specific rabbit anti-mouse brain serum and complement (RalphaMB+C) either before or after preculturing, suggesting that a T cell eas essential both to the generation of suppressor activity and to its expression. Suppressor activity was entirely absent in precultured nylon wool column-nonadherent spleen cells, a T cell-enriched population containing most of the RalphaMB+C-sensitive cells in the spleen. Precultured nylon column-adherent cells (T cell-depleted) did have suppressive activity, and a mixture of nylon-adherent and nylon-non-adherent cells was a suppressive after preculture as the precultured unseparated spleen. Moreover, the ability of nylon-adherent spleen cells to generate suppressive activity during preculturing was abrogated by treatment with RalphaMB+C. Thus, the "spontaneous" generation of CML-suppressive activity was dependent upon a limited subpopulation of splenic T cells isolated in the nylon column-adherent fraction. The relationship of these data to a previously described synergy between subpopulations of normal spleen in the generation of CML is discussed, and the findings related to other suppressor systems described in the literature.

Animals↗

PubMed1992

[In vivo and in vitro antitumoral effects of an extract from Vibrio cholerae in different murine models. II. Antitumoral effect in vitro of an extract from Vibrio cholerae: inhibition of 3LL cell growth and stimulation of murine splenocytes].

A bacterial extract from Vibrio cholerae, the DGZ, has been separated into two fractions by gel filtration. The effects of these two fractions and that of the crude extract are investigated and compared by two different in vitro tests. It appears that these extracts exert direct inhibition over the growth of 3LL cultured cells and that they trigger splenocytes in vitro proliferation.

Animals↗

PubMed1990

Tolerance to endotoxin in vitro: independent regulation of interleukin-1, tumor necrosis factor and interferon alpha production during in vitro differentiation of human monocytes.

Bacterial lipopolysaccharide (LPS)-induced production of three known endogenous pyrogens, interferon alpha (IFN-alpha), interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) was studied during in vitro differentiation of human peripheral blood monocytes. In freshly seeded cells, secretion of IL-1 and TNF-alpha, but not IFN-alpha were readily induced by LPS. After 24 hr and for up to 14 days of culture, monocytes became irreversibly tolerant to LPS for the release of IL-1. IFN-alpha secretion was not induced by LPS in cells cultured for up to 8 days. Monocytes also became tolerant to LPS for TNF-alpha production 48 hr after an initial stimulation with LPS. This tolerant state, however, was transient, lasting from 6 to 8 days, after which competence for TNF secretion resumed. These observations demonstrate that regulation of production of IL-1, TNF-alpha and IFN-alpha by human mononuclear phagocytes is mutually independent, related to the stage of cell differentiation and modulated by cell stimulation. Since in vitro tolerance to LPS mimics the in vivo tolerance to LPS with respect to fever, we speculate that they are closely related.

Biological Factors↗

PubMed1989

Enhanced viability after in vitro fertilization of bovine oocytes matured in vitro with high concentrations of luteinizing hormone.

The purpose of this study was to better understand requirements for oocyte maturation to yield viable embryos after bovine in vitro fertilization (IVF). High proportions (95% to 100%) of cumulus-surrounded oocytes matured in vitro in all treatments, but subsequent development was enhanced after maturation with high concentrations of purified bovine luteinizing hormone (LH). Beneficial effects of undisturbed cumulus cells were demonstrated. Improved IVF followed insemination of cumulus-surrounded oocytes, but not denuded oocytes, after maturation with high LH (100 micrograms/ml) versus low LH (10 micrograms/ml), implicating cumulus cells in mediating hormonal enhancement. Oocytes matured with high LH resulted in embryos of superior viability, as reflected by cleavage to 4- to 8-cell stages. Pregnancy resulting from transcervical embryo transfer further documented embryonic viability. These findings should be useful in further development and implementation of reproductive and genetic technologies.

Animals↗