Development of in vitro matured,in vitro fertilized domestic cat embryos following cryopreservation,culture and transfer

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.     

In vivo veritas, in vitro artificia

Recently, the understanding of dynamic cellular changes that occur in vivo has advanced significantly, both at the extracellular and intracellular levels. These changes might fluctuate with daily, circadian, weekly, or monthly intervals, and the approaches used to understand these changing conditions in vitro should parallel in vivo studies. In addition, the in vitro milieu should be optimized and better defined, so that artifacts due to in vitro culture systems would not pose dangers for the proper interpretation of results. In this article, we discuss some of these issues and propose solutions.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Liver mitochondrial aldehyde dehydrogenase: in vitro expression, in vitro import, and effect of alcohols on import

An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.     

SNARE-driven, 25-millisecond vesicle fusion in vitro

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k(dock) and the unimolecular fusion rate k(fus). On low t-SNARE-density bilayers at 37 degrees C, docking is efficient: k(dock) = 2.2 x 10(7) M(-1) s(-1), approximately 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D(lipid) = 0.6 microm2 s(-1)); approximately 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k(fus) = 40 +/- 15 s(-1) (tau(fus) = 25 ms). This is 10(3)-10(4) times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains approximately 40 times slower than the fastest, submillisecond presynaptic vesicle population response.     

Automated,in vitro harvest of somatic embryos

This work has demonstrated the aseptic, automated harvest of somatic embryos from a bioreactor suspension culture. Machine vision, emulating the selection criteria of an experienced biologist, classified embryos as harvestable or non-harvestable as they flowed through a 3 mm glass conduit. Embryos classified as harvestable were separated in a sealed harvest chamber. The system harvested 60% of the embryos at a rate of 2.4 embryos/h and incorrectly harvested less than 1% of the non-harvest objects. The low harvest rate precludes the applicability of this technique to research and commercial tissue culture laboratories. The suspension feed-rate, culture population density and culture homogeneity were identified as the most important factors influencing embryo harvest rate. The performance of this technique on more densely populated cultures was projected using anticipated improvements in suspension feedrate. It was concluded that, under the conditions of this analysis, the harvester would be of limited value in a commercial propagation environment but could be beneficial to many research labs working with plant somatic embryos.     

Proteolipid and human aorta calcification, in vitro

The objective of this study was to determine whether in vitro calcification of human aorta is proteolipid dependent. Homogenates were prepared from tissue with no gross pathologic manifestations. Samples were examined for calcifiability in a metastable calcium phosphate solution before and after lipid extraction. Fractions of the extracted lipid were similarly examined. The tissue calcified before but not after lipid extraction. Calcifiability was restricted to the proteolipid portion of the lipid extract. Under the conditions employed, therefore, proteolipid is required for calcification of human aorta, in vitro.     

Barriers,nephrotoxicology and chronic testing in vitro

In many organs of the human body, there are effective physiological barriers which contribute to regulation of the uptake, transport and secretion of endogenous and exogenous materials. ECVAM is involved in the development of several in vitro models for detecting damage to various barriers, for example, the renal epithelium, the intestinal barrier, and the blood-brain barrier, after acute and chronic exposure to chemicals and products of various kinds. Long-term toxicity testing is an important issue in toxicology. At present, there are no generally accepted in vitro models available for replacing chronic testing in animals. Under chronic exposure conditions, the cellular response is larger than that which can be predicted in the standard cytotoxicity models. Therefore, the approach to predicting chronic toxicity will need to involve more-complex in vitro models. Several in vitro long-term toxicity systems currently available are under evaluation.     

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.     

Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos

To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages.     

ACTH,cyclic nucleotides,and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated³²P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH_1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10^–10 to 10^–4 M. In a number of protein bands, a biphasic effect of ACTH_1–24 was observed: in concentrations of 10^–4–10^–5 M, a reduced amount of³²P was found; in concentrations of 10^–6–10^–7 M, hardly any effect could be detected, whereas consistently at concentrations around 10^–8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH_1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.     

Retinoblastoma, chromosome 13, and in vitro cellular radiosensitivity.

Five diploid fibroblast strains from patients with deletions mapping in the long arm of chromosome 13 and three strains bearing partial trisomies of chromosome 13 were assayed for clonogenic survival following X-irradiation. Results of these experiments suggest the existence of a region on 13q14 which is related to increased sensitivity to cell killing in vitro by X-rays. This locus seems to be distinct from but close to the retinoblastoma locus, which has been associated with the same band.     

The first dromedary (Camelus dromedarius) offspring obtained from in vitro matured, in vitro fertilized and in vitro cultured abattoir-derived oocytes

Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 degrees C; 5% CO2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 microg/mL FSH, 1 microg/mL E2 and 500 microM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 x 10(6) spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 degrees C, 5% CO2 and 90% N2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts (n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves (n = 4) were born later on. The remaining pregnant females (n = 8) are due to calf within the next months. In conclusion, this is the first reported offspring in camelids obtained by transfer of embryos produced by IVM, IVF and IVC using abattoir-derived oocytes, fresh semen and culture in a semi-defined medium.     

DNA stress and strain, in silico, in vitro and in vivo

A vast literature has explored the genetic interactions among the cellular components regulating gene expression in many organisms. Early on, in the absence of any biochemical definition, regulatory modules were conceived using the strict formalism of genetics to designate the modifiers of phenotype as either cis- or trans-acting depending on whether the relevant genes were embedded in the same or separate DNA molecules. This formalism distilled gene regulation down to its essence in much the same way that consideration of an ideal gas reveals essential thermodynamic and kinetic principles. Yet just as the anomalous behavior of materials may thwart an engineer who ignores their non-ideal properties, schemes to control and manipulate the genetic and epigenetic programs of cells may falter without a fuller and more quantitative elucidation of the physical and chemical characteristics of DNA and chromatin in vivo.