Isolation of rat gonocytes by velocity sedimentation at unit gravity

A method for obtaining enriched populations of gonocytes from rat embryos at 18 days p.c. has been developed. Single cell suspensions with high cell yield and good viability of the cells were obtained by a collagenase/trypsin digestion of the testes. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600,000 gonocytes (70-75% purity), sedimenting at about 12 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing. Purities were determined by Nomarski microscopy and verified in fixed preparations and by Coulter volume measurements.     

Development of a new method for isolating zebrafish oocytes (Danio rerio) from ovary tissue masses

In order to study cryopreservation of zebrafish (Danio rerio) oocytes, large numbers of oocytes need to be isolated from the ovaries for experimental use. Zebrafish oocytes have been previously separated from ovaries mechanically and the method is laborious and time consuming. The aim of the present study was to develop a simple and rapid method for obtaining large number of morphologically and functionally intact zebrafish oocytes at different stages of development. Zebrafish ovaries were treated with three enzymes-trypsin, collagenase or hyaluronidase at different concentrations (0.2-1.6 mg/ml) for different time periods (5-20 min). Membrane integrity and development competence of the oocytes isolated mechanically and enzymatically from zebrafish ovary were assessed. The results showed that when optimal concentration and treatment time combinations were used, high separation and viability rates of oocytes could be obtained with all the three enzymes. The best conditions were confirmed as 1.6 mg/ml hyaluronidase treatment for 10 min or 0.4 mg/ml collagenase treatment for 10 min at 22 degrees C. Oocytes survivals after optimal hyaluronidase enzymatic separation were 96.6+/-0.7%, 92.9+/-1.3% and 94.6+/-0.9% for stages I, II and III oocytes, respectively using trypan blue staining. Successful enzymatic separation of zebrafish oocytes is reported here for the first time. The method developed in this study will undoubtedly assist investigations on cryopreservation of zebrafish oocytes and studies on zebrafish oocytes in general.     

Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons ofAplysia californica

1. The influences of enzyme treatments (trypsin and collagenase) on responses to perfused acetylcholine were examined on physically isolated single Aplysia neurons, using the voltage-clamp, internal perfusion, and rapid external perfusion technique. 2. During treatment with trypsin (0.025 to 0.1%) for 10 to 30 min at room temperature (22 to 25 degrees C), the peak amplitude of the Na current induced by acetylcholine increased in a time- and dose-dependent manner, and the decay in the continued presence of acetylcholine was slowed. This effect of trypsin treatment was irreversible after washing for 60 min without enzyme. 3. Edrophonium, a cholinesterase inhibitor, has previously been shown to augment the Na acetylcholine response in this preparation by inhibition of acetylcholinesterase. After treatment of the neuron with trypsin, the augmentation after edrophonium was abolished. Furthermore, in the presence of edrophonium, trypsin also failed to increase the response. The dose-response curve for acetylcholine after treatment of trypsin was similar to that in the presence of edrophonium. These results suggest that the modification of the current response by trypsin is a result of removal of cholinesterase activity from the membrane. 4. In contrast to the effects of trypsin, collagenase (0.03 to 0.1%) for 10 to 60 min did not change the current amplitude of the acetylcholine response. However, collagenase treatment did alter the kinetics of the acetylcholine response in a dose-dependent manner, in that the rate of decay was accelerated. A similar acceleration was seen in the acetylcholine responses on other neurons which were due to Cl or K currents, suggesting that the effect was independent on the type of channel. This effect of collagenase was reversible after 30 to 60 min of washing of the neuron. 5. In the presence of edrophonium or after the treatment with trypsin, collagenase still accelerated the current kinetics of the acetylcholine response, indicating that cholinesterase activity is not related to this effect. Furthermore, heated collagenase (presumably inactivated) had a similar action, suggesting that the enzymatic activity of collagenase is not related to the modification of the response. 6. These results suggest that Aplysia acetylcholinesterase is sensitive to trypsin but not to collagenase. However, the preparation of a collagenase used in these studies contains some factor which alters the response to acetylcholine, but this effect is reversible and unrelated to enzymatic activity.     

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.

A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.     

Bovine herpesvirus-1 associated with single, trypsin-treated embryos was not infective for uterine tubal cells

It has been reported that bovine herpesvirus-1 (BHV-1) remains associated with in vitro-produced (IVP) bovine embryos after exposure to the virus and either washing or trypsin treatment. However, it is not known if the quantity of virus associated with an exposed IVP embryo is likely to infect a recipient cow after transfer. The specific objective of this study was to determine if IVP embryos that were exposed to BHV-1 would infect uterine tubal cells (UTC) in a co-culture system. In vitro-produced Day 7 embryos were exposed to BHV-1 and then washed or trypsin treated according to the IETS guidelines. These embryos were then co-cultured individually or in groups with UTC in microdrops of tissue culture medium 199 (TCM 199) supplemented with 10% equine serum. Following co-culture for 48 h, virus isolation was attempted on the embryos and the UTC from each drop. Virus was detected in washed individual embryos, groups of washed embryos, groups of trypsin-treated embryos and the UTC co-cultured with each of these treatments. However, BHV-1 was not detected in the individual, trypsin-treated embryos or the UTC co-cultured with them. It is concluded that trypsin treatment might effectively prevent infection of recipients if individual, Day 7, exposed embryos were transferred into the uterus.     

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Evaluation of virus decontamination techniques for porcine embryos produced in vitro

The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

The influence of bud length,age of the tree and culture media on androgenesis induction inAesculus carnea Hayne anther culture

Statistical analyses of the data revealed very significant differences in androgenesis induction ofA. carnea Hayne anther culture depending on the bud length, nutrient medium composition and age of the parental tree. Significant mutual influence of all these factors was also observed. The highest number of androgenic anthers was obtained when 4 mm long buds were used. Older trees (60 and 100 yrs) gave a higher number of androgenic anthers than the younger ones (20 and 40 yrs). MS medium supplemented with 2,4-d and Kin (1 mg l^–1, each) was the most favourable for androgenesis induction. Pollen embryos (haploids and aneuploids) were formed by the division of uninuclear microspores.The highest percentage of germinated embryos and further synchronous development of the shoot and root was achieved in MS medium supplemented with IAA, GA₃ (1 mg l^–1) and activated charcoal (1%). When other germination media were used, malformations of androgenic embryos were observed.Abbreviations AC activated charcoal - H casein hydrolysate - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - GA₃ gibberelic acid - Kin 6-furfurylaminopurine - MS Murashige and Skoog - T thidiazurone - N phenyl-N'-1,2,3-thiadiazol-5-ylurea - Z zeatin-6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine     

Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.     

The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei)

Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.     

Effect of hypoxia on the immune response of giant freshwater prawn Macrobrachium rosenbergii and its susceptibility to pathogen Enterococcus

Giant freshwater prawns Macrobrachium rosenbergii (14-19 g) were challenged with Enterococcus (3 x 10(5) cfu prawn(-1)) previously incubated in TSB medium for 24 h, then placed in water having concentrations of dissolved oxygen (DO) at 7.75, 4.75, 2.75 and 1.75 mg l(-1). Onset of mortality occurred after 6 h exposure to 1.75 mg l(-1) DO, and after 12 h exposure to 2.75 mg 1(-1) DO. Cumulative mortality of prawns at 1.75 mg l(-1) DO was significantly higher than that at 4.75 and 2.75 mg l(-1) DO, and cumulative mortality of prawns at 4.75 and 2.75 mg l(-1) DO was significantly higher than that at 7.75 mg l(-1) DO after 96 h. The prawns (20-30 g) which had been placed in water for 0 to 120 h at 7.75, 4.75, 2.75 and 1.75 mg l(-1) DO were examined for the THC (total haemocyte counts), DHC (differential haemocyte counts), phenoloxidase activity, respiratory burst, percentage phagocytosis and clearance efficiency. No significant difference in semi-granular cells and granular cells of prawns was observed among four treatments. The prawns following 120 h exposure to 2.75 mg l(-1) DO decreased significantly the hyaline cells and THC by 39% and 36%, respectively. Phenoloxidase activity and respiratory burst decreased significantly by 33% and 11% when the prawns were exposed to 2.75 mg l(-1) DO after 24 h, respectively. Percentage phagocytosis and clearance efficiency to Enterococcus decreased significantly by 44% and 54% for the prawns following 12 h exposure to 2.75 mg l(-1) DO, respectively. It is concluded that DO as low as 2.75 mg l(-1) and 4.75 mg l(-1) causes depression in immune system of M. rosenbergii, and increases its susceptibility to Enterococcus infection.     

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies

The aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 10(6.16) TCID(50)/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR-FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles. It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.     

Comparative effects of trypsin, collagenase and mechanical harvesting on cell membrane lipids studied in monolayer-cultured endothelial cells and a green monkey kidney cell line

Experiments are presented in which membrane lipids of endothelial cells in monolayer culture were labelled with [14C]linoleic acid. Approx. 90% of the radioactive label were incorporated into phospholipids. A comparison of various harvesting methods showed that during the disruption of the labelled endothelial cell monolayer, 0.25% trypsin and 0.125% trypsin (+0.01% EDTA) released 650 and 470% more radioactivity, respectively, than did 0.01% collagenase (+0.01% EDTA). Parallel studies were performed on a green monkey kidney cell line. In this case, 0.25% trypsin released 520% more radioactivity than did 0.1% collagenase (+0.01% EDTA), although 0.125% trypsin in the presence of EDTA (0.01%) was much less traumatic than trypsin alone, the released radioactivity being of the same order of magnitude as that for collagenase. Morphological studies on endothelial cell cultures failed to reveal any distinctive differences in surface morphology following the various enzyme treatments. The results suggest that collagenase treatment of endothelial cell monolayers is the least traumatic harvesting or subculturing method as far as the integrity of the lipids in the cell membrane is concerned.     

A simple non-enzymatic method for the isolation of high yields of functional rat hepatocytes

A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra-acetic acid (EDTA)-containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91 +/- 4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37 degrees C (by pre-incubation) or at 4 degrees C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.