Synthesis and turnover of collagen precursors in rabbit skin

1. The rate of synthesis of [(14)C]hydroxyproline by rabbit skin was studied in vitro and in vivo. 2. The soluble collagen fractions were shown to have a very rapid turnover. The 0.15m-sodium chloride-extractable collagen showed t((1/2)) values of 1.2hr. in vitro and 12hr. in vivo. The 0.5m-sodium chloride-extractable collagen exhibited a t((1/2)) value of 20hr. in vivo. 3. Under the conditions used it was not possible to obtain radioactive insoluble collagen in vitro. 4. A significant amount of soluble collagen is lost before it becomes insoluble. 5. These observations may help to explain why large amounts of peptide-bound hydroxyproline appear in the urine during periods of rapid collagen synthesis.     

Differential development of rabbit embryos following microinsemination with sperm and spermatids

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.     

The source of progesterone in preimplantation rabbit blastocysts.

In vitro and in vivo synthesis of progesterone, sequestration of progesterone from the surrounding medium, and its subsequent conversion to metabolites was investigated in 146 hr post coitus preimplantation rabbit blastocysts. No significant conversion of 3H-pregnenolone to 3H-progesterone was observed throughout the 8 hr incubation. Progesterone content in blastocysts and culture medium did not change during the course of an 8 hr incubation. This suggests that the failure to detect incorporation of label into progesterone was not due to the presence of a large endogenous pool of pregnenolone. Significant uptake (p less than 0.05) of 3H-progesterone from the incubation medium was observed as was significant conversion of the 3H-progesterone to unidentified metabolites. Therefore it would appear that the preimplantation rabbit blastocyst is not capable of de novo synthesis of progesterone from pregnenolone prior to implantation but sequesters progesterone from the surrounding medium and converts it to progesterone metabolites.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.     

Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.     

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.     

Xenogenous fertilization of laboratory and domestic animals in the oviduct of the pseudopregnant rabbit

Xenogenous fertilization was accomplished using bovine, porcine, and hamster follicular oocytes. The xenogenous fertilization rates for bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit were 13.4% and 2.0%, respectively. Temperatures of ovary, during transport to the laboratory, of 0 degrees or 37 degrees C had no effect on xenogenous fertilization rates of bovine oocytes. In vitro culture in 50 mug/ml FSH did not alter the xenogenous fertilization rates of bovine oocytes. Fertilization was observed with oocytes recovered 40 to 75 hr after insemination. Two cell embryos were recovered 70 to 75 hr after insemination. Ligation of the rabbit oviduct, number of ova deposited and sperm concentration did not affect the xenogenous fertilization rates of hamster ova. Cleavage of xenogenously fertilized hamster oocytes occurred between 28 and 29 hours after insemination.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture.

The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.     

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.     

Pulmonary biotransformation of insulin in rat and rabbit.

In vitro biodegradation of insulin in rabbit and rat lung homogenates was investigated. Insulin can be sequentially metabolized into two primary fragments in rabbit lung homogenate by an aminopeptidase. The amino acid sequences of the fragments were found to be the des-Phe-InsulinB1 (Metabolite I) and des-Phe-Val-InsulinB1-2 (Metabolite II). However, only the former metabolite (Metabolite I) was identified in the rat lung homogenate. The km and Vm values associated with rabbit lung homogenate were 0.29 +/- 0.14 mM and 16.4 +/- 6.9 microM/hr/mg protein, respectively, whereas those for a rabbit lung preparation containing both microsomes and cytosol were 0.22 +/- 0.07 mM and 17.9 +/- 5.4 microM/hr/mg protein, respectively. The km and Vm associated with the cytosolic fraction of rabbit lung were 0.32 +/- 0.16 and 20.6 +/- 6.1 microM/hr/mg protein, respectively. The results indicate that the lung aminopeptidase may be a cytosolic enzyme. The degradation of dimeric insulin in the lung homogenate was faster than that of hexameric insulin due to the difference in collision frequency between the enzyme and insulin aggregates. The major metabolites in the lungs reportedly retain almost the same bioactivity of insulin, suggesting that the pulmonary route of insulin delivery will not adversely affect its hypoglycemic activity.     

Studies on Ca2+-channel distribution in maturation arrested mouse oocyte

The present study was carried out to identify the existence of voltage-dependent Ca2+-channels (P/Q-, N-, and L-type) and their distributional differences in germinal vesicle (GV) and GV breakdown (GVBD)-arrested mouse oocytes which includes GVBD to telophase I of meiosis I and matured oocytes (MII, metaphase of meiosis II) by using the immunocytochemical method and a confocal laser scanning microscope. (1) Comparison between follicular oocytes (GV) and GV-arrested oocytes after 17 hr of in vitro culture. In follicular oocytes, P/Q-, N-, L (anti-alpha1C anti-alpha1D)-type Ca2+-channels showed both localized and uniform staining. In contrast, GV-arrested oocytes, after in vitro culture for 17 hr, showed no presence of Ca2+-channels in most oocytes. (2) Comparison between GVBD oocytes after culture in vitro for 3 hr and GVBD-arrested oocytes after culture in vitro for 17 hr. In GVBD oocytes, P/Q-, N-, L (anti-1C, anti-alpha1D)-type Ca2+-channels showed both localized and uniform staining. In contrast, in GVBD-arrested oocytes, none of the three types of Ca2+-channels were identified in 72-86% of oocytes. The present study demonstrates that in most GVBD-arrested oocytes that do not mature to MII, there is no Ca2+-channel identified. Therefore, most of the GVBD-arrested oocytes seem to have defects in Ca2+-channel expression/translation. Also, distributional changes of Ca2+-channels take place depending on the maturation progress in GV oocytes and MII stage oocytes (ovulated and 17 hr cultured MII stage oocytes). In addition, we found evidence that a functional voltage-dependent Ca2+-channel (L-type) exists in mouse oocytes (ovulated and cultured MII staged oocytes by a confocal laser scanning microscope).     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

Maintenance of plasma-derived proteins at much lower concentrations in the uterine lumen of the rabbit: a kinetic study of passage.

Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.     

A study of the heat-shock response in Neurospora crassa

1. Neurospora crassa was grown at 28 degrees C for 12 hr and transferred to higher temperatures for 2 hr. 2. Cultures labelled with [35S]methionine showed the synthesis of several new proteins in response to heat-shock at 46 to 48 degrees C. 3. Major polypeptides of approximate Mr 105,000, 99,000, 78,000, 43,000 and 23,000 were detectable in one-dimensional SDS-polyacrylamide slab gel electropherograms. 4. 2-D analysis using isoelectric-focussing in the first dimension and electrophoresis in SDS-polyacrylamide gels in the second led to the resolution of some of the heat-induced polypeptide into multiple spots differing in pI values. 5. mRNA from heat-shocked cells was translated poorly in Wheat Germ extract and rabbit reticulocyte lysate in vitro translation systems.     

Culture of in vitro fertilized rabbit ova

Embryonic development of in-vitro fertilized rabbit ova was assessed following in-vitro culture in four different serum supplemented media. A mixture of Basal Medium Eagle (BME) and Ham's F10 medium (1:1) provided better support for in-vitro development than Ham's F10, BME, or regular acidic saline (RAS). In-vitro embryonic development in the BME/Ham's F10 mixture was synchronous with in-vivo development through at least 55 hr of culture. After 54 hr of culture, embryos transferred to the oviduct of a synchronous pseudopregnant recipient were able to implant at the same rate as simultaneously transferred embryos grown in vivo. BME/Ham's F10 supplemented with 10% newborn calf serum was highly supportive of rabbit embryo development following in-vitro fertilization.