Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.     

Evidence for an inhibitory influence of rat placental lactogen on prolactin release in vitro

Incubation of placental tissue from Day 11 pregnant rats for increasing periods of time resulted in proportionately more rat placental lactogen (rPL) release. The amount of placental tissue incubated correlated directly with the amount of rPL released into the medium. When placentas were coincubated with anterior pituitaries from ovariectomized rats, prolactin release was significantly inhibited. When media from incubations which had contained varying numbers of Day 11 placentas for 24 h were added to vials containing anterior pituitaries, prolactin release was inhibited, proportionate to the amount of rPL in the media. Media from incubations of Day 9 placentas, which contained very little rPL, had no effect on prolactin release. When medium containing anterior pituitary tissue was incubated for 24 h, pituitaries removed, and the medium incubated with placental tissue for an additional 24 h, there was no difference in prolactin levels compared to incubation medium not containing placental tissue. Addition of a trypsin inhibitor to the medium containing placental tissue did not augment the amount of prolactin remaining after a 24-h incubation. Thus it would appear that the placenta does not release a substance into the medium that destroys prolactin. This suggests that secretions from the placenta, presumably rPL, can exert a negative feedback on prolactin secretion at the level of the anterior pituitary.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Intraluminal androgen binding protein alters 3H-androgen uptake by rat epididymal tubules in vitro

Previous experiments have shown that androgen binding protein (ABP) and androgens exist in high concentrations in the tissue and the lumen of the rat caput epididymis. The present experiments were performed to determine whether or not intraluminal APB affects tubule net uptake of androgens. Caput epididymal tubules were dissected into 2-cm segments, subjected to microperfusion into the tubule lumen, and incubated for 2.5 h in 35 degrees C minimum essential medium (MEM) containing 2.0 ng tritiated testosterone (3H-T) per ml. 14C-polytheylene glycol [PEG] was included as a contamination marker. In the first series of experiments, caput tubules were perfused with a control, artificial perfusion (MKB) containing no ABP or fresh rat rete testis fluid (RTF), which is known to contain ABP. Tubules incubated while containing RTF took up 138% of the tritiated androgens taken up by control tubules. In the second series of experiments, tubules were perfused with fresh caput epididymal lumen content, MKB alone, MKB containing either 5.0 ng purified rat ABP/microliters or 50 ng ABP/microliters. Tubules incubated while containing perfused MKB took up only 47% of the tritiated androgens taken up by tubules containing perfused native lumen content. Increasing intraluminal ABP concentrations in the MKB medium increased 3H-androgen uptake in a stepwise fashion. Intraluminal ABP at a concentration of 50 ng/microliters was associated with a 71% return of 3H-androgen uptake towards that amount of 3H-androgen taken up by tubules perfused with native lumen content. Intraluminal ABP enhances net androgen uptake by caput epididymal tubules from their surrounding medium in vitro.     

Restriction of patching of bound concanavalin A after incorporation of arachidonic acid into the plasma membrane of virally transformed fibroblasts

Topographical distribution of concanavalin A binding sites (CABS) was studied in two lines of virally transformed fibroblasts as a function of fatty acid composition. Fatty acid composition was manipulated by incubating cells in fatty acid, ATP, CoA, and delipidated fetal calf serum (FCS). VLM cells grown in medium containing 5% FCS have a clustered CABS distribution. Plasma membrane vesicles (PMVs) derived from these cells have an arachidonate content of 1.7%. Elevation of PMV arachidonate to 15.8% results in a marked restriction of CABS patching, while elevation to 6.8% is associated with intermediate restriction of patching. Restriction of patching is associated with increased microviscosity. CABS of Rous sarcoma virus-transformed chicken embryo fibroblasts (RSV-CEF) are also responsive to arachidonate enrichment medium. Whereas untreated cells have a clustered CABS distribution, cells incubated for 24 h in arachidonate enrichment medium have predominantly a dispersed CABS distribution. In both VLM cells and RSV-CEF, ATP, CoA, and delipidated FCS alone have no effect upon CABS mobility. Inhibition of CABS patching is also observed when aspirin is included in the arachidonate enrichment medium but not when the cells are incubated in prostaglandins, thus suggesting that the restriction of CABS mobility is not mediated by prostaglandins. Other fatty acids (palmitate, oleate, nonadecanoate) failed to restrict CABS movement. The inhibition of CABS mobility is independent of cell shape change.     

Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C

Growth of pathogen bacterium. Enterococcus was not affected in tryptic soy broth (TSB) medium containing ammonia-N concentration in the range of 0-5.14 mg l(-1). Giant freshwater prawn Macrobrachium rosenbergii (8-12 g) were challenged with Enterococcus which had been incubated for 24 h in TSB medium containing different concentrations of ammonia-N at 0-5.14 mg l(-1) Cumulative mortality of M. rosenbergii was higher for the bacteria incubated in TSB medium having ammonia-N at 0 and 0.26 mg l(-1), than those incubated in TSB medium having 1.28, 2.57 and 5.14 mg l(-1) ammonia-N after 24 h of challenge. However, cumulative mortality of prawn was significantly higher for the bacteria incubated in TSB medium with no ammonia added after 120 h of challenge. The prawns (8-12 g) were challenged with Enterococcus previously incubated in TSB medium for 24 h, then placed in water having concentrations of ammonia-N at control (0.06 mg l(-1)), 0.55, 1.01, 1.68 and 3.18 mg l(-1). Mortality of prawns increased directly with ammonia-N concentrations after 72 h challenge. The pranws (20-30 g) which had been exposed to control, 0.55, 1.68 and 3.18 mg (-1) ammonia-N for 7 days were examined for the total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity and respiratory burst of haemocytes. Phenoloxidase activity decreased when the prawns were exposed to ammonia-N greater than 0.55 mg l(-1). The respiratory burst increased significantly at 0.55 mg l(-1) but decreased significantly at 1.68 and 3.18mg (-1) ammonia-N. No significant difference in haemocyte count was observed among the prawns at different ammonia-N concentrations. It is suggested that ammonia in water decreases the virulence of Enterococcus, and reduces the immune resistance of M. rosenbergii.     

In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Evaluation of culture systems containing bovine oviduct epithelial cells or granulosa cells to mature and maintain the developmental competence of bovine oocytes in vitro

The effects of estrous cow serum (ECS), bovine oviduct epithelial cells (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium (CM; TCM 199 + 25 mM HEPES + 100 mug gentamicin sulfate/ml) or in CM medium supplemented or conditioned with 20% ECS, BOEC +/- 20% ECS or GC + 20% ECS. Supplemented media were incubated for 2 h before addition of OCCs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS +/- GC for 24 or 48 h before addition of OCCs. The developmental competence of oocytes after TVM was assessed by insemination with glass wool separated, frozen-thawed bovine spermatozoa in microdroplets of modified medium (TALP) containing heparin (5 mug/ml) and BOEC for 18 h. The presumptive zygotes were cultured in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to assess embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (66 to 80%) were high, irrespective of the IVM conditions. Only after the IVM of OCCs in CM medium alone was the percentage of oocytes undergoing IVF significantly lower (66%; P<0.05). The proportion of IVF oocytes developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in supplemented or conditioned CM medium containing ECS and/or somatic cells (18 to 28%) compared with those in CM medium alone (9%). When the CM medium was supplemented or conditioned with GC + 20% ECS, the proportion of fertilized oocytes developing to blastocysts increased significantly (28%; P<0.05). These results indicate that the potential of immature OCCs to be fertilized and to complete embryonic development to the blastocyst stage in vitro is enhanced by maturation in CM medium containing 20% ECS and/or BOEC or GC.     

Critical importance of moisture content of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state fermentation system

Summary A large reduction (about 30%–78%) is observed in the production of alpha-amylase by Bacillus licheniformis M27 in standardized wheat bran medium under solid-state fermentation when the moisture content of the medium is higher than the standardized value (65%). However, a surge in enzyme production in the first 24 h of fermentation is observed in media with 75% and 85% moisture. The role of decreased oxygen transfer in reducing enzyme tires by about 78% in the medium containing 95% moisture is evident, since the enzyme tire can be effectively increased by agitating the medium during fermentation. No such limitation in oxygen transfer is evident in medium containing 65% moisture even where incubated under static conditions or when the flask is capped by aluminum foil. The data indicate the critical importance of the moisture content of the medium in -amylase production by B. licheniformis M27 in solid-state fermentation systems. The results also have several implications of scientific and techno-economic importance and are useful in explaining some of the advantages of a solid-state fermentation system over the submerged fermentation process. Offprint requests to: B. K. Lonsane     

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.     

Effect of increasing selenite concentrations, vitamin E supplementation and different fetal calf serum content on GPx1 activity in primary cultured rabbit hepatocytes

Primary rabbit hepatocytes from 6 week old female New Zealand White rabbits (3.0 x 10(6) viable hepatocytes per treatment) were incubated for 24 h or 48 h with two basic variants of the selenium and vitamin E free DMEM/F12-HAM nutrition medium containing 2.5% or 10% fetal calf serum (FCS). Selenium and vitamin E concentrations of the media were varied by the addition of 0, 10, 50 and 100 ng Se/mL medium as sodium selenite and 100 microg alpha-tocopheryl acetate/mL. Lactic dehydrogenase (LDH) leakage of the hepatocytes was not influenced by the various selenium concentrations of the media, whereas vitamin E addition significantly inhibited LDH release. The activity of cellular glutathione peroxidase (GPx1) was markedly induced by increasing the selenium supplementation of the culture media. Vitamin E supply further enhanced GPx1 induction. In hepatocytes cultivated at the lower serum concentration (2.5% FCS), increasing the selenite concentration of the media raised GPx1 and reduced the intracellular levels of the reduced tripeptide glutathione (GSH). No vectored relation between the selenium concentration of the media and the activity of superoxide dismutase (SOD) could be observed. After both incubation periods (24 h and 48 h) SOD activity was significantly higher in the cytosol of hepatocytes grown in media containing 10% FCS as compared to cells incubated at the 2.5% FCS level. Furthermore, SOD activity was reduced by the addition of vitamin E to the media. In conclusion the results indicate an effective metabolism of rabbit hepatocytes for selenite even in amounts as low as nanograms. A general cytoprotective role for vitamin E can be shown by its ability to decrease LDH leakage and by the reduction of SOD activity.     

Production of intracellular 35S-glutathione by rat and human hepatocytes for the quantification of xenobiotic reactive intermediates

The quantification and identification of xenobiotic reactive intermediates is difficult in the absence of highly radiolabeled drug. We have developed a method for identifying these intermediates by measuring the formation of adducts to intracellularly generated radiolabeled glutathione (GSH). Freshly isolated adherent rat and human hepatocytes were incubated overnight in methionine and cystine-free ('thio-free') medium. They were then exposed to 100 microM methionine and 10 microCi 35S-labeled methionine in otherwise thio-free medium to replete cellular GSH pools with intracellularly generated 35S-labeled GSH. After 3 h, acetaminophen was added as a test compound and the cells were incubated for an additional 24 h. Intracellular GSH and its specific activity were quantified after reaction with monobromobimane followed by HPLC analysis with fluorescence and radiochemical detection. Radiolabeled GSH was detectable at 3 h and maintained high specific activity and physiological concentrations for up to 24 h. Incubation medium from acetaminophen treated and nontreated hepatocytes were analyzed for radiolabeled peaks by HPLC using radiochemical detection. Radiolabeled peaks not present in nontreated hepatocytes were identified as acetaminophen GSH adducts by LC-MS. Formation of acetaminophen 35S-GSH adducts by rat hepatocytes containing endogenously synthesized 35S-GSH was increased with acetaminophen concentrations ranging from 500 to 2 mM.     

An adrenal cell system suitable for long term study of factors affecting steroidogenesis.

Cell suspensions of normal adult rad adrenals have been prepared by trypsinization and incubated in Ham's nutrient mixture F10 containing horse serum, fetal calf serum, and lima bean trypsin inhibitor. In most experiments culture medium was not changed during incubation. In this system the number of cells fell to 50% after 2 days, then slowly declined to 20% after 1 month of incubation. A corticosterone (B) response was seen to as little as 5 muU of ACTH per millilitre, a concentration which is within the range found in normal rat serum. With maximal stimulation (100 mU ACTH/ml) the rate of accumulation was highest during the first 24 h then slowly decreased over the following 9 days. When in separate experiments ACTH was added after various times of incubation up to 3 weeks, there was a B response which continued for as long as 1 week after the ACTH was added; the later the time at which ACTH was added the lesser was the initial B response and the longer the lag period before a substantial response occurred. In cell suspensions in medium containing 5.0 mequiv. of K+ per litre, aldosterone content increased for approximately 24 h, then showed little or no change over the next 9 days. With increased K+ concentration, aldosterone was found in greater amounts and accumulation continued for longer periods, both without and with ACTH. This adrenal cell system appears suitable for long term study of factors affecting steroidogenesis.     

Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay.

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4 degrees C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 x 10(6) sperm/ml) were preincubated for 1.0 h (38 degrees C, 5% CO2 in air) and then co-incubated (2 x 10(5) sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P greater than 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 +/- 5.4%; leopard cat, 38.6 +/- 2.8%) or stored ZP (domestic cat, 32.4 +/- 4.2%; leopard cat, 27.6 +/- 2.3%). Sperm incubated in protein-free medium (TLP-PVA) were less capable (P less than 0.05) of ZP penetration (domestic cat, 14.6 +/- 5.9%; leopard cat, 7.9 +/- 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 +/- 5.9%; leopard cat, 58.4 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus

Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0-20 mg l(-1). White shrimp Litopenaeus vannamei (7-12 g in the intermolt stage) were challenged with V. alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5. 10 and 20 mg l(-1)). There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l(-1)ammonia-N after 120 h of challenge. The shrimps were challenged with V. alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l(-1)(control), 1.10, 5.24, 11.10 and 21.60 mg l(-1). Mortality of shrimp in 5.24, 11.10 and 21.60 mg l(-1)was significantly higher than those in the control solution (0.01 mg l(-1)) after 48-168 h. Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l(-1)ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus. No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations. Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l(-1)ammonia-N and greater after 7 days. The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l(-1)ammonia-N. With shrimps exposed to 11.21 and 21.22 mg l(-1)ammonia-N for 7 days, phagocytic activity and clearance efficiency to V. alginolyticus significantly decreased. It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L. vannamei from the V. alginolyticus infection.     

Cardioprotective effect of chronic hyperglycemia: effect on hypoxia-induced apoptosis and necrosis

It is generally accepted that mild forms of diabetes render the heart resistant to an ischemic insult. Because myocytes incubated chronically in medium containing high concentrations of glucose (25 mM) develop into a diabetes-like phenotype, we tested the hypothesis that high-glucose treatment diminishes hypoxia-induced injury. In support of this hypothesis, we found that cardiomyocytes incubated for 3 days with medium containing 25 mM glucose showed less hypoxia-induced apoptosis and necrosis than cells exposed to medium containing 5 mM glucose (control). Indeed, whereas 27% of control cells became necrotic after 1 h of chemical hypoxia with 10 mM deoxyglucose and 5 mM amobarbital (Amytal), only 11% of the glucose-treated cells became necrotic. Similarly, glucose treatment reduced the extent of apoptosis from 32% to 12%. This beneficial effect of glucose treatment was associated with a 40% reduction in the Ca(2+) content of the hypoxic cell. Glucose treatment also mediated an upregulation of the cardioprotective factor Bcl-2 but did not affect the cellular content of the proapoptotic factors Bax and Bad. Nonetheless, the phosphorylation state of Bad was shifted in favor of its inactive, phosphorylated form after high-glucose treatment. These data suggest that glucose treatment renders the cardiomyocyte resistant to hypoxia-induced apoptosis and necrosis by preventing the accumulation of Ca(2+) during hypoxia, promoting the upregulation of Bcl-2, and enhancing the inactivation of Bad.     

Removal of gaseous n-valeric acid in the air by Rhodococcus sp. B261 immobilized onto ceramic beads

n-Valeric acid, one of the main malodorous pollutants from livestock houses was eliminated with a biofilter prepared with Rhodococcus sp. B261 immobilized onto ceramic beads. The strain was isolated from composted pig faeces and grown in an artificial medium containing volatile fatty acids as a carbon source. The cells were immobilized onto ceramic beads in vacuo. The beads were aseptically incubated at 37 °C, pH 8.0, for 24h for activation of the cells. The beads with immobilized cells (3.36×10⁹ c.f.u./g ceramic beads) and moisture content of 35% (w/w) were packed into a glass column equipped with a water jacket to keep the temperature constant. One hundred-seventy ppm of gaseous n-valeric acid were removed for 11 days at 30h ^-1 (space velocity) and 37 °C.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture.

Bovine oocytes cultured for 12-20 h in TC-199 were incubated for 24 h in fertilization medium, Brackett and Oliphant medium with bovine serum albumin (10 mg ml-1), caffeine (5 mmoll-1) and heparin (10 micrograms ml-1), with or without frozen-thawed spermatozoa. High penetration rates (93-96%) and significantly (P < 0.001) higher maturation rates were obtained in oocytes incubated with (93-100%) than without (62-72%) spermatozoa. However, when oocytes at the germinal vesicle stage were cultured for 44 h fertilization medium, maturation of oocytes to metaphase II was reduced. However, all oocytes that were first cultured for 20 h and further for 24 h with spermatozoa were penetrated and 40% of the penetrated oocytes reached metaphase II. All of the remaining oocytes that did not mature arrested at the stages of condensed germinal vesicle (39%) or prometaphase I (22%). These results indicate that oocytes at metaphase I at and after sperm penetration are stimulated by sperm penetration to complete maturation.