Bicarbonate Medium Research Articles

Reduction of embryotoxicity by protein in embryo culture media

Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentration, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.

Effect of cell-free synchronous uterine flushings and microsurgery on the development of porcine embryos in vitro

Experiment I was designed to determine if cell-free synchronous uterine flushings contain an embryotoxic substance that is normally screened by the intact zona pellucida. Sixty 4-cell embryos were allocated to three treatment groups: 1) control embryos (n = 20) were cultured in Modified Kreb's Ringer Bicarbonate medium + 10% bovine calf serum (mKRB-BCS), 2) UF embryos (n = 20) were cultured in 80% mKRB-BCS + 20% sterile dialyzed uterine flushings (UF), 3) MicroUF embryos (n = 20) received a microsurgical incision in the zona pellucida and were cultured in 80% mKRB-BCS + 20% UF. Following 72 h in culture at 37 degrees C under a 90% N2, 5% CO2, and 5% O2 atmosphere, the number of nuclei/embryo and the incidence of protrusion of the trophoblast through the zona pellucida (PTZ) were recorded. Addition of UF had no effect on embryo development. A greater (P less than .005) proportion of MicroUF embryos exhibited PTZ as compared to UF and control embryos. Experiment II was devised to further characterize the occurrence of PTZ in Micro porcine embryos. Thirty-three 4- to 10-cell embryos and 14 morulae were distributed across two treatments: 1) control embryos (n = 16 and 6, respectively) were cultured as described in Experiment I; and 2) micro embryos were treated similarly to MicroUF embryos in Experiment I but were cultured in mKRB-BCS only. At the onset of PTZ, embryos were immediately fixed and examined. The proportion of embryos exhibiting PTZ was greater (P less than .007) for Micro versus control embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of glutamine synthetase in rat corpus striatum in vitro by methionine sulfoximine increases the neurotoxic effects of kainate and

Coronal slices of rat brain were incubated in Krebs bicarbonate medium containing kainate (300 microM), or N-methyl-D-aspartate (500 microM). Degeneration of striatal neurons by both these toxins was apparent after 40 min incubation, and was accompanied by a 33% (kainate) and 21% (N-methyl-D-aspartate) reduction in striatal glutamine synthetase activity. Pre-incubation of the slices with 500 microM L-methionine sulfoximine, an inhibitor of glutamine synthetase, for 20 min prior to the exposure to either kainate or N-methyl-D-aspartate, again showed extensive degeneration of striatal neurons, and a supra-additive reduction in glutamine synthetase activity in the tissue. The activity of the neuronal marker enzyme, neuron-specific enolase, was also reduced by pre-incubation of the slices with L-methionine sulfoximine before the addition of kainate or N-methyl-D-aspartate, but to a much lesser extent than glutamine synthetase. The results are discussed in terms of a possible mechanism of interaction between either kainate or N-methyl-D-aspartate, and glial cell metabolism.

Development of porcine embryos following microsurgical blastomere transplantation

The objective of these studies was to characterize the in vitro and in vivo development of porcine embryos following microsurgical blastomere transplantation. In Experiment 1, 60 four-cell embryos recovered on Day 3 of gestation (Day 0 = onset of estrus), were subjected to either blastomere transplantation (micromanipulated; n = 30) or no microsurgical procedure (control; n = 30). Four-cell embryos were cultured in modified Kreb's Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin and 10% bovine calf serum. After 72 h in culture, embryos were stained and the number of nuclei per embryo was determined. Blastomere transplantation had no effect on embryonic development in vitro as the numbers of micromanipulated and control embryos that reached the 12–24 and ≥25 nuclei stages were similar ( P<0.40). In Experiment 2, 64 four- to eight-cell embryos recovered on Day 3 of gestation were distributed across two treatments: control ( n=33) or micromanipulated ( n=31). Micromanipulated embryos each received a single blastomere from a donor embryo at the same stage of development. After culture for 4–5 h, seven recipients which exhibited estrus within 24 h of the donors, received either control or host embryos. Seventy-two hours later, the transferred embryos were recovered from the recipient females, stained and the number of nuclei per embryo was determined. The proportion of micromanipulated and control embryos recovered was not different. In comparison with control embryos, a greater ( P<0.02) proportion of micromanipulated embryos contained <50 nuclei. The mean number of nuclei present in embryos which reached the >100 nuclei stage was greater ( P<0.0004) for micromanipulated than for control embryos (173±20 and 125±4, respectively). While blastomere transplantation appears to have no detrimental effect on development of porcine embryos in vitro, it may reduce the proportion of embryos developing beyond the 50 cell stage in vivo.

Effects of media buffer systems on growth and electrophysiologic characteristics of cultured sweat duct cells

Primary cultures of human reabsorptive sweat duct cells were grown in MCDB 170 medium buffered with either HEPES, bicarbonate, or a mixture of HEPES and bicarbonate buffers. Cultures grown in MCDB media containing bicarbonate seemed to differentiate into a multilayered, keratinized epithelium and began senescing after 1 wk in culture. In contrast, cultures grown in media containing HEPES as the only buffer seemed to undergo a selection process, resulting in the outgrowth of cells that did not multilayer or keratinize extensively for up to 3 or 4 wk in culture. Despite marked differences in growth, cells grown in both bicarbonate and HEPES-buffered media retained electrophysiologic characteristics appropriate to the progenitor. Mean resting potentials were -21.8 +/- 0.8 mV (n = 82), -23.3 +/- 1.3 mV (n = 70) and -18.2 +/- 0.8 mV (n = 82) for duct cells grown in HEPES, bicarbonate, and HEPES-bicarbonate media, respectively. Substitution of Cl- with the impermeant anion gluconate in the bathing medium caused membrane potential depolarization in all media, revealing the presence of a Cl- conductance. Administration of the Na+ conductance inhibitor amiloride hyperpolarized the mean resting potential of cells grown in HEPES medium (-6.8 +/- 0.6 mV, n = 68), bicarbonate medium (-6.9 +/- 0.5 mV, n = 60), and HEPES-bicarbonate medium (-5.9 +/- 0.6 mV, n = 69), demonstrating expression of a Na+ conductance. We observed some but minimal variation with age in any of these conditions.

Culture of the one- and two- cell porcine embryo: Effects of varied osmolarity in Whitten's and Kreb's Ringer Bicarbonate media

Culture of the one- and two- cell porcine embryo: Effects of varied osmolarity in Whitten's and Kreb's Ringer Bicarbonate media

Regulation of lipoprotein lipase activity and mRNA content in rat epididymal adipose tissue in vitro by recombinant tumour necrosis factor

Tumour necrosis factor (TNF) has previously been shown to decrease lipoprotein lipase (LPL) activity and mRNA levels in 3T3-L1 cells and in adipose tissue from rats and guinea pigs when injected in vivo, but not to alter LPL activity in human adipocytes incubated in vitro. The effect of recombinant human TNF on LPL activity and mRNA levels in rat epididymal adipose tissue incubated in vitro was examined. LPL activity and mRNA levels fell in adipose tissue taken from fed rats and incubated in Krebs-Henseleit bicarbonate medium with glucose. The addition of insulin and dexamethasone prevented these falls. TNF (400 ng/ml) produced a fall of approx. 50% in LPL activity after 2 h of incubation and of approx. 30% in LPL mRNA levels after 3 h. TNF did not decrease LPL activity in isolated adipocytes. These results demonstrate that rat adipose tissue incubated in vitro is responsive to TNF whereas isolated adipocytes are not.

Evidence for a Short Feedback of Prolactin on the Tubero‐Infundibular Endings: Differential Effect on the Release of [3 H]Gamma‐Aminobutyric Acid and [3 H]Dopamine from Superfused Median Eminence

Abstract Release experiments were undertaken on the tubero-infundibular terminals of the hypothalamic median eminence. Isolated median eminences were preloaded with [(3) H]y-aminobutyric acid (GABA) or [(3) H]dopamine in such conditions that non-specific uptake was pharmacologically impaired. Each median eminence was continuously superfused with Krebs bicarbonate medium and tritium releases were evoked with high potassium solutions. The evoked release was estimated by measuring the total radioactivity of which actual dopamine and GABA represented more than 60% and 80%, respectively. The experiments were done by applying two successive 5-min pulses of potassium, the second in the presence of prolactin. These stimulations were submaximal: for each, a KCl concentration was chosen that gave about half the maximal response (45 mM for GABA and 30 mM for dopamine); the responses were suppressed by calcium-free medium. Rat prolactin induced a significant increase of dopamine-evoked release in a dose-dependent manner with a maximum (30%) at 25 mug prolactin/ml of medium. Over the same range of concentration a significant but still weak (10%) increase of evoked GABA was observed only with the highest prolactin concentration (25 mug prolactin/ml). At the same concentrations, ovine prolactin induced a markedly reduced effect on dopamine release and had no effect on GABA release. Spontaneous releases of dopamine and GABA were not affected by prolactin. These results show that prolactin enhances acutely and directly, but differently, the evoked release of GABA and dopamine by the median eminence. This differential effect may reflect either a differential regulation or a greater heterogeneity of GABAergic endings. Our data give support for a short feedback exerted by prolactin directly on the tubero-infundibular neurons.

Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing.

Electroejaculate traits, testicular volume, and circulating FSH, LH, and testosterone concentrations were compared between two populations of domestic cats consistently producing either a high (greater than 60%, normospermic) or low (less than 40%, teratospermic) incidence of structurally normal spermatozoa/ejaculate. The effects of semen dilution in Biggers, Whitten and Whittingham (BWW) or modified Krebs Ringer bicarbonate (mKRB) medium and swim-up processing on sperm viability and duration of motility in vitro also were assessed. Ejaculate volume, percent sperm motility, sperm progressive motility, motile spermatozoa/ejaculate, testes volume, and mean serum FSH and LH concentrations were similar (P greater than 0.05) between normospermic and teratospermic cats. However, sperm concentration/ml of ejaculate was greater and circulating testosterone levels were lower in teratospermic males. Swim-up processing increased (P less than 0.05) percent sperm motility, progressive motility, and the number of structurally normal sperm cells recovered and also prolonged the duration of sperm motility in both cat populations. In teratospermic ejaculates, swim-up separation increased the proportion of morphologically normal spermatozoa recovered by more than two-fold. Diluting cat semen with either BWW or mKRB increased flagellar bending in both normospermic and teratospermic cats. The sperm motility characteristics of only the teratospermic ejaculates were influenced by medium type; mKRB increased percent sperm motility and progressive motility whereas BWW had no effect. Compared with undiluted raw ejaculates, the duration of sperm motility was improved 18- to 24-fold by diluting semen in either BWW or mKRB medium followed by swim-up processing. This study demonstrates that the electroejaculate characteristics of domestic cats vary markedly and that some males consistently produce high proportions of morphologically abnormal spermatozoa. Diminished serum testosterone concentrations and normal pituitary secretion of FSH and LH in teratospermic males suggest that there is an inverse relationship between gonadal androgen production and pleiomorphic spermatozoa in the domestic cat. The swim-up procedure is effective for recovering motile, structurally normal spermatozoa from teratospermic cats.

Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse spermatozoa

The purpose of this study was to determine whether sulfogalactosylglycerolipid (SGG) was desulfated during mouse sperm capacitation. Levels of [35S]SGG were determined in freshly retrieved caudal epididymal sperm, motile capacitated sperm, and immotile sperm, after feeding mature male mice with [35S]sulfate-laced chow for 32 days. Caudal epididymal sperm and coisolated epididymal cells were separated into pellet and interphase fractions by centrifugation through a two-step Percoll gradient (45 and 90%). Upon resuspension in Krebs-Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin, the Percoll-gradient pellet fraction consisted mainly of motile capacitated sperm, whereas the interphase fraction comprised largely immotile sperm and fragmented epididymal epithelial cells. The level of [35S]SGG in the Percoll-gradient-pelleted sperm appeared to be much higher than that in the Percoll-gradient interphase sperm. Percoll-gradient-pelleted sperm were further incubated in the culture medium for 2 h. The level of [35S]SGG showed little or no change after 1 h, but was reduced appreciably after 2 h. At this time point, sperm motility was also decreased. Reduction of sperm SGG is correlated with sperm immotility and (or) senescence and may have no direct relation to the capacitation process.

Effect of Mass, Oxidation State and Duration of Chronic Ingestion on Plutonium Absorption in Fed Rats

The effects of time, mass and oxidation state on plutonium gastrointestinal absorption and tooth adsorption were studied during and after chronic ingestion of plutonium-238 (IV) or (VI) (1.55-15.60 kBq/ml) in 6.5 mM bicarbonate medium by fed rats via drinking water for 8 days to 3 months. Animals were killed during the ingestion to follow the kinetics of whole-body storage and clearance of plutonium. At 1.55 kBq/ml the amount of plutonium retained in the skeleton increased continuously during the 85 days of ingestion and reached a plateau thereafter. This plutonium retention was therefore dependent on the total mass administered but not proportional to this mass, as the fraction of administered plutonium retained decreased during the first 22 days of ingestion and then stabilized. This is reflected by the gastrointestinal transfer (f1), which had risen to (3.80 +/- 0.82) x 10(-5) on Day 3 of ingestion and then decreased to a stabilized value of (1.07 +/- 0.06) x 10(-5) from Day 30 to the end of the ingestion period. In the liver, the amount of plutonium retained reached a plateau, which lasted from Day 30 to the end of ingestion. The kidneys and spleen were also found to be retention sites. By Day 3 of ingestion, for a mass ingested of 5 x 10(-7) g/kg of body mass, the maximum mean value of f1 we found was smaller than the 10(-4) recommended by ICRP Report 30. The oxidation state had no effect on f1. Large plutonium deposition was observed on the teeth. For both oxidation states (IV) and (VI), about 0.10% of the administered dose was deposited on the teeth after 3 days of ingestion, whatever the plutonium concentration administered. However, whereas the amount of plutonium (IV) deposited did not change throughout the ingestion period, tooth deposition of plutonium (VI) decreased.

Development of porcine embryos from one- and two-cell stages to blastocysts in culture medium supplemented with porcine oviductal fluid.

Oviductal fluid (OVF) was harvested chronically from 5 sows beginning on Day 1 of the estrous cycle (Day 0 of estrous cycle = day of detected estrus) and used for embryo culture (Day 3 OVF only). Two experiments were conducted to investigate in vitro development of 1-cell and 2-cell porcine embryos in a modified Kreb's Ringer bicarbonate medium (culture medium, CM), early luteal phase OVF or CM supplemented with OVF (CM-OVF, 25% OVF v/v in CM) with or without transfer to fresh CM. In Experiment 1, 1-cell and 2-cell embryos were harvested from sows (n = 7) approximately 44 h after detected estrus. In Experiment 2, 1-cell embryos were collected from 5 sows treated with altrenogest and gonadotropins, approximately 50 h after injection of human chorionic gonadotropin. The volume of OVF (ml) declined progressively throughout the 4 days of collection (24 h, 8.44 +/- 0.28; 48 h, 6.88 +/- 1.78; 72 h, 4.96 +/- 0.35; 96 h, 4.64 +/- 0.25 after onset of estrus; p less than .01). In both experiments, development to blastocyst stage was lowest among embryos cultured in OVF and highest among those cultured in CM-OVF (Experiment 1: CM, 27.3; OVF, 10; CM-OVF, 63.6; Experiment 2: CM, 26.7; OVF, 0; CM-OVF, 82.4; % blastocyst formation).(ABSTRACT TRUNCATED AT 250 WORDS)

An endogenous digitalis-factor derived from the adrenal gland: studies of adrenocortical tumor cells.

The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.

Net calcium efflux from live bone during chronic metabolic, but not respiratory, acidosis.

In vivo chronic metabolic acidosis induces bone mineral dissolution. Whether the dissolution is due to alterations in physicochemical factors alone, as in acute metabolic acidosis, or requires participation of bone cells is not clear. The effect of chronic respiratory acidosis on bone has also not been established. To determine the effects of chronic metabolic and respiratory acidosis on net calcium flux from bone, we cultured live and dead neonatal mouse calvariae for 99 h in control medium or in medium acidified (pH approximately equal to 7.1) either by lowering the bicarbonate concentration (Met) or by increasing the PCO2 (Resp). We measured net calcium flux (JCa) over 0-48, 48-96, and 96-99 h. Over the first 48 h, there was greater net calcium efflux from live and dead Met than from both Resp groups. All four acidic groups had greater net calcium efflux than controls. Over the last 51 h of the chronic 99 h culture, there was net calcium efflux only from live Met (JCa = 285 +/- 129 nmol.bone-1.3 h-1) and not from any of the other groups (live control, JCa = -183 +/- 24; live Resp, JCa = -110 +/- 22; dead control, JCa = -256 +/- 12; dead Met, JCa = 11 +/- 78; dead Resp, JCa = -27 +/- 47; each P less than 0.02 vs. live Met). There is net calcium efflux from live cultured neonatal mouse calvariae during chronic metabolic, but not respiratory, acidosis. During chronic acidosis, decreased medium bicarbonate, and not just a fall in pH, is necessary to enhance net calcium efflux from live bone.

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.

Development of pig blastocysts in vitro is altered by serum, bovine serum albumin and amino acids and vitamins

We tested the effects of the amino acids and vitamins in minimum essential medium (MEM) and Eagle's medium (BME) on pig blastocyst development and nuclei number. Embryos were recovered either 5 or 6 d after first detected estrus and were cultured for 96 h in U-bottomed wells (0.2 ml). In Experiment 1, addition of MEM amino acids and vitamins to modified Krebs-Ringer bicarbonate (MKRB) medium containing either bovine serum albumin (BSA, 4 mg/ml) or lamb serum (10%, v/v) resulted in fewer (P<0.001) nuclei and smaller (P<0.05) embryo volumes at the end of culture as compared to embryos cultured in MKRB without MEM-supplements. Addition of MEM-amino acids without glutamine (Experiment II) depressed blastocyst volume and rate of hatching, but glutamine (2 mM) had no effect on embryo development. Dialysis (molecular weight > 12,000 retained) of fetal bovine serum (Experiment III) did not affect blastocyst expansion but reduced (P<0.05) the number of nuclei/blastocyst at the end of the culture. Embryos cultured in MKRB with dialyzed serum and the amino acids and vitamins in BME were smaller (P<0.05) and had fewer (P<0.05) nuclei than embryos cultured in MKRB with dialyzed serum but without the BME-supplements. We conclude that, under our culture conditions, MEM and BME amino acids and vitamins are detrimental to the development of early pig blastocysts and that this effect is not due to glutamine. Also, dialysis of fetal bovine serum removes some component(s) that are important for cell division by pig embryos, but it does not affect blastocyst expansion.

Ionized calcium plays a pivotal role in controlling blood pressure during haemodialysis.

The influence of dialysate bicarbonate concentration on ionized plasma calcium as well as on haemodynamics have been studied prospectively. In 3 consecutive weeks, 7 chronic patients were dialysed with a dialysate containing low bicarbonate (30 mEq/l), medium bicarbonate (35 mEq/l) and high bicarbonate (39.5 mEq/l), respectively. Notwithstanding the same considerable influx of calcium during high, medium and low bicarbonate dialysis (Bc-D), a significant increase in ionized calcium from 1.09 to 1.41 mmol/l was observed during low Bc-D only. During medium and high Bc-D no significant increase in ionized calcium occurred. In multivariate analysis the only significant predictive variable for the changes in ionized calcium was the change in total CO2 (r = 0.70; p less than 0.001). During low Bc-D the mean arterial pressure remained almost the same, while during medium and high Bc-D a significant decrease of mean arterial pressure was observed. Multivariate analysis revealed that only the change in ionized calcium had a significant predictive effect on the changes of blood pressure (r = 0.69; p less than 0.001). In conclusion, during dialysis an increasing ionized plasma calcium level was counteracting the negative effect of fluid removal on blood pressure. The results of this study underline the pivotal role of ionized plasma calcium for the regulation of blood pressure.

Pig blastocyst development in vitro is affected by amino acids.

Pig embryos were removed 5 d after onset of estrus and cultured in modified Krebs-Ringer bicarbonate (mKRB) medium containing bovine serum albumin (BSA; 1 mg/ml). Media in Exp. 1 and 2, but not in Exp. 3, contained 10% heat-inactivated lamb serum (LS). In Exp. 1, embryos were cultured in 1) mKRB + LS or mKRB + LS supplemented with 2) glutamine (2 mM); 3) phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); or 4) phenylalanine, methionine, isoleucine and glutamine. Embryos cultured in media with supplemental phenylalanine, methionine and isoleucine attained larger (P less than .05) volumes during culture and initiated hatching at an increased (P less than .05) frequency compared with embryos cultured without additional amino acids. In contrast, adding glutamine depressed (P less than .05) the maximum volumes observed during culture. In Exp. 2 mKRB + LS was compared with mKRB + LS plus phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); methionine and isoleucine; phenylalanine and isoleucine, or phenylalanine and methionine. Embryos cultured in media supplemented with phenylalanine and methionine attained larger (P less than .05) volumes than embryos cultured in either media without added methionine. Fewer (P less than .05) embryos cultured without methionine initiated hatching (56%) compared with embryos provided methionine (89%). In Exp. 3, we evaluated the addition of glutamine (1 mM) with or without phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM) to serum-free mKRB. Adding glutamine alone, but not in combination, increased (P less than .05) blastocyst volumes on d 3 and maximum volume attained during culture compared with embryos cultured in mKRB alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel effect of vasoactive intestinal polypeptide and peptide histidine isoleucine: Inhibition of in vitro secretion of prolactin in the tilapia, Oreochromis mossambicus

The effects of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) on the in vitro secretion of two prolactins (PRL) from the rostral pars distalis (RPD) and of growth hormone (GH) from the proximal pars distalis (PPD) of the pituitary of the tilapia ( Oreochromis mossambicus) were studied. RPDs were incubated for 20 hr in hypoosmotic (280–300 mOsm) or hyperosmotic (340–350 mOsm) Krebs-Ringer bicarbonate medium with added peptide concentrations of 0 (control), 0.3, 3.0, 30, and 300 n M; similarly, PPDs were incubated with the same peptide concentrations in isoosmotic (325 mOsm) medium supplemented with cortisol. PRL and GH in the tissue and secreted into the medium were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by soft laser densitometry of the protein band(s). Neither VIP nor PHI has a detectable effect on the secretion of GH. Secretion of the two PRLs is significantly inhibited by VIP and PHI in both hyperosmotic and hypoosmotic medium. In hyperosmotic medium, 300 n M VIP inhibits secretion of both PRLs by 47%, whereas in hypoosmotic medium, 300 n M VIP inhibits their secretion by 27%. PHI inhibits their secretion by ca. 65% in hyperosmotic medium and by 40% in hypoosmotic medium. There is preliminary immunocytochemical evidence for some VIP-like immunoreactivity (IR), but no conclusive indication of PHI-like IR, in the hypothalamo-hypophysial area. The inhibitory actions of VIP and PHI on PRL secretion in tilapia are in contrast to the known stimulatory actions of VIP and PHI on PRL secretion in tetrapods.

Effect of intestinal factors on extraction of insulin in perfused rat liver.

To study the direct effect of intestinal factors on hepatic extraction of insulin, an investigation was made into the extraction of insulin from isolated rat liver perfused with portal venous effluent (PVE) obtained from the isolated perfused rat intestine. Rat intestine was perfused with Krebs-Ringer bicarbonate medium for 45 min, and the PVE was collected from glucose-, lipid-, or NaCl-treated and untreated control intestines. The PVE, after adjustment of its glucose (180 mg/dl) and insulin (200 microU/ml) concentrations, was used as the perfusing medium for the liver of a different rat. The liver was perfused without recirculation with the PVE not containing insulin for 15 min and then perfused with the PVE containing insulin for the next 30 min. Insulin removal from liver perfused with PVE from lipid- or NaCl-treated intestine (52.6 +/- 5.4 or 46.6 +/- 4.1%) was similar to that from comparable controls (49.7 +/- 2.8 or 48.2 +/- 2.9%), respectively. However, that from glucose-treated intestine (39.7 +/- 6.2%) was significantly (P less than 0.01) lower than that from control intestine (51.1 +/- 2.5%). These results indicate that an intestinal factor secreted after glucose ingestion significantly reduces hepatic extraction of insulin and that at least a part of the incretin phenomenon is due to a decreased hepatic extraction of insulin after oral glucose administration.