Regenerating (reg) and insulin genes are expressed in prepancreatic mouse embryos.

The pancreatic regenerating (reg) gene is proposed to be involved in pancreatic beta-cell growth. Up- or down-regulation of reg gene expression has been shown to parallel variations in beta-cell mass and function in the adult pancreas. In several species at least two nonallelic reg genes have been identified. In this study we investigated the expression of each individual reg gene (reg-I and reg-II) during embryogenesis in the mouse. Single mouse embryos were harvested at 8.5, 9, 10, and 12 days of development, homogenized and subjected individually to reverse transcription (RT)-PCR, with a single primer pair to amplify both reg-I and -II mRNAs. Southern blot analysis of the RT-PCR products revealed the presence of reg mRNA at day 9 of embryogenesis, just before the beginning of pancreatic organogenesis. Slot-blot analysis with internal oligonucleotide probes that specifically recognize reg-I or -II sequences demonstrated that only reg-I mRNA was present in day 9 and day 10 prepancreatic embryos. Reg-II mRNA was not detected until day 12, a stage corresponding to late organogenesis. RT-PCR for insulin mRNA from the same samples used for the amplification of reg mRNA showed that the earliest insulin expression occurred at day 8.5, and coincided with the onset of reg-I expression. Hybridization with gene-specific oligonucleotide probes revealed that only insulin-II mRNA was detectable at this time. Insulin-I mRNA was not detectable until day 12 and coincided with early reg-II expression. These results suggest that the two nonallelic reg genes and the two insulin genes are expressed differentially during early embryogenesis. Differential expression of reg-I and -II suggests that they may be induced by different and independent stimuli and have distinct functions.

Catecholamines modulate chicken immunoglobulin M and immunoglobulin G plaque-forming cells.

Norepinephrine (NE) and epinephrine (E) function as chemical messengers in the central nervous and the endocrine systems of the chicken. The effects of in vivo and in vitro exposure of NE and E on IgM and IgG splenic plaque-forming cell (pfc) formation were determined to the antigen SRBC. Six-week-old Line UNH 105 New Hampshire chickens were injected i.v. with NE (500 micrograms/kg BW) or E (100 micrograms/kg BW) followed by 1 mL of 5% SRBC 30 min later. Five days after antigen injection, IgM and IgG pfc were assayed. Compared with controls, in vivo NE suppressed (P < .05) IgM and IgG pfc formation. In vitro NE treatment of splenic lymphocytes reduced (P < .05) IgM pfc but did not affect IgG pfc numbers. In vivo treatment with E increased (P < .05) IgM pfc whereas in vitro E exposure increased (P < .05) IgM pfc. Immunoglobulin G pfc were suppressed (P < .05) by both in vivo and in vitro E exposure. The presence of surface receptors for NE and E on splenic lymphocytes was determined using in vitro incubation with antagonists to alpha and beta receptors. These data suggest that there are alpha and beta receptor sites on lymphocytes for NE and E, and that these catecholamines have a regulatory role in plaque cell proliferation.

Diabetic teratogenesis. In vitro evidence for a multifactorial etiology with little contribution from glucose per se.

To determine the extent to which elevated glucose and 3-hydroxybutyrate (3OHB) concentrations contribute to the embryotoxic properties of diabetic serum, we tested the effects of serum from untreated or acutely insulin-treated diabetic rats on the development of mouse embryos during neurulation in vitro. Male Sprague-Dawley rats (n = 143) with streptozocin-induced diabetes for 1 week received infusions of insulin (n = 105) or saline (n = 38) for up to 120 min. The insulin-infused animals were exsanguinated when serum glucose concentrations fell to between 5.6 and 8.3 mM. Saline-infused animals were exsanguinated after a similar duration of infusion. Serum samples were tested for embryotoxic effects on 3-6 somite mouse embryos cultured in vitro for 24 h. Of embryos cultured in serum from untreated diabetic animals (glucose: 24 +/- 1 mM; 3OHB: 2.0 +/- 0.3 mM), 36% (31 of 87) exhibited gross malformations, mostly of the neural tube. Only 16% (10 of 62) of embryos grown in serum from acutely insulin-treated animals (glucose: 7.4 +/- 0.2 mM; 3OHB: 0.20 +/- 0.06 mM) were malformed. This rate that was less than half the rate caused by exposure to diabetic serum (P < 0.01), but a rate that remained much greater than the rate associated with culture in normal serum (0% in this study; < 2% historically). In vitro addition of glucose to serum from insulin-treated animals to re-establish hyperglycemia in the diabetic range (25 mM) resulted in a 17% (12 of 70) malformation rate, nearly identical to the 16% rate caused by normoglycemic serum from insulin-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the biguanide class of oral hypoglycemic agents on mouse embryogenesis.

The incidence of birth defects among offspring of mothers with non-insulin dependent diabetes mellitus (NIDDM) is 2-3-fold higher than among infants of non diabetics. Since mothers with NIDDM are frequently given oral hypoglycemic agents, including sulphonylureas and biguanides, to control their disease and since these agents have been associated with the occurrence of congenital malformations in humans and animals, the embryotoxic effects of the most commonly employed biguanides, metformin and phenformin, were evaluated in whole embryo culture. Neurulating mouse embryos were exposed to therapeutic concentrations (metformin 500-2,550 mg per day; phenformin 50-400 mg per day, respectively) of the compounds for 24-48 h. Concentrations of metformin in culture ranged from 0.15 to 1.8 mg/ml and phenformin from 2.5 x 10(-5) to 0.4 mg/ml. Cultures were terminated and scored for gross morphological alterations and total protein content. Metformin produced no alterations in embryonic growth and no major malformations. Approximately 10% of all embryos exposed to metformin regardless of dose, exhibited open cranial neuropores after 24 h of culture. However, this anomaly appeared to represent a delay in closure as opposed to an overt defect, since no embryos exposed to the highest concentration of the drug and cultured for 48 h showed open neural tubes. In contrast, phenformin produced dose dependent changes in incidence of malformations, protein content, and embryolethality. Malformations included neural tube closure defects, craniofacial hypoplasia, and reduction in size of the first and second visceral arches. Doses above 0.1 mg/ml produced embryolethality and all embryos were killed at the 0.4 mg/ml concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of altered maternal metabolism during gastrulation and neurulation stages of embryogenesis.

In summary, many congenital malformations are produced during gastrulation and neurulation stages of embryogenesis at a time when no definitive chorioallantoic placenta has been established. In rodents, altered maternal metabolism may have a direct impact on the embryo or an indirect impact via disruption of the nutritive function of the visceral yolk sac. If similar mechanisms operate in human embryos, these factors probably alter functions of the trophoblastic shell. In any case, it is crucial to remember that the metabolic status of the embryo is rapidly changing and during early stages of organogenesis may respond to alterations in nutrients quite differently during the first four weeks of gestation than at later stages of organogenesis and the fetal period.

Modulation of chicken plaque-forming cells by serotonin and dopamine.

Serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) are endogenous components of the central nervous and endocrine systems of the chicken. To determine the effects of these monoamines on antibody-mediated immunity. New Hampshire chickens of Line UNH 105 were injected intravenously with 5-HT (100 micrograms/kg of body weight) and DA (1 mg/kg of body weight). One milliliter of a 5% SRBC suspension was injected intravenously 30 min later. Both IgM and IgG splenic plaque-forming cells were assayed 5 days after antigen injection. For in vitro studies, spleen lymphocytes from SRBC-primed chicks were incubated with DA and 5-HT followed by quantitation of IgM and IgG plaque-forming cells. The in vivo incubation of splenic lymphocytes with specific antagonists was used to ascertain the presence of monoamine receptors on lymphocytes. The 5-HT significantly enhanced IgM plaque-forming cells compared with controls following in vivo [550 +/- 85 (SE) cells/10(6) splenic lymphocytes versus 359 +/- 44] but not in vitro exposure. The IgG plaque-forming cells were not affected by 5-HT. The DA significantly suppressed IgM plaque-forming cells responses following in vivo (284 +/- 46 versus 499 +/- 66) and in vitro (254 +/- 57 versus 451 +/- 51) exposure. Significant suppression of IgG plaque-forming cells was found in vivo (287 +/- 40 versus 462 +/- 75) and in vitro (153 +/- 36 versus 371 +/- 81) following treatment. Specific DA antagonists, apomorphine and metoclopramide, did not alleviate the in vitro suppressive effect of DA.

Phenylalanine and its metabolites induce embryopathies in mouse embryos in culture.

The aim of this study was to determine the teratogenicity of phenylalanine (Phe) and Phe metabolites in neurulating mouse embryos. Therefore, the system of whole embryo culture was employed and D9 (neurulating) mouse embryos were exposed to Phe, phenylethylamine (PEA), phenylpyruvic acid (PPA), phenylacetic acid (PAA), 2-OH phenylacetic acid (2-OH PAA), and phenyl-lactic acid (PLA) at concentrations ranging from 0.01 mM to 10 mM for 24 hours. After 24 hours, embryos were examined for morphological abnormalities and protein content by the Lowry method. Phe at 1 and 6 mM concentrations was not teratogenic; however, 10 mM inhibited cranial neural tube closure in 82% of the embryos. PEA was the most toxic factor and concentrations of 1 and 10 mM were embryo-lethal, whereas neural tube closure defects (NTDs) were observed in 67% of the embryos at 0.1 mM. 2-OH PAA was the second most toxic metabolite with concentrations of 1 and 10 mM producing NTDs in 10 and 100% of the embryos, respectively. PLA and PAA produced no NTDs at concentrations of 1 mM, 60% at 5 mM, and 100% at 10 mM. Finally, PPA produced approximately 50% NTDs at both 1 mM and 10 mM concentrations. PLA, PAA, 2-OH PAA, and PPA produced a significant reduction in embryonic protein, and PEA and 2-OH PAA reduced yolk sac protein values. PEA, 2-OH PAA, PPA, PAA, and PLA also produced craniofacial abnormalities, i.e., incomplete expansion of the forebrain, collapse of the optic vesicle, and hypoplasia of the mandible and/or the maxilla.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoamines alter in vitro migration of chicken leukocytes.

The effect of the biogenic amines serotonin (5-HT), dopamine (DA), and norepinephrine (NE) on peripheral blood leukocyte (PBL) migration was studied in two populations derived from line UNH 105 New Hampshire chickens. Maximum migration from capillary tube migration chambers was achieved in 1 hr. An age effect in both populations was indicated by significantly larger migration areas found in leukocytes from 7-week-old chickens compared to those of 4-week-old chicks. Thirty min after intravenous monoamine injection, line UNH 105 PBL migration was unaffected by exogenous monoamines. In the second population, B24/B24 chicks, NE enhanced migration at 4 weeks of age but DA suppressed migration at 7 weeks of age. In vitro exposure of PBL to the biogenic amines also affected leukocyte migration. Migration was augmented by 100 ng 5-HT but suppressed by 1 microgram 5-HT in UNH 105 chicks. Furthermore, DA suppressed PBL migration and NE enhanced migration in the same population. PBL from B24/B24 chicks were not affected by in vitro exposure to 5-HT, however, DA enhanced migration whereas NE suppressed migration. Specific antagonists for 5-HT, DA, and NE blocked the effects of each monoamine suggesting that receptors are present on chicken leukocytes. These receptors mediate action of the monoamines on leukocyte migration activity.