Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary. In both the cases, oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of meiosis. The in vivo study revealed a significant decrease in the number of ovulated mouse oocytes and an increase in meiosis I-arrested and hyperploid metaphase II oocytes at a single i.p. dose of 70 mg/kg body weight of nocodazole. A significant increase was also observed in the number of meiosis I-arrested and hyperploid mouse oocytes from preantral follicle culture, when they were cultured in the presence of >or=30 nM nocodazole during the final stages of maturation. This concentration is slightly lower than that previously shown to induce nondisjunction in denuded mouse oocytes or in cultured human lymphocytes. The higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be related to a synergistic adverse influence of nocodazole on the oocyte, on somatic cell integrity and on cell-cell communication, which possibly also affects ovulation in vivo. When expressed in molarity relative to the mouse weight, the effective dose of the acute exposure in vivo is 3-4 orders of magnitude higher than the lowest effective concentration employed continuously in vitro. Reduced bioavailability of nocodazole to the target cells due to its poor water solubility may contribute to this difference. Preantral follicle culture can be helpful in analysing mechanisms in chemically induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.     

The effect of lysozyme on the complement-dependent bactericidal action of different antibody classes.

Preparations of rabbit, dog and sheep IgA, IgA and IgM were examined for their antibacterial effects using a complement-dependent bactericidal assay. IgM and IgG were effecient bactericidal antibodies in the presence of complement; IgA, however, contained negligible activity. Except for sheep IgG no enhancement of bactericidal activity was observed in the presence of added lysozyme.     

Identification of cyclophilin A from human decidual and placental tissue in the first trimester of pregnancy

The protein patterns of tissue homogenates from human deciduaand placenta of first trimester pregnancies were investigated.Particular attention was paid to the low molecular weight componentsof these tissues, since substantial evidence has accumulatedthat some of these smaller proteins show a characteristic cyclicand pregnancy expression. Two specific bands were purified fromhomogenates of first trimester decidua and placenta using gelfiltration and anion exchange chromatography. These bands wereseparated by gel electrophoresis and blotting onto polyvinylidendifluoridemembrane. Partial amino acid sequencing of both proteins revealedsequences identical to human cyclophilin A. One protein wassequenced V-N-P-T-V-F-F-D-I-A-V-D-G-E-P-L-G-R-(X)-S-F-E-L-F-A-D-K-V-Pand identified as the 17 kDa isoform of cyclophilin A. The otherprotein was sequenced V-N-P-T-V-F-F-D-I-A and identified asthe 18 kDa isoform of cyclophilin A. cyclophilin A/decidua/placenta/progesterone/progesterone receptor     

Influence of various macrophage populations on Con-A induced T-cell proliferation.

The effect of different macrophage populations and supernatants derived from these macrophages on Con-A induced lymphocyte activation in macrophage depleted lymph node cells has been investigated. All macrophage populations tested were able to restore the lymphocytic response to Con A. Macrophages of different origin and differently elicited were heterogeneous with respect to the lymphocyte-activating and inhibitory activity. Bone marrow macrophages, cultured from 6 to 14 days or obtained from animals stimulated in vivo by BCG were quite inefficient in restoring the lymphocyte proliferation and showed no inhibitory activity up to the percentage per culture tested. Normal or BCG or proteose-peptone induced peritoneal macrophages were quite efficient stimulators, thioglycollate-broth elicited peritoneal macrophages were less efficient in this respect, but very efficient inhibitors. This is reflected to some degree in the supernatants derived from these macrophages which contain T-lymphocyte activating factor (TAF) and inhibitory factors, most likely low molecular weight inhibitory factors. Bone-marrow derived macrophages do not produce either factors in amounts measurable in this assay. These results demonstrate that macrophages, depending on their origin and possibly influenced by external activation signals, are heterogeneous with respect to their function in T-cell proliferation.     

Production of prostaglandin E2 in monocytes stimulated in vitro by Chlamydia trachomatis, Chlamydophila pneumoniae, and Mycoplasma fermentans

Chlamydia trachomatis (CT) as well as Chlamydophila pneumoniae (CP) cause chronic inflammatory diseases in humans. Persistently infected monocytes are involved in the pathogenesis by inducing mediators of inflammation. An in vitro system of chlamydial persistence in human peripheral blood monocytes (HPBM) was used to investigate prostaglandin E(2) (PGE(2)) production and the expression of the key enzyme for prostaglandin production, cyclooxygenase-2 (COX-2). PGE(2) production was determined by PGE(2)-ELISA of HPBM-culture supernatants. Cox-2 mRNA expression was measured by real-time RT-PCR of total RNA isolated from HPBM. Both, CT and CP, stimulated PGE(2) production of HPBM in vitro. Equivalent numbers of CT per host cell induced a higher PGE(2)-response compared to CP. The amount of synthesized PGE(2) depended on the chlamydial multiplicity of infection (MOI). Even at an MOI of 10 the amount of CT- and CP-induced prostaglandin, respectively, was lower than the amount of prostaglandin induced by E. coli lipopolysaccharide (LPS) at a concentration of 10microg/ml. In contrast to stimulation with LPS, Chlamydia-induced PGE(2) production as well as cox-2 mRNA decreased after day 1 post infection (p.i.). These data indicate that Chlamydia stimulate PGE(2) production in human monocytes. Since Chlamydia are often contaminated by mycoplasma, the influence of mycoplasma on the prostaglandin production was investigated additionally. Mycoplasma fermentans (MF) also stimulated PGE(2) production. The co-infection of mycoplasma and Chlamydia resulted in an additive effect in the production of PGE(2). Thus it is important to use host cells and Chlamydia free of mycoplasma contamination for the analysis of Chlamydia-induced prostaglandin production.     

Immunohistochemical demonstration of migration inhibitory factor (MIF) in experimental allergic contact dermatitis.

The kinetics of appearance of MIF+ cells was investigated in experimental contact dermatitis using a monoclonal antibody (7D10) against murine MIF which was reacted with cryostat sections of tissues and detected by the indirect immunoperoxidase test. Four groups of BALB/c mice were investigated: (1) sensitized with 2,4-dinitrofluorobenzene (DNFB); (2) unsensitized controls; (3) tolerized; (4) unsensitized. A challenge dose of DNFB was applied to the ear of animals of groups 1-3 and of croton oil to those of group 4. Three phases could be distinguished in group 1: (a) an initial vascular and exudative reaction; (b) an early cellular phase; and (c) a late cellular phase. At zero time rarely any T lymphocytes (Lyt 1+; Lyt 2+) were seen in all four groups. Within less than 30 min venous endothelial cells became strongly MIF+. This was followed by an influx of monocytes/macrophages reaching a maximum of 72 h in group 1 and a slight peak at 12 h in groups 2 and 3. At 16-24 h in all groups the endothelial reaction weakened while many 7D10+ macrophages appeared in group 1. By double-labelling it was shown that lymphocytes were 7D10-. The influx of lymphocytes, part of which carried the T cell receptor, began at 12 h, reaching a maximum at 72 h in group 1. In groups 2 and 3 only a weak lymphocytic infiltrate developed which declined at 24 h. Group 4 developed an inflammatory reaction after the initial phase with similar kinetics as in group 1. The data suggest that an immune inflammatory reaction is preceded by a nonspecific reaction of the vascular endothelium and the mononuclear phagocytic system and that MIF is playing a central role in these events.     

An unusual bandlike web in an infant with lethal multiple pterygium syndrome

We report on a patient with a lethal multiple pterygium syndrome who also had an unusual, bandlike web across one axilla and partial intestinal atresia. Umbilical cord wrapping with subsequent vascular compromise appears to be the most likely pathogenetic mechanism for the additional anomalies.     

DNA-Analysis of Human Testicular Samples by Cytofluorometry

Germ cells in human testicular samples have been estimated by histology and quantitated by flow cytometry. There is an excellent relationship between the two techniques. These samples were classified histologically as "normal", "reduced number of germ cells" or "no germ cells". A definition of "normalcy" as determined by flow cytometry as having at least 38% of the cells in the haploid state, is proposed. Testicular samples from prepubertal boys with infiltrating leukemic cells often show an increase in the percentage of S-phase cells indicating the presence of tumor. These results indicate the value of flow cytometry in the identification of reproductive and neoplastic disorders.     

Role of muscarinic cholinergic mechanisms in the substantia nigra pars reticulata in mediating muscular rigidity in rats

Summary Bethanechol chloride (5–25 g), when injected into the substantia nigra pars reticulata (SNR) of rats, produced muscular rigidity in a dose-dependent way, and in addition, catalepsy and ipsilateral posture. The effects of bethanechol in the dose of 25 g were prevented by coadministration of 10 g scopolamine hydrochloride. Injections of 25 g betanechol or 10 g scopolamine into the reticulata only slightly affected the muscular rigiditiy produced by 15 mg/kg i.p. morphine hydrochloride. The results suggest that muscarinic cholinergic mechanisms in the substantia nigra pars reticulata, although effective by themselves, affect by expression of at least one striatal functional alteration, the muscular rigidity, in a less effective way than GABAergic or endogenous opioid mechanisms do.