The relationship between fertility potential measurements on cryobanked semen and fecundity of sperm donors

Sperm penetration assay (SPA) scores obtained from cryobanked semen were correlated with therapeutic insemination (TI) fecundity in a group of established sperm donors, thereby evaluating the efficacy of the SPA in screening donors for sperm banking. While the SPA has been used to separate fertile from infertile males, we altered assay conditions to use frozen semen and to distinguish performance among fertile donors. Three frozen ejaculates from 11 pregnancy-proven donors were analysed. Of 905 TI cycles, 275 recipients achieved 95 pregnancies. There were no significant relationships between fecundity and donor semen, washed sperm parameters, sperm recoveries or recipient age. A significant relationship was revealed between mean SPA scores (range 8.7-66.6 penetrations/ovum) and donor fecundity (range 0.04-0.16, P < 0.03). Sperm concentration was varied in an effort to establish the most sensitive test condition. Using 0.25x10(6) motile spermatozoa/ml, a highly significant relationship was observed (P < 0.002). The four donors with the lowest SPA scores achieved the four lowest fecundities. It is concluded that a modified SPA can be used on frozen donor semen to estimate donor fertility potential. If applied routinely in donor semen banking, poor quality applicants could be excluded, thereby increasing pregnancy rates while decreasing donor screening costs.     

Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Enu mouse mutagenesis: generation of mouse mutants with aberrant plasma IgE levels

BACKGROUND: The ENU Mouse Mutagenesis Project aims at a large-scale, systematic production of mouse mutants using the alkylating agent ethyl-nitrosourea (ENU). Offspring of mutagenized mice are subjected to a multiparameter screen to detect alterations in various phenotypes with the ultimate goal of identifying novel genes relevant for the expression of the phenotype. Using this approach, we have analyzed plasma IgE concentrations to identify mouse mutants with aberrant plasma IgE levels. METHODS AND RESULTS: ENU-mutagenized male C3HeB/FeJ were mated to wild-type females to produce F1 offspring. F1 animals were analyzed for alterations in their plasma IgE concentrations that showed a dominant mode of inheritance, or bred further to screen for recessive phenotypes. Plasma IgE concentrations were determined by ELISA and a normal range for plasma IgE was established using C3HeB/FeJ wild-type animals. So far we have tested 6568 F1 animals. Repeated testing confirmed a stable aberrant IgE phenotype in 124 animals. To confirm the genetic basis of the observed phenotype, these mice were subjected to confirmation crossing. Currently we have established 9 independent mutant mouse lines (3 with high plasma IgE and 6 with plasma IgE below detection limit) that have been genetically confirmed and additional 24 variant mouse lines are currently undergoing confirmation testing. CONCLUSION: ENU mouse mutagenesis allowed us to generate and identify mouse mutants with aberrant plasma IgE levels, which may be used to characterize novel genes involved in IgE regulation and may serve as animal models for IgE-mediated diseases.     

A study of lectin bindings to the fetal membranes.

Human tissues contain carbohydrates for a main component, functioning as a source and reservoir of energy, connective and supporting element, recognition site and related tasks. Our main interest is to reveal the synthesis and distribution of carbohydrate elements in human fetal membranes. The aim of our work was to clarify, which kinds of elements containing carbohydrates, existed in the fetal membranes. Therefore we applied a lectin-binding study using the following FITC labelled lectins: ConA, WGA, PNA, LCA, RCA. This lead to the result, that ConA, LCA, WGA and RCA produced a positive reaction in the amnion epithelium, which was negative when using PNA. The basement membrane I showed an intense fluorescence when we used ConA, LCA and WGA, using RCA it was weaker and using PNA fluorescence was nearly missing. The examination of the amniotic fibroblast and intercellular substance showed a positive reaction with all lectins, but the intercellular substance lead to weaker fluorescence. The chorionic fibroblasts, intercellular substance and basement membrane II produced fluorescence using ConA, LCA, WGA and PNA, but no reaction could be examined, when using RCA. The trophoblastic cells did not react with LCA and RCA. The intercellular substance reacted positively with all lectins.     

Receptors for immunoglobulin isotypes (FcR) on murine T cells. I. Multiple FcR expression on T lymphocytes and hybridoma T cell clones

T cell receptors for the Fc portion of the various isotypes of mouse immunoglobulins (FcR) were examined by rosette formation, using as indicator cells erythrocytes coated with monoclonal antibodies of all known isotypes of serum immunoglobulins. Three populations of mouse T cells were studied: normal thymocytes, activated T cells (ATC), generated by educating thymocytes in lethally irradiated allogeneic hosts, and hybridoma T cells, derived from somatic hybridization of ATC with the FcR-negative thymoma BW.5147. We found that many different FcR could be distinguished by their specificity for a single isotype or for a combination of several isotypes; ATC and hybridoma T cells expressed several such receptors that, at least in cloned cells, could be demonstrated to be borne by individual cells; hybridoma T cells of independent origin bore indistinguishable receptors whereas ATC expressed markedly different FcR and upon overnight incubation at 37 degrees C, immunoglobulins were found to bind onto the cell surface, even though no corresponding constitutive FcR was detected. The same was observed with hybridoma T cells and with thymocytes. It follows that a single T cell can express several FcR. Altogether, these FcR are capable of binding all known isotypes of serum immunoglobulins. They differ from one T cell to another.     

Purification and properties of Herpesvirus saimiri DNA

³H-Thymidine-labeled Herpesvirus saimiri (HVS) was purified from supernatant and cells of infected owl monkey kidney monolayer cultures. Pronase/SDS-extracted HVS DNA was characterized in neutral sucrose gradients. Cocentrifugation of this DNA with ¹⁴C-labeled T4-phage DNA resulted in s⁰₂₀, w = 58 ± 1.5 S as the sedimentation constant, corresponding to a molecular weight of 91 ± 5 × 10⁶ daltons. Unsheared HVS DNA banded in cesium chloride at 1.709 g/ml, but it broke down during the different manipulations to at least two double-stranded DNA molecules of largely different base composition which shared no sequence homologies. One part (42% of the total viral genome) had a density of 1.729 g/ml, corresponding to 70% cytosine plus guanine content, the other one (representing 58% of the intact molecule) banded at 1.694 g/ml, corresponding to 35% cytosine plus guanine.     

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.