Production of offspring after sperm chromosome screening: an experiment using the mouse model.

STUDY QUESTION: Is it possible to produce offspring after sperm chromosome screening? SUMMARY ANSWER: It is possible to produce zygotes after examining the genome of individual spermatozoa prior to embryo production. WHAT IS KNOWN ALREADY: Chromosomal aberrations in gametes are a major cause of pregnancy loss in women treated with assisted reproductive technology. However, to our knowledge, there are no reports on the successful genomic screening of spermatozoa, although some attempts have been made using the mouse as a model. STUDY DESIGN: To prevent the transmission of chromosomal aberrations from fathers to offspring, we performed sperm chromosome screening (SCS) prior to fertilization using the mouse as a model. The production of offspring after SCS consists of (i) replication of the sperm chromosomes, (ii) analysis of one copy of the replicated sperm chromosomes, (iii) construction of a zygote using another set of chromosomes and (iv) production of a transferable embryo. MATERIALS, SETTING, METHODS: A single spermatozoon of a male mouse, with or without a Robertsonian translocation, was injected into an enucleated oocyte to allow the replication of sperm chromosomes. One of the sister blastomeres of a haploid androgenic 2-cell embryo was used for chromosome analysis. The other blastomere was fused with an unfertilized oocyte, activated and allowed to develop to a blastocyst before transfer to a surrogate mother. MAIN RESULTS AND ROLE OF CHANCE: With high efficiency, we were able to analyze sperm chromosomes in a blastomere from the androgenic 2-cell embryos and culture zygotes, with and without aberrant chromosomes, to the blastocyst stage before embryo transfer. The karyotypes of the offspring faithfully reflected those of the blastomeres used for SCS. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using a mouse model; whether or not the method is applicable to humans is not known. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that it is possible to produce zygotes without any paternally inherited aberrations by examining the genome of individual spermatozoa prior to embryo production.

Osteomyelitis due to trimethoprim/sulfamethoxazole-resistant Edwardsiella tarda infection in a patient with X-linked chronic granulomatous disease.

Edwardsiella tarda, a catalase-positive bacillus widely distributed throughout nature, is generally susceptible to trimethoprim/sulfamethoxazole. We describe osteomyelitis due to trimethoprim/sulfamethoxazole-resistant E. tarda in a patient with chronic granulomatous disease (CGD). Once E. tarda acquires antibiotic resistance, infected CGD patients may develop severe infections with unforeseeable consequences.

Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes.

BACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated. RESULTS: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3-7 days before freeze-drying, 85-95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P < 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution. CONCLUSIONS: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes.

Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.

Adult murine neurons: their chromatin and chromosome changes and failure to support embryonic development as revealed by nuclear transfer.

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered.

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo.

The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

Chromosome-type aberrations induced in chromosome 9 after treatment of human peripheral blood lymphocytes with mitomycin C at the G(0) phase.

To determine the fate of chromosome aberrations induced primarily by clastogenic chemicals, aberrations of chromosome 9 in cultured human peripheral blood lymphocytes were analyzed after exposure to mitomycin C (MMC) at G(0) phase. Chromosome 9 painting by fluorescence in situ hybridization revealed that the translocation of 9p or 9q to another chromosome and the centric fragment representing the entire length of 9p were characteristically generated from chromatid-type aberrations involving the centromeric region of chromosome 9. These changes were not observed at 48 h after culture initiation, but persistently appeared at later stages (72-120 h postinitiation). Induction of centric fragments of 9p and micronuclei without the alpha satellite DNA of chromosome 9 suggested that most of the breaks were induced near the alpha satellite DNA locus on 9q. Modified patterns of chromosome 9 aberrations were also observed, being related to the copy number of the short or long arm of the chromosome. Such unbalanced karyotypes could remain in the lymphocyte genome over further cell divisions for at least 120 h after culture initiation, indicating that these aberrant cells can survive and that they could pose a health risk.

Diffuse cutaneous mastocytosis responding to cyproheptadine.

A 3-month-old male infant was referred to our department with a generalized brown, thickened leathery skin and blisters which had been present since birth; the blisters developed recurrently at sites of minor trauma, healing without scar formation, although Darier's sign was positive. Microscopically, biopsy specimens showed a dense dermal band-like infiltrate with mast cells, whereas a biopsy from a vesicle showed subepidermal bulla formation. Physical examination did not reveal any systemic involvement with mastocytosis and the patient was treated with cyproheptadine, which has considerable anti-serotonin activity and, interestingly, reduced the degree of blistering; this treatment might therefore be tried in other cases.

Acute monocytic leukemia presenting as cutaneous involvement.

Specific cutaneous lesions appearing during acute monocytic leukemia (AMoL) are more frequent than those associated with other types of leukemia. However, skin involvement preceding the presence of leukemic cells in the peripheral blood is quite rare. In this paper, we describe a case where a 25-year-old male had multiple infiltrative erythemas and nodules on his arms. Histologically, the nodules were formed by masses of tumor cells in the dermis. Peripheral-blood tests revealed no abnormalities, but bone marrow aspiration from the sternum led to a diagnosis of AMoL. The diagnosis of specific cutaneous lesions of AMoL was confirmed by the results of cytochemical studies of bone marrow smears, and cutaneous nodules of cutaneous biopsy specimens led to early diagnosis. Complete remission was achieved with combination chemotherapy and peripheral blood stem cell transplantation.

Production of D-Glutamate from L-Glutamate with Glutamate Racemase and L-Glutamate Oxidase.

We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the α-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 µmol of D-glutamate was produced and isolated from 10 µmol of L-glutamate, about 90% of the theoretical yield.

p53 gene mutations in squamous cell carcinoma occurring in scars: comparison with p53 protein immunoreactivity.

We determined the relationship between p53 expression and p53 gene mutations in squamous cell carcinoma occurring in scars and unrelated to UV light irradiation. We analyzed biopsy specimens obtained from three patients with squamous cell carcinoma. A monoclonal antibody against p53 (DO-7) was used for the immunohistochemical analysis. p53 gene mutations were detected by the polymerase chain reaction and single-strand conformation polymorphism analysis and direct DNA sequencing. p53 overexpression was observed in atypical squamous cells of one case. Those of two other cases, however, showed negative immunoreactivity to p53. Exon 6 of the p53 gene in all three cases and exon 7 in one case showed electrophoretic mobility shifts in polymerase chain reaction and single-strand conformation polymorphism analysis. DNA sequencing analysis showed a missense mutation and a silent mutation in exon 6 of the case with p53 overexpression, a three-base deletion in exon 6 of one case with no p53 overexpression, and a three-base deletion in exon 6 and a missense mutation in exon 7 of another such case. Although immunohistochemical overexpression of p53 has been thought to result from p53 gene mutations, our results suggest that negative immunoreactivity to p53 also can result from p53 gene mutations, for example, short gene deletions.

New types of liposidomycins that inhibit bacterial peptidoglycan synthesis and are produced by Streptomyces. I. Producing organism and medium components.

Liposidomycins are atypical lipid-bearing nucleoside antibiotics that inhibit bacterial peptidoglycan synthesis. A producing strain was identified as a Streptomyces sp. from its cultural characteristics and physiological properties. It produced new types of liposidomycins that lacked sulfate and/or 3-methylglutaric acid moieties present in known liposidomycins by changing medium components. Sucrose and malt extract were particularly suitable sources for specific production of the new types of liposidomycins.

Comparative studies of MCL-5 cells and human lymphocytes for detecting indirect-acting clastogens.

The MCL-5 cell line was established from human lymphoblastoid TK+/- cells transfected with cDNAs of human cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) and microsomal epoxide hydrolase. The TK+/- cells constitutively express a relatively high level of endogenous CYP1A1. To study metabolic activities to indirect-acting clastogens, MCL-5 cells were treated with four clastogens, i.e. aflatoxin B1 (AFB1), diethylnitrosamine (DEN), cyclophosphamide (CPA), and 7,12-dimethylbenz[a]anthracene (DMBA). Human lymphocytes from peripheral blood were used as control cells under the assay conditions with or without induced rat liver metabolic activation (S9). All chemicals tested without S9 induced chromosomal aberrations (CA) in MCL-5 cells but not in human lymphocytes. All chemicals induced CA in both cell types in the presence of S9.

Reassessment of gastric lymphoma in light of the concept of mucosa-associated lymphoid tissue lymphoma: analysis of 53 patients.

BACKGROUND: The concept of mucosa-associated lymphoid tissue (MALT) lymphoma is now widely accepted. However, precise characterization of the features of MALT lesions in the stomach is needed. For this, extensive analysis of resected gastrectomy specimens from a large number of patients with MALT lymphoma is essential. METHODS: Fifty-three patients who underwent gastrectomy for the treatment of primary gastric lymphoma were studied. In the histologic examination, a mean of 46 specimens per case were analyzed; the distribution of lymphomatous lesions was plotted on maps of gastrectomy specimens, input into a computer, and used to measure the size of lesions. RESULTS: The median age of the patients was 56 years, and the male-to-female ratio was 27:26. Stage of disease was I(E) for 35 patients, II(E) for 15, and III for 3. Histologically, 25 patients had low grade lesions, 18 had combined high and low grade lesions, and had 10 high grade lesions. Macroscopically, low grade MALT usually had multiple instances of superficial spreading of lesions without ulceration, whereas high grade MALT exhibited a solitary tumor-forming lesion. All of the superficial spreading type without ulceration were low grade MALT. The higher the grade of tumor, the larger the tumor size. Twenty-four patients received chemotherapy. The 5-year survival rate was 67.2%. Multivariate analysis revealed that only the clinical stage was a significant factor in prognosis. CONCLUSIONS: Low grade MALT can be differentiated from other types of MALT by macroscopic findings. When the MALT concept is adopted, stage of disease is important in relation to survival.

Establishment and characterization of an epithelioid sarcoma cell line with an autocrine response to interleukin-6.

A novel epithelioid sarcoma (ES) cell line, designated as ES-OMC-MN, was established from a 44-year-old woman with recurrence and metastasis of ES. The cells were spindle-shaped or polygonal and were positive for cytokeratin and vimentin on immunohistochemical staining. Electron microscopy revealed desmosome-like structures between the cells. These characteristics were also noted in the original tumor. Southern blot analysis of HindIII digests showed an additional 8.0 kb band and an 8.8 kb band in DNA from the cultured cells and the original tumor as well as the peripheral blood cells of the patient, while only an 8.8 kb band was observed in control human placental DNA. There were no point mutations of N-ras codons 12, 13, and 61, suggesting that the abnormality of N-ras was not due to mutation but to polymorphism. Interleukin-6 (IL-6) was secreted into the culture medium at high levels. Recombinant IL-6 augmented the proliferation of these cells, while cell growth was inhibited by incubation with an anti-IL-6 antibody. The cells also expressed surface IL-6 receptors, indicating that IL-6 acted on this cell line in an autocrine manner. IL-6 and IL-6 receptors were also found in the original tumor. These results demonstrate that ES-OMC-MN cells retained all the morphological and biochemical characteristics of the original tumor and suggest that an autocrine effect of IL-6 may be involved in the development of ES.

Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.