The supplementation of culture medium with protease improves the hatching rate of mouse embryos

Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition. These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus. In order to evaluate the effects of proteases on embryonic development and hatching, 2-cell mouse embryos were cultured for 72 h with or without proteases. The addition of 1.0 microg/ml pronase (PE) and/or 0.1 microg/ml proteinase K (PK) did not affect embryonic development up to the blastocyst stage (94.1% versus 88.2%; 92.2% versus 90.2%, respectively) but significantly increased the hatching rate (60.4% versus 39.2%, 71.8% versus 35.3%, respectively). However, the addition of alpha-chymotrypsin (Chymo) was detrimental to embryonic development and hatching. Changes in the structure of the zona pellucida (ZP) structure of embryos which had been cultured in human tubal fluid (HTF) medium with PE and PK were assessed by fluorescein isothiocyanate-conjugated (FITC)-casein. Embryos cultured in HTF-PE and PK were not stained with FITC-casein. When these embryos were cultured within oviducts, their perivitelline space (PVS) became strongly stained with FITC-casein which was easily removed by phosphate- buffered saline washing. This suggests that PE and PK altered the structure of the ZP. We suggest that the addition of PE and PK to culture media may accelerate the hatching of embryo, by structurally altering the ZP and PVS. This may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.      

Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene

DFFRY (the Y-linked homologue of the DFFRX Drosophila fat-facets related X gene) maps to proximal Yq11.2 within the interval defining the AZFa spermatogenic phenotype. The complete coding region of DFFRY has been sequenced and shows 89% identity to the X-linked gene at the nucleotide level. In common with DFFRX , the potential amino acid sequence contains the conserved Cys and His domains characteristic of ubiquitin C-terminal hydrolases. The human DFFRY mRNA is expressed in a wide range of adult and embryonic tissues, including testis, whereas the homologous mouse Dffry gene is expressed specifically in the testis. Analysis of three azoospermic male patients has shown that DFFRY is deleted from the Y chromosome in these individuals. Two patients have a testicular phenotype which resembles Sertoli cell-only syndrome, and the third diminished spermatogenesis. In all three patients, the deletions extend from close to the 3' end into the gene, removing the entire coding sequence of DFFRY. The mouse Dffry gene maps to the Sxrb deletion interval on the short arm of the mouse Y chromosome and its expression in mouse testis can first be detected between 7.5 and 10.5 days after birth when type A and B spermatogonia and pre-leptotene and leptotene spermatocytes are present.      

Improved prognosis for patients with mediastinal lymphoblastic lymphoma

Patients with diffuse lymphoblastic lymphoma (which includes convoluted lymphocytic lymphoma) with mediastinal involvement have predictable progression of disease to a leukemic phase that is cytologically indistinguishable from acute lymphoblastic leukemia (ALL). Therefore we treated 12 patients with diffuse lymphoblastic lymphoma involving the mediastinum with therapy that is effective in ALL. Treatment consisted of intermittent combination chemotherapy with adriamycin and preventive central nervous system therapy (craniocervical irradiation and intrathecal methotrexate). Mediastinal irradiation was given either for initial respiratory distress or to patients who had incomplete regression of disease following induction chemotherapy. Eleven patients achieve complete remission. With a median follow-up of 41 mo, and using life table analysis, 86% of these patients have remained in continuous complete remission. The results of this study demonstrate the efficacy of treating diffuse lymphoblastic lymphoma with mediastinal presentation as a disseminated lymphoid malignancy.     

Monoclonal antibody Ki-B3 detects a formalin resistant antigen on normal and neoplastic B cells

A new monoclonal antibody Ki-B3 produced by a fusion with leukemic cells of a centroblastic/centrocytic lymphoma (m.l. follicular) is introduced. This antibody predominantly recognizes B cells of follicular mantle and germinal center cells, as well as plasma cells in normal lymphoid tissue. Furthermore, 80% of all low- and high-grade B cell lymphomas are stained, whereas among T cell lymphomas, only four of 15 T lymphoblastic lymphomas were positive to Ki-B3. All peripheral T cell lymphomas showed a negative reaction. Additionally, Ki-B3 detects a small percentage of monocytes and some myelomonocytic leukemias. All epithelial tissues as well as all sarcomas tested were invariably negative. Ki-B3 precipitates a 220 kiloDalton (kD) molecular weight antigen similar to the leukocyte common antigen. Presumably Ki- B3 detects a subtype of the leukocyte common antigen that is predominantly expressed on mature and immature B cells. As the antigen is formalin resistant, Ki-B3 can be used in routine hematology on paraffin sections for the detection and differential diagnosis of B cell lymphomas.     

Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.