OBJECTIVE : The sperm acrosome reaction is a Ca2+ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of Ca2+ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. METHOD : Human semen samples were obtained from healthy donors with nomal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 micrometer Ca2+ A23187 (Ca2+i); Group 3 where spermatozoa were exposed 5 micrometer Ca2+i and mibefradil; Group 4 where spermatozoa were exposed 5 micrometer Ca2+i and nifedipine, and Group 5 where spermatozoa were treated with 5 micrometer Ca2+i and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at 37degrees C. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total >100 spermatozoa/side. RESULT AND CONCLUSION : We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 micrometer mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The Ca2+i-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.
Acrosome Reaction* ; Acrosome* ; Calcimycin ; Calcium Channels, T-Type ; Fluorescence ; Germ Cells ; Humans* ; Incidence* ; Male ; Mibefradil* ; Nifedipine ; Semen ; Spermatozoa* ; Tissue Donors ; Zona Pellucida

BACKGROUND: Heat shock proteins (HSPs), or stress proteins, are immunodominant antigens of many microorganisms. In this study, we have detected the anti-HSP 70 antibody and tried to explain the role of the antibody with respect to the pathogenesis of SLE. Furthermore, we have attempted to find out the possibility to link the presence of the autoantibody with the monitoring and diagnosis of systemic lupus erythematosus (SLE). METHODS: A total of 80 samples from 55 SLE patients were screened for the presence of anti-HSP 70 antibodies. Simultaneously 59 healthy people were tested as a control group. The anti-HSP 70 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and confirmed by western blot in anti-HSP 70 antibody ELISA positive samples. The activity of disease state was confirmed by the patients' medical record and systemic lupus activity measure (SLAM). RESULTS: The mean optical density (O.D.450) of ELISA in healthy controls and SLE patients were 0.15+/-0.18 (mean+/-S.D.) and 0.13+/-0.14. The correlation of SLAM Score and ELISA O.D. was r2=0.19, P=0.014. And, the mean O.D. value of ELISA was 0.18+/-0.02 and 0.11+/-0.01 before and after treatment (P <0.05). We compared samples with SLAM Score. The O.D. of anti-HSP 70 ELISA in these patients were 0.20+/-0.02 and 0.08+/-0.002 before and after treatment respectively (n=10, mean+/-S.D., P <0.01). CONCLUSIONS: Anti-HSP 70 antibody was not a clinically useful diagnostic marker in SLE patients. However, the titer of anti-HSP 70 antibody can be used for the monitoring of the therapeutic effectiveness in these patients.
Antibodies ; Blotting, Western ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Heat-Shock Proteins ; Humans ; Immunodominant Epitopes ; Lupus Erythematosus, Systemic* ; Medical Records

The earliest stages of mammalian embryogenesis are governed by the activity of maternally inherited transcripts and proteins. Cytoplasmic polyadenylation of selected maternal mRNA has been reported to be a major control mechanism of delayed translation during preimplantation embryogenesis in mice. The presence of cis-elements required for cytoplasmic polyadenylation (e.g., CPE) can serve as a useful tag in the screening of maternal genes partaking in key functions in the transcriptionally dormant egg and early embryo. However, due to its relative simplicity, UA-rich sequences satisfying the canonical rule of known CPE consensus sequences are often found in the 3'-UTR of maternal transcripts that do not actually undergo cytoplasmic polyadenylation. In this study, we developed a method to confirm the validity of candidate CPE sequences in a given gene by a multiplex comparison of 3'-UTR sequences between mammalian homologs. We found that genes undergoing cytoplasmic polyadenylation tend to create a conserved block around the CPE, while CPE-like sequences in the 3'-UTR of genes lacking cytoplasmic polyadenylation do not exhibit such conservation between species. Through this cross-species comparison, we also identified an alternative CPE in the 3'-UTR of tissue-type plasminogen activator (tPA), which is more likely to serve as a functional element. We suggest that verification of CPEs based on sequence conservation can provide a convenient tool for mass screening of factors governing the earliest processes of mammalian embryogenesis.
Animals ; Consensus Sequence ; Cytoplasm ; Embryonic Development ; Embryonic Structures ; Female ; Mass Screening ; Mice ; Ovum ; Polyadenylation ; Pregnancy ; RNA, Messenger, Stored ; Tissue Plasminogen Activator