The new mAbs recognizing the C-terminal domain of human ACE are useful tools for quantification of tACE expression on human live and fixed spermatozoa and further adequate analysis of the tACE role in reproduction.
a-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1±10 mm AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 mm AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5±17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.
SUMMARYAntisperm antibodies (ASA) are a cause of male infertility. ASA are often found in varicocele patients. The study objective was to assess the ASA role in fertility recovery after varicocelectomy. The longitudinal study involved 99 patients with varicocele. Patients were examined according to the WHO recommendations; ASA level was measured using the direct method of Sperm MAR test: 66 patients were ASA-negative, 33 had MAR-IgG ≥ 10%. All patients underwent microsurgical varicocelectomy. Student's t-test, Wilcoxon test, Chi-squared test and signed rank test were used for data analysis. The retrospective analysis of all operated patients data showed that the patients without spermiogram improvement after varicocelectomy had higher ASA levels. 3 months after the surgery, the initially ASA-negative varicocele patients demonstrated 2.5 times increase in number of progressive motile spermatozoa in the ejaculate (p < 0.001), accompanied by 6% decrease in abnormal sperm count (p < 0.05); the spermiogram parameters improved in 77% of cases (p < 0.01). After the surgery, ASA developed in 16% of cases (Max -MAR-IgG = 12%). The patients who were initially ASA-positive demonstrated ASA decrease only in half of the cases (16 of 33; p > 0.05). The main outcome in this group was a favourable response to the surgery (ASA level decrease) vs. no reduction in autoimmune process. The improvement in the ASA-positive group was demonstrated in the patients with higher varicocele grade (median -2 vs. 1; p < 0.05) and lower ASA level (MARIgG = 48% vs. 92%; p < 0.01). The pregnancy rate within a year after surgery was 2.8 times more frequent in couples with ASA-negative men: 39% (25 of 65) in the ASA-negative group compared to 14% (4 of 28) in the ASA-positive group (p < 0.05). Thus, antisperm immune response decreases the varicocelectomy efficacy for reproductive function recovery: the higher percentage of ASA and lower grade of varicocele are associated with an unfavourable prognosis.
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