Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.
The effect of pentoxifylline on the spontaneous and follicular fluid (hFF)-induced acrosome reaction (AR) was studied in an attempt to identify those patients who would benefit from the use of pentoxifylline as an AR-stimulating agent. Pentoxifylline (1 mg/ml) produced no significant increase over spontaneous AR at any of the time points studied (60 min: 6.2% +/- 2.0 spontaneous AR vs 7.4% +/- 2.0 pentoxifylline AR; 120 min: 7.1% +/- 1.6 spontaneous vs 9.0% +/- 2.5 pentoxifylline; 180 min: 7.0% +/- 1.8 spontaneous vs 10.9% +/- 4.0 pentoxifylline, n = 6). On the other hand, hFF produced a significant increase in AR (6.9% spontaneous AR vs 11.2% hFF AR, p < 0.001, n = 39) and this effect was enhanced by pre-incubation with pentoxifylline (15.0% pentoxifylline + hFF AR vs 11.2% hFF AR, p < 0.001). There were no differences in percentage AR induced by hFF or pentoxifylline + hFF between patients with normal (n = 16) and abnormal (n = 25) semen samples. Individual analysis of the effects of hFF and pentoxifylline + hFF on AR showed great variability between patients and enabled us to classify them into four categories: inducible-responder (15%), non-inducible-responder (23%), inducible-non-responder (41%) and non-inducible-non-responder (21%). An induction index (II) (induction produced by hFF divided by spontaneous AR) and a response index (RI) (induction produced by pentoxifylline + hFF divided by hFF) were calculated for each sample. Patients previously classified as inducible exhibited an II > 0.30 and patients previously classified as responders showed an RI > 0.26. There was no correlation between II and RI values (Spearman, r = -0.185, p = 1). Our results show that pentoxifylline is an enhancer of the induced AR only in some patients. We propose an 'AR response assay' to identify responder patients and thus enable rational use of this drug.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.