The objective of this study was to assess the effects of pentoxifylline (PTX) and Ca2+ ionophore (CI) A12387 treatment on some biological characteristics of sperm cells in oligoasthenoteratozoospermia (OAT) patients. After processing, each sample was divided into four groups: 1, control; 2, exposed to 3.6 mM PTX; 3, exposed to 5 μm calcium ionophore (CI); and 4, exposed to both PTX and CI; 30 min at 37°C. Sperm motility was measured before and after preparation. Acrosome reaction (AR), status of sperm vacuoles, mitochondrial membrane potential (MMP) and DNA fragmentation were assessed using PSA-FITC staining, motile sperm organelle morphology examination (MSOME), JC-1 staining and sperm chromatin dispersion (CSD) test, respectively. Treatment with PTX and CI led to increased and decreased sperm motility, respectively (P < 0.05). Furthermore, vacuole status and rates of sperm DNA fragmentation were not significantly different among groups (P > 0.05). Moreover, the data showed that the rates of AR and disrupted MMP were significantly different between groups (P < 0.05). In conclusion, in vitro application of PTX not only did not have any adverse effects on sperm cell biology characteristics, but also can rectify the harmful effect of CI.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6–8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

The ability of mouse oocytes to become activated after exposure too the calcium ionophore A23187 has been investigated at different stages of meiotic maturation. The potential to respond to ionophore has been studied in relation to the time since resumption of meiotic maturation, the chromosomal conformation of the DNA within each cell and the protein synthetic profile of the maturing oocyte. Our studies demonstrate that when maturing oocytes from an MF1 strain of mice were treated with A23187 activation occured only in oocytes which had reached second meiotic metaphase (MII). However, development of the ability to respond to ionophore was not dependent on an orderly progression through normal chromosomal rearrangements such as separation at metaphase I (MI) and subsequent polar body extrusion, since there process could be prevented and the capacity to be activated became apparent in such oocytes at a time when control cells had reached MII. These data suggest that the ability to respond to ionophore depends on the development of a cytoplasmic or complex capable of monitoring the time since initiation of germinal vesicle breakdown. Metabolic radiolabelling of oocytes which were able to respond to calcium ionophore, even though they had been prevented from undergoing normal chromosomal rearrangements, showed them to be synthesising a group of proteins known as the 35 kDa complex.

The present study examined the inhibitory effects of various pretreatment concentrations (0–100 μM) of the calcium ionophore A23187 on polyspermic fertilization and then examined the effect of the maturation period and the time between calcium ionophore treatment and fertilization on the inhibitory effect of calcium ionophore on polyspermic fertilization. In experiment 1, a high concentration of calcium ionophore (100 μM) increased the rate of activated oocytes, but the rate of fertilization declined. On the other hand, when oocytes were treated with a low concentration of calcium ionophore (10 μM), monospermic fertilization was significantly increased (10 μM; 31.3%) (p < 0.05). In experiment 2, oocytes were cultured for various times (0, 0.5, 3, 6 h) after calcium ionophore treatment (10 μM) before fertilization. The highest rate of monospermic fertilization was detected in the oocytes cultured for 6 h after calcium ionophore treatment before fertilization. In experiments 3 and 4, we examined the effect of the maturation period (40 h or 44 h) on the rate of fertilization and blastulation of oocytes pretreated with calcium ionophore. The treatment of oocytes with calcium ionophore significantly decreased the rate of polyspermic fertilization regardless of the maturation period (44 h: with calcium ionophore 26.25% vs without 78.8%; 40 h: with calcium ionophore 37.5% vs without 77.5%); however, calcium ionophore treatment increased the rates of monospermic fertilization and blastulation of the oocytes matured for 44 h, but not those matured for 40 h. In conclusion, activation with a low concentration of calcium ionophore (10 μM) and a further 6 h of culture before fertilization improved the rate of monospermic fertilization and blastulation.

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 μg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

Damage to skeletal muscles may be mediated via free radicals or intracellular calcium overload. To look for inter-relationships between these pathways we have examined the effect of intracellular Ca overload on muscles from rats fed on either a vitamin E-deficient or vitamin E-sufficient diet and assessed the non-enzymic lipid peroxidation in these muscles by examining the production of thiobarbituric acid reactive substances by homogenates. Vitamin E-deficient muscles were more susceptible to Ca-induced intracellular enzyme efflux and this was acutely corrected by supplementation of the external medium with 230 μmol α-tocopherol/l. Vitamin E-deficient muscles showed increased levels of basal lipid peroxides and were more susceptible to iron-catalysed lipid peroxidation. Addition of the Ca ionophore A23187 increased lipid peroxidation in vitamin E-deficient muscle homogenates, but had the opposite effect in vitamin E-sufficient muscles. These results demonstrate that vitamin E-deficient muscle has an increased susceptibility to intracellular Ca overload, but that this effect cannot be explained by a direct stimulatory effect of the ionophore on non-enzymic lipid peroxidation.