Long-term melatonin administration attenuates low-LET gamma-radiation-induced lymphatic tissue injury during the reproductively active and inactive phases of Indian palm squirrels (Funambulus pennanti).

A comparative analysis of low linear energy transfer (LET) gamma-radiation-induced damage in the lymphatic tissue of a tropical seasonal breeder, Indian palm squirrel (Funambulus pennanti), during its reproductively active phase (RAP) and inactive phase (RIP) was performed with simultaneous investigation of the effects of long-term melatonin pre-treatment (100 microg/100 g body weight). A total of 120 squirrels (60 during RAP and 60 during RIP) were divided into 12 groups and sacrificed at 4, 24, 48, 72 and 168 h following 5 Gy gamma-radiation exposure; control groups were excluded from exposure. Total leukocyte count and absolute lymphocyte count (ALC) and melatonin only of peripheral blood, stimulation index, thiobarbituric-acid-reactive substances (TBARS) level, superoxide dismutase (SOD) activity, and the apoptotic index of spleen as analysed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) noted at observed time-points were significantly reduced in melatonin pre-treated groups during RAP and RIP. Long-term melatonin pre-treatment mitigated radiation-induced alterations more prominently during RIP, as assessed by ALC, TBARS, SOD, TUNEL and caspase-3 activity, at some time-points. Our results demonstrate an inhibitory role of melatonin on caspase-3 activity in splenocytes during RAP and RIP following gamma-radiation-induced caspase-mediated apoptosis. Hence, we propose that melatonin might preserve the viability of immune cells of a seasonal breeder against background radiation, which is constantly present in the environment.

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs.

Non-specific L-type calcium channel blockers, such as verapamil (> or =50 microM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca(2+)-deficient medium. However, the effects of extracellular Ca(2+) on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca(2+)-deficient medium. The initiation of apoptotic features was observed at 50 microM of verapamil. Extracellular Ca(2+) (1.80 mM) reduced intracellular H2O2 level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 microM, but not higher concentrations of > or =100 microM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca(2+) ions have a role during SEA and protect against verapamil induced apoptosis in aged rat eggs.

Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis.

The objective was to find out the functional roles of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H(2)O(2), total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H(2)O(2) induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H(2)O(2), reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H(2)O(2) and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide.

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro.

Hydrogen peroxide (H2O2) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H2O2 with or without caspase-3 inhibitor for various times. The treatment of H2O2 induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H2O2 treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H2O2 treatment. The shrinkage was induced by H2O2 in a dose- and time-dependent manner in both immature and mature oocytes. Although, H2O2-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H2O2 after 1.0 hr of culture. Treatment of H2O2 induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H2O2 induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.

Effect of styrene maleic anhydride (SMA) on viability and integrity of isolated rat oocytes in vitro.

In vitro spermicidal property of the polymer, styrene maleic anhydride (SMA), a compound under clinical trial as an intravasal male contraceptive, is already established. The present study was designed to evaluate the effect of the polymer (SMA) on the viability and integrity of rat oocytes in vitro. The pH-lowering effect of this polymer was evident following precipitation in Medium 199. Oocytes became nonviable with clearly resolved granulated cytoplasm following incubation with undiluted polymeric first wash medium. Dilution of polymeric wash medium prolonged the integrity and viability of oocytes under incubation. The potency of the first wash medium in terms of affecting the viability of oocytes was highest among different washes and dilutions. Bulging of cytoplasm and an enlarged zona pellucida envelope were observed in a few oocytes treated with the first wash. These findings have shown that, like sperm, oocytes also lose their integrity and viability when coincubated with the polymer or medium containing the polymer. However, developing an appropriate technology for delivery is crucial to exploit this potency of the polymer for development of a female contraceptive.

Effect of cyclosporine on human sperm motility in vitro.

Cyclosporine affects motility and viability of human sperm when incubated together in vitro. Sperm motility was almost reduced to nil following 10 min of incubation with cyclosporine at a concentration of 1 mg/mL. However, 200 micrograms/mL of the drug has no effect on motility and viability when tested for up to 60 min under standard laboratory conditions. Cyclosporine effect on sperm was both dose and time dependent. Sperm sensitivity and susceptibility to cyclosporine even to lower doses increased significantly following withdrawal of bovine serum albumin from the incubating medium. Compared to untreated controls, lactate dehydrogenase was estimated higher by more than 2 to 4 times in the sperm-free incubating media, suggesting an altered membrane porosity in the affected spermatozoa.

The in vitro effects of forskolin, IBMX and cyanoketone on meiotic maturation in follicle-enclosed catfish (Clarias batrachus) oocytes.

The effects of an adenylate cyclase activator (forskolin, FK), phosphodiesterase inhibitor (3-isobutyl-l-methyl-xanthine, IBMX) and an inhibitor of steroidogenesis (cyanoketone, CK) on germinal vesicle breakdown (GVBD) in the catfish (Clarias batrachus) were investigated in vitro. In most of the experiments GVBD was induced by using 1 microgram/ml 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP), which is the maturation-inducing steroid (MIS) for this species. Adenosine 3':5'-cyclic monophosphate (cAMP) levels were also measured in the control, MIS-induced and/or FK- and IBMX-treated follicle-enclosed oocytes. MIS-induced GVBD was inhibited by FK (> or = 0.5 microM) or IBMX (> or = 1.0 mM), but oocyte exposed to 0.1 microM FK or 0.5 mM IBMX, after MIS stimulation, underwent GVBD. However, an inhibition of GVBD was recorded when the MIS-induced folliculated oocytes were preincubated with CK (1 microgram/ml) and subsequently treated with 0.1 microM FK. In the time course study, when the oocytes were stimulated by MIS for various time intervals and then treated with 1.0 microM FK or 1.0 mM IBMX, both the substances blocked maturation if they were added up to 12 hr after MIS. The extent of inhibition was gradually decreased and was completely removed after 30 hr of post-MIS stimulation. The stimulatory dose of 17 alpha,20 beta-DP (1 microgram/ml) not only induced GVBD (83.2 +/- 1.50%) in vitro but also reduced oocyte cAMP level (65.3 +/- 2.85 pmol/100 micrograms protein) significantly after 6 hr of incubation. However, FK (10.0 microM) or IBMX (1.0 mM) countered these effects and promoted the accumulation of cAMP in the oocytes; FK being more potent. On the other hand, when unstimulated full-grown but immature oocytes were cultured in vitro in the presence of different concentrations of FK, an induction of oocyte maturation was recorded in dose- and time-dependent manner. These results strongly suggest the involvement of cAMP in the regulation of catfish oocyte maturation.