OBJECTIVETo investigate the characteristic and effect of cadmium on ATP-activated currents (I(ATP)) mediated by P2X4 purinoceptors.

METHODSTranscribe cDNA coding for the rat P2X4 receptor to cRNA in vitro. Inject the cRNA to oocytes of an xenopus laevis using the microinjection technique. Reveal the effect of cadmium on I(ATP) mediated by P2X4 receptor using the two-electrode whole-cell voltage clamp technique.

RESULTS(1) Within a certain concentration range, cadmium was found to reversibly magnify I(ATP) mediated by P2X4 receptors expressed in oocytes of an xenopus. When the concentration of cadmium reached 30 micromol/L, the increase of I(ATP) was the most significant. I(ATP) turned to decrease when the concentration of cadmium was more than 30 micromol/L; (2) The concentration-response curve was shifted to left by applying cadmium at 10 micromol/L; the EC50 was reduced from (17.1 +/- 1.5) micromol/L to (9.8 +/- 1.8) micromol/L (n = 6, P < 0.01) and the Hill coefficient was increased from 1.14 +/- 0.13 to 1.57 +/- 0.36; (3) The effect of cadmium on I(ATP) showed no dependence on membrane voltage; (4) The magnifying effect on I(ATP) reached maximum when preincubating cadmium for 120 seconds.

CONCLUSIONThe increase I(ATP) by cadmium is reversible, concentration-dependent, time-dependent, and voltage-independent. One reason of this augment effect could be the allosteric modulation on P2X4 receptors.

Adenosine Triphosphate ; metabolism ; Animals ; Cadmium ; toxicity ; Microinjections ; Oocytes ; drug effects ; metabolism ; physiology ; Rats ; Receptors, Purinergic P2X4 ; metabolism ; Xenopus laevis

OBJECTIVETo investigate the possible pathways for ovarian injury after administration of cyclophosphamide in rats.

METHODSAdult SD rats received a single injection of saline vehicle or chemotherapeutic agent cyclophosphamide, and 8 weeks later, the ovaries were removed, fixed and serially sectioned for pathological examination and ovarian follicle counting. The expression of stem cell factor (SCF) protein was evaluated by immunohistochemistry and immunoreactive score, and SCF mRNA expression determined by RT-PCR in rat ovaries.

RESULTSCyclophosphamide had a detrimental effect on ovarian stromal function and lead to primordial follicle loss. Immunoreactive SCF antigens were expressed on the oocytes in the primordial and primary follicles of rat ovaries, and also in the granulosa cells of the secondary follicles and early antral follicles. There was a higher granulosa SCF, lower oocyte SCF and higher SCF mRNA level in the ovaries of the rats exposed to cyclophosphamide as compared with those in control rat ovaries (P <0.05).

CONCLUSIONAltered SCF expression in the ovaries of rats exposed to cyclophosphamide can be helpful for understanding the mechanisms for chemotherapeutic drug-induced ovarian damage.

Animals ; Cyclophosphamide ; adverse effects ; Female ; Gene Expression ; drug effects ; Granulosa Cells ; drug effects ; metabolism ; Oocytes ; drug effects ; metabolism ; Ovary ; cytology ; drug effects ; injuries ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Factor ; genetics ; metabolism

Parthenogenetic activation is a procedure that an oocyte at meiosis II stage is activated into mitosis by some chemical or physical stimulation other than a sperm and the embryo is formed in the absence of any contribution from a male gamete. The activation of oocyte is the result of calcium ion oscillations and deactivation of some cytokines such as maturation promoting factor, mitogen-activated protein kinase and cytostatic factor. Parthenogenetic activation is artificially induced by various kinds of physical and/or chemical methods. The main activation method of human oocyte is chemical methods. The rates of activation and cleavage depend on the age, origin,and culture conditions of the oocyte.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Calcium ; metabolism ; Cycloheximide ; pharmacology ; Cytokines ; metabolism ; Female ; Humans ; Oocytes ; drug effects ; growth & development ; metabolism ; Parthenogenesis ; drug effects

OBJECTIVETo observe the effects of telmisartan on Kv1.3 and Kv1.5 potassium channels expressed in Xenopus oocytes.

METHODSKv1.3 and Kv1.5 potassium channel currents expressed in Xenopus oocytes were recorded and observed in the absence and presence of telmisartan using standard two-microelectrode voltage clamp techniques.

RESULTSTelmisartan resulted in a concentration- and voltage-dependent inhibition effect on Kv1.3 channel current (IC(50) 2.05 micromol/L)and on Kv1.5 channel current (IC(50) 2.37 micromol/L).

CONCLUSIONSTelmisartan blocks open-state Kv1.3 channel which could be one of the mechanisms related to its immunomodulatory and anti-atherosclerosis effect. Telmisartan also blocks open-state Kv1.5 channel which might partly account for its effect on reducing the incidence of atrial fibrillation.

Animals ; Benzimidazoles ; pharmacology ; Benzoates ; pharmacology ; In Vitro Techniques ; Kv1.3 Potassium Channel ; drug effects ; Kv1.5 Potassium Channel ; drug effects ; Oocytes ; drug effects ; metabolism ; Patch-Clamp Techniques ; Xenopus

In this paper, membrane current properties of the fully-grown oocytes from toad, Bufo bufo gargarizans, were studied by using two-microelectrode voltage clamp technique. Axion of adult female toad was destroyed, and then ovarian lobes containing oocytes in stage I to VI were removed and incubated in Ca(2+)-free ND96 solution with collagenase (1.5 mg/ml) for 1 h. Subsequently, the oocytes were washed in Ca(2+)-free ND96 solution for 10 min to completely remove the follicular layer. For the experiments only the oocytes in stage V and VI were selected and used during 1 to 5 d. The membrane was depolarized from a holding potential of -80 mV to +60 mV in 10 mV step. It was found that a sustained outward current was elicited by depolarization. Potassium channel blockers (tetraethylammonium chloride, TEA, 10 mmol/L and 4-aminopyridine, 4-AP, 10 mmol/L) reduced the outward current to (23.4+/-0.72)% of the maximum. However, further addition of chloride channel blocker (5-nitro-2, 3-phenypropylamino benzoate, NPPB, 30 micromol/L) could almost completely block the outward current to (2.1+/-0.08)% of the maximum. In the presence of TEA and 4-AP, removal of extracellular Ca(2+) or adding verapamil (40 micromol/L), could also reduce the outward current to (2.2+/-0.04) % and (3.1+/-0.15) % of the maximum, respectively. It is concluded that calcium-dependent chloride channels exist in plasma membrane of Bufo bufo gargarizans oocytes, besides potassium channels.
4-Aminopyridine ; toxicity ; Animals ; Bufo bufo ; Calcium ; metabolism ; Cell Membrane ; metabolism ; Chloride Channels ; drug effects ; physiology ; Female ; Nitrobenzoates ; pharmacology ; Oocytes ; metabolism ; Tetraethylammonium Compounds ; pharmacology ; Verapamil ; pharmacology

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.
Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Ethylene Glycol ; pharmacology ; Female ; Fluorescent Dyes ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; physiology ; Mice ; Microscopy, Fluorescence ; Mitochondria ; drug effects ; metabolism ; Oocytes ; drug effects ; physiology ; Temperature ; Vitrification

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.
Animals ; Antioxidants/administration & dosage/*pharmacology ; Dose-Response Relationship, Drug ; In Vitro Oocyte Maturation Techniques/*veterinary ; Oocytes/cytology/*drug effects/physiology ; Quercetin/administration & dosage/*pharmacology ; Reactive Oxygen Species/*metabolism ; *Swine

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 microg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 microg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 microg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 microg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.
Animals ; Antioxidants/administration & dosage/*pharmacology ; Dose-Response Relationship, Drug ; In Vitro Oocyte Maturation Techniques/*veterinary ; Oocytes/cytology/*drug effects/physiology ; Quercetin/administration & dosage/*pharmacology ; Reactive Oxygen Species/*metabolism ; *Swine

For studying the effect of integrin on the [Ca(2+)](i) of mouse eggs and its transmembrane signaling mechanism, zona-free mouse eggs were loaded with calcium probe Fluo-3/AM and the intensity of fluorescence of the eggs treated with different factors was measured through laser confocal microscopy. The results showed that the [Ca(2+)](i) of zona-free mouse eggs was increased when the eggs were treated with RGD peptide, fibronectin (Fn) and anti-mouse integrin subunit alpha(6) and beta(1) monoclonal antibodies, respectively. The [Ca(2+)](i) of the mouse eggs was also increased when the eggs were placed in calcium-free medium and treated with RGD peptide or Fn. The changes in the mouse egg [Ca(2+)](i) caused by RGD and Fn were similar to those caused by sperm. However, the concentration of Ca(2+) of the zona-free mouse eggs pretreated with tyrosine kinase inhibitor was not increased when the eggs were treated in the same way, and, neither was the intracellular calcium increased in those eggs pretreated with PKC inhibitor when the eggs were treated with RGD peptide. It is therefore suggested that the occupancy of integrins on the membrane of mouse eggs by their ligands mediates the release of Ca(2+) and then the increase in the [Ca(2+)](i) of eggs, which is one of the early events of egg activation. The tyrosine kinase signaling pathway and PKC are involved in this process as well.
Animals ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Female ; Integrins ; physiology ; Ion Transport ; drug effects ; Mice ; Oligopeptides ; pharmacology ; Oocytes ; metabolism ; Ovum ; metabolism ; Protein Kinase C ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.
Animals ; Embryonic Development/*drug effects ; Female ; In Vitro Oocyte Maturation Techniques/veterinary ; Nuclear Transfer Techniques/*veterinary ; Oocytes/growth & development ; *Parthenogenesis ; Sirolimus/*pharmacology ; Sus scrofa/*growth & development/metabolism