Koji Ashizawa

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress.

In the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocytes reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

Development of a repeated-dose liver micronucleus assay using adult rats: An investigation of diethylnitrosamine and 2,4-diaminotoluene

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.

PRESENCE OF PROTEIN PHOSPHATASE TYPE 1 AND ITS INVOLVEMENT IN TEMPERATURE-DEPENDENT FLAGELLAR MOVEMENT OF FOWL SPERMATOZOA

Even in the presence of ATP, the motility of demembranated fowl spermatozoa was negligible at the avian body temperature of 40°C. Motility could be restored by the addition of calyculin A, okadaic acid, specific inhibitors of phosphatase type 1 (PP1) and PP‐2A, and inhibitor 1 or inhibitor 2, which are specific inhibitors of protein phosphatase type 1 (PP1). Demembranated spermatozoa, stimulated by calyculin A or okadaic acid, lost their motility following the addition of 1 mM CaCl2, but this was restored gradually by the stepwise addition of EGTA. Immunoblotting of sperm extract using an antibody to PP1 revealed a major cross‐reacting protein of 36–37 kDa, which corresponded to the molecular weight of the known catalytic subunit of PP1. These results suggest that PP1 present in the fowl sperm axoneme may be involved in the inhibition of fowl sperm motility at 40°C via Ca2+‐dependent regulatory systems.

The role of mitochondrial transition pores on bovine oocyte competence after heat stress, as determined by effects of cyclosporin A

This study was undertaken to investigate the effects on the nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS) when treating bovine oocytes before in vitro maturation (IVM) with 1 µM cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening. Mitochondrial activity, reactive oxygen species (ROS), and apoptosis levels of the oocytes were also assessed. Nuclear maturation rates of both the HS‐exposed oocytes treated with or without CsA groups (HS + CsA or HS group) were significantly lower (P < 0.05) than that of the control group, while the rate of the HS + CsA group was significantly higher (P < 0.05) than that of the HS group. Furthermore, although the cleavage and blastocyst formation rates of the HS group were significantly lower than those of the control groups (P < 0.05), both rates of the HS + CsA group recovered to the same level as those of the control group. The HS group showed a significantly higher ROS level, lower mitochondrial activity in the oocytes, and TUNEL‐positive cumulus cells, but not oocytes, compared with those of the control group (P < 0.05), whereas the TUNEL‐positive and mitochondrial activity levels of the HS + CsA group recovered to those of the control group. These results indicate that 1 µM CsA treatment before IVM may mitigate reduced mitochondrial activity, increase number of apoptotic cumulus cells under HS, and improve the nuclear maturation and developmental competence of bovine oocytes. Mol. Reprod. Dev. 79:31–40, 2012.

Improvement of the post-thaw qualities of Okinawan native pig spermatozoa frozen in an extender supplemented with ascorbic acid 2-O-α-glucoside ☆

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native pig Agu. The objective of the present study was to determine whether ascorbic acid 2-O-alpha-glucoside (AA-2G), a stable ascorbate derivative, is capable of improving the quality of cryopreserved Agu spermatozoa. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 400 or 800 microM AA-2G was thawed, and then evaluated the sperm motility and other qualities. Treatment with 200 microM AA-2G has the most beneficial effect on the sperm motility and the plasmalemma integrity after frozen-thawing among the concentrations tested (P<0.05). In particular, the incidences of total motile sperm and rapid progressive motility at 1 and 3h after incubation were markedly increased by treatment with AA-2G at 200 microM. The addition of AA-2G during cooling and freezing efficiently protected spermatozoa against the lipid peroxidation and the DNA damage. Spermatozoa frozen in the presence of AA-2G possessed significantly higher levels (P<0.05) of ATP even after thawing than those frozen without AA-2G, implying that sperm viability was effectively conserved. Furthermore, higher sperm penetrability to matured oocytes in vitro was maintained in sperm treated with AA-2G during cryopreservation. These effects were observed for all sperm derived from three individuals. These findings demonstrate that the addition of AA-2G to the freezing extender efficiently improves the post-thaw qualities of fragile Agu sperm through the protection of spermatozoa against cell damage caused by oxidative stress during cryopreservation.

Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa

Phosphorylation of fowl sperm proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after incubating the demembranated spermatozoa with [gamma-32P]ATP at 30 degrees C or 40 degrees C. A marked difference of phosphorylation between 30 degrees C and 40 degrees C was observed in a 43 kDa protein. This protein was slightly phosphorylated at 40 degrees C, but strongly phosphorylated at 30 degrees C in a cAMP-independent manner. The motility of demembranated spermatozoa was negligible at 40 degrees C, but vigorous movement was observed at 30 degrees C. These results showed that phosphorylation of a 43 kDa protein is likely to be a regulatory step in the maintenance of fowl sperm motility.

Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5‐dihydroxycinnamate (2,5‐MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose‐dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5‐MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5‐MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca2+ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5‐MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/109 cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR‐SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998.

Intracellular signal transduction pathways in the regulation of fowl sperm motility: evidence for the involvement of phosphatidylinositol 3-kinase (PI3-K) cascade.

The possible role of PI3‐K in the reversible temperature‐dependent immobilization of fowl sperm motility was investigated by using PI3‐K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3‐K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40°C but was maintained vigorously when the temperature was decreased to 30°C. At 30°C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl2 and 10 µM LY294002 or LY303511: around 70–80% of spermatozoa remained motile. In contrast, at 40°C, the motility of spermatozoa was activated immediately after the addition of Ca2+, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca2+‐supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca2+ or LY303511 + Ca2+ were almost the same values compared with those of Ca2+ alone at 40°C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility‐regulating cascade. Immunoblotting of sperm extract using an antibody to PI3‐K revealed a major cross‐reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3‐K. These results suggest that PI3‐K may be positively involved in the calcium‐regulated maintenance of flagellar movement of fowl spermatozoa at 40°C. Mol. Reprod. Dev. 76: 603–610, 2009.

Stimulation of the motility and oxygen consumption of fowl spermatozoa by bicarbonate at 40°C

Abstract Attempts were made to investigate the effects of bicarbonate on the reversible, temperature-dependent immobilization of fowl spermatozoa at 40°C. Washed fowl spermatozoa became almost immotile at 40°C, but motility increased significantly by the addition of bicarbonate. The oxygen consumption was also stimulated. In contrast, bicarbonate did not restore the motility of demembranated spermatozoa at 40°C. The addition of 10 μM cAMP with bicarbonate also did not effect motility. These results suggest that the action of bicarbonate for restoration of fowl sperm motility at 40°C may not be directly on the axoneme, but mediated by solubilized substances which have been removed by demembranation of spermatozoa. However, restoration of motility by bicarbonate may not be related to the bicarbonate-adenylate cyclase-cAMP system.

Prediction of the estrous cycle and optimal insemination time by monitoring vaginal electrical resistance (VER) in order to improve the reproductive efficiency of the Okinawan native Agu pig.

The present study was conducted to determine whether vaginal electrical resistance (VER) can be exploited to improve the low reproductive efficiency of the rare Okinawan native Agu pig, in which estrous signs are difficult to ascertain by visual observation. The lowest VER (272.0+/-12.4 units, n=5) and the preovulatory LH surge were detected at 57.6+/-5.3 and 36.8+/-9.6h before the onset of estrus, respectively. The initiation of gradual increase in VER was found after 9.6+/-4.7h following the peak LH, and the higher levels of VER were plateaued during the luteal phase. These VER fluctuations were correlated with changes in plasma LH (P<0.05) and progesterone (P<0.001), but not estrogen. Moreover, the conception rate (41%, n=32) was dramatically improved by artificial insemination at 24 and 34 h after the beginning of the VER increase when compared with insemination at the conventional time (12 and 24h after detection of estrus, 20%, n=45), widely used in commercial pigs (P<0.05). These data suggest that VER fluctuation can be used to estimate the stage of the estrous cycle, and the scheduling artificial insemination according to increase in VER as an index for the preovulatory LH surge could improve Agu reproductive efficacy.