The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 µM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.

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.

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.

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 oocyte's 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.

Role of acidification elicited by sialylation and sulfation of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions.

The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO(3) treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.

Effect of the temperature-humidity index on body temperature and conception rate of lactating dairy cows in southwestern Japan.

In the present study, we investigated the relationship between the temperature-humidity index (THI) and the conception rate of lactating dairy cows in southwestern Japan, one of the hottest areas of the country. We also investigated the relationship between measurement of the vaginal temperature of lactating dairy cows as their core body temperature at one-hour intervals for 25 consecutive days in hot (August-September, n=6) and cool (January-February, n=5) periods and their THI. Furthermore, we discussed the above relationship using these vaginal temperatures, the conception rates and the THI. As a result, when the conception rates from day 2 to 0 before AI were classified into day 2, 1 and 0 groups by the six maximum THI values in each group (mTHI; <61, 61-65, 66-70, 71-75, 76-80, >80), only the conception rate for the mTHI over 80 at 1 day before AI group was significantly lower (P<0.05) than the other groups. The conception rate for days 15 to 17, but not days 19 to 22 and 30 to 35, after AI in the cows that experienced average mTHI over 80 (amTHI>80) was significantly lower (P<0.05) than that of the cows that did not experience amTHI>80. There was a significant positive correlation (P<0.01) between the mTHI and the mean daily vaginal temperature, but not during the cool period. When the mTHI reached 69, the vaginal temperature started to increase. As for the relationship between the conception rates and vaginal temperatures for all mTHI classes, in the mTHI>80 at 1 day before AI group, the vaginal temperature increased by 0.6 C from 38.7 C, resulting in a reduction of 11.6% in the conception rate from 40.5%. In conclusion, these results suggest that one of the causes of the fall in conception rate of lactating dairy cows during the summer season in southwestern Japan may be an increase in their core body temperature with a higher mTHI than the critical mTHI of 69 at 1 day before AI.

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 degrees C but was maintained vigorously when the temperature was decreased to 30 degrees C. At 30 degrees C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl2 and 10 microM LY294002 or LY303511: around 70-80% of spermatozoa remained motile. In contrast, at 40 degrees C, the motility of spermatozoa was activated immediately after the addition of Ca(2+), but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca(2+)-supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca(2+) or LY303511 + Ca(2+) were almost the same values compared with those of Ca(2+) alone at 40 degrees 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 degrees C.

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

Differential expression patterns of Wnt and beta-catenin/TCF target genes in the uterus of immature female rats exposed to 17alpha-ethynyl estradiol.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.

Regulation of fowl sperm motility: evidence for the indirect, but not direct, involvement of dynein-ATPase activity on the reversible temperature-dependent immobilization.

Potential mechanisms of the reversible temperature-dependent immobilization of fowl sperm were investigated. At 30 °C, motility of demembranated fowl sperm was inhibited by adding 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), but restored immediately after the subsequent addition of 2 mM CaCl(2), whereas at 40 °C, such additions did not appreciably affect motility (which remained almost negligible). With intact sperm, 10(-9) to 10(-3) M Ca(2+) had no effect on motility at 30 °C, which remained high. In contrast, intact sperm at 40 °C were almost immotile below 10(-5) M Ca(2+), and then gradually recovered motility at higher Ca(2+) concentrations. The negligible motility of demembranated sperm at 40 °C, and at 30 °C in the presence of EGTA, was stimulated by addition of 100 nM of the protein phosphatase inhibitor calyculin A. Dynein-ATPase activities of sperm at 40 °C in the presence of 2 mM EGTA, 2 µM CaCl(2), 2 mM CaCl(2,) or 100 nM calyculin A were higher than those at 30 °C. Therefore, stimulation of fowl sperm motility by temperature, Ca(2+), and phosphatase inhibition was not simply associated with an increase of flagellar dynein-ATPase activity. Furthermore, Ca(2+) was essential, at the axonemal level, for initiation of the 'intrinsic' motility of fowl sperm at 30 °C, but this Ca(2+)-dependent mechanism might be different from that involved in restoration of motility of intact sperm at 40 °C. In addition, perhaps inhibition of protein phosphatase activity was involved in initiation of sperm motility, but acting at a location different from Ca(2+) on the axoneme.

Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein.

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10(8) sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.

Temperature-dependent regulation of sperm motility of Ijima's copper pheasants (Syrmaticus soemmerringii ijimae), one of 'near threatened' species.

In order to conserve the copper pheasants, one of the Japanese 'near threatened' species, the knowledge of the sperm characteristics is the inevitable issue. Therefore, temperature-dependent regulation of copper pheasant sperm motility was investigated in comparison with that of domestic fowl spermatozoa. Motility of intact spermatozoa from both species was markedly affected by temperature. During incubation at 30 degrees C, copper pheasant spermatozoa showed around 60-70% motility, but became almost immotile when the temperature was raised to 40 degrees C. Then, when the temperature of the sperm suspension was subsequently cooled to 30 degrees C, the spermatozoa regained their motility. The motility of domestic fowl spermatozoa showed a similar pattern. Temperature also affected the motility of both demembranated copper pheasant and domestic fowl spermatozoa in the same way. The motility of intact copper pheasant and domestic fowl spermatozoa at 30 degrees C was unaffected following the addition of 2 mM CaCl(2), 100 nM calyculin A, an inhibitor of protein phosphatase-type 1 (PP1), or 4 mM diB-cAMP, respectively, compared with those with no effectors. However, the presence of 10 microM ML-7, a selective inhibitor of myosin light chain kinase (MLCK), inhibited motility of spermatozoa from both species. At 40 degrees C, the presence of CaCl(2) or calyculin A restored the motility of spermatozoa from both species, but the addition of diB-cAMP or ML-7 could not prevent the immobilization of spermatozoa. At 30 degrees C in the presence of ATP, the motility of demembranated copper pheasant spermatozoa was over 60% but was inhibited following the addition of 10 microM ML-7; a similar pattern was found with demembranated domestic fowl sperm motility. The motility of demembranated spermatozoa from both species was inhibited following the addition of 2mM EGTA to the reactivation medium at 30 degrees C, but restored by the subsequent addition of 4 mM CaCl(2). These results suggest that copper pheasant sperm motility might be regulated by similar mechanisms to that of domestic fowl spermatozoa: i.e., the balance of Ca(2+)/MLCK or an MLCK-like protein-dependent phosphorylation and PP1-dependent dephosphorylation. The similarity in physiological regulation of spermatozoa from both species shows that extensive technology developed for artificial breeding of the domestic fowl might be applicable to captive breeding of copper pheasants.

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.

Characteristics of Okinawan native agu pig spermatozoa after addition of low-density lipoprotein to freezing extender.

Technical refinement of boar sperm cryopreservation is indispensable for effective breeding of the rare Okinawan native pig, the Agu. The objective of the present study was to determine whether addition of low-density lipoprotein (LDL) extracted from hen egg yolk to the freezing extender improves the characteristics of cryopreserved Agu spermatozoa. Ejaculated Agu sperm frozen in extender supplemented with 2, 4, 6, 8 or 10% LDL instead of egg yolk was thawed, and the post-thaw sperm characteristics were evaluated. Treatment with 4-8% LDL during cooling and freezing significantly increased the intracellular cholesterol content, as compared to that of sperm frozen in extender containing 20% egg yolk (P<0.05). Higher potential resistance to cell damage from cryoinjury was also observed in sperm frozen in extender supplemented with LDL: the integrities of plasmalemma and DNA, mitochondrial activity and proteolytic activity of the acrosomal content in the post-thaw sperm were superior to those of sperm that were not treated with LDL. Moreover, the percentages of total motile sperm and the extent of rapid progressive motility at 1 and 3 h after incubation were markedly higher in sperm treated with 4 or 6% LDL, and these sperm also had more ATP. However, LDL did not inhibit in vitro sperm penetrability, even though the cholesterol content of post-thaw sperm was higher after treatment with LDL. These findings indicate that addition of 4-6% LDL instead of egg yolk to the freezing extender improves the post-thaw characteristics of Agu sperm by protecting sperm against cold shock damage during cryopreservation.

Antihyaluronidase action of ellagic acid effectively prevents polyspermy as a result of suppression of the acrosome reaction induced by sperm-zona interaction during in vitro fertilization of porcine oocytes.

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.

The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17alpha-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ERalpha-selective agonist), diarylpropionitrile (DPN, an ERbeta-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ERalpha-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ERbeta-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.