Involvement of N-glycosylation of zona glycoproteins during meiotic maturation in sperm-zona pellucida interactions of porcine denuded oocytes.

The present study was conducted to delineate whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N-glycosylation played a role in sperm-ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM on sperm-ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N-glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm-ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP-bound sperm. However, the inhibition of N-glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N-glycosylation was involved in the initial stage of fertilization. We conclude that 24-44 h of N-glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.

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.

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.

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.

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.

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.

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes.

The present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2-10 microg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 microg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 microg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 microg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.

Anti-hyaluronidase oligosaccharide derived from chondroitin sulfate a effectively reduces polyspermy during in vitro fertilization of porcine oocytes.

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 microg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1-100 microg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 microg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10-100 microg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25-30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 microg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 microg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.

Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid.

To elucidate the beneficial effects of porcine follicular fluid (pFF) added to maturation medium on the sustenance of cytoplasmic maturation responsible for the subsequent developmental competence after in vitro fertilization (IVF) of porcine oocytes, we focused on the antioxidative role of pFF in its function of protecting oocytes from reactive oxygen species (ROS)-induced cell damage. Porcine follicular fluid collected from small (2-6 mm) follicles had about 7.2-fold higher levels of superoxide dismutase (SOD) activity than that of fetal bovine serum (FBS), and this activity was markedly blocked by the CuZn-SOD inhibitor, diethyldithiocarbamate (DETC). The interruption of meiotic progression and the increasing intracellular glutathione (GSH) content throughout the maturation period, as well as an outbreak of DNA damage in oocytes and cumulus cells were difficult to detect in oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system, whereas cell damage encompassed by ROS was prominent in oocytes cultured with 10% FBS and 10% pFF plus 100 microM DETC. Similarly, significant enhancement to the degree of transformation of the sperm nucleus into the male pronucleus (MPN) after in vitro fertilization was shown by the addition of pFF to the maturation medium. The presence of DETC during in vitro maturation reduced the ability of oocytes to promote MPN formation to the same extent as oocytes matured with FBS. The proportion developing to the blastocyst stage was increased in oocytes that matured with pFF, but this developmental competence was significantly lowered by treatment with DETC (P < 0.05). These findings suggest that pFF plays a critical role in protecting oocytes from oxidative stress through a higher level of radical scavenging activity elicited from SOD isoenzymes, resulting in the enhancement of cytoplasmic maturation responsible for developmental competence postfertilization.