Embryo cryopreservation is one of the important technologies in various research and clinical areas. Very recently, carboxylated ε-poly-L-lysine (COOH-PLL) has been developed as a new cryoprotective regent, and we previously demonstrated that combined treatment of COOH-PLL with ethylene glycol (EG), which is a commonly used cryoprotective regent, is effective for vitrification of unfertilized mouse oocytes and pronuclear embryos. In this study, we examined different concentration of COOH-PLL and tried to vitrify the embryos at the different stages beyond the 2-cell stage using the same protocol. Embryos at the pronuclear, 2-cell, 4-cell, 8-cell, morulae, and blastocyst stages were treated with 7.5% (vol/vol) EG and 7.5% (vol/vol) COOH-PLL for 3 min and then vitrified with 15% EG (vol/vol) and 15% (vol/vol) COOH-PLL (E15P15) for 1 min by the Cryotop. The embryos except for pronuclear embryos showed low rates of survival, blastocyst, and hatched blastocysts. To improve the survival and developmental ability of the vitrified embryos, blastocysts were vitrified with different concentrations of COOH-PLL. At the results, the survival and developmental rates of vitrified blastocyst in E20P10 group were higher (98.0 ± 2.5% and 93.3 ± 4.0%) than those of the E30, E25P5, and E15P15 groups (P < 0.05). When 2-cell, 4-cell, and 8-cell embryos or morulae were vitrified with E20P10, survival, blastocyst, and hatched blastocyst rates were dramatically improved (more than 86.0%). Taken together, vitrification with 20% COOH-PLL and 10% EG is available for vitrification of mouse pre-implantation embryos at the different stages.

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.

Although cryopreservation of pronuclear-stage embryos has increasing importance in the production of genetically modified animals via microinjection, it is well known that cryopreserved porcine embryos show low developmental ability. Cryopreservation causes damage to organelles such as mitochondria that are involved in metabolism and play important roles in embryonic development. To reduce the damage in embryo cryopreservation, we focused on carboxylated ε-poly-l-lysine (COOH-PLL), which has been recently developed as a new cryoprotective agent. Carboxylated ε-poly-l-lysine has antifreeze protein properties, such as inhibition of ice recrystallization (Matsumura and Hyon 2009). In this study, we examined the effect of COOH-PLL on development and mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Porcine follicular oocytes were matured (in vitro) and then fertilized (IVF) in vitro. At 10 h after IVF, the presumptive embryos were centrifuged to visualise the pronuclei. Embryos with 2 or 3 pronuclei (PN embryos) were used for vitrification. The PN embryos were exposed to equilibration solution for 10 min and then exposed to vitrification solution for 1 min before being plunged into LN on Cryotop®. Vitrification solution was phosphate buffered saline supplemented with 30% (vol/vol) ethylene glycol + 0.5 M sucrose + 20% (vol/vol) fetal calf serum + 0 or 20% (wt/vol) COOH-PLL (P0 or P20). Development to the blastocyst stage of the vitrified PN embryos was observed after in vitro culture for 158 h. Nonvitrified embryos were used as a control (fresh). Fresh and vitrified with P0 or P20 PN embryos were stained with MitoTracker® Red CMXRos. The embryos stained with MitoTracker® Red CMXRos were investigated by using a laser-scanning confocal microscope. Mitochondrial fluorescent intensity of embryos was evaluated by measuring the mean numbers of pixels in the fluorescent area (mean/area) using the NIH ImageJ software. The blastocyst rate of embryos vitrified with P20 (19.4%) was significantly (P < 0.05) higher than that of P0 (1.3%), and there were no significant differences in blastocyst rates between P20 and fresh group (28.4%; P > 0.05). The mitochondrial fluorescent intensity of embryos vitrified with P20 (2.8 mean/area) was significantly higher than that of P0 (1.5 mean/area), but the intensity of embryos vitrified with P20 was significantly lower than that of fresh (4.2 mean/area; P < 0.05). Our results suggest that supplementation of COOH-PLL with vitrification solution improved development and also reduced mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Further studies are required to clarify the effect of COOH-PLL on various kinds of damage caused by cryopreservation such as other organelles, including endoplasmic reticulum damage, cytoskeleton damage, or DNA fragmentation of vitrified or warmed embryos in pigs.

Temperature, humidity, oxygen concentration, bacteria, and airborne volatile organic compounds (VOCs) have been reported to influence the development of mammalian embryos during in vitro culturing. Identifying and managing culture embryos, we are able to use a pen or labels for writing patient's information on the plastic ware. There has been concern that the VOCs originated from the ink of felt-tip pens, which are too small to be removed with the HEPA filter, might cause the decrease of the developmental rate of culture embryos. On the other hand, verification report is less about the labels. This study was conducted to investigate effects of affixing labels on culture dishes on development of the embryos in the mouse. Prospective experimental Animal study. We performed ovarian hyperstimulation on 8-week-old female BDF1 mice by administering eCG 7.5 IU or hCG 7.5 IU. After rearing these females in the same cage with males of the BDF1, fertilized eggs were collected from oviducts of the plug-positive females. Plastic dishes (Falcon) with a 35-mm aperture were prepared with five 20-μL drops of M16, covered with mineral oil (Vitrolife), placed in a 60-mm dish (Falcon), and then cultured at rest in an incubator (HC-3100 ASTEC). Five to ten embryos were cultured per drop. Labels using acrylic adhesive were affixed to the inside of the lid of the 60-mm dish, and culturing was performed at 37°C, 5% CO2, 5% O2, 90% N2, and saturated humidity. The number of affixed labels totaled 0 (control group), 1 (group A), 7 (group B), or 13 (group C), and development of the cultured mouse embryos from the 1-cell stage was investigated for 96 hours. Statistically analysis between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons. Following the approval by the Animal Care and Use Committee of Azabu University, we used 263 mouse embryos in this study, which was performed at the Azabu University in the duration from September 2015 to December 2015. The cleavage rate of the control group and groups A, B, and C were 97.5%, 97.1%, 12.0%, and 9.5%, respectively, with significant (P<0.001) decreases in groups B and C compared with the control; the developmental rate to the blastocyst stage were 88.6%, 0%, 0%, and 0%. As increasing the number of label, the incidences of cleavage of the cultured embryos were reduced, and there was no embryo developed to the blastocyst stage in all of the label-affixed groups. Affixing labels using acrylic adhesive on culture dishes have detrimental effects on the development of mouse embryos at 1-cell stage. It is concluded that careful consideration should be given to the adhesive's components when using label stickers for culture dish management.

Unfertilized oocytes are one of the most desired germ-cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including IVF and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. We have recently reported that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence (Kohaya et al. 2011 J. Reprod. Dev. 57, 675–680). Since the previous study was carried out using ICR mice (closed colony), we examined whether our protocol can be applied for C57BL/6J mice (inbred strain), which are commonly used for production of transgenic and knockout mice. The effect of cumulus cells on the ability of C57BL/6J mouse oocytes to be fertilized and develop in vitro was examined. Cumulus oocyte complexes (COC) derived from female mice with super ovulation were collected by flushing. Cumulus cells were removed for a portion of the oocytes (DO) using hyarulonidase. Oocytes from both treatment groups (COC and DO) were then vitrified according to the protocol we previously reported (Kohaya et al. 2011). After warming, vitrified COC and DO were used for IVF. All percentage data were subjected to arcsine transformation before statistical analysis. Data were analyzed by one-way ANOVA and Tukey’s test. Significance was considered at P < 0.05. The pronuclear formation rate of vitrified DO after IVF (20/58, 33.3%) was reduced compared with vitrified COC (55/90, 62.1%). Vitrified COC showed significantly (P < 0.05) higher developmental ability to develop into the 2-cell (50/90, 57.0%) and blastocyst stages (42/90, 45.9%) compared with vitrified DO [24.8% (16/58) and 18.4% (11/58), respectively]. The vitrified COC developed to term at a high success rate (51/90, 56.7%) being equivalent to the rate obtained with IVF using fresh COC (52/90, 57.8%). Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence. These findings will contribute to improve oocytes vitrification in not only experimental animals but also in clinical application in human infertility.

In rats, successful IVF with cryopreserved rat sperm has never been reported. The objective of the present study was to establish and improve the IVF protocol using epididymal and ejaculated rat spermatozoa after cryopreservation. At first, we examined whether cryopreserved ejaculated spermatozoa would be useful for IVF in Wistar rats. Capacitation-associated tyrosine phosphorylation was accelerated in frozen-thawed ejaculated sperm in a time-dependent manner. These frozen-thawed spermatozoa were co-cultured with cumulus–oocyte complexes in modified R1ECM for 10 h. The putative zygotes were transferred to R1ECM and then cultured up to 144 h. Although the rates of insemination and 2 pronucleus (2PN) formation were low (26.5 and 23.0%, respectively), most of 2PN oocytes were developed to the 2-cell stage (91.0%). A total of 44 embryos at the 2-cell stage derived from frozen-thawed ejaculated sperm were transferred to 5 recipient females and 21 pups (47.7%) were delivered. Next, we used frozen-thawed epididymal spermatozoa for IVF in Wistar rats. After thawing, intracellular cyclic adenosine monophosphate (cAMP), free cholesterol levels and capacitation-associated protein tyrosine phosphorylation levels of the sperm were assayed. Intracellular cAMP and free cholesterol levels in frozen-thawed epididymal sperm were maintained at a low level, suppressing capacitation-associated tyrosine phosphorylation. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX) dramatically increased cAMP and capacitation-associated tyrosine phosphorylation levels in frozen-thawed epididymal sperm. When the IBMX-treated frozen-thawed sperm was used for IVF, the proportions of 2PN and the development to blastocysts were significantly higher (approximately 40 and 20%, respectively) than those of frozen-thawed epididymal sperm treated without IBMX (approximately 10 and 3%, respectively). These embryos were developed to term at a high success rate (49%) equivalent to the rate obtained with IVF using fresh sperm (58%). Moreover, we tried to apply our IVF system to inbred rat strains [Fischer 344 (F344) and Brown-Norway (BN)]. We examined whether the IVF protocol was available for F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, capacitation-associated tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Taken together, we developed an IVF protocol using cryopreserved rat sperm and our data suggest that the IVF system can be applied not only to Wistar rats but also to the F344 strain. This research was also partially supported by a research project grant awarded by the Azabu University Research Services Division.

In mammalian oocytes, artificial activation is essential for further embryonic development of somatic cell nuclear transferred or round spermatid-injected oocytes. Unlike the protocol for mice, the optimal activation protocol for rat oocytes is not well established. Recently, we showed for the first time that a combined treatment with ethanol and 6-dimethylaminopurine (6-DMAP) efficiently induced activation of rat oocytes, resulted in a higher proportion of blastocyst formation as compared with other combined treatments or with the 6-DMAP treatment alone (Sano et al. 2009 Zygote 17, 29–36). Although the ethanol treatment has been used for oocyte activation not only in rats, but also in other species to date, it is unclear why the ethanol treatment is effective, especially in activating rat oocytes. Because it has been reported that alcohol alters intracellular pH via exchangers, transporters, or both on the membrane surface in the myocardium, it leads us to the possibility that the ethanol treatment regulates such exchangers, transporters, or both in rat oocytes. The aim of the present study was to clarify the role of Na+/H+ exchanges (NHE) in activating rat oocytes treated with ethanol. First, we examined which types of NHE are expressed in rat ovaries, oocytes, and parthenogenetic embryos. Expression of the NHE1 protein was also confirmed in both oocytes and parthenogenetic embryos, but not in NHE2 and NHE3. Treatment with 5-(N-ethyl-N-isopropyl)amiloride hydrochloride, an NHE inhibitor, did not affect the development of ethanol-activated oocytes to the 2-cell stage; however, it decreased the rate of blastocyst formation in a dose-dependent manner. The 5-(N-ethyl-N-isopropyl)amiloride hydrochloride treatment did not decrease development to either the 2-cell or the blastocyst stage of oocytes activated by 6-DMAP alone. Taken together, we conclude that ethanol treatment was involved in NHE1 activation, resulting in high developmental ability of rat oocytes. This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS (KAKENHI; 21780253) to J. I. The work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities, to J. I. and N. K.

Although cloned animals have been reproduced in many species via somatic cell nuclear transfer (SCNT), only 1 previous report showed successful generation of cloned rats. Some researchers evaluated developmental competence of reconstructed rat embryos and showed that these reconstructed embryos were never progressed beyond the 2-cell stage because of inadequate formation of the mitotic assembly and nuclear organisation or improper transcription of cytoskeleton genes. Even though our group also improved the proportion of 2 pronuclear formation in rat reconstructed embryos by the treatment with a proteasome inhibitor, MG132 (Nakajima et al. 2008 Cloning and Stem Cells 10, 461–468), no embryos were developed beyond the 2-cell stage. Recently, it has been demonstrated that a histone deacetylase inhibitor, trichostatin A (TSA) treatment dramatically improves efficiency of cloning in the mouse, although the precise effect of TSA is unclear in SCNT. However, in the case of rats, availability of TSA has not been tested. The aim of the present study was to clarify whether TSA treatment was also effective in rat SCNT. According to our previous reports, rat oocytes were collected and the spindles of the oocytes were removed in medium supplemented with MG132. After enucleation, nuclei of cumulus cells were injected in the oocytes and the reconstructed embryos were cultured in the medium supplemented with 0 (control), 5, 50, or 500 nM TSA. More than 100 embryos are used for each treatment group. Pronuclear formation, development to the 2-cell stage and blastocyst formation were evaluated. Our results demonstrated that TSA treatment affected neither the proportion of pronuclear formation nor development to the 2-cell stage in rat reconstructed embryos. However, the treatment with higher concentrations of TSA treatment (50 nM and 500 nM) compared to the concentration (5 nM) which was usually used for mouse SCNT enabled the reconstructed embryos to develop to the blastocyst stage but at low rate (1.4 to 2.2%). In both the 5 nM treatment and control groups, none of the rat reconstructed embryos developed to blastocyst. Taken together, our data suggests that TSA treatment also seems to be effective in rat SCNT. These findings will be useful for improvement of the protocol in rat SCNT. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities to J. I. and N. K.

Primordial follicles act as stores of ovarian follicles and are potential sources of oocytes for medical, agricultural, and zoological purposes. Ovarian xenografting seems to be advantageous for maturing the oocytes in primordial follicles (primordial oocytes) and useful for the conservation and reproduction of domestic or endangered animals. We have generated viable embryos from porcine primordial oocytes xenografted into nude mice (Kaneko et al. 2006 Reproduction). Xenografting of ovarian tissues after cryopreservation would be a very powerful tool for this purpose. Recently, Moniruzzaman et al. 2009 Theriogenology) reported that follicles were able to develop to the pre-antral stage in ovarian tissues after cryopreservation and xenografting, but that oocytes were not obtainable from them. In the present study, we vitrified the tissue after different immersion periods in a cryoprotectant, ethylene glycol (EG), and evaluated the possibility of oocyte collection, and also their maturation and fertilization abilities. Ovarian tissue from piglets approximately 20 days old was minced into cubes of about 1.0 to 2.0 mm. After equilibration in 4% EG in IVC-PyrLac (Kikuchi et al. 2002 Biol. Reprod.) as a base solution (BS) for 15 min, they were immersed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BS) for 45 s or 7 min (45-s and 7-min immersion groups, respectively), then dropped with about 4 μL of vitrification solution into liquid nitrogen (LN2). After storage in LN2, microdroplets were transferred in warming solution (0.4 M trehalose in BS) at 37°C for 2 min, then consecutively transferred for 2-min periods into 0.2 M, 0.1 M, or 0.05 M trehalose in BS.As described previously (Kaneko et al. 2006 Reproduction), 20 to 30 pieces of tissue were grafted into kidney capsules of ovariectomized nude mice. The host mice were treated with porcine FSH (62.5 U mL-1 in osmotic pomp) for 12 days before assessment of the survival of the ovarian grafts. When antral follicles were evident in the grafts, oocytes were collected using a surgical blade. They were then matured and fertilized in vitro (Kikuchi et al. 2002 Biol. Reprod.). Ovaries containing antral follicles were obtained between 62 and 125 days after grafting from 6 out of 12 mice in the both 45-s and 7-min groups. When a total of 39 and 49 fully grown oocytes, respectively, had been collected from these groups and cultured, the maturation rates calculated on the basis of 1st polar body extrusion were 18% (7/39) and 33% (16/49), respectively. The corresponding rates for sperm-penetrated oocytes were 83% (5/6) and 88% (14/16), respectively. All the oocytes formed male and female pronuclei at 10 h after insemination. The rates in the 2 immersion groups did not differ significantly by chi-square test, with or without Yates’ correction. In conclusion, fully grown porcine oocytes can be collected from primordial follicles that have been cryopreserved and xenografted into nude mice. The period of immersion before vitrification may not affect oocyte maturation or fertilization ability.

In rats, the success of in vitro fertilization (IVF) was reported almost 40 years ago. Although it had been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported. Very recently, we reported establishment of a successful IVF system using frozen-thawed spermatozoa treated with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), in Wistar rats (Seita et al. 2009 Biol. Reprod.). The objectives in this study were (1) improvement of the IVF system to a more convenient and simple protocol and (2) a preliminary study for applying our IVF system to inbred rat strains Fischer 344 (F344) and Brown-Norway (BN). In experiment 1, we examined the effect of preincubation time for frozen-thawed sperm on fertilization (2 pronuclei formation). Frozen-thawed sperm were preincubated up to 6 h and then used for IVF according to our previous report. Data showed that sperm preincubated for 5 h contributed to higher fertility than those for other preincubation times. In experiment 2, we examined the effect of co-culture time up to 10 h for IVF on fertility and embryonic development in vitro. The oocytes co-cultured with sperm beyond 6 h showed higher fertilization and blastocyst formation rates than those in 2 and 4 h. In experiment 3, we examined the effect of initial culture period in fertilization medium (310 mOsm modified R1ECM; O h et al. 1998 Biol. Reprod.) on embryonic development in vitro. After IVF, oocytes were cultured in fertilization medium for 6, 12, or 24 h and further cultured in R1ECM up to 120 h. Cleavage rates were not affected by initial culture time in fertilization medium. However, oocytes cultured in fertilization medium for 12 h showed higher blastocyst formation rate than those for 0, 6 and 24 h. In experiment 4, we examined whether the IVF protocol can be applied to F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Our data demonstrate that (1) a more efficient IVF system using frozen-thawed rat sperm was developed and (2) the IVF system can be applied to at least F344 strain. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

In mammals, repetitive increases of the intracellular Ca2+ level, known as Ca2+ oscillations, are observed in oocytes immediately after sperm-oocyte fusion, which is a prerequisite event for oocyte activation. Previous studies indicate that phospholipase C zeta (PLCζ), a strong candidate sperm factor for triggering Ca2+ oscillations, is localized in the sperm head of several mammalian species. We have reported that the rate of pronucleus formation in oocytes injected with a sperm head is lower than that for oocytes injected with a whole spermatozoon (Nakai et al. 2009 IETS). This has given rise to a hypothesis that not only the sperm head but also the tail play a role in inducing oocyte activation in pigs. In this study, we attempted to detect the localization of PLCζ in the pig sperm tail and also its ability to activate porcine oocytes after injection. To clarify the localization of PLCζ in pig sperm, frozen-thawed ejaculated pig sperm were immunostained using an anti-PLCζ antibody that has been reported previously (Kurokawa et al. 2005). Western blotting was also carried out to examine whether PLCζ (72 kDa) was present in the sperm tail. Sperm tails were detached from the head by sonication and then collected after centrifugation in a Percoll density gradient. We also confirmed whether the sperm tail itself had the ability to trigger oocyte activation using the following 4 injection groups: (1)1 sperm head (Head), (2) 1 sperm tail (Tail), (3) 1 sperm head and 1 tail (Head + Tail), and (4) Sham. The nuclear status of the injected oocyte was evaluated at 10 h after injection. In the present study, we used 3 sperm samples that were prepared from different boars. In pig sperm, the acrosome, tail, and post-acrosomal regions were stained by the PLCζ antibody. The signals in both the post-acrosomal and tail regions disappeared after pretreatment with antigenic peptide, but that in the acrosome region was retained. Furthermore, we confirmed the presence of a band of approximately 72 kDa from the sperm tail and also confirmed its disappearance upon pretreatment with antigenic peptide. The rates of oocytes released from metaphase-II arrest in the Head, Tail, and Head+Tail groups were significantly higher than that in the Sham group (P < 0.05 by ANOVA andTukey test). However, most of the oocytes in the Tail group failed to form pronuclei and showed other meiotic stages (anaphase-II, telophase-II, or metaphase-III). In conclusion, we have shown that PLCζ is expressed in the post-acrosomal and tail region of pig sperm. It is suggested that, in the pig, the sperm tail participates in the triggering of oocyte activation. The authors thankRafaelA. Fissore (Department ofVeterinary and Animal Sciences, University of Massachusetts Amherst) for providing the antigenic peptide for PLCζ. This study was supported in part by JSPS Fellowship (71310042 to M.N.) from the Japanese Society for Promotion of Science (JSPS).

Cryopreservation of reproductive organs is an important technology for preservation of genetic resources of experimental, domestic, and wild animals. In addition, cryopreservation of the ovary could be applied to restore the fertility of young women diagnosed with cancer because it could not only provide future fertility, but could also decrease the emotional consequences of cancer therapy for women afflicted with such devastating diseases. Cryopreservation of whole ovary particularly would enable such females to be pregnant by natural mating after transplantation. The aim of the present study was to evaluate the possible of vitrification for young rat ovaries as a possible model for human ovaries using a Cryotop method. Whole ovaries were collected from 10-day-old female rats (Brown-Norway × Wistar) and then washed several times in PB1 medium containing 20% (v/v) fetal calf serum (FCS). The ovaries were submerged in equilibration solution [10% (v/v) dimethyl sulfoxide (DMSO), 10% (v/v) ethylene glycol (EG), and 20% (v/v) FCS in PB1] for 1, 3, 5 or 10 min and then vitrified in vitrification solution (20% DMSO, 20% EG, and 20% FCS in PB1) for 5 min, being plunged into liquid nitrogen on Cryotops. Vitrified ovaries were later warmed in 37°C in PB1 containing 1 M sucrose and 20% FCS for 5 min and then PB1 containing 0.5 M sucrose and 20% FCS for 5 min. Eight ovaries were embedded in paraffin and cut into 4 μm slices. The slices were stained by hematoxylin-eosin (HE) and integrity of nuclei and cytoplasm in the follicles was histologically evaluated. In each group, 3 ovaries were used for the staining of live/dead (Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, #L3224). Follicles were separated from the ovary with enzyme and then classified into 2 categories by staining. Follicles possessing more than 50% of green-stained and red-stained granulosa cells were divided as live and dead, respectively. Data were analyzed by Tukey’s test. Results of the HE staining showed that integrity of nuclei and cytoplasm in ovaries equilibrated for 10 min (11%) was lower than those for 1 min (25%), 3min (42%), and 5 min (30%). As for evaluation by live/dead staining, most of follicles in ovaries equilibrated for 10 min (25%) were stained with red (dead). In vitrified ovaries equilibrated for 1 min, 3 min, and 5 min (18%, 16%, and 18%, respectively), there were no significant differences in red-stained follicles. These results suggest that optimal equilibration time is 3 to 5 min for vitrification of whole ovaries of 10-day-old rats. Using this equilibration time, we are now trying to evaluate the capability of vitrified/warmed ovaries after transplantation to recipient rats. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J. I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

Contrary to experimental animals, it is well known that pig oocytes show low developmental competence after IVM, fertilization and culture despite of the attempts to improve the IVM technology. One of the reasons causing such low developmental ability of porcine oocytes seems to be the culture condition, especially the gas phase for IVM because there is a large differences in oxygen tension between in vitro and in vivo conditions. Indeed, our preliminary study revealed that oocytes matured in vivo had larger perivitelline space than oocytes matured in vitro, which could be affected by in vitro culture condition. The present study was conducted to evaluate the effect of oxygen tensions on nuclear maturation, cumulus expansion and glutathione synthesis of porcine oocytes during IVM. COCs at the germinal vesicle stage were collected from ovaries of prepubertal gilts and cultured in modified NCSU37 either under 2, 5, or 20% O2 for 44 h (Group 2%, Group 5%, and Group 20%, respectively). Five percent CO2 was used for all groups. After culture, the cumulus expansion was morphologically evaluated by classification to three grades (Grade 1: excellent [the length of expanded cumulus cells was longer than the diameter of the oocyte], Grade 2: good [the length of expanded cumulus cells was less than the diameter of oocyte], Grade 3: poor [oocyte having partial or single layer of expanded cumulus cells]). All experiments in this study were replicated more than 5 times. Data were analyzed by ANOVA and then shown as mean ± SD%. The rate of Grade 1 in Group 2% (16.8 ± 8.3%, 32/189) was significantly lower than those in Group 5% (68.2 ± 11.2%, 149/228) and Group 20% (78.6 ± 6.9%, 162/201) (P < 0.05). As for rates of Grade 2 and Grade 3, there were no significant differences between the groups. After evaluation of cumulus expansion, cumulus cells were removed and oocytes were stained by aceto-orcein for evaluation of nuclear maturation. The rates of metaphase II-stage oocytes were 41.0 ± 12.4% (86/210), 47.6 ± 20.5% (119/263) and 47.7 ± 12.9% (100/199) in Group 2%, Group 5%, and Group 20%, respectively. There were no significant differences among the groups. In order to clarify the effect of oxygen concentration on cytoplasmic maturation, COCs were cultured for 44 h and glutathione level of the oocytes was measured by 5,5′-dithio-bis-2-nitro-benzonic acid-glutathione disulfide reductase recycling method. Regardless the oxygen concentration, glutathione level was increased from the start of culture (6.2 ± 3.9 pmol/oocyte). But there were no significant differences in the glutathione level among groups. These results suggest that oxygen concentration during IVM could affect cumulus expansion but not nuclear maturation and cytoplasmic glutathione level in pig oocytes. This work was supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

In pigs, the intracytoplasmic sperm injection (ICSI) procedure alone is insufficient to induce oocyte activation for embryonic development. Artificial activation can be accomplished, such as by electrical pulse-enhanced in vitro development to the blastocyst stage (Nakai et al. 2006). It is well known that the sperm factor (phospholipase Cζ; PLCζ) in spermatozoa, which triggers oocyte activation, is diffused into ooplasm when sperm fuse with oocytes. Our previous study showed that the activation rate of porcine oocytes injected with one sonicated sperm head was significantly lower than that of oocytes injected with a whole spermatozoon or with 3 sonicated sperm heads (Nakai et al. IETS 2007). These results suggest that the sonication treatment per se may affect the quantity of PLCζ in sperm. Furthermore, various pretreatments of sperm besides sonication have been conducted (e.g. removal of the sperm membrane) to increase the efficacy of ICSI. In this study, we investigated the effect of pretreatments (sonication, Triton X-100, and repeated cycles of freezing–thawing without cryoprotectant) on the quantity of PLCζ in porcine sperm. Cryopreserved-thawed boar-ejaculated sperm were used for 3 experimental groups: (1) sperm were sonicated for 10 s in pig-fertilization medium (pig-FM; Suzuki et al. 2002; Soni group), (2) freezing–thawing was repeated 3 times in pig-FM without cryoprotectant (3-F/T group), or (3) sperm were incubated in pig-FM supplemented with 0.1 or 1% Triton X-100 at 37°C for 1 min (0.1 and 1% Triton X-100 groups, respectively). Cryopreserved-thawed whole sperm without any treatment was used as a control. Results from staining with fluorescein diacetate and propidium iodide showed that almost all sperm were propidium iodide positive (dead sperm) immediately after the each treatment. In the control group, approximately 40% of sperm were fluorescein diacetate positive (live sperm) after thawing. The presence of PLCζ (72 kDa) was examined by Western blotting using the antibody against the N-terminal 19-mer sequence of porcine PLCζ (Kurokawa et al. 2005). A band corresponding to porcine PLCζ was not detected in any treatment group in any culture period (from 0 to 135 min). In contrast, PLCζ was detected in the control group and in all culture periods. These results strongly suggest that PLCζ in porcine sperm was lost immediately after the pretreatments, such as by sonication, incubation with 0.1 or 1% Triton X-100, and repeated cycles of freezing–thawing. The decrease in PLCζ protein by pretreatment may be one of the causes of incomplete activation of oocytes in porcine ICSI. This work was supported by a Grant-in-Aid for JSPS Fellows.

In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca(2+)-dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca(2+)](i). However, little information has been known on events that occur following Ca(2+) induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen-activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular-signal regulated kinase 1 (ERK1) were also phosphorylated in a time-dependent manner. However, the total ERK1 level did not change during the culture. Pre-treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose-dependent manner. Additionally, pre-incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.

During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal [Ca2+] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca2+]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP3R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP3R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP3R1 phosphorylation played a crucial role in the induction of [Ca2+]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP3R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP3R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca2+ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP3R1. According to our previous report in the mouse, IP3R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP3R1 phosphorylation and total IP3R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP3R1 was abundantly expressed at the GV stage. The total level of IP3R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP3R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP3R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP3R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP3R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP3R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34cdc2 kinase and MAPK will be carried out to clarify the relationship between IP3R1 phosphorylation and M-phase kinase(s).

The rat, as well as the mouse, is one of the most valuable experimental animals for biomedical and physiological research. There are numerous valuable mutant rats including transgenetic strains. Cryopreservation of rat oocytes and sperm as haploid germ cells is a key technology for banking the genetic resources efficiently. The aim of the present study was to examine survival of vitrified/warmed oocytes and developmental competence of resultant zygotes in the rat. Rats used in the present study were all Wistar rats. Epididymal spermatozoa were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256). After thawing, spermatozoa were sonicated to obtain sperm heads for intracytoplasmic sperm injection (ICSI). Oocytes were collected from immature females superovulated with eCG and hCG. Oocytes were equilibrated in 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) dimethylsulfoxide (DMSO) + 20% (v/v) FCS in PB1 for 5 min and then transferred into 15.0% EG (v/v) + 15.0% DMSO (v/v) + 20% FCS + 0.5 m sucrose in PB1 (vitrification solution) for 1 min at room temperature (22–24C). During exposure to the vitrification solution, oocytes were loaded on a Cryotop (Kitazato Supply Co., Tokyo, Japan). At warming, the film of Cryotop was directly immersed into PB1 containing 0.5 m sucrose and 20% FCS at 37.5C. The warmed oocytes were washed three times and put into a HEPES-buffered (22 mm) modified R1ECM (310 mOsm) medium. The sperm heads were microinjected intracytoplasmically into the warmed oocytes. Then, presumptive zygotes were transferred surgically into the oviducts of recipient females (Day 0), and Caesarean section of the recipients was performed on Day 22. After vitrification and warming, 245 of 275 (88%) oocytes survived morphologically, 240 of the warmed oocytes were injected, and 156 oocytes (65%) were morphologically normal after the injection. To confirm development to term of zygotes derived from cryopreserved oocytes and sperm, 143 injected oocytes were transferred to 9 recipients, resulting in 3 pregnancies and the generation of one live pup. The results indicate that rat zygotes derived from cryopreserved oocytes and sperm through ICSI can develop to term, and full developmental competence can be preserved in rat oocytes after cryopreservation.