岡田 幸之助

Japanese field vole (Microtus montebelli) is a wild-derived rodent and have unique characteristic. Thus, these species have been expected as model animal. This study was performed to develop novel superovulation procedure for Japanese field vole. First, when 30 IU pregnant mare's serum gonadotropin (PMSG) and 30 IU human chorionic gonadotropin (hCG) were administrated 48 hours apart, females showed higher response to hCG compared with three concentrations of PMSG. Second, to effectively induce ovulation on females after vaginal opening, they were mated with vasectomized male instead of hCG administration. Average number of ovulated oocytes using PMSG mating (13.9 +/- 1.9 oocytes) was higher than PMSG-hCG (control; 6.9 +/- 23 oocytes) or PMSG-hCG mating (6.8 +/- 0.8 oocytes). Finally, we attempted superovulation using GnRH agonist (GnRHa). With this treatment, we speculated that GnRHa might induce endogenous luteinizing hormone releasing to cause ovulation. Such superovulation was performed with 30 IU PMSG and different concentration of 20% polyvinylpyrrolidone-GnRHa (15, 30, 45, and 60 mu g/kg). As results, average number of ovulated oocytes was highest with 30 mu g/kg GnRHa (14.5 +/- 4.1 oocytes). The numbers of ovulated oocytes of other concentrations were 5.0 +/- 1.4 (15 mu g/kg), 12.8 +/- 2.7 (45 g/kg), and 8.8 +/- 3.7 oocytes (60 g/kg). Nuclear status of most collected oocytes was the second meiotic division (range, 94.3%-100%). These superovulation procedures will be useful for development of in vitro culture systems and assisted reproductive technologies for not only Japanese field vole but also other voles. (C) 2016 Elsevier Inc. All rights reserved.

Pentobarbital sodium (Somnopentyl) can induce surgical anesthesia with a strong hypnotic effect that causes loss of consciousness. Animals have been known to die during experimental surgery under anesthesia with Somnopentyl, causing it to be declared inadequate as a general anesthetic for single treatment. An anesthetic combination of 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam and 5.0 mg/kg butorphanol (M/M/B:0.3/4/5) was reported to induce anesthesia for a duration of around 40 min in ICR mice; similar anesthetic effects were reported in both male and female BALB/c and C57BL/6J strains of mice. However, the anesthetic effects of this combination in Japanese field vole, Microtus montebelli, remain to be evaluated. In the present study, we assessed the effects of Somnopentyl and different concentrations of anesthetic combination (M/M/B:0.3/4/5, 0.23/3/3.75 or 0.15/2/2.5) in Japanese field voles, by means of anesthetic scores. We also examined effect of these anesthetics on production of offspring. Death of the animals was observed only with Somnopentyl. The anesthetic score of Somnopentyl was lower than those of the other anesthetics, although there were no significant differences in duration, body weight and frequency of respiratory among the evaluated anesthetics. Abortion rate with Somnopentyl was significantly higher than that with the M/M/B:0.23/3/3.75 combination, although there was no significant difference in the number of offspring between two. In conclusion, results of this study provide basic information for achieving appropriate anesthetic concentrations in addition to indicating a new, safe and effective surgical anesthetic for Japanese field voles.

This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 mu g GnRH (gonadotropin-releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 mu g GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 13 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.

Diagnosis of corpus luteum (CL) function by rectal palpation (RP) has been widely used for recipient selection of embryo transfer (ET), a technology essential for genetic improvements in cattle. To examine the accuracy of RP diagnosis method, the relationship between RP-based CL function and reproductive performance was compared in this study. In Experiment 1, CL of Holstein heifers on day 7 after estrus was classified into functional or hypoplastic by RP, and the results were compared with ultrasonographic (US) images and plasma progesterone (P4) levels. As a result, heifers with functional CL judged by RP had a mean maximum CL diameter of 20.1 +/- 3.1 mm on US and a mean P4 concentration of 8.1 +/- 2.3 ng/mL. These values were significantly greater than those of heifers with hypoplastic CL (12.4 +/- 5.4 mm, 4.0 +/- 2.8 ng/mL) (P < 0.001). In Experiment 2, the length of the estrus cycle was examined between functional CL and hypoplastic CL. The rate of heifers with a normal estrus cycle length with 1825 days was significantly lower with hypoplastic CL than with functional CL (16/24 vs. 43/46, P < 0.01). In Experiment 3, 543 inseminated heifers were similarly classified by CL function by RP 7 days after estrus. The heifers with functional CL showed higher pregnancy rate compared with the heifers with hypoplastic CL (75.2 vs. 47.9%, P < 0.0001). Finally, the CL function of 66 heifers was examined by RP on day 7 post-estrus, and ET was performed in 49 (74.2%) heifers with functional CL. As a result, 27 (55.1%) of them became pregnant. Taken together, these results reconfirm that RP on day 7 after estrus is useful for selection of heifers with functional CL.

The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (CYTOKERATIN 18, LAMINS A/C, TRANSFERRIN, a-FETOPROTEIN and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies.

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in in vivo produced porcine zygotes, whereas in vitro produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two in vitro techniques commonly used for in vitro embryo production (in vitro fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after in vitro fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that in vitro matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the in vitro technique for embryo production.

The positive effect of strontium ions (Sr(2+)) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr(2+) on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE /DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl(2)), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr(2+)-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p > 0.05). After the microinjection of spermatozoa incubated with SrCl(2), 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr2+-treated groups (27%, p < 0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl(2) (n = 51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n = 48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine "in vivo" produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200-300 cells and is not caused by the difference in the cell volume of ICM and TE cells.

Chromosome separation in meiosis I is different from those in mitosis and meiosis II in that homologs separate from each other in the former while sisters do so in the latter. We show here that meiosis-specific cohesin subunit Rec8 in mouse oocytes shows essentially the same pattern of localization to those reported in yeasts and mammalian spermatocytes; Rec8 along chromosome arm ( armRec8) is lost at the metaphase I-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until the onset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8 antibody into the oocytes inhibits homolog separation but not the first polar body emission ( cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase II oocytes prevents sister separation in anaphase II after oocyte activation. These data demonstrate that the loss of armRec8 and cenRec8 is required for separation of homologs and sisters, respectively, but both are not required for other late mitotic events such as spindle elongation and cytokinesis in mouse oocytes. Further, by using some inhibitors for spindle assembly, proteasome and Topoisomerase II and overexpression of Securin, we propose that loss of armRec8 ( homolog separation) and cytokinesis are suppressed until anaphase I by Securin whose destruction is regulated by spindle checkpoint-proteasome pathway, and that Topoisomerase II is required for homolog separation independently from such pathway.

The aim of present study was to optimize culture conditions for pig embryos. Initially, we evaluated three different basic culture conditions. When embryos from electro-activation (parthenotes) or in vitro fertilization (IVF-embryos) were cultured in PZM supplemented with 3 mg/ ml bovine serum albumin (PZM-3) in 4-well dishes, in medium covered with oil in 4-well dishes or in droplets under oil, 0%, 33% and 20% of the parthenotes, and 11%, 23% and 20% of the IVF-embryos developed to blastocysts. Subsequently, we examined the development of embryos when they were Cultured in 4-well dishes in medium covered with oil continuously for 7 days or cultured under the same conditions but with a change to fresh medium on Days 2 and 4. In this experiment, 23% (no medium change) and 34% (change) of the parthenotes developed to blastocysts, respectively. When IVF-embryos were cultured under similar conditions, 33% and 38% of the embryos developed to blastocysts. Further improvement was achieved when PZM was supplemented with FBS from Day 4. In this experiment, 47% of the parthenotes developed to blastocysts with an average cell number of 57 +/- 7.7. in IVF-embryo group, 49% of the embryos developed to blastocysts with a mean cell number of 60 +/- 6.1. These results indicate that a change to fresh medium and inclusion of FBS in the medium during the late stages of culture can generate a higher proportion of high-quality blastocysts.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

This work was undertaken to investigate the activation of pig oocytes after microinjection of crude sperm extract prepared from ejaculated boar spermatozoa. The results were compared with those of electro-stimulated oocytes. When in vitro-matured pig oocytes were microinjected with sperm extract and others were electro-stimulated, 100% and 92%, respectively, of the oocytes were released from arrest at metaphase II (MII) and formed female pronuclei. To test their developmental ability, the injected oocytes were treated with cytochalasin B and then cultured in NCSU23 medium. After 168 h, 30% and 44% of the oocytes that had been microinjected with sperm extract and electro-stimulated, respectively, developed to the blastocyst stage. At 6-8 h after the microinjection of sperm extract or electro-stimulation, cortical granules were released from the oocytes. In addition, Cdc2 kinase activity declined to a low level in the treated oocytes. These results indicate that microinjection of crude sperm extract induces the release of in vitro-matured pig oocytes from MII-arrest and leads them into a series of events related to oocyte activation. © 2004, JAPANESE SOCIETY OF OVA RESEARCH. All rights reserved.

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Call. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ball on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ Concentration ([Ca2+],) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+](i). The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ball, like that of Call, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca2+ in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5degreesC) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 muM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 muM). However, the promoting effects of these reagents were blocked when spermatozoa. were pretreated with a cell-permeable Ca2+ chelator (BAPTA-AM, 25 muM), whereas the same pretreatment with a cell-impermeable Ca2+ chelator (BAPTA, 25 muM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca2+-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 muM. When 4 muM thapsigargin and 10 muM cBiMPS were examined for their effects on free Ca2+ levels in sperm heads by using a cell-permeable Ca2+ indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca2+-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca2+ is involved in sperm head-to-head agglutination induced by a cAMP analogue.

In the present study, we derived parthenogenetic porcine fetuses from in vitro-matured oocytes following a simple activation process in order to evaluate their developmental limitations in-vivo. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h. They were subjected to a single square pulse of direct current for 100 mu sec at 1,500 V/cm and then treated with 5 mu g/mL cytochalasin B for 4 h to obtain activated diploid oocytes. The diploids were cultured in modified Whitten's medium until transfer. Diploids which had cleaved to the 2- and 3- to 4-cell stages were transferred to oviducts of recipients. Live and/or dead parthenogenetic fetuses were recovered in 6 of 8 trials at 17, 18, 19, 24 and 29 d post activation. The total proportion of fetuses to transferred diploids was 31.3% (62/198). When fetuses were recovered at 19 d post activation, the proportion of development into fetuses was 71% (15/21). Our results, however, suggest that periods of gestation longer than 19 d resulted in a decrease of these proportions to 45% (18/40) at 24 d and to 18% (7/40) at 29 d. The hearts were beating in nearly all of the fetuses recovered at 19, 24 and 29 d post activation. Thus, parthenogenetic porcine diploids developed to at least the stage of limb-bud formation beyond the early heart-beating stage. Abnormalities were also externally visible on some fetuses. Formation of cyst-like structures in the heart and liver, and insufficient development of the head region and acephali were observed in some cases. (C) 2000 by Elsevier Science Inc.