Masao Shino

Viable Piglets Generated from Porcine Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Head Injection

Abstract Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 μM Ca-I was prolonged for 30–120 min (Ca-I treated; 55.6–78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30–120 min (control; 45.3–58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77–150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55–100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection.

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

Offspring Derived from Intracytoplasmic Injection of Sonicated Rat Sperm Heads

ABSTRACT The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no difference was observed in the development in vitro between rat oocytes injected with sperm heads separated by sonication or by Piezo-pulse.

Determination of α2-macroglobulin concentrations in healthy rats of various ages and rats inoculated with turpentine oil by an enzyme-linked immunosorbent assay

Summary An enzyme-linked immunosorbent assay (ELISA) for rat α2-macroglobulin (α2M) was developed, and the concentrations of α2M in sera in normal Crj:CD and Crj:WI rats as well as in turpentine oil injected Crj:WI rats were determined. Anti-rat α2M serum was prepared by immunization of rabbits with the α2M fraction separated from rat acute phase serum using Sephacryl S-300. The specificity of the rabbit anti-rat α2M serum was confirmed by immunoelectrophoresis and Western blotting. The α2M concentrations in 180 normal Crj:CD (90 males and 90 females) and 180 Crj:WI (90 males and 90 females) rats were 14.5–94.9 μg/ml (mean 36.8 ± 13.3) and 13.6–65.8 μg/ml (mean 34.9 ± 12.4), respectively. There were no significant differences between male and female Crj:CD and Crj:WI rats, respectively (p

Effects of FSH, LH and PGF2α on the cell number and steroid secretion by bovine granulosa and luteal cells in vitro

1) FSHは,その添加量が多くなるにしたがい培養 GC及びLCの増殖を促進し,なかんずくLCに対して顕著であった。ホルモン産生面では,GCに対しては,プロジェステロン産生を刺激する傾向がみられたがそれほど強くはなかった。エストロジェン産生については,明瞭な傾向は把握できなかった。LCに対しては,添加量に平行してプロジェステロソの産生を促進した。エストロジェン産生は,若干刺激されるのではないかと思われた。以上はPMSの抑制的なのと反対の成績である。2)LHは,培養GC及びLCの増殖を顕著に促進した。ホルモソ産生面では,GCに対しては,プロジェステロソの産生を,少量では促進,大量では抑制した。エストロジェソ産生については,はっきりした傾向は認められなかった。LCに対しては,プロジェステロソの産生を抑制し,エストロジェソ産生に対しては,はっきりした傾向は認められなかった。3)GC及びLCの培養液中にLHを添加するとHCG, Prolactinを添加した場合と同様に,添加量を増すにつれて細胞形態が次第に線維芽化することが認められた(図1~12)。4)PGF2αは, GCの増殖を抑制し,五Cに対しては若干その増殖を刺激するように思われた。卵胞由来ではない且ela-s3細胞もその増殖が抑制された。ホルモン産生面では,GCに対しては,プロジェステロソ産生を顕著に促進した。エストロジェソ産生については,はっきりした傾向は認められなかった。LCに対しては,プロジェステロソ産生については促進的な傾向がみられた。エストロジェソ産生については,はっきりした傾向は認められなかった。

279 LIVE RAT OFFSPRING FROM CRYOPRESERVED EJACULATED SPERMATOZOA THROUGH IN VITRO FERTILIZATION

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0°C at 0.5°C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150°C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0°C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 × 106 sperm/mL into a 200-¼L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 ± 6%, 24 ± 5%, and 18 ± 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.

109 A BRIDGE OF SPERM TAIL BETWEEN BLASTOMERES ENHANCED PROTEIN MIGRATION IN THE RAT TWO-CELL STAGE EMBRYOS

The aim of the present study was to examine transient expression of transgene injected into nuclei of rat 2-cell stage embryos. We also investigated the relationship between expression in both blastomeres and tail position of penetrated spermatozoa in rat 2-cell stage embryos. Rat 2-cell stage embryos were recovered from superovulated Wistar females mated with same strain mature males at 48 h after hCG injection. DNA fragments, as the transgene containing the EGFP (enhanced green fluorescent protein) gene controlled under the CMV-IE promoter, were microinjected into one nucleus of 2-cell stage embryos. After microinjection, embryos were cultured in KRB at 37.0°C in a 5% CO2 and 95% humidified air until observation. First, transient EGFP expression in 151 injected embryos was observed using a fluorescence microscope at 6 h intervals until 48 h after injection. At 6 h after microinjection fluorescent embryos were detected, and the proportion of fluorescent embryos increased over time. The rate reached maximum (84%, 52/62) at 24 h after microinjection, and several fluorescent patterns of fluorescent blastomeres in the embryos were observed. There were blastomeres with the same or different fluorescence levels and a single fluorescent blastomere. Second, to assess tail position of the penetrated sperm in the fluorescent embryos, 75 whole mount specimens were observed by inverted phase-contrast microscopy at 24 h after the injection. Also, parthenogenetic 2-cell stage embryos that never contained sperm tail were microinjected with the transgene and observed in the same manner. To obtain parthenogenetic 2-cell embryos, 80 ovulated ova were collected from non-mated females, and incubated with 2 mM 6-DMAP for 4 h. The ova were additionally cultured for 20 h in KRB at 37.0°C in a 5% CO2 and 95% humidified air. In embryos with both blastomeres fluorescent (94%, 33/35), the sperm tail existed in both blastomeres like a bridge between blastomeres. In contrast, in one embryo with a single fluorescent blastomere (4%, 1/24), the sperm tail existed in both blastomeres, and in other embryos with a single fluorescent blastomere (75%, 18/24), the sperm tail was positioned in the one blastomere. On the other hand, in 63 parthenogenic rat 2-cell embryos in which there was no sperm tail, most embryos (86%, 54/63) had a single fluorescent blastomere at 24 h after microinjection. The results indicated that the sperm tail position in the 2-cell embryos makes the protein migration variable. In conclusion, when the CMV-IE/EGFP gene was microinjected into nuclei of rat 2-cell embryos, at 24 h after the microinjection the EGFP was detected in most embryos; however, fluorescent patterns in blastomeres varied. It seems that EGFP derived from the transgene injected into one blastomere may move into another blastomere in rat 2-cell stage embryos, and that the presence of a sperm tail in both blastomeres may influence EGFP distribution.

255 PRONUCLEUS FORMATION IN RAT ZONA-FREE OOCYTES CO-CULTURED WITH HOMOLOGOUS POST-THAW SPERMATOZOA

The aim of the present study was to develop an IVF system with frozen/thawed rat spermatozoa. We examined the effect of cooling rate to 5.0°C on post-thaw sperm motility and membrane integrity, and also investigated the ability of post-thaw spermatozoa to form pronuclei. Under room temperature, epididymal spermatozoa of Wistar rats were collected in 2.0 mL of egg yolk medium containing 8.0% (w/v) lactose monohydrate and 0.7% (v/v) Equex Stem. Samples were loaded into 0.25-mL straws and cooled to 5.0°C in the chamber of a programmed freezer. For cryopreservation, the samples were exposed to liquid nitrogen (LN) vapor for 10 min and then plunged into LN. Straws were thawed in a 37.0°C water bath for 10 s. Ovulated oocytes were collected and the zona pellucidae were removed with 0.1% pronase. One-hundred μL of thawed samples were put into a droplet of 400 μL R1ECM and pre-incubated for 1 h. R1ECM solution was added to the droplet to adjust to 0.5–1.5 × 106 sperm mL-1. The zona-free oocytes were then transferred into the droplet and co-cultured for 10 h. Oocytes were observed for pronuclei formation by means of an inverted phase contrast microscope. In Experiment I, the influence of sperm cooling rate to 5.0°C on sperm motility and membrane integrity was evaluated. Portions of samples were cooled at 54.0°C/min, 0.9°C/min, 0.5°C/min, and 0.3°C/min. The remainders were then frozen. The non-cooled samples were designated as controls. In Experiment II, we examined whether post-thaw spermatozoa have the ability to form pronuclei in vitro or not. All percentage data were arc-sine transformed and then analyzed by the Students t-test. In Experiment I, the membrane integrity between the spermatozoa cooled at 0.5°C/min and the non-cooled spermatozoa was not different (38.1% vs. 37.2%; P > 0.05), but the integrity of these was higher than in spermatozoa cooled directly at 54.0°C/min (38.1% vs. 25.3%; P < 0.05). After culture for 1 h, the motility of spermatozoa cooled at 0.5°C/min was higher than that of those cooled at 54.0°C/min (61.3% vs. 53.3%; P < 0.05). At 2 h post-thaw the motility of spermatozoa cooled at 0.5°C/min was higher than that of spermatozoa cooled at 54.0°C/min and at 0.9°C/min (11.0% vs. 4.5%, 4.9%; P < 0.05). The membrane integrity of post-thaw spermatozoa cooled at 0.5°C/min was also higher compared to that of spermatozoa cooled at 54.0°C/min (22.5% vs. 8.4%; P < 0.01). In Experiment II, 28 (26.2%) of 107 oocytes had pronuclei when the post-thaw spermatozoa cooled at 0.5°C/min were used. The results indicated that the frozen/thawed spermatozoa cooled to 5.0°C at 0.5°C/min showed higher sperm motility and membrane integrity, and that spermatozoa can form pronuclei in homologous zona-free oocytes in vitro. Although in the rat sperm damage occurred during cooling to 5.0°C, and sperm motility and membrane integrity were also decreased by the cold shock, it is possible to decrease the damage by cooling slowly to 5.0°C at 0.5°C/min.

Viability after liquid storage at 15°C of boar sperm induced lipid peroxidation.

硫酸鉄 (FeSO4) とビタミンC (Ascorbic acid: VC) により脂質過酸化誘起したブタ精子の15℃液状保存後の精子生存指数を調べた。さらに Hypotaurine (HT) を脂質過酸化誘起処置液に添加し, その後の液状保存後の精子生存指数も調べた。11頭の種雄豚より採取した精液の精漿を除去して, 脂質過酸化誘起処置液で精子数を3.0×108/mlに希釈し, 38℃で1時間インキュベートすることによって脂質過酸化誘起処置をおこなった。この精子脂質過酸化誘起処置液は, VC 0.5mMとFeSO4を0.01, 0.1もしくは1mM含む Modena を用いた。そして, 脂質過酸化誘起処置直後の Malonaldehyde (MDA) 生成量を分析した。これらの脂質過酸化誘起処置精子を15℃で液状保存し, 精子生存指数を処置直後, 保存後48, 96, 144, 192, 240および288時間目に調べた。また, FeSO4 0.1mMを含む脂質過酸化処置液にHTを50mM添加して脂質過酸化誘起処置をおこない, 処置直後のMDA生成量および精子生存指数を調べた。さらに, この誘起処置精子を15℃液状保存し, その精子生存指数を処置直後, 保存後48, 96, 144, 192, 240および288時間目に調べた。0.1もしくは1mMのFeSO4とVCによる脂質過酸化誘起処置によりMDA生成量が有意に増加した (P<0.001)。これらの脂質過酸化誘起処置精子の15℃液状保存後の精子生存指数は, 保存48時間目以降および処置直後から, 無処置精子よりも有意に低下した (P<0.05)。HT 50mMを含む脂質過酸化誘起処置においては, その精子生存指数は無処置精子と比較して有意な低下が認められなかった。しかし, HT 50mMを含む処置精子のMDA生成量はHT 50mMを含まない処置精子のものと比較し有意な差は認あられなかった。以上のことから, ブタ精液15℃液状保存前に脂質過酸化が生じた精子は, 保存後の精子生存指数の低下が認められた。また, 15℃液状保存前のFeSO4 0.1mM脂質過酸化誘起処置液への50mMのHT添加は, 15℃液状保存後の精子生存指数の低下を抑制することが明らかになった。