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We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pβ-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken β-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.

Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinal-vesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesicle-stage oocytes.

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P

The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 mum (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 microm) required longer (P

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P

In this study, some characteristics of cryoprotectant, polyalcohol used for vitrification such as toxicity, glass-forming tendency and stability of the amorphous state were estimated and the relationship of those characteristics with cryoprotection for vitrified rat morula was discussed. Glycerol was not harmful for rat morulae exposed for 5 minutes at room temperature to the examined concentration up to 50%. Both of 50% 1, 2-propandiol and 50% 1, 3-butandiol showed detrimental effect on the embryonic survival after exposure for 1 minute. However, combination of 25% propandiol or butandiol with 25% glycerol sustained the survival of embryos at 40-60% even after exposure for 5 minutes. Out of the three cryoprotective solutions, a vitrification solution (VS) of propandiol was easiest to transform into amorphous condition during cooling process. Devitrification (initial) temperature of VS was -92, -77 and -77C for 50% of propandiol, glycerol and butandiol, respectively. Seventy five percent of rat morulae in the solution containing more than 40% glycerol survived when directly plunged into liquid nitrogen, but none of the embryos survived in 50% propandiol or 50% butandiol. Both cryoprotectant, 25% propandiol or 25% butandiol combined with 25% glycerol had a comparable effect with 40-50% glycerol. In 5 percent percoll or 5% PVP supplemented to VS, the survival rate of embryos was improved.

The sex of embryos was determined by the polymerase chain reaction (PCR) method using the DNA fragment of a sex-related gene or Y-chromosome specific nucleotide sequence. By using the DNA fragment of the human sex determinant (SRY), cells of male humans, rabbits, rats, mice and goats can be distinguished from the female cells of each animal. By PCR using DNA of the mouse sex determinant (Sry), male cells of mice and rats were selected from the cell population. The sex of cattle cells and goat cells could be determined by PCR using BOV97M (cattle Y-specific repeated DNA sequence). The DNA preparation of rabbit or cattle embryos was amplified by the primer of either SRY or BOV97M, and the embryonic sex was successfully determined. The sex of the embryos predicted using the H-Y antibody or sex chromosome was confirmed by PCR analysis for biopsy of the trophoblastic cells. The bovine growth hormone gene (bGH) was used as an internal standard of cattle-specific DNA fragment. High efficiency and accuracy for embryo sexing was attained from biopsied embryos. Three of the 8 (38%) biopsied embryos transferred into the recipient resulted in pregnancy, and the sex of 2 calves born was the same as expected.

ラット,ヤギおよびウシの胚盤胞を眼科用メスの先端をさらに研磨し改良した金属刃を使って切断2分離を行い,体外培養後0.5時間から3時間にその分断胚の発育状態を検討した。1.ラット,ヤギおよびウシのいずれの胚も高率に切断2分離することができた。しかし,ヤギの初期胚盤胞は透明帯が硬く,均等分断が困難であった。2.ヤギにおいては透明帯が柔軟になる後期胚盤胞の時期に分断した場合,内細胞塊を著しく損傷させることなく切断2分離できることが明らかになった。3.培養後の2分離胚の発育率は,ヤギ(6/6,100%),ウシ(10/13,77%)の胚にくらべてラットでは低かった(29/62,47%)。以上の結果より,本実験に用いた金属刃による透明帯上からの切断2分離は,透明帯除去および胚の吸引保定の必要性がなく,簡易で再現性の高い方法であって,ヤギやヴシ胚で透明帯が軟化した時期に適用する場合良好な結果が得られることが示唆された。

卵胞卵の培養において,供試卵の状態や培養方法が培養成績に大きく影響すると考えられる。本実験ではブタ及びウシの卵胞卵を用いて培養条件と卵の成熟の関係についてしらべた。1) 屠場での卵巣の採取から採卵までの卵巣の最適保存温度及び保存時間の限界について検討した。33~35°C,20~25°C,4~10°Cに調整した生理食塩液に浸漬して実験室にもちかえった場合,33~35°C保存でもっとも良い培養成績が得られた。33~35°Cでは保存時間はブタでは3時間が限界と考えられた。ウシでは8.5時間保存した場合でも高率に成熟したが,さらに保存時間が長くなると退行変性する卵が著しく増加した。2) 各種完全合成培養液で卵胞卵を培養したところ,ウシ,ブタともにエネルギー基質の添加されていないKRB液中では成熟しなかったが, TCM 199, Ham F12,Eagle MEM及びグルコース,ピルビン酸,乳酸を含む修正KRB中で成熟した。培養液に添加する高分子物質として,ウシ血清アルブミン,ウシ血清アルブミン分画V及びフェトインを用いても高率に成熟したがポリビニルピロリドンでは幾分成熟率が低下した。またブタ卵胞卵の成熟に対して培養液に添加されるウシ血清アルブミンの最適濃度は1 mg/mlであった。またブタ卵胞卵では至適pHは7.2~7.4であって, PH7.0以下では卵核胞崩壊率が低下し,またpH7.6以上になると染色体異常凝縮,細胞崩壊などの退行卵が高率に出現した。ウシ卵胞卵ではpH 6.8~7.6で高率に成熟し,ブタ卵胞卵の場合にくらべて至適pH域の広いことが明らかにされた。ブタ卵胞卵は316,355milliosmols区で高率に成熟したが,より低張ないし高張の液では退行卵が高率に出現した。ウシ卵胞卵でも316,355milliosmols区で高率に成熟したが,234~388milliosmolsの範囲で30%以上中期IIへ移行しブタ卵胞卵にくらべ広い浸透圧範囲で成熟しうる傾向を示した。3) 機能的な黄体の存在する卵巣から採取した卵胞卵を培養した場合,退行卵の出現率が高かった。白体の存在する卵巣から採取した卵はすべて成熟した。未成熟個体の卵巣からの卵では,培養後種々の成熟ステージのものが観察され,性成熟個体からの卵にくらべて分裂の同調性の点で劣っていた。卵丘細胞の付着していない卵では付着している卵にくらべて成熟率は低下した。また卵黄実質の直径が50~90μの発育不良と思われる卵では成熟せず退行卵が高率に出現した。