Protein phosphorylation is essential for formation of male pronucleus in bovine oocytes

This study examined the association between the morphological and protein phosphorylation events following sperm penetration of in vitro matured and in vitro fertilized bovine oocytes. Oocytes were labeled with [³²P]‐orthophosphate at 3 hr intervals from 3 to 18 hr of following insemination. The phosphorylation of protein complexes of 23 kDa and 18 kDa specifically increased with the formation of male and female pronuclei. In addition, oocytes were treated with 6‐Dimethylaminopurine (6‐DMAP) or Okadaic acid (OA) at 0, 3, 6, and 9 hr respectively following insemination. Although the formation of female pronucleus was not affected by 6‐DMAP, the male pronuclear formation was completely inhibited by the presence of 6‐DMAP at 0 and 3 hr of post insemination. The formation of both pronuclei was inhibited by the presence of OA at any time following insemination. These results suggest that the male pronuclear formation is associated with protein phosphorylation and that the formation of the male and the female pronuclei may involve different factors in bovine zygotes since they respond differently to the kinase modulations. Mol. Reprod. Dev. 52:43–49, 1999. © 1999 Wiley‐Liss, Inc.     

Detailed analysis of pronucleus development in bovine zygotes in vivo: Ultrastructure and cell cycle chronology

The ultrastructural development of pronuclei and cytoplasm was studied in bovine zygotes developed in the oviducts. The timing of the morphological events was related to sonographically detected ovulation and to the progress of the cell cycle determined by double labelling (³H and ¹⁴C-thymidine) of newly synthesized DNA combined with autoradiographic detection. The onset of the S-phase occurred at 11–12 hr after the estimated time of ovulation (EO), and this phase of the cell cycle lasted for 7–9 hr. During the G1-phase, the pronuclei contained spheres of compact, electron-dense fibrillar material classified as nucleolus precursor bodies. Early in the S-phase (13 hr aver EO) spherical fibrillogranular bodies containing larger rounded electron-dense components were detected in the periphery of the pronuclei as well. At 15 hr, the latter bodies had become connected through electron-dense material with spherical multivacuolated fibrillar bodies of the same electron density as the nucleolus precursor bodies. At 17 hr, similar compact spherical bodies, now presenting a single large vacuole, were observed on some occasions, while in other zygotes the morphology remained unchanged throughout the rest of the S and G2-phases. © 1996 Wiley-Liss, Inc.     

Temporally different poly(adenosine diphosphate-ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of ³H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with ³H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased ³H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation. These results also indicate that a poly(ADP-ribosylation) signal is required for cell division; such signal is temporally different from that related to S phase initiation and occurs at the exit of S phase. © 1993 Wiley-Liss, Inc.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo

Here we describe the efficient generation of eGFP-transgenic rats using a lentiviral approach. Analysis of the founder generation demonstrated that 46% of the offspring had stably integrated the provirus into the genome and of those 92% expressed eGFP in all blood-derived leukocytes. In contrast to their offspring, all founder rats were mosaic with regard to eGFP-expression, suggesting delayed viral transduction after injection. The expression level of eGFP in the F1 generation is influenced by and segregates with the site of proviral integration. Interestingly, a single copy of the transgene is sufficient for reliable detection by flow cytometry, irrespective of the leukocyte subtype analyzed. Adoptive transfer of purified CD4(+) T-lymphocytes from transgenic rats and subsequent reisolation from various organs further demonstrated that expression of the lentiviral transgene is maintained in a foreign host and therefore allows for efficient tracking of transferred cells. Taken together, lentivirally generated eGFP-transgenic rats are a powerful tool for various applications in immunology and presumably also many other fields.     

Fine structure of the flagellar apparatus of gametesin situ and motile zygotes of the green algaUlothrix flacca var.Roscoffensis (Ulotrichales) (Chlorophyta)

Summary This fine structural study ofUlothrix flacca (Dillw.) ThuretRoscoffensis variety (Berger-Perrot), a marineUlothrix, describes in detail the flagellar apparatus configuration of gametesin situ in the gametangia and in motile zygotes. The gametes's flagellar apparatus shows two basal bodies overlapping at their proximal end at a 30° angle, in an 11/5 o'clock configuration or with a counterclockwise absolute orientation. The basal bodies are interconnected by a non-striated band or capping plate. They are wrapped in their proximal part by an electron-dense sheath and obtured by a bilobed terminal cap. A cruciate microtubular root system having a 4-2-4-2 alternation pattern is present. A striated microtubule associated component (S.M.A.C.) or system I fibres accompany the two membered root R₂. The system II fibres or rhizoplasts along with striated bands associated to the microtubular roots, were not observed and are presumed to be absent.In the motile zygotes, the basal bodies are paired in a cruciate pattern. During the fusion process, two basal bodies, one of each pair, slide in a face to face position with a slight displacement into the 11/5 o'clock direction; the other two make a 30° counterclockwise rotation, thus making a 60° angle between the two basal bodies of each pair instead of 30° in the gamete.After comparison with the flagellar apparatus of other green alga gametes, it is concluded that the taxonomic affinities ofUlothrix flacca var.Roscoffensis, lie with theUlvophyceae sensuStewart andMattox 1978.Abbreviations CP capping plate - ER endoplasmic reticulum - G Golgi body - LG lipid globule - M mitochondria - MS presumed mating structure - N nucleus - R ₂,R ₄ microtubular roots - SH sheath - SMAC striated microtubule associated component - TC terminal cap - V vacuole - Ve vesicles in the anterior papilla - 1, 2, 1, 2 basal bodies numerotation     

DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes

Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1 h of denaturation with 4 N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1 h of denaturation with 4 N HCl in this study. After this treatment, DAPI (4′,6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained.     

Analysis of preimplantation golden hamster conceptuses resulting from spermatozoa aged in utero

The effects of aging golden hamster spermatozoa in the female reproductive tract on the percentage of ova fertilized, the stage of development, and the chromosome complement of the resulting zygotes were studied. Females were inseminated artificially with cauda epididymal sperm at 6 h (control), 10, 15,18, or 21 h before the estimated time of ovulation. A decrease in the percentage of ova fertilized was found as the time spermatozoa were aged in utero prior to ovulation increased. The zygotes collected at 56 h postovulation from females inseminated 15 or 18 h prior to ovulation were delayed in development, as judged by the number of blastomeres. Although an increase of chromosomally abnormal zygotes was not found, a possibility exists that mosaicism may have been present, as evidenced by zygotes with unequal sized blastomeres, and went undetected.     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。