Effects of oligo sequence and chemistry on the efficiency of oligodeoxyribonucleotide-mediated mRNA cleavage.

Using the endogenous histone H4 mRNA of Xenopus oocytes as a target, we have previously shown that 20mer oligos complementary to different parts of this sequence vary in their effectiveness at causing mRNA cleavage in vivo, and that some of the RNA can never be cleaved. In this paper we show that the resistant RNA is not localised within one part of the oocyte, and that the relative resistance in vivo of endogenous or synthetic H4 mRNA to the different oligos is preserved in an in vitro assay system using deproteinised RNA. If an prior annealing step is included in vitro, all resistance is abolished. Chemical modification of one oligo by end substitution with methylphosphonate or phosphorothioate residues did not improve cleavage efficiency. Oligos with complete phosphorothioate substitution cause slower cleavage in vivo but persist for longer. Consequently phosphorothioate oligos are effective at lower doses than phosphodiester ones, provided that the incubation time is long enough (24 hours). Increasing oligo length from 20nt to 30nt increases phosphorothioate oligo efficiency over long reaction times in vivo, but decreases efficiency during short in vitro assays. Similar increases in length did not affect phosphodiester oligo performance in vivo, but caused a decrease in efficiency in vitro which was overcome by an annealing step.     

Cytogenetic analysis of caprine 2- to 4-cell embryos produced in vitro

Prepubertal goat in vitro matured/in vitro fertilised oocytes produce only a small percentage of blastocysts. The present study examines the incidence of chromosomal anomalies in 2- to 4-cell embryos in vitro produced (IVP) from prepubertal oocytes fertilised with the semen of two males. Cumulus-oocyte complexes were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% heat inactivated Donor Bovine Serum (DBS), 10 microg/ml FSH + 10 microg/ml LH + 1 microg/ml 17beta-oestradiol for 27 h at 38.5 degrees C in 5% CO2 in air. IVM oocytes were inseminated with the sperm from two males prepared using the swim-up and heparin-capacitation procedures. At 24 h postinsemination (hpi) the oocytes were transferred to 100 microl drops of SOF medium for a further 24 h. At 17 hpi a sample of oocytes was stained with lacmoid to evaluate the nuclear stage after fertilisation. The cleavage rate was determined at 24, 36 and 48 hpi and chromosome slides were prepared according to the gradual-fixation technique and stained with Leishman. A total of 1070 2- to 4-cell embryos from prepubertal goat oocytes were studied, but it was only possible to analyse 241 cytogenetically. Of these, 40% exhibited a normal diploid chromosome complement, 59% were haploid and 1% were triploid. There were significant differences between the two males in sperm oocyte penetration and oocyte cleavage but no differences were found in chromosomal anomalies. In conclusion, the low number of embryos karyotyped and the high number of haploid embryos found in this study suggested a high incidence of abnormal fertilised embryos and deficient cytoplasmic maturation of the oocyte which inhibits sperm head decondensation.     

Localized synthesis of the Vg1 protein during early Xenopus development

The Xenopus Vg1 gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-beta family of small secreted growth factors. We have raised antibodies to recombinant Vg1 protein and used them to show that Vg1 protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vg1 protein is synthesized until the gastrula stage. The embryonically synthesized Vg1 protein is present only in vegetal cells of an early blastula. We find that Vg1 protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vg1 protein is cleaved to give a small peptide of M(r) = approximately 17 x 10(3). These results support the proposal that the Vg1 protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo.     

The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.

The properties of antisense phosphorothioate and unmodified oligodeoxynucleotides have been studied in Xenopus oocytes and embryos. We find that phosphorothioates, like unmodified oligodeoxynucleotides, can degrade Vg1 mRNA in oocytes via an endogenous RNase H-like activity. In oocytes, phosphorothioate oligodeoxynucleotides are more stable than unmodified oligodeoxynucleotides and are more effective in degrading Vg1 mRNA. In embryos, neither unmodified nor phosphorothioate deoxyoligonucleotides were effective in degrading Vg1 message at sub-toxic doses.     

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.     

VgRBP71 stimulates cleavage at a polyadenylation signal in Vg1 mRNA,resulting in the removal of a cis-acting element that represses translation

Translation of Vg1 mRNA is repressed in Xenopus oocytes until it is localized to the vegetal cortex. Localization and translational repression are mediated by separate elements in the 3'UTR of the mRNA. VgRBP71 binds to the 3' end of the localization element and stimulates cleavage at an adjacent polyadenylation signal. The protein has an RNA strand-separation activity that likely underlies this event. Polyadenylation occurs at this site in Vg1 mRNA with the consequence of removing the downstream translational repressor element. Ectopic expression of VgRBP71 in stage II oocytes results in cleavage of the mRNA and premature expression of Vg1 protein. These results support a model in which VgRBP71 activates translation of Vg1 mRNA by promoting the removal of a cis-acting repressor element.     

七星瓢虫卵黄原蛋白基因的表达mRNA的体外转译

七星瓢虫成熟雌虫脂肪体总RNA和poly(A)~+RNA中可转译mRNA的水平约为雄虫和不成熟雌虫的两倍,其中所含的卵黄原蛋白mRNA可在体外转译系统中指导卵黄原蛋白多肽的合成。 雌虫取食人工饲料时,其脂肪体RNA中可转译mRNA的水平很低,不能指导卵黄原蛋白多肽的合成。保幼激素类似物能诱导可转译卵黄原蛋白mRNA的出现。     

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.     

Mouse oocytes and early embryos express multiple histone H1 subtypes

Oocytes and embryos of many species, including mammals, contain a unique linker (H1) histone, termed H1oo in mammals. It is uncertain, however, whether other H1 histones also contribute to the linker histone complement of these cells. Using immunofluorescence and radiolabeling, we have examined whether histone H10, which frequently accumulates in the chromatin of nondividing cells, and the somatic subtypes of H1 are present in mouse oocytes and early embryos. We report that oocytes and embryos contain mRNA encoding H10. A polymerase chain reaction-based test indicated that the poly(A) tail did not lengthen during meiotic maturation, although it did so beginning at the four-cell stage. Antibodies raised against histone H10 stained the nucleus of wild-type prophase-arrested oocytes but not of mice lacking the H10 gene. Following fertilization, H10 was detected in the nuclei of two-cell embryos and less strongly at the four-cell stage. No signal was detected in H10 -/- embryos. Radiolabeling revealed that species comigrating with the somatic H1 subtypes H1a and H1c were synthesized in maturing oocytes and in one- and two-cell embryos. Beginning at the four-cell stage in both wild-type and H10 -/- embryos, species comigrating with subtypes H1b, H1d, and H1e were additionally synthesized. These results establish that histone H10 constitutes a portion of the linker histone complement in oocytes and early embryos and that changes in the pattern of somatic H1 synthesis occur during early embryonic development. Taken together with previous results, these findings suggest that multiple H1 subtypes are present on oocyte chromatin and that following fertilization changes in the histone H1 complement accompany the establishment of regulated embryonic gene expression.     

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.     

Differences in membrane properties between unfertilised and fertilised single rabbit oocytes demonstrated by electro-rotation. Comparison with cells from early embryos

The apparent membrane capacity of tubular rabbit oocytes increases from 1.7-2.0 microF/cm2 before fertilisation to 3.7-4.0 microF/cm2 after fertilisation. The membrane conductivity measured on single cells was also increased by fertilisation from less than 1 mS/cm2 to 14 mS/cm2. Cells obtained from 2-, 4- or 8-cell embryos exhibited intermediate values of membrane capacity (2.3-2.8 microF/cm2) and conductivity (5-22 mS/cm2). The values quoted are those effective between 1 and 10 kHz, the frequency of the rotating field used. The large apparent capacities are probably due to the presence of structures such as microvilli which cause the actual membrane area to exceed the smooth sphere area. It must be assumed that these structures change in form or number on fertilisation, and that they persist in embryos, at least up to the 8-cell stage. No difference was apparent between cells fertilised in vitro or in vivo. Comparison of the above zona-free data with measurements on zona-complete oocytes indicate how fertilised and unfertilised rabbit eggs may be distinguished from one another, even in the presence of the zona pellucida.     

Vg1 has specific processing requirements that restrict its action to body axis patterning centers

Unlike most transforming growth factor-beta (TGF-beta) superfamily members, Vg1 has been shown not to produce gross phenotypic alterations in Xenopus embryos when overexpressed by mRNA injection. Experiments with artificial chimeric constructs and a recently identified second allele of Vg1 suggest that this may be due to unusually stringent requirements for proteolytic processing. We provide biological and biochemical evidence that cleavage by two distinct proteolytic enzymes is required for effective activation of Vg1. We demonstrate a tightly restricted overlap in expression patterns of Vg1 with the proteases required to release the mature peptide. The data presented may account for the long-standing observation that the vast majority of Vg1 protein, in vivo, is present in its unprocessed form. Taken together, these observations provide a plausible mechanism for local action of Vg1 consistent with requirements imposed by current models of pattern formation in the developing body axis.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Expression of DNA ligases I and II during oogenesis and early development of Xenopus laevis.

We have analyzed the expression of DNA ligase I protein during oogenesis and early development of Xenopus laevis. The protein is already present in stage I oocytes and then accumulates throughout oogenesis to reach a steady state level by stage VI. It remains at this level at least until tadpole stage. In stage VI oocytes DNA ligase I protein is almost exclusively localized in the germinal vesicle. We have partially purified a DNA ligase II activity from stage VI oocytes, unfertilized eggs, and stage 8 embryos. An 80-kDa polypeptide can be specifically adenylated in all three purified extracts. It is not recognized by antibodies directed against DNA ligase I and is active on oligo(dT)-poly(rA) substrate. It could therefore represent DNA ligase II protein. The presence of both DNA ligases I and II in oocytes and embryos is inconsistent with the DNA ligase model that had been previously proposed for amphibia.     

The effect of partial dissection of the zona pellucida on the fertilization of bovine oocytes in vitro and on the subsequent development of the embryos outside the body

Influence of partial zona dissection (PZD) on fertilization and cleavage of cow oocytes and on pre-implantation development of embryos obtained by this method was investigated. Decreased concentration of spermatozoa in less degree influenced on rates of fertilization and cleavage of oocytes with partial zona dissection than on intact eggs. The embryos obtained by method PZD can develop in vitro to blastocyst stage. However, their development is slowed down and presence of dissection in zona pellucida can result in premature hatching such blastocysts.     

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3′-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.     

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.