Effect of mutations in domain 2 on the structural organization of oocyte 5 S rRNA from Xenopus laevis.

In order to test and refine the molecular model of Xenopus laevis 5 S rRNA proposed in a previous work, we have synthesized, by site-directed mutagenesis and in vitro transcription, four mutants in the internal loop B and in the hairpin loop C of domain 2. The conformations of these mutant 5 S rRNAs have been tested using a variety of enzymatic and chemical structure-specific probes and computer modeling. The mutations induce conformational changes restricted to the mutated regions. Our results demonstrate unambiguously that the three helical domains of the Y-shaped structure are independent and that loop C possesses an intrinsic conformation, which is not involved in any tertiary long-range interaction. They point to the crucial role of invariant nucleotides in maintaining the intrinsic conformation of the loop and to the effect of sequence on the stability of loop regions.

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. II. Inhibin and aromatase inhibitor activity.

The inhibin content and aromatase inhibitor activity (AIA) of 72 follicular fluids (FF) obtained from 42 women undergoing in vitro fertilization (IVF) and embryo transfer (ET) were studied as a function of IVF ET outcome. Inhibin levels were determined by bioassay (BA) and RIA; AIA was measured by BA. The inhibin content of follicles characterized as immature by their estradiol (E2) levels and E2/progesterone (P) ratios was significantly lower (P less than 0.05) than that of mature follicles (i.e. leading to pregnancy). The mean AIA for mature follicles were significantly lower than AIA in groups where pregnancy was not obtained. AIA for follicles from which a pregnancy was obtained for each ET was also significantly lower than that in FF characterized as immature of hypermature. The highest E2/AIA and inhibin BA/AIA ratios were associated with the highest incidence of successful IVF ET outcome. No correlation was found between AIA and inhibin, on the one hand, and E2, delta 4-androstenedione, E2/P, and PRL, on the other. However, a positive correlation was found between inhibin (RIA and BA) and P, reflecting the production of inhibin by granulosa cells during luteinization. These studies allowed us to conclude that FF inhibin levels do not differ according to IVF ET outcome, but are an index of follicular maturation. AIA not only constitutes an index of follicular maturation and granulosa cell luteinization, but is of predictive value for IVF ET outcome as E2/AIA and inhibin BA/AIA ratios.

Resistance of human follicular oocytes to parthenogenetic activation: DNA distribution and content in oocytes maintained in vitro.

Sixty-nine human oocytes were subjected to various parthenogenetic stimuli but no activation was observed. Analysis of Feulgen-stained DNA (F-DNA) distribution and content showed anomalies of chromosomal material in 36% of oocytes at 24-75 h after retrieval. A significant loss of F-DNA was noticed in apparently normal metaphase II oocytes remaining in culture for 2-3 days.

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes.

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.

Antispectrin antibodies stain the oocyte nucleus and the site of fertilization channels in the egg of Discoglossus pictus (Anura).

In Discoglossus pictus eggs, only the dimple contains ionic channels active at fertilization; in particular, chloride channels are found in the central portion of the dimple, which is also the site of sperm penetration. Moreover the dimple hosts an imposing cytoskeleton, consisting of a cortical network and bundles of microfilaments extending from the microvilli. Since spectrin cross links actin and is connected through ankyrin to anion transporters in the plasma membrane of erythrocytes as well as to anion channels in other cells, we studied, in D. pictus egg, the relationship between the localization of spectrin and the high polarization of ionic channels and cytoskeletal organization. By means of immunocytochemistry, we localized spectrin exclusively in the egg dimple. In an attempt to trace back the source of spectrin localization, we immunostained sections of D. pictus ovary and localized spectrin in the nuclei of previtellogenic oocytes, where actin is also present. Antispectrin staining remained until germinal vesicle breakdown. By contrast, a cortical localization was found only when the oocytes divided into two hemispheres and into the germinative area (GA), which, after germinal vesicle breakdown, gives rise to the dimple. At this stage the antispectrin signal was particularly strong in the GA. Using Rho-pialloidin, we also established that spectrin is generally present where F-actin is found. However, spectrin and F-actin do not have the same pattern of fluorescence. In conclusion, our data suggest that spectrin may play a role in oocyte and egg polarity. In eggs, it could be instrumental in anchoring to the cytoskeleton membrane proteins such as receptors and ionic channels, including chloride-permeable channels.

Purification and mechanism of action of a nucleosome assembly factor from Xenopus oocytes.

Chromatin with nucleosomes spaced at 180-base pair intervals can be formed in vitro on circular DNA molecules using a Xenopus oocyte S-150 extract, but the ability to form a periodic chromatin structure is lost upon fractionation of this extract. To identify factors other than the known ones involved in chromatin assembly, we have first depleted the extract by incubating it in batch with charged resins, and we have subsequently reconstituted it with purified fractions. Studies performed with the fractionated components indicate that formation of periodically spaced nucleosomes on the relaxed, closed circular DNA proceeds in two steps and does not require DNA topoisomerases. In a first step, histones H3/H4 are transferred from the endogenous H3/H4-N1 complex to the DNA, forming a nascent chromatin structure. This structure can then be rapidly complemented in a subsequent and independent step with a stoichiometric amount of histone H2A/H2B dimers. Under these experimental conditions, excess histone H2A/H2B dimers inhibit DNA supercoiling and nucleosome formation. We describe the purification of a factor from the Xenopus oocyte S-150 which permits DNA supercoiling and nucleosome formation under conditions of excess histone H2A/H2B. The activity purifies as a complex of five nonacidic polypeptides with apparent molecular masses ranging between 56 and 62 kDa. This factor prevents the binding of excess histone H2A/H2B to the DNA, and it can also remove excess histone H2A/H2B already bound to the DNA, thus ensuring that stoichiometric amounts of all four nucleosomal histones associate with the DNA.

[The relative content of oocyte and somatic 5S rRNA at different stages of embryonic development as an index of the replacement of maternal ribosomes by ribosomes of the embryo in the loach]. / Otnositel'noe soderzhanie ootsitnoi i somaticheskoi 5S-rRNK na raznykh stadiiakh émbrional'nogo razvitiia kak pokazatel' zameny materinskikh ribosom ribosomami zarodysha u v'iuna.

Relative content of the oocyte and somatic 5S rRNA in loach Misgurnus fossilis L. during development was determined electrophoretically. Embryos before hatching contain 70% and swimming larvae no less than 50% of the oocyte 5S rRNA. We assume that the relative content of 5S rRNA fractions reflects the proportion between ribosomes synthesized during oogenesis and those synthesized in embryos and larvae. We calculated using previous data (Timofeeva, Kafiani, 1964) the rates of maternal ribosome decay and ribosome synthesis in the embryo. During organogenesis these rates appear to be 1.17-1.09 x 10(6) and 1.7 x 10(6) molecules/sec per embryo, respectively.

Nucleolin from the multiple nucleoli of amphibian oocyte nuclei.

When fixed preparations of newt germinal vesicle (GV) contents are treated with RNase and are then probed with radiolabeled single-stranded DNA in 0.1-2.0 X SSC, the extrachromosomal nucleoli bind the probe non-specifically. DNA/protein blot analysis of proteins from newt GVs shows that gv95, an acidic protein (pI = 5.0) of Mr = 95,000, is the most prominent non-specific DNA-binding protein. Immunocytochemical analysis with affinity purified antibody directed against gv95 shows that it is located in the multiple nucleoli. We used an antibody directed against rat nucleolin to show that newt gv95 and two similar Xenopus GV proteins are the amphibian versions of nucleolin, a nucleolar ribonucleoprotein originally identified in mammalian cells. We show that mAb 3A10, directed against newt histones H1 and H5, labels gv95 on protein immunoblots and the multiple nucleoli in cytological preparations. These results suggest that histone H1 and nucleolin share a cross-reacting epitope.

ATP and ADP in human pre-embryos.

Spare human oocytes and pre-embryos from an in-vitro fertilization (IVF) programme were individually analysed for ATP and ADP content using a bioluminescence method employing the firefly luciferin-luciferase reaction. The ATP content of oocytes that failed to fertilize in vitro was 1.71 +/- 0.28 pmol (n = 10), pronuclear stage ova 1.93 +/- 0.08 (n = 6), 2-cell stage 1.78 +/- 0.20 (n = 7), 4-cell stage 1.73 +/- 0.15 (n = 6), 6-8 cell stage 2.76 +/- 0.53 (n = 8), morula stage 2.36 +/- 0.68 (n = 8), early blastocyst stage 2.08 +/- 0.25 (n = 7) and expanded blastocyst stage 2.26 +/- 0.15 (n = 3) pmol. The ADP content of these pre-embryos was low in all stages with a small increment in the 2-4 cell stages and the blastocyst stage. This gave an elevated ATP/ADP ratio (range 19-92) indicating a good energy status. The sensitive luciferin-luciferase assay may be a tool for studying the energy status of spare oocytes and pre-embryos under different incubation conditions in human IVF programmes.

Amplification of a beta-haemoglobin sequence in individual human oocytes and polar bodies.

A 680 base-pair sequence of the human beta-haemoglobin gene was reproducibly amplified in individual unfertilised human oocytes and in first polar bodies isolated from them. Specificity and sensitivity of amplification were achieved by two sequential reactions with two sets of primers, amplifying first a 725 base-pair sequence and secondly a 680 base-pair sequence from within the first amplified fragment. A restriction enzyme digest of the DNA amplified from a single oocyte with the endonuclease Dde I confirmed the identity of the amplified beta-haemoglobin fragment; this technique provides a diagnostic test for the genetic defect responsible for sickle cell anaemia. Analysis of the DNA from the first polar body may enable detection of such defects in unfertilised eggs from carrier women. Selection of eggs without the defect for fertilisation may therefore obviate the need for diagnostic procedures on embryos.

New type of POU domain in germ line-specific protein Oct-4.

Members of a family of murine octamer-binding proteins interact specifically with the octamer motif, a transcription regulatory element found in the promoter and enhancer regions of many genes. Oct-4 is a maternally expressed protein that is also present in the pre-implantation mouse embryo. Although many regulatory proteins are expressed in post-implantation embryos, transcription factors regulating pre-implantation processes have remained elusive. The Oct-4 gene is therefore a prime candidate for an early developmental control gene. Here we report the complementary DNA cloning of the mouse Oct-4 gene, and the characterization of the encoded protein(s) by sequential in vitro transcription, translation, DNA-binding and protease-clipping analysis. Deletion analysis shows that the DNA-binding activity is mediated by a POU domain encoded in an open reading frame corresponding to a 324-amino-acid protein. Sequence comparison with known POU domains reveals that Oct-4 is a novel member of the POU-family.

Different cytoskeletal organization in two maturation stages of Discoglossus pictus (Anura) oocytes: thickness and stability of actin microfilaments and tropomyosin immunolocalization.

In Discoglossus pictus oocytes, the germinative area (GA) contains long and irregular microvilli where actin microfilaments are located. In the egg, the funnel-shaped dimple that originates by invagination of the GA is present. In the dimple both microvilli and microfilament bundles have a very orderly appearance. This report extends previous observations (Campanella and Gabbiani, Gamete Res 3:99-114, 1980) and shows that GA microfilaments are thinner (36 A average) than dimple microfilaments (60 A average), as measured in ultrathin section. Moreover, the interfilament distance is smaller in GA bundles than in the dimple bundles. To get an insight into actin organization in oocytes and eggs, we used an actin-depolymerizing factor (ADF) in which cryostat sections were incubated prior to immunofluorescent staining with antiactin antibodies. The microfilaments of the GA microvilli and partially of the oocyte cortex are resistant to ADF when compared to those in the dimple and the rest of the egg cortex. We also investigated immunocytochemically the presence of tropomyosin and found that this protein is localized in the dimple and in the cortex of oocytes and eggs but is absent in the GA.

First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo.

This paper describes an analysis of the first cell cycle of mouse oocytes aged postovulation and fertilized in vivo. For this purpose, we developed a procedure for inducing ovulation in vivo that allows accurate timing of ovulation. The method is based on a luteinizing hormone (LH)-releasing hormone (LHRH) administration at proestrus. This ovulation procedure had no detectable effect on the rate of ovulation or postimplantation embryonic death. We used this method of ovulation induction in an analysis of the separate stages of the first cell cycle of in vivo fertilized postovulation aged oocytes. All stages assessed were shorter in aged oocytes (12 hr postovulation) than in zygotes from unaged oocytes (1 hr postovulation): 1) the time interval between insemination and penetration of the aged oocytes was 1.5 hr shorter than the time interval of the unaged oocytes; 2) pronuclear formation in the fertilized aged oocytes was somewhat quicker than pronuclear formation in fertilized unaged oocytes; 3) in zygotes from aged oocytes, the time between formation of pronuclei and the pronuclear membrane breakdown was 1 hr shorter than in zygotes from unaged oocytes; 4) the first cleavage division was 3 hr advanced in zygotes from aged oocytes compared with the moment of the first cleavage division in zygotes from unaged oocytes. We also determined the glutathione (GSH) content of unaged and aged oocytes to investigate a possible relationship between the rate of pronuclear formation and GSH. The level of GSH was two times lower in oocytes aged postovulation for 12 hr than in unaged oocytes.2+ level of GSH in fertilized, unaged oocytes was half that in

A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA.

In an effort to understand how polarity is established in Xenopus oocytes, we have analyzed the process of localization of the maternal mRNA, Vg1. In fully grown oocytes, Vg1 mRNA is tightly localized at the vegetal cortex. Biochemical fractionation shows that the mRNA is preferentially associated with a detergent-insoluble subcellular fraction. The use of cytoskeletal inhibitors suggests that (1) microtubules are involved in the translocation of the message to the vegetal hemisphere and (2) microfilaments are important for the anchoring of the message at the cortex. Furthermore, immunohistochemistry reveals that a cytoplasmic microtubule array exists during translocation. These results suggest a role for the cytoskeleton in localizing information in the oocyte.

Absence of substantial bending in Xenopus laevis transcription factor IIIA-DNA complexes.

The extent and location of DNA-bending induced in the Xenopus laevis transcription factor IIIA-oocyte 5S RNA gene complex was determined by the gel retardation method. The electrophoretic mobilities of TFIIIA complexed with restriction fragments of 160, 177, 282 and 300 bp that contain the sequence of the major oocyte 5S RNA gene were compared. In these fragments the 120-bp gene is positioned either in the middle or at the end. Minor differences in the mobility of the complexes indicate that the degree of DNA bending is only slight. To determine the bending angle more precisely, a bending vector system, pBend3, was used to examine the complex of TFIIIA with the internal control region (ICR) of the 5S RNA gene. A 61-bp synthetic duplex corresponding to the ICR sequence was cloned into pBend3. Duplicated circular permuted restriction sites allow several 186-bp fragments to be generated in which the position of the ICR can be varied. Gel retardation of TFIIIA-DNA complexes with the ICR sequence contained in pBend3 indicates a bending angle of only 30 degrees and shows that interaction in the ICR could account for all of the bending found in the complete oocyte 5S RNA gene.

High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization.

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.

Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.

Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes.

PTPA, a specific phosphotyrosyl phosphatase activator of the PCSH2 and PCSL protein phosphatases, was purified up to apparent homogeneity from Xenopus laevis ovaries and rabbit skeletal muscle and highly purified from dog liver. PTPA appears as a 40-kDa protein in gel filtration, as well as in sucrose gradient centrifugation, and as a 37-39-kDa protein doublet in SDS-PAGE. Its estimated cellular concentration of 0.75 microM in oocytes or 0.25 microM in rabbit skeletal muscle is suggestive of an important role in the regulation of the cellular PTPase activity. The PTPase activation reaction of the PCSL phosphatase is time-dependent, ATP and Mg2+ being essential cofactors [A50(ATP) = 0.12 mM in the presence of 5 mM MgCl2]. With RCM lysozyme as substrate, the specific activity of the PTPA-activated PCSL phosphatase is 700 nmol of Pi/(min.mg). The pH optimum of the PTPase shifts from 8.5-9 in basal conditions to a neutral pH (7-7.5), and the A50 for the essential metal ion Mg2+ is decreased (3 mM). The activation is rapidly reversed in the presence of the substrate, and more slowly after removal of ATP.Mg. The PTPA-activated PCSL phosphatase represents a major PTPase activity in the cytosol of X. laevis oocytes (at least 50% of the measurable PTPase with RCM lysozyme phosphorylated on tyrosyl residues). The PTPA activation is specific for the PTPase activity of the PCSL and PCSH2 phosphatases, without affecting their phosphoseryl/threonyl phosphatase activity. However, effectors of the phosphorylase phosphatase activity, such as polycations and okadaic acid, also influence the PTPase activity. Phosphorylase alpha inhibits the activated PTPase activity (I50 = 5 microM). The PTPase activity of the other oligomeric PCS phosphatases (PCSH1 and PCSM) is not influenced, suggesting an inhibitory role for some of their subunits. This activation is compared with the recently described PTPase stimulation of the PCS phosphatases by ATP/PPi [Goris, J., Pallen, C. J., Parker, P. J., Hermann, J., Waterfield, M. D., & Merlevede, W. (1988) Biochem. J. 256, 1029-1034] and by tubulin [Jessus, C., Goris, J., Cayla, X., Hermann, J., Hendrix, P., Ozon, R., & Merlevede, W. (1989) Eur. J. Biochem. 180, 15-22].

Measurement of HPRT activity in the human unfertilized oocyte and pre-embryo.

The hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) activities in individual non-fertilized human eggs and in human pre-embryos (4-cell to blastocyst stage) have been analysed. A wide spread of activities was observed, the mean values of which decline with time post-ovulation for both eggs and advancing pre-embryonic stages. The variation in activities was less in groups of eggs or pre-embryos recovered from a single ovulatory cycle. The activity of HPRT, but not of APRT, was readily detectable in single 4-cell and 8-cell blastomeres. When pre-embryos at various preimplantation stages were exposed to alpha-amanitin, to block transcription of mRNA from the pre-embryonic genome, no clear effect on HPRT activity was observed. It is concluded that the HPRT and APRT activities measured in the pre-embryos studied here are likely to be maternally inherited, and that use of a direct assay for HPRT activity for the pre-implantation diagnosis of Lesch-Nyhan disease would be premature.