Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos

Between 1 and 4% of human oocytes fertilized in vitro are tripronuclear. It has been reported that these tripronuclear oocytes can develop to grossly normal-appearing morulae and that chromosomally, these embryos could be triploid, diploid, or severely depleted. The etiology and proportion of apparently diploid and aneuploid embryos deriving from tripronuclear human oocytes is unknown. This study provides evidence for the first time that most (18 of 29) tripronuclear human oocytes cleave directly to 3-cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4 of 29) tripronuclear human oocytes cleave to 2-cells plus an extrusion, and these embryos are diploids, whereas some (7 of 29) cleave to 2-cells, and these embryos are triploid after the first cleavage division. These findings demonstrate that most tripronuclear human oocytes have an altered cleavage pattern at the first cleavage division, that most tripronuclear human oocytes (76% in this study) do not develop into triploid embryos, and that a correlation exists between the pattern of the first cleavage division and the subsequent karyotype of these embryos. Insight into the mechanisms by which these oocytes fail to develop into triploid embryos is also provided.     

Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.     

Ontogeny of the circadian system during embryogenesis in rainbow trout (Oncorhynchus mykyss) and the effect of prolonged exposure to continuous illumination on daily rhythms of per1, clock, and aanat2 expression

It is widely held that the development of the circadian system during embryogenesis is important for future survival of an organism. Work in teleosts has been, to date, limited to zebrafish, which provides little insight into the diversity of this system within such a large vertebrate class. In this study, the authors analyzed the diel expression of per1, clock, and aanat2 in unfertilized rainbow trout oocytes and embryos maintained under either a 12:12-h light:dark (LD) cycle or continuous illumination (LL) from fertilization. 24-h profiles in expression were measured at fertilization as well as 8, 21 42, and 57 days postfertilization (dpf). Both per1 and clock were expressed in unfertilized oocytes and all embryonic stages, whereas aanat2 expression was only measureable from 8 dpf. A reduction in both per1 and clock mean expression levels between unfertilized oocytes/0-1 dpf embryos and 8-9 dpf embryos was suggestive of a transition from maternal RNA to endogenous mRNA expression. Although aanat2 expression was not clearly associated with photic conditions, photoperiod treatment did alter the expression of per1 and clock expression/rhythmicity from as early as 8 dpf (per1), which could suggest the presence and functionality of an as yet unidentified "photoreceptor." As a whole, this work demonstrates that clock systems are present and functional during embryonic development in rainbow trout. Further studies of their expression and regulation will help understand how the environment interacts with embryonic development in the species.     

The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.     

Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Characterization of mouse-hamster-human cross-reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona-free mouse oocytes

Several intrasplenic immunizations with batches of ～15 or ～30 zona-free, unfertilized mouse oocytes resulted in 200–300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ～370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ～116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley-Liss, Inc.     

Structure and expression of the human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single oocyte

Oocyte-specific histone H1 is expressed during oogenesis and early embryogenesis. It has been described in mice and some nonmammalian species, but not in humans. Here, we identified the cDNA in unfertilized human oocytes using direct RT-nested PCR of a single cell. Sequencing of this cDNA indicated an open reading frame encoding a 347-amino acid protein. Expression was oocyte-specific. Homology was closest with the corresponding gene of mouse (H1oo; 42.3%), and, to lesser extent, with that of Xenopus laevis (B4; 25.0%). The gene, named osH1, included five exons as predicted by the NCBI annotation project of the human genome, although the actual splicing site at the 3(') end of exon 3 was different by 48 nucleotides from the prediction. The presence of polyadenylation signals and successful amplification of cDNA by RT-PCR using an oligo(dT) primer suggested that the osH1 mRNA is polyadenylated unlike somatic H1 mRNA. Our technique and findings should facilitate investigation of human fertilization and embryogenesis.