Giant Oocyte Research Articles

"Lost and Found": snoRNA Annotation in the Xenopus Genome and Implications for Evolutionary Studies.

Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.

Giant Oocytes with Two Meiotic Spindles and Two Polar Bodies: Report of Two Cases

With the advent of IVF technology, the terms normal and abnormal oocytes have been defined and one type of abnormal oocyte is the “giant oocyte”. Giant oocytes are defined to have a 30% larger diameter and twice the volume of normal oocytes[1,2]. Giant oocyte is a rarery observed phenomenon among humans and embryos may develop from these oocytes [2,3].The first hypothesis for the mechanism of their formation is cytoplasmic fusion of two oogonia and the second one is the lack of cytokinesis during mitotic divisions in an oogonium [4]. Fertilization and progression of a giant oocyte is suspected to be the cause of digynic triploidy, which is defined as triploidy with two maternal and one paternal complements [5]. In this case report, we present two giant oocytes, each shown to have two meiotic spindles via visualization by polarization microscope. Because giant oocytes can develop into embryos that are morphologically normal, but genetically abnormal, an embryologist has to be aware of this phenomenon. For this reason, the scientific aim of this report is to present the polarization microscopic properties of giant oocytes and increase the awareness of such oocytes among embryologists.

Giant oocytes in human in vitro fertilization treatments.

Giant oocytes are potential sources of chromosomal abnormalities and should thus never be used in in vitro fertilization and embryo transfer (IVF-ET) procedures. The presence of giant oocytes may indicate the efficiency of the ovarian stimulation and can refer to the quality of sibling oocytes. IVF cycles performed between January 2008 and November 2013 (n = 1521) were divided into two groups: Giant Oocyte Group (GO Group) contained cycles with at least one giant oocyte in the cohort of the retrieved oocytes (n = 37), Normal Group contained cycles with no giant oocytes (n = 1484). In the second part of the study, cycles from GO Group and Normal Group were matched according to patient age, number of retrieved oocytes and stimulation protocol, and thus 30 pairs were formed. Clinical and embryological data were analyzed. The incidence of giant oocytes was 0.3 %. The average patient age was lower (33.5 ± 3.9 vs. 35.3 ± 4.9, p = 0.02); estradiol (E2) levels (1954 ± 903 vs. 1488 ± 909 pg/l, p < 0.01) and number of retrieved oocytes (12.7 ± vs 8.1 ± 5.1, p < 0.01) were significantly higher in the GO Group. There was no difference in clinical pregnancy rates (37.8 vs. 37.4 %, p = 1.00) between the two groups. No major differences in the embryo qualities were found. In the second part of the study, fertilization rate in the matched GO Group was lower (50.6 ± 21.9 vs. 61.9 ± 22.4 %, p = 0.04). Clinical pregnancy rates (36.7 vs. 36.7 %, p = 1.00) did not differ between the matched cycles. Our data suggest that the stimulation protocol does not affect the incidence of giant oocytes. Giant oocytes present in cycles with higher number of retrieved oocytes in younger women. The presence of these gametes does not refer to the quality of sibling oocytes and embryos, or the outcome of the treatment.

Pearls are novel Cajal body-like structures in the Xenopus germinal vesicle that are dependent on RNA pol III transcription

We have identified novel nuclear bodies, which we call pearls, in the giant oocyte nuclei of Xenopus laevis and Xenopus tropicalis. Pearls are attached to the lampbrush chromosomes at specific loci that are transcribed by RNA polymerase III, and they disappear after inhibition of polymerase III activity. Pearls are enriched for small Cajal body-specific RNAs (scaRNAs), which are guide RNAs that modify specific nucleotides on splicing snRNAs. Surprisingly, snRNAs themselves are not present in pearls, suggesting that pearls are not functionally equivalent to Cajal bodies in other systems, which contain both snRNAs and scaRNAs. We suggest that pearls may function in the processing of RNA polymerase III transcripts, such as tRNA, 5S rRNA, and other short non-coding RNAs.

Nuclear actin and transcriptional activation.

Differentiated cells do not revert to an embryonic state in normal development. However, the method called nuclear reprogramming enables these differentiated cells to be reversed to an embryonic state. One essential event in the reprogramming process is reactivation of embryonic genes such as Oct4 (also known as Pou5f1). This reprogramming of transcriptional programs can be achieved by transplantation of mammalian somatic nuclei to the giant Xenopus laevis oocyte nucleus, referred to as the germinal vesicle (GV). Factors and mechanisms responsible for this transcriptional reprogramming have not been elucidated. Recently, we have found that a polymerized form of actin is abundantly present in nuclei transplanted into the Xenopus oocyte nucleus and plays an important role in transcriptional reactivation of Oct4. This study emphasizes a significant contribution of nuclear actin in transcriptional activation. Here, we discuss possible roles of nuclear actin in Xenopus oocytes and in other cell types in the context of transcriptional activation.

A giant oocyte in a cohort of retrieved oocytes: does it have any effect on the in vitro fertilization cycle outcome?

To investigate an association between the presence of a giant oocyte and outcome for the cohort in in vitro fertilization (IVF). Retrospective study. Hospital-based academic medical center. IVF and IVF/intracytoplasmic sperm injection (ICSI) cycles having at least one giant oocyte (study group; n = 105), matched with cycles without a giant oocyte (external control group). A further subanalysis compared giant oocyte cycles with the next closest cycles within patients (internal control group; n = 65). Standard IVF protocols. Incidence of giant oocytes, fertilization rate, quality of cohort embryos, and pregnancy outcome. Out of 97,556 oocytes, 117 (0.12%) were giant. Compared with the external control group, cleavage in the study group was abnormal (increased percentage of 1-cell and 10-cell embryos and decreased percentage of 6-cell and 7-cell embryos), although there was no difference for percentage of embryos with perfect symmetry, fragmentation, or implantation, clinical pregnancy, or ongoing/delivered rate. The study group had two cases of pregnancy termination due to chromosomal abnormalities (4.4%) versus none in external controls. No striking differences in embryo quality existed between the study and internal control groups. A giant oocyte is associated with abnormal cleavage in cohort embryos, but not with implantation or pregnancy rates. Giant oocytes occur sporadically and appear not to reflect quality of remaining oocytes in the ovary.

Examining the contents of isolated Xenopus germinal vesicles

One can manually isolate the giant oocyte nucleus or germinal vesicle (GV) of Xenopus from a living oocyte with nothing more complicated than jewelers’ forceps and a dissecting microscope. Similarly, one can remove the nuclear envelope by hand and allow the lampbrush chromosomes and other nuclear organelles to spread on a microscope slide. After centrifugation, the nuclear contents adhere tightly to the slide, where they can be subjected to immunostaining or fluorescent in situ hybridization for visualization by conventional or confocal microscopy. Preparations of isolated GV contents reveal details of nuclear structure that are almost impossible to attain by more conventional techniques.

346 ULTRASTRUCTURE OF A HUMAN GIANT MII OOCYTE: CASE REPORT

The occurrence of giant oocytes has been reported with considerable frequency during IVF procedures in women of all ages and was associated with an increased response to gonadotrophin therapy. Previous studies suggested that giant oocytes, larger in diameter than normal oocytes and either at the GV, MI, or MII stage, might be a source of human digynic triploidy. However, to our knowledge there are no previous reports concerning the ultrastructure of human giant oocytes, and therefore the objective of this study was to characterize a human giant mature metaphase II oocyte from the ultrastuctural point of view. A giant oocyte with a visible polar body was used under informed consent, after controlled superovulation during a IVF treatment cycle. The oocyte was fixed with Karnovsky’s fixative, post-fixed in 2% OsO4 in 0.15 M cacodylate buffer, pH 7.2, dehydrated and embedded in Epon Semithin and ultrathin sections were cut with a diamond Diatome knife (Diatome, Hatfield, PA, USA) in a LKB ultramicrotome. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were collected on 200 mesh copper grids (Taab Laboratories Equipment Ltd, Aldermaston, UK) and stained with 3% aqueous uranyl acetate (20 min) and Reynolds lead citrate (10 min). Contrast ultrathin sections were observed in a JEOL 100XII transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 60 Kv. Cytogenetic analysis was not performed in this oocyte. At the time of collection, the giant oocyte was 1.4-fold larger than average size oocytes and contained a fragmented polar body. The zona pellucida had a normal loosely fibrillar structure and the perivitelline space was normal containing remnants of follicular cells. The cytoplasmic organelles were no uniformly dispersed with a large organelle-free zone (lake) observed in the periphery of the oocyte cortex and smaller focal lakes visible in the subcortex. Within the cortex the dense cortical vesicles were reduced in number and did not form one or two continuous rows beneath the oolemma. Smooth endoplasmic reticulum (SER) aggregates of tubules were also scarce but underdeveloped SER aggregates of tubules were present in the cortex. The distribution of the SER large and middle vesicles was abnormal: these organelles were nearly absent in the cortex but present in the inner cytoplasm. The content of some vesicles was denser than normal. We also observed the presence of several large secondary lysosomes filled with multiple small and medium lipid droplets (lipofuscin bodies), corresponding to retractile bodies located in the cortex. Large dense vesicles of different densities and volumes were found in the cortex and subcortex. The polar body showed very few cortical vesicles and did not contain mitochondria and SER large or small vesicles. The condensed chromosomes were aligned on the metaphase II plate, in the cortex of the oocyte. The ultrastructural alterations observed in this oocyte corroborated the conclusions of previous genetic studies and confirmed that giant oocytes are immature cytoplasmatically and would be associated with a higher frequency of abnormal development following fertilization. Thus, giant oocytes should not be used in IVF treatments. ASL was supported by FCT, Portugal.

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.

Morphological Analysis of Giant Oocytes to Assess Their Relevance for the Origin of Triploid Zygote Formation

We studied the frequency, morphological characteristics, and clinical significance of giant oocytes. The union of gametes during normal fertilization is characterized by the formation of two pronuclei (2PN) at syngamy. Development of triploidy (3PN) zygotes is considered to be abnormal fertilization mechanisms. Main explanations for the occurrence of 3PN include the failure of the second polar body extrusion, dispermic penetration, or the entry of one diploid sperm. Here we report the observation of giant oocytes, which can offer an additional explanation for the origin of 3PN. Observational Study This study includes 10 giant oocytes obtained from 10 patients who underwent conventional IVF or ICSI. For IVF cases, giant oocytes were identified during the fertilization assessment. As for ICSI cases, unfertilized giant oocytes could be identified during a course of cumulus cell removal just before the ICSI procedure. They were examined for the number of the pronucleus and polar bodies(PB)18-20 hours after IVF/ICSI. Fertilized giant oocytes were further cultured to assess the developmental potential. Results: A total of 2564 oocytes were collected during the study period. 10 of them (10/2564 oocytes: 0.39%) were classified as giant oocytes. When considering only those patients having giant oocytes, the frequency of giant oocytes was found to be 9.6%(10/104 oocytes). Diameter of all giant oocytes was measured to be around 150 μm. The volume of giant oocytes was calculated to be about twice as large as that of a normal sized oocyte. For ICSI cases(n=4), all giant oocytes had apparently the two distinct first-PBs at the 9 o’clock and 12 o’clock position in perivitelline space in relation to the holding pipette. Afterward 3PN were confirmed and the second-PB extrusion was clearly recognized adjacent to the respective first-PB. All 3PN zygotes showed subsequent early cleavage to reach blastocyst stage. For IVF cases, half of giant oocytes(n=3) showed 3PN formation with extrusion of two second-PBs, and the rest of them(n=3) remained unfertilized. 1) An additional explanation for the occurrence of 3PN zygote after ICSI/IVF can be drawn from this observation. Most giant oocytes seems to have two sets of metaphase II(MII)/PB structures. It is possible that chromosomal content in these giant oocytes with two first-polar bodies is diploid, so that giant oocytes can cause digynic triploidy. 2) Considering the volume of a giant oocyte, one mechanism of its formation might be the fusion of two oocytes. 3) The early recognition of 3PN embryo is of considerable clinical significance. Majority of 3PN embryos arising from giant oocytes are thought to be composed of all triploid blastomeres. No risks should be taken and these embryos are always excluded from transfer.

Morphologic and cytogenetic analysis of human giant oocytes and giant embryos.

Objective: Giant oocytes occur with considerable frequency in human ovaries and are implicated in triploid conceptions and development of mature ovarian teratomas. There is general belief that the majority of giant oocytes undergo atresia in the fetal and early neonatal periods, however, some appear to remain in the adult ovary, to varying degrees. The aim of the present study was to determine the incidence and cytology of giant oocytes aspirated from patients undergoing IVF.Design: A prospective analysis of giant oocytes and embryos encountered over a 3 year period in our IVF program.Materials/Methods: Over a period 3 years, we collected data on the presence or absence of giant oocytes in our IVF program. Each giant oocyte and resulting embryo was morphologically assessed. When possible, giant embryos were analysed cytogenetically by FISH.Results: Out of a total of 14,827 oocytes examined, 43 giant oocytes were found. This gave an overall incidence of 0.29%. There were no cases in which more than one giant oocyte was retrieved from the same patient. The average number of retrieved oocytes and the mean estradiol (E2) value on the day of hCG were each significantly higher in the group of patients that had giant oocytes (19.6 oocytes, E2 = 2647 pg/ml) than in the group without (9.9 oocytes, E2 = 2167 pg/ml) (p = .01; p = .01 respectively). Giant oocytes were, on average, 28% larger in diameter (mean 147 um; without the zona pellucida) than normal oocytes (115 um) and had about twice the normal volume. Two different morphological patterns were observed among giant unfertilized and fertilized oocytes. Unfertilized oocytes either contained one diploid or two haploid chromosomal sets, with one or two polar bodies respectively. Ten giant oocytes fertilized and were observed to have either 2 or 3 pronuclei, with either 2 or 4 polar bodies respectively. Six of these giant embryos were analyzed by FISH. Four of the 6 giant embryos analyzed had 2 pronuclei. All 6 of the embryos analyzed were chromosomally abnormal.Conclusions: These observations suggest that embryos resulting from fertilization of giant oocytes are unlikely to be genetically normal. Therefore, our results would suggest that giant embryos originating from either 2 or 3 pronuclear zygotes should be excluded from uterine transfers. Objective: Giant oocytes occur with considerable frequency in human ovaries and are implicated in triploid conceptions and development of mature ovarian teratomas. There is general belief that the majority of giant oocytes undergo atresia in the fetal and early neonatal periods, however, some appear to remain in the adult ovary, to varying degrees. The aim of the present study was to determine the incidence and cytology of giant oocytes aspirated from patients undergoing IVF. Design: A prospective analysis of giant oocytes and embryos encountered over a 3 year period in our IVF program. Materials/Methods: Over a period 3 years, we collected data on the presence or absence of giant oocytes in our IVF program. Each giant oocyte and resulting embryo was morphologically assessed. When possible, giant embryos were analysed cytogenetically by FISH. Results: Out of a total of 14,827 oocytes examined, 43 giant oocytes were found. This gave an overall incidence of 0.29%. There were no cases in which more than one giant oocyte was retrieved from the same patient. The average number of retrieved oocytes and the mean estradiol (E2) value on the day of hCG were each significantly higher in the group of patients that had giant oocytes (19.6 oocytes, E2 = 2647 pg/ml) than in the group without (9.9 oocytes, E2 = 2167 pg/ml) (p = .01; p = .01 respectively). Giant oocytes were, on average, 28% larger in diameter (mean 147 um; without the zona pellucida) than normal oocytes (115 um) and had about twice the normal volume. Two different morphological patterns were observed among giant unfertilized and fertilized oocytes. Unfertilized oocytes either contained one diploid or two haploid chromosomal sets, with one or two polar bodies respectively. Ten giant oocytes fertilized and were observed to have either 2 or 3 pronuclei, with either 2 or 4 polar bodies respectively. Six of these giant embryos were analyzed by FISH. Four of the 6 giant embryos analyzed had 2 pronuclei. All 6 of the embryos analyzed were chromosomally abnormal. Conclusions: These observations suggest that embryos resulting from fertilization of giant oocytes are unlikely to be genetically normal. Therefore, our results would suggest that giant embryos originating from either 2 or 3 pronuclear zygotes should be excluded from uterine transfers.

Giant diploid oocytes as a cause of digynic triploidy in mammals.

Giant oocytes and zygotes twice the normal size from the Chinese hamster were studied embryologically and cytogenetically in order to clarify their significance as a cause of digynic triploidy. There were 6 (0.62%) giant eggs among 973 primary oocytes, 5 (1.37%) among 365 secondary oocytes from mature follicles missing ovulation, 3 (0.44%) among 677 secondary oocytes from oviducts, 4 (0.45%) among 886 one-cell zygotes, and 3 (0.49%) among 608 two-cell zygotes. The results indicated that follicles containing a giant oocyte might have a tendency to miss ovulation. But once ovulated, the giant eggs were fertilized normally and developed normally. Nuclear and chromosomal aspects were studied in 6 primary and 18 secondary oocytes, and 6 one-cell and 5 two-cells zygotes. There were mononuclear and binuclear giant oocytes, but the latter state was rarely maintained to metaphase II. The primary oocytes showed a diploid number of bivalents (22 tetrads) with normal chiasma configuration, and the secondary oocytes had 22 dyads with normal features, indicating normal segregation. The one-cell zygotes exhibited one set of diploid female pronuclear chromosomes and one set of haploid male pronuclear chromosomes in the late prophase. The two-cell zygotes were all digynic triploids; the tail of a fertilizing spermatozoon was attached. There were five XXX and six XXY giant triploids, giving a ratio of 1:1, as expected. It was suggested that the nuclear-cytoplasmic ratio of giant triploid eggs may be more favorable at least for preimplantation development than that of any other type of triploidy. Giant diploid oocytes may be an important source of human digynic triploids, in spite of the prevailing view from studies of marker chromosomes that suppression of first polar body extrusion is the predominant cause.