Chromosomal analysis of unfertilized human oocytes prepared by a gradual fixation-air drying method

Two hundred and sixty-five unfertilized human metaphase II (MII) oocytes from an in vitro fertilization program were studied cytogenetically using our chromosomal technique, a gradual fixation-air drying method. Of the 265 oocytes, 185 (70%) were successfully karyotyped. There were 21 aneuploids (11.4%) consisting of 8 hyperhaploids (4.3%), 11 hypohaploids (5.9%) and 2 complex cases (1.1%). There were also 9 structural anomalies (4.9%) and 18 diploids (9.7%). In aneuploidy, the loss or gain of dyads (so-called nondisjunction) occurred more frequently than the loss or gain of monads (so-called predivision). The frequency of abnormally behaved chromosomes (segregation errors) due to nondisjunction, anaphase lag and predivision was studied among the seven chromosomal groups (A-G) and compared with the frequency expected from an equal probability of segregation errors in each of the 23 chromosomes. The observed frequency was somewhat higher than the expected frequency in groups E and G but the difference was not statistically significant in either group. These results were discussed in relation to previous studies on human M II oocyte chromosomes.     

Cytogenetic analysis of unfertilized human oocytes

The results of morpho-functional and cytogenetic analyses of 341 oocytes unfertilized in the course of extracorporal fertilization programme are presented. The causes of the "failure" during in vitro fertilization of the oocytes are discussed. Relation of the frequency of oocyte chromosome abnormalities (40.2%) on the patient age and cell maturity has been shown.     

Cytogenetics of unfertilized human oocytes

During an in-vitro fertilization programme 150 oocytes from 62 women with a mean age of 31 years (range 24-39) remained unfertilized. Successful chromosome analysis was carried out on 96 oocytes by Q-banding: 59 (61.5%) oocytes bore a normal haploid complement, 8 (8.3%) were diploid and 3 (3.1%) tetraploid. In 26 (27.1%) oocytes aneuploidy was observed; these included 9 (9.4%) nullisomic, 5 (5.2%) double nullisomic, 4 (4.2%) triple nullisomic and 2 (2.1%) disomic oocytes. The remaining 54 (36.0%) oocytes could not be evaluated. A nearly uniform rate of aneuploidy was found for unfertilized oocytes among different donor age groups.     

Features of chromosome segregation in unfertilized human oocytes

Mechanisms of abnormal chromosomal segregation in female meiosis and factors exerting influence on unfertilized human oocytes have been analysed in vitro. The differences in results of cytological studies on the rate of aneuploidy have been reviewed. The advanced maternal age is primarily associated with disturbances in oocyte meiosis.     

Electric fusion of unfertilized starfish oocytes

An electric power device to fuse starfish oocytes was constructed. The outputs were supplied to a pair of parallel platinum wires in a solution of mannitol to which small amounts of salts were added. Oocytes of the starfish Asterina pectinifera , deprived of the vitelline envelope, were fused by several repetitions of a combination of 2.5 MHz AC field for a few seconds and a 50 μs DC pulse. Observation of fusing pairs of immature oocytes revealed that: (i) the oocyte placed near to the cathode forms a bulge at the surface facing the anode when subjected to DC pulses and, with subsequent DC pulses, a similar bulge is formed on another oocyte; and (ii) the pair of bulges then fuse together leading to fusion of the main body of the two oocytes. The conjugate of maturing oocytes soon became a single sphere, usually within 10 min, but this process toward spherical form paused when the oocyte was extruding its polar bodies. The conjugate of immature oocytes took 1 h to become a single sphere. The fusion did not disturb the progress of meiotic events, but electric pulses at an intensity suitable for the fusion often activated maturing oocytes and mature eggs.     

The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro

Summary To detect structural alterations in human oocytes that may give rise to predisposition to aneuploidy, unfertilized human oocytes from an IVF programme were processed for indirect anti-tubulin immunofluorescence. The spindle of oocytes aged for 2 days is rather small, and bi- or multipolar. Chromosomes are no longer aligned at the spindle equator but are scattered all over the degenerating spindle. This implies that human oocytes aged for 2 days may no longer be able to develop into a chromosomally balanced, normal embryo. In oocytes aged for 3–4 days the chromosomes become more decondensed and form a restitution nucleus. Microtubules radiate out from the latter towards the cell periphery and form a network of fibres in the cytoplasm. A similar alignment of tubules is found in unfertilized, activated oocytes. Oocytes with an aberrant cytoskeleton and chromosomal array were predominantly obtained from aged females. They include two binucleated oocytes with two sets of chromosomes and two oocytes with displaced chromosomes one of which had a tripolar spindle.     

Induced chromosome aberrations in unfertilized oocytes of mice

Summary Just before ovulation we treated 10–12-week-old female mice of the strains C3H and (101×C3H)F₁ with 2.3.5.-triethyleneimonobenzoquinone-1.4 (T), cyclophosphamide (C) and methotrexat (M). The chromosome analysis took place shortly after ovulation at the time of metaphase II. The method described in detail seems to be appropriate to examine the induction of genetic defects during oogenesis by chemical agents.

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Zusammenfassung Wir behandelten 10–12 Wochen alte weibliche Mäuse der Stämme C3H und (101×C3H)F₁ kurz vor der Ovulation mit 2.3.5.-Triethyleniminobenzochinon-1.4 (T), Cyclophosphamid (C) und Methotrexat (M) Die Chromosomenanalyse erfolgte kurz nach der Ovulation zum Zeitpunkt der Metaphase II. Die im Detail beschriebene Methode erscheint geeignet, die Auslösung genetisch bedingter Eireifungsstörugen durch chemische Agentien zu untersuchen.

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This work was sponsored by the Deutsche Forschungsgemeinschaft.     

Cryopreservation of unfertilized rat oocytes by ultrarapid freezing]

Unfertilized rat oocytes were placed in a highly concentrated solution of cryoprotectant (DAP 224:2M dimethylsulphoxide, 2M acetamide, 4M propylene glycol in PB1) in 0.5 ml sampling tubes and then immediately immersed into liquid nitrogen; thawing was conducted in a 37 degrees C waterbath. After thawing, 630 out of 968 oocytes (65.1%) were morphologically normal. After insemination in vitro of cryopreserved oocytes, the proportion of pronuclear oocytes with spermatail (s), male (s) and female pronuclei (8-10 h post insemination), and 2-cell embryos with two identical blastomeres (28-30 h post insemination) was 60.8% (152/250) and 29.8% (39/131), respectively. One hundred and fifty oocytes that were judged as pronuclear oocytes under the inverted microscope 8-10 h after insemination were transferred to the oviducts of pseudopregnant recipients; 18.7% (28/150) of the oocytes developed to normal young.     

High survival rate of unfertilized mouse oocytes after vitrification

Unfertilized mouse oocytes were cooled rapidly by directly plunging them into liquid nitrogen, immediately after exposure to a highly concentrated solution (modified VS1: 2.53 M-dimethyl sulphoxide, 2.36 M-acetamide, 1.19 M-propylene glycol, 5.4% (w/v) polyethylene glycol (Mr 8000) in PB1), and later warmed in a 37 degree C waterbath. After warming, 305 out of 348 oocytes (87.6%) were morphologically normal. After fertilization in vitro of cryopreserved oocytes, the proportions of pronuclear oocytes and 2-cell embryos 5 and 24 h after insemination were 81.6% (124/152) and 78.4% (120/153), respectively. All 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 45.8% (55/120) developed to normal young.     

Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes

The effect of maternal age on the incidence of chromosomal abnormalities was investigated on a large sample of 3,042 in vitro unfertilized human oocytes II obtained from 792 women aged 19-46 years and participating in an in vitro fertilization program for various indications. The chromosomal analysis combined a gradual fixation of oocytes and an adapted R-banding technique. A total of 1,397 interpretable karyotypes were obtained. Various types of numerical aberration were observed, involving conventional chromosome nondisjunction (3.5%), single-chromatid nondisjunction (5.9%), complex (0.8%) or extreme aneuploidy (0.5%), diploidy (5.4%), and set of single chromatids (3.8%). No significant difference was found in the mean age of women according to the various types of chromosomal abnormalities. A positive relationship was found between maternal age and the global rate of aneuploidy, in agreement with the findings of epidemiological studies. The incidence of both whole-chromosome nondisjunction and precocious chromatid separation were correlated to maternal aging but the most significant correlation was found between maternal aging and single-chromatid nondisjunction. The rate of diploidy was also correlated to a slight extent to maternal aging, whereas no correlation was found between maternal age and the rate of single-chromatid sets. These data reveal that single-chromatid malsegregation is an essential factor in the age-dependent occurrence of nondisjunction in human oocytes. Disturbance in sister-chromatid cohesion might be a causal mechanism predisposing to premature chromatid separation and subsequently to nondisjunction in female meiosis.     

Chromosomal analysis of mammalian oocytes matured in vitro with various culture systems

The objective of this study was to evaluate oocyte maturation in vitro. Ten virgin CD-1 mice were used with 3 replications for in vitro with 4 different culture media. Media were minimal essential medium (MEM) with Earl's salt, Waymouth MB 752/1 (MB 752/1), BGjb medium (BGjb), and tissue culture medium-199 (TCM-199). The oocyte chromosomes were C-banded to enable an objective analysis of the chromosome abnormality and number. There was a percentage of blockage at metaphase I (M I), in matured oocytes in all culture media. Metaphase II (M II) was reached by 70.9 to 87.3% of oocytes in 4 different culture media. The frequencies of hyperploid M II oocytes were 0.0, 1.1, 2.8 and 2.6% for TCM-199, MEM, MB 752/1 and BGjb, respectively. A small proportion of oocytes was also found to be polyploid in 4 different culture media. There was an occurrence of premature centromere separation among oocytes. It was concluded that the chromosomes of the oocytes matured in vitro were not all in the normal condition (being at M II). The media used in this study for oocyte maturation caused maturation delay (being blocked at M I), premature centromere separation, polyploidy, and aneuploidy (such as, hyperploid, hypoploid).     

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na⁺-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na⁺-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x^?_c and X^?_AG, respectively. Here we report the detection of Na⁺-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na⁺-dependent aspartate transport was not detected in pig oocytes. Mediated Na⁺-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X^?_AG is similar in both the pig and the mouse, system x^?_c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species. © 1993 Wiley-Liss, Inc.     

Cryopreservation of unfertilized mouse oocytes from inbred strains by ultrarapid freezing

Unfertilized mouse oocytes from inbred strains (BALB/c, C3H/He and C57BL/6) were frozen ultrarapidly by direct plunging into liquid nitrogen, immediately after exposure to a highly-concentrated solution (DAP 213: 2 M dimethyl sulphoxide, 1 M acetamide, and 3 M propylene glycol in PB 1), and were later thawed in a 37 degrees C waterbath. After thawing, 76.8-90.9% of recovered oocytes were morphologically normal. Following fertilization in vitro of cryopreserved oocytes, the proportion of 2-cell embryos 24 h after insemination ranged from 70.7% to 83.4%. Nearly all 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 31.0-43.0% of 2-cell embryos developed into normal young.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na⁺-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B^O,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na⁺-dependent in the oocyte; this Na⁺-dependent component of transport becomes undetectable by the blastocyst stage. The Na⁺-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B^O,+. In both oocytes and blastocysts Na⁺-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na⁺-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na⁺-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na⁺-dependent system B^O,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley-Liss, Inc.     

Chromosomal axis formation and meiotic progression in chicken oocytes: a quantitative analysis

The assembly and disassembly of the synaptonemal complexes (SCs) correlate with the progression of meiotic prophase I. Using immunostaining of the cohesin component SMC3, which is present in the axial elements of the SC, we characterized the synaptic process in chicken oocytes and quantified the frequency of the different prophase stages at hatching and at 3 different ages after hatching. The analysis provides detailed quantitative data regarding the meiotic stages in the chicken ovary showing that the maximum amount of pachytene oocytes is found around hatching and that oocytes reach the diplotene stage 5 days after entering into meiosis. We confirmed the asynchrony of the meiotic development in the female chicken gonad showing that the ovary has a composite population of cells at different stages from day 17 before hatching and for several days after hatching. The significance of these results is discussed in relationship to functional experimental procedures that involve avian oocytes.     

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.