Can we predict oocyte maturation prior to denudation for intracytoplasmic sperm injection?

Intracytoplasmic sperm injection (ICSI) was introduced in 1992 as a method to treat couples with severe male infertility. However, in the last two decades, the use of ICSI has increased substantially even among patients without male factor infertility. In ICSI the oocytes are scrutinized for maturity upon insemination and the immature oocytes are discarded. The aim of the present study was to assess the ability of an experienced embryologist to identify the maturity of the oocytes prior to their denudation.In the present prospective observational study, four experienced embryologists examined the oocytes prior to their denudation and decided whether the oocytes were mature or immature. Later, the oocytes were denudated and the embryologist again examined the oocytes to confirm their prior assumptions.483 oocytes were examined by four embryologists. Three hundred and fifty one of the oocytes were mature (72.7%) and 132 were immature (27.3%). The embryologists were able to correctly identify oocytes maturation status in 85.3% of cases. The embryologists were able to correctly identify 90% of the mature oocytes and 72.7% of the immature oocytes. When they assumed that the oocytes were mature they were correct in 89.% of the cases, while only 74.6% of their prediction that the oocytes were immature were true. To conclude, the embryologists are able to identify the oocytes maturation status before denudation at the majority of the cases. Whenever the oocytes are suspected to be immature, further consideration should be made whether to proceed to ICSI or not.

Actin-microtubule interplay coordinates spindle assembly in human oocytes.

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.

Morphometric assessment of in vitro matured dromedary camel oocytes determines the developmental competence after parthenogenetic activation.

The aim of the current study was to improve the selection method of camel oocytes after in vitro maturation by reducing exclusion criteria that were based only on the presence of the first polar body. A combined nuclear and morphometric assessment of camel oocytes after in vitro maturation was included to perform a judgment. The nuclear status of the oocytes, including the presence of the first polar body, meiosis I stage, and lack of nuclear materials, was investigated. The morphometric criteria that comprised the dimensions of each oocyte were as follows: diameter of the whole oocyte, including the zona pellucida (ZPO), zona pellucida thickness (ZPT), ooplasm diameter (OD), the perivitelline space (PVS) area, and PVS diameter. Among the oocytes with different nuclear status, there were no differences in ZPO and ZPT. However, oocytes with no nuclear material showed a significant reduction in OD (110.19 ± 1.4 µm) and a significant increase in PVS area (2139 ± 324.6 µm2) and PVS diameter (13.9 ± 1.96 µm) when compared with oocytes in the meiosis I stage (117.41 ± 2.85 µm, 1287.4 ± 123.4 µm2, and 8.56 ± 0.65 µm, respectively). To simplify the selection, the major difference between meiosis I and degenerated oocytes was the diameter of the PVS, which was greater than the ZPT in degenerated oocytes. Therefore, three groups were morphologically differentiated into oocytes with polar bodies (PB1), meiosis I (MI) oocytes, and degenerated oocytes. MI oocytes were able to extrude the polar body after activation but were not able to develop into blastocysts. In contrast, MI oocytes were able to develop into blastocysts after a biphasic activation protocol in which the oocytes were electrically activated and treated with ionomycin after 2 h. In conclusion, the results obtained by the morphometric assessment allowed us to develop a simple and objective classification system for in vitro matured dromedary camel oocytes, which will lead to accurate oocyte selection for the support of subsequent embryonic development.

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

[Dynamics of morphofunctional changes in aging bovine ova during the prolonged culture in vitro].

In the absence of activating stimuli, aging processes are initiated in matured mammalian oocytes, which negatively affect the quality of ova and their capacity for further development. On the model of the prolonged culture of bovine oocytes, the dynamics of a number of morphofunctional changes associated with the postovulatory aging was investigated in the present work. In cumulus-enclosed oocytes, migration of the first polar body relative to metaphase II chromosomes started between 18 and 22 h of maturation. The angle of the body deviation from the metaphase plate rose as the culture time increased to 30 h. By 32 h of culture, a gain in the rate of ova with the abnormal chromosome morphology was observed that continued up to 56 h. Furthermore, after 56 h, signs of spontaneous parthenogenetic activation were revealed in 16 percent of matured oocytes. During the prolonged culture of oocytes deprived of cumulus cells after 20 h of maturation, an increase in the frequency of chromosomal abnormalities was found only by 44 h. At the same time the cumulus elimination did not affect the maintenance of the meiosis II blockade in aging ova. Meanwhile, destructive chromosomal changes in oocytes were attended by a gradual rise in the level of apoptotic degeneration and reduction in the proliferative activity of surrounding cumulus cells. The results obtained point to the various temporal dynamics of distinct morphofunctional changes and to the participation of cumulus cells in modulation of the speed of metaphase chromosome destructive modifications in aging bovine ova.

Dynamin 2 regulates actin-mediated spindle migration in mouse oocytes.

BACKGROUND INFORMATION: During meiosis, a bipolar spindle forms in the central cytoplasm of an oocyte and then moves to the cortex to extrude the first polar body. This is dependent on the regulation of actin and actin-related molecules. Dynamin 2, a large guanosine triphosphatases (GTPase) known to regulate clathrin-mediated endocytosis, is involved in actin recruitment and actin-based vesicle mobility. In this study, we investigated the role of Dynamin 2 in oocyte meiosis. RESULTS: Dynamin 2 was localised at the cortex and around the spindles of oocytes. Disrupting Dynamin 2 activity by RNAi or an inhibitor resulted in polar body extrusion failure. Using time-lapse microscopy to monitor aberrant oocyte cytokinesis, the chromosomes were first separated, but then re-joined. Actin expression in oocytes was decreased; and actin cap formation was disrupted, which was confirmed by the disappearance of cortical-granule-free domains. In addition, live cell imaging showed that spindle migration had failed and that spindles were arrested centrally in oocytes. This may have been due to the Dynamin-binding protein Profilin and actin-related protein 2/3 (ARP2/3) complexes, which exhibited dispersed signals after disrupting Dynamin 2 activity. CONCLUSIONS: Thus, our results indicate that Dynamin 2 regulates spindle migration and polar body extrusion during mouse oocyte meiosis through an actin-based pathway.

MKlp2 inhibitor paprotrain affects polar body extrusion during mouse oocyte maturation.

BACKGROUND: Mammalian oocyte meiotic maturation involves a number of important processes, including spindle assembly and migration, cortical reorganization and polar body extrusion. Numerous proteins contribute to these processes, but it is unknown whether MKlp2 (mitotic kinesin-like protein 2; also called KIF20A), a microtubule-associated protein that regulates cytokinesis during mitosis, is involved in oocyte maturation. METHODS: Confocal microscopy, time lapse microscopy, inhibitor treatment were adopted to examine the roles of MKlp2 in mouse oocyte. RESULTS: Immunostaining results showed that MKlp2 localized to oocyte microtubules. Time-lapse microscopy showed that disrupting MKlp2 expression with paprotrain, a specific inhibitor of MKlp2, resulted in polar body extrusion failure. This could be rescued after rescuing oocytes from paprotrain in fresh medium. Cell cycle analysis showed that most oocytes were arrested at metaphase I or telophase I. However, oocyte spindle structure and chromosome alignment were not disrupted after the inhibition of MKlp2 by paprotrain. CONCLUSIONS: This study demonstrated that MKlp2 is crucial for oocyte maturation by regulating polar body extrusion.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes.

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two-cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early Caenorhabditis elegans embryo.

This chapter is an update of the previously published book chapter "Correlative Light and Electron Microscopy of Early C. elegans Embryos in Mitosis" (Müller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007). Here, we have adapted and improved the protocol for the isolated meiotic embryos, which was necessary to meet the specific challenges a researcher faces while investigating the development of very early Caenorhabditis elegans embryos ex-utero. Due to the incompleteness of the eggshell assembly, the meiotic embryo is very fragile and much more susceptible to changes in the environmental conditions than the mitotic ones. To avoid phototoxicity associated with wide-field UV illumination, we stage the meiotic embryos primarily using transmitted visible light. Throughout the staging and high-pressure freezing, we incubate samples in an isotonic embryo buffer. The ex-utero approach allows precise tracking of the developmental events in isolated meiotic embryos, thus facilitating the comparison of structural features between wild-type and mutant or RNAi-treated samples.

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 µM of milrinone, the enucleation rate was significantly improved by 100 µM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 µM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 µM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes.

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 µm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.

A clone of elusive parents: gonadotroph adenoma-female type.

A 69-year-old woman presented with visual disturbance. Perimetry testing revealed a bitemporal hemianopia. Brain MRI demonstrated a 2.2-cm gadolinium-enhancing pituitary mass. Previously she had been treated for hypothyroidism, hypertension, and dyslipidemia. She had hyperprolactinemia. Endoscopic transsphenoidal debulking improved her visual field defects. Histology showed a chromophobic adenoma. Electron microscopy showed elongated, polar cells with long, slender processes. The small uniform secretory granules were peripherally disposed, collecting heavily within cell processes. Based on electron microscopical characteristics the tumor is consistent with an ACTH-negative female gonadotroph adenoma. The parent cell of this rare variant of a pituitary adenoma is yet unknown.

Five chromosome segregation in polar bodies and the corresponding oocyte.

For a comprehensive picture of the meiotic process and to follow up its products, five chromosomes were tested by fluorescent in-situ hybridization in both polar bodies (PB) and corresponding 145 oocytes. Results were obtained in 143 sets and the prediction of euploidy or aneuploidy based on PB analysis was confirmed by direct analysis in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes, while in the remaining eight, at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in PB: 58% in PB1 and 42% in PB2. Premature separation of chromatids occurred in 89% of aneuploid PB1, whereas only 11% of errors derived from bivalent non-disjunction. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division. In conclusion, the testing of PB predicted reliably the oocyte's chromosome condition. Although limited to five chromosomes, the follow up of meiosis by fluorescent in-situ hybridization provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte.

Polar body morphology is not predictive of its cell division origin.

PURPOSE: This study sought to determine whether morphology of simultaneously biopsied polar bodies is predictive of their cell division origin. METHODS: Levels of heterozygosity were measured using single nucleotide polymorphism (SNP) microarrays in sequentially biopsied polar bodies to establish the predictive value on samples with known cell division origins. The validated method of predicting cell division origin of polar bodies using heterozygosity rates was then applied to simultaneously biopsied polar bodies which had origin predictions made by morphological assessment. RESULTS: SNP microarray heterozygosity analysis was proven to be 94% predictive when tested against the known origin of sequentially biopsied polar bodies (n = 133). This methodology subsequently demonstrated that morphology was only 63% consistent when tested on simultaneously biopsied polar bodies (n = 455; P < 0.0001). Predictions of the origins of aneuploidy using morphology assignment were also significantly different than with heterozygosity assignment of polar body cell division origin (P = 0.0003). CONCLUSIONS: Studies of the origin of aneuploidy utilizing morphological assignment of polar body cell division origin without heterozygosity analysis may be inaccurate and should be interpreted with caution.

Following the course of pre-implantation embryo patterning by non-linear microscopy.

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

Pre-treatment of sperm reduces success of ICSI in the pig.

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

The relationship between meiotic spindle imaging and outcome of intracytoplasmic sperm injection: a retrospective study.

Meiotic spindle analysis with a non-invasive technique, the PolScope, is used to protect the meiotic spindle from damage during microinjection. To evaluate the predictive feature of PolScope, we have designed a retrospective study to analyse the correlation between the meiotic spindle visualisation with regard to spindle location and outcomes of assisted reproductive technologies (ART), including patient age, previous cycles, the number of the collected oocytes, fertilisation rates (FR), pronuclear scoring (PNS) and embryo scoring of the days from two to five. All of the data belonging to 1496 oocytes from 190 patients were statistically analysed. We found that the oocytes having PolScope visualised spindle have higher FR, and also observed that when the spindle located at 0°-30° according to the first polar body, gave the highest FR. PNS gave higher scores in the spindle visualised group, but spindle angle did not affect PNS outcomes. Although a correlation was found between spindle visualisation and developed embryo qualities, particularly at day 2 and 3, spindle angles did not affect embryo quality. We conclude that PolScope microscopy has an efficiency to estimate FR, and cleavage stage embryo development.