In Vitro Fertilization (IVF) is generally accepted as the most effective treatment for infertility. Its success depends on the correct and meticulous implementation of each stage in the procedure. The process of systematically examining embryos is standardized through the use of internationally recognized criteria. On the other hand, the evaluation of oocyte quality continues to be conducted more arbitrarily. A morphologically good quality mature human oocyte is universally described as one that shows a homogeneous cytoplasm, has a single polar body (PB), an approprate zona pellucida (ZP) thickness and a proper perivitelline space (PVS). An abnormality in one or more of these features are very common in IVF cycles and may be related to several factors that are extrinsic and intrinsic to the patient. There has been extensive speculation over whether specific anomalies in the structure of oocytes can suggest a reduced developmental capacity. The most notable among the dysmorphisms of oocytes are the severe morphological deviations, such as smooth endoplasmic reticulum clusters, cytoplasm granularity, and giant oocytes that are related to genetic abnormalities, and extra-cytoplasmic parameters such as PB morphology, the PVS and ZP abnormalities that may indicate oocyte ageing. This paper acknowledges the significance of oocyte morphology grading as an important and practical predictor of a successful IVF outcome and it can serve as a supplementary measure to embryonic assessment in order to optimize efficacy of assisted reproductive technology (ART). It discusses the fundamental knowledge that infertility specialists and embryologists should possess to enable its routine application in the ART laboratory.

Fertilization In Vitro ; Sperm Injections, Intracytoplasmic ; Reproductive Techniques, Assisted ; Embryonic Development ; Meiotic Spindle ; Spindle Apparatus

Spindle assembly checkpoint (SAC) is a protective mechanism for cells to undergo accurate mitosis. SAC prevented chromosome segregation when kinetochores were not, or incorrectly attached to microtubules in the anaphase of mitosis, thus avoiding aneuploid chromosomes in daughter cells. Aneuploidy and altered expression of SAC component proteins are common in different cancers, including lung cancer. Therefore, SAC is a potential new target for lung cancer therapy. Five small molecule inhibitors of monopolar spindle 1 (MPS1), an upstream component protein of SAC, have entered clinical trials. This article introduces the biological functions of SAC, summarizes the abnormal expression of SAC component proteins in various cancers and the research progress of MPS1 inhibitors, and expects to provide a reference for the future development of lung cancer therapeutic strategies targeting SAC components.
.
Humans ; Cell Cycle Proteins/metabolism* ; Spindle Apparatus/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; M Phase Cell Cycle Checkpoints/genetics* ; Lung Neoplasms/metabolism*

In assisted reproductive techniques, the operator attempts to select morphologically best embryos to predict embryo viability. Development of polarized light microscope, which evaluates the oocytes' spindles according to birefringence of living cells, had been helpful in oocyte selection. The aim of this study is evaluating the relationship between meiotic spindles visualization and intracytoplasmic sperm injection (ICSI) outcomes in human oocytes. In this study, 264 oocytes from 24 patients with an average age of 30.5±7.5 years with infertility duration of 1 to 10 years were collected. The oocytes were randomly allocated to the control injection group (n=126) and the oocyte imaging group (spindle-aligned group) (n=138). In the spindle-aligned group, the meiotic spindle was identified by means of polarized light microscope to align the spindle at 6 or 12 o'clock. Then the spindle-aligned group was divided into three sub-groups based on spindle morphology: fine, average, and (poor). After ICSI, embryos were checked every 24 hours and scored; 72 hours later, high-grade embryos were transferred intravaginally to uterus. This study showed that the fertilization rate in the spindle-aligned group was higher than the control group (P<0.05). After cleavage, a positive correlation was observed between spindle morphology and embryo morphology. Among the sub-groups of spindle-aligned group, the embryos' morphology of the fine group was better than the other subgroups and embryos of the poor group had lower quality and more fragmentation. The results revealed that the selection of oocytes based on meiotic spindle imaging can significantly improve the rate of fertilization and embryo cleavage and certainly increase the rate of implantation.
Birefringence ; Embryonic Structures ; Fertilization ; Humans* ; Infertility ; Microscopy, Polarization ; Oocytes* ; Reproductive Techniques, Assisted ; Sperm Injections, Intracytoplasmic* ; Spindle Apparatus* ; Uterus

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles.
Humans ; Kinetochores ; M Phase Cell Cycle Checkpoints ; Metaphase ; Microtubules ; Phosphotransferases ; Protein Kinases ; Ribosomal Protein S6 Kinases ; Ribosomal Protein S6 Kinases, 90-kDa* ; Siblings ; Spindle Apparatus*

The aim of the present study is to observe the dynamic changes of γ-tubulin in mouse somatic nuclear transferred (SCNT) embryos. The γ-tubulin was detected and analyzed in the enucleated oocyte and SCNT embryos by immunofluorescence and laser confocal microscopy. The results showed that γ-tubulin distributed in the cortex of the enucleated MII oocytes, and decreased in this area during the activation of oocytes. Meanwhile cytoplasmic asters appeared, but there was no spindle formed. Spindle formation could be observed in the enucleated oocytes which were injected with cumulus cells and activated by SrCl2. The spots-like γ-tubulin signals spread between chromosomes at the prophase, and the signals arrayed with spindle or aggregated at two poles of the spindle at the early metaphase. Furthermore, γ-tubulin signals were localized between the segregated sister chromatids at anaphase or telophase. Some reconstructed embryos exhibited advanced activation, showing abnormal spindles and aberrant distribution of γ-tubulin and chromosomes. Two spindles would be formed when the cumulus cell was injected into an intact oocyte, and the distribution of γ-tubulin was similar to that of the normal SCNT. Moreover, advanced activation also occurred in this case and formed either two small spindles or one big barrel-shaped spindle. These results suggest that γ-tubulin plays a pivotal role in spindle assembling in mouse SCNT embryos. The reconstructed oocytes were easily to be activated, and aberrant distribution of γ-tubulin is associated with formation of abnormal spindles and chromosome misalignment.
Animals ; Embryo, Mammalian ; metabolism ; Fluorescent Antibody Technique ; Metaphase ; Mice ; Microscopy, Confocal ; Nuclear Transfer Techniques ; Oocytes ; cytology ; Spindle Apparatus ; metabolism ; Telophase ; Tubulin ; metabolism

BubR1 gene is a homologue of the mitotic checkpoint gene Mad3 in budding yeast which is highly conserved in mammalian. BubR1 protein is a key component mediating spindle assembly checkpoint activation. BubR1 safeguards accurate chromosome segregation during cell division by monitoring kinetochore-microtubule attachments and kinetochore tension. There is a dose-dependent effect between the level of BubR1 expression and the function of spindle assembly checkpoint. BubR1-deficient would lead to mitotic progression with compromised spindle assembly checkpoint because cells become progressively aneuploid. Recently, it has been reported that BubR1 also plays important roles in meiotic, DNA damage response, cancer, infertility, and early aging. This review briefly summarizes the current progresses in studies of BubR1 function.
Cell Cycle Proteins ; genetics ; metabolism ; physiology ; Chromosome Segregation ; genetics ; physiology ; Kinetochores ; metabolism ; physiology ; Mitosis ; genetics ; physiology ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; physiology ; Saccharomycetales ; genetics ; physiology ; Spindle Apparatus ; genetics ; metabolism ; physiology

Stem cells use asymmetric and symmetric cell division to generate progeny. Symmetric cell division is defined as the generation of daughter cells that are destined to acquire the same fate. Stem cells divide asymmetrically to generate one daughter with a stem-cell fate and one daughter with different fate. Disruption of the machinery that regulates asymmetric division may be a reason for the generation of cancer. The asymmetric mechanism is maintained by cell polarity factors, cell fate determinants, and the spindle apparatus. The mutation or dysregulation of these factors may change stem cells from asymmetric to symmetric cell division, then leading to tumorigenesis. Therefore, further study is needed on the mechanisms of stem cell control between asymmetric and symmetric cell division, as well as the relationships among stem cells, cancer stem cells, and tumor cells. It may bring us a new approach for the resistance, recurrence, and metastasis of tumors.
Animals ; Cell Division ; physiology ; Cell Polarity ; Cell Transformation, Neoplastic ; Drosophila ; cytology ; Humans ; Neoplasms ; pathology ; Neoplastic Stem Cells ; pathology ; Neurons ; cytology ; Spindle Apparatus ; metabolism ; Tumor Suppressor Proteins ; metabolism

Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.
Animals ; Embryo, Mammalian ; Embryonic Development ; Female ; Meiosis ; Mice ; Mitosis ; Oocytes ; cytology ; Parthenogenesis ; Spindle Apparatus ; physiology ; Tubulin ; physiology

The Spindle-view, a specialized instrument for observing spindle image, was applied to observe the meiotic spindles of vitro matured porcine oocytes at 36, 42, 44, 48h, and enucleation from porcine, comparing to the previously methods (McGrath-Solter's method and two-step-squeezing method) in the enucleated. The results showed that: (1) there was no noticeable differences at vicinity of spindle images and 1st polar body among in vitro matured porcine oocytes at 40-48 h under the instrument; (2) Spindle-view is suitable for the observation of meiotic spindles of matured oocytes and enucleation from porcine; the modified Spindle-view method for enucleation is significantly better than McGrath-Solter' s method and two-step-squeezing method in the enucleated rates (95.5%, 42.1%, 74.2%, P < 0.0l) of absolutely removing nuclei matter; (3) the spindle images could be used to monitor the oocyte qualities.
Animals ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; Cytological Techniques ; Female ; Nuclear Transfer Techniques ; Oocytes ; cytology ; Spindle Apparatus ; ultrastructure ; Swine

OBJECTIVETo study the effect of maternal age on meiotic spindle and chromosome configuration of oocytes.

METHODSSpindle and chromosome configuration was examined in day 1 unfertilized human oocytes after in vitro fertilization (IVF) and intracytoplasmic sperm injection(ICSI) by immunocytochemistry and visualized by laser confocal microscopy.

RESULTSStatistically significant differences were observed on normal spindle and chromosome configurations of oocytes between 25-29 maternal age group (33% and 31%, respectively), and 30-34 age group (P< 0.05) as well as 35-40 age group(0%, P<0.01). The incidence of abnormal spindle and chromosome configurations of oocytes from 30-34 and 35-40 maternal age groups was much higher than that of oocytes from 25-29 age group (P<0.01, P<0.05).

CONCLUSIONIncidence of abnormal spindle and chromosome configuration of oocytes is related to maternal age. It could be an important reason of age related oocyte aneuploidy.

Adult ; Age Factors ; Chromosomes, Human ; metabolism ; Female ; Fertilization in Vitro ; Humans ; Immunohistochemistry ; Microscopy, Confocal ; Oocytes ; metabolism ; Sperm Injections, Intracytoplasmic ; Spindle Apparatus ; metabolism