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Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.
Heide Schatten, Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA,
Sun Qing-Yuan, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
The formation of the meiotic spindle is a critical process to assure accurate chromosome segregation and subsequent embryo development. Coordinated formation and organization of microtubules, centrosomes, and chromosomes is important for meiotic spindle formation at the oocyte's center after germinal vesicle breakdown (GVBD), for the formation of the MI (meiosis I) spindle to segregate homologous chromosomes, and for the formation of the MII (meiosis II) spindle to segregate chromatids, resulting in oocyte haploidy. The human oocyte is particularly susceptible to errors in chromosome segregation which may be related to defective centrosome and microtubule organization and to defective chromosome attachment to kinetochore microtubules and loss of molecular surveillance factors. The present chapter is focused on (1) formation of central, MI and MII spindle, with focus on microtubules and centrosomes; (2) chromosome dynamics and segregation during MI and MII, with focus on molecular aspects and surveillance mechanisms; and (3) spindle abnormalities, environmental influences, and possible treatments to restore spindle integrity with implications for assisted reproductive technologies (ART).
The formation of the meiotic spindle is a critical step during oocyte maturation and begins when the germinal vesicle breaks down (GVBD) as a result of stimulation by luteinizing hormone (LH). Spindle formation in most mammalian oocytes takes place at the oocyte's center and involves significant restructuring of the cytoskeleton that will impact subsequent cellular and molecular functions that are also important for later development . Coordinated formation and organization of microtubules, centrosomes, and chromosomes begins directly after GVBD with remodeling of these major spindle components in the oocyte's center to form the meiotic spindle.
Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.
Recent developments in scanning electron microscopy (SEM) have resulted in a wealth of new applications for cell and molecular biology, as well as related biological disciplines. It is now possible to analyze macromolecular complexes within their three-dimensional cellular microenvironment in near native states at high resolution and to identify specific molecules and their structural and molecular interactions. New approaches include cryo-SEM applications and environmental SEM (ESEM), staining techniques and processing applications combining embedding and resin-extraction for imaging with high resolution SEM, and advances in immuno-labeling. New developments include helium ion microscopy, automated block-face imaging combined with serial sectioning inside an SEM chamber, and Focused Ion Beam Milling (FIB) combined with block-face SEM. With chapters written by experts, this guide gives an overview of SEM and sample processing for SEM and highlights several advances in cell and molecular biology that greatly benefited from using conventional, cryo, immuno and high-resolution SEM.
The prospect of utilizing stem cells for clinical applications has generated an enormous amount of enthusiasm in the stem cell research community and has led to a wealth of new data that offer the possibility for practical applications into clinical translation. Recent advances in stem cell imaging has contributed greatly to the ultimate goal to identify and culture specific cell types for regeneration of tissue that had been affected by disease such as Parkinson's, Alzheimer's, heart disease, muscle diseases, and others.
The role of centrosomes in stem cell division has recently been highlighted and further ascribes important functions to centrosomes in stem cell maintenance, cellular differentiation, and development. Advanced cell and molecular studies coupled with immunofluorescence, electron microscopy, and live cell imaging of specific centrosome proteins have contributed greatly to our knowledge of centrosome composition, structure, and dynamics and have uncovered new insights into mechanisms of centrosome functions in asymmetric cell division. The establishment of asymmetry and differential positioning of mother and daughter centrosomes during stem cell mitosis is important for allowing one cell to maintain stem cell characteristics while the sibling cell undergoes differentiation. Another key role for centrosomes has been revealed in primary cilia of embryonic stem cells that play significant roles in cellular signaling and are therefore critically important for stem cell decisions. Studies of signaling through primary cilia may contribute important information that may aid in the production of specific cells that are suitable for tissue repair and regeneration in the field of regenerative medicine.
Two-photon excitation microscopy (also referred to as multiphoton laser scanning microscopy) has gained increasing popularity during the past few years because of the distinct advantages over single-photon microscopy, which includes increased penetration depth and low out-of-focus photobleaching and photodamage. It allows superior imaging of thick specimen compared to single-photon microscopy, and it excels at imaging live cells either single or within intact tissue. This highly valuable tool has been used with great success to gain important new insights into brain tissue, embryos, whole organs, entire animals, and it has been most useful in numerous other applications.