| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gamete Biology |
a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gamete biology, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The oocyte offers an alternative model for cell polarity research in mammals. Similar to the budding yeast, an oocyte is a single cell with a spatially asymmetric arrangement of membranes and cytoplasmic components. Different from budding yeast and somatic cells, the metaphase plate in an oocyte is located near the plasma membrane. As was shown for the mouse metaphase II (MII) oocyte, the asymmetrically localized domains include 1) a cortical actin-rich domain; 2) a meiotic spindle, including chromosomes, localized beneath the actin-rich domain; 3) a cortical granule-free domain overlying the meiotic spindle; 4) a microvilli-free domain of the plasma membrane overlying the meiotic spindle; and 5) an organelle-free area with few detectable cellular components, such as mitochondria, Golgi complex, and endoplasmic reticulum [5–7]. Typically, asymmetric meiotic divisions in oocytes result in the formation of two very different types of progeny: a very large oocyte and small polar bodies that are approximately 1% of the oocyte's size [8, 9]. Each of them, however, possesses the same maternal DNA content. The size difference between these two cell types is a direct result of the asymmetric positioning of the spindle [7, 9].
In our previous study [10], we found that after a 3% (w/v) (90 mM) sucrose treatment, the region including the MII spindle and chromosomes in mouse oocytes could be visualized as a projection under standard light microscopy. A similar phenomenon has also been found in bovine oocytes [11]. Although the original purpose of previous studies was to find a simple but efficient method for locating and removing the chromosomes, the sucrose-induced changes provided a new and improved model with which to investigate cell polarity, because cellular asymmetry was enhanced by sucrose treatments. Furthermore, the maintenance and dynamics of cell polarity by the cytoskeleton can also be studied with this model, in which the formation of the projection can be used as a marker for interactions between different components of the cytoskeleton on treatments of the oocytes by cytoskeletal drugs.
The objectives of the present study were, first, to identify the induction factor(s) for changes occurring in MII oocytes when incubated in media containing sucrose or other hypertonic media and, second, to investigate the role(s) of the cytoskeleton (microtubules and microfilaments) in these changes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All media were prepared fresh and filter-sterilized through a 0.2-µm filter (Acrodisc; Pall Gelman Laboratory, Ann Arbor, MI). The osmolalities of all media and solutions were measured with a Vapor Pressure Osmometer (Wescor, Inc., Logan, UT). Cytochalasin B (CCB; 15 µM), which disrupts actin filaments, nocodazole (NOC; 10 µM), which disrupts microtubules, and taxol (TAX; 12 µM), which causes polymerization of tubulin molecules, were prepared as 1-mg/ml stocks in dimethyl sulfoxide (DMSO) and diluted to final concentrations in M2 (Sigma; M7167) [12] plus 10% fetal bovine serum (FBS). These concentrations are in the effective ranges for these drugs as shown in previous studies [7, 13, 14]. The same amount of DMSO, which never exceeded 140 mM in treatment media, was added in control media.
Oocyte Collection
All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Connecticut. Germinal vesicle (GV)-stage oocytes were obtained from untreated CD1 female mice (age, 4–5 wk; Charles River Laboratories, Wilmington, MA). Oocytes were extracted from large ovarian follicles in M2 medium with a 27-gauge needle. Subsequently, they were cultured in maturation media (M2 + 10% FBS) containing a microfilament polymerization inhibitor (CCB), a microtubule polymerization inhibitor (NOC), or a microtubule depolymerization inhibitor (TAX). The MII-stage oocytes were collected from CD1 mice at 13–17 h post-hCG after superovulation with 10 IU of eCG followed 48 h later by 10 IU of hCG and were placed in M2 plus 10% FBS. Oocytes were freed of their cumulus cells by brief exposure to 300 IU/ml of hyaluronidase at 37°C and gentle pipetting. Denuded oocytes were incubated at 37°C in a humidified atmosphere with 5% CO2 in air or at room temperature in different media as shown below (see Experimental Design).
Immunohistochemistry and Laser-Scanning> Confocal Microscopy
To visualize DNA, oocytes were stained in 1.5 µM Hoechst 33342 for 10 min and observed with a Nikon inverted microscope (Eclipse TE300; Nikon, Tokyo, Japan) under normal and/or ultraviolet (UV) light. For immunohistochemistry, oocytes were fixed in 2% formaldehyde at 37°C for at least 30 min. They were then washed in washing buffer (PBS containing 3 mM NaN3, 0.01% [w/v] Triton X-100, 0.2% [w/v] nonfat dry milk, 2% [v/v] normal goat serum, 0.1 M glycine, and 2% [w/v] BSA) three times (15 min each) and left in washing buffer overnight at 4°C for blocking and permeabilization. Oocytes were then triple-stained to visualize microtubules, microfilaments, and DNA. Briefly, samples were incubated in mouse anti-
-tubulin (1:200) for 4 h at 37°C or overnight at 4°C. After three washes in washing buffer, the oocytes were incubated in fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (1:200) for 1 h at 37°C. The oocytes were washed and incubated with rhodamine-phalloidin (1:1000) at 37°C for 30 min to stain for actin filaments. Finally, the oocytes were washed, stained for DNA with 7.5 µM propidium iodide or 15 µM Hoechst 33342, mounted on glass slides, and examined with a laser-scanning confocal microscope (TCS SP2; Leica, Mannheim, Germany).
Experimental Design
Experiment 1: Effect of sucrose concentration This experiment was designed to define the optimal concentration of sucrose for initiating projection formation in mouse oocytes. Oocytes at the MII stage were cultured in M2 plus 20% FBS containing 0, 50, 100, 200, 400, or 800 mM sucrose at 37°C for 1 h. Projections were recorded under an inverted microscope at 400x magnification and separated into two grades: grade A, a very smooth-curved distended area on the surface of oocytes that appears as a distortion of their shape (Fig. 1a), and grade B, a pointed protrusion on the oocytes that is smaller in area but more pronounced than grade A and easily distinguished from other domains (Fig. 1a).
|
Experiment 2: Effect of serum concentration Experiment 2 was designed to investigate whether serum concentration has any effect on the formation of projections. This experiment capitalized on the most effective concentration of sucrose as determined during experiment 1, which was 400 mM. The MII-stage oocytes were washed and equilibrated in M2 plus 400 mM sucrose containing 0, 10, 20, 50, or 100% (v/v) FBS.
Experiment 3: Effect of various standard media To detect the effect, if any, of the standard media, oocytes at the MII stage were incubated in different media containing 400 mM sucrose and 10% FBS. The media used were M2, M199 (catalog no. 12458-014; Gibco, Grand Island, NY), DPBS+ (Ca2+- and Mg2+-containing; catalog no. 14287-080; Gibco), DPBS- (Ca2+- and Mg2+-free; catalog no. 12377-016; Gibco), Beltsville Embryo Culture Medium (BECM) [15], and KSOM (catalog no. MR-121-D; EmbryoMax, Phillipsburg, NJ) [16].
Experiment 4: Effect of sugars other than sucrose To determine if other common sugars have the same effect on oocytes as sucrose, oocytes were incubated in M2 medium plus 10% FBS containing 400 mM sucrose (757 mOsm), maltose (746 mOsm), trehalose (754 mOsm), sorbitol (717 mOsm), mannitol (715 mOsm), or glucose (720 mOsm). To locate the chromosomes under UV light, cultured media contained a final concentration of 1.5 µM Hoechst 33342 dye.
Experiment 5: Effect of osmolality Oocytes were incubated and balanced in media with different osmolalities generated by 0, 85 (or 0.5%), 170 (or 1%), 340 (or 2%), and 680 mM (or 4%) NaCl in M2 medium with 0.1% BSA and 1.5 µM Hoechst 33342. The oocytes were observed under normal and/or UV light and recorded as grade A or B as described above.
Experiment 6: Effect of cytoskeletal drugs on in vivo-matured oocytes Oocytes at the MII stage, recovered from superovulated mice, were placed in M2 plus 10% FBS containing cytoskeletal drugs as follows: 1) 15 µM CCB, 2) 10 µM NOC, 3) 12 µM TAX, 4) 15 µM CCB plus 10 µM NOC, 5) 15 µM CCB plus 12 µM TAX, or 6) 140 µM DMSO as control. After a 3-h incubation at 37°C in a humidified atmosphere of 5% CO2, oocytes were placed in media as above with the addition of 1.5 µM Hoechst 33342 in the presence or absence of 400 mM sucrose. The oocytes with one, two, or more projections were observed and graded under an inverted microscope. After each experiment, the oocytes were fixed for immunohistochemistry and observed under a laser-scanning confocal microscope.
Experiment 7: Effect of cytoskeletal drugs on immature oocytes The GV-stage oocytes were matured in M2 plus 10% FBS containing cytoskeletal drugs as follows: 1) 15 µM CCB, 2) 10 µM NOC, 3) 12 µM TAX, or 4) 140 µM DMSO as control. After incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% air for 18 h, which is the time needed for oocyte maturation, oocytes were placed in the same media as above with Hoechst 33342 (1.5 µM) in the presence or absence with 400 mM sucrose. The oocytes were observed under normal and/or UV light with an inverted microscope to record projections and then fixed for immunohistochemistry and laser-scanning confocal microscopy.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Six different concentrations of sucrose were used, and projection formation by in vivo-matured oocytes was compared. In the control group (without sucrose), only 4% of oocytes had a projection (Fig. 1). When incubated in 50 mM sucrose, 11% of oocytes had a projection. However, when incubated with more than 100 mM sucrose, more than 90% of oocytes had a projection. Generally, the projections were either smooth-curved (grade A) or point-shaped (grade B) (Fig. 1a). The chromosomes were often observed inside the projections in treated oocytes. Although the percentages of oocytes with a projection in the last four treatment groups were comparable, the proportion of grade A or B projections in each group was different. Most of the oocytes in 100 or 200 mM sucrose had grade A projections, whereas most in 400 or 800 mM sucrose had grade B projections (Fig. 1b). This experiment demonstrated that the formation and shape of projections were affected by different concentrations of sucrose.
Experiment 2: Effect of Serum Concentration
In experiment 1, 400 mM sucrose was shown to be the lowest concentration to induce the most grade B projections. Therefore, we used 400 mM sucrose in this and subsequent experiments to effectively induce projection formation. The MII oocytes were incubated in M2 plus 400 mM sucrose supplemented with 0, 10, 20, 50, or 100% (v/v) FBS, and no significant differences were found among the five groups (Fig. 2a). This experiment indicated that projection formation was not affected by serum concentration.
|
Experiment 3: Effect of Standard Medium
Based on the results of experiments 1 and 2, the 400 mM sucrose and 10% FBS (a common concentration) were selected. Six commercial or standard media (M2, M199, DPBS+, DPBS-, BECM, and KSOM) were tested. The osmolalities of the six media supplemented with 400 mM sucrose and 10% FBS were similar (745–768 mOsm). Oocytes incubated in these six media had comparable percentages of projection formation (Fig. 2b). These results illustrated that the standard medium was not involved in the induction of projections in oocytes.
Experiment 4: Effect of Sugars Other than Sucrose
Experiment 4 was designed to investigate if projection-induction in oocytes was sucrose-specific. Five mono- or disaccharides at a concentration of 400 mM were added to M2 plus 10% FBS and compared with 400 mM sucrose in M2 plus 10% FBS. The osmolality of the media with 400 mM disaccharides was approximately 750 mOsm, whereas that of those with monosaccharides was approximately 720 mOsm. However, no significant differences in the percentages of oocytes with projections were found among these six groups (Fig. 2c). This experiment suggested that the induction of projections was not sucrose-specific and that other sugar solutions with comparable osmolality could also induce projections in mouse MII oocytes.
Experiment 5: Effect of Osmolality
Experiment 5 was designed to investigate the effect of hypertonicity on projection formation. Oocytes were incubated in five concentrations of NaCl (0, 85, 170, 340, or 680 mM) in M2 medium supplemented with 0.1% BSA. In the control group (0 mM NaCl at 276 mOsm), no projections were found. However, 96% of oocytes incubated in medium containing 85 mM NaCl (470 mOsm) had projections, among which 54% were grade A and 42% grade B (Fig. 2d). When the concentration of NaCl increased to 170 mM (627 mOsm), the percentage of oocytes with projections of grades A and B combined was similar to that found with 85 mM NaCl, but the percentage of grade B projections significantly increased. When oocytes were incubated in 340 or 680 mM NaCl, 100% had grade B projections. This experiment demonstrated that the projections of mouse oocytes were induced by hypertonic osmolality and that their morphology was dependent on osmolality concentrations.
Experiment 6: Effect of Cytoskeletal Drugs> on In Vivo-Matured Oocytes
This experiment was designed to examine the roles of cytoskeleton on sucrose-induced projection formation. Drugs that disrupt actin filaments (CCB) or microtubules (NOC) or that polymerize tubulin molecules (TAX) were used either alone or in combination. None of the cytoskeletal drugs alone abolished the sucrose-induced projection formation (Fig. 3). In both the control and sucrose-treated oocytes, the chromosomes were located at the periphery and the spindle was attached to the actin-rich domain, as shown in light or confocal microscopic images (Fig. 4, a–d). Ninety-three percent of matured oocytes treated in 15 µM CCB for 3 h and then in 400 mM sucrose had projections. Surprisingly, most of the oocytes (74%) had two projections (Fig. 3; Fig. 4, g and h). Immunohistochemistry revealed that the MII spindle was detectable but that the two poles were asymmetric, with one attaching to the actin-rich cortex and the other orienting itself to the inner ooplasm (Fig. 4, f and h). Many more cytoplasmic microtubule asters were found in oocytes incubated in 400 mM sucrose, with or without CCB treatment, compared with those, if any, in isotonic sucrose-free media (Fig. 4, b, d, f, and h). The formation of the microtubule asters may have resulted because the hypertonic treatment induced the shrinkage of oocytes and, hence, increased the concentration of free tubulin molecules in the cytoplasm. This, in turn, increased microtubule polymerization and formation of microtubule asters [17].
|
|
When oocytes were incubated in 10 µM NOC for 3 h, the spindle was depolarized, yet chromosome clusters were still located near the plasma membrane and attached to the actin-rich domain in the cortex (Fig. 4, i and j). After incubation in 400 mM sucrose, 87% of oocytes had one or more projections at the locations of chromosome clusters and the actin-rich domain (Fig. 3; Fig. 4, k and l).
After exposure to 12 µM TAX for 3 h, many cytoplasmic microtubules were detected, but no obvious spindles were found (Fig. 4, m and n). Chromosomes were dispersed into multiple clusters (Fig. 4n). The actin-rich domain was observed in the area where the chromosomes were located. When incubated in 400 mM sucrose, 93% of oocytes had projections, among which 7% had two or more projections (Fig. 3; Fig. 4, o and p). If oocytes were exposed to both CCB and NOC, the actin-rich domain still existed, but no spindles or projections were detectable even with sucrose treatment (Fig. 3; Fig. 4, q–t). When oocytes were incubated in both CCB and TAX, the chromosomes in most oocytes were dispersed into multiple clusters, and most of them moved to the center of the oocytes (Fig. 4, u and v). After being placed in 400 mM sucrose, only 17% of oocytes had one or more projections at the locations of the chromosomes that remained close to the cortical actin-rich domain (Fig. 3; Fig. 4, w and x). None of the treatment combinations abolished occurrences of actin-rich domain in oocytes (Figs. 3 and 4). The persistence of the actin-rich domains was not affected by higher concentrations of cytoskeletal drugs. Media containing as much as 10-fold these concentrations also failed to disrupt the actin-rich domains, and most of oocytes still have an obvious actin-rich domain overlying the MII spindle (Fig. 4, f inset and h inset).
Experiment 7: Effect of Cytoskeletal Drugs> on Immature Oocytes
In the control group in which GV oocytes were incubated in 140 µM DMSO for 18 h, 42 of 49 oocytes (86%) extruded the first polar body, indicating proper in vitro maturation (Fig. 5). Whereas the majority of oocytes treated with cytoskeletal drugs could undergo GV breakdown, only 2% in the CCB group, 0% in the NOC group, and 9% in the TAX group extruded the first polar body. When incubated in 400 mM sucrose, 88% in the control group, 66% in the NOC group, and 58% in the TAX group had one or more projections (Fig. 5). In the CCB-treated group, however, chromosomes were located in the center of the oocytes, and only 11% of the oocytes had actin-rich domains. None of the oocytes treated by CCB had a projection, which differed from the results with in vivo-matured oocytes, in which 100% had actin-rich domains and 93% had one or two projections (Figs. 5 and 6).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sucrose has been widely used in embryo and oocyte cryopreservation [18–23] as well as micromanipulations, such as intracytoplasmic sperm injection and nuclear transfer [24–27]. Unable to permeate through the plasma membrane, sucrose induces water molecules to move out of the oocytes and, thus, effectively cause them to shrink. The shrinking of the oocytes brings the spindle-chromosome complex closer to the plasma membrane. Projections are then formed due to the bulging of the rigid spindle-chromosome complex through the plasma membrane. Because many other sugars, such as glucose, trehalose, and maltose, are also used in embryo cryopreservation [28–30], it is not surprising that oocytes show similar changes when incubated in isosmotic concentrations of these sugars.
The projections in mouse oocytes are induced by hypertonicity, the common role for sucrose in cryopreservation. This is evidenced by the experiment in which oocytes developed projections on hypertonic NaCl treatment. That different projection morphology (smooth vs. sharp-tipped projection) was observed on treatment of sucrose at different concentrations further demonstrated the effect of osmolality concentration on projection formation. Higher osmolality causes more shrinking of the oocytes and, thus, more bulging of the rigid structures under the plasma membrane. It is important to point out that the sucrose-induced shrinkage of the oocytes does not significantly reduce the viability of the oocytes or the subsequent development of the embryos, as shown by our studies [10, 11, 24, 25] and by those of others [18–23, 26, 27]. The electrolyte NaCl, on the other hand, has a deleterious effect on the viability of oocytes and is not suitable for the induction of projection for purposes of micromanipulation [31, 32].
In the present study, the projection was always colocalized with the spindle-chromosome complex or chromosome clusters, suggesting the importance of a submembrane, rigid structure in the formation of projections. The formation of projections not only requires the presence of spindle and/or chromosomes, it also requires that these rigid structures be connected to the cortex of the oocytes. This is shown by experiments in which drugs that can specifically disrupt either microtubules or microfilaments were used to test the roles of cytoskeleton on projection formation. On CCB treatment of mature oocytes, one pole of the spindle detached from the cortex and the other remained anchored to the cortex. Two projections were observed in these oocytes, one from the end of the spindle still attached to the cortex and the other from the chromosome cluster. That the chromosome cluster induced formation of a projection despite one end of the spindle being free from attachment suggests that the chromosomes themselves were also attached to the cortex. These attachments may contain CCB-insensitive elements, such as microtubules. Furthermore, in immature oocytes treated with CCB during maturation, no actin-rich domain was established and the chromosomes were located near the center of the oocytes. No projections were therefore formed. These results demonstrated the important roles of connections between spindle or chromosome clusters and the cortex by nonspindle microtubules in the formation of projections.
Additionally, our observations also indicated that the two poles of the meiotic spindle are asymmetrical. The proximal pole appears to be attached to the cortex by CCB-sensitive component(s) (i.e., microfilaments), whereas the distal pole's connection to the cortex is CCB-insensitive (i.e., nonspindle microtubules). Spindle-pole asymmetry has also been reported in budding and fission yeasts [33–35], as well as in mammalian oocytes [36–38], in which different protein components were found in the two spindle poles [33].
The connection between chromosomes and the oocyte cortex may include more elements than nonspindle microtubules. In the present study, NOC treatments disrupted the preformed spindles in mature oocytes and caused the metaphase chromosomes to disperse into several clusters. Each of the chromosome clusters caused the formation of a projection on hypertonic treatment of the oocytes, because the chromosomes were still attached to the cortex. These results suggest that the chromosomes not only are secured to the cortex by the spindles and nonspindle microtubules, but that they are also attached to the cortex by elements of a nonmicrotubule nature. When NOC was combined with CCB, which disrupted both microtubules and actin filaments, no projections were formed in oocytes treated in hypertonic medium. These results suggest that some of the chromosomes' connection to the cortex is actin-based. Actin cables have been found to connect the spindle to the cortex in yeasts [39–41]. They may also be responsible for the attachment of the spindle and chromosomes in mammalian oocytes. That disruption of these cables was associated with the lack of formation of oocyte projections demonstrated that the connections between chromosomes and the cortex by actin cables and nonspindle microtubules were necessary components in the process of projection formation. Formins, a family of highly conserved proteins that share a common domain organization, are required for the assembly of actin cables in budding yeast [42–44]. Whether these proteins are also involved during actin-cable assembly in mouse oocytes remains to be studied.
Previous studies have shown that two cortical domains exist in the mouse MII oocyte. A small domain overlies the spindle area rich in actin and myosin II, devoid of microvilli, and has a reduced affinity for lectin and concanavalin A. The remainder of the oocyte is covered by a large domain rich in microvilli, lectin, and concanavalin A-binding sites and poor in actin and myosin II [6, 8, 45, 46]. Cytochalasin has been shown to block the formation of the actin-rich domain during maturation of mouse immature oocytes [5, 13], but it may or may not disrupt the pre-existing actin-rich domain in mature oocytes depending on the exposure period, cell types, concentration, and/or the type of cytochalasin used [5, 47, 48]. In the present study, exposure to CCB at a frequently used concentration (15 µM) or at a concentration as high as 150 µM for 3 h did not disrupt the pre-existing actin-rich domain in mature oocytes. Furthermore, the presence of an actin-rich domain always coincided with the formation of projections on sucrose treatments of mature oocytes. In immature oocytes, however, the prevention of actin-rich domain formation by CCB abolished the sucrose-induced projections. These observations suggest a critical role for actin-rich domain in the process of projection formation.
Our explanation of the mechanisms involved in hypertonicity-induced projection formation and its disruption by drugs in the mouse oocytes can be summarized as follows and is schematically diagrammed in Figures 7 and 8. First, when the mouse MII oocyte is shrunk by hypertonicity, the actin-rich domain, together with attached spindle and chromosomes, becomes more rigid than other cytoplasmic areas and bulges through the plasma membrane, resulting in the observed projections or area of protrusion. Because both poles of the spindle as well as the chromosomes between the poles are attached to the cortex, three projections should theoretically be formed. However, because of the balance between the two poles and the close proximity of the three structures, primarily only the projection caused by the middle bulging part of the spindle—where the chromosomes are located—is discernable, whereas the two projections caused by the spindle poles are often, if not always, undetectable. Second, when the actin filaments are disrupted by CCB, one pole of the spindle becomes detached from the cortex but the other remains attached to the actin-rich domain. When incubated in hypertonic medium, the cytoplasm shrinks and an area protrudes from the cell at the site where one spindle pole attaches. Moreover, because the chromosomes themselves are attached to the cortex by nonspindle microtubules, a second area of protrusion is often formed at the site of the chromosomes. Third, if the spindle and nonspindle microtubules are depolymerized by NOC, chromosome clusters remain connected to the actin-rich domain(s), possibly by actin cables, and cause projection formation when subjected to hypertonic conditions. Fourth, when TAX treatment decreases the critical concentration of tubulin for polymerization, the spindle is destroyed. The chromosomes then become dispersed into several clusters but remain attached to the cortical actin-rich domain by actin cables and cause formation of projections. Fifth, if the connections of chromosomes to the cortex by both the actin cables and the nonspindle microtubules are disturbed by treatment with CCB plus NOC or TAX, no projections are formed in most oocytes. Sixth, if immature oocytes are exposed to CCB during maturation, the actin-rich domain does not form and spindles do not position themselves at the cortex. It is no surprise that no hypertonic-induced projections are formed in such cases. Seventh, during maturation, both NOC and TAX can block the formation of the spindle, but they do not block the formation of actin-rich domains or actin cables that attach the chromosomes to the cortex. Therefore, most oocytes treated with NOC and TAX do form a projection if subsequently incubated in hypertonic sucrose medium.
|
|
In summary, three factors are necessary for projection formation in mature mouse oocytes on hypertonic treatment: 1) an asymmetric cortical domain rich in actin; 2) a rigid submembrane constituent, either spindle or chromosome clusters; and 3) a solid connection between the cortical domain and its submembrane constituents, such as actin cables and nonspindle microtubules. Any disruption of one of these factors will block the hypertonicity-induced projection formation. The present study also demonstrated that projection formation in mouse oocytes is a good model with which to study the roles of different components of the cytoskeleton in the maintenance of cellular asymmetry.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: X. Jerry Yang, Department of Animal Science, University of Connecticut, 1390 Storrs Rd., U-163, Storrs, CT 06269-4163.> FAX: 860 486 0534; jyang{at}canr.uconn.edu
Received: 19 March 2002.
First decision: 12 April 2002.
Accepted: 1 July 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Q.-Y. Sun and H. Schatten Regulation of dynamic events by microfilaments during oocyte maturation and fertilization Reproduction, February 1, 2006; 131(2): 193 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Liu, H. Kusakabe, C.-C. Chang, H. Suzuki, D. W. Schmidt, M. Julian, R. Pfeffer, C. L. Bormann, X. C. Tian, R. Yanagimachi, et al. Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits Biol Reprod, June 1, 2004; 70(6): 1776 - 1781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Chang, Z. P. Nagy, R. Abdelmassih, X. Yang, and X. C. Tian Nuclear and Microtubule Dynamics of G2/M Somatic Nuclei During Haploidization in Germinal Vesicle-Stage Mouse Oocytes Biol Reprod, March 1, 2004; 70(3): 752 - 758. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
