| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Research Laboratories of Schering AG, Berlin, Germany
b Health Care Discovery of Novo Nordisk A/S, Copenhagen, Denmark
c Department of Anatomy and Reproductive Biology, School of Medicine, RWTH University of Aachen, D-52057 Aachen, Germany
d Fakultät für Biologie, Gentechnologie/Biotechnologie, University Bielefeld, D-33501 Bielefeld, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Importantly, the potential of the oocyte to become fertilized and to support successful pre- and postimplantation development not only depends on meiotic/chromosomal maturation events but is also critically influenced by the quality and maturity of the ooplasm and the plasma membrane. Usually and ideally, the oolemma and cytoplasm prepare for fertilization and subsequent embryonic development in parallel with nuclear maturation. However, for the human there is accumulating evidence that protein synthesis patterns differ qualitatively and quantitatively between naked oocytes maturing in vitro in the absence of follicle cells and those matured in vivo inside a follicle, after appropriate hormonal stimulation [10]. Cytoplasmic maturation is difficult to define and to monitor. It is often described as oocyte polarity and spatial patterning of cell organelles, especially of cortical granules [11, 12], as well as changes in transcriptional [13], translational, and posttranslational activities [14, 15]. When cytoplasmic maturation does not occur properly, the oocyte will fail to fertilize and develop successfully [16].
Concerning activation of meiotic maturation, Byskov et al. [17] recently discovered a group of endogenous meiosis-activating sterols occurring naturally in the biosynthetic pathway between lanosterol and cholesterol. One sterol isolated from human follicular fluid (4,4-dimethyl-5
-cholesta-8,14,24-trien-3ß-ol, FF-MAS) was obtained from women undergoing treatment for infertility by in vitro fertilization. This sterol induces resumption of meiosis in cumulus-enclosed mouse oocytes in vitro in a dose-dependent manner, in contrast to lanosterol and cholesterol, which were inactive in this respect. FF-MAS appears to originate from the cumulus cells surrounding the oocyte after direct oocyte-follicle cell interactions and signaling events [18]. In agreement with the meiosis-promoting effect with extracted FF-MAS, Grøndahl et al. [19] were able to show that synthetic FF-MAS is dose-dependently capable of mediating resumption of meiosis in vitro in both naked and cumulus-enclosed mouse oocytes arrested in meiosis with Hx, isobutylmethylxanthine (IBMX), or dibutyryl cAMP (dbcAMP).
The activity of FF-MAS to induce maturation has so far been studied exclusively in mouse oocytes arrested in dictyate stage for 24 h by agents acting at or downstream of cAMP signaling (Hx, IBMX, or dbcAMP). It was therefore of interest to see whether the action of agents such as forskolin, mediating meiotic arrest by inducing adenylate cyclase [20], was also overcome by addition of FF-MAS in vitro. Since oocytes rapidly resume maturation when cAMP levels drop, it was important to see whether FF-MAS influences the kinetics of meiotic maturation in mouse oocytes overcoming the meiotic block. It is also still completely unknown whether FF-MAS affects cytoplasmic in addition to nuclear maturation events in mammalian oocytes. The present study was therefore performed to investigate the effect of FF-MAS on the microtubular cytoskeletal and chromosomal organization in mouse oocytes via double-labeling fluorescence. Finally, we analyzed the influence of FF-MAS on cytoplasmic maturation of oocytes by using electron microscopy to compare the ultrastructure of oocytes maturing spontaneously to that of FF-MAS-matured oocytes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immature, 21- to 23-day-old female mice (C57BL/6J x DBA/2J F1; Charles River, Sulzfeldt, Germany) weighing 13–16 g were kept under controlled temperature (20–22°C), light (lights-on 0600–1800 h), and relative humidity (50–70%) with food and water ad libitum. All experiments on animals were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Source of FF-MAS
The C29-sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol (FF-MAS) was synthesized by the Department of Medicinal Chemistry of the Schering AG as described by Dolle et al. [21]. The purity of the finally described FF-MAS was checked and determined to be 99.3% by HPLC (column: Symmetry C18, 5 µm; column size: 150 x 3.9 mm; eluent: acetonitrile:water 95:5 containing 0.5 g/L ammonium acetate; detection: photodiode array at 214 nm).
Mouse Oocyte Assay
Fully grown, GV-intact oocytes were obtained from ovaries after priming with an i.p. injection of 0.2 ml recombinant FSH (Gonal-F; Serono, Randolph, MA) containing 20 IU FSH. Forty-eight hours later, the animals were killed by cervical dislocation. The ovaries were dissected out and placed in -minimum essential medium (-MEM without ribonucleosides; Gibco BRL, Gaithersburg, MD; cat. #22561) supplemented with 8 mg/ml human serum albumin (State Serum Institute, Copenhagen, Denmark), 0.23 mM pyruvate (Sigma Chemical Co., St. Louis, MO; cat. #S-8636), 2 mM glutamine (Flow Labs, McLean, VA; cat. #16–801), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Flow Labs; cat. #16–700). The medium contained 3 mM Hx (Sigma; cat. #H-9377) or 5 µM IBMX (Sigma; cat. #I-5879) to prevent GVB; these media are designated Hx-medium and IBMX-medium, respectively. The oocytes were collected in either Hx- or IBMX-medium under a stereomicroscope by puncturing follicles using 27-gauge needles. Only spherical naked oocytes displaying an intact GV were used. Naked oocytes were rinsed three times in Hx- or IBMX-medium and cultured without any oil in 4-well multidishes (Nunclon, Wiesbaden, Germany) in which each well contained 0.4 ml of the respective medium and 40–50 oocytes. Dilutions of FF-MAS were prepared from a stock solution containing 1 mg/ml dissolved in ethanol. Control groups were cultured in a corresponding amount of ethanol (3.8 µl/ml). The oocytes were cultured for various times in a humidified atmosphere of 5% CO2 in air at 37°C.
To evaluate whether FF-MAS-induced meiotic maturation is comparable to spontaneous in vitro maturation, naked oocytes were cultured for 0.5, 1, 2, 3, 4, 5, 6, 10, 14, 18, and 22 h in Hx-free medium, Hx-medium alone, or Hx-medium supplemented with 10 µM FF-MAS.
Naked oocytes were also cultured for 0, 6, 10, 14, 18, and 22 h either with 10 µM forskolin (Fo; Sigma; cat. #F-6886) alone or with 10 µM Fo supplemented with 10 µM FF-MAS in order to analyze the effect FF-MAS on these meiotically arrested oocytes. Fo is a rapid and reversible activator of adenylate cyclase [19]. For each experimental group, a minimum of three experiments were conducted. Data were pooled and are presented as mean ± SEM.
By the end of the respective culture period, the numbers of oocytes with GV, GVB, and polar bodies (PB), respectively, were counted using a Leica MZ 12 stereomicroscope (Leica, Heerbrugg, Switzerland). The %GVB, defined as percentage of oocytes undergoing GVB per total number of oocytes, was calculated as %GVB = (number of GVB + number PB/total number of oocytes) x 100.
Processing of Mouse Oocytes for Fluorescence Analysis
For fluorescence analysis, naked mouse oocytes after 1) spontaneous meiotic maturation in vitro in medium without Hx and 2) after meiotic maturation in Hx-medium containing 10 µM FF-MAS were removed from the culture dish 20 h after start of culture. Each group consisted of 40–50 oocytes derived from 4 to 5 experiments. In order to investigate chromosome and microtubule dynamics in oocytes, indirect anti-tubulin immunofluorescence was used in combination with fluorescein isothiocyanate (FITC)-labeled antibodies and DNA staining with 4'–6-diamidino-2-phenylindole (DAPI). Naked oocytes were aspirated with a mouth-operated micropipette [22] from the culture dish and placed into prewarmed M2 medium (Sigma cat. #M 7167) [23] containing 4 g/L BSA. All oocytes were freed from their zonae pellucidae by brief exposure to 0.7% Pronase (Boehringer, Mannheim, Germany) in M2 medium. After washing in M2 medium, oocytes were extracted for 45 min in a prewarmed buffer that preserves microtubules and chromosomes (containing 50 mM KCl, 5 mM EGTA, 0.5 mM MgCl2, 25 mM Hepes, 20 µM PMSF, 25% glycerin, and 2% Triton X-100). They were then attached to poly-L-lysine-coated slides prewashed with KCl-free extraction buffer, fixed for approximately 7 min in -20°C cold methanol, rehydrated in PBS at room temperature, and labeled with antibody. For conventional immunolocalization of tubulin, optimal results were obtained using an incubation for 45 min at 37°C with a monoclonal anti-tubulin antibody (Sigma; cat. #T 9026, clone DM 1A; 1:400 in PBS) that is specific for -tubulin and binds to all microtubular fibers in oocytes [24]. After washing in prewarmed PBS, the oocytes were further incubated at 37°C for 45 min in a 1:60 dilution of the second antibody, an FITC-labeled rabbit anti-mouse IgG (Sigma; cat. #F 7506). After washing in PBS, staining of chromatin was performed for 15 min with DAPI. Slides were finally immersed in antifade (diamino-bicyclooctane; Dabco, Sigma cat. #D 2522) in 20% glycerol/PBS. Labeled oocytes were viewed and photographed using a Zeiss (Oberkochen, Germany) Axiophot II fluorescence microscope equipped with fluorescein (Zeiss 487902) and DAPI (Zeiss 487909) selective filter sets and a 50W mercury arc lamp using a x63 Neofluor (1.25 NA) objective lens. Images were recorded on Kodak (Eastman Kodak, Rochester, NY) T-MAX 400 pro film using uniform exposure times.
Statistical Evaluation
In the mouse oocyte assay, the percentages of oocytes showing GVB were examined by one-way ANOVA followed by pair-wise comparison (Student's t-test) between treatment groups and controls. A P value less than 0.05 was considered significant.
In oocytes processed for fluorescence analysis, microtubular asters were counted in metaphase I and II oocytes, respectively. The mean number of microtubular asters per oocyte was calculated in both experimental groups. Statistical significance between spontaneously matured and FF-MAS-matured oocytes was examined by one-way ANOVA. A P value less than 0.05 was considered significant.
Electron Microscopical (EM) Investigations of Mouse Oocytes
For EM studies, naked oocytes were divided into four experimental groups. Group I consisted of oocytes GV arrested in vivo that were immediately processed for EM after puncture from the ovary. Oocytes from groups II–IV were cultured for 20 h in -MEM medium supplemented with 8 mg/ml human serum albumin, pyruvate/glutamine, and penicillin/streptomycin as described above. Group II consisted of oocytes cultured in Hx-medium to maintain oocytes in the GV stage. In group III, meiotic maturation was spontaneously induced by culturing oocytes in Hx-free medium. In group IV, meiotic maturation was induced by addition of 10 µM FF-MAS to Hx-medium and culture for 20 h.
At the end of culture, 10 oocytes per group were fixed and processed for EM as described by Hegele-Hartung et al. [25]. Briefly, oocytes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) for 1 h at room temperature, shortly afterward washed in 0.1 M cacodylate buffer, and postfixed for 1 h in 0.1 M cacodylate-buffered OsO4 at room temperature. After rinsing in cacodylate buffer, the oocytes were dehydrated with ethanol and propylene oxide and embedded in Araldite (Ladd Research, Burlington, VT) epoxy resin. Semithin and ultrathin sections were cut from the midregion of oocytes on a Reichert (Reichert Jung, Nossloch, Germany) OM U3 microtome using glass or diamond knives. Semithin sections were mounted on glass slides. Ultrathin sections were mounted on Formvar (Polysciences, Eppenheim, Germany)-coated copper grids and poststained with uranyl acetate and lead citrate.
The light microscopical observations were carried out on a Zeiss Axiophot II at x400–630 magnification. The oocytes were classified with regard to nuclear maturation as immature (GV stage), metaphase I (MI; GVB), anaphase I, telophase I, or metaphase II (MII; GVB plus PB). The EM observations were performed on a Zeiss EM 10A at 60–80 kV with x1250–31 500 magnification.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Oocytes cultured in modified -MEM (without Hx and FF-MAS) initiated GVB at 2 h. At 3 h, 62% of the oocytes had already undergone GVB. After 5–6 h, nearly all oocytes (96%) had resolved their nuclear membrane and resumed maturation (Fig. 1A). In contrast to controls, nearly all oocytes treated with 3 mM Hx-medium remained arrested in dictyate stage with an intact GV during the entire culture time (Fig. 1A).
|
Treatment of oocytes with 10 µM FF-MAS led to a 11-h delay in resumption of meiosis as detectable by GVB. Thus the first significant increase in the rate of oocytes with GVB (28%) was observed after 10 h. At 14, 18, and 22 h, a further increase occurred, reaching values of 53%, 84%, and 96%, respectively (Fig. 1A).
Effect of FF-MAS on Meiotically Arrested, Fo-Treated Mouse Oocytes
Fo at 10 µM concentration was found to be a potent inhibitor of spontaneous maturation. The inhibition was transient, since we found a dramatic increase in the percentage of oocytes with GVB at 18 h (54%). At 22 h, 73% of oocytes had undergone GVB in spite of the presence of Fo (Fig. 1B).
The addition of 10 µM FF-MAS to oocytes reversed the meiotic inhibition induced by Fo. After 6 h, 36% of oocytes had already resolved their GV. At 10, 14, 18, and 22 h, 56%, 78%, 86%, and 95% of all oocytes, respectively, had undergone GVB (Fig. 1B).
Spindles and Chromosomes in Spontaneously Matured Mouse Oocytes
Seventy-four percent of the oocytes processed for immunofluorescence at 20 h of maturation in vitro were at the MI stage. Twenty-four percent of the oocytes from this experimental group had reached MII, or were in the process of chromosome segregation, at anaphase I or telophase I when cultured in Hx-free medium. Only one oocyte among the controls, possibly derived from a preantral follicle, was incompetent to resume meiosis since it still had an intact GV. Characteristically, the spindles of both meiosis I and II oocytes were barrel-shaped (Fig. 2A) or slightly pointed at the flat spindle poles. A dense network of microtubules seemed to fill the region between the poles and the chromosomes, and the spindle apparatus appeared compact with very few astral fibers extending away from the poles. In addition to spindle microtubules, the cytoplasm of MI oocytes contained 3.2 ± 0.4 (mean ± SD) aster-like microtubular aggregates per oocyte, asters presumably associated with cytoplasmic microtubule organizing centers (MTOCs) (Fig. 2A). Microtubules of these asters were generally very short. Chromosomes were all lined up as metaphase bivalents at the spindle equator in MI spindles (Fig. 2B). Hardly any oocytes had chromosomes displaced from the equator. A few oocytes in late telophase (Fig. 2C) were found with a typical midbody tubulin pattern. Chromosomes in the PB or oocyte were sometimes arranged in a half circle, which is typical for this stage and no indicator of nondisjunction. Lagging of chromosomes in the interpolar space was not found. Again, a few cytoplasmic asters were observed. MII oocytes possessed typical anastral spindles with flat poles (Fig. 2D) and well-aligned MII chromosomes aligned perpendicular to the cleavage plane. Characteristically, only 1.8 ± 0.2 (mean ± SD) microtubular asters were found in the cytoplasm of MII oocytes.
|
Spindles and Chromosomes in FF-MAS-Matured Mouse Oocytes
Eighty-six percent of FF-MAS-treated oocytes processed for immunofluorescence after 20 h of culture were at the MI stage. Fourteen percent were at anaphase I or telophase I or at the MII stage after 20 h of culture in Hx-medium. Similar to those of the spontaneously matured oocytes, the spindles of MI oocytes were barrel-shaped and still brightly fluorescent (Fig. 2E). However, spindle poles appeared sometimes more fusiform, and in several cells, microtubules also radiated out from polar MTOCs. In contrast to observations in spontaneously matured oocytes, the number of small cytoplasmic aster-like microtubular aggregates was significantly elevated to 12.6 ± 1.2 (mean ± SD) per MI oocyte (Fig. 2E). Still the bivalent chromosomes were properly aligned at the spindle equator in meiosis I oocytes (Fig. 2F), comparable to observations in the untreated controls. Anaphase and telophase oocytes possessed normal anaphase/telophase spindles in which all chromosomes had migrated to the spindle poles (Fig. 2G), and no lagging as indicator of nondisjunction was observed. In these oocytes, hardy any microtubular asters were found in the cytoplasm. Oocytes in MII exhibited the typical barrel-shaped spindle with flat spindle poles (Fig. 2H), and we noticed no disturbances in chromosome alignment at the equator. Microtubular asters in MII oocytes were not statistically increased (2.1 ± 0.4; mean ± SD) compared to those in spontaneously matured control oocytes.
Ultrastructure of In Vivo GV Oocytes and Hx-Arrested Mouse Oocytes
The ultrastructure of all investigated GV oocytes at recovery that were not arrested with Hx in vitro showed well-preserved cytoplasmic cell organelles as well as a large, centrally located nucleus with an electron-dense nucleolus (Fig. 3). No difference was observed on comparison of in vivo GV oocytes with in vitro oocytes arrested with Hx.
|
Oocytes cultured in the presence of Hx remained arrested at the GV stage. The nuclei of all oocytes were located in the center of the cell (Fig. 4A). The nuclear envelope appeared folded, which is characteristic for dictyate stage (Fig. 4A). A prominent dense compact nucleolus was found in the middle of all nuclei.
|
When GVB was inhibited by Hx, oocytes did not become polarized and did not develop a microvillus-free area at one side of the cell periphery. The surface of all GV oocytes was covered by many homogenously distributed microvilli. Along the bases of microvilli, a relatively dense meshwork of microfilaments was found (Fig. 4B). Beneath this cytoskeleton of microfilaments only a few cortical granules (CGs), spherical membrane-bound electron-dense vesicles, were randomly distributed in the oocyte cortex (Fig. 4B). A population of CGs was also present in the interior of the oocyte. In the vicinity of microvilli, small electron-dense aggregates were observed within the plasma membrane (Fig. 4B).
Cytoplasmic organelles such as fibrillar lattices, vesicular smooth endoplasmic reticulum, mitochondria, Golgi complexes, and lysosomes were common features (Fig. 4, A and C). Whereas mitochondria were uniformly distributed all over the cytoplasm, smooth endoplasmic reticulum of the vesicular type was not very distinct in GV oocytes (Fig. 4C). Typical Golgi complexes consisting of smooth-surfaced cisternae and vesicles were usually seen at the GV stage perinuclearly. In contrast, multivesicular complexes were often found at the periphery of the GV oocytes (Fig. 4A).
Ultrastructure of Spontaneously Matured Mouse Oocytes
In total, seven oocytes at meiosis I and three at meiosis II were sectioned and processed for EM observation. They all exhibited an intact ultrastructure without any signs of degeneration. Therefore they will be described together.
The surface of the MI oocytes was polarized, showing a well-defined microvillus-free area (Fig. 5A). This region was associated with the meiotic spindle and a dense aggregation of microfilaments (Fig. 5A). The microvillus-free area was sharply demarcated by the appearance of numerous microvilli that projected into the perivitelline space. The basis of the microvillous area was not covered by a prominent layer of microfilaments. In 8 of 10 oocytes investigated, CGs were aligned closely beneath the oolemma (Fig. 5B) and could be found in the microvillous area opposite from the spindle. Characteristically CGs were absent from the microvillus-free area in all oocytes. In the perivitelline space, CG release was observed as identified by fine and electron-dense material (Fig. 5B). In addition, a number of electron-dense spots appeared occasionally (in 3 of 10 oocytes) in and under the oolemma of the microvilli-containing oolemma (Fig. 5B).
|
Cytoplasmic organelles were randomly distributed within the cytoplasm (Fig. 5A). They consisted of fibrillar lattices (Fig. 5B), endoplasmic reticulum of the smooth type (Fig. 5, B and C), spherical mitochondria with transverse or peripheral cristae (Fig. 5C), Golgi complexes, and multivesicular complexes. Whereas mitochondria were expressed in the middle of the oocyte, Golgi and multivesicular complexes were located in the periphery of the cytoplasm. In all oocytes investigated, only a few primary or secondary lysosomes were present.
Ultrastructure of FF-MAS-Matured Mouse Oocytes
In the FF-MAS group, 8 of the 10 oocytes investigated were at and after the MI stage, and 2 were at the MII stage. Oocytes in MI were polarized. The microvillus-free area could be clearly distinguished from the microvilli-containing area (Fig. 6A), similar to what was observed in spontaneously matured oocytes. Structure and distribution of cytoplasmic organelles (Fig. 6, A and C) were also comparable to those of spontaneously matured oocytes. In some FF-MAS-matured oocytes (4 of 10 oocytes), a homogenous increase in electron density of the entire cytoplasmic matrix was evident. However, clumping or vacuolization of cell organelles, an effect known to be associated with oocyte culture, did not occur.
|
Changes in the FF-MAS-exposed group as compared to spontaneously matured oocytes involved the oolemma and the underlying oocyte cortex in the microvillous area (Fig. 6B): in 7 of 10 oocytes, the oolemma of the microvilli-containing area contained many electron-dense aggregates protruding into the perivitelline space (Fig. 6B). Although the number of CGs appeared to be unchanged compared to that in spontaneously matured oocytes, CGs were generally located further away from the oolemma (Fig. 6B). This observation was made in all oocytes under investigation. Obviously, CGs failed to be transported from the Golgi complex to the oolemma (compare Fig. 6B with 5B).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
[19, 27] or other signaling pathways, influences the production or activity of such crucial factor(s), downstream of or separate from the lowered cAMP levels and transient inactivation of protein kinase A. The reversal of the Hx-induced meiotic block by FF-MAS is dependent on protein synthesis [19] and therefore may require a prolonged period of synthesis before critical concentrations of signaling proteins are high enough to cause meiotic resumption and downstream activation of MPF that drives oocytes into GVB. Exposure of oocytes to inhibitors of phosphatase PP1 and PP2A also causes resumption of meiosis and GVB in mammalian oocytes [28]. However, spindle formation is aberrant under the influence of such inhibitors, and oocytes do not progress beyond meiosis I when phosphatase inhibitors are present throughout maturation [28]. In contrast, mouse oocytes induced to resume maturation by FF-MAS and also exposed to this sterol throughout the entire culture period have normal spindles and are able to undergo anaphase I, emit a PB, and form a normal MII spindle. Therefore, FF-MAS appears to have a more physiological influence than phosphatase inhibitors, as may be expected from its expression in follicles in vivo. With respect to the normal appearance of the spindle apparatus and the progression to and arrest at MII, our observations show that FF-MAS-induced maturation exhibits all the characteristic features of meiosis in vivo, and therefore appears to induce activation of MPF as well as c-Mos kinase and mitogen-activated protein kinases, components of cytostatic factor [5, 29]. Meiosis is known to be associated with characteristic modifications of centrosomes and microtubules within the spindle region [30]. Multiple cytoplasmic microtubule asters have been observed in mouse oocytes resuming maturation [31–33]. Many of these asters and the cytoplasmic MTOCs get recruited by chromosomes and align at the flat spindle poles during prometaphase I, while cytoplasmic MTOCs at the cell periphery gradually lose microtubular fibers. Accordingly, increased numbers of cytoplasmic asters are observed at prometaphase I of meiosis, whereas a reduction in the number of centrosome-nucleated cytoplasmic microtubule assembly is observed at MI and MII [34]. This increase or decrease in aster formation is inversely correlated with high or low MPF levels, respectively [5, 35], and mitogen-activated protein kinase activity [36]. This indicates that the phosphorylation status of centrosomal and spindle components determines the assembly and disassembly dynamics during meiosis. We found a marked increase in cytoplasmic microtubular aster formation at MI after FF-MAS treatment. Concomitantly, spindle fluorescence appeared very strong. Overall protein content and concentration of several distinct proteins appear increased in oocytes after in vivo maturation when compared to these values in naked oocytes [10]. On the basis of the knowledge that both FF-MAS and Hx are physiological constituents of follicular fluid [2, 17], it may therefore be suggested that the late and slow increase in GVB in FF-MAS-treated oocytes represents a rather more physiological situation in comparison to the spontaneous maturation conditions in vitro, and that the quality of oocytes progressing to MII in FF-MAS is superior to that of spontaneously in vitro-matured, naked mouse oocytes. This could also explain why FF-MAS increases the number of fertilizable and activated naked and cumulus-enclosed oocytes and their developmental capacity [37].
High quality of oocytes is also apparent from the correct alignment of chromosomes at the spindle equator in FF-MAS-matured oocytes, since dispersal and displacement of chromosomes are often signs of predisposition to nondisjunction in response to spindle damage and aging [32, 38, 39]. However, chromosomes of FF-MAS-exposed oocytes are typically located at the equator.
Mammalian oocytes have a short fertilizable life span [40, 41], especially in vitro. This is linked with the proper development of CGs during meiotic maturation; CGs are important indicators of oocyte cytoplasmic maturation. CGs are small, membrane-bound vesicles in the oocyte cortex that undergo exocytosis in response to elevated cytoplasmic calcium upon fertilization [42]. The contents of CGs are released into the perivitelline space and appear to modify the zona pellucida (ZP), especially the ZP glycoproteins ZP2 and ZP3. The functional consequence of these modifications is that sperm can no longer bind to and penetrate the ZP, giving rise to an extracellular ZP block to polyspermy [43]. CGs migrate to the periphery of the oocyte during meiotic maturation and, once anchored immediately under the oolemma, remain at the cortex until fertilization [44]. However, loss of CGs is not observed only during fertilization in the mouse. In both in vitro- and in vivo-matured oocytes, release of CGs clearly starts at MI. About 30% are lost at MI, and maximal loss is observed at MII [41]. As far as can be deduced from EM sections, FF-MAS leads to a slow movement of CG toward the oolemma as well as to an increase in reuptake of CG material from the perivitelline space. This is expressed by a centripetal arrangement of electron-dense CGs as well as an increase in electron-dense particles within the oolemma. It is therefore suggested that FF-MAS can reduce the "spontaneous cortical granule release" that is observed under in vitro and in vivo conditions in mouse oocytes in the absence of sperm penetration [45], thereby preventing precocious modification of the ZP, e.g., zona hardening [46]. Precociously released CGs appear to be a major contributory factor to ZP hardening [47], which is associated with a decrease in fertilization rate. In addition, immature mammalian oocytes, as well as aged oocytes, have a higher incidence of abnormal fertilization [48]. To improve the results of in vitro fertilization procedures, especially in humans, an optimal window for fertilization of oocytes is important in order to improve the incidence of fertilization. To increase the fertilizable life span of the oocyte in vitro, one approach may be to synchronize the kinetics of nuclear and cytoplasmic maturation. FF-MAS in the presence of Hx appears to reduce precocious CG release.
The dynamics of microtubule- and actin-mediated organelle movement is well established for processes such as extension of endoplasmic reticulum [49], changes in mitochondrial distribution [50], and movement of CGs to the cell surface [47]. Our studies with FF-MAS clearly show that it is possible to delay the kinetics of nuclear maturation and also to delay cytoplasmic maturation, especially CG migration, in the mouse under physiological conditions with respect to the presence of Hx without disturbing or delaying the general cortical polarization in the microvillus-free and microvilli-containing areas of the plasma membrane. In addition, FF-MAS treatment of mouse oocytes not only delays the migration of CGs to the oolemma but also appears to increase the reuptake of CG material. Although FF-MAS may not be an indispensable component at the initiation of meiotic resumption in vivo [51], our observations suggest that it has beneficial effects on in vitro maturation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Christa Hegele-Hartung, FC/HT Research of Schering AG, Müllerstrasse 170–178, D-13342 Berlin, Germany. FAX: 49 30 46818056; christa.hegelehartung{at}schering.de
Accepted: June 25, 1999.
Received: April 6, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Nature 1996; 383:728–731.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  M. Zhang, H. Ouyang, and G. Xia The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption Mol. Hum. Reprod., July 1, 2009; 15(7): 399 - 409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cukurcam, I. Betzendahl, G. Michel, E. Vogt, C. Hegele-Hartung, B. Lindenthal, and U. Eichenlaub-Ritter Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes Hum. Reprod., March 1, 2007; 22(3): 815 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M Mehlmann Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation Reproduction, December 1, 2005; 130(6): 791 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Coticchio, G. Rossi, A. Borini, C. Grondahl, G. Macchiarelli, C. Flamigni, S. Fleming, and S. Cecconi Mouse oocyte meiotic resumption and polar body extrusion in vitro are differentially influenced by FSH, epidermal growth factor and meiosis-activating sterol Hum. Reprod., December 1, 2004; 19(12): 2913 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Cao, S. H. Pomerantz, M. Popliker, and A. Tsafriri Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis? Biol Reprod, December 1, 2004; 71(6): 1807 - 1812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bergh, A. Loft, K. Lundin, S. Ziebe, L. Nilsson, M. Wikland, C. Grondahl, J.-C. Arce, and for the CEMAS II Study Group Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) Hum. Reprod., September 1, 2004; 19(9): 2109 - 2117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cukurcam, C. Hegele-Hartung, and U. Eichenlaub-Ritter Meiosis-activating sterol protects oocytes from precocious chromosome segregation Hum. Reprod., September 1, 2003; 18(9): 1908 - 1917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Grondahl, J. Breinholt, P. Wahl, A. Murray, T. H. Hansen, I. Faerge, C. E. Stidsen, K. Raun, and C. Hegele-Hartung Physiology of meiosis-activating sterol: endogenous formation and mode of action Hum. Reprod., January 1, 2003; 18(1): 122 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baltsen Gonadotropin-Induced Accumulation of 4,4-Dimethylsterols in Mouse Ovaries and Its Temporal Relation to Meiosis Biol Reprod, December 1, 2001; 65(6): 1743 - 1750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Faerge, B. Terry, J. Kalous, P. Wahl, M. Lessl, J.L. Ottesen, P. Hyttel, and C. Grondahl Resumption of Meiosis Induced by Meiosis-Activating Sterol Has a Different Signal Transduction Pathway than Spontaneous Resumption of Meiosis in Denuded Mouse Oocytes Cultured In Vitro Biol Reprod, December 1, 2001; 65(6): 1751 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.L. Cavilla, C.R. Kennedy, M. Baltsen, L.D. Klentzeris, A.G. Byskov, and G.M. Hartshorne The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries Hum. Reprod., March 1, 2001; 16(3): 547 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hegele-Hartung, M. Grützner, M. Lessl, C. Grøndahl, J. L. Ottesen, and M. Brännström Activation of Meiotic Maturation in Rat Oocytes After Treatment with Follicular Fluid Meiosis-Activating Sterol In Vitro and Ex Vivo Biol Reprod, February 1, 2001; 64(2): 418 - 424. [Abstract] [Full Text]
|
|
|
|
|
|
I. Faerge, C. Grøndahl, J.L. Ottesen, and P. Hyttel Autoradiographic Localization of Specific Binding of Meiosis-Activating Sterol to Cumulus-Oocyte Complexes from Marmoset, Cow, and Mouse Biol Reprod, February 1, 2001; 64(2): 527 - 536. [Abstract] [Full Text]
|
|
|
|
|
|
S. M. Downs, B. Ruan, and G. J. Schroepfer Jr Meiosis-Activating Sterol and the Maturation of Isolated Mouse Oocytes Biol Reprod, January 1, 2001; 64(1): 80 - 89. [Abstract] [Full Text]
|
|
|
|
|
|
K. M. Vaknin, S. Lazar, M. Popliker, and A. Tsafriri Role of Meiosis-Activating Sterols in Rat Oocyte Maturation: Effects of Specific Inhibitors and Changes in the Expression of Lanosterol 14{{alpha}}-Demethylase During the Preovulatory Period Biol Reprod, January 1, 2001; 64(1): 299 - 309. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. P. Bagowski, J. W. Myers, and J. E. Ferrell Jr. The Classical Progesterone Receptor Associates with p42 MAPK and Is Involved in Phosphatidylinositol 3-Kinase Signaling in Xenopus Oocytes J. Biol. Chem., September 28, 2001; 276(40): 37708 - 37714. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
