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Gamete Biology |
Follicle Biology Laboratory, University Hospital and Medical School, Vrije Universiteit Brussel, B-1090 Brussels, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicular development, gamete biology, ovum, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cryopreservation of isolated mouse ovarian follicles has been demonstrated to be possible [2–5]. Both equilibrium cooling and nonequilibrium cooling methods were assessed. Carroll et al. [2, 3] and Cortvrindt et al. [4] used a conventional slow cooling/rapid thawing protocol, and dela Pena et al. [5] demonstrated for the first time that mouse preantral follicles could be vitrified. Follicle survival rates after thawing/warming obtained in these studies were comparable and in an acceptable range (approximately 80%). An evident way to further assess follicle viability and quality is to follow its growth potential after cryopreservation. Complete in vitro follicle culture of cryopreserved preantral follicles yielded an oocyte maturation rate similar to freshly isolated control follicles [4, 5]. It was even demonstrated that the cryopreservation process did not affect in vitro fertilization and developmental competence of the follicle-enclosed oocytes [5].
These data demonstrate that, from the point of view of oocyte maturation and developmental capacity, mouse preantral follicles can tolerate cryopreservation fairly well. However, the quality of cryopreserved follicles, in terms of normal folliculogenesis (growth and differentiation pathways), could still be improved. In vitro growth of conventionally frozen-thawed follicles is retarded and the estradiol and inhibin production is reduced compared with fresh controls [4]. These alterations limit the use of cryopreserved follicles as standardized starting material for toxicology testing. To elucidate the origin of these alterations, more fundamental research is required on physiological changes induced during the cryopreservation process.
Essential steps in a conventional freezing procedure are the addition and removal of cryoprotective agents, the cooling of the sample to nonphysiological temperatures before freezing, and their warming after thawing. Each phase of this process is a possible source for cell damage. In a first step to clarify the mechanism of cryopreservation-induced damage, this study aimed to analyze the effects of chilling.
Direct chilling injury, or cold shock, can be defined as irreversible damage or death following exposure of cells to low nonphysiological temperatures (close to 0°C) before the nucleation of ice. It is a widespread cellular reaction and one of the major limiting factors for achieving optimal cryopreservation [6].
In the field of reproduction, chilling injury has been described for oocytes, spermatozoa, and embryos. Potential cellular sites for chilling injury are the elements of the cytoskeleton. This has been extensively studied for the meiotic spindles, microtubules, and microfilaments of mammalian fully grown oocytes [7–10]. Other targets for chilling injury are the cytoplasmic membranes, as has been shown for bovine oocytes [11, 12] and ram and fowl spermatozoa [13]. Cooling can also be responsible for modifications of ooplasmic organelles [14, 15].
In the present study, we determined the effects of chilling to 0°C on several subcellular elements (cytoskeletal organization, geminal vesicle [GV] chromatin configuration, and ultrastructural morphology) of isolated ovarian follicle units in a mouse model. The kinetics, as well as the degree of chilling injury was defined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (Bornem, Belgium).
Nocodazole (stock 10 mM in dimethyl sulfoxide [DMSO] stored at 4°C), which disrupts microtubules, was used at concentrations of 1, 5, and 10 µM. Cytochalasin D (stock 1 mg/ml in DMSO stored at -20°C), which disrupts actin filaments, was used at concentrations of 0.5 and 5 µg/ml. These concentrations are within the effective range for these drugs [7]. Dilutions to final concentrations were made in Leibovitz-glutamax medium, supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 µg/ml streptomycin, and 100 U/ml penicillin (referred to as L15* medium; all obtained from Invitrogen, Merelbeke, Belgium). In preliminary experiments, equivalent dilutions of DMSO carrier were tested and no effect on the cytoskeleton was observed (data not shown).
Animals and Isolation of Early Preantral Follicles
All mice used in this study were F1 hybrid mice (C57Bl/6j x CBA/ca; Harlan, The Netherlands), housed and bred according to national legislation, and all experimental procedures were done after obtaining written consent from the Institutional Ethical Commission of the Vrije Universiteit Brussel. The animals were killed by cervical dislocation. Ovaries from 14-day-old females were dissected and collected in L15* medium at 37°C.
Follicles were freed mechanically from the ovaries with fine insulin needles (261/2-G; Becton Dickinson, Erembodegem, Belgium). The follicles used in the experiments were selected during three washing steps in L15* medium according to their intactness, diameter, and a clear, centrally located oocyte. The selected follicles had a diameter of between 100 and 130 µm (type 3b-4 from the Pedersen and Peters classification [16]). This is the class of follicles that can be grown and matured completely in vitro in 12 days, with a reproducible high yield [1]. Thirty to 40 good-quality preantral follicles could be recovered per ovary. After isolation, follicles were subjected to various treatments as described below.
Experimental Design
Preliminary experiment: Effect of cytoskeletal drugs on preantral follicles This experiment was designed to determine if induced modifications on the tubulin and actin components of the follicle could be detected with current immunocytochemical staining techniques.
For the detection of the disruption of microtubules, follicles were collected and incubated at 37°C for 1 h in the presence or absence of nocodazole. Three different concentrations of nocodazole were used: 1, 5, and 10 µM. After treatment, the follicles were fixed for immunocytochemical staining. To detect the recovery of the microtubular system after nocodazole treatment, follicles were incubated for 1 h in the presence of the highest concentration of nocodazole (10 µM) and were subsequently washed in L15* medium. They were kept in this medium for 10, 30, or 60 min before fixation. Between 11 and 36 follicles were included per experimental group.
To visualize the disruption of microfilaments, follicles were incubated for 1 h at 37°C in L15* medium containing 0.5 or 5 µg/ml cytochalasin D. After this treatment, follicles were fixed immediately. To check the reversibility of the treatment, follicles were exposed to the highest concentration of cytochalasin D (5 µg/ml) for 1 h and were subsequently washed in L15* medium. They were then cultured further in this medium for 10, 30, or 60 min before fixation. Between 15 and 32 follicles were included per experimental group.
Cooling of early preantral follicles
Effects of cooling follicles to 0°C for varying periods was examined by using a similar experimental set-up as described by Songsasen et al. [10] (see Fig. 1). Isolated follicles were randomly divided into 10 groups (10–15 follicles/group; two to three animals/replicate). One group was fixed immediately after isolation (control 1). Four groups of follicles were rapidly cooled by expelling them in an Eppendorf test tube containing 300 µl L15* medium precooled to 0°C. The Eppendorf test tubes were plunged in a bath containing a mixture of water and ice. At the end of the cooling period (1, 5, 10, or 30 min), the chilled follicles from each group were separated into two sets. The first set was transferred immediately into a fixative solution without being warmed. The second set of follicles was warmed quickly by placing them immediately in a pre-equilibrated culture dish at 37°C containing microdroplets (30 µl) of 
-minimal essential medium (-MEM; Invitrogen) supplemented with 5% heat-inactivated FCS (Invitrogen), 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium (ITS), and 10 mIU/ml recombinant FSH (Gonal-F; kindly donated by Ares Serono International, Geneva, Switzerland). The follicles were incubated in this culture medium at 37°C in a humidified atmosphere of 5% CO2 in air for 60 min before being fixed. To exclude iatrogenic effects by culture, a second control group was included in the design (control 2). Follicles from this group were held in culture medium at 37°C inside an incubator and were fixed at the end of the experiment (which was around 90 min). Four independent replicates were carried out.
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Fixation, Fluorescence Labeling, and Confocal Microscopy
Follicles were simultaneously fixed and extracted in a microtubule-stabilizing buffer (0.1 M PIPES, pH 6.9, 5 mM MgCl2O.6H2O, 2.5 mM EGTA, 0.01% [v/v] aprotinin, 1 mM dithiothreitol, 0.1% [v/v] triton X-100, 1 µM taxol, 50% [v/v] deuterium oxide [D2O], and 2.0% [v/v] formaldehyde) for 45 min at 37°C. Fixed follicles were washed three times (15 min each) at 37°C in a blocking solution consisting of PBS with 0.2% sodium azide, 2% normal goat serum, 1% BSA, 0.2% powdered milk, 0.1 M glycine, and 0.01% triton X-100 [17]. Follicles were stored in the same blocking solution at 4°C for up to 4 wk until immunocytochemical staining.
To visualize microtubules, follicles were labeled with a 1:1 mixture of mouse monoclonal antibodies specific for -tubulin and ß-tubulin (1:100 dilution in blocking solution) by incubating overnight at 4°C. Then the follicles were washed three times for 15 min in blocking solution at 37°C followed by incubation in Alexa fluor conjugated goat-anti-mouse immunoglobulin (dilution 1:100 in blocking solution; Molecular Probes, Eugene, Oregon) for 3–4 h at 37°C. The nuclei of granulosa cells, theca cells, and oocytes were counterstained with ethidium homodimer-2 (dilution 1:100 in blocking solution; incubation overnight at 4°C; Molecular Probes).
To visualize filamentous actin (F-actin), follicles were incubated with Oregon Green 488-phalloidin (dilution 1:40 in blocking solution; Molecular Probes) at room temperature for 90 min.
Labeled follicles were washed and mounted in 90% glycerol-PBS solution containing 0.2% 1,4-diazabicyclo[2.2.2]octane as an antifading reagent. To prevent squeezing, follicles were mounted in microdroplets (± 10 µl) in chambered cover slips (Sanbio, Uden, The Netherlands). Preparations were stored in the dark at 4°C until microscopical analysis.
Labeled follicles were observed using a laser scanning confocal microscope (LSCM Fluoview IX70; Olympus Omnilabo N.V., Aartselaar, Belgium) equipped with a krypton/argon (488/568) laser as a light source and selective filter sets for Alexa fluor/Oregon Green 488 and ethidium homodimer-2. For consistency and repeatability for image analysis, all images in this study were obtained using a 20x (numerical aperture 0.70) objective with a laser intensity of 20% and a 3.5x zoom software setting. In case of drastically reduced fluorescence intensity, the voltage of the photomultiplier (PMT) detector was increased to obtain a more accurate image.
Transmission Electron Microscopy
Follicles were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). After postfixation with 1% osmium tetroxide in H2O, block staining with uranyl acetate and dehydration, follicles were embedded in Spurr. Serial semithin sections of 1 µm were cut throughout the follicle. Ultrathin sections (0.1 µm) for electron microscopic evaluation were collected at the point where the nucleolus of the oocyte was visible. Evaluation was done on a Zeiss (Oberkochen, Germany) E109 transmission electron microscope (TEM). A total of 40 follicles were examined, which included four follicles per experimental group.
Data Analysis
Differences in tubulin organization among chilled follicles are presented as the mean percentage of four independent replicates. Variations between experiments are indicated with the standard deviation (SD; error bars on graph). For evaluation of the differences between groups, data were subjected to arcsine transformation and one-way ANOVA. When a significant F ratio was defined by ANOVA, groups were compared with the Tukey posttest. When the P value was <0.05, the difference was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of nocodazole Figure 2 shows confocal images of a control follicle and follicles exposed to nocodazole. In the controls (follicles not treated with nocodazole), tubulin labeling was most prominent in the granulosa cell layer. A dense network composed of microtubular arrays and asters was present throughout the cytoplasm of the follicle-enclosed oocyte (Fig. 2A).
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After exposure to nocodazole (1, 5, or 10 µM) for 60 min, the fluorescence intensity of the antitubulin staining in the granulosa cells and the oocyte decreased substantially compared with nontreated follicles (Fig. 2B, inset). In the follicle-enclosed oocyte, the elaborated cytoplasmic microtubule network disappeared and three major types of tubulin configurations could be distinguished. The first pattern was the localization of tubulin foci or asters exclusively in the proximity of the GV (Fig. 2B). There was a dose-dependent effect: foci or asters became smaller when higher concentrations of nocodazole were used. The second pattern of altered tubulin configuration, which was only occasionally observed, was the presence of asters in the periphery just underneath the oocyte cortex. The asters were interconnected via short microtubules, forming a ring-like structure in the pericortical cytoplasm (Fig. 2C). In a third pattern, tubulin structures in the oocyte were completely disassembled. Neither tubulin asters, foci, nor fibers could be detected at any place in the cytoplasm (Fig. 2D). Concomitant with the depolymerization of tubulin structures in the oocyte was the presence of nicks in the nuclear envelope (see arrows in Fig. 2, C and D). This feature was present in only a minority of the follicles (20%) and was not dose-dependent.
The percentages of follicles showing one of these altered tubulin configurations in their oocytes are summarized in Table 1. While a dense network of tubulin was present in 100% of control follicles, tubulin structures were completely disassembled in the oocytes of approximately half of the follicles treated with 5 µM and 10 µM nocodazole (52% and 40%, respectively).
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The nocodazole-induced depolymerization of tubulin was reversible (Table 1). At early time points (10 min) after removal of nocodazole, the intensity of the tubulin staining in the complete follicle (oocyte + granulosa cell layers) was still lower compared with control follicles, but the majority of the follicle-enclosed oocytes exhibited tubulin polymerization around the GV. Thirty minutes after nocodazole removal, perinuclear tubulin asters were clearly larger in size and long microtubules were radiating from these foci throughout the cytoplasm. One hour after removal from nocodazole, an extensive and uniformly distributed microtubule network was reconstituted in all follicle-enclosed oocytes and the intensity of the tubulin staining in the granulosa cell layer was comparable with controls.
Effects of cytochalasin D Figure 3A demonstrates a control follicle labeled for filamentous actin (F-actin). A prominent layer of microfilaments is apparent in the oocyte cortex and many actin-filled transzonal processes (TZPs) were observed. A weak staining is present in the granulosa cell layers surrounding the oocyte.
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After treatment with 0.5 µg/ml cytochalasin D, the staining became very weak, irregular, and speckled (Fig. 3B). Large foci of F-actin staining became present around the GV. A similar pattern, but more pronounced, was visible after exposing follicles to 5 µg/ml cytochalasin D.
The first 10 min after removal of cytochalasin D, spots of F-actin staining were still visible around the GV, but the staining throughout the follicle was more homogeneous and intense. Sixty minutes after treatment, the effect of cytochalasin D was completely reversed.
Cooling of Early Preantral Follicles
Having established the technical parameters to study modifications in the cytoskeletal organization, we examined the effect of cooling the follicles to 0°C for varying periods.
Effect of chilling on microtubules As soon as 1 min after chilling follicles, the fluorescence intensity of the antitubulin staining in the oocyte and in the surrounding granulosa cells decreased substantially. When follicles were chilled for longer periods (5, 10, or 30 min), tubulin alterations in the follicle-enclosed oocytes could be detected and follicles were classified accordingly: 1) dense network of microtubules throughout the cytoplasm of the oocyte (Fig. 4A); 2) altered tubulin organization, defined as microtubule assembly (foci or asters) solely in association with the GV (Fig. 4B) or located near the cortex of the oocyte (Fig. 4C); 3) absence of tubulin organization (Fig. 4D).
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A time-dependent change was noticeable (Fig. 5). A dense network of microtubules was present in 100% of the follicles in control 1 (n = 59) and control 2 (n = 54). Cooling follicles for 5, 10, or 30 min resulted in a progressive reduction of follicle-enclosed oocytes showing this tubulin pattern, which was significantly different from the controls (P < 0.05). Thirty minutes after exposure to 0°C, all follicles lost their physiological microtubule organization.
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Altered tubulin organization (aster formation at GV or, occasionally, at oocyte cortex), was present in more than half of the follicles chilled for 5, 10, or 30 min. Asters became smaller with increasing exposure time to 0°C.
The proportions of follicle-enclosed oocytes with no tubulin organization in their cytoplasm increased from 5% in the follicles chilled for 1 min to 35% of follicles chilled for 30 min (P < 0.05) (Fig. 5).
Another consequence of exposure to low temperature was an effect on the integrity of the nuclear membrane in the oocyte. With prolongation of the chilling time, an irregular, wavy shape of the GV (Fig. 4D, arrow) became more severe. Extension of exposure to 0°C increased the mean percentage of follicles exhibiting a convoluted nuclear membrane from 7% for 1 min of chilling to 43% for 30 min of chilling (P < 0.05) (data not shown in graph format).
Warming up chilled follicles to 37°C and incubation at this temperature for 60 min in culture medium resulted in repolymerization of tubulin. The fluorescence intensity of the antitubulin staining increased to a similar level as in control groups. The oocyte cytoplasm had reestablished a network of numerous microtubules. This was observed for all follicles (n = 189; 100%; P < 0.05), even those chilled for 30 min (Figs. 5 and 6A).
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Effect of chilling on microfilaments Chilling had only minor effects on the actin-containing elements of the cytoskeleton. Except for a slight reduction in fluorescence intensity of the staining, there was no difference in configuration and localization of the actin filaments between controls and chilled follicles. Transzonal projections rich in actin, which determine the connections between the oocyte and the granulosa cells, were still abundantly present after exposure of follicles to 0°C for various periods. Rewarming follicles (37°C, 1 h) established the fluorescence intensity to a similar level as observed in controls (data not shown).
Effect of chilling on chromatin configuration within the GV To check if chilling might have an influence on the chromatin configuration of the follicle-enclosed oocytes, the degree of heterochromatin association with the nucleolus was evaluated. The stage I–IV GV classification of Mattson and Albertini [17] was used. Control follicles contained oocytes with predominantly stage II GV (75%), which is defined by the presence of two to five foci of chromatin bordering the nucleolus (Fig. 6B). Stage I GV (dispersed chromatin) and stage III GV (partial ring of chromatin around the nucleolus) were also present for 18% and 7%, respectively. When follicles were chilled to 0°C for various periods up to 30 min, there was no effect visible on the status of the chromatin configuration.
Effect of chilling on ultrastructure The ultrastructure of chilled follicles, evaluated by electron microscopy, was similar to control follicles. The only pronounced change was the breakdown of the characteristic cytoplasmic fibrillar lattices in the follicle-enclosed oocytes. While these fibrillar structures were distributed all over the ooplasm in control follicles (Fig. 7A), they appeared to be absent in chilled follicles (1–30 min; Fig. 7B). After rewarming follicles to 37°C (1 h) the fibrillar lattices reappeared. Although precise quantitative information is not available, the density after 1 h at 37°C seemed to be somewhat reduced compared with controls (Fig. 7C). The localization and the morphology of other cytoplasmic components in the oocyte (e.g., mitochondria, Golgi complexes, elements of smooth endoplasmic reticulum, multivesicular bodies, lipid globules) remained unchanged after chilling. The nuclear membrane of the GV was occasionally highly convoluted after chilling, but no breaks were detected. The nucleolus was spherical and had a reticulated type of structure. Numerous short microvilli were uniformly distributed over the surface of the oocyte.
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In the granulosa cells, no apparent ultrastructural signs of damage were visible.
Electron microscopy allowed accurate observation of the connections between the oocyte and the granulosa cells. Variously shaped cytoplasmic processes from adjacent follicle cells traversing the zona pellucida were preserved in cooled follicles. Some of these projections were reaching the oocyte. This could be detected even when follicles were exposed to 0°C for as long as 30 min.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microtubules are known to depolymerize under the influence of reduced temperatures [18]. This study demonstrated that chilling to 0°C for only 1 min causes depolymerization of microtubules in both granulosa cells and the oocyte. Cooling for more extended periods was associated with time-dependent alterations of the tubulin organization in the follicle-enclosed oocytes. Observed changes included a reduction in the length and number of interphase arrays of microtubules, an overall localization of tubulin asters to a perinuclear or, occasionally, a cortical position and sometimes complete disappearance of tubulin structures. These modifications matched nocodazole-induced depolymerization of microtubules used as a positive control. Transient formation of asters can be explained by an increase in the cytoplasmic concentration of free tubulin due to the dissolution of interphase microtubules and/or a decrease in the critical concentration for tubulin polymerization by the effect of cooling [7]. Consequently, tubulin foci nucleate at microtubule organizing centers. Previous studies have demonstrated that stable, nucleation-competent centrosomes in growing oocytes are present around the GV and at the cortex [17, 19]. In a further stage of pronounced depolymerization of microtubules, tubulin asters disappear as well. A complete breakdown of the microtubular component of the cytoskeleton results in cell fragility. This could be one explanation for the highly irregular, convoluted shape of the germinal vesicle, which was observed in some oocytes. Effects of chilling on microfilament organization were less dramatic.
The structural transitions in the microtubule complex observed in this study are comparable with the changes occurring naturally near the end of the growth phase of oogenesis, when the prophase I oocyte acquires meiotic competence [19]. During this transition, interphase microtubules in the cytoplasm undergo a conversion to M-phase microtubule organization. This is characterized by a progressive reduction in immunodetectable tubulin polymers in tandem with the appearance of prominent tubulin foci around the GV and alterations in GV chromatin. The chromatin becomes packed in a ring around the nucleoli [17, 19, 20]. A similar correlation between tubulin and chromatin configuration in chilled follicles could not be observed. A type II GV was predominantly present in studied follicles, which is consistent with previous findings for the same class of preantral follicles [17].
Literature has put an emphasis on the chilling sensitivity of fully grown mammalian oocytes because the meiotic spindles are highly susceptible to disruption by temperature fluctuations [7–10]. Immature growing oocytes, which are arrested in prophase of the first meiotic division, do not contain any prominent microtubular structure. Theoretically, the microtubular organization of this type of oocyte may be less prone to the damage of cold shock. However, these oocytes are biosynthetically very active. An extensive spatial patterning of organelles is taking place during the growth phase. These rearrangements require an intact cytoskeleton where membrane-limited organelles translocate and assume defined positions within the growing oocyte [17, 21]. A change in the cytoskeletal architecture under the influence of chilling-induced depolymerization of microtubules could disturb a correct organization and trafficking of molecules and organelles. However, ultrastructural investigation of chilled follicles did not reveal rearrangements or clustering of organelles concomitant with the localization of microtubules to a perinuclear site.
Except for the disappearance of fibrillar lattices in the oocyte, no chilling-induced damage on cytoplasmic organelles could be detected. Studies from others on chilling mouse MII oocytes found that membranous organelles such as endoplasmic reticulum, Golgi, and nuclear envelope are susceptible to cooling [14, 15]. Although a larger sample would be necessary for a more conclusive statement, the present TEM study did not show clear evidence of such changes. Oocytes surrounded by two to three layers of tightly packed granulosa cells might be more protected from this damage.
This study focused on oocyte-granulosa cell interactions as well. Direct communication between follicle cells and the oocyte by gap junctions and/or paracrine factors are important for preantral follicle development [22–25]. Cytoskeletal elements are required for somatic-germ cell interactions at the level of the plasma membrane. Evaluation of the physical integrity of the connections was performed using confocal and electron microscopy. Microfilament staining of preantral follicles revealed that a dense array of F-actin containing follicle cell extensions were abundantly present after chilling, although the intensity of the staining was slightly reduced. Microtubules within transzonal processes, which are present in mouse follicles at the preantral stage and play a role in directed organelle movement [25], could not be reliably demonstrated due to resolution characteristics of our microscope. Granulosa-zona pellucida anchoring projections, required for TZP orientation, and zona-traversing projections could be detected by TEM, even after chilling the follicles for 30 min. Although TEM observation gives a topographically selected kind of information, these findings combined with the findings of confocal microscopy testify that the close contact between the somatic cells and the oocyte is present upon chilling.
The few chilling-induced alterations were reversible. Rewarming follicles to 37°C and allowing them to recover for 1 h restored the normal tubulin and actin organization to the same level as observed in noncooled control follicles. The presence of fibrillar lattices in the oocyte was not completely restored to normality because the density seemed to be lower compared with controls. It is, however, possible that the fibrillar lattices return to a characteristic high density only after several hours at 37°C, as has been noted previously for vitrified GV mouse oocytes during postthaw culture [26].
These observations altogether led us to conclude that early preantral mouse follicles are relatively tolerant to direct chilling injury. By allowing follicles to recover upon cooling for a sufficient time, any effect of temperature drop on structural changes will be restored. This indicates that small follicles can withstand the cooling-warming steps during conventional cryopreservation procedures fairly well. The reason that cryopreservation led to altered follicle growth and hormone production during in vitro culture might perhaps be attributed to induced defects within the somatic cell compartment rather than to injury on the follicle-enclosed oocyte. It might be the reflection of a reduced number of intact granulosa cells (and/or theca cells) caused by an initial cell death during the cryopreservation process or a delayed proliferation rate of the granulosa cells after thawing [4, 27]. It is plausible that granulosa and/or theca cells have different cryopreservation optima than oocytes. Further systematic studies of other cryobiological variables (e.g., effects of cryoprotective additives) on similar and other endpoints might increase our understanding of the transient functional deficit that was documented after cryopreservation of mouse preantral follicles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Leen Vanhoutte, Follicle Biology Laboratory, University Hospital and Medical School Infertility Center, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. FAX: 0032 2 477 50 60; leenvanhoutte{at}hotmail.com; leen.vanhoutte{at}ugent.be
3 Current address: Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
Received: 3 July 2003.
First decision: 3 August 2003.
Accepted: 25 November 2003.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
