Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice

I. Demeestere1,4, A. Delbaere2, C. Gervy3, M. Van den Bergh1,2, F. Devreker2 and Y. Englert1,2

1 Research Laboratory on Human Reproduction, 2 Fertility Clinic and 3 Laboratory of Chemistry, Erasme Hospital, French Speaking Free University of Brussels, Route de Lennik 808, 1070 Brussels, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. METHODS: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. RESULTS: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. CONCLUSIONS: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

Key words: follicle/isolation/in-vitro culture/mouse/theca


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In-vitro follicle culture is an essential tool in understanding the underlying mechanisms of oocyte growth and differentiation. Various culture systems for follicles have been developed, at different stages of follicle development and in several species. Primordial follicles represent the earliest form of ovarian follicles and consist of an oocyte surrounded by a single layer of flattened somatic cells (Gougeon, 1996Go). These follicles constitute the storage form of follicles, defined at birth in mammals. Mechanisms regulating the activation of primordial follicles have not been elucidated so far but are apparently gonadotrophin-independent (Picton, 2001Go). Nevertheless, the consequence of this activation is the differentiation and proliferation of follicular somatic cells forming a multilaminar granulosa layer, together with the initiation of oocyte growth, subsequently associated with the differentiation of theca-like cells. The follicle has then reached the preantral stage (Eppig et al., 1996Go; Van den Hurk et al., 2000).

Because primordial follicles potentially represent a large source of oocytes in humans and large mammals, with their possible applications in clinics and agriculture, efforts have been focused on developing culture systems for follicles at that stage.

Mouse primordial follicles have been grown in organ culture to preantral stages, then further matured after enzymatic isolation and fertilized, leading to the birth of one live mouse (to date the only animal produced from a primordial follicle), which later proved to present multiple malformations (Eppig and O'Brien, 1996Go; Eppig and O'Brien, 1998Go). In humans, it has been shown that 71% of isolated primordial follicles were viable by live–dead staining (Oktay et al., 1997Go). Moreover, human isolated primordial follicles have been shown to be able to grow up to multilaminar stages after 24 h of culture in collagen gel (Abir et al., 1999Go). Recently, the in-vitro development of fully or partially isolated human primordial follicles has been assessed in different culture conditions (Abir et al., 2001Go). Only fully isolated follicles cultured within a matrix of collagen gel were able to grow in vitro.

Human primordial follicles can also initiate their growth in vitro in ovarian tissue cultures (Hovatta et al., 1997Go, 1999Go; Wright et al., 1999Go). While ovarian tissue culture preserves follicle–follicle interactions as well as interactions between follicles and the surrounding stroma cells, culture of follicles as isolated units removes potential paracrine signals from stroma cells or between follicles, allows an easier diffusion of the nutrients and facilitates the analysis of the follicular morphology and hormonal secretions (Gosden et al., 1993Go; Van den Hurk et al., 2000).

Different in-vitro culture systems of preantral follicles from rodents have been developed for many years and have produced fully competent oocytes and viable offspring (Roy and Greenwald 1985Go; Eppig and Schroeder 1989Go; Nayudu and Osborn 1992Go; Cortvrindt et al., 1996Go). Based on these experiments, many attempts have been made to develop preantral follicle culture systems in humans (Roy and Treacy 1993Go; Abir et al., 1997Go) and in large mammals (Telfer et al., 2000Go; Van den Hurk et al., 2000). The common step of these different preantral follicular culture systems is the isolation of the follicles from the ovarian cortex before culture.

Follicles can be isolated mechanically or by enzymatic digestion. Mechanical microdissection of preantral follicles has been performed in the mouse (Nayudu and Osborn, 1992Go; Spears et al., 1994Go; Cortvrindt et al., 1996Go), rat (Daniel et al., 1989Go), cat (Jewgenow and Stolte, 1995Go), sheep (Cecconiet al., 1999Go), bovine (Guttierrez et al., 2000Go) and human (Abir et al., 1997Go). This technique maintains the integrity of the follicular structure: the basement membrane is still present after the procedure and the theca–granulosa–oocyte interactions are retained. However, microdissection of the preantral follicles is time-consuming and laborious when it is performed on dense ovarian cortex, as in humans and large mammals.

Enzymatic isolation of preantral follicles has been performed in the mouse (Eppig and Schroede, 1989), hamster (Roy and Greenwald, 1985Go), pig (Hirao et al., 1994Go), bovine (Figueiredo et al., 1993Go) and human (Roy and Treacy, 1993Go). This technique presents the advantage of yielding a large number of preantral follicles and is easier to perform on dense ovarian cortex. The proteolytic digestion of the extracellular matrix removes most if not all of the theca cells and degrades the basement membrane so that after the procedure, the follicle is reduced to an oocyte–cumulus complex.

After mechanical isolation, follicles can be cultured in a spherical culture system preserving the three-dimensional follicular structure, either by using a culture support (hydrophobic membrane) to which cells do not attach (Nayudu and Osborn, 1992Go), or by transposing the follicles every day (Boland et al., 1993Go; Hartshorne et al., 1994Go; Johnson et al., 1995Go). Alternatively, follicles can be cultured in a non-spherical system associated with the rupture of the basement membrane and the attachment of the granulosa cells to the support (Cortvrindt et al., 1996Go). This latter technique does not keep the follicular structure intact.

After enzymatic isolation, the culture system has to be adapted to avoid the migration of the granulosa cells away from the oocytes by facilitating the attachment of the granulosa cells. For this purpose, some authors have cultured oocyte–granulosa cell complexes on collagen membrane or serum-coated dishes. In these systems, the complexes lose their spherical structure and the oocytes can be directly collected in the medium after maturation (Eppig et al., 1996Go).

Oocyte–granulosa cell complexes have also been cultured completely embedded within a collagen matrix in different species (Torrance et al., 1989Go; Roy and Treacy 1993Go; Hirao et al., 1994Go). These culture systems preserve their spherical morphology until the antral stage but the recovery of the oocytes appears to be more difficult.

Follicles can be cultured as a group or as isolated units. While group cultures retain interfollicular communication and control (Spears et al., 1996Go; Mizunuma et al., 1999Go) individual follicle culture allows a careful evaluation of the follicular development as assessed by morphological changes as well as hormonal secretions (Gosden et al., 1993Go). Regardless of the culture conditions, the impact of the isolation technique on the subsequent follicular morphology and steroidogenesis, on oocyte maturation and embryo development are not known due to a lack of comparative studies between the two techniques.

The aim of this study was to compare the effect of the mechanical and enzymatic isolation of mouse preantral follicles on the subsequent follicular and oocyte development using an individual in-vitro culture system.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 16 separate experiments was been performed: nine experiments were carried out to assess the follicular morphology and hormonal secretion as well as the oocyte maturation rate; four experiments were carried out to evaluate the fertilization and embryo development rates; three experiments were performed for cytochemical staining of the theca cells.

Isolation of preantral follicles
All experiments were conducted using female hybrid (C57bl/8jxCBA/ca) mice housed and bred in a temperature- and light-controlled room and provided food and water ad libitum. Approval was obtained for this study from the Local Animal Ethics Committee.

For all experiments, mice aged 12–14 days old were killed by cervical dislocation. Ovaries were removed aseptically, separated from the connective tissue and placed in 2.5 ml of Leibovitz's L15 medium (with L-Glutamine) supplemented with 10% of fetal calf serum (FCS) (both provided by Life Technologies, Merelbeke, Belgium), 100 mIU/ml penicillin and 100 mg/ml streptomycin (Sigma Aldrich, Bornen, Belgium) at 37°C (Cortvrindt et al., 1996Go).

For each mouse, one ovary was dissected mechanically using a 26 gauge needle and the other was disassociated by adding crude collagenase type I (1 mg/ml, >=125 collagen digestion units per mg) and DNase (18 IU/ml) (Sigma Aldrich) to L15 medium for 1 h at 37°C. Every 10 min, the ovaries incubated in the enzymatic solution were triturated by gentle pipetting in order to facilitate proteolytic digestion (Eppig and Schroeder, 1989Go).

Culture of preantral follicles
Mechanically and enzymatically isolated follicles were rinsed three times in 30 µl of culture medium composed of Minimal Essential Medium (MEM GlutaMAX, Life Technologies) supplemented with 100 mIU/ml rFSH (kindly donated by Organon, Oss, The Netherlands), 10 mIU/ml rLH (Sigma Aldrich), 5% FCS and ITS (insuline 5 µg/ml; transferrine 5 µg/ml; selenium 5 ng/ml: Sigma Aldrich) (Cortvrindt et al., 1997Go, 1998Go).

An intact preantral follicle was defined as a round follicular structure containing a visible oocyte centrally located and surrounded by more than one granulosa cell layer (generally two layers) with at least one completed granulosa cell layer. Follicles were measured under 400x magnification with a precalibrated ocular micrometer. Twenty intact follicles of 100–130 µm were selected from each ovary and individually cultured for 12 days in Falcon Culture Dishes (Becton Dickinson, Le Pont de Claix, France) in 20 µl of culture medium under 5 ml of mineral oil (Sigma Aldrich) at 37°C in a humidified atmosphere of 5% CO2 in air as described previously (Cortvrindt et al., 1996Go).

Every 2 days, 10 µl of culture medium from each drop was replaced by fresh medium and the culture media of surviving follicles was pooled and frozen for subsequent hormonal assay. After 12 days of culture, 1.5 IU/ml rHCG (kindly donated by Ares Serono; Geneva, Switzerland) and 5 ng/ml epidermal growth factor (EGF) (Boehringer Mannheim; Brussels, Belgium) were added to the culture medium in order to induce oocyte maturation (Smitz et al., 1998aGo).

Fourteen to sixteen hours post rHCG/EGF, oocyte–cumulus complexes (OCC) were mechanically denuded to evaluate the oocyte nuclear maturation stage.

In-vitro fertilization of in-vitro grown oocytes
OCC collected 16 h post rHCG/EGF from four different experiments were rinsed and placed in a 500 µl drop of modified simplex optimized medium with potassium (KSOM) (Devreker and Hardy, 1997Go) supplemented with 1 mg/ml of bovine serum albumin fraction V (BSA; Sigma Aldrich). After induction of capacitation for 2 h in modified Tyrode's medium (Summers et al., 1995Go) supplemented with 15 mg/ml BSA, sperm from 3–6 month old hybrid mice was added to the OCC drops to give a final motile sperm concentration of 1–2x106/ml. After 4 h incubation, fertilized oocytes were washed three times in modified KSOM medium supplemented with BSA and further cultured for 120 h in 30 µl droplets of the same culture medium (5–6 embryos per droplet).

Control was performed using in-vivo grown oocytes collected from superovulated F1 hybrid mice by i.p. injection of 10 IU of equine serum gonadotrophin (Folligon; Intervet, Mechelen, Belgium) and 10 IU of chorionic gonadotrophin (Chorulon; Intervet, Mechelen, Belgium) 48 h later.

In-vivo grown OCC were fertilized in vitro for 16 h post-chorionic gonadotrophin injection in the same conditions as those obtained after in-vitro culture and maturation.

Hormonal measurements
In nine experiments, conditioned media from surviving follicles was pooled within each experiment at different times of culture. Surviving follicles were defined as the attached follicles which had retained their oocytes in the granulosa cell mass.

Pooled media was frozen and hormonal measurements were performed at the end of each experiment. Estradiol and testosterone were measured in the conditioned media collected every 2 days as from day 4 of culture. Progesterone was measured in the media at days 10, 12 and 13 (16 h post rHCG/EGF).

Estradiol and progesterone were measured using electrochemiluminescence immunoassay (ECLIA; Roche-Mannheim, Germany) and testosterone was measured using coated tube radioimmunoassay (Biosource, Belgium). Intra-assay variation coefficients were 3, 4, and 2.4% for estradiol, testosterone and progesterone respectively. Interassay variation coefficients were 5.5, 8.3 and 5.4% for estradiol, testosterone and progesterone respectively.

Because most of the estradiol values in the collected medium were off the scale (>4.8 ng/ml), dilution of 1:4 was performed for all samples, with human steroid-free serum routinely used to dilute samples assayed with the same immunoassay kits. All samples were thus handled in the same conditions. For values of estradiol >4.8 ng/ml in the 1:4 medium, samples were diluted 1:8.

Cytochemical staining of theca cells
As alkaline phosphatase activity is localized specifically in the theca interna of growing follicles and not in granulosa cells, it has been used successfully to evaluate the presence of theca cells in cultured follicles (Sangha and Guraya, 1989Go; Cortvrindt et al., 1998Go). Three separate experiments were performed for cytochemical staining of the theca cells: half of the follicles were stained for cytochemical analysis at day 0 and the other half were fixed and stained after 4 days of culture.

Follicles were isolated mechanically and enzymatically as described previously and were individually cultured in 96-well plates (Life Technologies) in 80 µl of culture medium. All the following reactives were provided by Sigma Aldrich except those mentioned. At day 0 and 4 of in-vitro culture, follicles were rinsed with phosphate buffered saline (PBS) pH 7.2 (Life Technologies) and fixed in 4% formaldehyde supplemented with 1% CaCl2 for 15 min at room temperature.

The fixed follicles were then rinsed again and incubated for 45 min in darkness in a histochemical solution composed of 0.25 mg/ml naphthol AS-BI phosphate acid sodium salt and 0.75 mg/ml Fast Blue BB salt in 0.2 mol/l Tris-HCl buffer pH 9.3. Follicles were counterstained with 0.5% Nuclear Fast Red. Follicles were rinsed again and directly observed on an inverted microscope. Staining was evaluated and graded as negative (–), weak (+), moderate (++) and strong (+++).

Statistical analysis
Differences in morphological development and theca cells staining between groups were assessed using {chi}2-test. Differences in follicular hormonal production between both groups and according to the duration of the culture were analysed by one-way ANOVA followed by the Mann-Whitney test. Only P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Morphological follicular development and hormonal assessment
Follicles isolated mechanically were defined as group M while those isolated enzymatically were defined as group E (Figure 1A,BGo). The mean follicular size at day 0 was not significantly different in group M (117 ± 11.9 µm; n = 154) and group E (118.4 ± 10.2 µm; n = 153). After 2 days of culture, basement membrane disruption had occurred in 34.4% of group M and 100% of group E (Figure 1C,DGo). Follicles from group M with an intact basement membrane at day 2 underwent basement membrane disruption within the next 2 days of culture.



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Figure 1. Different phases of mouse preantral follicles cultured in vitro (Hoffman Contrast Phase Microscope) (A) Follicle isolated enzymatically (day 0). The basement membrane is degraded and no longer visible (original magnification x200). (B) Follicle isolated mechanically (day 0). The basement membrane is present with few theca cells attached (original magnification x200). (C) Follicle isolated enzymatically (day 2). The granulosa cells are attached to the dish and the basement membrane is completely ruptured (original magnification x300). (D) Follicle isolated mechanically (day 2) with a rupture of the basement membrane at one point (original magnification x300). (E,F) Follicles isolated enzymatically (E) and mechanically (F) with antral-like cavity formation within the granulosa cell mass (day 12) (original magnification x100).

 
After 12 days of culture, the follicle survival rate was higher in group M than in group E (94.1 and 76.4% respectively, P < 0.05) (Table IGo). In this regard, premature release of the oocyte was more frequently observed in group E during the first days of culture.


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Table I. Morphological characteristics of preantral follicles isolated mechanically (Group M) or enzymatically (Group E) and cultured in vitro for 12 days. The selected follicles at day 0 have a diameter of 100–130 µm (mean ± SD)
 
After 12 days of culture, antral-like cavity formation, defined as a clear space appearing within the granulosa cells (Figure 1E,F), was observed in 24.1% of surviving follicles in group M and 62.3% of group E surviving follicles (P < 0.05). In group E, granulosa cells displayed a larger and earlier expansion. However, the rate of mucification after rHCG/EGF was similar in both groups (Table IGo).

Sixty-nine and 75% of the oocytes collected 14–16 h post-rHCG/EGF reached metaphase II in groups M and E respectively. Ten and 14% of the remaining oocytes underwent germinal vesicle breakdown (GVBD) in groups M and E respectively (NS; P > 0.05). At day 13 of in-vitro culture, matured oocytes reached the same size in groups E (n = 21) and M (n = 31) (Table IGo).

In-vitro fertilization rate
Four independent experiments were carried out to evaluate the capacity of the oocytes to be fertilized and develop until blastocyst stage. OCC from group M (n = 59) and E (n = 55) were directly inseminated with capacitated spermatozoa. After 24 h, 33.8 and 45.2% of stage II embryos were observed in group M and E respectively (NS; P > 0.05). Fifty and 40% of these 2-cell stage embryos reached the blastocyst stage after 5 days of culture (NS; P > 0.05). Oocyte fertilization and embryo development rates were significantly lower than those observed after in-vivo growth and in-vitro oocyte fertilization (Table IIGo).


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Table II. Two-cell stage and blastocyst stage embryos obtained after IVF of in-vitro grown and matured oocytes derived from preantral follicles isolated either mechanically (Group M) or enzymatically (Group E). These results were compared with the number of 2-cell stage and blastocyst stage embryos obtained after IVF of in-vivo grown and matured oocytes (Control)
 
Hormonal assays
Estradiol and testosterone were measured in conditioned medium at days 4, 6, 8, 10 and 12 of culture (Figure 2Go). The secretion rate per 48 h of both hormones was significantly higher in group E than in group M throughout the entire culture period (Figure 2AGo). The difference observed between both groups in estradiol secretion was significant except at day 6.



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Figure 2. Levels of estradiol (A) and testosterone (B) secreted per 48 h by preantral follicles isolated either mechanically (Group M) or enzymatically (Group E). Measurement was performed on pooled culture media of surviving follicles from nine different experiments. Results are expressed as the mean ± SEM.

 
The mean testosterone secretion per 48 h remained stable throughout the entire culture period in group M while it increased between day 4 and day 12 in group E (Figure 2BGo). Testosterone secretion was higher in group E than in group M from day 6 to the end of the culture (P < 0.05). Progesterone levels increased after induction of oocyte maturation with rHCG/EGF in both groups (day 13 of culture).

Levels were not statistically different between groups M and E [0.66 ± 0.2 to 2.7 ± 0.3 ng/ml in group E at day 12 and 13 respectively and 0.27 ± 0.02 to 1.8 ± 0.1 ng/ml in group M at day 12 and 13 respectively (mean ± SEM)].

Theca cells staining
Three separate experiments were performed to evaluate the presence of theca cells at day 0 and day 4 of culture in vitro (Table IIIGo). For each experiment, half of the selected follicles were fixed at day 0, the other half were cultured for 4 days before cytochemical analysis.


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Table III. Presence of theca cells at day 0 and day 4 of culture as assessed by alkaline phosphatase reaction staining according to the follicle isolation technique (mechanically, Group M or enzymatically, Group E)
 
At day 0, 73.4 and 83.8 % of the follicles isolated enzymatically (n = 34) and mechanically (n = 31) respectively displayed positive alkaline phosphatase reaction (NS; P > 0.05). However, the intensity of the staining of theca cells (weak, moderate and strong) differed between the groups (P < 0.05). Following enzymatic isolation 72% of the follicles were classified as having a moderate or strong staining, while after mechanical isolation only 6% were classified as having a moderate or strong staining (Figure 3Go). At day 4 of culture, most follicles were positive for alkaline phosphatase (86% positive reaction in group M, n = 43 and 90% in group E, n = 42), but no significant difference appeared between groups regarding the intensity of the reaction.



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Figure 3. Cytochemical staining of the theca cells of follicles isolated enzymatically (left) or mechanically (right). At day 0, follicles isolated enzymatically presented a strong phosphatase alkaline reaction while follicles isolated mechanically had a weak phosphatase alkaline reaction (bar = 100 µm).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Both enzymatic and mechanical preantral follicle isolation techniques have been described previously (Roy and Greenwald, 1996Go). The use of various follicular culture systems and the differences in the follicle selection make the impact of the isolation technique difficult to evaluate. This study compared the effect of two preantral follicle isolation techniques (mechanical or enzymatic) on the subsequent follicular development in vitro in an individual culture system. The culture system used in the present study was developed after mechanical follicular isolation (Cortvrindt et al., 1996Go). This system offers several advantages, follicles are cultured individually in microdrops, providing an easy access to the conditioned medium and allowing a careful morphological and metabolic assessment of individual follicles.

Follicles isolated enzymatically were previously cultured in a group on a collagen matrix and individually in agar or collagen-gel matrix to avoid the premature release of the oocyte related to the degradation of the basement membrane (Eppig and Schroeder, 1989Go; Torrance et al., 1989Go; Roy and Treacy, 1993Go). The present study shows that follicles isolated enzymatically were also able to grow individually in vitro without collagen membrane support. The survival rate after 12 days of follicular culture was ~70% for enzymatically isolated follicles compared to a survival rate of >90% for mechanically isolated follicles cultured in the same conditions. Other authors observed a follicular survival rate of ~90% after 10 days of culture of enzymatically isolated follicles using other culture conditions (Eppig et al., 1998).

Although the present culture system may not lead to the optimal follicular survival rate after enzymatic isolation of the follicles, it has the advantage of allowing a precise hormonal and morphological follicular follow-up.

Morphological differences between follicles isolated mechanically or enzymatically were assessed with respect to the potential damage of the enzymes to the basement membrane surrounding the granulosa cells and to the theca cells. A rapid disruption of the basement membrane was observed in the enzymatically isolated follicles group (100% in group E compared with 34% in group M at day 2). In-vivo, the basement membrane plays an essential role to maintain the spherical follicular structure and its surface increases enormously during follicular growth (30 000-fold in mice) (Gosden et al., 1993Go). In a non-spherical culture system as used in this study, the disruption of the basement membrane within the first days of culture in vitro was necessary to allow the granulosa cells to attach with subsequent follicular development. The degradation of the basement membrane did not compromise the subsequent follicular development. However, it could explain the lower survival rate of the enzymatically isolated follicles, compared to the mechanically isolated follicles in relation to the frequent premature release of the oocyte after enzymatic isolation in this culture system.

Enzymatically isolated follicles secreted more estradiol and testosterone compared with mechanically isolated follicles during the entire culture period. Estradiol production during follicular development is dependent on the presence of androgen precursors secreted by the theca cells and aromatized to estradiol by the granulosa cells. We initially expected that the enzymatic dissection of the follicles could damage the theca cells and therefore induce a defect in hormonal secretion. In fact, some authors had previously demonstrated that the absence of the theca cells at day 0 had a negative impact on the estradiol production during follicle culture (Cortvrindt et al., 1998Go). In this study, the higher secretion of estradiol in the enzymatically isolated follicle group suggested the presence or the rapid differentiation of theca cells during the first days of follicle culture. Alkaline phosphatase staining was used to evaluate the presence of theca cells at day 0 and day 4 of culture. Surprisingly, an intense positive reaction at day 0 demonstrated that theca cells or their precursors were present in >70% of the follicles immediately after enzymatic isolation. After mechanical isolation, 84% of the follicles had few theca cells attached to the basement membrane. The staining of mechanically isolated follicles was more homogeneous than staining of enzymatically isolated follicles: immediately after enzymatic isolation, follicles presented weak, moderate, strong or no phosphatase alkaline reaction in comparable proportions while most of the mechanically isolated follicles presented a weak staining. After 4 days, this difference was no longer significant. Theca cells are thought to originate from fibroblast-like precursor cells located in the ovarian tissue. The preantral follicles secrete theca differentiating factors regulated by FSH, allowing the development and the differentiation of the theca cells (Magoffin and Magarelli, 1995Go). Our results suggest that theca cells or their precursors are still present after enzymatic isolation. These cells are probably developing and differentiating under the stimulation of different paracrine factors and assume subsequent steroidogenesis. Our experiments demonstrate that the enzymatic isolation technique does not affect either theca cells development or the subsequent steroidogenesis, since steroidogenesis was improved after enzymatic isolation of the preantral follicles compared to mechanically isolated follicles.

However, development of the somatic component of the follicles was dissociated from the oocyte competence to undergo both nuclear and cytoplasmic maturation. Indeed, no significant differences were observed between both groups in terms of maturation, oocyte fertilization and embryo development rates. Thus, culture conditions that apparently promote some morphological and hormonal aspects of follicular development are not necessarily associated with a beneficial effect on oocyte development. Similar observations have been previously described with different growth factors that enhanced the follicular development without having an effect on oocyte maturation (Smitz et al., 1998bGo).

Acquisition of the full development competence occurs in the oocyte at the final growth stage. Oocytes have to reach their full size to support future completion of development. At day 13 of culture in vitro, oocytes reached the same size in both groups suggesting that the isolation technique has no effect on the oocyte growth and on its capacity to mature in vitro. Moreover, these results are in agreement with previous studies reporting that the mean diameter of in-vivo grown oocytes from adult mice (75 µm) was larger than that of in-vitro grown oocytes obtained after culture of preantral follicles (Hu et al., 2001Go).

In both groups, oocyte fertilization and embryo development rates of the in-vitro grown oocytes were lower than those observed after in-vitro fertilization of in-vivo grown oocytes. The lower blastocyst development rate observed in this study confirms previous results described in the same culture system (Cortvrindt et al., 1996Go).

In a different culture system, the developmental competence of the in-vitro grown oocytes was similar to that of in-vivo grown oocytes, but when the culture medium was supplemented with FSH the embryo developmental rate was significantly reduced (Eppig and O'Brien, 1998Go). The requirement for FSH is surprisingly different depending on the culture system used. The presence of rFSH in the culture system used in this study has been shown to be essential for follicle survival. Without rFSH, only 11% of the follicles survived for 12 days (Cortvrindt et al., 1997Go).

This study compared two preantral isolation techniques in a defined culture system. It demonstrated that most of the follicles isolated enzymatically had a larger quantity of stroma or theca cells surrounding the granulosa cell layers compared with the mechanically isolated follicles. These attached cells allowed the secretion of a higher concentration of steroid hormones. Whatever isolation technique is used, the developmental capacity of the in-vitro grown oocytes was lower than that of the in-vivo grown and matured oocytes.

It is generally thought that mechanical isolation produces less damage to the preantral follicles than enzymatic isolation and could therefore produce better quality follicles (Gosden et al., 1993Go). The present study shows that this is not necessarily the case and should encourage researchers to continue developing enzymatic as well as mechanical isolation methods. This could be particularly important when considering the isolation of human follicles, since they are released from a dense and tough stroma from which mechanical isolation is very laborious.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by the Belgian National Fund for Scientific Research (Grant-Televie).


    Notes
 
4 To whom correspondence should be addressed. E-mail: idemeest{at}ulb.ac.be Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 22, 2001; resubmitted on February 26, 2002; accepted on May 2, 2002.