Differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence

I. Adriaens1,2,3, R. Cortvrindt1 and J. Smitz1

1 Free University Brussels, Center for Reproductive Medicine – Follicle Biology Laboratory, Laarbeeklaan 101, 1090 Brussels and 2 Belgian Nuclear Research Centre at Mol, Radiation Protection Research – Radiobiology, Boeretang 200, 2400 Mol, Belgium

3 To whom correspondence should be addressed at: Belgian Nuclear Research Centre in Mol, Radiation Protection Research – Radiobiology, Boeretang 200, 2400 Mol, Belgium. e-mail: iadriaen{at}sckcen.be


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
BACKGROUND: We investigated at what stage early cultured preantral mouse follicles become dependent on a minimal effective FSH dose (10 mIU/ml) and analysed the influence of implementing FSH at several time-points during in vitro culture. METHODS: Two-layered mouse follicles were cultured for 12 days under seven different FSH exposure regimens and ovulated on day 13 by hCG/EGF. Ovulated cumulus–oocyte complexes were fertilized and embryos were cultured up to the blastocyst stage. RESULTS: When FSH was absent or added only once at the start of culture, follicle survival was significantly reduced (22 and 52% respectively versus 95% when FSH was continuously present, P < 0.01) and oocyte quality was compromised, providing few oocytes for embryo culture (19 and 7% versus 71% in continuous presence of FSH, P < 0.01). Optimal follicle survival rates can be ensured by implementing FSH at the latest from day 4 of culture. By introducing FSH later than day 4, follicle survival rates and number of ovulated oocytes decreased. Estradiol production and luteinization were strongly related to the moment of introducing FSH in culture. Fertilization and preimplantation embryo development rate were found to be highest in cultures where FSH support was implemented during the preantral stage. CONCLUSION: Exposure to FSH before formation of the antral-like cavity had a positive effect on follicle survival and oocyte robustness.

Key words: follicle/follicular development/FSH/IVF/oocyte development


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
From physiological studies using purified gonadotrophin preparations and involving knockout or transgenic animals, it has become clear that FSH is the essential driver of antral follicle growth in vivo. It is less well known how FSH affects the preantral growth stages in vivo and additionally how FSH should be implemented in in vitro follicle growth regimens to obtain developmentally competent oocytes. In GnRH-deficient hypogonadal (hpg) mice, serum levels of gonadotrophins are undetectable and the gonads fail to develop postnatally (Cattanach et al., 1977Go). Kumar et al. (1997Go) indicated that FSH-{beta} knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. Early antral follicles depend on the presence of FSH for further survival and differentiation.

Although early preantral follicles are independent of FSH for their initial growth, FSH receptors are present on the granulosa cells (GC) of these follicles (O’Shaughnessy et al., 1996Go). The best known FSH receptor is FSH-R1 (Babu et al., 2001Go). However, several events occur well before the expression of the stimulatory G-protein (Gs)-coupled FSH-R1 receptor. These suggest that other FSHr motifs might be functional to play a role during development. It was proven that a new splice variant, FSH-R3, is present in immature ovaries and that there is a positive correlation between follicle differentiation and the expression rate of this receptor (Babu et al., 2001Go). In the human, one-third of the primary and two-layered follicles produce FSH-R mRNA and all multilaminar follicles are positive for FSH-R mRNA (Oktay et al., 1997Go). Human ovarian xenografts placed under the kidney capsule of mice homozygous for severe combined immunodeficiency (SCID) and hypogonadism (hpg) contain follicles progressed beyond the secondary stage only when animals are treated with FSH (1 IU) (Oktay et al., 1998Go).

In mice aged <3 days, FSH receptor transcripts are just detectable, but none of the transcripts are full length. The first full length transcripts can be found on neonatal day 5. It has also been demonstrated that the initial development of FSH-R mRNA is independent of the gonadotrophins (O’Shaughnessy et al., 1996Go). FSH receptor knockout (FORKO) mice have a disordered estrous cycle, ovulatory defects and an atrophic uterus. The females of these knockout animals are infertile (Danilovich et al., 2000Go; Burns et al., 2001Go).

At the moment of first antrum formation in juvenile mice (between neonatal days 10 and 15), the follicles become dependent on the presence of FSH. FSH activates the proliferation of the GC, stimulates aromatase enzyme activity and induces expression of LH receptors on GC. The presence of LH receptors is essential for the induction of ovulation and for the formation of a corpus luteum after the midcycle LH surge (Baird, 2000Go; Zeleznik, 2000Go). Granulosa cells in FSH-{beta} knockout mice do not accumulate LH receptor mRNA (Burns et al., 2001Go). This means that there is no terminal differentiation as seen in luteinizing cells of follicles from wild-type animals. Follicle growth or atresia during the antral growth phase results from a delicate balance between gonadotrophins and paracrine factors. Follicular growth is supported by factors such as insulin-like growth factor (IGF-I), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), interleukin-1{beta} (IL-1{beta}) and activin (Markström et al., 2002Go). Factors inducing follicle atresia comprise tumour necrosis factor-{alpha} (TNF-{alpha}), GnRH, androgens and free radicals (Kaipia and Hsueh, 1997Go). Small, slow-growing follicles do not depend upon gonadotrophins to prevent GC death in vitro (McGee et al., 1997Go). This suggests that the apoptosis of preantral follicles is regulated differently from that of larger follicles (O’Shaughnessy et al., 1996Go).

Several culture systems for the in vitro development of preantral mouse follicles have been described (Eppig, 1977Go; Nayudu and Osborn, 1992Go; Cortvrindt et al., 1996Go). These culture systems have been used for physiological studies and have contributed to our understanding of the mechanics of folliculogenesis and ovulation. In most of the systems, the presence of FSH appeared to be essential for the survival and the development of the follicles (Nayudu and Osborn, 1992Go; Cortvrindt et al., 1996Go). In the in vitro system developed by Cortvrindt et al. (1996Go), which starts off with follicles between 100 and 130 µm, only 8% of the follicles can survive the 12 day culture period when no gonadotrophins were added to the culture medium. The minimal effective dose of rFSH in this system was titrated out and appears to be 10 mIU/ml of the recombinant gonadotrophin (unpublished observations from R.Cortvrindt et al.) Additions of FSH and LH were essential to permit oocyte nuclear maturation up to the metaphase II stage (Cortvrindt et al., 1998aGo,b). Nayudu and Osborn (1992Go), who worked with follicles >150 µm in diameter, grew them to a diameter of ~275 µm without FSH. However, despite clear growth, no antrum formation was noticed and also no secretion of estradiol could be measured. However, when FSH was added to the medium, the preantral follicles grew to morphologically normal antral follicles (400–500 µm diameter) which produced estradiol. When culturing intact follicles, a dose-dependent relationship was demonstrated between the FSH concentration in the medium and the estradiol secretion (Nayudu and Osborn, 1992Go).

While previous studies clearly emphasized that FSH plays a central role in folliculogenesis, it is less clear how exposure to FSH might impact upon oocyte quality. Inappropriately timed administration of FSH could—despite securing an optimal survival of somatic cell components—disturb the delicate crosstalk between oocyte and granulosa cells and lead to oocyte incompetence for further development (Eppig and O’Brien, 1997Go; Albertini et al., 2001Go). Albertini et al. (2001Go) have identified specialized connections between granulosa cells and oocytes characterized by uniquely structured transzonal projections (TZP) that are regulated by FSH. The type and density of TZP might be regulated by locally delivered FSH concentrations throughout the follicle growth process. Observations on density of TZP during follicle growth suggested that these connections might be the effectors for bidirectional polarized secretion and endocytosis of factors synthesized within the follicle itself (Albertini et al., 2001Go). It is important to know how physiological amounts of FSH might effect growth and differentiation of very small follicles in in vitro culture and how this follicle-promoting effect might impact on oogenesis and early embryonic development.

This study investigated at which precise moment of in vitro follicle development follicles become dependent on the presence of a minimal effective dose of rFSH (10 mIU/ml) for normal follicle and oocyte development. The purpose was also to search for the appropriate time sequence for FSH administration in order to obtain an ideal combination between follicle survival and oocyte developmental competence.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Animal model for follicle recruitment
Early preantral follicles (Type 3b in the Pedersen classification) (Pedersen and Peters, 1968Go) were isolated from ovaries of 14 day old females (F1 hybrids: C57BLxCBAca; Harlan, The Netherlands). Adult females, 6 week old, were used to collect in vivo-matured oocytes in order to create a control group for the IVF. Adult males with proven fertility were used for the collection of sperm. Animals were killed by cervical dislocation.

All animals were housed and bred according to national legislation and with the consent of the ethical commission (Project number: 01-395-1).

Culture conditions and media
Collection medium.
All tissues and follicles were collected in L15 Leibovitz medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 IU/ml penicillin and 100 µg/ml streptomycin. All products are from Invitrogen (Belgium). During the operating procedures the medium was always kept at 37°C.

Follicle culture medium.
The follicle culture medium consisted of {alpha}-minimal essential medium ({alpha}-MEM) supplemented with 5% heat-inactivated FCS, 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml selenium (ITS; Sigma, Belgium). Recombinant FSH (Gonal-F®; kindly donated by Serono, Switzerland) was added according to the different experimental conditions (final medium concentration of FSH was always 10 mIU/ml). Earlier data from our laboratory showed that 10 mIU/ml of this FSH preparation is the minimal effective dose for obtaining meiotically mature oocytes in this culture system (I.Adriaens, R.Cortvrindt and J.Smitz, unpublished observations). The culture dishes were kept in the incubator at 37°C, 100% humidity and 5% CO2 in air.

Ovulation induction stimulus.
Ovulation induction was obtained by supplementing 1.5 IU/ml recombinant hCG (Ovidrel®; kindly donated by Serono) and 5 ng/ml recombinant epidermal growth factor (rEGF; Boehringer Mannheim, Germany) to the follicle culture medium (Smitz et al., 1998Go).

Sperm capacitation and IVF medium.
Sperm capacitation and IVF was performed in KSOM [containing NaCl (95.00 mmol/l), KCl (2.50 mmol/l), glucose (5.56 mmol/l), KH2PO4 (0.35 mmol/l), MgSO4 (0.20 mmol/l), lactate (2.27 ml/l), pyruvate (0.20 mmol/l), NaHCO3 (25.00 mmol/l), L-glutamine (1.00 mmol/l), CaCl2H2O (1.71 mmol/l), EDTA (0.01 mmol/l), Phenol Red (0.01 g/l), penicillin (100 IU/ml) and streptomycin (100 µg/ml)] supplemented with 3% bovine serum albumin (BSA) (all products from Sigma, Belgium).

The capacitation and the IVF occurred at 37°C, 100% humidity and 5% CO2 in air.

Embryo culture medium
The embryos were cultured in KSOM with 0.5% BSA. Embryo development took place under reduced oxygen conditions (37°C, 90% N2, 5% CO2 and 5% O2).

Follicle culture
This culture system was previously described (Cortvrindt et al., 1997Go); for the sake of clarity a brief description is given from the essential steps of the bioassay.

Early preantral follicles were mechanically isolated from the ovaries. The follicles were washed in the culture medium before being plated singly in 10 µl culture droplets. Twenty follicles per culture dish (60 mm Petri dishes, Falcon; Becton Dickinson, Belgium) were covered with 5 ml of embryo-tested mineral oil (Sigma, Belgium).

Each dish was considered as one single unit in an experiment. Follicles were randomly distributed in the different experimental conditions.

At day 1 of the culture (i.e. 24 h after the isolation) the quality of the follicles was ascertained. Follicles were measured, the connection between oocyte and granulosa cells evaluated and the presence of theca cells recorded. This evaluation was done under an inverted microscope with a Hoffman contrast-modulation system at a magnification of x400 (Nikon inverted microscope, Japan). For these experiments a strict follicle selection was applied: only the follicles measuring between 100 and 130 µm on day 1 were included in the experiment. At day 2 of the culture, 10 µl medium was added to the culture droplets. Every other day, follicles were evaluated under a stereo microscope and half of the culture medium was refreshed. The collected medium was kept at –20°C for further analyses.

A representative example of the morphological changes that follicles undergo throughout the in vitro culture period in continuous presence of FSH is given in Figure 1. Individual small preantral follicles, surrounded by the basal membrane and theca cells are put into culture (‘follicular stage’). Initially, theca cells grow out to the culture dish and granulosa cell proliferation is limited. The follicles remain in this stage until day 4. From day 4 up to day 6 or day 8, a pronounced granulosa cell proliferation leads to their protrusion through the basal membrane and formation of large preantral follicles (‘diffuse stage’). During the last phase of the culture (from day 8 until day 12), follicles start to differentiate and reach the pre-ovulatory stage. This stage is defined by the presence of theca cells attached to the bottom of the dish, a ring of large mural granulosa cells which enclose the antral-like cavity, a cumulus oophorus located centrally with small cumulus cells in close contact with the oocyte (‘antral, pre-ovulatory stage’). The ovulatory stimulus was given at day 12 and 18 h later the in vitro-released mucified cumulus–oocyte complexes (COC) were collected for fertilization.



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Figure 1. Development and differentiation of a representative follicle cultured for 12 days in the continuous presence of 10 mIU/ml FSH. Early preantral follicles (follicular stage) are put into culture and after 4 days the follicles develop to late preantral follicles (diffuse stage). During the second half of the culture period the follicle mural and cumulus cell differentiation becomes apparent, resulting in the formation of antral-like cavities (antral stage). The follicles differentiate into pre-ovulatory follicles. Eighteen hours after the ovulation stimulus (given on day 12), mucified cumulus–oocyte complexes are free-floating in the culture droplet.

 
Experimental set-up.
Seven independent experiments were carried out. The same batch of media and added products was used throughout this study. In each of these experiments, follicles were collected from eight ovaries (i.e. four mice) and distributed at random among the different experimental groups.

The different conditions are referred to as: positive control: 10 mIU/ml FSH was continuously present during culture (from day 0 onwards); negative control: no FSH was added during culture; only added on day 0: 10 mIU/ml FSH was only added in the medium at the start of culture and never again after that.

From day 2, 4, 6, 8: 10 mIU/ml FSH was introduced into the medium from these days onwards. The number of follicles used in each experiment is indicated in Table I.


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Table I. Experimental set-up.
 
Evaluation parameters
Every other day the development of the in vitro-cultured follicles was evaluated morphologically and the follicle survival was recorded (Cortvrindt et al., 1996Go). In this culture system survival is defined as those follicles which could retain their oocyte completely embedded within the granulosa cell mass and this was expressed as a percentage of follicles initially included in the experiments (at the moment of plating).

Follicular secretory products (progesterone and estradiol) were measured in the conditioned medium collected on different days (i.e. day 4, 6, 8, 10, 12 and 13).

IVF of in vitro-grown and -matured oocytes
Sperm from two males with proven fertility was capacitated for 2 h. The COC were collected just before insemination and placed in 30 µl droplets of KSOM with 3% BSA (10 COC/droplet). Sperm were added to the droplets at a final concentration of 2x106 sperm cells/ml. Fertilization was allowed to occur for 2.5–3 h. Embryo culture was done in 30 µl droplets of embryo culture medium. The 2-cell rate was scored after 24 h and the blastocyst rate at day 5.

The fertilization procedure was controlled by performing an IVF on in vivo-matured oocytes. Mature oocytes were retrieved from adult females (minimum 6 weeks old) primed with 5 IU Folligon and stimulated with 5 IU Chorulon (respectively 48 and 18 h before retrieval). Both compounds were from Intervet (Belgium).

Hormone measurements
The concentrations of 17{beta}-estradiol and progesterone were measured in spent medium from the different experimental conditions. Measurements of steroids were done with radioimmunoassay extensively validated for this medium as described previously (Cortvrindt et al., 1996Go).

Statistics
Results were statistically analysed by one-way analysis of variance and Tukey test (Statistica 6, Statsoft Benelux, The Netherlands). P < 0.01 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Folliculogenesis
Survival (Figure 2).
In this culture system, follicles loose their spherical structure as they attach to the culture dish. Follicle survival was therefore assessed as the number of follicles that could retain their oocyte enclosed within the granulosa cell mass divided by the total number of follicles at the start of culture/culture dish. Our system differs in this aspect from other culture systems developed for preantral follicles. In the system developed by Eppig et al. (1996Go), oocyte–granulosa cell complexes collected after enzymatic digestion from 12 day old animals were cultured in groups on collagen-impregnated membranes. After 10 days of culture, oocytes were removed from the membrane and placed in maturation medium (15–17 h). Others preferred to select somewhat larger follicles (between 150 and 200 µm) to culture and to keep the spherical structure by actively prohibiting the follicle from attaching by active transposition. In this way the basement membrane retained theca cells at the external surface, which formed theca cell layers during culture (Nayudu et al., 1992Go; Spears et al., 1994Go).

Figure 2 illustrates the relationship between FSH supplementation at a critically low dose of 10 mIU/ml (the minimal effective dose) and follicle survival rates in vitro. The first clear indication of the importance of FSH in the culture medium to sustain in vitro follicle growth can be appreciated at observation day 8 of culture (Figure 2B). All cultures that received FSH for the first time on day 6 or later had a significantly lower survival rate (P < 0.01) compared to the positive control. At this point, no significant difference was noticed between FSH administration from day 2 or day 4 and the positive control. Evaluation of the follicle survival rates on day 10 (Figure 2C) shows that follicles growing in either continuous presence of FSH or after FSH addition from day 2 or 4 onwards performed equally well. In the condition where FSH was only added at the start of culture, a reduction in survival rate compared to these cultures became apparent. In all other conditions a stepwise drop in survival rate was related to the later input of FSH supplementation.



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Figure 2. Follicle survival expressed as the percentage of surviving follicles in comparison to the number of follicles at the start of culture. The survival rate at the different days of culture is shown (mean + SD bars). Groups with no letter in common are significantly different from each other (P < 0.01).

 
At day 12 of culture there was a strong relationship between FSH implementation time and follicle survival rate.

Follicle differentiation and morphology under different FSH exposure conditions (Figures 3 and 4).
The influence of FSH on in vitro follicle development is shown by observation of the different experimental conditions. On day 2 of culture, all conditions had theca cell outgrowth to the bottom of the dish, attaching the follicular structures (not shown). At day 4 of culture, no differences were seen between the conditions (not shown). At day 6, it was noticed that the follicles from conditions which had FSH in the medium (positive control, FSH only on day 0, FSH from day 2 and from day 4) showed an outgrowth of the granulosa cells beyond the boundaries of the basal membrane, defined as the ‘diffuse stage’ (not shown). At day 8 of culture (Figure 3A), it became obvious by simple morphological observation that granulosa cell proliferation was directly correlated with FSH supplementation. When FSH was absent in the culture medium or when FSH was added only at the start of culture (i.e. ‘negative control’ and ‘FSH only added once on day 0’), a large percentage of the follicles remained in the ‘follicular stage’ (testifying to a limited granulosa cell proliferation). In all conditions where FSH was present for ≥4 days, a differentiation into the antral stage was observed (Figure 3B). A strong relationship between the period of FSH exposure and antrum formation rate was found. Follicles never exposed to FSH or only at the start of culture (FSH only added on day 0) were poorly developed and a large percentage remained in the early preantral stage. Granulosa cell proliferation was limited and granulosa cell differentiation at day 12 with the development of an antral-like cavity was never observed (Figures 3C and 4).



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Figure 3. The bars represent the average percentages of the surviving follicles at the follicular (F) and the antral-like stage (A). SD bars are indicated above the bars. Percentage of follicles in the transition states follicular/diffused and diffused/antral are not shown in this graph. The average is calculated from seven independent experiments. Both stages are represented for each condition and on several days of replenishing FSH (days 8, 10 and 12). The results from the statistical analyses are represented above the bars. Groups with no letter in common are significantly different from each other; P < 0.01.

 
On day 12 (Figure 3C), the influence of FSH on follicle growth and development is clearly demonstrated. Follicles receiving FSH during their entire growth period (positive control) had left the preantral stage and had reached full differentiation to the pre-ovulatory stage. The different conditions of FSH supplementation demonstrated that to complete follicular development up to the pre-ovulatory stage, FSH needs to be present at least from day 4, i.e. from the late preantral stage. If FSH was only added from day 6, follicles still differentiated but the mural granulosa cell layer was not fully developed and the antral-like cavity was hardly visible, due to the low number of granulosa cells formed during the preantral stage. When FSH was added on day 8, follicles were poorly developed, granulosa cell proliferation was limited and differentiation was not very obvious. If no FSH was added during culture, follicles remained within the preantral stage, with some granulosa cell proliferation but no differentiation. When FSH was only administered at day 0, granulosa cell proliferation was initiated, most follicles left the early preantral stage but granulosa cell coherence was lost with progression of culture.

Conditions became significantly different for the various parameters (follicular, diffuse and antral stage) when FSH supplementation was not present in the medium for ≥4 days.

Steroid production (Figure 5).
Follicles were only able to produce estradiol after addition of FSH to the culture medium. E2 levels on day 4 were still below the limit of sensitivity of the E2 assay (10 ng/l). At day 6, E2 was present in culture medium sampled from positive controls and in the conditioned media where FSH was added from day 2. In all other conditions, E2 remained undetectable. At day 8, all conditions where FSH had been added at the latest on day 6 demonstrated E2 production. Estrogen concentrations were proportional to the duration of FSH exposure. At day 10, E2 had increased in all six conditions containing FSH and was below sensitivity in conditioned medium from the negative control. On day 12, the amount of E2 had increased exponentially except when FSH had been omitted from the culture medium.

Progesterone levels were low (<1 µg/l) until day 12 when the follicles were stimulated by hCG/EGF. Eighteen hours after the ovulation trigger, there was a marked increase in progesterone production in all conditions containing FSH. Figure 6 shows the mean percentage increase of progesterone on day 13: [(progesterone day 13 – progesterone day 12)/progesterone day 12]x100. There is a clear relationship between FSH exposure time and the percentage increase in progesterone.



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Figure 6. Percentage increase in the different conditions (mean + SE bars): [(progesterone day 13 – progesterone day 12)/progesterone day 12]x100.

 
Oogenesis
Meiosis resumption of cultured oocytes.
Surviving follicles (which left the follicular stage) in all conditions with FSH released mucified COC after in vitro ovulation induction. The size of the COC was positively correlated to the observed morphology and granulosa cell proliferation in the different experimental conditions. The largest number of oocytes irreversibly blocked in the germinal vesicle stage was found in the negative control or when FSH was only present at the start of the culture. In all other conditions, 90–100% of the oocytes extruded the first polar body (not shown).

IVF of in vitro-grown and -matured oocytes (Figure 7).
The COC were fertilized in groups of 10. As an internal control for the IVF procedure, we fertilized in vivo-grown oocytes of pregnant mare’s serum gonadotrophin/hCG-treated mice. Not all oocytes that survived the 12 day culture period also survived the IVF procedure (Figure 7). In the positive control, 71% viable oocytes, from the surviving follicles, were retrieved after the stress of the IVF procedure. In the negative control this was only 11%. When FSH was added from day 2, 4, 6 or 8 onwards, respectively, 51, 80, 45 and 45% viable oocytes, from the total number of surviving follicles, were collected after the IVF procedure.



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Figure 7. Results from the IVF and the embryo culture (mean + SD bars). (A) IVF results (scored by the 2-cell stage). (B) Percentage of 2-cell embryos that developed into blastocysts.

 
When no FSH was present during the culture period or when added only once on day 0, few oocytes survived the culture period and were available for scoring fertilization.

In vitro embryo culture (Figure 7).
Surviving follicles in the different FSH exposure groups yielded oocytes with a fertilizing ability averaging between 11 and 65 % (i.e. percentage 2-cell stage). This value is significantly lower than those from in vivo-produced oocytes obtained from superovulated adult controls (91%). After fertilization, from 0 to 50% of the embryos developed to blastocyst, a value that is marginally lower than in the in vivo control group (66%) (Figure 7). Although few oocytes obtained without or with minimal FSH exposure had been fertilized. These were unable to develop further into blastocysts.

Overview of significant results by timed FSH exposure (Figure 8)
Follicle development and oocyte developmental quality from the different experimental conditions is represented percentage-wise in Figure 8. Follicle development combined with a decrease in oocyte quality was found in conditions implementing FSH from day 6 and later, in the ‘negative control’ and the ‘FSH only added on day 0’ group. No statistically significant differences in oocyte and embryonic development were noticed between the positive control and the conditions with FSH supplementation from day 2 or 4 onwards. There was a tendency for highest fertilization and embryonic development rates in the conditions with FSH from day 4. While follicle survival showed a progressive decrease with later FSH implementation, fertilization and embryonic development kept steady unless FSH was administered after day 4.



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Figure 8. Overview of oocyte developmental competence. The numbers above the graph (N) indicate the total number of follicles enclosed in the different conditions.

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In vivo, FSH is essential for normal development of the gonads in hpg mice and for full folliculogenesis in FSH knockout mice. FSH receptor knockout (FORKO) mice can develop preantral follicles up to the early antral stage but not further (Kumar et al., 1997Go; Dierich et al., 1998Go; Burns et al., 2001Go). In in vitro cultures the needs for FSH might be dependent on the culture system, the medium composition, the developmental stage at which the follicles are put in culture and the gonadotrophin pre-treatment of the animal used for follicle harvesting. While many studies dealing with in vitro systems for follicle growth in rodents indicate development-promoting effects of FSH, it is still unclear whether different gonadotrophin exposures might influence oocyte fertilization and capacity for further embryonic development. Our experiments used follicles at a single stage (3b) to study the exact moment at which follicles cultured in an in vitro system for preantral mouse follicles became FSH dependent for their survival, and we determined the effects of timed FSH implementation in relation to final oocyte quality. The follicle culture system used here supports folliculogenesis from the early preantral stage onwards, covering the growth (preantral) and differentiation (antral) phase (Cortvrindt et al., 1996Go). A follicle bioassay from Vitt et al. (1998Go) reported that isoforms of a human rFSH preparation exerted different effects on follicle development. The least acidic isoforms induced fastest preantral growth, producing the largest follicles at the lowest dose. The less- and mid-acidic isoforms had more impact on stimulation of estradiol production and antral formation than the acidic isoforms. The rFSH used in this study had a previously characteristic activity and stable mixture of isoforms (as determined by the manufacturer Serono). It allows the study of proliferation and differentiation of the cultured follicles in a single experimental set-up that was repeated seven times. FSH was used at a final concentration of 10 mIU/ml, which proved previously to be the minimal essential dose in this culture system for obtaining a maximum number of oocytes resuming meiosis (R.Cortvrindt, unpublished observations). Use of the minimal effective dose allowed us to assess eventual effects of a brief exposure to FSH at a moment of follicle development where FSH is not supposed to bring support in follicle survival: at the start of culture, i.e. when follicles are at the early preantral stage 3b.

When FSH was omitted from the culture (negative control), follicle survival at the end of culture was extremely low and normal follicle differentiation was non-existent. Most follicles were kept spherically shaped (‘follicular’) and granulosa cell proliferation was limited. Administration of the minimal effective dose of FSH at the start of culture supported follicle survival to a certain extent, clearly proving the in vitro responsiveness of preantral follicles from juvenile mice to FSH. However, under this condition FSH was only able to induce granulosa cell proliferation, but was not sufficient to induce granulosa cell differentiation at a later point (cf. no antrum formation/no E2 production on day 12). These results show that FSH promotes granulosa cell proliferation in the early preantral phase. This timed study also shows that a minimal essential dose of rFSH is required at least from day 4 to further allow normal folliculogenesis in an equally effective way as in the positive control. No significant differences in follicle development and survival were noticed in the conditions with FSH administration at different time-points during the early preantral stage of follicle development (i.e. in the positive control, FSH from day 2 or day 4). A lower survival rate was noticeable when FSH was only implemented in culture during the late preantral/early antral stage of development (i.e. from day 6 or day 8 of culture). The data presented here confirm that the presence of FSH during the preantral phase could be important to ensure a critical number of granulosa cells to allow final survival of the follicle structure (Herlands and Schultz, 1984Go). Our results are consistent with those of McGee et al. (1997Go), who demonstrated that FSH supplemented with 8-bromo-cGMP in rat preantral follicle cultures significantly augmented the survival rate by suppressing apoptosis. Other investigators have also shown that in mouse follicle cultures, FSH stimulates follicle growth rate in a manner related to the initial follicle diameter and with increasing concentrations of FSH with a maximum of 100 mIU/ml (Nayudu et al., 1992Go; Hartshorne et al., 1994Go). Atresia in small follicles could be prevented by adding FSH to the medium (Hartshorne et al., 1994Go).

During the first 4 days of culture the granulosa cells responded to FSH supplementation with proliferation; the presence of FSH during the second half of culture (from day 6) induced granulosa cell differentiation (e.g. antrum formation and increasing E2 production). This switch in response upon FSH exposure was independent of the number of granulosa cells present (e.g. FSH treatment during the preantral follicle phase). In the cultures which did not receive FSH during the preantral phase, the later addition resulted in immediate differentiation of GC, and, as a result of a lower granulosa cell number, antral-like cavities were less well-defined. Later implementation rendered follicles more fragile. When FSH was only added during the antral stage, follicle survival was significantly lower than in the other FSH-containing conditions and only very few follicles could develop an antral-like cavity, showing that FSH promotes follicle survival during the preantral stage.

In this experiment, support of LH-dependent action on follicle growth and differentiation was excluded by working with a recombinant product and a gonadotrophin-free serum supplement (FCS at 5%). Estradiol production was lower than in previously published data using the same in vitro culture system (Cortvrindt et al., 1996Go, 1997). This difference in E2 production is due to the lower rFSH concentration (10 versus 100 mIU/ml) used in the present experiments, which demonstrates the relationship between FSH, induction of aromatase and E2 output. This relationship between E2 and FSH has also been demonstrated during in vitro culture of sheep preantral follicles (Cecconi et al., 1999Go).

Production of estradiol in an LH-free and androgen-free culture medium could be explained by the fact that a constitutive androgen production by the theca cells in this culture system provided sufficient substrate for a continuous estradiol production. Preantral follicles produce paracrine theca cell-differentiating factors that promote androgen production by an LH-independent mechanism (Magoffin and Magarelli, 1995Go). Recent data in rat ovarian theca cells suggest that insulin-like growth factor-I (IGF-I) and stem cell factor (SCF) might be important components of the physiological theca-differentiating signal (Huang et al., 2001Go). Neither IGF-I nor SCF alone can mimic the effects of follicle-conditioned medium on theca cells. However, a combination of both factors in physiological concentrations was able to reproduce theca differentiation and androgen production.

Oestrogen production in our culture only became detectable after adding FSH to the medium. Although follicles could be rescued even though FSH was added as late as from day 8 of culture, E2 production was entirely dependent on FSH supplementation. It was demonstrated that upon binding of FSH to its G-protein-coupled receptor in the granulosa cell membrane, intracellular cAMP levels rise (Michael et al., 1995Go, 1997). This, in turn, will enhance the binding of steroidogenic factor-1 (SF-1) and cAMP response element binding protein (CREB)—two critical transcription factors—to the promoter II of the aromatase gene. This trigger will result in the activation of aromatase expression in the granulosa cells and ensure conversion of androgens (produced by the theca cells) to estrogens. The proportional production of E2 to the duration of FSH exposure can be explained by the correlation between the time of FSH exposure and number of GC.

This culture system used hCG to trigger final oocyte maturation and luteinization. Expression of LH receptors and their ability to respond to stimulation was indirectly measured by challenging the cultured follicles with hCG stimulation. Eighteen hours after the ‘ovulation’ trigger, progesterone levels were found dramatically increased in all cultures exposed to FSH, proving that these conditions supported LH receptor expression. The granulosa cells are converted from predominantly estrogen to predominantly progesterone synthesizing cells. This conversion will up-regulate synthesis of LH receptors, resulting in an augmented progesterone level and a down-regulation of the affinity of granulosa cells for E2 and FSH.

Like estrogen production, progesterone production was also proportional to the duration of previous FSH exposure. However, the proportionally decreased progesterone concentrations observed in the groups with the latest introduction of FSH in culture could be explained by the lower final cell number in these follicles.

The results concerning antral formation, E2 and progesterone production (as a reflection of LH-receptor expression) clearly demonstrate that the presence of FSH during the preantral stage is beneficial for normal follicle differentiation.

Mucification of the COC in response to the ovulatory stimulus was noticed in all conditions except in the condition ‘FSH only added on day 0’. A large number of COC from the other conditions, i.e. the negative control and FSH only once on day 0, did not mucify after stimulation by hCG/EGF. Poor mucification is a direct reflection of poor oocyte quality in these conditions. Completion of nuclear maturation was observed in most of the follicles cultured in the presence of FSH. When FSH was not present or only added on day 0, surviving oocytes rarely extruded the first polar body (PB): most oocytes remained blocked in the germinal vesicle stage. The lack of mucification and the poor meiotic maturation rate is the reflection of an inadequate oocyte cytoplasmic maturation by absence or lack of FSH. In the conditions with FSH added at the latest on day 8, all surviving follicles responded to the hCG stimulus by forming an expanded COC. Nearly all oocytes enclosed in these complexes reached complete nuclear maturation (metaphase II).

Experiments described in the present paper yielded high PB extrusion rates (90–100%) in oocytes exposed to FSH before day 6 of culture. Such an extremely high meiotic maturation rate using a minimal effective FSH concentration has not been reported so far by any other long-term in vitro culture system of early preantral follicles (Hartshorne et al., 1994Go).

Oocyte quality in this culture system was studied by performing IVF and blastocyst culture. The IVF results from cultured oocytes were not as good as from in vivo-grown oocytes. The lower IVF results could be due to a complete absence of any LH in this culture set-up. Westergaard et al. (2000Go) suggested that low concentrations of LH have a detrimental effect on the outcome of IVF and that circulating LH concentrations above a certain critical level are required for optimal oocyte maturation. Biochemical compounds or physiological factors normally provided during in vivo growth and lacking in this culture system could be responsible for a lower cytoplasmic maturation. Mature oocytes from different FSH exposures were subjected to fertilization experiments. The loss of oocytes during the manipulation of the IVF procedure was mostly due to oocyte degeneration during the period of incubation with sperm cells (2.5–3 h). This degeneration seems to be linked to oocyte quality. The loss was most pronounced in cultures without FSH or when FSH was only briefly present during follicle culture, i.e. the negative control, FSH only added once on day 0 and FSH added from day 8 onwards. Lack of sufficient FSH exposure led to oocyte fragility. Follicle survival rates improved in relation to the earlier onset of exposure to FSH in the preantral growth phase. There was no increase in embryonic quality proportional to follicle survival with an earlier onset of FSH exposure than on day 4. Postponing FSH supplements beyond day 4 clearly decreased follicle yield and proportionally decreased oocyte competence. Eppig et al. (1997Go) reported that when early preantral follicles were cultured in groups in the presence of 5% fetal calf serum, FSH addition to medium containing insulin promoted an inappropriate differentiation of granulosa cells leading to compromised blastocyst formation rate. Results from a completely different culture set-up in this study emphasizes that FSH is an important developmental factor for early follicles enabling competence for further development. FSH ensures robustness and developmental quality of the oocyte when implemented rather too early than too late.

In conclusion, a minimal effective dose of FSH plays a key role during follicle differentiation and prolonged presence of this gonadotrophin is required to provide acceptable rates of robust oocytes for fertilization purposes. Fertilization and blastocyst development rates were not statistically significantly different for FSH exposures during the preantral stages. Omission of FSH during the early preantral stage in this culture system tends to compromise a maximal oocyte developmental competence.


    Acknowledgements
 
Lotte Arentoft and Isabelle Hellinckx are thanked for their technical help in the laboratory. This research was funded by the IWT (International Scientific and Technological Cooperation; Grant no. 980343) and by the LINK (Grant no. BRGEOZ15). The authors thank Serono International, Geneva, Switzerland for the gift of recombinant gonadotrophin.



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Figure 4. The morphological appearance of representative follicles out of each experimental condition on day 12 of culture (magnification x400).

 


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Figure 5. Steroid measurements in the conditioned medium of the different FSH exposure regimens collected on the different days of culture replenishment.

 

    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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Submitted on July 11, 2003; accepted on October 15, 2003.