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Gamete Biology |
Research Laboratory on Human Reproduction and Fertility Clinic3
Laboratory of Chemistry,4 Erasme Hospital, French Speaking Free University of Brussels, 1070 Brussels, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, follicle, in vitro fertilization, insulin-like growth factor receptor, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The involvement of IGF-I in follicular development is suggested by the presence of IGF-I receptor and IGF-I mRNA in the ovary. In mice, low IGF-I mRNA levels were detected in primary follicles, but transcription increased to maximum during the late preantral and early antral stages [1, 2]. Studies on IGF-I knockout mice further demonstrated the inability of the follicles to reach the antral stage, inducing infertility because of lack of ovulation [3], confirming the essential role of IGF-I during folliculogenesis.
The IGF-I system has also been linked to the follicular survival capacity as suggested by the inverse correlation between the level of IGF-I mRNA and follicular health [2, 4]. The action of IGF-I is controlled by the secretion of binding proteins (IGFBPs) by granulosa cells. IGFBPs promote follicular atresia by the sequestration of IGF-I. The incapacity of IGF-I to interact with its receptor allows the activation of the Fas antigen-mediated cell death in the follicles through apoptotic mechanisms [5]. Transcription of IGFBPs usually precedes the decrease of IGF-I expression in the atretic follicles [2, 6].
The effect of IGF-I on folliculogenesis during in vitro culture was previously studied by using follicular cells from antral follicles. IGF-I was shown to stimulate the proliferation and steroidogenesis of granulosa cells cultured in vitro in different species [7–9]. These findings also support evidence that IGF-I acts in synergy with FSH in the induction of granulosa cell development by the regulation of FSH receptor expression. Moreover, FSH was reported to increase IGF-I receptors in granulosa cells cultures [10, 11], suggesting that some effects of FSH could be mediated via IGF-I [7].
When added during in vitro culture of preantral follicles, IGF-I has been shown to stimulate follicular growth in human [12], bovine [13], rat [14], and mouse [15] in synergy with FSH. In the rat, IGF-I added during in vitro preantral follicular culture increased significantly the follicular diameter and DNA content. Observation of these follicles using a transmission electron microscope showed a well-developed endoplasmic reticulum and mitochondria in the granulosa cells. The microvilli between the oocyte and the granulosa cells and the gap junctions between follicular cells were also increased when the follicles were cultured in the presence of IGF-I, suggesting that IGF-I promotes the functional integrity of the follicles [14].
However, information about the effects of IGF-I during preantral follicular growth on subsequent oocyte maturation, fertilization, and embryo development is not yet available. To answer this question, we modified a previously described in vitro preantral culture system in mice [16]. The present study used a simplified culture medium to avoid, as much as possible, interactions between supplemented IGF-I, and other medium components.
First, insulin, which is a classical supplement of follicular culture media, was not added to the medium: supraphysiological concentrations of insulin, as provided by commercial insulin-transferrin-selenium (ITS) supplements, may indeed interact with the IGF-I system [17, 18]. Moreover, previous experiments showed that the presence of insulin was not essential to support follicular growth [19, 20].
Second, the concentration of fetal calf serum (FCS) was reduced. Previous data suggested that IGF-I is the factor in serum-containing media that blocks Fas-mediated apoptosis [21]. In in vitro cultures of pig granulosa cells with media supplemented with 1% serum, the proportion of apoptotic cells decreased in the presence of IGF-I to about 50% and the presence of FSH in the medium did not modify the proportion of apoptotic cells [9]. These results suggested that the antiapoptotic effect of IGF-I was easily detectable when the medium was supplemented with 1% serum. We used a concentration of 1% FCS in the follicular culture medium, providing less than 1 ng/ml of IGF-I [22].
Using this modified culture medium, experiments were designed to investigate the effect of IGF-I on preantral follicular growth, steroidogenesis, and oocyte maturation and to evaluate the developmental capacity and the quality of the embryos obtained after in vitro fertilization of in vitro– grown oocytes cultured in the presence of IGF-I.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sigma Aldrich Co. (Bornem, Belgium) supplied all chemicals with the exception of those noted here. The 
-minimal essential medium GlutaMAX (-MEM), the Leibovitz L15 medium, and the FCS were purchased from Life Technologies (Merelbeke, Belgium). Sodium selenite and epidermal growth factor (EGF) were purchased from ICN (Asse Relegem, Belgium) and Boehringer Mannheim (Brussels, Belgium), respectively. Recombinant human FSH (r-FSH) and recombinant hCG (r-hCG) were kindly provided by Organon (Oss, The Netherlands) and Serono (Geneva, Switzerland), respectively.
Animals
All experiments were conducted with female hybrid (C57bl/8j x CBA/ ca) mice housed and bred in a temperature- and light-controlled room and provided food and water ad libitum. This study obtained the agreement of the Local Animal Ethics Committee.
Preantral Follicle Isolation and Culture
For all experiments, two mice from 12 to 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 L15 medium (with l-glutamine) supplemented with FCS, penicillin, and streptomycin at 37°C as described previously [16].
For each mouse, both ovaries were dissected mechanically using a 26- gauge needle. Only intact preantral follicles, with diameters between 100 and 130 µm and characterized by at least one complete granulosa cell layer and a visible, centrally located oocyte were selected for culture.
The culture medium was composed of MEM Glutamax supplemented with 100 mIU/ml r-FSH, 1% FCS, 5 µg/ml transferrin, and 5 ng/ml selenium. Each selected follicle was rinsed and transferred in groups of five follicles in 96-well tissue culture plates (Nunc, Merks Eurolab, Leuven, Belgium) containing 100 µl of culture medium under 85 µl of mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air. After 2 days of culture, 100 µl of fresh medium was added in each well. Every other 2 days until Day 12 of culture, 100 µl of medium was collected and replaced by fresh medium. The collected culture media from the wells containing five surviving follicles were pooled and stored at –20°C until hormonal assays.
Hormonal Assays
17ß-estradiol and progesterone were assayed using electrochemiluminescence immunoassays (Roche-Mannheim, Germany) that were validated as described previously [23].
Oocyte Maturation
After 12 days of culture, oocyte maturation was induced by replacing the culture medium with the maturation medium. The maturation medium consisted of MEM-Glutamax supplemented with 5% FCS, 100 mIU/ml r-FSH, 1.5 IU/ml r-hCG, and 5 ng/ml EGF and ITS (insulin 5 µg/ml, transferrin 5 µg/ml, selenium 5 ng/ml). Eighteen hours post r-hCG/EGF, oocyte-cumulus-complexes (OCCs) were collected and either mechanically denuded to evaluate the oocyte nuclear maturation stage or in vitro fertilized.
In Vitro Fertilization
OCCs collected 18 h post r-hCG/EGF were rinsed and transferred in 500-µl drops of modified simplex optimized medium with potassium (KSOM) [24] supplemented with 4 mg/ml BSA and 5.56 mM glucose. After induction of capacitation for 2 h in Whittingham medium [16] supplemented with 30 mg/ml BSA, epididymal sperm from 3- to 6-mo-old hybrid mice was added to the OCC drops to give a final motile sperm concentration of 1–2 x 106/ml. After 3
h, fertilized oocytes were washed three times in modified KSOM medium supplemented with 1 mg/ml BSA and 0.2 mM glucose. The embryos were further cultured for 120 h in 30- µl droplets of the same culture medium under oil (five to six embryos per droplet) in an incubator at 37°C and 5% CO2 in air.
Differential Labeling of Blastocysts
Expanded blastocysts obtained after 120 h of embryo culture were stained according to the differential labeling technique [24]. The embryos were washed in M2 medium supplemented with 4 mg/ml BSA (M2+BSA). The zona pellucida was removed by incubating the blastocysts in acid Tyrode solution (pH 2.0–2.4). The zona-free embryos were washed and transferred for 15 min in rabbit anti-mouse serum diluted 1: 10 before thorough washing in M2+BSA. Embryos were then incubated 15 min at 37°C in guinea pig complement serum dilution 1:10 and containing 0.01 mg/ml propidium iodide to specifically label trophectoderm (TE) nuclei. Embryos were observed for antibody-mediated complement lysis and fixed in 0.05 mM bisbenzemide in absolute alcohol for at least 48 h at 4°C. This fluorochrome labeled both inner cell mass cells (ICMs) and TE nuclei.
Labeled embryos were washed in absolute alcohol before examination. Differentially labeled embryos were mounted in glycerol and nuclei were counted under fluorescence microscope after gentle disaggregation of the embryo. The ICM nuclei appear green and the TE cell nuclei appear orange. The number of dead cells was estimated on the basis of the presence of apoptotic nuclei characterized by discrete clusters of nuclear fragments. The dead cell index was defined as follows:
Effect of Different Concentrations of IGF-I on Follicular Steroidogenesis and Oocyte Maturation
In this experiment, IGF-I was added to the culture medium during the whole culture period to define the optimal IGF-I concentration regarding the follicular steroidogenesis and the oocyte maturation rate. For each experiment, preantral follicles were selected and cultured in medium supplemented with different concentrations of IGF-I: control without IGF-I (n = 150); IGF-I 10 ng/ml (n = 150); IGF-I 50 ng/ml (n = 150) or IGF-I 100 ng/ml (n = 145). IGF-I was also added in the maturation medium at the same concentrations. Every 48 h, collected media were pooled in each group (30 follicles per group) and assayed for estradiol and progesterone as previously described [23]. After in vitro maturation, OCCs were collected and denuded to record nuclear maturation stage. This experiment was repeated five times.
Effect of IGF-I Added at Different Times of Follicular Culture on Subsequent Oocyte Fertilization Rate and Embryo Development
For each experiment, 120 follicles were selected and cultured according to four different conditions (30 follicles per group): control group without IGF-I (control); group exposed to IGF-I 50 ng/ml from Day 0 to Day 12 of culture and during final oocyte maturation (fully exposed); group exposed to IGF-I 50 ng/ml from Day 0 to Day 12 of culture and not during final oocyte maturation (D0–D12); group exposed to IGF-I 50 ng/ml only during maturation and not during follicular growth (maturation). The OCCs collected after in vitro maturation were fertilized and the blastocysts obtained were stained for differential labeling after 5 days of culture. Degenerated oocytes were excluded from embryo culture. This experiment was repeated eight times.
Statistical Analysis
The proportion of matured and fertilized oocytes and of embryos reaching the blastocyst stage was compared using chi-square analysis. Differences in follicular hormonal production between groups and according to the duration of the culture were analyzed by one-way ANOVA followed by the Mann-Whitney U-test. The differences between distributions of cell numbers in the TE and ICM lineages were also analyzed by the Mann- Whitney U-test. Only P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preantral follicles were cultured and matured in the presence of four different concentrations of IGF-I (control without IGF-I; IGF-I 10 ng/ml; IGF-I 50 ng/ml; or IGF-I 100ng/ ml).
The follicular estradiol secretion per 48 h increased in the presence of IGF-I (Fig. 1, A and B). Estradiol levels were significantly higher in the culture media from follicles cultured in the presence of IGF-I than in the control group at the beginning of the culture (Day 6). Follicular estradiol secretion was comparable in the different groups cultured in the presence of IGF-I (10, 50, or 100 ng/ml), except at Day 4 in which estradiol secretion was higher when the follicles were cultured in the presence of 50 ng/ml IGF-I than when the follicles were cultured in the presence of 100 ng/ml IGF-I (Fig. 1, A and B).
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Follicular progesterone secretion after oocyte maturation was higher in the presence of IGF-I (8.8 ng/ml ± 0.63, 13.28 ng/ml ± 4.01, 12.72 ng/ml ± 1.42, and 10.88 ng/ml ± 2.1 for 0, 10, 50, and 100 ng/ml IGF-I, respectively, at Day 13). However, the difference with the control group reached significance only for the group cultured with 50 ng/ml IGF-I.
The total number of OCCs collected at 18 h after hCG was comparable between groups (Table 1). The rate of oocytes at the metaphase II and germinal vesicle breakdown stages varied from 56% to 69% and 15.1% to 24.8 %, respectively, with no significant differences among groups (Table 1). The percentages of oocytes at the germinal vesicle stage were not statistically different among the groups cultured in the presence of IGF-I (Table 1). The control group presented a significantly lower rate of germinal vesicle stage oocytes, compared with the group of follicles cultured in the presence of 10 ng/ml IGF-I (12.7% and 23.5%, respectively).
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Effect of IGF-I Added at Different Times of Follicular Culture on Subsequent Oocyte Fertilization Rate and Embryo Development
This experiment was carried out to evaluate the effect of IGF-I (50 ng/ml) added at different times of preantral follicular culture and oocyte maturation on subsequent fertilization and embryo development.
The number of degenerated oocytes, observed after in vitro fertilization and defined by a broken zona pellucida or a dark shrunken cytoplasm, was reduced when follicles had been exposed to IGF-I during the whole culture and oocyte maturation period (fully exposed), compared with the group of follicles not exposed to IGF-I (control) or exposed to IGF-I during oocyte maturation period only (maturation; Table 2).
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The percentage of two-cell stage embryos obtained 24 h after in vitro fertilization of the OCCs collected at Day 12 was comparable in the different groups whether exposed or not to IGF-I (Table 2).
However, the blastocyst development rate after 120 h of embryo culture assessed by the blastocyst/two-cell stage embryos was enhanced in the group exposed to IGF-I during the whole follicular culture period (D0–D12), compared with the control group (Table 2): 40% of the two-cell stage embryos reached the blastocyst stage 120 h post hCG in the group in which the follicles were cultured in the presence of IGF-I (D0–D12), compared with 21.7% in the control group. A similar trend was observed when IGF-I was added during follicular culture and oocyte maturation (fully exposed) or when it was added during final oocyte maturation only (maturation) but the blastocyst:two-cell stage embryos ratios did not statistically differ from the control group. After 140 h of culture, the blastocyst/two-cell stage rate did not statistically differ among groups (Table 2).
Differential Labeling of the Blastocysts
The blastocysts were stained using differential labeling after 5 days of embryo culture (140 h post hCG). Nineteen percent of the blastocysts were only partially differentially labeled with some TE nuclei being labeled with only bisbenzemide. In these embryos, only the total cell number was considered. The presence of IGF-I increased the total cell number by 41.6%, 43.2%, and 37%, respectively, in the fully exposed group (fully exposed), the group exposed during the follicular culture period only (D0–D12), and the group exposed during the final maturation period only (maturation), compared with the control group (Table 3). This increase in the total cell number was significantly higher in the group exposed to IGF-I during the follicular culture period only (D0–D12) and the group exposed to IGF-I during the final maturation only (maturation) compared to the control group. Regarding the two different cell types, the ICM:TE ratios were of 0.66, 0.77, 0.56 and 0.57 respectively in the control group, the fully exposed group, the group exposed during follicular culture period only (D0– D12) and the group exposed during the final maturation only (maturation). The differences between those ratios were not statistically significant. No difference was observed in the dead cell index among groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our culture conditions, IGF-I had a stimulatory effect on follicular steroidogenesis. A stimulatory effect of IGF-I on granulosa cell proliferation in follicular culture was previously reported in different species [13, 14, 25]. In mouse preantral follicular cultures, a significant increase in follicular diameter was seen when IGF-I was added to the culture medium together with FSH. Moreover, a synergistic effect of IGF-I and FSH was also observed on the follicular secretion of inhibin [15].
The stimulating effect of IGF-I on estradiol secretion was previously reported in bovine granulosa cell cultures [26]. In the rat, the presence of IGF-I during granulosa cell culture potentiated the action of FSH on cholesterol side- chain cleavage cytochrome P450 expression, catalyzing the conversion of cholesterol to pregnenolone [27].
Follicular progesterone levels produced after oocyte in vitro maturation were also significantly higher in the presence of 50 ng/ml of IGF-I, compared with the control group. In other species, IGF-I was shown to enhance progesterone production by granulosa cells in culture, but the effect was absent or limited on OCCs or ovarian fragments [10, 28]. In the mouse, however, treatment of cultured granulosa cells as well as cultured whole ovarian fragments with FSH or IGF-I produced an increment in progesterone accumulation, and the cotreatment with IGF-I and FSH resulted in the amplification of FSH hormonal action [1].
When higher doses of IGF-I (100 ng/ml) were added during preantral follicular culture, no additional benefit was observed on steroidogenesis. These results suggest a saturation effect of IGF-I for a dose greater than 50 ng/ml in the presence of FSH. This observation is in accordance with the findings of Zhao et al [14], who demonstrated that IGF- I, in the presence of FSH, induced a significant increase in follicular diameter when it was added during in vitro culture of rat preantral follicles at concentrations of 1 and 10 ng/ml, whereas a concentration of 100 ng/ml had no additional effect. Other previous studies showed that the rate of apoptosis in porcine granulosa cells during in vitro culture decreased in the presence of IGF-I or FSH [9]. However, the antiapoptotic effect of IGF-I was shown to be maximal at the dose of 50 ng/ml, and no additional effect was observed between 50 and 250 ng/ml [9]. Moreover, the addition of FSH and IGF-I to the culture medium induced an increase in the progesterone production by cultured granulosa cells, reaching a plateau at 50 ng/ml of IGF-I [9]. The absence of dose-dependent effects of IGF-I seems to be related to the presence of FSH. Previous experiments on bovine granulosa cells in culture demonstrated a dose-dependent effect of IGF-I on the secretion of various hormones (estradiol, inhibin A, activin A, progesterone) in the absence of FSH, whereas in the presence of FSH, the stimulation of hormone production decreased when high doses of IGF-I were added to the culture medium [26]. The mechanism of suppression of the dose-dependent stimulation effect of IGF-I in in vitro follicular culture in the presence of FSH is not clearly understood. One of the hypotheses is a precocious luteinization of the granulosa cells induced by an increase of FSH action by IGF-I and associated with a decline of aromatase activity. Early differentiation of the granulosa cells may result in retardation of growth and proliferation as suggested previously [29], inducing a decrease of progesterone production in response to hCG.
In our study, the total number of OCCs collected after oocyte maturation was unchanged in the presence of IGF- I. However, the number of degenerated oocytes observed after OCC fertilization was reduced when follicles were both cultured and matured in the presence of IGF-I, suggesting that besides a stimulating effect on the follicular steroidogenesis, IGF-I could play a part in oocyte survival. This could be mediated through the granulosa cells, possibly by increasing the density of gap junctions essential for the functional integrity of the follicles [14].
In different species, IGF-I was shown to improve nuclear maturation in oocytes surrounded by compact cumulus cells [28, 30, 31]. However, no improvement of nuclear maturation rate by IGF-I was observed in mouse [32] or sheep [33]. In our study, no difference was observed in the oocyte maturation rate whether or not the medium contained IGF- I. It was shown previously that factors enhancing follicular steroidogenesis did not necessarily have an impact on further oocyte maturation [23].
When added to the in vitro oocyte maturation medium, IGF-I was reported to enhance embryonic cleavage rate and development in pig and bovine [10, 28]. In the mouse and the buffalo, the addition of IGF-I to the embryo culture induced an increase in cell number in the produced blastocyst [34, 35]. The effect of IGF-I during in vitro follicular growth on subsequent embryo development had not been studied so far. In the present study, the embryo development rate was slower than the one observed in another study [36] also using in vitro grown and matured oocytes. This could be related to the follicular culture conditions: we used a simplified medium (with 1% FCS and no insulin) that could explain the lower developmental rate. We observed comparable embryo cleavage rates whether the follicles were cultured, matured, or both, with or without IGF- I. However, an acceleration of embryonic development was observed when IGF-I was added during the follicular growth, compared with the control group. The cumulative effect of IGF-I added during both follicular growth and oocyte maturation (fully exposed) had no additional benefit, compared with IGF-I added during follicular growth only (D0–D12).
The possibility that the effect of IGF-I during maturation could be modified by interfering with the maturation medium because of the higher concentration of serum or the presence of insulin must be considered. However, preliminary experiments showed that the oocyte maturation rate was dramatically reduced when a simplified culture medium supplemented with hCG/EGF was used as maturation medium (data not shown).
The total cell number of the blastocysts was also significantly enhanced after the follicular culture (D0–D12) or after the oocyte maturation (maturation) in the presence of IGF-I. The total number of cells per blastocyst was also higher when IGF-I was added to the culture medium during both follicular growth and oocyte maturation (fully exposed), compared with the control group, but the difference did not reach statistical significance.
In mice, the presence of a well-defined ICM was correlated with embryo viability post transfer, and an excessive reduction in either lineage diminishes the embryonic developmental potential [24]. However, it would be interesting to confirm the implantation potential of the embryos obtained after follicular culture in our conditions by analyzing their ability to develop to term after replacement in recipient mice.
In conclusion, the present study reports the effects of IGF-I during culture of mouse preantral follicles on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in a well-known culture system but using a simplified culture medium. IGF-I had a stimulatory effect on follicular steroidogenesis, without any effect on the oocyte maturation rate. However, the embryo development and the blastocyst cell numbers were enhanced when the follicles were cultured in the presence of IGF-I.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: I. Demeestere, Research Laboratory on Human Reproduction, Erasme Hospital, 808 Route de Lennik, 1070 Brussels, Belgium. FAX: 0032 2 5554520; idemeest{at}ulb.ac.be
Received: 17 September 2003.
First decision: 7 October 2003.
Accepted: 30 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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