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a Dipartimento di Scienze e Tecnologie Biomediche, 67100 L'Aquila, Italy
b Istituto di Fisiologia Veterinaria, Facoltà di Medicina Veterinaria, 64020 Teramo, Italy
c Dipartimento di Sistemi per Economia, 67100 L'Aquila, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By contrast, the complete development in vitro of preantral follicles obtained from human or domestic animal ovaries is more difficult to achieve. This difficulty is especially due to the greater follicle dimension and to the presence of a thick theca, which would restrict the adequate supply of nutrients as well as gas and waste product exchange during the long-term culture period required for follicle culture.
The first attempt to isolate and culture human preantral follicles was performed by Roy and Treacy [5]. These authors isolated preantral follicles by enzymatic digestion of premenopausal ovaries and cultured some of them until they reached the antral stage. Preantral bovine follicles isolated by an enzymatic [6] or by a mixed enzymatic/mechanical procedure [7] were also cultured in vitro. In these studies, as for other mammals [8], the presence of FSH seemed essential for normal growth of in vitro-cultured bovine preantral follicles, as well as for the maintenance of a physiological pattern of estradiol secretion. However, from the results presented by Wandji and coworkers [6], it was evident that the dissociation technique adopted to isolate bovine preantral follicles was inadequate, since it adversely affected culture outcome, as demonstrated by the degeneration of most of the follicle-enclosed oocytes at the end of the culture period.
The oxygen concentration used during the in vitro culture of preantral follicles represents another important parameter in determining proper culture conditions. However, the effects exerted by high- or low-gas atmospheres on mammalian oocyte growth are controversial. As far as mouse oocytes are concerned, Eppig and colleagues [9] reported that in 5% O2, oocyte developmental competence was promoted, while Smitz and coworkers [10] found that this specific O2 concentration caused an unphysiological development of cultured preantral follicles, as evidenced by a reduced follicular growth rate and by oocyte degeneration.
On the basis of these results, the aim of the present study was to identify a culture system capable of supporting in vitro development of preantral follicles mechanically isolated from the ovaries of prepubertal sheep. To this end, we investigated the effects of FSH and of two different oxygen concentrations on the growth, antral cavity formation, and estradiol release of singly cultured preantral follicles. In addition, the effect of oxygen concentration on the acquisition of oocyte meiotic competence was evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the chemicals used in this study were purchased from Sigma (Sigma Chemical Co., St. Louis, MO), unless otherwise indicated.
Animals
Ovaries from prepubertal sheep were collected within 15 min of slaughter, freed from ligaments, and rinsed several times in PBS supplemented with antibiotics (75 mg/L penicillin-G, 50 mg/L streptomycin sulfate). Ovaries were kept at 37°C during transportation from the slaughterhouse to the laboratory.
Isolation and In Vitro Culture of Preantral Follicles
After medulla removal, ovaries were transferred in Hepes-buffered Minimum Essential Medium Eagle (MEM) supplemented with antibiotics (75 mg/L penicillin-G, 50 mg/L streptomycin sulfate), and cut into small pieces using a surgical blade. Intact preantral follicles were mechanically isolated from the cortex of the ovaries, with a clump of stromal tissue attached, and sorted on the basis of their diameters into three classes: small (mean diameter: 130 ± 10 µm), medium (185 ± 14 µm), and large (250 ± 10 µm) follicles. Preantral oocyte and follicle diameters were measured by using an ocular micrometer (x40 magnification) inserted into an inverted-phase microscope. Follicles were individually transferred to 96-V-well microtiter plates (Greiner Labortechnik, Frickenhausen, Germany). In each well containing 25 µl MEM alpha modification (
MEM) supplemented with 10% fetal calf serum (FCS), 1% ITS (insulin, transferrin, and selenium; I1884 Sigma), and antibiotics (75 mg/L penicillin-G, 50 mg/L streptomycin sulfate), follicles were seeded onto 75 µl pre-equilibrated mineral oil (density = 0.84 g/ml) and then overlaid by 25 µl oil [11]. Increasing porcine FSH (pFSH, biological potency = 7 U/mg; F2293; Sigma) doses, ranging from 0.01 to 1 µg/ml, were used to culture preantral follicles under 5% O2 (gas mixture: O2, 5% CO2, 90% N2) or 20% O2 (gas mixture: 20% O2, 5% CO2, balance air) at 38.5°C for 6 days. Preliminary experiments demonstrated that under the above-described culture conditions, a period longer than 6 days had a deleterious effect on follicle cell survival. Culture media were always pre-equilibrated with 5% or 20% O2, depending on experimental procedures, and replaced every other day. From each follicle well, 15-µl samples were taken and stored at -70°C for estradiol determination.
Morphological Evaluation of Follicle Development
By the end of culture, final follicle diameters were recorded together with the presence or absence of an antral cavity, which was defined as a visible translucent area within the granulosa cell mass comprising about half of the follicle. Follicles were scored for their morphological aspect, and those showing morphological signs of degeneration (i.e., darkness of oocytes and surrounding cumulus cells, confirmed by the trypan blue dye exclusion test), or those with misshapen oocytes were discarded. Follicle growth was expressed as increase in diameter (mean ± SE), or as relative volumetric increase (RVI) (mean ± SE), using the formula (Vf - Vi)/Vi, in which Vi and Vf indicated the initial and the final follicle volumes, respectively. RVI values were then sorted into 4 different classes indicating 1) absent (-V; RVI 
0.8), 2) low (+V; RVI 0.81–5.0), 3) medium (++V; RVI 5.1–10), and 4) maximum (+++V; RVI > 10) increase in volume.
The diameters of oocytes obtained from in vitro-grown (IVG) follicles as well as from freshly collected early antral and preovulatory follicles were also recorded.
In Vitro Maturation of IVG Oocytes
At the end of culture, IVG antral follicles were carefully opened, and the released cumulus-oocyte complexes (COCs) were evaluated for their morphological aspect. COCs presenting continuous and compact layers of cumulus cells and corresponding oocytes, observed after cumulus cell removal, without signs of cytoplasmic degeneration were classified as healthy. Oocyte meiotic competence was assessed by in vitro maturation of COCs derived from preantral follicles that had been cultured in the presence of 1 µg/ml FSH under both oxygen concentrations. To this end, COCs were transferred to drops of Medium 199 supplemented with 10% FCS, 1 µg/ml FSH, and 1 µg/ml porcine LH (USDA-pLH-B-1; LH potency = 2.3 U/mg; NIDDK-NIH Bethesda, MD) under oil, and incubated for 24 h under 5% CO2 in air. At the end of the maturation period, oocytes were fixed in acetic acid:ethanol (1:3), and the assessment of chromatin configuration was performed after lacmoid staining.
Estradiol Determination
The levels of estradiol-17ß (E2) secreted by preantral follicles during a 6-day culture period were determined. E2 released in culture media was measured immunoenzymatically by means of Biopharm Ridascreen (Darmstadt, Germany) assay kits for E2. The sensitivity of the assay, defined as the amount of steroid giving a 10% drop in the binding of the enzyme-conjugated estradiol, was 30 ng/L. The recovery of known amounts of estradiol (200–2000 ng/L) added to samples of culture medium was 87.1 ± 2.2% (mean ± SE of 8 determinations). Intraassay and interassay precision, expressed as the coefficient of variation for replicate determinations of a pool of medium samples, was 3.8% (10 replicates) and 5.7% (8 assays), respectively. The cross-reactions of anti-estradiol antibody were as follows: E2, 100.0%; estradiol-3-benzoate, 50.0%; estradiol-17, 0.9%; estrone, 0.7%; testosterone < 0.25%; estriol, < 0.1%; androstenedione, < 0.1%.
Statistical Analysis
A total number of 568 preantral follicles were analyzed in 4 independent experiments. Comparisons between oocyte diameters were made using t-tests. Estradiol data were analyzed by two-tailed t-tests. The maturational competence of IVG oocytes was assessed in 3 independent experiments, and differences in the proportions were compared by the chi-square test; p < 0.05 was considered as the limit of significance. Statistical analyses used to evaluate preantral follicle development were multiple correspondence analysis (SPSS-CATEGORIES) [12], and segmentation analysis (SPSS-CHAID). By correspondence analysis, it was possible to represent multidimensional information (categorical variables) in a low-dimensional approximation. Output included a plot of the category quantifications, which gave insight into the relationships between the variables in the analysis. The category quantifications were centroids of the objects that shared that category. Segmentation analysis created classification systems displayed in decision trees. If data were divided into classes, this analysis used them to build rules that could be used to classify cases with maximum accuracy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the roles exerted by FSH and by 5% or 20% O2 concentrations on sheep preantral follicle growth, the three classes of preantral follicles (small, medium, and large; Fig. 1A) were cultured in the absence (control) or in the presence of increasing FSH concentrations, ranging from 0.01 to 1 µg/ml. Cultures were carried out in an atmosphere of either 20% or 5% O2 for 6 days. Under either oxygen concentration, little or no increase in final diameter was observed for any of the classes of preantral follicles cultured as control, even if no morphological signs of cell degeneration were evident at the end of the culture period. In comparison with these conditions, FSH stimulated follicle growth at all concentrations tested. A slight improvement of follicle growth was observed at 1 µg/ml FSH (Tables 1 and 2; Fig. 2). Among the size classes considered, the class of small preantral follicles showed the highest increase compared with the other follicular classes (Fig. 3). The effect of 5% or 20% O2 atmosphere on follicle growth is reported in Figure 4. From these results, it is evident that a reduced oxygen concentration during the culture period exerted only a limited effect on follicle growth as compared with 20% O2.
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Roles of FSH and Oxygen Concentration on Antral Cavity Development and COC Formation
At the end of the 6-day period, each follicle was assessed for the presence or absence of an antral cavity (Fig. 1B) and, after the mechanical rupture of the follicle, for the quality of the related COC (Fig. 1C). Under either 5% or 20% O2 concentration, antral cavities were observed in a very low proportion of FSH-free cultures, while they became clearly visible in a high number of follicles after addition of FSH (Fig. 5). The percentage of healthy COCs that formed also increased in FSH-stimulated follicles (Fig. 6). In Figure 7, results of segmentation analysis are reported. In this analysis, antral cavity formation was the dependent variable, while the presence of healthy COCs, FSH concentrations, and oxygen concentrations were the predictors. It was evident that a high FSH concentration (1 µg/ml) and, next in importance, 5% oxygen concentration were fundamental to obtaining the highest proportion (100%) of follicles showing the presence of antra and, at the same time, of good-quality COCs.
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In Figure 8, all the results obtained in our experiments are schematically represented in a factorial map. In this map, the x axis represents culture conditions (stimulative on the left, unstimulative on the right) determining follicle development, while the y axis represents follicle size classes. From this figure, it was confirmed that the most appropriate culture conditions for obtaining sheep preantral follicle development in terms of increase in volume, antrum, and COC formation, were represented by the use of high gonadotropin concentrations and low oxygen concentration throughout the whole culture period.
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Morphological Evaluation and Maturational Competence of IVG COCs
At the end of the culture period, oocyte final diameter and maturational competence were determined for COCs released from follicles cultured in the presence of 1 µg/ml FSH and under 5% or 20% O2. Based on our results, oxygen concentration had no significant effect on oocyte growth, since oocyte diameters increased from a mean value of 70 ± 10 µm recorded at the beginning of culture (Day 0) to a final value of 110 ± 5 µm and 115 ± 6 µm (Day 6) for 20% and 5% O2, respectively (p < 0.01). These values were similar to that recorded for oocytes obtained from early antral follicles (111 ± 11 µm), but lower than the mean diameter of 140 ± 13 µm recorded for fully grown oocytes (p < 0.05).
Oocyte meiotic competence was assessed by in vitro maturation of COCs obtained from preantral follicles that had been cultured in the presence of 1 µg/ml FSH and under the two different oxygen concentrations. Results obtained showed that 5 out of 20 under 20% O2, and 12 out of 28 under 5% O2 of the in vitro-matured oocytes underwent germinal vesicle breakdown (p > 0.05) and that similar low proportions of them reached the metaphase II stage (MII; 5% MII in 20% O2 vs. 4% MII in 5% O2).
Estradiol Determination
During the entire culture period, the follicles cultured in the absence or presence of 0.01 and 0.1 µg/ml FSH secreted small amounts of E2, regardless of the oxygen gassing conditions (Fig. 9, a and b). With 1 µg/ml FSH, only follicles cultured under 5% O2 released progressively increasing amounts of estradiol, with a sharp rise in production from Day 2 to Day 4 (p < 0.0001). By contrast, when preantral follicles were cultured under 20% O2, the high FSH dose was unable to stimulate a significant estradiol production throughout the whole culture period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our culture system, preantral follicles with intact theca have been isolated by mechanical dissection of the ovary, with a procedure similar to that used for the isolation of mouse preantral follicles [2, 3, 13, 14]. The culture of single intact follicles into a small volume of medium (25 µl) as well as the presence of 10% serum may contribute to the formation of a microenvironment capable of sustaining oocyte and granulosa cell survival, and may also reduce eventual deleterious effects determined by a high-oxygen atmosphere. Another advantage of this culture system is the possibility of evaluating complete follicle development up to the formation of the antral cavity. Other studies have reported the possibility of isolating preantral follicles from the ovaries of farm animals such as cattle by enzymatic digestion of the ovary [6, 7]. The results presented by Wandji and colleagues [6] showed that the somatic component of the bovine preantral follicle could grow and produce steroids despite the high incidence of oocyte death. Therefore, in the bovine ovary, the regulative roles of the oocyte in specific follicle functions, like those described for rodents [15–19], would be lost, and, as a consequence, the follicle would develop without the maintenance of the bidirectional dialogue between oocyte and surrounding granulosa cells [20].
The fundamental role of FSH in in vitro development of mammalian preantral follicles is demonstrated by the finding that a very high number of such follicles can be efficiently grown in vitro only under gonadotropin stimulation [1, 5, 6, 14, 21–23]. Our results demonstrated that sheep preantral follicles can also be efficiently grown in vitro under the effect of FSH. In addition, for the class of small follicles, a greater increase in final volume has been observed in response to gonadotropin. However, in our study, follicle growth is not considered representative of a physiological development. In fact, even if follicle growth appears to be only slightly stimulated by the use of a low-oxygen concentration, a reduced oxygen atmosphere significantly improves the capacity of cultured follicles to further differentiate into antral follicles. Indeed, only when sheep follicles were cultured under 5% O2 and stimulated by the high FSH concentration (1 µg/ml) could we obtain the highest proportion of estradiol-secreting antral follicles containing healthy COCs. Thus, our conclusions are in agreement with those obtained by Eppig and Wigglesworth [9], since these authors demonstrated a significant increase of mouse oocyte survival when cultures were carried out under 5% O2.
Regarding the kinetics of estradiol released by gonadotropin-stimulated preantral follicles cultured under 5% O2, we found that after a rapid increase up to Day 4, the kinetics of E2 production showed a rapid decline, probably because culture conditions became insufficient for sustaining a complete antral development of these follicles. However, our results demonstrate that under both oxygen concentrations used here, sheep preantral follicles grow to the same extent, despite the completely different estradiol secretion. This indicates that estradiol synthesis and the process of follicle growth are not strictly dependent on each other, and confirms previous results obtained by Schoot and colleagues [24] for human oocytes, and by Boland and Gosden [25] for mouse oocytes.
It is well demonstrated that, in mammals, oocytes derived from large antral follicles are more developmentally competent than those from small antral follicles [21, 26], and that their capacity to resume and complete meiosis is acquired in a step-wise fashion [27]. From our experiments, it is evident that the low gaseous oxygen concentration and the high FSH concentration used, while significantly improving the percentage of healthy COC formation, had no evident effect on the proportion of oocytes resuming meiosis in vitro. Thus, the observations that the mean final diameter of sheep IVG oocytes is lower than that recorded in vivo for fully grown oocytes, and that a low percentage of the oocytes resuming meiosis reach MI/MII stages, indicate that our culture conditions are insufficient to guarantee germ cells with high developmental potential.
In conclusion, in the present study we have described a suitable culture system for in vitro development of sheep preantral follicles. We demonstrated that the presence of an appropriate FSH concentration together with low oxygen concentration throughout the 6-day culture period are beneficial to improve antral cavity formation, to stimulate estradiol production, and to obtain elevated numbers of healthy COCs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 39 861 558819; mattioli{at}ifv.vet.unite.it
Accepted: September 22, 1998.
Received: December 9, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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