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a Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, United Kingdom
b IERM, Division of Biological Sciences, School of Agriculture Building, University of Edinburgh, Edinburgh EH9 3JG, United Kingdom
c School of Biological Sciences, Division of Agriculture and Horticulture, University of Nottingham, Sutton-Bonington Campus, Loughborough, Leicstershire LE12 5RD, United Kingdom
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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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Early follicular development is a lengthy process [3, 4], and its regulation remains largely unknown. Nonetheless, it is clear that successful culture of murine oocytes, fertilization, and further developmental competence are achieved only if the three-dimensional organization of the granulosa cells around the oocyte is maintained throughout culture [5]. Indeed, the oocyte is dependent on the surrounding granulosa cells from which it receives its nutrients [6]. Nutrient transport between the granulosa cells and the oocyte is achieved by extensive gap junction communication [7, 8]. Granulosa cell contact is essential for maintaining the oocyte in meiotic arrest, since their removal [9], or disruption of the gap junction communications between granulosa cells and the oocyte, results in spontaneous germinal vesicle breakdown [10].
We recently described a long-term granulosa cell culture system whereby terminal differentiation does not occur, thereby ensuring that the granulosa cells retain their in vivo phenotype and remain responsive to physiological concentrations of FSH, insulin, and insulin-like growth factor (IGF)-I [11]. Furthermore, granulosa cells cultured in this system form cell aggregates with extensive gap junctions between cells [12]. In the work described here, this granulosa cell culture system is adapted for the culture of bovine preantral follicles. This follicle culture system can sustain long-term culture of preantral follicles. Follicles maintained their cellular organization and general architecture, and follicle growth and enhanced antrum formation can be stimulated by FSH, epidermal growth factor (EGF), and IGF-I. This culture system may provide a valuable approach for study of the regulation of early follicular development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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McCoy's 5a medium with sodium bicarbonate, penstrep (containing 10 000 IU penicillin and 10 mg streptomycin per milliliter), BSA, tissue culture grade transferrin, selenium, bovine insulin, androstenedione, and ethidium bromide were purchased from Sigma Chemical Co. (Poole, Dorset, UK). Hepes, amphotericin, and L-glutamine were purchased from Gibco BRL, Life Technologies Ltd. (Paisley, Renfrewshire, UK). Bovine FSH (USDA-bFSH-I-2; bioactivity potency 854 IU·mg-1) was generously donated by the U.S. Department of Agriculture. IGF-I analogue and long-R3 IGF-I (LR3-IGF-I, which has significantly reduced binding to IGF-binding proteins), were purchased from Gropep Pty Ltd. (Adelaide, Australia). EGF was purchased from Toyoba Co. Ltd. (Osaka, Japan).
Preantral Follicle Isolation
Bovine preantral follicles were dissected from ovaries at random stages of the estrous cycle as described by Ralph [13]. Briefly, ovarian cortical slices (1–2 mm thick) were cut from the ovarian surface. Preantral follicles were visualized under the dissecting microscope and manually isolated using 25-gauge needles. Between 16 and 28 preantral follicles that appeared normal were obtained for each culture. These follicles were characterized as having a spherical oocyte, surrounded by granulosa cells and limited by an intact basement membrane and an outer thecal-stromal layer (see Fig. 2; Day 0).
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Preantral Follicle Culture and Treatments
Preantral follicles were cultured in a system developed for bovine granulosa cells [11]. Briefly, the culture medium was McCoy's 5a medium with bicarbonate containing 20 mM Hepes, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 3 mM L-glutamine, 0.1% BSA, 10-7 M androstenedione, 2.5 µg/ml transferrin, and 4 ng/ml selenium [11]. Follicles were cultured individually in 96-well plates with 250 µl of culture medium supplemented with 10 ng/ml of insulin. Follicles were incubated in a humidified atmosphere with 3.8% CO2 at 37°C, and medium was replaced every 6 days. Hormonal treatments were prepared in sterile 96-well plates and were equilibrated in the incubator for 3 h before the medium was changed.
On five occasions, between 16 and 28 normal-appearing bovine preantral follicles were isolated and cultured with treatments including bFSH (1 ng/ml) and the growth factors EGF (0.5 ng/ml) and long-R3 IGF-I (1 ng/ml), either alone or in combination. Preantral follicles were also cocultured with granulosa cells obtained from antral follicles (2–4 mm in diameter) and treated with IGF-I/FSH. Cultures were originally intended to last for 6 days. In the first two cultures, 100% of the follicles maintained a normal and healthy appearance for 6 days. Subsequently, cultures were extended for up to 28 days (cultures 3–5).
Assessment of Follicular Growth and Health
After isolation, follicles were selected for their integrity. Only follicles classified as normal (with oocyte and granulosa cells completely surrounded by the basement membrane and thecal-stromal layer) were cultured. Follicles selected in this way showed no signs of atresia [14] and, when examined under a transmission electron microscope, were found to be morphologically similar to follicles within the ovarian cortex [15]. Follicle degeneration was marked by breakdown or rupture of the basement membrane, opacity of granulosa cells, or extrusion of the oocyte from within the follicle. Follicles were fixed with 4% paraformaldehyde in Dulbecco's PBS for 30 min at room temperature and stained with 5 mg/ml ethidium bromide in Dulbecco's PBS for 15 min at room temperature. After staining, the follicles were examined by confocal microscopy (Bio-Rad MRC-500; Bio-Rad Laboratories Ltd., Hertfordshire, UK) to confirm the presence of an antrum. Follicle and oocyte diameters were measured using a calibrated eyepiece fitted in an inverted microscope under brightfield.
Statistical Analyses
The effects of treatment and day of culture on follicular and oocyte diameter were analyzed by regression analysis testing for homogeneity of regression among treatments and allowing for variation between cultures. The effects of treatment on antrum development or follicle degeneration were analyzed by generalized linear mixed models, allowing for the random effect of experiment (see [16]). The cumulative effect of treatment versus control was assessed using Bonferroni's adjustment [17]. Follicle degeneration was considered to have occurred in those follicles that deteriorated without forming an antral cavity. The effects of treatment on the day of antrum formation or degeneration of follicles that did not form an antrum were tested by survival analysis. Preantral follicles not developing an antrum by the end of each culture, but classified as normal (4 of 27, 3 of 24, and 3 of 18 for culture 3, 4, and 5, respectively) were considered as right-censored observations in the survival analysis. The effects of oocyte diameter, follicle diameter before culture, and the day of antrum formation or follicle degeneration were also tested.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mean preantral follicle diameter was 166 ± 2.15 µm at the beginning of the cultures. Regression analysis showed an increase in follicle diameter with time in culture (Fig. 1), with a significant (P < 0.01) quadratic effect of the day of culture over time (R2 = 0.74). This indicated a rapid increase in follicle diameter during the first week of culture, but the growth rate then slowed after 8–10 days in culture. Treatment with FSH, IGF-I, FSH/IGF-I, and EGF enhanced (P < 0.05) the growth rate of preantral follicles. In contrast, the presence of granulosa cells inhibited (P < 0.05) FSH/IGF-I stimulation (Fig. 1).
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As observed for follicle diameter, oocyte diameter increased with time in culture (Day 0 = 55.8 ± 0.8 µm vs. Day 14 = 65.74 ± 1.8 µm; regression y = 57.12 (± 3.22) + 0.62 (± 0.11) day; R2 = 0.47). However, there was no effect of treatment (P > 0.10) on oocyte growth.
Follicle Morphology
Immediately after isolation, preantral follicle granulosa cells surrounded a spherical oocyte. The granulosa cells were limited by an intact basement membrane and an outer thecal layer (Fig. 2; Day 0). This basic morphology of the follicle was preserved throughout the culture period. Follicles remained in an oval or spherical shape and did not attach to the well surface. As follicle diameter increased with time in culture, there was an increase in the volume of the follicle that appeared to be accompanied by an increase in granulosa cell number (Fig. 2). After an extended period of culture (range 10–28 days), antral spaces appeared within some follicles. These antral spaces joined to form antral cavities (A; Figs. 2 and 3).
Antrum Formation and/or Follicle Degeneration
The mean probability of antrum formation or degeneration for each treatment is shown in Table 1. There were no differences (P > 0.05) in the mean probability of antrum formation among treatments. However, the cumulative mean probability of antrum formation for treated follicles was higher (P < 0.05) than for control follicles. The estimated probability for follicle degeneration by treatment did not differ (P > 0.05), nor did the cumulative probability for treated follicles versus control follicles (P > 0.05).
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Survival analysis showed that the time from the beginning of culture to antrum development was not affected by the treatment applied (P > 0.05), or by oocyte or follicle diameter at the time of dissection. Similarly, treatment did not affect (P > 0.05) day of follicle degeneration.
A common characteristic of follicle degeneration was extrusion of the granulosa cells from the follicle (Fig. 2b, Day 25) after bursting of the basement membrane. This extrusion was observed whether or not follicles had previously formed an antral cavity. The granulosa cells extruded from within the follicular walls remained in close proximity, forming clumps of viable cells organized around the oocyte.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Long-term culture requirements have hindered the advance of preantral follicle cultures. To date, no culture system has been reported that can maintain follicular architecture for longer than 12–16 days [23]. Thus the adaptation of the granulosa cell culture system that maintains phenotypically nonluteinized granulosa cells [11, 12] in long-term culture has been applied to culture bovine preantral follicles for up to 28 days, allowing studies of follicle growth and maturation (antrum formation) while keeping follicular architecture intact.
Preantral follicles grew in culture, even in the absence of trophic hormones. This growth was probably promoted by the presence of insulin in the culture media. We have shown that insulin promotes the proliferation of granulosa cells under similar cultural conditions [11, 12, 25]. Follicular growth may also be regulated by substances produced within the follicle. Indeed, numerous putative autocrine/paracrine follicle mediators have been investigated. Activin is a granulosa cell product that increases thymidine uptake by mouse preantral follicle granulosa cells [26]. Similarly, granulosa cell produced-inhibin can stimulate estradiol and androstenedione production by granulosa and theca cells, respectively [27]. Granulosa cells of preantral follicles produce a thecal differentiation factor [28] that stimulates androstenedione production by thecal cells. We have found IGF-I mRNA in thecal cells and IGF-II mRNA expression in both thecal and granulosa cells of bovine antral (3–8 mm) follicles [29]. Indeed, Armstrong et al. [30] found IGF-II mRNA expression in the thecal layer of bovine follicles from 1–2 mm in diameter. Thecal cells also produce EGF/transforming growth factor 
[31], and these were shown to stimulate proliferation of thecal and granulosa cells in both mice and sheep [25, 31].
Follicular growth may also be influenced by the interference from an inhibitory factor within the ovary. Compelling evidence for a local inhibitory factor(s) within the ovary is the activation in vitro of primordial follicles in ovarian tissue slices [32, 33]. This inhibitory factor(s) acts on follicles of different size ranges. When murine preantral follicles were cultured in groups [34], or in contact with each other [34, 35], the growth of some follicles was inhibited. In the current study, the addition of FSH, EGF, and/or IGF-I stimulated higher rates of follicular growth compared to that in control follicles, confirming the stimulatory effects of FSH and growth factors. In contrast, coculture of preantral follicles with granulosa cells from antral (2–4 mm) follicles in this study inhibited the stimulatory action of FSH/IGF-I on follicle growth. However, although smaller than their counterparts, these follicles otherwise appeared healthy. Whether attenuation of follicular growth was due to a direct effect of a putative inhibitory factor secreted by more mature granulosa cells, or due simply to competition for nutrients and stimulatory hormones, could not be determined.
The probability of antrum formation in follicles treated with FSH, IGF-I, or EGF was over 0.50 and was associated with a significant increase in follicular diameter. Antrum formation may regulate acid-base balance and respiratory gases [36] once the follicle becomes too large for nutrients and oxygen to reach the cells by passive diffusion (around 200 µm in diameter). Follicle development is halted in murine follicles, grown in vitro, at the preantral stage in the absence of FSH [19]. No differences in follicle growth or in the probability of antrum formation were observed in this study between growth factor treatments, even in the presence or absence of FSH. Although we cannot discount the effects of gonadotrophic priming of the follicles prior to their isolation, these results may indicate that the threshold of gonadotropin dependence varies between species, depending on the size of the preovulatory follicle. Accordingly, in the absence of FSH, preantral follicle growth stops at the preantral stage in hypophysectomized mice [37], at 2.5 mm in diameter in hypophysectomized and gonadotropin-depleted sheep [38, 39], and at around 3–4 mm in gonadotropin-suppressed cattle [40]. The use of this culture system will enable study of the regulation of early follicular growth and antrum formation in preantral follicles.
In addition to growth of follicles, there was continuous growth of the oocyte within the follicle. In murine culture systems it has been shown that only oocytes that maintain contact with granulosa cells are capable of maturation and subsequent fertilization and embryonic development [5, 41]. Here again, species differences may dictate specific culture requirements. In the mouse, the preantral follicle has to grow for a further 4 days to attain preovulatory size [23], and the oocyte is almost fully developed in the preantral follicle. In sheep [24], it has been demonstrated that it takes 3–4 mo for animals to reinitiate estrous cycles after ovariectomy and autotransplantation with ovarian grafts. In these autografts, all antral follicles were shown to have degenerated within 1 wk of the grafting. In bovine preantral follicles, it is estimated that a further 40 days are needed for the follicle to reach the preovulatory size, and the oocyte has to double its size during this time [23]. In this study, the growth of the oocytes in culture was parallel to the growth of the follicle, and the size reached by the oocyte after culture was within the expected size range for early antral follicles in vivo [42]. Therefore, oocyte maturity in murine preantral follicles may permit the culture of granulosa-oocyte complexes (GOC) for a short period yielding viable oocytes, but this may not be comparable to what occurs in larger species. In the current study, follicles in which the GOC were extruded after bursting of the follicular wall (Fig. 2b) maintained the three-dimensional architecture of the granulosa cells surrounding the oocyte. Thus, further studies are needed to evaluate whether these complexes are able to survive an extended culture period and to evaluate oocyte growth and competence.
Follicular growth is accompanied by extensive tissue remodeling of the theca interna and basement membrane surrounding the granulosa cells. In this study, the 30% increase in follicle diameter corresponded to a 72% increase in follicle surface area. After culture of preantral follicles, thecal tissue was thinner and appeared to be stretched (Fig. 2) compared to that of follicles before culture (Fig. 2; Day 0). Furthermore, some follicles grew by rupturing the basement membrane and thecal layer, thereby extruding the GOC. Basement membrane remodeling involves the selective degradation and synthesis of extracellular matrix (ECM) proteins. ECM degradation involves complex interactions between proteolytic enzymes (e.g., matrix metalloproteinases) and their regulators [43]. The secretion of gelatinase activity has been demonstrated in thecal cells in serum-free culture [44]. In addition, both granulosa and thecal cells produce ECM [43]. However, whether follicular cells in vitro are able to produce the enzymes and substrates needed to assemble the basement membrane—at a rate of growth required to maintain the integrity of the basement membrane of growing follicles for long-term periods in culture—needs further investigation.
In conclusion, sustained growth of bovine preantral follicles for relatively long periods in culture has been achieved. Under these culture conditions, follicles maintained their cellular organization and were responsive to stimulatory effects of FSH, EGF, IGF-I, and possibly insulin. For the first time, the growth of bovine preantral follicles up to the antral stage is described. This culture system should provide a valuable model for study of the regulation of early follicular growth and antral development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by CONACYT (Mexico), Ministry of Agriculture Fisheries and Food (UK), and Meat and Livestock Commission (UK). 
2 Correspondence and current address: C.G. Gutierrez, Departamento de Reproducción, Facultad de Medicina Veterinaria, Cd. Universitaria 04510, Mexico D.F., Mexico. FAX: 52 5 550–8697; ggcarlos{at}servidor.unam.mx
Accepted: December 8, 1999.
Received: April 13, 1998.
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REFERENCES |
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