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a Institute of Ecology and Resource Management,
b School of Agriculture and Department of Reproductive and Developmental Sciences, Obstetrics and Gynaecology Section, Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH9 3JG, United Kingdom
ABSTRACT
Satisfactory development of bovine follicles in vitro remains elusive. This study used a serum-free system to evaluate the effects of insulin-like growth factor-1 (IGF-1) on bovine preantral follicles in culture and to identify the activity of gelatinase matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in vitro to assess their potential as markers of development. Preantral follicles were cultured for 6 days in serum-free medium containing insulin and IGF-1 (10 ng/ml). No difference was observed in follicular growth, health, or antrum formation between IGF-1-treated follicles and controls. However, IGF-1 had a negative effect (P < 0.01) on oocyte size and granulosa cell proliferation. When MMP-9 was secreted, the probability of follicles having healthy granulosa or theca cells at the end of the culture period was 0.85 and 0.60, respectively. If TIMP-1 was released, the probability of follicles having healthy somatic cells was 0.79. When TIMP-2 was detected, the probability of granulosa and theca cell health was 0.78 and 0.67, respectively. These results demonstrate no positive effects of IGF-1 on bovine follicles in this system. Furthermore, MMP-9 and TIMPs are related to follicular health and, therefore, can be used as markers of follicular development.
follicle, granulosa cells, ovary, theca cells
INTRODUCTION
The number of oocytes in the ovary at birth varies enormously between species, ranging from tens of thousands in mice to millions in humans and domestic species [1]. Of these oocytes, less than 1% will eventually ovulate [2]. In cattle, procedures for in vitro maturation and fertilization have progressed substantially during the past decade, leading to increased availability of zygotes, embryos, and calves for breeding and research purposes [3]. However, this technology has limitations, because oocytes cannot be harvested from the large population of hormone-insensitive, preantral follicles. Development of a culture system that can support preantral and, eventually, primordial follicles to a stage at which the oocytes can be matured and fertilized in vitro would help to provide large amounts of homogeneous oocytes. Such a system would also allow the identification of factors necessary for normal follicular development and acquisition of oocyte developmental competence.
Culture systems developed for rodent follicles [4–8] have led to the production of developmentally competent oocytes after culture of whole preantral follicles or their granulosa-oocyte complexes. Advances have been made in transferring aspects from the rodent models to develop culture systems for porcine preantral follicles [9], but progress with other domestic species has been much slower. Recently, a culture system for sheep preantral follicles was described [10] that achieved antrum formation and estradiol production and maintained healthy oocytes and cumulus cells during a 6-day culture period. However, this system failed to produce germ cells with high developmental potential. Similarly, for bovine follicles, culture has been terminated long before the preovulatory stage was reached [11–14]. In cattle, large ovaries, less densely packed follicles, fibrous stromal tissue, large follicle size, and slow follicular growth have all played a role in delaying the development of successful isolation and culture techniques. Recently, however, accelerated growth of bovine preantral follicles to the antral stage has been achieved over a period of as many as 28 days [11], with FSH, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) having a positive effect on follicular growth and development.
Insulin-like growth factors, and particularly IGF-1, have been identified as being important paracrine regulators of ovarian function. Previous studies have helped to determine the actions of IGFs in antral follicles, which are thought to vary according to species and the interaction of gonadotropins [15]. For example, granulosa cells from bovine antral follicles have increased proliferation and estradiol production in response to IGF-1 [16]. However, the precise role of the IGF system during the earlier stages of follicular development is poorly understood.
Previously, culture systems for cattle preantral follicles have resulted in loss of theca cells [11, 13] or rupture of the basement membrane [11] in some follicles. During follicular growth, turnover and reconstruction of the basement membrane is facilitated by matrix metalloproteinases (MMPs), which are zinc- and calcium-dependent enzymes that can degrade the protein components of the extracellular matrix [17]. The MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs) and are also responsible for reconstruction of the basement membrane at the time of ovulation and during corpus luteum formation [18]. The MMPs involved in the breakdown of collagen IV (i.e., a major constituent of the basement membrane) are the gelatinases MMP-2 and MMP-9. The main follicular sources of MMPs and TIMPs are thought to be the granulosa and theca cells [18, 19], but TIMP protein has also been found in oocytes [20].
The present study used a serum-free culture system [11] to investigate the effects of IGF-1 on several aspects of bovine preantral and early antral development in vitro. In addition, secretion of MMPs and TIMPs by cultured follicles was identified, and using morphological comparisons, these factors were assessed for use as markers of follicular development.
MATERIALS AND METHODS
Isolation of Preantral Follicles
Bovine ovaries from random stages of the estrous cycle were obtained from an abattoir and transported at 25–30°C. Beneath a laminar flow hood, ovaries were rinsed with 70% alcohol, and fine slices of ovarian cortex were taken using a scalpel and placed in Liebovitz's medium (GIBCO BRL, Life Technologies Ltd., Paisley, Renfrewshire, UK) supplemented with sodium pyruvate (2 mM), glutamine (2 mM), BSA (3 mg/ml), penicillin G (75 µg/ml), and streptomycin (50 µg/ml). All chemicals were from Sigma Chemicals (Poole, Dorset, UK) unless otherwise stated. In a petri dish under the dissecting microscope, preantral follicles (100–200 µm) were isolated from the cortical slices using fine, 25-gauge needles attached to syringe barrels. Follicles with an intact basement membrane and an even distribution of granulosa and theca layers were selected for culture.
Culture of Preantral Follicles
For the control group, preantral follicles were cultured individually in 96-well plates (Bibby Sterilin Ltd., Stone, Staffs, UK) in 250 µl of culture medium (McCoy's 5a medium with bicarbonate [Sigma]) supplemented with Hepes (20 mM), BSA (0.1%), L-glutamine (3 mM), penicillin (100 IU/ml), streptomycin (0.1 mg/ml), transferrin (2.5 µg/ml), selenium (4 ng/ml), androstenedione (10-7 M) and insulin (10 ng/ml). For the treatment group, 10 ng/ml of the analogue Long R3 IGF-1 (Gropep Pty Ltd., Adelaide, Australia), which does not bind to IGF-binding proteins, was added to the control medium. Plates were incubated for 6 days in a sterile, humidified air atmosphere with 5% CO2 at 37°C. Follicular diameters were measured under the dissection microscope on Days 0, 2, 4, and 6. Half the medium was replaced every second day, and this conditioned medium was stored at -20°C for subsequent MMP/TIMP analysis.
Histological Assessment of Follicles
At the end of the culture period, follicles were fixed overnight in Bouin's solution and dehydrated in ethanol. Absolute ethanol was replaced with cedar wood oil for a minimum of 24 h, and then the oil was cleared from the follicles using toluene for 30 min. Follicles were embedded in paraffin wax (60°C), with changes every hour for 4 h to remove all traces of toluene. The samples were sectioned (6 µm), mounted on gelatin-coated slides, and then allowed to dry overnight at 37°C before staining with hematoxylin-and-eosin.
Histological measurements and observations were made under the light microscope with a crossed micrometer (Graticules Ltd., Tonbridge, Kent, UK). The section containing the oocyte nucleolus or, if this was absent, the largest cross-section of the oocyte was used for observations and measurements. Follicular and oocyte sizes were measured, and proliferation was assessed by counting the number of granulosa and theca cell layers. Granulosa cell death was measured by counting the number of pyknotic cells and then expressing them as a percentage of the total number of granulosa cells; atretic follicles were defined as being those with more than 5% pyknotic nuclei [21]. Theca cell health was assessed in two ways: by the level of pyknosis, as defined earlier; and by the number of layers present, with less than three layers indicating theca cell degeneration. Oocyte quality was assessed on a scale of 0–2, with 0 indicating an absent or severely misshapen oocyte with no germinal vesicle and obviously degenerate, 1 indicating a misshapen oocyte, and 2 indicating a morphologically normal oocyte with an intact germinal vesicle.
Detection of MMP-2 and MMP-9 Secretion by Gelatin Zymography
The MMP-2 and MMP-9 activities were detected by gelatinase zymography according to the method described by Riley et al. [22]. In brief, 100-µl samples of culture medium were dialyzed against distilled H2O in small tube-O-dialyzers (Chemicon International, London, UK) before being lyophilized and reconstituted in 7.5 µl, 0.1% SDS in H2O. Samples were separated by SDS-PAGE using 7.5% gels containing 1 mg/ml gelatin on a minigel apparatus (Bio-Rad, Hemel Hempstead, UK). Gels were washed (twice for 15 min in 2.5% (v/v) Triton X-100, and twice for 2 min in 10x Tris-buffered saline [TBS]), then incubated overnight at 37°C in digestion buffer (50 mM Tris; 0.2 M NaCl; 5 mM CaCl2, 1 M ZnCl2; 0.02% [v/v] Brij-35) Gels were stained for 3 h at 23°C with 0.5% Coomassie Blue R250 in 30% methanol/10% glacial acetic acid in H2O and then destained (staining solution with Coomassie Blue omitted). Destaining revealed white bands where gelatin was degraded by gelatinase activity. The MMP-2 and MMP-9 were identified by comparison with molecular weight markers and control standards of human amniotic fluid collected during labor at term [22].
Detection of TIMP-1 and TIMP-2 Secretion by Reverse Zymography
The TIMP activity was detected using a commercial kit (University Technologies Inc., Calgary, Canada) according to the method described by Riley et al. [22]. Samples were lyophilized and reconstituted as before and separated according molecular weight by PAGE using 12% gels containing gelatin (1 mg/ml) and a preparation of MMP-2 (conditioned medium from BHK-21 cells that constitutively express MMP-2; University Technologies). The gels were washed (50 mM Tris, 5 mM CaCl2, 2.5% [v/v] Triton X-100) for 2.5 h at 23°C, then incubated in digestion buffer (50 mM Tris, 5 mM CaCl2) overnight at 37°C. After staining and destaining (as in zymography), TIMPs were represented by discrete, dark bands on the gel where inhibition of MMP-induced degradation of the gelatin substrate had occurred. The TIMPs were identified by comparison with molecular weight markers and control standards of conditioned medium containing mouse TIMP-1, -2, and -3 expressed by transfected BHK cells (University Technologies).
Statistical Analyses
Mean follicular diameters from the two experimental groups on Day 6 were compared using a 2-sample t-test. In addition, mean follicular growth rates from Days 0 to 6 were compared within groups using the same test. Oocyte diameters from freshly isolated and cultured preantral follicles were compared using a one-way ANOVA, with subsequent t-tests to allow for individual comparisons between groups. Oocyte diameters are also represented here graphically using a box and whisker plot. The numbers of follicular granulosa layers at the end of culture were compared between groups using a chi-square test. With probability estimates for the occurrence of MMPs and TIMPs having been calculated, Baye's Theorem [23] was applied to estimate the probability of granulosa or theca cell health given that MMPs and TIMPs were present.
RESULTS
Follicle Growth
Follicles were cultured for 6 days in the presence (n = 72) or absence (n = 61) of IGF-1, with follicular diameters being measured every second day. For analysis of growth, follicles were separated into two size classes: preantral (<150 µm; n = 60), and large preantral (150–200 µm; n = 73). As Figure 1 illustrates, significant follicular growth occurred over 6 days in the presence and the absence of IGF-1 in both preantral and large preantral follicles (P < 0.01). No significant effect of IGF-1 on follicular growth was found in either size group (P > 0.05).
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Histological Assessment
Histological observations were made using freshly isolated follicles (n = 10), IGF-1-treated follicles (n = 24), and control follicles (n = 22), all of which were 150–200 µm before culture. A significant difference in oocyte diameter between the three groups was detected by ANOVA (P < 0.01), and follicles treated with IGF-1 had significantly smaller oocytes than controls and freshly isolated follicles (P < 0.01) (Fig. 2). Follicles cultured with IGF-1 also had a reduced number of granulosa cell layers (P < 0.01) by Day 6 of culture (Table 1). However, IGF-1 had no effect on the percentage of follicles with degenerating oocytes and granulosa cells, the thickness and level of pyknosis of the follicular theca cell layers, or the percentage of follicles that formed an early antrum in vitro (Table 1). Examples of follicles with healthy and degenerating somatic cells are shown in Figure 3.
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Secretion of MMP-2, MMP-9, TIMP-1, and TIMP-2 Activities
Culture medium from 32 follicles was analyzed for gelatinase activity, and 24 samples were analyzed for TIMPs. Examples of a zymogram demonstrating MMP-2 and MMP-9 activity and of a reverse zymogram showing TIMP-1 and TIMP-2 secretion by follicles into the culture medium are shown in Figure 4. The MMP-2 (72 kDa) was released by 87.5% of follicles, whereas the MMP-9 (92 kDa) was secreted by 62.5% of follicles (Fig. 4). Comparisons between zymographic analysis and morphology showed that if MMP-9 was secreted during culture, the probability of these follicles having healthy granulosa cells at the end of the culture period was 0.85. The probability of theca cell health (as defined by the number of layers) was 0.60. When TIMP-1 (28 kDa) was released in vitro, (58% of follicles), the probability that the follicles would have healthy granulosa or theca cells was 0.79. When TIMP-2 (21 kDa) was produced (75% of follicles), the probability of the follicles having healthy granulosa or theca cells was 0.78 and 0.67, respectively.
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DISCUSSION
The development of bovine preantral follicles to the early antral stage in a serum-free culture system was achieved, and secretion of MMPs and TIMPs correlated with follicular health during a 6-day period. We also demonstrated, to our knowledge for the first time, that IGF-1 has a negative effect on oocyte size and granulosa cell proliferation during this early stage of follicular development.
The IGF system has been well characterized in large antral follicles, but fewer studies have examined the effects of IGFs during earlier stages of follicular development. Wandji et al. [24] detected low IGF-1 mRNA levels in primary follicles from immature mice, but transcription increased to a maximum during the late preantral and early antral stages. In addition, apoptotic follicles had lower levels of IGF-1. This indicates that IGF-1 is associated with the growth and survival of rapidly expanding, large preantral and early antral follicles in the mouse [24, 25]. However, differences in the temporal and spatial production of IGF-1 between rodents and domestic species suggests different mechanisms of action. In sheep, the influence of IGF-1 on granulosa cells from small (1–3 mm) sheep follicles is proliferative, whereas in more mature follicles (5–7 mm), steroidogenic effects predominate [26]. Regarding the source of IGF-1, mRNA has been detected during the early antral stage in both granulosa and theca cells of sheep follicles [27] and in granulosa cells of porcine follicles [28], but no IGF-1 expression has been found during the preantral stage. In cattle, IGF-1 mRNA has been detected at low levels in granulosa cells of antral follicles, with an increased level after selection [29], and also in theca cells [30], indicating that IGF-1 may be important during the later stages of folliculogenesis (e.g., in relation to LH responsiveness).
Although IGF-1 attenuates spontaneous apoptosis in porcine granulosa cell cultures [31], it is not produced by ovine or bovine granulosa cells in vitro in the absence of luteinization [32, 33]. Receptors for IGF-1 on bovine granulosa cells do not fully develop until the antral stage [34], and this indicates a paracrine rather than an autocrine role for IGF-1 in the control of granulosa cell function, with IGF-1 playing a role later in the follicular cycle among domestic species than among rodents. Therefore, any positive effects on follicular development should be limited, which we have confirmed in the present study. Previously, Gutierrez et al. [11] reported that IGF-1 had a stimulatory effect on follicular and oocyte growth and antrum formation in an extended culture using confocal microscopy. They did not, however, present any data on follicular health. In addition, their study had limitations regarding the detection of an early antral cavity or early signs of atresia. Therefore, in the present study, we used histological analysis and a shorter culture period to detect the effects of IGF-1 during the early stages of development in more detail. A longer culture period may allow granulosa cells to differentiate sufficiently for IGF-1 to function as a stimulator of development, as reported by Gutierrez et al. [11]. Thus, the present study is novel, in that its results suggest the action of IGF-1 is strictly regulated according to developmental stage, and that treatment of immature follicles with IGF-1 may result in precocious differentiation, retarding growth and proliferation.
Regarding the preantral and early antral stages of follicular development, very little is known concerning the contribution of the various MMPs and associated regulators to follicular remodeling. We demonstrated that when MMP-9 and TIMPs are secreted in vitro, a follicle has a higher probability of being healthy at the end of the culture period. Several studies have investigated the distribution of MMPs and TIMPs at different stages of follicular development. In the ovary of neonatal rats, activity of MMP-2, but not MMP-9, has been detected, with visualization of TIMP-1 in the oocyte [20]. In eCG-primed ovaries of rats and mice, MMP-2 is detected in the granulosa and theca cells, MMP-9 is restricted to thecal and interstitial cells, and TIMP-1 is located in the blood vessels and theca cells [20, 35, 36]. In addition, MMP-9 is restricted to the theca cells layers in goat follicles of less than 3 mm [37], and it has been detected in the differentiating granulosa cells in culture [18] and, subsequently, in the developing corpus luteum of rats [20]. Collectively, the results of these studies implicate the granulosa and theca cells as sources of MMPs and TIMPs during the follicular stage of development.
In vitro, secretion of MMP-2 and MMP-9 from bovine thecal cells increases in response to LH [17]. This observation may imply that theca cells have a role in remodeling of the basement membrane, or that the remodeling of vascular tissue present within the culture may be under gonadotropin control. The regulation of MMP activity is complex and may be controlled at the level of transcription by growth factors, cytokines, and hormones, with subsequent activation of secreted proforms and inhibition by TIMPs [36]. The mechanisms controlling remodeling at the gonadotropin-independent stages of follicular development are unknown; thus, further investigations are required. The MMPs and TIMPs also may be regulated depending on their specific spatial and temporal functions. The distribution of MMP-9 in the steroidogenic cells of the theca layer [20], interstitium, and corpus luteum as well as its absence in the neonatal ovary [20] and the timing of expression [38] suggest a role in the remodeling and vascularization associated with corpus luteum formation. The detection of MMP-2 and MMP-9 during our study in cattle suggests a role for these enzymes at a much earlier stage than, to our knowledge, has been studied previously, and that MMP-9 and TIMPs can now be used as noninvasive markers for assessing follicular quality in vitro.
The TIMPs have been implicated in other processes involving cell growth. For example, TIMP-1 has been associated with erythroid-potentiating activity [39] and TIMP-1 and -2 have growth factor-like activities in some cell types [40]. The increased probability of follicular health when TIMPs were present in our study agrees with this function. Results of other reports suggest that TIMP-1 is a facilitator of steroidogenesis [41–43]. Differences in TIMP activity between atretic and nonatretic follicles during the late preantral and early antral stage may be a marker for the shift in steroidogenic capability and future selection of healthy follicles at this stage of development. The significance of the location of TIMP-1 in the oocyte [20] is unclear, and during the early stages of folliculogenesis, a potential function for TIMPs as oocyte growth factors remains uninvestigated. Future experiments should assess if the correlation between MMP and TIMP secretion with follicular quality in vitro also applies to oocyte health and development. This will allow us to better understand interactions between the oocyte and somatic cells regarding the coordination of oogenesis and folliculogenesis.
In our study, follicles that did not maintain a differentiated thecal layer in vitro tended to be less healthy overall than follicles with thick, nonpyknotic theca cell layers. Maintaining interplay between the oocyte and the surrounding somatic cells is vital to achieve normal folliculogenesis. Growth differentiation factor-9 (GDF-9), which is an oocyte-derived factor from the transforming growth factor-ß superfamily, is required for sustaining follicular growth and differentiation after the primary (i.e., one-layer) follicular stage [44]. Studies using knockout mice [44, 45] have shown that in the absence of GDF-9, follicles are incompetent to emit a signal that recruits theca cell precursors to surround the basement membrane [45]. Our observations confirm the importance of maintaining healthy theca cells and their connections with the granulosa layers in bovine follicular culture, and our observations also support a role for these cell types in basement membrane remodeling during the early stages of folliculogenesis.
In conclusion, we have reported bovine follicular development from the preantral to the antral stage in a serum-free culture system. Use of IGF-1 as a treatment had no positive effects on follicular development during the preantral and early antral stages. We detected MMP and TIMP activity in vitro and identified MMP-9, TIMP-1, and TIMP-2 as markers of follicular health. Elucidation regarding the action of MMPs and TIMPs and the role of growth factors during follicle growth and antrum formation in an extended culture will provide insight into the complex processes involved in follicular and oocyte growth and development. Improving our knowledge of the interactions between follicular cells and the extracellular matrix is necessary for maintaining the integrity of preantral follicles in vitro, thus sustaining follicular growth and viability.
ACKNOWLEDGMENTS
The authors thank Mr. D. Thomas for advice on the statistical analysis and Mr. J. Binnie for help with the histological preparation.
FOOTNOTES
First decision: 30 November 1999.
1 Supported by the Ministry of Agriculture, Fisheries, and Food. 
2 Correspondence. FAX: 44 0 131 667 2601; f.mccaffery{at}ed.ac.uk
Accepted: March 1, 2000.
Received: November 1, 1999.
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