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Biology of Reproduction 67, 1099-1105 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Growth, Antrum Formation, and Estradiol Production of Bovine Preantral Follicles Cultured in a Serum-Free Medium1

Takehiro Itoha, Masayuki Kacchia, Hiroyuki Abea, Yutaka Sendaia, and Hiroyoshi Hoshi2,a

a Research Institute for the Functional Peptides, Yamagata 990-0823, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17ß (E2) production in preantral follicles of bovine ovaries. Large preantral follicles (145–170 µm in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5°C in 5% CO2 in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E2 secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E2 production earlier than insulin-containing medium alone. Furthermore, maximal E2 secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.

estradiol, follicle-stimulating hormone, follicular development, insulin, luteinizing hormone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, a large number of oocytes are present during the primordial stage to the preantral follicle stage. However, most oocytes present in the follicles at these stages undergo atresia and only a few oocytes develop to the ovulatory stage in vivo [1]. If preantral follicles could be efficiently isolated from the ovaries and be grown in vitro to reach meiotic competence, they would provide a large potential source of genetic material. For instance, oocytes from preantral follicles would be useful in animal reproduction [2], transgenesis [3], and conservation of endangered species [4].

Many culture systems have been used to demonstrate that oocytes grow to full size from preantral follicles in mice [5–9], rats [10, 11], and hamsters [12]. Eppig and O'Brien [13] reported a complex two-step culture system in which a live mouse pup was obtained from the culture of oocyte-granulosa cell complexes isolated from neonatal primordial follicles. However, it has been more difficult to establish a complete in vitro culture system for preantral follicles obtained from domestic animal ovaries. Problems may arise because these preantral follicles require a longer growth period and different culture conditions, which correspond to different growth and developmental stages, in order to achieve complete growth and development. In addition, enzyme treatments used in rodent preantral follicles are unsuitable for isolating intact preantral follicles from ovaries of domestic animals because of the hard fibrous nature of the ovaries from larger mammals. A number of methods have been discussed for isolating preantral follicles from domestic animal species [14] and a microdissection technique appears to be preferable for obtaining relatively large (120–220 µm), high-quality preantral follicles [14–17].

A few reports have described successful in vitro development of preantral follicles in domestic animal species (pigs [18, 19]; sheep [20], and bovine [15, 21]). When preantral follicles from prepubertal sheep ovaries were cultured in FSH and serum-containing medium, the follicles formed an antral cavity and produced estradiol (E2), which is normally synthesized at the antral follicle stage in vivo [20]. The authors showed that a limited number of oocytes from these follicles could achieve meiotic competence up to metaphase II after in vitro maturation. In bovine, Gutierrez et al. [15] recently reported that preantral follicles (166 ± 2.15 µm in diameter) could be maintained in a serum-free medium for a long-term period and reported the formation of antral cavities. However, that report did not demonstrate follicular cell function and differentiation associated with antrum formation. Gutierrez et al. incubated the preantral follicles directly in culture medium with no matrix, and occasionally, the follicles burst the basement membrane and extruded their granulosa cells during long-term culture. Recently, larger porcine preantral follicles (200–310 µm in diameter) were reported to grow to the antral stage after a short, 4-day culture period in a serum-containing medium, and the oocytes from these in vitro-matured preantral follicles acquired meiotic competence and could undergo fertilization and embryonic development [19].

In this study, our goals were to 1) establish a serum-free culture system that would be able to maintain the three-dimensional structure of large bovine preantral follicles for a long-term period, and 2) demonstrate the requirements and biological roles of insulin and insulin growth factors (IGFs) and their effects combined with FSH and LH on follicular and oocyte growth, antrum formation, and E2 production of preantral follicles using a serum-free culture system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Large Preantral Follicles

Ovaries from adult Japanese Black cows were collected at a local meat processing plant. Ovaries without a corpus luteum were selected and brought to the laboratory in a thermal flask at room temperature within 1 h, and were then rinsed several times in calcium-free and magnesium-free PBS. Ovaries were cut into small pieces using a surgical blade in Hepes-buffered Hanks salt tissue culture medium 199 (TCM-199; Life Technologies Inc., Rockville, MD) supplemented with BSA (1 mg/ml; Intergen Co., Purchase, NY), bovine pancreas aprotinin (50 U/ml; Sigma Chemical Co., St. Louis, MO) and gentamicin sulfate (10 µg/ml; Sigma). Large preantral follicles were isolated with a 27-gauge needle (Terumo Co. Ltd., Tokyo, Japan) fitted to a 1 ml syringe barrel under a stereomicroscope (Stemi 2000; Carl Zeiss, Jena, Germany) with a small amount of adherent stromal tissue. Follicle isolation was carried out at room temperature in a sterile environment. Follicles with diameters of 145–170 µm were collected with a mouth-operated fine pipette and washed twice with HP-M199 (Research Institute for the Functional Peptides, Yamagata, Japan) containing 1 mg/ml BSA. HP-M199 medium is identical to TCM-199 except that it is free of Tween-80 and para-aminobenzoic acid, but it contains 5 mM taurine, 5 nM selenium, and 10 µg/ml gentamicin sulfate. Taurine and selenium may act as free radical scavengers in serum-free medium. Morphology was assessed microscopically and only follicles with an intact round structure and a spherical, centrally located oocyte with no antrum were used in these experiments.

Culture of Large Preantral Follicles

Large preantral follicles with normal morphology were embedded within collagen gels and cultured up to 13 days at 38.5°C in an atmosphere of 5% CO2 and 95% air. Type I collagen gel solution was prepared by mixing 0.3% acid-soluble bovine type I collagen solution (Cellgen; Koken Co. Ltd., Tokyo, Japan) and 2x HP-M199 at a ratio of 1:1 (v:v). This solution usually gelled within 5 min at room temperature. To embed isolated follicles in the collagen gel, five follicles in 5 to 10 µl of HP-M199 were placed into 0.25 ml of collagen gel matrix in 24-well culture plates (Becton Dickinson Co., Ltd., Franklin Lakes, NJ). Immediately after the collagen gelled, culture medium was added to the wells. Basal control medium is a serum-free medium that contains HP-M199 with 1 mg/ml BSA, 50 U/ml bovine pancreas aprotinin (Sigma), and 2 mM hypoxanthine (Kohjin Co. Ltd., Tokyo, Japan). Hypoxanthine, a naturally occurring cAMP-phosphodiesterase inhibitor, is reported to maintain the association between a mouse oocyte and surrounding granulosa cells in vitro [22]. Single or combined supplementation of 20 ng/ml human recombinant insulin-like growth factor (IGF)-I (Biomedical Technologies Inc., Stougton, MA), 20 ng/ml human recombinant IGF-II (Biomedical Technologies), 20 ng/ml bovine insulin (Sigma), 50 ng/ml bovine FSH (bFSH-H058/H; Biogenesis Ltd., England) or 50 ng/ml bovine LH (bLH-i055; Biogenesis) were evaluated in the basal control medium.

Follicular diameter and the presence of antral formation was determined every other day using an inverted microscope (IMT-2; Olympus Optical Co. Ltd., Tokyo, Japan) equipped with an image processor (ARGUS-10; Hamamatsu Photonics K.K., Hamamatsu, Japan). Antral cavity formation was defined as a visible translucent area within the granulosa cell mass. At 48-h intervals, 500 µl of the conditioned medium was collected and fresh medium was added. After culture, the cumulus-oocyte complexes were microsurgically dissected from the follicles using a 27-gauge needle, and the diameter of oocytes were measured using an inverted microscope (IMT-2) equipped with an image processor (ARGUS-10). The conditioned media were stored at -30°C until E2 assays were performed.

Steroid Measurement

17ß-Estradiol concentration in the spent medium was measured with an E2 correlate enzyme immunoassay kit (Assay Designs Inc., Ann Arbor, MI) at 405 nm using a microplate reader (Model 3550; Bio-Rad Laboratories, Hercules, CA). The cross-reactions of anti-E2 antibody were as follows: E2, 100%; estrone, 4.64%; estriol, 0.53%; progesterone, 0.06%; testosterone, 0.02%; dehydroisoandrosterone, <0.001%; and dehydroisoandrosterone-3-sulfate, <0.001%.

Histological Assessment

Follicles were fixed for 1–3 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at 0–4°C. After fixation, the follicles were washed in PB and then postfixed for 1 h with 1% osmium tetroxide in PB at 0–4°C. All samples were dehydrated in ascending concentrations of ethanol solutions, substituted in propylene oxide, and embedded in epoxy resin (Taab Laboratories Equipment Ltd., Barkshire, U.K.). Semithin sections were cut with a sapphire knife on an ultramicrotome (Reichert Ultracuts, Leica, Heerbrugg, Switzerland). Semithin sections were placed on glass slides, stained with 1% toluidine blue, and examined using an Olympus BH2 light microscope.

Statistical Analyses

The mean follicle diameter of each treatment group at Days 0 through 13 of culture was analyzed by paired t-test. The mean oocyte diameter in each treatment group after 13 days of culture was analyzed by ANOVA. 17ß-Estradiol concentrations for each gel matrix (five follicles) were expressed as least squares means ± SEM. 17ß-Estradiol concentrations in each treatment in the same culture period were analyzed by ANOVA. All statistical analyses were performed with a Power Macintosh G4 computer and Stat View 4.0 statistical software (Abacus Concepts, Berkeley, CA). Antrum formation rates are represented as the number of antral-formed follicles divided by the total number of cultured follicles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Addition of insulin or IGF-I (P < 0.0001) and IGF-II or FSH (P < 0.0005) both showed increases in follicular diameters. However, the mean diameters of follicles cultured with LH showed a decrease (P < 0.005) after 13 days of culture (Table 1). The mean oocyte diameter of freshly isolated preantral follicles used in this study was 59.3 ± 1.8 µm (n = 25) at the onset of the culture period. Differences in mean oocyte diameters were observed between the follicles cultured in the basal control medium and those cultured with all hormones tested (P < 0.01) after 13 days of culture. In addition, differences in oocyte diameters were observed between IGF-I and IGF-II or LH treatments (P < 0.01).


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  		TABLE 1. Effects of insulin, IGFs, FSH, and LH on the growth of preantral follicles

The combined effects of FSH and LH with insulin (Table 2) or IGF-I (Table 3) were examined for their effects on the growth of preantral follicles and oocyte diameters as described in Table 1. All combinations showed that follicle diameters increased after 13 days of culture (P < 0.0001). In the presence of insulin, no significant difference in oocyte diameter was observed with the addition of FSH or LH alone. However, after treatment with a combination of insulin, FSH, and LH, oocyte diameters were larger than they were with insulin alone or insulin and LH in combination (P < 0.01). On the other hand, no differences in oocyte diameters were observed between treatments with IGF-I and FSH, or LH, or both.


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  		TABLE 2. Combination effects of FSH and LH on the growth of preantral follicles in the presence of insulin


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  		TABLE 3. Combination effects of FSH and LH on the growth of preantral follicles in the presence of IGF-I

Preantral follicles grown in the control basal medium throughout the entire culture period failed to form an antral cavity. However, the single addition of insulin, IGF-I, or FSH initiated antrum formation by 7 days of culture, and 56.7% (17 of 25), 60.0% (15 of 25), and 32.0% (8 of 25) of the follicles formed an antral cavity by 13 days of culture, respectively (Fig. 1A). When each factor was added individually, insulin induced E2 secretion significantly more than the other factors did after 9 days of culture (Fig. 1B).



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  		FIG. 1. Effect of insulin (Ins), IGF-I, IGF-II, FSH, or LH on antrum formation (A) and E2 production (B) of preantral follicles during 13 days in culture. Antrum formation rates are represented as the number of antral-formed follicles divided by the total number of cultured follicles. Twenty-five preantral follicles were used for each culture condition. Values for E2 production are expressed as the mean of five determinations ± SEM. Asterisks show significance to other values (P < 0.05) in the same culture period

The combination of insulin and FSH induced earlier antrum formation than did follicles cultured with insulin alone (Fig. 2A). When LH was present with insulin and FSH, even earlier antrum formation took place. Insulin combined with FSH and LH resulted in 6.7% and 30.0% of preantral follicles forming an antral cavity by Days 3 and 5 of culture, respectively. The maximal rates of antrum formation after 13 days of culture were similar whether the preantral follicles were grown in basal control medium containing only insulin or in a combination of all three hormones (Fig. 2A). 17ß-Estradiol production of cultured follicles in the presence of insulin was unaltered by the addition of FSH (Fig. 2B). However, when FSH and LH were added with insulin, significant increases in E2 secretion were observed after 5 days of culture compared with insulin alone, and insulin and FSH in combination. 17ß-Estradiol secretion reached a maximal level on Day 7 and was maintained through Day 13 of culture.



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  		FIG. 2. Combination effects of FSH and LH with insulin on antrum formation (A) and E2 production (B) of preantral follicles during 13 days in culture. Antrum formation rates are represented as the number of antral-formed follicles divided by the total number of cultured follicles. Thirty preantral follicles were used for each culture condition. Values for E2 production are expressed as the mean of six determinations ± SEM. Asterisks show significance to other values (P < 0.05) on the same culture period

Immediately after isolation, preantral follicles were oval or spherical in shape and contained one spherical oocyte with a germinal vesicle surrounded by at least two to three layers of granulosa cells (Fig. 3, a and c). The granulosa cells were limited by an intact basement membrane and an outer thecal layer (Fig. 3c). These basic morphological features of the follicle were preserved in these culture conditions. When preantral follicles were cultured in basal control medium containing insulin, FSH, and LH for 13 days, follicle and oocyte diameters became larger, and more granulosa and thecal cells and the antral cavity were all visible within the follicle (Fig. 3, b and d).



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  		FIG. 3. Light micrographs of whole (a and b) and semithin sections (c and d) of bovine follicles after isolation (a and c) and after culture (b and d). The representative follicle shown was cultured in basal control medium containing insulin (20 ng/ml), FSH (50 ng/ml), and LH (50 ng/ml) for 13 days. The follicle retained its oval shape, its volume increased, and these observations were accompanied by an apparent increase in granulosa (G) and thecal (T) cell numbers. An antrum (A) was visible in the cultured follicle. B, basal membrane; T, thecal cell layer. Bars = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated that bovine large preantral follicles embedded in collagen gels could be successfully maintained for a long period of time in a serum-free medium without losing their normal three-dimensional structure. This improved culture environment allowed investigation of the important physiological roles of insulin-related factors and gonadotropins on the growth, development, and differentiation of preantral follicles to the antral stage. Gomes et al. [23] revealed that the flat/adhesive cultures of mouse preantral follicles led to distortion of follicular morphology and frequent follicle disruption, but three-dimensional collagen gel cultures were able to support normal follicular structure and higher growth of preantral follicles in response to FSH. In our study, a three-dimensional collagen gel culture system with the use of a serum-free medium was able to maintain healthy follicles for the long term. This culture system allowed bovine preantral follicles and their oocytes to respond to growth factors and gonadotropins, which resulted in better growth, development, and function. Gutierrez et al. [15] identified antrum formation in follicles with confocal microscopy, but they found that it was difficult to discern an early antral cavity or early signs of follicular atresia. Therefore, we performed a histological analysis and measured E2 secretions to more accurately evaluate the effects of growth factors and gonadotropins on the growth, development, and differentiation of follicles.

In vivo, folliculogenesis is known to be regulated by both endocrine and intraovarian autocrine and paracrine factors. The ovarian follicle is an important structural and functional unit of the ovary and provides the microenvironment necessary for oocyte growth and development [24, 25]. There is growing evidence that endocrine growth factors, paracrine growth factors, or both play key roles in follicular development and that they modulate survival, proliferation, and differentiation of follicular cells, acting in concert with gonadotropins [26]. Insulin is commonly added to culture medium for in vitro growth of mammalian preantral follicles. In the present study, physiological concentrations of insulin and IGF-I significantly stimulated follicle and oocyte growth of bovine preantral follicles in a serum-free medium. The single addition of insulin or IGF-I induced high percentages (near 60%) of antrum formation after 13 days in culture, but E2 production was enhanced only by insulin.

In similar studies, bovine preantral follicles could grow even in the absence of gonadotropins when they were cultured in the presence of a physiological dose (10 ng/ml) of insulin [15]. Insulin led to enhanced cell proliferation in rat granulosa [27] and thecal cells [28]. Stimulation of follicular growth may be primarily due to the larger numbers of granulosa cells. The physiological roles of IGFs have been well characterized in large antral follicles, but fewer studies have shown the effects of IGFs during earlier stages of follicular development. Wandji et al. [29] demonstrated low levels of IGF-I mRNA expression in primary follicles of immature mice, but the expression increased to maximum levels during the late preantral and early antral stages. Moreover, apoptotic follicles had lower levels of IGF-I gene expression. IGF-I may be involved in the growth and survival of rapidly growing, large preantral and early antral follicles in mice [29, 30]. A recent report demonstrated that rat preantral follicles (140–160 µm in diameter) cultured in the presence of IGF-I in a serum-free medium led to a larger size, and that follicles maintained their normal morphology [31].

Estrogens are well-known endocrine and intrafollicular autocrine mitogenic compounds [32]. In mammals, these steroids are generally synthesized in granulosa cells of antral follicles. In our study, insulin strongly induced E2 production, which was accompanied by follicle antrum formation, but IGF-I showed no stimulation. Insulin has been found to stimulate ovarian steroidogenesis by both granulosa and thecal cells, thereby increasing production of androgens, estrogens, and progesterones [33]. Moreover, insulin has been shown to affect ovarian steroidogenesis by binding to its own receptor rather than cross-associating with IGF receptors [34, 35]. However, IGF-I and insulin both enhanced E2 production of bovine granulosa cells derived from large antral follicles when these cells were cultured in a serum-free medium [36]. The different sensitivity of IGF-I may depend on the granulosa cells derived from the different developmental stages of the follicles. Further study must be undertaken to elucidate the different response between insulin and IGF-I on the E2 secretion of follicles during antrum formation.

Follicle-stimulating hormone is known to have divergent effects on follicular growth and differentiation in mammals, both in vivo and in vitro [37, 38]. More than one group of researchers have shown that FSH is involved in growth and development of bovine preantral follicles in vitro [15, 39]. In the present study, FSH alone enhanced antrum formation, but it could not increase E2 secretion. This suggests that FSH may contribute to follicle development with a combined action of one or more other factors.

Addition of LH in the presence of FSH had a beneficial effect on growth by enhancing antrum formation and supporting the development of early antral follicles. When FSH and LH were added to the culture in the presence of insulin, stimulation of follicular and oocyte growth was augmented. In addition, antrum formation was initiated earlier, and higher levels of E2 production were maintained after antrum formation took place. Gutierrez et al. [15] reported that FSH led to a larger follicular diameter and antrum formation in bovine preantral follicles in insulin-containing medium. Our study also showed that FSH resulted in larger bovine follicles cultured in the presence of insulin. In large preantral human follicles, FSH has been reported to promote antrum formation and estrogen production in vitro [40]. Cecconi et al. [20] reported that sheep preantral follicles formed antral cavities and secreted high levels of E2 into serum-containing medium when they were cultured in the presence of high concentrations (1 µg/ml) of FSH in a low oxygen environment (5% O2). However, E2 production showed a rapid decline after 4 days in culture, probably because culture conditions were insufficient for sustaining adequate antral development of these follicles. It has also been reported that FSH promoted follicular survival and antrum formation in early preantral follicles in mice [41]. Because FSH has also been reported to suppress apoptosis in serum-free culture in rat preantral [42] and antral [43] follicles, it is possible that the physiological role of FSH may be to prevent atresia in preantral and antral follicles. Qvist et al. [8] revealed a positive effect of LH on large antral cavity formation when it was present in combination with FSH in culture. In addition, LH could also increase follicular survival, antrum formation, and estrogen secretion of mouse preantral follicles in culture when thecal cells were present in the follicles [44]. These results suggest an important role for thecal cells in regulating gonadotropin action in vitro.

In conclusion, the present study demonstrated that the normal three-dimensional structure of bovine preantral follicles could be maintained for up to 13 days in vitro in an improved serum-free culture system. This system allowed identification of the requirements and interactions of insulin and IGF-I with gonadotropins (i.e., FSH, LH) on follicular and oocyte growth, antrum formation, and follicular function (i.e., E2 secretion). This culture system should be useful for studying the regulation of early follicular growth, development, and function.


   FOOTNOTES
 
1 This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (PRO BRAIN). Back

2 Correspondence: Hiroyoshi Hoshi, Research Institute for the Functional Peptides, 4-3-32, Shimojo, Yamagata 990-0823, Japan. FAX: 81 23 646 2526; hoshih{at}func-p.co.jp Back

Received: 26 September 2001.

First decision: 17 October 2001.

Accepted: 26 April 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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