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J. Anim. Sci. 2004. 82:1967-1975
© 2004 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro1

J. Mao*, M. F. Smith*, E. B. Rucker*, G. M. Wu*, T. C. McCauley*, T. C. Cantley*, R. S. Prather*, B. A. Didion{dagger} and B. N. Day*,2

* Department of Animal Sciences, University of Missouri-Columbia, Columbia 65211; and and {dagger} Monsanto, St. Louis, MO 63198


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth’s (Exp. 1) or {alpha}-minimum Eagle’s essential medium (Exp. 2 and 3) supplemented with 10 µg/mL of transferrin, 100 µg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

Key Words: Apoptosis • Cell Proliferation • Epidermal Growth Factor • Insulin-Like Growth Factor I • In Vitro • Preantral Follicle


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Advances in reproductive biotechnology have increased the demand for large quantities of fertilizable oocytes. Current methods of in vitro embryo production depend on a supply of developmentally competent oocytes from large antral or preovulatory follicles, whose number is relatively small. Thus, research has focused on developing a preantral follicular culture system that can be used to study early folliculogenesis and to serve as a source of oocytes for in vitro embryo production. To date, fertilizable oocytes have been obtained from in vitro culture of primordial follicles (Eppig et al., 1996Go) and early preantral follicles in mice (Cortvrindt et al., 1996Go). In contrast, conditions for complete in vitro development of human and porcine preantral follicles have not been established. This difficulty is likely due to the greater follicle diameter and a thicker follicular wall, which restricts the transport of nutrients and gases during the long-term culture of follicles. In addition, growth of murine oocytes is complete by the preantral stage, whereas oocytes in porcine preantral follicles have not completed the growth phase (Morbeck et al., 1992Go). Consequently, culture systems for porcine preantral follicles must promote both somatic cell and oocyte growth. To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted a series of experiments designed to accomplish the following objectives: 1) characterize granulosal cell proliferation and apoptosis in cultured preantral follicles and 2) determine the effect of IGF-I and/or epidermal growth factor (EGF) on preantral follicular growth, antrum formation, and granulosal cell proliferation and apoptosis in both serum-free and serum-containing media.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Materials
Epidermal growth factor, recombinant human IGF-I, transferrin, L-ascorbic acid, and BSA (fraction V) were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal calf serum, Waymouth’s MB 752/1, medium-199 (M199), and {alpha}-minimum Eagle’s essential medium ({alpha}-MEM) culture media were purchased from Life Technologies (Gaithersburg, MD). Ninety-six-well plates were obtained from Fisher Scientific (Pittsburgh, PA). The in situ terminal deoxynucleotidyl transferase dUTP nick end-label (TUNEL) staining kit (ApopTag) was purchased from Intergen Co. (Purchase, NY). Anti-proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (PC-10) was obtained from Roche Diagnostics Corp. (Indianapolis, IN). Normal donkey serum and secondary donkey anti-mouse immunoglobulin (Ig)-G-fluorescein isothiocyanate (FITC) conjugate were purchased from Jackson ImmunoResearch Labs (West Grove, PA). Highly purified ovine FSH (NIDDK-oFSH-20; bioactivity potency = 4,453 U/mg) was kindly provided by the National Hormone and Pituitary Program of National Institute of Diabetes and Digestive and Kidney Diseases and A. F. Parlow of Harbor-UCLA Medical Center (Los Angeles, CA). On each day of the experiment, over 30 prepubertal gilt ovaries without large follicles (>3 mm in diameter) on the surface were collected at a local abattoir between February and June of 2001 and between September 2001 and February 2002 and transported to the laboratory within 3 h at 25 to 28°C.

Preantral Follicle Isolation and Selection
Ovarian cortical tissue (<1 mm thickness) was sliced from the ovarian surface with a hand microtome. Preantral follicles with a diameter of 250 to 300 µm (Mao et al., 2002Go) were visualized under the dissecting microscope and manually isolated with a 28-gauge needle and surgical blade. A HEPES-buffered Tyrode’s medium containing 0.01% polyvinyl alcohol (wt/vol) was used for collection and holding of isolated follicles (Mao et al., 2002Go). After isolation, follicles were pooled, washed three times in culture medium, and healthy follicles were selected based on their morphology as described by Gutierrez et al. (2000)Go. Briefly, follicles in which the oocyte and granulosal cells were completely surrounded by the basement membrane, thecal cells, and stromal tissue were classified as healthy. As demonstrated in cattle (Telfer, 1998Go), follicles selected according to the preceding criteria were not atretic.

Culture of Preantral Follicles
Culture medium was equilibrated to 39°C and 5% CO2 for 3 h before use. After isolation, preantral follicles were randomly allocated to treatment groups and placed individually in 200 µL of culture medium in 96-well plates (not tissue culture treated). The plates were incubated at 39°C under 5% CO2 in air. Half the medium (100 µL) was replaced with freshly prepared medium every 48 h. Follicular diameter was measured at X and Y dimensions (90° angle) between the basement membranes with an ocular micrometer at the beginning of and during culture.

Experimental Design
Various concentrations of IGF-I (1 to 100 ng/mL) and 10 ng/mL of EGF were chosen based on the previous studies (Gospodarowicz and Bialecki, 1979Go; Hsu et al., 1987Go; Guthrie et al., 1998Go; Sirotkin et al., 2002Go). In the preceding studies, 10 and 50 ng/mL of IGF-I and 10 ng/mL of EGF alone effectively stimulated granulosal cell proliferation and inhibited cellular apoptosis. Furthermore, EGF concentration in small follicles was approximately 13 ng/mL, which is higher than the large antral follicles (<6 ng/mL; Hsu et al., 1987Go).

Experiment 1.
To determine the incidence of granulosal cell proliferation and apoptosis in cultured follicles and to determine the effects of various IGF-I concentrations on preantral follicular growth and recovery rate of cumulus cell-oocyte complexes (COC), a total of 240 preantral follicles (six replicates) were cultured in Waymouth’s MB 752/1 medium supplemented with 10 µg/mL of transferrin, 100 µg/mL of L-ascorbic acid, 7.5% fetal calf serum, 2 mU/mL of ovine FSH, and various concentrations of IGF-I (0, 1, 10, and 100 ng/mL) for 8 d. On d 8, half of the follicles (n = 30 follicles per treatment group) were carefully opened with two fine needles (28 gauge), and the isolated oocytes were morphologically examined without prior knowledge of the treatment groups. A COC was defined as an oocyte covered with granulosal cells on more than 50% of the zona pellucida surface area. The recovery rate of COC from follicles was calculated based on the number of follicles cultured in each treatment group. Oocyte diameter, without the zona pellucida, was recorded. The remaining follicles were fixed in 10% (vol/vol) neutral buffered formalin (Sigma) overnight, embedded in paraffin, and serial sections (5 µm thickness) were prepared on saline-coated glass slides. The percentage of proliferating and apoptotic granulosal cells was assessed by immunohistochemical staining, as described below.

Experiment 2
. In Exp. 1, Waymouth’s medium was used and few preantral follicles developed an antrum. Consequently, a preliminary experiment was performed to compare the proportion of follicles that formed an antrum in the following media: Waymouth’s medium, M199, and {alpha}-MEM. The proportion of preantral follicles that developed an antrum was higher when cultured in M199 and {alpha}-MEM compared with Waymouth’s medium, but no difference existed between M199 and {alpha}-MEM. Therefore, {alpha}-MEM was used in Exp. 2 and 3.

The objective of Exp. 2 was to determine the effects of EGF and IGF-I on follicular growth, and the proliferation and apoptosis of granulosal cells in a defined, serum-free culture system. Basal culture medium was {alpha}-MEM supplemented with 0.1% BSA, 10 µg/mL of transferrin, 100 µg/mL of L-ascorbic acids, and 2 mU/mL of ovine FSH. Treatment groups consisted of the following: control, EGF (10 ng/mL), IGF-I (50 ng/mL), and EGF (10 ng/mL) + IGF-I (50 ng/mL). A total of 120 follicles (n = 30 follicles per treatment group, 3 replicates) were assigned randomly to each group and cultured individually, as indicated in Exp. 1, for either 4 or 8 d. The follicles that formed an antrum were recorded and follicular diameter between basement membranes was measured. On d 4, half the follicles from each group were fixed in 10% (vol/vol) neutral buffered formalin overnight at 4°C. The remaining half of the follicles was fixed on d 8 to assess granulosal cell proliferation and apoptosis in follicles.

Experiment 3.
In Exp. 2, proliferation of granulosal cells decreased precipitously in serum-free culture medium on d 4 and 8. Therefore, the objective of Exp. 3 was to determine the effect of EGF (10 ng/mL), IGF-I (50 ng/mL) or EGF + IGF-I on granulosal cell proliferation and apoptosis in follicles cultured in serum-containing medium for shorter periods (1, 2, 3, and 4 d). Culture medium was {alpha}-MEM supplemented with 7.5% fetal calf serum, 10 µg/mL of transferrin, 100 µg/mL of L-ascorbic acid, and 2 mU/mL ovine FSH, with or without the appropriate growth factor. A total of 240 preantral follicles (n = 15 follicles per group, per day; 5 replicates) was cultured for and fixed at 24, 48, 72, and 96 h. Then, follicles were processed as described above to determine granulosal cell proliferation and apoptosis. Follicular diameter and antrum formation were recorded during culture. Granulosal cell proliferation and apoptosis in freshly isolated preantral follicles were also determined before culture.

In Situ Terminal Deoxynucleotidyl TUNEL Assay
Sections of follicles were deparaffinized in xylene and rehydrated in a graded series of ethanol (100 to 70%, vol/vol). In situ TUNEL assay was conducted according to the manufacturer’s protocol. Briefly, tissue sections were incubated with proteinase K (20 µg/mL), treated with terminal deoxynucleotidyl transferase (TdT), incubated with antidigoxigenin antibody conjugated to FITC for 30 min, washed four times in PBS (pH 7.4), and mounted with VectaShield mounting medium containing 0.3 µg/mL propidium iodide (Vector Laboratories Inc., Burlingame, CA). Slides were examined under a fluorescent microscope (Nikon Eclipse 800, Nikon Instruments, Inc., Melville, NY). Images were captured with a CoolSnap HQ CCD camera and processed with MetaMorph software (Version 4.6, Universal Imaging Corp., West Chester, PA). For negative controls, the TdT enzyme was substituted with the same volume of PBS. Biologically negative and positive control slides were prepared from morphologically healthy and atretic follicles isolated from a prepubertal gilt ovary on the basis of their opacity, vascularization, and the integrity and uniformity of the granulosal cell layer. Follicles with a bright translucent appearance, good vascularization, and a regular granulosa were classified as healthy. Atretic follicles were classified as opaque, poorly vascularized, and had an irregular or detached membrane granulosa. Representative sections of in vivo healthy and atretic follicles are shown in Figures 5A and BGo, respectively.



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  		Figure 5. Representative images of morphologically healthy (A, negative control) and atretic (B, positive control) antral follicles freshly isolated from pig ovary, and follicles cultured for 4 d in serum-supplemented medium with epidermal growth factor (EGF) (C and D; observed by in situ terminal deoxynucleotidyl transferase dUTP nick end-label staining) in Exp. 3. A) Healthy follicle with intact and well-organized multilaminar granulosal cell layer and no apoptotic cells. B) Atretic follicle showing considerably thinned granulosal cell layer. Granulosal cells detached from the basement membrane with numerous degenerated and apoptotic granulosal cells. C and D) Follicles from EGF treatment group without or with antrum formation, demonstrating higher apoptosis in antral follicles than nonantral follicle and more apoptotic granulosal cells adjacent to the antrum than those in periphery area of the follicle. Basement membrane = BM. Granulosal cell = GC. Apoptotic granulosal cells are yellow in the merged images with propidium iodide staining (red) as indicated by arrowheads. Scale bar represents 50 µm.

 
Cell Proliferation Assay
As described above, slides were deparaffinized in xylene and rehydrated in a graded series of ethanol. Sections were subsequently incubated at room temperature (20 to 21°C) with blocking buffer containing 1% (vol/vol) normal serum for 30 min, and with anti-PCNA primary antibody (diluted 1:200, vol/vol) at 4°C overnight. For negative controls, adjacent tissue sections were incubated with the same amount of PBS. The slides were washed three times in PBS (5 min each) and incubated for 60 min at room temperature (20 to 21°C) with donkey anti-mouse IgG-FITC conjugate. Tissue sections were washed in PBS and mounted with VectaShield mounting medium containing 0.3 µg/mL of propidium iodide (Vector Laboratories Inc.). Slides were examined and processed as described above. The percentage of proliferating granulosal cells was expressed as the number of PCNA-positive nuclei per 100 granulosal cells.

To determine whether the proportion of proliferating or apoptotic cells was similar throughout the follicles, cell proliferation and TUNEL assays were performed on three sections, 30 µm apart from the same follicle tissue in the freshly isolated and cultured follicles (five follicles for each). The CV among sections was 4.1 and 5.9% for the TUNEL and cell proliferation assays (PCNA staining), respectively. For each immunohistochemistry assay, three sections from the same sample follicle tissue were processed for TUNEL and cell proliferation staining, and data from these sections were analyzed.

Statistical Analysis
All dependent variables were analyzed for normality using the Wilk-Shapiro test of the SAS (SAS Inst., Inc., Cary, NC). Heterogeneity of variance was detected in the cell proliferation data; consequently, these data were logarithmically transformed. Treatment effects on follicular diameter on different days of culture were analyzed using repeated measures GLM procedure of SAS. The percentages of intact follicles and recovery rate of COC were calculated based on total follicles in each treatment group (Exp. 1) and analyzed with the SAS GLM procedure. For analysis of oocyte diameter (Exp. 1) and percentage of apoptotic and proliferating granulosal cells, the SAS GLM procedure was also used. In the presence of a significant treatment effect, the comparison among the treatment groups was made by Duncan’s multiple-range test. An alpha level of 5% was used to indicate significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Experiment 1: Effect of IGF-I on Preantral Follicular Growth and Granulosal Cell Proliferation and Apoptosis
Follicular diameter at the beginning of culture (D0) and changes during culture on d 2, 4, 6, and 8 are presented in Figure 1Go. Follicular diameter did not differ among the treatment groups at the beginning of culture (mean diameter = 245.4 ± 2.0 µm). There was no main effect of IGF-I on follicular diameter on different days of culture; however, there was a significant interaction between IGF-I treatment and day of culture (P < 0.01). Further analysis of the follicular growth curves showed that 0 and 1 ng/mL of IGF-I did not sustain follicular growth after d 4, whereas 10 and 100 ng/mL of IGF-I did sustain follicular growth. Follicular diameter on d 8 was 255.1 ± 5.6, 268.8 ± 5.9, 281.6 ± 5.9, and 287.6 ± 5.6 for the control, 1, 10, and 100 ng/mL of IGF-I groups, respectively. Follicular diameter in the 10 and 100 ng/mL groups was higher on d 8 compared with the control group (P < 0.05).



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  		Figure 1. Changes in the diameter of follicles cultured with 0, 1, 10, or 100 ng/mL IGF-I during the 8 d culture period in Exp. 1. An interaction (P < 0.01) between IGF-I concentration and day of culture was detected. Follicular diameters did not change from d 4 to 8 in the 10 and 100 ng/mL groups, and decreased for the 0 ng/mL IGF-I group. Letters (a, b, c) indicate that follicular diameters on d 8 bearing different letters are significantly different (P < 0.05).

 
Morphologically intact follicles were defined as follicles whose oocyte and granulosal cells were completely surrounded by the basement membrane. A total of 120 intact preantral follicles was placed in culture. The proportion of intact follicles during culture, the recovery rate of COC isolated from follicles, and the diameters of oocytes on d 8 of culture are summarized in Table 1Go. High concentrations of IGF-I (10 and 100 ng/mL) increased the percentage of intact follicles and the recovery rate of COC compared with the control group (P < 0.05). The average oocyte diameter was 72.8 ± 1.2 µm at the beginning of culture and did not increase after culture. In addition, no differences were found in oocyte diameter among the treatment groups on d 8.


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  		Table 1. Effect of various concentrations of IGF-I (0, 1, 10, and 100 ng/mL) in culture medium on the proportion of intact follicles, recovery rate of cumulus cell–oocyte complexes (COC) isolated from follicles and oocyte diameter without zona pellucida on d 8 in Experiment 1
 
The proportion of proliferating granulosal cells, indicated by PCNA-positive cells, was 24.6 ± 1.5% in freshly dissected preantral follicles. However, after 8 d of culture, the proportion of proliferating granulosal cells was 4.1 ± 1.6, 0.1 ± 1.4, 12.1 ± 1.7, and 10.5 ± 1.3% for the 0, 1, 10, and 100 ng/mL of IGF-I groups, respectively. The overall granulosal cell proliferation from all treatment groups on d 8 was 10.4 ± 1.1% (P < 0.01). Granulosal cell proliferation in the 10 and 100 ng/mL IGF-I groups was higher compared with the control group (P < 0.01). There was no difference between the 0 and 1 or between the 10 and 100 ng/mL of IGF-I treatment groups.

The percentage of apoptotic granulosal cells in freshly isolated preantral follicles was 0.1 ± 0.04%. After 8 d of culture, the overall percentage of apoptotic granulosal cells increased to 7.5 ± 0.7% (P < 0.05). The percentage of apoptotic granulosal cells in the 10 and 100 of ng/mL IGF-I groups (4.1 ± 0.6 and 2.0 ± 0.2%) was lower (P < 0.01) than the 0 and 1 ng/mL of IGF-I groups (14.7 ± 1.9 and 14.1 ± 1.7%). No difference existed between 0 and 1 or between 10 and 100 ng/mL of IGF-I treatment groups.

Experiment 2: Effect of IGF-I and EGF on Preantral Follicular Growth and Granulosal Cell Proliferation and Apoptosis in a Serum-Free Culture Medium
There was no difference among treatment groups in follicular diameter at the beginning of culture (mean diameter = 254.7 ± 1.8 µm). Overall, effects of treatment and day of culture and treatment x day interaction on follicular diameter were detected (P < 0.01). In the control group, follicular diameter increased during the first 4 d of culture and subsequently decreased. Follicular diameters in the EGF, IGF-I, and EGF+IGF-I treatment groups increased during the first 4 d of culture and were sustained, which resulted in the follicular diameters on d 4, 6, and 8 of culture being larger than the control group (P < 0.01; Figure 2Go). An effect of treatment on antrum formation was also observed (P < 0.05). There was no difference between the IGF-I (25.0 ± 6.3%) and the control group (19.3 ± 7.1%) or between EGF (44.8 ± 7.9%) and the EGF+IGF-I group (45.0 ± 7.4%). However, compared with the control group, the proportion of antral follicles after culture was increased by EGF and EGF+IGF-I supplementation (P < 0.01).



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  		Figure 2. Changes in the diameter of follicles in the control, epidermal growth factor (EGF, 10 ng/mL), IGF-I (50 ng/mL), and EGF+IGF-I groups of Exp. 2. There was an interaction (P < 0.01) between the growth factor and day of culture. Compared with the control, EGF, IGF-I, and EGF+IGF-I stimulated and sustained preantral follicular growth, resulting in follicular diameters on d 4, 6, and 8 of culture in the EGF, IGF-I, and EGF+IGF-I groups that were larger (**P < 0.01) than the control group.

 
Granulosal cell proliferation on d 4 and 8 was low (1 to 5%) in serum-free medium as shown in Figure 3AGo irrespective of whether EGF and/or IGF-I were present On d 4 of culture, granulosal cell proliferation in the IGF-I group was higher (P < 0.01) compared with the control group. However, no difference was found among the EGF, EGF+IGF-I, and control groups. On d 8, no difference was detected in granulosal cell proliferation among the four groups.



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  		Figure 3. Effect of EGF (epidermal growth factor, 10 ng/mL), IGF-I (50 ng/mL), and EGF + IGF-I on granulosal cell proliferation (A) and apoptosis (B) on d 4 and 8 in serum-free culture medium (Exp. 2). Columns represent the mean (± SEM). Letters (a, b, c) indicate that means bearing different letters are different (P < 0.05).

 
Granulosal cell apoptosis was lower in the EGF and IGF-I groups compared with the control group on d 4 of culture (P < 0.05), but it did not differ from the control group on d 8 (Figure 3BGo). The proportion of apoptotic granulosal cells was higher in the presence of both EGF and IGF-I than with either factor alone on d 4 and 8 (P < 0.05). Analysis of the pattern of apoptotic granulosal cells in cultured follicles indicated that more granulosal cells adjacent to the antrum were apoptotic compared to those in the periphery of the follicle section.

Experiment 3: Effect of IGF-I and EGF on Preantral Follicular Growth and Granulosal Cell Proliferation and Apoptosis in a Serum-Supplemented Culture Medium
The mean diameter of follicles at the beginning of culture was 252.6 ± 1.9 µm. The growth pattern of follicles during the 4-d culture period was similar to that in Exp. 2 for the first 4 d, resulting in follicular diameters in the EGF, IGF-I, and EGF+IGF-I groups being larger than those of the control group (P < 0.05). The proportion of antral follicles in the IGF-I group (18.0 ± 5.8%) did not differ from the control group (22.0 ± 7.3%). However, a higher proportion of antral follicles was observed in the EGF (68.8 ± 9.1%) and EGF+IGF-I (62.0 ± 14.2%) groups compared with the control group (P < 0.01).

Effects of treatment, day, and treatment x day interaction on granulosal cell proliferation were detected (P < 0.01; Figure 4AGo). The beneficial effect of IGF-I on stimulating granulosal cell proliferation was observed on all days of culture. An effect of EGF on granulosal cell proliferation was not detected during the first 3 d of culture, but it was observed on d 4. The percentage of proliferating granulosal cells in the EGF+IGF-I group was lower than that observed for the EGF and IGF-I groups (P < 0.01). Granulosal cell proliferation was high in all follicles for the first 3 d of culture, but fell dramatically on d 4 in all media except that containing IGF-I. Effects of treatment and treatment x day interaction on granulosal cell apoptosis were detected (P < 0.02; Figure 4BGo). Compared with the control group, addition of EGF, IGF-I, or EGF+IGF-I suppressed apoptosis of granulosal cells (P < 0.01). Similar to Exp. 2, more granulosal cells adjacent to the antrum were apoptotic than those in the periphery of the follicles (refer to Figure 5DGo).



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  		Figure 4. Effect of EGF (epidermal growth factor, 10 ng/mL), IGF-I (50 ng/mL), and EGF + IGF-I on granulosal cell proliferation (A) and apoptosis (B) on d 1, 2, 3, and 4 in serum-containing medium (Exp. 3). Columns represent the mean (± SEM). Letters (a, b, c) indicate that means bearing different letters are different between treatment groups within day of culture (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Current experiments examined the role of IGF-I and EGF in regulating preantral follicular growth and antrum formation, granulosal cell proliferation and apoptosis in cultured preantral follicles. These experiments provided some new and direct evidence to show that EGF and IGF-I sustained preantral follicular growth, suppressed granulosal cell apoptosis, and stimulated granulosal cell proliferation and follicular antrum formation. This is also the first report to show that IGF-I increased the recovery rate of COC from cultured follicles.

Selection of culture conditions is critical for the growth of preantral follicles in vitro. Our previous study compared simple culture medium with complete medium and concluded that complete medium containing AA and vitamins was suitable for long-term culture of preantral follicles (Mao et al., 2002Go). Waymouth’s and {alpha}-MEM media are both complete media and were used in the current experiments. However, more preantral follicles formed an antrum when they were cultured in {alpha}-MEM compared with Waymouth’s medium in Exp. 2. In agreement with the current study, previous studies by Hirao et al. (1994)Go and Shuttleworth et al. (2002)Go reported only 19 and 29% of cultured pig preantral follicles formed an antrum in Waymouth’s media. The reason for the difference between media is not clear. We speculated that some ingredients in Waymouth’s medium might have negative effects on antrum formation. A long-term goal is to develop a defined culture system (serum free) for porcine preantral follicles; however, serum is an important component of culture medium, which provides growth factors and improves pig preantral follicular growth (Telfer et al., 2000Go). In the current study, granulosal cell proliferation was low on both d 4 and 8 in serum-free culture medium in Exp. 2. When serum was present in the medium (Exp. 3), cellular proliferation was high during the first 3 d of culture, but dropped on d 4, implying that the concentration of growth factors in the serum was not high enough to sustain cellular proliferation for a prolonged period or that serum had a negative effect on follicular growth after 3 d of culture.

High granulosal cell proliferation, low granulosal cell apoptosis, follicular fluid accumulation, and an increase in oocyte size each contribute to follicular growth. In the current study, IGF-I and EGF stimulated and sustained follicular growth in both serum-free and serum-containing culture media. The importance of IGF-I in regulating follicular growth has been studied for many years. Initial evidence came from the discovery that the IGF-I receptor was expressed by granulosal cells (Veldhuis and Furlanetto, 1985Go; Zhou et al., 1996Go). Furthermore, primary culture of granulosal cells isolated from antral follicles has shown that IGF-I stimulated porcine granulosal cell replication (Baranao and Hammond, 1984Go; May et al., 1988Go) and prevented spontaneous apoptosis of granulosal cells in serum-supplemented medium (Guthrie et al., 1998Go). Using a whole-follicle culture technique, the current study has confirmed those discoveries and extended them to preantral follicles by demonstrating that IGF-I consistently supported preantral follicular growth, stimulated cellular proliferation, and suppressed apoptosis of granulosal cells, as well as enhanced COC recovery. These beneficial effects predominated when preantral follicles were cultured in serum-supplemented medium. Since IGF-I did not increase oocyte diameter or stimulate follicular antrum formation, its stimulatory effect on follicular growth was achieved mainly by increasing granulosal cell proliferation and inhibiting cellular apoptosis, as shown in those experiments with or without serum supplementation.

In addition to the well-documented effects of IGF-I on granulosal cells, it seems likely that IGF-I also has an effect on oocytes, as mRNA expression of IGF-I and its receptor was observed in the growing oocytes of human infant ovaries and in mature oocytes of adult ovaries (Zhou and Bondy, 1993Go). Furthermore, IGF-I receptors were detected in the porcine oocyte of preantral follicles (Quesnel, 1999Go), further suggesting that IGF may be involved in the process of oocyte growth. In fact, IGF-I increased the number of oocyte cortical granules in the rat (Zhao et al., 2001Go). In the present study, more COC were retrieved from follicles cultured with IGF-I; however, IGF-I had no effect on oocyte growth, which is in agreement with the observation in cattle (Gutierrez et al., 2000Go). These results indicate that, although IGF-I may play an important role in follicular growth, additional factors acting alone or in combination with IGF-I may be required for oocyte growth. In support of this hypothesis, Wu et al. (2002)Go showed that co-culture of pig preantral follicles with cumulus cells isolated from large antral follicles (>3 mm in diameter) stimulated oocyte growth. Therefore, some factor(s) secreted by antral follicle cumulus cells exert(s) beneficial effects on oocyte growth and development. More research is needed to identify those factors and study their roles in regulating oocyte growth.

The effect of EGF on preantral follicular growth and development, in culture media, with and without serum supplementation was also studied. A mitogenic effect of EGF on granulosal cells was not observed until d 4 in serum-containing medium. In the presence of EGF, granulosal cell apoptosis was suppressed and antrum formation increased in both serum-free and serum-supplemented culture media. Therefore, EGF has various effects on preantral follicles and its effect on follicular growth may be mainly due to the suppression of apoptosis and stimulation of antrum formation. EGF and its receptor are synthesized locally in the porcine ovary (Singh et al., 1995Go). The concentration of EGF is higher in the follicular fluid of small antral follicles (13.6 ± 1 ng/mL) than in the large preovulatory follicles (< 6 ng/mL; Hsu et al., 1987Go). Epidermal growth factor is thought to mediate its biological actions through a complex signal transduction pathway involving the activation of mitogen-activated protein kinases (Keel et al., 1995Go). Epidermal growth factor stimulates growth of preantral follicles in cows (Gutierrez et al., 2000Go), hamsters (Roy, 1993Go), mice (Boland and Gosden, 1994Go), and humans (Roy and Kole, 1998Go) in vitro. In combination with serum, EGF stimulated proliferation of cultured porcine theca cells (May et al., 1992Go). Thus, EGF has been implicated in regulation of preantral follicle development (Campbell, 1999Go; Roy, 1993Go). The FSH plays a major role in the initiation of antrum formation, whereas EGF increased FSH binding in porcine granulosal cells isolated from small follicles (May et al., 1987Go). This may explain why EGF enhanced antrum formation in the current study.

Epidermal growth factor suppressed apoptosis of granulosal cells and stimulated antrum formation in cultured preantral follicles. Interestingly, when follicles formed an antrum, granulosal cells adjacent to the antrum underwent apoptosis in both serum-free and serum-containing media as indicated in Figure 5DGo. These results indicate that EGF might play a role in follicular antrum formation in synergism with FSH; however, the microenvironment in the antrum might not be conducive for granulosal cell proliferation and survival. This suggestion is substantiated by the higher proportion of apoptotic granulosal cells adjacent to the antrum and lower cellular proliferation observed in the groups receiving EGF.

Both EGF and IGF-I stimulated granulosal cell proliferation and inhibited apoptosis of granulosal cells in cultured preantral follicles. Apoptosis has been identified as a mechanism of follicular atresia (Hsueh et al., 1994Go). Two patterns of atresia (Type A and B) have been described in canine follicles (Spanel-Borowski, 1981Go). Type A involved prominent necrotic changes in the oocyte and zona pellucida, whereas alterations of the granulosal cells were secondary. Type B atresia was typified by distinctive degenerative changes in granulosal cells with an almost unchanged oocyte and zona pellucida. Type A atresia predominated in the preantral follicles, whereas type B atresia occurred in antral follicles (Spanel-Borowski, 1981Go). To determine whether a follicle is healthy or atretic, the percentage of proliferating and apoptotic granulosal cells has been used as a marker in the mouse (Byskov, 1974Go) and in humans (Westergaard, 1982Go). In the rat, an incidence of 5% apoptotic granulosal cells was used as a sign of early atresia (Byskov, 1974Go). In the human, healthy nonovulatory follicles had a low rate of cell apoptosis and a high rate of cell proliferation, whereas atretic follicles contained many apoptotic nuclei. In the healthy human follicles, granulosal cell proliferation rate is higher than 16% (Westergaard, 1982Go). When the same criteria are applied to porcine preantral follicles in the current study, the relatively high proliferation rate and low incidence of apoptosis in granulosal cells in the freshly isolated preantral follicles indicate that the preantral follicles were nonatretic. However, after culture for a few days, many follicles were likely not healthy because the proliferation rate did not reach 16%. The results suggest that more work is needed to clearly define the regulation of preantral follicular growth.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Epidermal growth factor and insulin-like growth factor I sustained follicular growth by stimulating granulosal cell proliferation and follicular antrum formation and suppressed cellular apoptosis. Both growth factors seem to have an important role in the regulation of preantral follicular growth. Epidermal growth factor enhanced antrum formation and may be involved in the regulation of follicular antrum differentiation. The effect of IGF-I on suppressing apoptosis and promoting replication of granulosal cells strongly suggests that it may also play an important role in suppressing follicular atresia in porcine preantral follicles. These results may provide some information that is useful for developing an in vitro culture system for preantral follicles in which the oocytes are capable of fertilization and embryonic development.


   Footnotes
 
1 Financial support was provided by the collaborative animal research program between the Univ. of Missouri-Columbia, Dept. of Anim. Sci. and Monsanto Anim. Agric. Group: Development of Biotechnology Tools for Improved Genetic and Reproductive Performance in Swine. Back

2 Correspondence: 159 Animal Science Research Center, 920 East Campus Dr. (phone: 573-882-7555; fax: 573-884-7827; e-mail: DayB{at}missouri.edu).

Received for publication October 22, 2003. Accepted for publication February 18, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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