Avian Germinal Disc Region Secretes Factors That Stimulate Proliferation and Inhibit Progesterone Production by Granulosa Cells'

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1 BIOLOGY OF REPRODUCTION 54, (1996) Avian Germinal Disc Region Secretes Factors That Stimulate Proliferation and Inhibit Progesterone Production by Granulosa Cells' Shelley A. Tischkau and Janice M. Bahr 2 Departments of Animal Sciences and Physiology University of Illinois at Urbana-Champaign, Urbana, Illinois ABSTRACT Microscopic analysis of ovarian follicles in the domestic hen has revealed differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germinal disc, which contains the female gamete. These differences appear as a morphological gradient, which implies variations in granulosa cell function. This observation prompted us to hypothesize that the germinal disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions: 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of a preovulatory follicle? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis? Incorporation of 3H-thymidine was used as a measure of proliferation, and production of progesterone was used as a measure of steroidogenesis. In the first experiment, 8-mm-diameter sections were obtained from three morphologically distinct regions of the granulosa monolayer: 1) the GDR, 2) granulosa cells distal to the GDR (distal granulosa), and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa cells). The GDR incorporated the most 3 H- thymidine and produced the least progesterone. Distal granulosa cells incorporated the least 3 H-thymidine and produced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of 3 H-thymidine and produced an intermediate amount of progesterone. To answer the second question, conditioned medium was prepared from GDRs and distal granulosa cells (control) obtained from the F1 (largest preovulatory follicle) and F3 (the third-largest preovulatory follicle) follicles. Sections (8-mm in diameter) of the distal granulosa layer (F3 for 3 H-thymidine incorporation, F1 for progesterone production) were incubated in GDR-conditioned medium or granulosa cell-conditioned medium to determine whether factors secreted into the medium by the GDR and distal granulosa cells affect granulosa cell proliferation and/or steroidogenesis. Certain samples of GDR-conditioned medium and granulosa cell-conditioned medium were boiled, protease-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and protease-sensitive factors that promoted proliferation and inhibited progesterone production by granulosa cells. These data indicate that diametrically opposed gradients of proliferation and steroidogenesis are present within the granulosa layer of an individual preovulatory follicle. Furthermore, the GDR produces proliferation-stimulating and steroidogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cells. INTRODUCTION The avian preovulatory follicle undergoes a rapid growth phase in the 5-7 days prior to its ovulation. The oocyte more than triples in diameter during this period, primarily because of the incorporation of yolk from the blood. Additionally, granulosa and theca layers proliferate to accommodate the increase in size. The function of granulosa cells and their role in follicular development have been extensively studied [1-9]. These studies have primarily focused on maturational changes occurring within the granulosa layer as a whole. However, anatomical and physiological data suggest that the granulosa layer is not a uniform layer of cells. Granulosa cell morphology and function may be dependent upon the location of the granulosa cell relative to the germinal disc or female gamete. Light and electron microscopy [10, 11], flow cytometry [12], and 3 H-thymidine incorporation studies [13, 14] Accepted December 15, Received September 25, 'Supported in part by NSF and NIH-PHS-HDO Correspondence: Dr. Janice M. Bahr, Department of Animal Sciences. 326 Animal Sciences Laboratory., 1207 W. Gregory Dr., Urbana, IL FAX: (217) : jmbahr@uxl.cso.uiuc.edu have demonstrated that the columnar granulosa cells located near the germinal disc are in active stages of mitosis. The cuboidal granulosa cells located distally to the germinal disc contain lipid droplets, which are indicative of steroidsynthesizing cells [11]. Collectively, these data suggest that the germinal disc influences the physiology of the granulosa cell because granulosa cells associated with the germinal disc are immature and proliferate rapidly, and granulosa cells located distally to the germinal disc are more differentiated. Until recently, the role of the oocyte in regulating follicular function has received little attention. In the bird, the germinal disc is that portion of the oocyte that contains the female gamete and the majority of oocytic organelles; metabolic functions of the oocyte occur in this region. The germinal disc appears as a white plaque on the surface of the oocyte. Cytoplasmic processes from the granulosa cells, which form firm connections with the plasma membrane of the oocyte [10, 11, 15], are most prominent in the germinal disc region (germinal disc + overlying granulosa cells; GDR). Gap junctions provide a means of communication between the germinal disc portion of the oocyte and the overlying granulosa layer [16, 17]. A recent study has re- 865

2 866 TISCHKAU AND BAHR diameter) and, in certain experiments, the third largest preovulatory follicle (F3; approximately 25 mm in diameter) were removed, and the granulosa layer was isolated [19]. The germinal disc, which is tightly coupled to the overlying granulosa layer at this time [16], was removed with the granulosa layer. 3 H-Thymidine Incorporation FIG. 1. Structural differences in the granulosa layer are shown in this diagrammatic representation of a cross-section of a chicken preovulatory follicle. Diagram also shows site of samples taken for experiments. vealed the importance of the germinal disc in follicular growth. Destruction of the GDR of a follicle in the rapid growth phase blocked ovulation and lead to atresia [18]. The GDR has been proposed to be the "growth center" of the preovulatory follicle [10-12, 18]. Although it is apparent that a viable GDR is essential for completion of follicular maturation, the precise mechanism by which the GDR performs this function is unknown. We hypothesized that the GDR participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions: 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of an individual preovulatory follicle? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis? Animals MATERIALS AND METHODS Single-comb white Leghorn hens were caged individually and provided feed and water ad libitum. Hens were exposed to 17 h of light and 7 h of darkness with lights-on at 0400 h. Ovipositions were monitored by visual inspection at 30-min intervals from 0800 h to 1500 h, and again at 1700 h, to time late ovipositions. Hens in their first year of laying with regular clutches of at least six eggs were used for these experiments. Tissue Collection Hens were killed by cervical dislocation h before the expected time of ovulation of a midsequence follicle as determined from the time of previous ovipositions. The largest preovulatory follicle (F1; approximately 35 mm in Incorporation of 3 H-thymidine was determined according to a modification of the method of Dorrington et al. [20]. Briefly, tissues were incubated in 1 ml Dulbecco's Modified Eagle's Medium (DMEM) containing 1 ici 3 H-thymidine for 24 h at 37 C. Tissues were collected and sonicated in 0.5 ml Ca 2+- Mg2+-free Dulbecco's PBS (Sigma, St. Louis, MO); 0.5 ml of a 0.25% trypsin solution in Ca 2+ - Mg2+-free Dulbecco's PBS was added, and tissue was incubated at 37 0 C for 15 min. The preparation was frozen at - 20C, thawed, and filtered through Whatman (Clifton, NJ) DE-81 filter paper. Filters were washed twice with 2 ml distilled water, dissolved in 3 ml Cytoscint (Fisher Scientific Co., Pittsburgh, PA), and counted in a liquid scintillation counter to a 2% error. DNA was quantified spectrophotometrically. Progesterone and Protein Assays Progesterone content of culture medium was measured by RIA as previously described [21]. Progesterone production was measured in samples that were unstimulated or treated with 25 ng/ml ovine LH (NIADDK-oLH-26). The amount of protein in the tissue samples was determined by the Bio-Rad protein assay (Bio-Rad. Hercules, CA). Experiment 1: Are Physiological Gradients of Proliferation and Steroidogenesis Present Within the Granulosa Layer of a Preovulatory Follicle? Sections (8 mm in diameter) were obtained from three morphologically distinct regions of the granulosa layer (Fig. 1) by means of a circular metal punch: 1) GDR, 2) granulosa cells distal to the GDR (distal granulosa), and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa). Tissues were incubated in 1 ml DMEM for 2 h (progesterone production) or 24 h ( 3 H-thymidine incorporation) at 37 C. Medium was collected after a 2-h incubation and analyzed for progesterone content. Alternatively, tissues were collected after 24 h to measure the rate of incorporation of [methyl,1',2' -H]thymidine (Amersham, Arlington Heights, IL), which had been added to the culture medium (1 Ci/ml). Experiment 2a: Does the GDR Secrete Factors That Affect Granulosa Cell Proliferation? Sections (8 mm in diameter) of GDRs and distal granulosa cells (control) from F3 and F1 follicles were incubated individually in DMEM for 24 h at 37 C to produce condi-

3 GERMINAL DISC REGION REGULATES GRANULOSA CELL FUNCTION 867 tioned medium (GDR-conditioned medium [GDR-CM] and granulosa cell-conditioned medium [Gr-CM]). Gr-CM was obtained as a control. To determine whether the GDR-CM and Gr-CM altered proliferation of granulosa cells, distal granulosa cells from the F3 follicle were used as a bioassay because the F3 follicle is less differentiated than the F1 follicle, and thus F3 granulosa cells have a greater potential to respond to putative factors that promote proliferation. Therefore, on the following day, 8-mm-diameter sections of the distal granulosa cells from the F3 follicle were incubated for 24 h (37 C) in 0.5 ml GDR-CM or Gr-CM, each of which was supplemented with 0.5 ml fresh DMEM to which 1 ici 3 H-thymidine was added. As an additional control, distal granulosa cells from the F3 follicle were incubated in 1 ml DMEM + 1 Ci 3 H-thymidine for 24 h at 37 C. To determine whether proliferation of granulosa cells was dependent upon proteins present in conditioned medium, GDR-CM and Gr-CM were boiled for 10 min or treated with 0.5 units of insoluble pronase (a nonspecific protease linked to agarose; Sigma) overnight at room temperature prior to incubation with distal granulosa cells of the F3 follicle. Protease was removed from conditioned media by a 15-min centrifugation at X g. To determine whether steroids present in conditioned media promoted proliferation, conditioned media were treated with charcoal dextran (1 mg charcoal: 0.1 mg dextran) to remove steroids and other small molecules for 1 h prior to incubation with distal granulosa cells from the F3 follicle on Day 2. Experiment 2bh: Does the GDR Secrete Factors That Affect Granulosa Cell Steroidogenesis? To determine whether the GDR-CM and Gr-CM influenced progesterone production by granulosa cells, F1 granulosa cells were used as a bioassay because they are more differentiated and therefore, have a greater potential than less mature follicles to produce progesterone in response to a stimulus. GDR-CM and Gr-CM were stripped with charcoal dextran to remove progesterone produced by the GDR and distal granulosa cells during incubation to prepare conditioned medium. Distal granulosa cells (8-mm-diameter sections) were incubated in 0.5 ml GDR-CM or Gr-CM, each of which was supplemented with 0.5 ml DMEM. As an additional control, distal granulosa cells from the F1 follicle were incubated in 1 ml DMEM for 2 h at 37 C. After a 2-h incubation, media were removed, and progesterone content was measured. To determine the nature of the steroidogenesis inhibiting factor, certain conditioned medium samples were boiled for 10 min or treated with insoluble protease (as described for experiment 2a) prior to incubation with distal granulosa cells from the F1 follicle. Statistical Analysis All experiments were repeated three to five times. Data are presented as means + SEM. One-way analysis of vari- FIG H-Thymidine incorporation by F1 granulosa cells 1) associated with GDR, 2) located midway between GDR and distal granulosa cells (pgr), and 3) located distal to GDR (dgr). Data represent means ± SEM of five independent experiments. Different letters indicate statistically significant differences (p < 0.05) as determined by ANOVA and the least significant differences method. ance and the least significant differences method were used to determine statistically significant differences between groups; p < 0.05 was considered significant. RESULTS Experiment 1: Are Physiological Gradients of Proliferation and Steroidogenesis Present Within the Granulosa Layer of a Preovulatory Follicle? The GDR incorporated the most 3 H-thymidine, and distal granulosa cells incorporated the least 3 H-thymidine (Fig. 2). Proximal granulosa cells incorporated an intermediate amount of 3 H-thymidine. The GDR produced low amounts of progesterone and FIG. 3. Basal and LH-stimulated progesterone production by GDR, proximal granulosa cells (pgr), and distal granulosa cells (dgr) of an F1 follicle. Data represent means SEM of five independent experiments. Different letters indicate statistically significant differences (p < 0.05) as determined by ANOVA and the least significant differences method.

4 868 TISCHKAU AND BAHR FIG. 4. Effects of F3 and F1 GDR-CM on 3 H-thymidine incorporation by F3 granulosa cells. F3 granulosa cells were incubated in distal Gr-CM and fresh medium (medium) as controls. Data represent means SEM of five independent experiments. Letters indicate statistically significant differences (p < 0.05) within follicle type determined by ANOVA and the least significant differences method. FIG. 6. Effects of F3 and F1l GDR-CM on progesterone production by F1 granulosa cells. F1 granulosa cells were incubated in Gr-CM and fresh medium (medium) as controls. Data represent means SEM of three to five independent experiments. Letters indicate statistically significant differences (p < 0.05) within follicle type determined by ANOVA and the least significant differences method. was minimally stimulated by LH (Fig. 3). Proximal granulosa cells produced intermediate amounts of progesterone and had an intermediate response to LH. Basal progesterone secretion was significantly higher by distal granulosa cells than by GDRs and proximal granulosa cells and had a substantially greater response to LH. Experiment 2a: Does the GDR Secrete Factors That Affect Granulosa Cell Proliferation? F3 granulosa cells incubated in F3 or F1 GDR-CM incorporated significantly more 3 H-thymidine than F3 granulosa cells incubated in F3 or F1 Gr-CM or in control medium (Fig. 4). 3 H-Thymidine incorporation by F3 granulosa cells was equally stimulated by F3 GDR-CM and F1 GDR-CM. Boiling and protease treatment of F1 GDR-CM abolished its proliferation-promoting activity. Charcoal stripping of F1 GDR-CM did not reduce the stimulatory effect of F1 GDR- CM on proliferation of F3 granulosa cells (Fig. 5). Experiment 2b: Does the GDR Secrete Factors That Affect Granulosa Cell Steroidogenesis? F3 or F1 GDR-CM significantly suppressed progesterone production by F1 distal granulosa cells compared to Gr-CM and medium controls (Fig. 6). F3 and F1 GDR-CM were equally effective in suppressing progesterone production by granulosa cells. Boiling and protease treatment of F1 GDR-CM eliminated the suppressive effects of GDR-CM on progesterone production (Fig. 7). FIG. 5. Effects of boiling, protease treatment, and charcoal stripping of F1 GDR- CM, Gr-CM, and fresh medium on 3 H-thymidine incorporation by F3 granulosa cells. F3 granulosa cells were incubated in Gr-CM and fresh medium (medium) as controls. Data represent means + SEM of three to five independent experiments. Letters indicate statistically significant differences (p < 0.05) within treatment determined by ANOVA and the least significant differences method. FIG. 7. Effects of boiling and protease treatment of F1 GDR-CM, Gr-CM, and fresh medium on progesterone production by F1 granulosa cells. F1 granulosa cells were incubated in Gr-CM and fresh medium (medium) as controls. Data represent means + SEM of three to five independent experiments. *Statistically significant differences (p < 0.05) within treatment determined by ANOVA and the least significant differences method.

5 GERMINAL DISC REGION REGULATES GRANULOSA CELL FUNCTION 869 DISCUSSION The major findings in this study are as follows: 1) functional gradients of proliferation and steroidogenesis exist within the granulosa layer of the preovulatory follicle as demonstrated by 3 H-thymidine incorporation and progesterone production, respectively, and 2) the GDR secretes protein factors that promote 3 H-thymidine incorporation and inhibit progesterone production by granulosa cells. Granulosa cells associated with the germinal disc incorporated the highest amount of 3 H-thymidine. Furthermore, 3 H-thymidine incorporation by granulosa cells decreased progressively with distance from the germinal disc. These data are the first to establish the existence of a proliferation gradient within the granulosa layer that is centered in the GDR. Our results are supported by histological evidence indicating that granulosa cells associated with the germinal disc show a greater incidence of mitotic figures than their distal counterparts [10, 11]. Previous studies have shown that granulosa cells associated with the germinal disc have a higher mitotic index [12] and incorporate more 3 H-thymidine than granulosa cells not associated with the germinal disc [13]. Our results extend these findings by establishing the existence of a physiological gradient of proliferation within the granulosa layer. A similar situation has been demonstrated in the rodent preovulatory follicle: in rat antral follicles, cumulus granulosa cells incorporated the most 3 H- thymidine; and a gradient of proliferation was apparent within the multilaminar mural granulosa cell layer, with granulosa cells nearest the antrum displaying the highest mitotic index [22]. In light of the data presented in the current study, these findings suggest that granulosa cells associated with the germinal disc in the avian follicle may be similar to cumulus granulosa cells, whereas distal granulosa cells may be likened to mural granulosa cells. However, this observation is merely correlative, and further experimentation is necessary to make comparisons concerning cell analogies. In direct opposition to the proliferation gradient, the current data also demonstrate that a gradient of differentiation, as measured by progesterone production, is present in the granulosa cells of preovulatory follicle. Basal progesterone production was lowest for the GDR and increased progressively with distance from the germinal disc. Whereas all granulosa cells increased progesterone production in response to LH, the magnitude of the response was dependent upon the location of the granulosa cells relative to the germinal disc. Progesterone production by the GDR increased slightly in response to LH. Proximal granulosa cells had an intermediate response to LH, and distal granulosa cells had the greatest response to LH, supporting the existence of a gradient of differentiation within the follicle. In a previous study, Marrone et al. [12] reported that distal granulosa cells contain more P 4 50 cholesterol side-chain cleavage enzyme than granulosa cells associated with the germinal disc, which suggests that distal granulosa cells have a greater capacity to produce steroids than do granulosa cells associated with the germinal disc. The gradients of proliferation and steroidogenesis within the granulosa layer of an individual follicle imply that the morphology and physiology of a granulosa cell may be dependent upon its position in the follicle relative to the germinal disc. Thus, the GDR may produce factors that influence granulosa cell function. The results of the second experiment indicate that the GDR produces heat- and protease-sensitive factors that promote proliferation and inhibit progesterone production by granulosa cells. The GDR used for these studies consisted of the germinal disc and the overlying layer of granulosa cells because it is impossible to separate the germinal disc from its overlying granulosa cells. Therefore, on the basis of these data, it is impossible to determine whether the source of the proliferation-stimulating and steroidogenesis-inhibiting factors was the germinal disc or the granulosa layer, or the result of interactions of the two cell types. In the mouse, oocytes secrete unidentified factors that promote granulosa cell proliferation [23] and cumulus cell expansion [24], and regulate granulosa cell steroidogenesis [25]. Gonadotropins are the primary regulators of ovarian function. However, local production of intraovarian growth factors may mediate the gonadotropin response. The results of this study indicate that production of undefined factors by the GDR influences granulosa cell proliferation and steroidogenesis. F3 GDR-CM and F1 GDR-CM equally stimulated granulosa cell proliferation, which suggests that the GDR-derived factors might be constitutively expressed or at least are not dependent upon the state of maturation (or size) of the follicle. Decreased proliferation that occurs with follicular maturation [13] may result from altered responsiveness of granulosa cells to these factors, a process that could be regulated by gonadotropins. The nature of the proliferation-promoting and steroidogenesis-inhibiting factors is not known. Heat and protease sensitivity suggests that these factors are proteins. It is possible that these proteins are commonly recognized growth factors. Furthermore, it is probable that observed effects result from the actions of several factors. However, little information is available concerning the presence of growth factors in the avian ovary. Insulin-like growth factor I (IGF- I) mrna [26], epidermal growth factor (EGF) receptor, and transforming growth factor a (TGFa)-like and EGF-like peptides have been localized in both granulosa and theca cells [27, 281. EGF, IGF-I, and TGFa have been shown to influence granulosa cell function in the avian ovary in a manner similar to the results described in our experiments. For example, EGF [291 and TGFa [29, 30] inhibited progesterone production by granulosa cells. EGF [31], TGFa [30, 32], and IGF-I [30] stimulated proliferation of granulosa cells. IGF-I

6 870 TISCHKAU AND BAHR stimulated basal progesterone production and enhanced LH-stimulated progesterone production by granulosa cell [271. Additionally, EGF and TGFa inhibited androstenedione production and LH stimulated aromatase activity by thecal cells [32]. This investigation, in conjunction with previous studies, provides convincing evidence that granulosa cell function is not uniform throughout the entire granulosa layer but varies depending upon the proximity of the granulosa cell to the germinal disc. Furthermore, secretion of heat- and protease-sensitive factors by the GDR may provide a mechanism for paracrine regulation of granulosa cell function and follicular development. The role of these factors in regulating theca layer function has not been determined. Destruction of the GDR of a rapidly growing preovulatory follicle terminated follicular growth, blocked ovulation, and resulted in atresia [18]. Additionally, the accumulation of lipid droplets and development of inner mitochondrial structure in granulosa cells after destruction of the GDR indicated that steroidogenesis had been disrupted [33]. It is likely that destruction of the GDR disrupts production of factors necessary to promote further growth and development of the follicle. Thus, the secretion of specific proliferation-promoting and steroidogenesis-inhibiting factors by the GDR may provide a mechanism by which the GDR communicates with the entire follicle and participates in the regulation of proliferation and differentiation of the developing preovulatory follicle. REFERENCES 1. Calvo FO, Wang S-C, Bahr JM. LH-stimulable adenylyl cyclase activity during the ovu latory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticss. Biol Reprod 1981; 25: Bahr JM, Calvo FO. A correlation between adenylyl cyclase and responsiveness to go nadotropins during follicular maturation in the domestic hen. In: Cunningham FJ, Lake PA, Hewitt D (eds.), Reproductive Biology of Poultry. Harlow: British Poultry Science LTD; 1984: Hertelendy F. Olson DM, Todd H, Hammond RW, Toth M, Asboth G. Role of prostaglandins in oviposition and ovulation. In: Cunningham FJ, Lake PA, Hewett D (eds.), Reproductive Biology of Poultry. Harlow: British Poultry Science Ltd.; 1984: Shimada K, Saito N. Control of oviposition in poultry. Crit Rev Poult Biol 1989; 2: Johnson AL. Steroidogenesis and actions of steroids in the ovary. Crit Rev Poult Biol 1990; 2: BahrJM, Johnson PA. Reproduction in poultry. In: Cupps PJ (ed.), Reproduction in Domestic Animals. 4th ed. New York: Academic Press: 1991: Tilly JL, Kowalski KI, Johnson AL. Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells. Biol Reprod 1991; 44: TillyJL, Kowalski Kl, Johnson AL. Cytochrome P450 side-chain cleavage in the hen ovary I1. P450- messenger RNA, immunoreactive protein and enzyme activity in developing granulosa cells. Biol Reprod 1991; 45: Nitta H, Mason JI, Bahr JM. Localization of 3-hydroxysteroid dehydrogenase in the chicken ovarian follicle shifts from the theca layer to granulosa layer with follicular maturation. Biol Reprod 1993; 48: Perry MM, Gilbert AB, Evans AJ. The structure of the germinal disc region of the hen's ovarian follicle during the rapid growth phase. J Anat 1978; 127: Bakst MR. Scanning electron microscopy of hen granulosa cells before and after ovulation. Scanning Electron Microsc 1979; 3: Marrone BL, Jammaluddin M, Hertelendy F. Regional pattern of cell maturation anti progesterone biosynthesis in the avian granulosa cell layer. Biol Reprod 1990: 42: Tilly JL Kowalski KI, Li Z. Levorse LM, Johnson AL. Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period. Biol Reprod 1992; 46: Tischkau SA, Jackson JA, Bahr JM. The germinal disc region is the proliferative center of the follicle. Biol Reprod 1993; 48(suppl 1):152 (abstract 374). 15. Gilbert AB. Female genital organs. In: King AS, McLelland J (eds.&, Form and Function in Birds. New York: Academic Press: Yoshimura Y. Okamoto 1T, Tamura T. Ultrastructural changes of oocyte and follicular wall during oocyte maturation in the Japanese quail (Cbturnir coturnti japonica). J Reprod Fertil 1993; 97: Yoshimura Y, Okamoto T, Tamura T. Electron microscopic observations on LH-induced oocyte maturation in lapanese quail (Coturnix coturnixjaponica). J Reprod Fertil 1993: 98: Yoshimura Y, Tischkau SA, Bahr JM. Destruction of the germinal disc region of an immature preovulatory follicle suppresses follicular maturation and ovulation. Biol Reprod 1994: 51: Gilbert AB, Evans AJ, Perry MM. A method for separating the granulosa cells, the hasal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticss. J Reprod Fertil 1977; 50: Dorrington JH, Bendell JJ, Chuma A. Transforming growth factor f and follicle stimulat ing hormone promote granulosa cell proliferation. Endocrinology 1988: 123: Bahr JM, Wang S-C, Huang MY, Calvo FO. Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen (Gallus domesticus). Biol Reprod 1983; 29: Hirshfield AN. Patterns of [3H]thymnidine incorporation differ in immature rats and mature, cycling rats. Biol Reprod 1986; 34: Vanderhyden BC, Telfer EE, EppigJJ. Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro. Biol Reprod 1992: 46: i. 24. Vanderhyden BC, Caron PJ, Buccione R. EppigJJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 1990; 140: Vanderhyden BC, Cohen JN, Morley P. Mouse oocytes regulate granulosa cell steroidogenesis. Endocrinology 1993; 133: Roberts RD. Boswell J Burt D, Sharp PJ Goddard C. IGF-I in the aian ovary. I Reprod Fertil 1990; Abstract Series 6, Abstract Peddle MJ. The role of EGF and other growth factors in the local regulation of steroid production in ovarian follicles. In: Proceedings of te Fifth International Symposium on Avian Endocrinology. Edinburgh: AFRC Roslin Institute: 1992: S10/ Onagbesan OM, Gullick W, Woolveridge 1, Peddle MI. Immunohistochemical localization of epidermal growth factor receptors, epidermal-growth-factor-like and transforminggrowth-factor-a like peptides in chicken ovarian follicles. J Reprod Fertil 1994: 102: Pulley DD. Marrone BL. Inhibitory action of epidermal groxvth factor on progesterone biosynthesis in hen granulosa cells during short-term culture: two sites of action. Endocrinology 1986; 118: Onagbesan OM Peddle MJ. Effects of insulin-like growth factor I and interactions A ith transforming growth factor a and LH on proliferation of chicken granulosa cells and production of progesterone in culture. J Reprod Fertil 1995; 104: Yoshimura Y, Tamura T. Effects of gonadotrophins, steroid hormones and epidermal growth factor on the in vitro proliferation of chicken granulosa cells. Poult Sci 1988: 67: Peddle M, Onagbesan M, Woolveridge I. The role of epidermal growth factor and other factors in the paracrine and autocrine control of ovarian follicular development in the domestic hen. In: Sharp PI (ed.), Avian Endocrinology. Bristol: I Endocrinol LTD: 1993: Yoshimura Y Bahr JM. Atretic changes of follicular wall caused by destruction of the germinal disc region of an immature preovulatory follicle in the chicken: an electron microscope study. J Reprod Fertil 1995: 105: