BIOLOGY OF REPRODUCTION 48, 110-116 (1993) Localization of 31-Hydroxysteroid Dehydrogenase in the Chicken Ovarian Follicle Shifts from the Theca Layer to Granulosa Layer with Follicular Maturation' HIROAKI NITTA, 3'4 J. IAN MASON, 5 and JANICE M. BAHR2' 4 Department of Animal Sciences,4 University of Illinois, Urbana, Illinois 61801 Cecil H. and Ida Green Centerfor Reproductive Biology Sciences,5 Departments of Biochemistry and Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 30-Hydroxysteroid dehydrogenase (3-HSD) catalyzes the conversion of pregnenolone to progesterone in the A4-3-ketosteroid metabolic pathway and dehydroepiandrosterone to androstenedione in the A5-3p-hydroxysteroid pathway. It has been suggested that small follicles of the chicken ovary that have not entered the follicular hierarchy metabolize steroids via the A530-hydroxysteroid pathway, whereas preovulatory follicles that have entered the hierarchy metabolize steroids via the A4 -3ketosteroid pathway. Our objective was to localize 3P-HSD in follicles of the chicken ovary by immunocytochemistry using an anti-human placental 3-HSD polyclonal antiserum to identify steroidogenic cells that convert pregnenolone to progesterone and/or dehydroepiandrosterone to androstenedione. Three groups of follicles of different maturities were examined: small follicles (1-10 mm in diameter and that have not entered the hierarchy), preovulatory follicles (10-35 mm in diameter and that have entered the hierarchy), and the most recent postovulatory follicle. Chicken ovaries were obtained 2 h after oviposition and fixed with Bouin's solution. Tissues were dehydrated with a series of ethanol, embedded in Paraplast (Brunswick Company, St. Louis, MO), and sectioned. Sections (4 jm) were immunostained for 3-HSD with a Rabbit ExtrAvidin Staining Kit (Sigma Chemical Co., St. Louis, MO). 3[-HSD was localized in the single theca layer of cortical follicles (approximately 1 mm in diameter), which are still embedded in the stromal tissue, and in the theca interna and externa of other small follicles (< 10 mm in diameter). No immunoreactivity was observed in the granulosa layer of the majority of small follicles. Weak staining of 3jP-HSD was occasionally present in the granulosa layer of mature small yellow follicles, which are the next follicles to enter the hierarchy. 3,-HSD was present in the theca interna of all preovulatory follicles; however, the intensity of the staining decreased as preovulatory follicles matured. In contrast, the staining of 3p-HSD in the granulosa layer became more intense as preovulatory follicles increased in size. Finally, the granulosa layer of the most recent postovulatory follicle obtained 2 h after oviposition stained intensively for 3-HSD. These results based upon the immunolocalization of 3-HSD in the chicken ovarian follicles suggest the following: 1) the theca layer can convert pregnenolone to progesterone and/or dehydroepiandrosterone to androstenedione, whereas the granulosa layer in the majority of small follicles lacks this ability; 2) both granulosa and theca layers of preovulatory follicles are production sites for progesterone and/or androstenedione; 3) the granulosa layer becomes more steroidogenically active for progesterone and/ or androstenedione production, whereas the theca layer loses its steroidogenic activity as preovulatory follicles mature; and 4) the granulosa layer retains its steroidogenic capability after ovulation. We conclude that the principal sites of progesterone and androstenedione shift during follicular maturation from the theca layer of small follicles to the granulosa layer of preovulatory follicles and postovulatory follicles. INTRODUCTION cording to size: cortical follicle (less than 1 mm in diameter) embedded in the ovarian stroma, small white follicles (approximately 1 mm in diameter), large white follicles (35 mm in diameter), and small yellow follicles (6-10 mm in diameter). Small follicles either enter the follicular hierarchy or become atretic. The steroidogenic capability of follicles changes with follicular development. The three largest preovulatory (F1F3) follicles are major sites of progestin production [1], and small follicles that have not entered the follicular hierarchy and the stromal tissue that contains cortical follicles are the principle sites of estrogen production [1-3]. It has been speculated that the steroidogenic pathway in follicles shifts from the A5-31-hydroxysteroid pathway in small follicles to the A4-3-ketosteroid pathway in preovulatory follicles with follicular development [1, 4]. Therefore, 3-hydroxysteroid dehydrogenase (3-HSD) is required for conversion of dehydroepiandrosterone to androstenedione in the A5-33-hydroxysteroid pathway of small follicles and for conversion The ovary of a mature chicken generally contains five preovulatory follicles (10-35 mm in diameter) arranged in a follicular hierarchy and classified according to size of the follicles, e.g., the largest (F1) follicle, the second largest (F2) follicle, and so on. The F1 follicle is the next one to ovulate after which the F2 follicle becomes the F1 follicle. The remaining follicular wall after ovulation becomes the postovulatory follicle. There are several stages of small follicles that have not entered the hierarchy and are classified acAccepted August 19, 1992. Received March 24, 1992. 'This work was supported in part by USDA-Ag-89-37240-4769 (J.M.B.) and NIHAG08175 0.I.M.). 2 Correspondence: Dr. Janice M. Bahr, 113C Animal Genetics Laboratory, Department of Animal Sciences, University of Illinois, 1301 W. Lorado-Taft Dr., Urbana, IL 61801. FAX: (217) 244-2871. 'Current address: Steroid Research Unit, Department of Obstetrics & Gynecology, L1221/0278 Women's Hospital, University of Michigan, Ann Arbor, MI 481090278. FAX: (313) 936-8617. 110 Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 ABSTRACT LOCALIZATION OF 3P-HSD IN FOLLICLES OF THE CHICKEN OVARY MATERIALS AND METHODS Animals Mature, single-comb White Leghorn hens were kept under the light schedule of 15L:9D (lights-on from 0600-2100 h). Oviposition time was recorded at hourly intervals in the morning and at 1700 h. Five chickens, 30-60 wk of age with regular laying sequences of 6 eggs or more, were selected, and ovarian tissues were obtained from chickens 2 h after oviposition of the first egg in the sequence. Alternate sections were treated with either normal rabbit serum (1: 200) as a control or the IgG-enriched fraction of the primary antiserum (1:200) at room temperature overnight in a moisture chamber. Sections were then processed with a Rabbit ExtrAvidinR Staining Kit (Sigma Chemical Co., St. Louis, MO), an avidin-biotin-peroxidase method. Sections were then reacted with a freshly prepared hydrogen peroxidase 3,3'-diaminobenzidine (Sigma) solution. At least 5 follicles of each size of small follicles (described in Results), all preovulatory follicles (F5-F1), and the postovulatory follicle from each ovary were examined. Intensity of immunoreactivity was assessed within cell populations, but not the number of positive cells. RESULTS Small Follicles Control sections of small follicles did not indicate any significant staining (Fig. 1, A-C). In cortical follicles, immunoreactivity for 3-HSD was present in interstitial cells of the single theca layer, but not in the granulosa layer (Fig. 1D). With follicular development, the theca interna and externa can be distinguished in the theca layer. Interstitial cells in both theca interna and externa layers of large white follicles (3-5 mm in diameter) stained for 3P-HSD (Fig. 1E). However, with follicular growth, the staining for 3f-HSD in the theca externa of small yellow follicles (6-10 mm in diameter) decreased (Fig. 1F). Weak immunoreactivity for 33HSD was occasionally found in the granulosa layer of small yellow follicles (photographs are not shown). Antibodies Preparation of the anti-33-HSD was described previously [7]. Briefly, placental 33-HSD, purified from the human placenta as reported by Thomas et al. [8], was injected into New Zealand White rabbits. An IgG-enriched fraction was obtained which contained antibodies against 3-HSD. Immunocytochemistry The most recent and largest postovulatory follicle, preovulatory follicles (F5-FI), and the remaining ovarian tissue containing various stages of small follicles were removed and processed for paraffin embedding. Tissues were fixed in Bouin's solution at room temperature overnight. Preovulatory follicles were washed with PBS 1 h after initial fixation to remove the yolk from the follicles, and follicular walls were placed again into the fixative. Fixed tissues were embedded in Paraplast (Brunswick Company, St. Louis, MO) after conventional dehydration with ethanol. Paraffin sections (4 pim) were placed on precleaned glass slides and slides were dried in an oven at 37 0C for 2 days. Deparaffinized sections were treated with 10% normal goat serum prior to incubation of the primary antibody to reduce background caused by the secondary antibodies raised in goats. PreovulatoryFollicles Control sections of preovulatory follicles were negative for 3-HSD (Fig. 2, A-C). The F5 follicle, the smallest preovulatory follicle, contained immunoreactivity for 3-HSD in granulosa cells and interstitial cells in the theca interna (Fig. 2D). In the F3 follicles, immunoreactivity was localized in granulosa cells and interstitial cells, similar to that observed in the F5 follicle (Fig. 2E). However, the immunoreactivity of 33-HSD in the granulosa layer of the F3 follicle was increased compared to the staining intensity of the 33-HSD of the F5 follicle. Finally, in the F1 follicle, immunoreactivity for 313-HSD in the granulosa layer was very intense whereas immunoreactivity in the interstitial cell of the theca layer was weaker when compared to less mature preovulatory follicles (Fig. 2F). Postovulatory Follicles Control sections of the largest and most recent postovulatory follicles were negative (Fig. 3A). Strong immunoreactivity for 33-HSD was observed in the granulosa layer whereas almost no immunoreaction was found in the theca layer (Fig. 3B). Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 of pregnenolone to progesterone in the A4 -3-ketosteroid pathway in the preovulatory follicles. Additionally, the activity of 3-HSD has been observed in interstitial cells, a steroidogenic cell population, of the theca layer of small follicles and preovulatory follicles. Activity of 31-HSD in the theca layer of preovulatory follicles decreases with follicular development [5]. Granulosa cells develop 3-HSD activity 7-8 days prior to ovulation [6]. However, the precise localization of 3P-HSD in follicles of the chicken ovary during follicular development has not been determined. Therefore, our purpose was to localize 3-HSD in follicles of the chicken ovary during follicular development by light microscopic immunocytochemistry using a polyclonal antiserum against human placental 3P3-HSD. The major advantage of this method is that 3-HSD can be precisely localized in paraffin sections; in contrast, the histochemical method makes use of freshly prepared frozen sections, in which clear cellular morphology is not preserved. 111 112 NITTA ET AL. Cortical Follicle Large White Follicle Small Yellow Follicle C 0 I C'r FIG. 1. Immunolocalization of 3P-HSD steroidogenic enzyme indicates conversion sites of pregnenolone to progesterone and/or dehydroepiandrosterone to androstenedione in small follicles (1-10 mm in diameter). Control sections of small follicles (A-C) indicated no significant staining. Cortical Follicle (approximately 1 mm in diameter): the theca layer is a single theca layer (Th) and interstitial cells (InC) in the theca layer are immunoreactive for 33-HSD (D); Large White Follicle (3-5 mm in diameter): interstitial cells (InC) staining for 3)-HSD were seen in both theca interna (Thl) and theca externa (ThE) layers (E); and Small Yellow Follicle (6-10 mm in diameter): fewer interstitial cell immunoreactive for 3-HSD were present in the theca externa (ThE) than in the theca interna (Thl) (F). Arrows indicate immunoreactivity. Bar = 25 iLm. DISCUSSION We are presenting for the first time the localization of 3P-HSD, a key steroidogenic enzyme, in follicles of the chicken ovary during follicular maturation. We found that 1) the localization of 31-HSD gradually shifted from the theca layer of small follicles to the granulosa layer of preovulatory and postovulatory follicles with follicular maturation; 2) interstitial cells located in theca interna and externa of small follicles (1-10 mm in diameter) contained 3-HSD, but staining for 3(-HSD in the theca externa decreased with follicular maturation; 3) weak staining of 31-HSD was present in the granulosa layer of some small yellow follicles, the next follicles to enter the follicular hierarchy. The localization of 31-HSD by immunocytochemistry in follicles of the chicken ovary confirms previous histochemical studies [6, 9]. Our immunocytochemical study of 33HSD in follicles of the chicken ovary demonstrated the shift of 33-HSD localization from the theca layer to the granulosa layer during follicular maturation. In small follicles, immunolocalization of 31-HSD indicated that the theca layer contained 31-HSD whereas the granulosa layer of the majority of small follicles lacked 33-HSD. In an earlier study, we found that the theca layer of small follicles, but not the granulosa layer, contained the cytochrome P450 steroidogenic enzymes necessary to convert cholesterol to estrogen [10]. These immunocytochemical studies indicate that the theca layer of small follicles is steroidogenically active Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 Oo LOCALIZATION OF 3-HSD IN FOLLICLES OF THE CHICKEN OVARY F5 F3 113 F1 -r' I FIG. 2. Immunolocalization of 31-HSD in preovulatory follicles. Control sections contained no nonspecific staining in the granulosa layer (Gr) and tneca interna (Thl) and theca externa (ThE) layers of preovulatory follicles (A-C). Fifth Largest Preovulatory Follicle (F5): immunoreactivity of 3p-HSD was present in interstitial cells (InC) of the theca layer and the granulosa layer (D); Third Largest Preovulatory Follicle (F3): interstitial cells (InC) in the theca interna and the granulosa layer contained immunoreactivity for 3P-HSD (E); and Largest Preovulatory Follicle (F1); strong immunoreactivity of 33-HSD was seen in the granulosa layer (F). Bar = 25 pLm. whereas the granulosa layer is inactive. The granulosa layer becomes steroidogenically active by some as yet unknown mechanism(s) when a small follicle has reached the appropriate size to enter the follicular hierarchy. It is well established that with follicular maturation there is a marked increase in steroidogenic activity of the granulosa layer of preovulatory follicles, which produces mainly progestins, whereas there is a concomitant decrease in steroidogenic activity in the theca layer, which produces androgens and estrogens [11, 12]. Our present study supported these findings in that preovulatory follicles had a decrease in immunoreactivity for 33-HSD in the theca layer and an increase in immunoactivity in the granulosa layer with follicular development. Finally, the granulosa layer of the largest and most recent postovulatory follicle, obtained 2 h after ovulation, stained very intensely for 3-HSD. This finding agrees with previous studies that demonstrated 3HSD activity in the granulosa layer of the postovulatory follicle [5]. We previously had measured an active adenylyl cyclase system in postovulatory follicles that was responsive to LH stimulation [13]. Moreover, Huang and Nalbandov [14] reported that granulosa cells of postovulatory follicles secrete progesterone. Therefore, the granulosa layer of the postovulatory follicle is a site of steroidogenesis. The biosynthetic site(s) of progesterone and/or androstenedione shifts from the theca layer to the granulosa layer during a preovulatory follicular stage. It has been speculated that there is a shift of steroidogenic metabolism from the A5-3-hydroxysteroid pathway in small follicles to the A4 -3-ketosteroid pathway in preovulatory follicles [1, 2]. The secretion of high amounts of dehydroepiandrosterone and low amounts of progesterone by small follicles suggests that these follicles use the A5 -33-hydroxysteroid pathway [1]. Granulosa cells of the preovulatory follicle are well known as the major production site of progesterone, an intermediate in the A4-3-ketosteroid pathway. Therefore, it appears that the 3-HSD in interstitial cells of the theca layer of small follicles catalyses primarily the conversion of dehy- Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 0 114 NITTA ET AL. Postovulatory Follicle I FIG. 3. Immunolocalization of 3-HSD steroidogenic enzyme in the largest and most recent postovulatory follicle. Control sections indicated the absence of immunoreactivity in the granulosa layer (Gr) and theca interna (Thl) and theca externa (ThE) layers (A). Strong immunoreactivity was observed in the granulosa layer (B). Arrows indicate immunoreactivity. Bar = 25 Irm. 5 droepiandrosterone to androstenedione in the A -33-hydroxysteroid pathway, whereas 3-HSD in granulosa cells of preovulatory follicles metabolizes pregnenolone to progesterone in the A4 -3-ketosteroid pathway. Thus, the change of 3P3-HSD from the theca layer to the granulosa layer during follicular maturation may be related to the shift of the A5 -33-hydroxysteroid metabolic pathway of small follicles to the A 4-3-ketosteroid pathway of preovulatory follicles arranged in the follicular hierarchy. Recently, a multiple cell theory for steroidogenesis in the theca layer of preovulatory follicles was proposed [15]. One important aspect of this theory is that the theca layer of preovulatory follicles can produce androgens and estrogens without dependence on the granulosa layer for substrate because interstitial cells in the theca interna can produce progestins and androgens from cholesterol. It has also been reported that turkey theca interna and theca externa cells produce androgen and estrogen, respectively [16]. In the present study, we found strong immunoreaction of 33HSD in the theca layer and weak immunoreaction in the granulosa layer of the less mature preovulatory follicle. This finding supports the importance of interstitial cells as a source of substrate for estrogen production by aromatase cells [15] in the theca layer. Therefore, contributions of the granulosa layer as a source of substrate for androgen and estrogen production in the theca layer of immature preovulatory follicles may be less important than in mature preovulatory follicles. Our immunolocalization of 3P3-HSD in small follicles of the chicken ovary showed that interstitial cells present in the single theca layer of cortical follicles contained 33-HSD whereas granulosa cells in these follicles contained no immunoreactivity. With follicular development, 33-HSD-positive interstitial cells were localized in the theca interna and externa of large white follicles. The majority of interstitial cells were found, however, in the theca interna of small yellow follicles, which are more mature than large white follicles. We are not able to provide reasons why the 3,BHSD steroidogenic enzyme is located in different cell layers, namely in the theca interna and externa, of small fol- Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 CO LOCALIZATION OF 3f-HSD IN FOLLICLES OF THE CHICKEN OVARY licles and disappears from interstitial cells in the theca externa with follicular development. We speculate, however, that interstitial cells in the theca externa are more important for estrogen production than those in the theca interna [10]. Interstitial cells in the theca externa that also contain steroidogenic enzymes for pregnenolone and androgen production, namely cholesterol side-chain-cleavage cytochrome P450 and 17a-hydroxylase cytochrome P450, are surrounded by estrogen-producing cells, i.e., aromatase cells, so that the substrate can directly diffuse into these cells [10]. It is possible that interstitial cells in the theca externa are a different cell type from interstitial cells in the theca interna and that interstitial cells in the theca externa differentiate with follicular development. Therefore, 313-HSDpositive cells in the theca externa may be regulated in a different manner than interstitial cells in the theca interna of small follicles. Furthermore, the production of 31-HSD ceases in the cells of the theca externa whereas interstitial cells in the theca interna retain 3P3-HSD expression. In small follicles that have not entered the follicular hierarchy, the theca layer is steroidogenically active whereas there is no evidence of steroidogenic activity in the granulosa layer. Davidson et al. [6] reported that the granulosa layer of follicles of the chicken ovary contains the 33-HSD activity 7-8 days prior to ovulation. In our study, there was no 313-HSD immunoreactivity in the granulosa layer of small follicles, with exception of a weak immunoreactivity in a few small yellow follicles. Tilly et al. [17] reported proges- terone secretion by isolated granulosa cells obtained from a pool of small yellow follicles. Furthermore, these granulosa cells metabolized exogenous pregnenolone to progesterone, which indicates 3-HSD activity. We speculate that one or a few follicles pooled for isolation of granulosa cells for this in vitro study [17] contained 3P-HSD, as seen in our present immunocytochemical study in which weak immunoreactivity of 31-HSD was observed occasionally in granulosa cells of some small yellow follicles. Thus, the granulosa layer of small follicles excluding some small yellow follicles acquire progesterone and/or androstenedione productive capability, as indicated by 3-HSD immunoreactivity, prior to entering the follicular hierarchy. On the basis of immunoreactivity of 3P-HSD in follicles of the chicken ovary, we conclude the following: 1) Localization of 3-HSD changes from the theca layer to the granulosa layer as small follicles become larger preovulatory follicles-which may be related to the suggested shift from the A5 -313-hydroxysteroid metabolic pathway in small follicles to the A4 -3-ketosteroid pathway in preovulatory follicles. 2) Interstitial cells staining for 31-HSD are distributed in both theca interna and externa of small follicles, and 3f-HSD-containing interstitial cells in the theca externa disappear with follicular maturation. 3) The appearance of 313-HSD in the granulosa layer of small follicles may occur prior to their entry into the hierarchy (Fig. 4). Thus, 33HSD is a pivotal key enzyme regulating steroid biosynthesis in developing and maturing follicles of the chicken ovary. REFERENCES 1. Robinson FE, Etches RJ. Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus). Biol Reprod 1986; 35:1096-1105. 2. Senior BE, Furr BJA. Preliminary assessment of the source of oestrogen within the ovary of the domestic fowl, Gallus domesticus. J Reprod Fertil 1975; 43:241247. 3. Armstrong DG. Ovarian aromatase activity in the domestic fowl (Gallus domesticus). J Endocrinol 1984; 100:81-86. 4. Lee KA. Steroidogenesis in small follicles (SF) in the domestic hen: regulated by 5 cAMP and A pathway. Biol Reprod 1990; 42(suppl. 1):113 (abstract 219). 5 5. Armstrong DG. 3-Hydroxy-A -steroid dehydrogenase activity in the rapidly growing ovarian follicles of the domestic fowl (Gallus domesticus). J Endocrinol 1982; 93:415-421. 5 6. Davidson MF, Gilbert AB, Wells JW. Activity of ovarian A -31-hydroxysteroid dehydrogenase in the domestic fowl (Gallus domesticus) with respect to age. J Reprod Fertil 1979; 61-64. 7. Doody KM, Carr BR, Rainey WE, Byrd W, Murry BA, Strickler RC, Thomas JL, Mason JI. 3-Hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortix of the human fetal adrenal gland. Endocrinology 1990; 126:24872492. 8. Thomas JL, Berko EA, Faustino A, Myers RP, Strickler RC. Human placental 31hydroxy-5-ene-steroid dehydrogenase and steroid 5--4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids. J Steroid Biochem 1988; 31:785-793. 9. Armstrong DG, Davidson MF, Gilbert AB, Wells JW. Activity of 30-hydroxysteroid dehydrogenase in the postovulatory follicle of the domestic fowl (Gallus domesticus). J Reprod Fertil 1977; 49:253-259. 10. Nitta H, Osawa Y, Bahr JM. Immunolocalization of steroidogenic cells in small follicles of the chicken ovary: anatomical arrangement and location of steroidogenic cells change during follicular development. Domest Anim Endocrinol 1991; 8:589-596. Downloaded from https://academic.oup.com/biolreprod/article-abstract/48/1/110/2762200 by guest on 14 June 2020 FIG. 4. A diagram of the changes in the localization and distribution of 31-HSD in the theca and granulosa layers during follicular development of the chicken ovary. Intensity of darkness indicates amount of immunoreactive 3p-HSD. Localization of 33-HSD shifted from the theca layer to the granulosa layer with follicular development. In cortical follicles (embedded in the ovary), interstitial cells in the single theca layer contained 3-HSD. As follicles grow, the theca layer is separated into externa and interna with interstitial cells present in both cell layers. The amount of 3P-HSD increased in the theca interna and decreased in the theca externa as follicles mature. The change in immunostaining was the result of a change in the number of interstitial cells of small follicles. However, intensity of immunoreactive 3P-HSD decreased in the theca interna of preovulatory follicles with follicular development. Appearance of 33-HSD in the granulosa layer of small follicles occurred prior to their becoming preovulatory follicles. Immunoreactivity of 313-HSD in the granulosa layer became more intense as follicles approached ovulation and was still present in the postovulatory follicle. 115 116 NITTA ET AL. 11. BahrJM, 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. Biol Reprod 1983; 29:326-334. 12. Etches RJ, Duke CE. Progesterone, androstenedione and oestradiol content of theca and granulosa tissues of the four largest ovarian follicles during the ovulatory cycle of the hen (Gallus domesticus). J Endocrinol 1984; 103:71-76. 13. Calvo FO, Wang S-C, Bahr JM. LH-stimulable adenylyl cyclase activity during the ovulatory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticus). Biol Reprod 1981; 25:805812. 14. Huang ES-R, Nalbandov AV. Steroidogenesis of chicken granulosa and theca cells: in vitro incubation system. Biol Reprod 1979; 20:442-453. 15. Nitta H, Osawa Y, Bahr JM. Multiple steroidogenic cell populations in the theca layer of preovulatory follicles of the chicken ovary. Endocrinology 1991; 129:20332040. 16. Poter TE, Hargis BM, El Halawani ME. Different steroid production between theca interna and theca externa cells: a three cell model for follicular steroidogenesis in avian species. Endocrinology 1989; 125:109-116. 17. 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; 44:305-314. 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