cm3-7227/92/1301-0186$03.00/0

Endocrinology Copyright 0 1992 by The Endocrine So&t>

Vol.

Printed

130. No. I in U.S.A

Epidermal Growth Factor Inhibits Follicular Response to Human Chorionic Gonadotropin: Possible Role of Cell to Cell Communication in the Response to Gonadotropin* KATSUHIDE TSUNG-CHENG

ENDO, SUSAN J. ATLAS, JANICE D. RONE, V. L. ZANAGNOLO, KUO, A. M. DHARMARAJAN, AND EDWARD E. WALLACH

Department of Gynecology and Obstetrics, Johns Hopkins Baltimore, Maryland 21205

University

ABSTRACT. Epidermal growth factor (EGF) affects follicular eteroidogeneaia and expression of gonadotropin receptors. The effects of EGF on hCG-induced e&radio1 and progesterone secretion and ovulation were examined in the &I t&o perfused rabbit ovary. We also examined the effects of EGF on hCGinduced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P < 0.05) reduced hCG-induced secretion of estradiol (control, 117 * 12 pg/min. follicle; 10 rig/ml EGF, 55 -C 10) and progesterone (control, 18.2 + 1.2 ng/min. follicle; 10 rig/ml EGF, 11.9 f 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency

School of Medicine,

(number of ovulated ova per number of mature follicles x 100) when EGF was given 36 min before hCG was reduced dosedependentlv from 58.2% with no EGF to 8.3% with 10 na/ml EGF (P < i).OOl). Ovulation was not inhibited by EGF wh;n it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(e) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication. (Endocrinology 130: 186-192, 1992)

E

The LH surge or administration of hCG causes marked changes in each follicular compartment; oocyte, granulosa cell layer, and thecal layer. Gonadotropin stimulates the transformation of granulosa cells into progesteronesecreting luteal cells (7). Prostaglandins, proteolytic enzymes such as tissue plasminogen activators, and other substances are activated and result in follicle rupture (8, 9). Granulosa cells in large preovulatory follicles express LH receptor on their surface membrane (10). However, the granulosa cell layer is avascular, and gonadotropins may be unable to reach the inner cells, which lack direct access to capillary circulation. The importance of cell to cell communication is well recognized in the maintenance of oocyte growth and meiotic arrest (11). Cell to cell communication, either within granulosa layers or between cumulus cells and the oocyte, is thought to be one of the means by which the gonadotropic signal is transmitted throughout the follicle. Communication between granulosa cells within the follicle is thought to be maintained in the perfused ovary, while it is reduced or interrupted when cells are isolated from the follicle, as in granulosa cell culture. This study was designed to investigate the effect of EGF on the follicular response to gonadotropic stimula-

PIDERMAL growth factor (EGF) inhibits FSHinduced development of LH receptors on granulosa cells without affecting receptor affinity (1, 2). EGF also inhibits FSH-induced granulosa cell aromatase activity (3). The concentration of EGF-like activity detected in follicular fluid, as determined by a receptor-binding assay, varies inversely with follicle size (4). An EGF-like substance is produced by thecal/interstitial cells (5), and immunohistochemical staining demonstrates a decrease in follicular EGF-like content during estrus (6). These observations suggest that EGF signaling is reduced in preovulatory follicles, whioh are prepared to respond to a LH surge. Received July 30, 1991. Address requests for reprints to: E. E. Wallach, M.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Houck 264A, Baltimore, Maryland 21205. Address all correspondence to: A. M. Dharmarajan, Ph.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 800 North Wolfe Street, Park B2-202. Baltimore, Maryland 21205. *Presented in part at the 38th Annual Meeting of Society for Gynecologic Investigation, San Antonio, TX, March 20-23, 1991. This work was supported by NIH Grant HD-19430, a SmithKline Beecham Fellowship (to K.E.), the Rockefeller Foundation (to A.M.D. and J.D.R.), and the Lalor Foundation (to V.L.Z.). 186

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tion and the role of cell to cell communication in this response. To this end, the in vitro perfused rabbit ovary was used to examine changes in steroid secretion and ovulation as indices of follicular response, while progesterone secretion by isolated granulosa cells was examined as an index of the granulosa cell response to hCG. The extent to which gonadotropins reach and directly activate granulosa cell layers was studied by examining the intrafollicular distribution of administered hCG with immunohistochemical methods.

Materials

and Methods

Animals

New ZealandWhite mature femalerabbits, weighing 3.5-4.5 kg, were used.All rabbits were cagedindividually for at least 3 weeksbefore use.Animals were given water and a diet of rabbit chow ad lib&urn. Rabbits were anesthetized with iv sodium pentobarbital(32 mg/kg), treated with heparin sulfate (120 U/ kg) for anticoagulation, and then subjectedto laparotomy. All studieswere performed in accordancewith the NIH Guide for the Care and Useof Laboratory Animals. In vitro perfusion

The cannulation procedure and perfusion apparatus have been describedin detail previously (12-14). Briefly, each ovarian artery and vein were isolated and cannulated in situ after ligation of major anastomotic connections. The ovary with its cannulated vascular pedicle and supportive connective tissue wasremovedand immediately placedin the perfusion chamber. The perfusion system consists of a chamber containing the ovary, an oxygenator, a reservoir, and a pulsatile roller pump that maintains flow at 1.5 ml/min, the approximate blood flow to the rabbit ovary (15). The oxygenator wasgassedwith 95% 02-5% CO,. Ovaries were perfused for 10.5 h at 37 C with 150 ml medium 199(M. A. Bioproducts, Walkersville, MD) supplementedwith heparin sulfate (200 U/liter), insulin (20 U/liter), streptomycin sulfate (50 mg/liter), and penicillin G (75 mg/ liter). Perfusate sampleswere collected 1 h after hCG administration from both arterial and venous cannulaefor determining steroid concentrations by solid phase RIA (Diagnostic Products, Los Angeles,CA). The ovary wasmonitored for fresh ovulation points every half-hour. Granulosa

cell collection

Dulbecco’sModified Eagle’sMedium (Sigma Chemical Co., St. Louis, MO) with 25 mM HEPES (Sigma) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), penicillin G (100 U/ml), and streptomycin sulfate (0.1 mg/ml) was usedfor collection and the suspensionmediumof the granulosa cells. Ovaries were removed from unstimulated rabbits, and mature follicles were punctured under a dissectingmicroscope using a 23-gaugeneedle in a 60-mm Falcon dish containing 8 ml medium. Oocyte-cumulus complexeswere expelled spontaneously after follicle puncture. Mural granulosa cells were removed from the follicle by gentle scraping with the needle (16). No attempt was madeto remove oocytes. The collected

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granulosacells were washedtwice with fresh medium(500 x g; 5 min) and then placed at 1 x 10 cells/O.7 ml mediumin 24well Falcon tissueculture plates for 1.5 h at 37 C with 5% C02. Experimental

design

Effect of EGF on hCG-induced estradiol and progesterone secretion and ouulation. Fourteen rabbits were usedfor this experi-

ment. Ovaries were perfused with or without EGF (0.1, 1.0, or 10 rig/ml; Sigma) from the onset of the perfusion. hCG (Pregnyl, Organon, West Orange, NJ; 50 IU) was administered to the ovary through the arterial cannula 30 min after the onset of perfusion. Since both estradiol and progesteronesecretion peaked between l-2 h after hCG administration (our unpublished data), arterial and venous samplesfor determining steroid secretion, an index of follicular response,were collected 1 h after hCG administration. Ovulatory efficiency wascalculated as (number of ovulated ova per number of mature follicles) x 100.Follicles with a diameter 1.5 mm were consideredmature. Effect of the order of administering EGF and hCG on estradiol and progesterone secretion and ovulation. Ten rabbits wereused

for this experiment. Ovaries wereperfusedwith mediumalone, hCG (50 IU) only, EGF (10 rig/ml) administered30 min before hCG, EGF administered30 min after hCG, or EGF alone.hCG or vehicle was given through the arterial cannula, while EGF (1.5 pg) or vehicle administeredafter hCG wasplaced into the reservoir. Estradiol and progesteronesecretion wasdetermined 1 h after hCG or vehicle administration, and ovulatory efficiency was calculated. The effect of EGF on progesterone secretion by granulosa cells in suspension. Twelve rabbits were used for six experiments.

hCG (0.5 IU/ml) was added 30 min after the onset of the culture, and EGF (10 rig/ml) or vehicle wasadministeredeither at the onset or 30 min after hCG to examine the effect of the order of administering hCG and EGF. Each group was measured in triplicate. The progesteroneconcentration in the medium 1 h after hCG administration was determined and compared with the results obtained by the perfusedovaries. Zmmunohistochemistry

Rabbit ovaries with arteries cannulated were perfused in vitro with medium 199 with or without EGF (10 rig/ml) and given hCG (50 IU) or vehicle 30 min after the onsetof perfusion. Immunohistochemical localization of hCG was examined 30 min after its administration to minimize the influence of receptor-ligand internalization. Thirty minutes after hCG administration, the ovaries were removed from the perfusion system, and 10 ml Bouin’s solution were infused through the arterial cannula for fixation. Four ovaries that had been exposedto hCG and two ovaries that had not received hCG were fixed in each group. The ovaries were maintained in Bouin’s solution for 24 h and then transferred into 70% ethanol until sectioned. Five-micron sectionsof the fixed ovarieswere stainedwith goat anti-hCG (O.E.M. Concepts,Inc., Toms River, NJ). After deparaffinization, specimenswere immersedin 3% H,Oa in Osborn-Weber PBS (OWPBS) for 30 min to block endogenous peroxidaseactivity and washedin OWPBS for 15 min. Sections were incubated with 1% heat-inactivated rabbit nonimmune

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serum diluted in 0.1% gelatin OWPBS for 15 min. The first antibody incubation wasperformed for 14-16 h in a humidified chamber at room temperature. Antibody was diluted 1:800 in 1% rabbit nonimmuneserumwith 0.1% gelatin OWPBS. Heatinactivated goat nonimmune serum and the anti-hCG preneutralized with excesshCG at 4 C overnight wereusedascontrols. The sections were rinsed in OWPBS for 15 min, incubated with biotinylated rabbit antigoat immunoglobulin G (Sigma) diluted 1:400with 1% rabbit nonimmune serumin 0.1% gelatin OWPBS for 45 min in a humidified chamber at room temperature, rinsed again in OWPBS for 15 min, and incubated with a Vectastain kit (avidin DH, biotinylated horseradishpreoxidaseH, Vector Laboratories, Inc., Burlingame, CA) for 45 min at room temperature. After rinsing in OWPBS, the color reaction was developedby incubating sectionsfor 3 min in 200 ml 0.1 M acetate buffer, pH 6.0, containing 70 mg diaminobenzidine, 2.5% nickel ammoniumsulfate, and 50 ~1H,O, (17). Data analysisand statistics Steroid secretionwasdeterminedas (V - A) x F/f (V, venous sampleconcentration; A, arterial sampleconcentration; F, flow rate; f, number of mature follicles). The resultswere expressed asthe difference betweensecretion at the time of hCG administration and 1 h later. x2 (ovulatory efficiency), analysis of variance, and Scheffe test or honestly significant difference test (steroid secretion, number of ovulations per ovary, and progesteroneconcentration in culture medium) were used to examinethe differences.P < 0.05 wasconsideredsignificant.

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0

Endo * 1992

Voll30.

0.1 EW

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No 1

10

@@ml)

FIG. 1. Effect of EGF on hCG-induced estradiol secretion by the perfused ovary. Ovaries were perfused with RGF (0, 0.1, 1.0, and 10 ng/ ml) and stimulated by hCG (50 IU) 30 min after theonsetof perfusion. Results are expressed as the difference between secretion at the time of hCG administration and that 1 h later. Results represent the mean rf SEM of 10 ovaries in the absence of EGF (control) and 6 ovaries in each EGF treatment. Values with different lettersare significantly different from one another (P < 0.06).

Results Estradiol and progesterone secretion by the in vitro perfused ovary

Estradiol secretion by the perfused ovary 1 h after hCG administration was reduced from 117 + 13 pg/min. follicle (mean + SEM) in the absence of EGF (control) to 55 + 11 in the presence of 10 rig/ml EGF (P < 0.05; Fig. 1). EGF did not inhibit secretion at doses of either 0.1 or 1.0 rig/ml. When EGF was administered before hCG, the secretion was reduced to 39% of that with hCG alone (P < 0.05; Fig. 2). When EGF was given after hCG, e&radio1 secretion was 60% of control (hCG alone) values, but the difference was not significant. Estradiol secretion was not significantly affected by EGF alone (Fig. 2). Progesterone secretion 1 h after hCG was reduced from 18.2 + 1.3 ng/min.follicle in the absence of EGF (control) to 11.9 f 0.9 with 10 rig/ml EGF (P < 0.05; Fig. 3). When EGF (10 rig/ml) was given before hCG, secretion was 47% of the level obtained with hCG alone (P < 0.01; Fig. 4). EGF given after hCG reduced progesterone secretion to 72% of control (hCG alone) values (P < 0.05; Fig. 4). EGF alone slightly induced progesterone secretion 1 h after hCG administration, but this value was not significantly different from that seen in the absence of stimulation (Fig. 4).

hCG

EGF-hCG

FIG. 2. Effect of the order of administering EGF and hCG on estradiol secretion by the perfused ovary. Results are expressed as the difference between secretion at the time of hCG administration and that 1 h later. Results represent the mean f SEM of four ovaries. Values with different ktters are significantly different from one another (P < 0.05).

Ovulation in the perfused ovary

Ovulatory efficiency stimulated by hCG was significantly reduced in a dose-dependent manner, from 58.2% without EGF to 8.3% with 10 rig/ml EGF, when EGF was present in the perfusate before hCG (Table 1). When EGF was given after hCG, EGF did not inhibit hCGinduced ovulation (Table 2). EGF alone did not induce ovulation. Progesterone secretion by isolated granulosa cells

The mean cell viability at the time of harvest was 70.5 + 4.7%. The administration of hCG induced progester-

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TABLE 1. Effect of EGF on hCG-induced perfused rabbit ovary

a

ovulation in the in vitro

EGF (rig/ml)

f b T

~,

_

0 (control)

0.1 1 .o EGF (nglml)

1 ~~

10

FIG. 3. Effect of EGF on hCG-induced progesterone secretion by the perfused ovary. Results are expressed as the difference between secretion at the time of hCG administration and that 1 h later. Results represent the mean + SEM of 10 ovaries in the absence of EGF (control) and 6 ovaries in each EGF treatment. Values with different letters are significantly different from one another (P < 0.05).

Perfused ovaries (no.) Mature follicles (no.) Ovulations (no.) Ovulatory efficiency (%) Ovulations per ovary (noJd Ovaries stimulated perfusion, letters are d Mean

0 10 67 39 58.2”

0.1 6 45 14 31.lb

1.0 6 53 10 18,9b~c

10 6 48 4 8.3’

3.9 + 0.7’ 2.3 f l.OeJ 1.6 f 0.7’J 0.7 f 0.7’

were perfused with EGF (0, 0.1, 1.0, and 10 ng/mI) and to ovulate by hCG (50 IU) 30 min after the on& of as described in Matmiuls and Methods. Values with different significantly different from one another (P < 0.05). + SEM.

TABLE 2. Effect of the order of administering ovulation in the in vitro perfud ovary

EGF and hCG on

Protocol

25 Perfused ovaries (no.) Mature follicles

Medium 199 (control) 4

hCG

EGF-hCG

hCG-EGF

EGF

4

4

4

4

29

27

30

28

37

0

17

2

14

0

0”

63.0b

6.3”

50.0b

0”

0”

4.3 + 1.1’

0.5 f 0.5d

3.5 f 0.9’

Od

(no.) Ovulation

c

(no.)

Ll

Ml99

hCG

EGF-hCG

t

L

G-EGF

Ovulatory efficiency (%I Ovulations per ovary (no.)’ EGF

FIG. 4. Effect of the order of administering EGF and hCG on progesterone secretion by the perfused ovary. Results represent the mean f SEM of four ovaries. Values with different letters are significantly different from one another (P < 0.05).

one secretion by isolated granulosa cells, and EGF given either before or after hCG did not affect progesterone secretion (Table 3). Intrafollicular

distribution

of administered

hCG

Intrafollicular hCG was specifically stained by goat anti-hCG in both EGF-exposed and unexposed (control) ovaries (Fig. 5). Staining for hCG was confined to the basement membrane, thecal layer, and a small fraction of the outer granulosa cell layer. The staining was seen in both large preovulatory follicles and atretic follicles. No staining was detected in small antral follicles. hCG was intensely and widely detected on granulosa cells in atretic follicles. There was no difference in the intensity

Ovaries were perfused in vitro with or without hCG (50 IU) in the presence or absence of EGF (10 q/ml). EGF was adminiatared 30 min before or after hCG, as described in Materials and hfethod~. VaIues with different letters are significantly different from one another (P < 0.05). ’ Mean f SEM. TABLE

3. Progesterone secretion by isolated granulosa cells 1 h after

hCG Protocol hCG EGF-hCG hCG-EGF Progesterone (rig/ml) 18.8 * 3.0 17.7 f 2.8 18.5 f 2.9 Progesterone (%) 100 93.6 f 2.8 98.1 f 3.3 Isolated granulosa cells from unstimulated mature foIlicles were plated at a density of 1 x 105 cells/ml and incubated with hCG (0.5 W/ml) in the presence or absence of EGF (10 rig/ml) for 1 h. EGF was administered before or after hCG, as described in Materials and Met& ads. Percentages indicate the ratio compared to progesterone production by hCG-treated granulosa cells. Values are shown as the mean f SEM of six experiments.

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FIG. 5. Immunohistochemical staining of ovarian follicles with goat anti-hCG. Ovaries were perfused with hCG (50IU) for 30min before fixation. A, Ovary perfused without EGF (control). Note the staining confined to the basement membrane (BM) and adjacent granulosa cells (GC). Magnification, x400. B, Ovary perfused with EGF. Staining was similar to that in A. Magnification, x400. C, Ovary perfused with EGF. Magnification, x200. D, Adjacent section to C, stained with antibody preneutralized with excess hCG. Magnification, x200. E, Ovary perfused without EGF. Note the extensive staining of the granulosa cell layer in this atretic follicle. Magnification, x400.

or the extent of hCG staining between control and EGFtreated ovaries. Discussion The follicular response to gonadotropic stimulation is initiated by binding of the ligand to specific receptors on

follicular cells. Either suppression of gonadotropin binding to its receptor or alterations in the transduction of the signal after binding results in reduced steroid secretion and inhibition of ovulation. The content of mRNA for P-450 aromatase (18) and P-450 side-chain cleavage enzyme (7), which play a key

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role in hCG-stimulated steroid production by granulosa cells, is regulated by CAMP. EGF has been reported to inhibit FSH-induced CAMP-mediated granulosa cell differentiation (2, 19). Administration of EGF to the perfused ovary may have affected CAMP-mediated alterations in steroidogenic enzymes after gonadotropin administration. The inhibitory effect was reduced when EGF was given after hCG, which may be due to the reduced exposure to EGF (30 min compared to 1 h). If EGF acts through direct alteration of intracellular CAMP content or if it affects alterations secondary to changes in CAMP, steroid secretion by granulosa cells in suspension should have been influenced by EGF. However, since no difference was detected between EGFtreated and control groups in hCG-induced progesterone secretion by isolated granulosa cells, EGF probably does not act by modulation of intracellular CAMP. While it is possible that isolated granulosa cells lost their EGF receptors during isolation or culture, this is unlikely, as they were not exposed to proteolytic enzymes. In the perfused ovary, the majority of granulosa cells are not directly exposed to hCG, and cell to cell communication is necessary to transmit the stimulatory signal throughout the granulosa cell layer. In contrast, all of the isolated granulosa cells are equally and directly exposed to hCG. EGF inhibits hCG-induced steroid secretion only when cell to cell communication is instrumental in generating the granulosa cell response. These observations suggest that EGF may affect cell to cell communication among granulosa cells. EGF is only effective in inhibiting ovulation when given before hCG. It is possible that EGF binding to the granulosa cell is influenced by previous exposure to hCG. EGF receptor expression in cultured granulosa cells is reported to be reduced by exposure to hCG (20). In the present study it is unlikely that the granulosa cell surface EGF receptor was degraded during the 30-min exposure to hCG. The initial alterations necessary to trigger follicle rupture had probably already been activated by the time of EGF administration. Once initial activation has been achieved, EGF is no longer effective. It is also possible that EGF inhibits hCG binding to its receptor on granulosa cells. However, EGF does not affect receptor affinity in differentiating granulosa cells (1). In the present study the immunohistochemical detection of hCG on granulosa cells in preovulatory follicles was not affected by EGF, although the staining was not quantitative. Follicular granulosa cells are avascular, and the majority of the cells do not have direct capillary access to the circulating ligand unless diffusion takes place across the basement membrane. Immunohistochemical localization of hCG was examined 30 min after its administration to minimize the influence of receptor-ligand internalization. In addition, this time sequence was considered to

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be sufficient because maximum alterations in steroid secretion were visible by 1 h after hCG administration. It must be kept in mind that immunohistochemical localization of hCG does not indicate the degree of receptor-bound hormone. It is unlikely that the follicular response to hCG is confined to the limited number of granulosa cells that exhibit hCG on their surface. Follicular granulosa cells communicate with one another and the oocyte by gap junctions (21,22). It may be postulated that the response of preovulatory follicles to gonadotropin is elucidated not only by direct binding of the ligand but also by secondary signals transmitted from the outer, initially activated, granulosa cells to those of the inner layer. Secondary signals may be propagated via gap junctions or by paracrine mechanisms. Our observations suggest that EGF may attenuate transmission of this secondary signal in preovulatory follicles by inhibiting the release of the signal from initially activated cells or reception of the signal by target cells. Staining with anti-hCG was intense on granulosa cells of atretic follicles. Since rabbits are reflex ovulators, each ovary contains follicles in various stages of maturity, ranging from newly matured ones to those that have matured several days before. Older mature follicles that have not been exposed to a gonadotropin stimulus may become atretic. The interstitial space between granulosa cells in atretic follicles may be increased as cell contact loosens, and hCG may more easily reach inner granulosa cells. Our observation suggests that the atretic process in the rabbit ovary may be different from that in the rat ovary in which atresia is characterized by a lack of LH receptors on granulosa cells (23). In summary, EGF inhibits hCG-induced estradiol and progesterone secretion and ovulation in the in vitro perfused rabbit ovary. In contrast, EGF does not inhibit hCG-induced progesterone secretion by isolated granulosa cells in which cell to cell communication is reduced or interrupted. Distribution of hCG in the preovulatory follicle, detected by immunohistochemical staining, is confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. It is suggested that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligandbound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication. Further investigation will be necessary to unveil the details of intrafollicular communication after gonadotropic stimulation. Acknowledgments The authors gratefully acknowledge the technical assistance of Ms. Beverly Smith and MS, Lillian Dasko, and the assistance of Ms. Fran Karas in the preparation of the manuscript.

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References 1. Mondschein JS, Schomberg DW 1981 Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211:1179-1180 2. Knecht M, Catt KJ 1983 Modulation of CAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 2582789-2794 3. Jones PBC, Welsh Jr TH, Hsueh AJW 1982 Regulation of ovarian progestin production by epidermal growth factor in cultured rat eranulosa cells. J Biol Chem 257:11268-11273 4. Hsu C-J, Holmes SD, Hammond JM 1987 Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement. Biochem Biophys Res Commun 147242-247 5. Skinner MK, Lobb D, Dorrington JH 1987 Ovarian thecal/interstitial cells produce an epidermal growth factor-like substance. Endocrinology 121:1892-1899 6. Roy SK, Greenwald GS 1990 Immunohistochemical localization of epidermal growth factor-like activity in the hamster ovary with a polyclonal antibody. Endocrinology 126:1309-1317 I. Goldring NB, Durica JM, Lifca J, Hedin L, Ratoosh SL, Miller WL, Orly J, Richards JS 1987 Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea. Endocrinology 120:1942-1950 8. Hedin L, Gaddy-Kurten D, Kurten R, Dewitt DL, Smith WL, Richards JS 1987 Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology 121:722731 9. Tsafriri A, Bicsak TA, Cajander SB, Ny T, Hsueh AJW 1989 Suppression of ovulation rate by antibodies to tissue type plasminogen activator and ap-antiplasmin. Endocrinology 124:415-421 10. Zeleznik AJ. Midelev Jr AR. Reichert Jr LE 1974 Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormone in uiuo. Endocrinology 95:818-825 11. Eppig JJ 1985 Oocyte-somatic cell interactions during oocyte -



12. 13. 14.

15. 16. 17. 18.

19. 20 21

22. 23.

TO hCG

growth and maturation in the mammal. In: Browder LW fed) Developmental Biology. Plenum Press, New York, vol 1:313-347 Lambertsen .Jr CJ, Greenbaum DF, Wright KH, Wallach EE 1976 In vitro studies of ovulation in the perfused rabbit ovary. Fertil Steril 27:178-187 Kobayashi Y, Wright KH, Santulli R, Wallach EE 1981 Ovulation and ovum maturation in the rabbit ovary perfused in uitro. Biol Reprod 24:483-490 Dharmarajan AM, Yoshimura Y, Sueoka K, Atlas SJ, Dubin NH, Ewine LL. Zirkin BR. Wallach EE 1988 Progesterone secretion by corpora lutea of the isolated perfused rabbit-ovary during pseudopregnancy. Biol Reprod 381137-1143 Ahren K, Janson PO, Selstam G 1972 Perfusion of ovaries in vitro and in oiuo. Acta Endocrinol [Suppl] (Copenh) 158285-309 Goodman AL 1984 In uitro evidence of a role for inhibin in female rabbits. Am J Physiol246:E243-248 Rone JD, Goodman AL 1987 Heterogeneity of rabbit aortic endothelial cells in primary culture. Proc Sot Exp Biol Med 184495 503 Hickey GJ, Chen S, Besman MJ, Shively JE, Hall PF, GaddyKurten D, Richards JS 1988 Hormonal regulation, tissue distribution, and content of aromatase q&chrome P450 messenger ribonucleic acid and enzyme in rat follicles and corpora lutes: relationship to estradiol biosynthesis. Endocrinology 122:14261436 Bendell JJ, Dorrington JH 1990 Epidermal growth factor influences growth and differentiation of rat granulosa cells. Endocrinology 127:533-540 Feng P, Knecht M, Catt K 1987 Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation. Endocrinology 120:1121-1126 Albertini DF, Anderson E 1974 The appearance and structure of the intercellular connections during the ontogeny of the rabbit ovarian follicle with special reference to gap junctions. J Cell Biol 63:234-250 Brower PT, Schultz RM 1982 Intercellular communication between granulosa cells and mouse oocytes: existence and possible nutritional role during oocyte growth. Dev Biol90:144-153 Daud AI, Bumpus FM, Husain A 1989 Angiotensin II: does it have a direct obligate role in ovulation? Science 245:870-871

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Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol...
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