localization of epidermal growth factor (egf) receptor in the rat corpus luteum, and egf and...

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ELSEVIER Molecular and Cellular Endocrinology 110 (1995) 95-102

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Localization of epidermal growth factor (EGF) receptor in the rat corpus luteum, and EGF and transforming growth factor-a stimulation of

luteal cell steroidogenesis in vitro

F.R. Tekpetey*, B. Singh, G. Barbe, D.T. Armstrong

Deparhnents of Obstetrics and Gynaecology and Physiology, The University of Western Ontario, 339 Windennere Road. London, ON N6A 5A5, Canada

Received 1 December 1994; accepted 21 February 1995

Abstract

Epidermal growth factor (EGF) and transforming growth factor-a CTGF-a) have potent mitogenic effects on granulosa and theta cells. However, their effects on steroidogenesis by these cells is controversial, and there is limited information regarding their effects on luteal cell steroidogenesis. The present study investigated the cellular distribution of the EGF receptor (EGF-R) in the rat corpus luteum (CL) by immunocytochemical staining, and the effects of EGF and TGF-a on progesterone and 2tk-dihydroprogesterone (20~~OH-P) production in cultures of luteal cells. Using a primary antibody directed against the human EGF-R peptide, specific EGF-R staining was obtained in the CL. Both small and large luteal cells had EGF-R staining. In initial cell culture experiments, treatment of freshly isolated luteal cells with EGF or TGFa (0.5-50 @ml) for 24 h had no effect on progesterone and 20a-OH-P accumulation. Addition of LH (250 q/ml) alone caused a 3.5fold increase in both progestins, but co-treatment with EGF or TGF-a produced no further enhancement of progestin accumulation. However, when cells were seeded overnight and the attached cells were washed prior to growth factor treatment for 3 days with media change every 24 h, both EGF and TGF-a caused dose-dependent increases in progesterone accumulation/24 h period (up to 2-fold at 50 @ml growth factor) on days 1 and 2 but not day 3 of treatment. 2Oo-OH-P accumulation was similarly stimulated (up to 2.5-fold) by EGF and TGF-a under these conditions. In addition, LH-induced increases in progestin accumulation were further enhanced dose-dependently by TGFu. These data confirm the presence of EGF-R in rat CL, and suggest a physiological role for EGF and TGF-a in luteal steroidogenesis.

Keywords: Rat; Epidermal growth factor receptor; Epidermal growth factor; Transforming growth factor-a; Luteal steroidogenesis

1. Introduction

The epidermal growth factor receptor (EGF-R) is a common receptor for EGF, transforming growth factor-

alpha (TGF-a), heparin-binding EGF, amphiregulin, cripto, and heregulin, all of which are members of the EGF-related family of growth factors (Rrigent and

Lemoine, 1992). The EGF-R is present in the theta and granulosa cells of the follicle (Vlodavsky et al., 1978; Fujinaga et al., 1992; Singh et al., 1995), as well as in the corpus I’uteum (CL) of several species (Vlodavsky et al., 1978; Chabot et al., 1986; Ayyagari and Khan-Dawood, 1987). However, there is limited information on the cellular distribution of EGF-R in the CL. Chegini et al. (1989)

*Corresponding author, Tel.: +l 519 6632983; Fax: +l 519 6633388.

have reported the presence of immunoreactive EGF-R in both the small (SLC) and large (LLC) bovine luteal cells, which are thought to originate from the theta and granulosa cells, respectively (Alila and Hansel, 1984). Re-

cently, we have also demonstrated mRNA and peptide expression for EGF-R in the pig SLC and LLC (Singh, Kennedy, Tekpetey and Armstrong; unpublished data).

Among the EGF-related family of growth factors, only EGF and TGF-a have been studied for their roles in ovarian function. Both growth factors are known to be synthesized in follicular granulosa and theta cells (Kudlow et al., 1987; Lobb et al., 1989; Chegini and Wil- liams, 1992; Singh et al., 1995). The two growth factors exert on granulosa and theta cell proliferation in several species (Gospodarowicz et al., 1977; Skinner and Coffey, 1988; May et al., 1988; May et al., 1992). Furthermore,

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96 F.R. Tekpeiey et al. I Molecular and Cellular Endocrinology I10 (1995) 95-102

EGF and TGF-a modulate steroid production by granulosa and theta cells, but these effects are inconsistent since both stimulatory (Jones et al., 1982; Roy and Greenwald, 1991; Yeh et al., 1993) and inhibitory (Hseuh et al., 1981; Bendell and Dorrington, 1990; Endo et al.,

1992) effects have been reported. The demonstrated presence of EGF and TGF-a

mRNAs (Kennedy et al., 1993; Lobb and Dorrington, 1993; Singh, Kennedy, Tekpetey and Armstrong, unpublished data) and peptides (Khan-Dawood, 1987; Chegini and Williams, 1992; Lobb and Dorrington, 1993; Singh, Kennedy and Tekpetey, unpublished data) in luteal tissue indicate a local synthesis of these growth factors in the CL as well. These findings together with the presence of EGF-R in the CL, suggest a potential autocrine and/or

paracrine role for these growth factors in the CL. While EGF has been shown to have no effect on luteal cell proliferation (Vlodavsky et al., 1978), there are no reports

regarding its effects on luteal steroidogenesis. Therefore the objective of this study was to investigate the cellular distribution of EGF-R in the rat CL, and to determine the effects of TGF-a on luteal steroidogenesis.

2. Materials and methods

2.1. Reagents and hormones Luteinizing hormone (LH; USDA-bLHB5) was gener-

ously donated by the National Hormone and Pituitary

Program (Baltimore, MD). Collagenase (type II) and de- oxyribonuclease I (DNase I) were obtained from Sigma

(St. Louis, MO) and Boehringer-Mannheim (Dorval, PQ, Canada), respectively. Recombinant human EGF and TGF-a, Hank’s balanced salt solution (HBSS; Ca*+ and Mg*+-free), Roswell Park Memorial Institute (RPMI) medium, penicillin, streptomycin and fetal calf serum (FCS; heat inactivated) were purchased from Gibco (Burlington, ON, Canada). Histostain-SP kit for immunocytochemistry was obtained from Zymed Laboratories

Inc. (San Francisco. CA).

2.2. Immunocytochemistry Corpora lutea used for the immunocytochemistry were

obtained from day-8 pseudopregnant Sprague-Dawley rats as previously described (Tekpetey and Armstrong, 1991). Freshly excised whole ovaries bearing CL were fixed in paraformaldehyde (4%) and gluteraldehyde (0.5%) fixative, dehydrated and embedded in paraffin. Tissue sections (5-7pm) mounted on positively charged slides were subjected to streptavidin-biotin immunostaining using a rabbit polyclonal antibody raised against a peptide corresponding to residues 1005-1016 of the hu-

man EGF-R peptide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). This antibody has previously been demonstrated to produce specific EGF-R immunostaining in pig theta and granulosa cells (Singh et al., 1995). The immunostaining procedure was as outlined in a Histo-

stain-SP kit with few modifications as previously described (Singh et al., 1995). Briefly the tissue sections

were first deparaffinized and endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 min at room temperature. Following three washes in D-PBS, the slides were incubated overnight at 4°C with anti-EGF-R (1:200 dilution in D-PBS containing 10% pig serum). For negative controls, the primary antibody was pre-absorbed with a lo-fold excess of a control peptide

(EGF-R; Santa Cruz Biotechnology) at room temperature for at least 2 h prior to the incubation. The slides were

then washed (3X) with D-PBS and the sections were incubated with biotinylated secondary antibody at room temperature for 10 min. After three washes with D-PBS, the sections were incubated with streptavidin peroxidase conjugate for 5 min at room temperature, followed by washing with D-PBS. The reactions were completed by the addition of freshly prepared substrate-chromogen mixture for 10-20 min at room temperature, two washes with distilled water, and counter-staining with haema- toxylin. After mounting the sections, the slides were ex- amined microscopically and the results were recorded by

photography.

2.3. Luteal cell preparation and culture Pooled CL from day-8 pseudopregnant Sprague-

Dawley rats were used for the preparation of Iuteal cells. The luteal tissue was dissociated by enzymatic digestion (0.25% collagenase (w/v) and 0.02% DNase (w/v) in HBSS + 0.1% BSA) followed by elutriation to remove contaminating non-steroidogenic cells, leaving a mixed cellular fraction of small (SLC) and large (LLC) steroidogenic luteal cells as previously described (Tekpetey

and Armstrong, 1994). Cell viability was assessed by try- pan blue exclusion test and averaged 95 + 1.3% (n = 3). We opted to use mixed luteal cells rather than separated

SLC and LLC because our immunocytochemistry experiment had localized EGF-R in both SLC and LLC (see Section 3). Furthermore, we have previously demonstrated that rat SLC and LLC both respond positively to LH (Tekpetey and Armstrong, 1991).

The isolated luteal cells were washed twice and seeded at 100 000 cells per well in 24-well plates (Falcon Plas- tics, Los Angeles, CA) containing RPM1 medium buff- ered with HEPES (25pM) and sodium bicarbonate (24 PM) and supplemented with antibiotics (50 III/ml

Fig. 1. EGF-R localization in rat CL by streptavidin-biotin immunostaining. For positive staining in (A) and (B), tissue sections were incubated with a

rabbit polyclonal primary antibody raised against a peptide corresponding to residues 1005-1016 of the human EGF-R peptide. For the negative control in (C). the primary antibody was pre-absorbed with a control peptide prior to incubation. Arrow and arrow heads point to SLC and LLC, respectively. (A)kT, connective tissue: CL, corpus luteum; bar = 80pm. (B.C) hm = 20pm.

F.R. Tekpetey et al. I Molecular and Cellular Endocrinology 110 (1995) 95-102 97

98 F.R. Tekpetey et (11. I Molecular and Cellular Endocrinology 110 (1995) 95-102

h 35

f 0 EGF

N 30 KiI LH + EGF

2 200 [x) LH + EGF

z ‘I E 150

\ :

”

0 0.5 5.0 50 0 0.5 5.0 50

Epidermal Growth Factor (ng/ml)

Fig. 2. Effect of EGF and LH on progesterone (upper panel) and 2Oa- OH-P (lower panel) accumulation in serum-free cultums of freshly isolated luteal cells after 24 h. Data represent the mean 2 SEM of three independent experiments.

penicillin; 50pg/ml streptomycin). In the first experiment, the hormone and growth factor treatments (see Section 3) were applied to freshly isolated cells and cultured for 24 h at 37°C in an atmosphere of 5% CO2 and

95% air. In the second experiment, the freshly isolated cells were first cultured overnight as described above in RPM1 medium containing 1% FCS. The cells were then washed, treatments applied in serum-free RPM1 medium and cultured for 24 h. Media and treatments were re- placed every 24 h for 3 days. At the end of the culture period in each experiment, media were harvested and stored at -20°C until assayed for secreted products.

2.4. RIA of secreted products Radioimmunoassays previously validated in our labo-

ratory for direct measurement of steroids in culture media were used for the determination of progesterone (Leung and Armstrong, 1979) and 20a-OH-pregn-4-en-3-one (2Oa-OH-P) (Morley et al., 1987).

2.5. Statistical analysis The cell culture data were analyzed by factorial analy-

sis of variance. Differences between group means were assessed by Duncan’s new multiple range test (Steel and Torrie, 1960).

3. Results

3.1. Localization of EGF-R in whole CL Localization of EGF-R in the CL is shown in Fig. 1.

Immunostaining for EGF-R was confined to the luteal tissue proper, with no staining in the surrounding inter- stitial connective tissue (Fig. 1A). At higher magnifica- tion, EGF-R staining was observed in both the SLC and LLC (Fig. IB). When the primary antibody was pre- absorbed with a control peptide prior to incubation with the tissue sections, no EGF-R staining was observed (Fig. IC), demonstrating the specificity of the primary antibody.

3.2. Steroid production by freshly isolated luteal cells in response to EGF, TGF-a and LH

Treatment with increasing concentrations of EGF (up to 50 @ml) had no effect on either progesterone or 2Oa- OH-P accumulation in cultures of freshly isolated luteal cells after 24 h (Fig. 2). The addition of LH alone signifi- cantly (P < 0.05) enhanced (3-I-fold) the accumulation of both steroids, but co-treatment with EGF caused no further increases in progesterone and 2Oa-OH-P accumu-

lation.

A 35

s 0 TGFa

c-4 30 m LH + TGFa

t eZa TGFa 1 7 200 [x3 LH + TGFa

z \ < 150

c”

4 100

& I 2s 50

z

0 0 0.5 5.0 50 0 0.5 5.0 50

Transforming Growth Factor-a (ng/ml)

Fig. 3. Effect of TGFa and LH on progesterone (upper panel) and 2Oa- OH-P (lower panel) accumulation in serum-free cultures of freshly isolated luteal cells after 24 h. Data represent the mean f SEM of three independent experiments.

F.R. Tekpetey et al. I Molecular and Cellular Endocrinology 110 (1995) 95-102 99

20

15

10

n5

G 0

Y -

r-

F

15 -

F 10.

u

2 0

:

0” 10

I;I

5

0

0 ECF

m LH + ECF

ab

0

d 0 0.5

Doy 1

b

b

1 r+

50 50 0 0 5 5.0 50

Day 2

d 0 05 50 50 0 0.5 5.0 50


Fig. 4. Effect of EGF and LH on progesterone accumulation in luteal

cell cultures on each of three days of treatment following overnight pre-

culture in the presence of serum. Data represent the mean f SEM of

three independent experiments. a-bSignificant differences (P < 0.05) between means within treatment group. *Significant difference

(P < 0.05) relative to the comparable histograph in the EGF only group

(open bars).

As shown in Fig. 3, TGF-a alone (up to 50 &ml) also caused no effect on progesterone or 20a-OH-P accumulation in the freshly isolated luteal cell cultures. Further- more, in combination with LH, TGF-a caused no further increase in LH-stimulated progesterone or 2Oa-OH-P

accumulation.

3.3. Steroid production by luteal cells in response to EGF, TGF-a and LH following overnight pre-culture in the presence of serum

When luteal cells were pre-cultured overnight prior to growth factor treatment, EGF alone caused significant dose-dependent increases (P < 0.05) in progesterone accumulation on days 1 and 2 of treatment (Fig. 4). These effects were diminished on day 3. The addition of LH alone enhanced progesterone accumulation, as previously

observed, and co-treatment with EGF caused further marginal increases in progesterone accumulation on days

1 and 2 that were not statistically significant (P > 0.05).

Epidermal growth factor also caused similar dose- dependent but more pronounced increases in 20a-OH-P accumulation on days 1 and 2, but not on day 3 of treatment (Fig. 5). In addition, EGF augmented the LH- stimulated 2Oa-OH-P accumulation dose-dependently on days 1 and 3 of treatment.

The addition of TGF-a alone caused dose-dependent increases (P < 0.05) in progesterone accumulation on day

1 of treatment following overnight pre-culture of the luteal cells (Fig. 6). These effects were diminished on days 2 and 3. In combination with LH, TGF-a had no

further effect on LH-stimulated progesterone accumulation on day 1, but at the highest dose (50 @ml) TGF-a

ezd ECF

[xl LH + ECF 150

100

50

0

A

% (\1 100 1 r Day 2

l

b

0 0.5 5.0 50 0 0.5 5.0 50

Doy 3 l

b

25


Fig. 5. Effect of EGF and LH on 2Ckz-OH-P accumulation in luteal cell

cultures on each of three days of treatment following overnight pre-

culture in the presence of serum. Data represents the mean _+ SEM of

three independent experiments. a*Significant differences (P < 0.05)

between means within treatment group. *Significant difference

(P < 0.05) relative to the comparable histograph in the EGF only group

(open bars).

F.R. Tekpetey et al. I Molecular and Cellular Endocrinology 110 (1995) 95-102

0 TCF6 = LH + TCFe

ob

b

I

b

; 10

5

0

Transforming Growth Factor-a (ng/ml>

Fig. 6. Effect of TGF-a and LH on progesterone accumulation in luteal


culture in the presence of serum. Data represents the mean + SEM of

three independent experiments. a-bSignificant differences (P c 0.05)

between means within treatment group. *Significant difference (P <

0.05) relative to the comparable histograph in the TGF-a only group

(open bars).

further enhanced the LH stimulation of progesterone accumulation on days 2 and 3.

As shown in Fig. 7, significant dose-dependent increases (P < 0.05) in 2Oa-OH-P accumulation in response

to TGF-a were achieved, particularly on days 1 and 2. Further, TGF-a enhanced dose-dependently, the LH- stimulated 20a-OH-P accumulation on all three days of treatment.

In all the situations where EGF and TGF-a caused further enhancement of LH- stimulated progestin accumulation in the luteal cell cultures, the combined effects were at best additive and not synergistic.

4. Discussion

In addition to the previously reported bioactive EGF-R

in the rat CL (Chabot et al., 1986), we have now demonstrated the presence of immunoreactive EGF-R in rat CL

in the present study. This finding is consistent with the demonstrated presence of immunoreactive EGF-R in other species such as the cow (Chegini et al., 1989) and pig (Singh, Kennedy, Tekpetey and Armstrong, unpublished data). Furthermore, the distribution of EGF-R in both SLC and LLC in this study is in agreement with those reported for the cow and pig. These data clearly indicate that the EGF receptor present in follicular theta and granulosa cells, which are the presumed precursors of SLC and LLC, respectively (Alila and Hansel, 1984), are

maintained and possibly enhanced (Vlodavsky et al., 1978) following luteinization.

In follicular theta and granulosa cells, EGF-R clearly has a physiological role in mediating the mitogenic and steroidogenic actions of EGF and TGF-a. However, the physiological role of EGF-R in the CL is uncertain. Vlo- davsky et al. (1978) have reported that spontaneous luteinization of cultured granulosa cells was associated with

Doy 1

LH + TGFu 100

50

0 0 0.5 5.0 50 0 0.5 5.0 50

A _c

Doy 2

c b

\ y 5O V

CL I 25

5

I 0 8 0 0.5 5.0 M 0 0.5 5.0 50

Doy 3 l

b

Transforming Growth Factor-a (ng/ml)

Fig. 7. Effect of TGF-a and LH on Xkz-OH-P accumulation in luteal


culture in the presence of serum. Data represents the mean k SEM of three independent experiments. a-cSignificant differences (P < 0.05)

between means within treatment group. *Significant difference

(P < 0.05) relative to the comparable histograph in the TGF-a only

group (open bars).

F.R. Tekpctey et al. I Molecular and Cellular Endocrinology 110 (1995) 95-102 101

an increase in the number of EGF-R per cell, but EGF had no effect on luteal cell mitogenesis. Although TGF-a effect on luteal cell mitogenesis has not been studied, TGF- a could potentially cause a paracrine stimulation of vas- cular endothelia cell proliferation since it is a potent angi- ogenic factor (Schreiber et al., 1986). In the present study, we have clearly demonstrated for the first time that both EGF and TGF-a can stimulate luteal progestin production in the absence of gonadotropin and also can augment LH- induced progestin production evident only when added following overnight pre-culture of the luteal cells in the presence of serum but without hormone or gonadotropin. A recent preliminary report by Tosbach et al. (1992) indicated that TGF-a can also promote progesterone production by a preparation of mixed bovine luteal cells. Our finding regarding EGF is in agreement with previous studies that have demonstrated EGF stimulation of progesterone production by granulosa cells (Jones et al., 1982; Trzeciak et al., 1987; Roy and Greenwald, 1991; Yeh et al., 1993), and of testosterone production by Ley- dig cells (Sordoillet et al., 1991).

In this study the inability of EGF and TGF-a, unlike LH, to stimulate progestin production in the freshly isolated luteal cells was somewhat surprising. Both the LH receptor (McFarland et al., 1989) and the prototypical EGF-R isolated from the carcinoma cell line A431 (Cummings et al., 1985), have extracellular ligand- binding domains that have high biochemical stability due to extensive glycosylation. Hence, it seems unlikely that biochemical perturbation of the EGF-R during the enzymatic digestion process is the cause of the lack of re- sponsiveness by freshly isolated luteal cells to EGF or TGF-a, unless the EGF-R on luteal cells is less glycosy- lated Oberg et al. (1990) have suggested that certain de- tails such as the structure of oligosaccharide side chains for EGF-R may differ in different cell types. An altema- tive explanation for the lack of EGF and TGF-a effect on freshly isolated cells may be that these cells secreted endogenous EGF and TGFa constitutively, which either masked the effect of the exogenous growth factors or down regulated the EGF-R on the freshly isolated cells. This scenario is plausible in view of the localization of EGF and TGF-a in luteal cells in other studies (Lobb and Dorrington, 1993; Singh, Kennedy, Tekpetey and Arm- strong, unpublished data). Furthermore, in studies with human granulosa cells undergoing luteinization, tyr- phostin, a putative inhibitor of the intrinsic EGF-R tyrosine kinase activity, inhibited both basal and EGF- stimulated progesterone ‘production indicating endogenous production of EGF/TGF-a in those cultures (Tekpetey et al., 1994). Under such circumstances, the overnight pre-culture of freshly isolated cells in the presence of serum permitted the removal of the endogenous growth factors and in addition some serum component(s) may, conceivably, have allowed for the replenishment of the cell-surface EGF-R thus rendering the cells responsive

to exogenous EGF and TGF-a. Studies with A-431 cells and human fibroblasts indicate that restoration of cell surface EGF-R following down regulation involves synthesis of new receptors which is negligible in the absence of serum but enhanced dramatically in the presence of serum (reviewed by Carpenter, 1983).

The signal transduction mechanisms involved in EGF and TGF-a action on gonadal steroidogenic cells are largely unknown. Classically, the interaction EGF/rCiF-a with EGF-R stimulates the hydrolysis of phosphatidyl inositol-45biphosphate leading to the transduction of second messenger signals via the protein kinase C (PKC) pathway (Abdel-Latif, 1986). However, EGF is also ca- pable of activating other signal transduction systems, in- cluding those mediated by phospholipase AZ and D (Bocckino et al., 1987; Teitelbaum, 1990). Furthermore, there is evidence for a positive cooperation between the protein kinase A (PKA) pathway and the activation of EGF-R-linked cellular events (Ball et al., 1990; Budnik and Mukhopadhyay, 1991, 1993). Epidermal growth factor has been shown to stimulate intracellular CAMP accumulation in pig oocyte-cumulus complexes (Singh, 1993) and in A-431 cells (Ball et al., 1990). In studies with rat granulosa cells, Knecht and Catt (1983) observed an inhibition of FSH-induced CAMP accumulation by EGF, while Trzeciak et al. (1987) have reported that EGF stimulated the synthesis of the cholesterol side-chain cleavage enzyme complex alone or together with FSH without enhancing CAMP formation. Luteal cell steroidogenesis is primarily regulated via the LH receptor- coupled PKA pathway (Richards et al., 1987), although luteal steroidogenic activity can also be stimulated by PKC activation (Brunswig et al., 1986). Recent reports have indicated that the EGF-R in bovine luteal cells lacks the intrinsic tyrosine kinase activity (Chakravorty et al., 1992), and that this receptor activates alternative signal- ling pathways (Chakravorty et al., 1993). Whether the luteal cell EGF-R of other species have similar defect is unknown. One report has indicated that EGF inhibited gonadotropin-sensitive adenylate cyclase activity in rat luteinized ovaries (Hafez and Ascoli, 1990). In contrast, other reports indicate that EGF enhances basal and LH or forskolin-stimulated adenylate cyclase activity in bovine luteal cells (Budnik and Mukhopadhyay, 1991, 1993). In the present study, the combined effects of LH and EGF or TGF-a on progestin production by luteal cells were at best additive and not synergistic, suggesting that the effects of these two agents may be mediated by converging intracellular signal transduction pathways, possibly via CAMP activation. Further studies are needed to verify this possibility.

In summary, we have localized immunoreactive EGF- R in rat small and large luteal cells, and demonstrated a physiological role for these receptors involving EGF and TGF-a stimulation of luteal cells steroidogenesis. These data provide further evidence to support the concept of a

102 F.R. Tekpetey et al. /Molecular and Cellular Endocrinology 110 (1995) 95-102

local autocrine and/or paracrine modulation of luteal steroidogenesis by growth factors.

Acknowledgements

We thank J.M. Rutledge for technical assistance and B. Lowery for secretarial assistance in the preparation of this manuscript. We also thank the National Hormone and Pituitary Program (Baltimore, MD) for the donation of LH. This work was supported by the Medical Research Council (MRC) of Canada. BS is an MRC fellowship awardee and DTA is an MRC career investigator.

References

Abdel-Latif, A.A. (1986) Pharmacol. Rev. 38227-272. Alila, H.W. and Hansel, W. (1984) Biol. Reprod. 31, 1015-1025. Ayyagari, R.R. and Khan-Dawood, F.S. (1987) Am. J. Obstet. Gynecol.

156,942-946. Ball, R.L.. Tanner, K.D. and Carpenter, G. (1990) J. Biol. Chem. 265,

1283612845. Bendell, J.J. and Dorrington. J.H. (1990) Endocrinology 127,533-540. Bocckino, S.B., Blackmore, P.F., Wilson, P.B. and Exton, J.H. (1987)

J. Biol. Chem. 262, 15309-15315. Brunswig, B., Mukhopadhyay, A.K., Budnik, L.T., Bohnet, H.G. and

Leidenberger, F.A. (1986) Endocrinology 118,743-749. Budnik, L.T. and Mukhopadhyay, A.K. (1991) J. Biol. Chem. 266,

13908-13913. Budnik, L.T. and Mukhopadhyay, A.K. (1993) Endocrinology 133,

265-270. Carpenter, G. (1983) Mol. Cell. Endocrinol. 31, 1-19. Chabot, J.-G., St-Amaud, R., Walker, P. and Pelletier, G. (1986) Mol.

Cell. Endocrinol. 44,99-108. Chakravorty, A., Joslyn, M.I. and Davis, J.S. (1992) Proc. IX Ovarian

Workshop, Chapel Hill, NC. Chakravotty, A., Imakawa, K. and Davis, J.S. (1993) Biol. Reprod. 48

(suppl. I), abstract 181. Cbegini, N., Lei, Z.M. and Rao, C.V. (1989) in Growth Factors in the

Ovary (Hirshfield, A.N., ed.), pp. 213-230, Plenum Press, New York.

Chegini, N. and Williams, R.S. (1992) J. Clin. Endocrinol. Metab. 74, 973-980.

Cummings, R., Soderquist, A. and Carpenter, G. (1985) J. Biol. Chem. 260,11944-l 1952.

Endo, K., Atlas, S.J., Rone, J.D., Zanagnolo, V.L., Kuo, T.-C., Dhar- marajan, A.M. and Wallach, E.E. (1992) Endocrinology 130, 186 192.

Fujinaga, H.. Yamoto, M., Nakano, R. and Shima, K. (1992) Biol. Re- prod. 46.705-709.

Gospodarowicz, D., 111, C.R. and Birdwell, C.R. (1977) Endocrinology 100,1108-l 120.

Hafez, M.M. and Ascoli, M. (1990) Endocrinology 127,394402. Hsueh, A.J.W., Welsh, T.H. and Jones, P.B.C. (1981) Endocrinology

108,2002-2004. Jones, P.B.C., Welsh, T.H. and Hsueh, A.J.W. (1982) J. Biol. Chem.

257,11268-l 1273.

Kennedy, T.G., Brown, K.D. and Vaughan, T.J. (1993) Endocrinology 132, 1857-1859.

Khan-Dawood, F.S. (1987) Fertil. Steril. 47,916919. Knecht, M. and Catt, K.J. (1983) 1. Bioi. Chem. 258.2789-2794. Kudlow, J.E., Kobrin, M.S., Pirchio, A.F., Twardzik, D.R., Hemandez,

E.R., Asa, S.L. and Adashi, E.Y. (1987) Endocrinology 121, 1577- 1579.

Leung, P.C.K. and Armstrong, D.T. (1979) Endocrinology 104. 1411- 1417.

Lobb. D.K., Kobrin, MS., Kudlow, J.E. and Dorrington, J.H. (1989) Biol. Reprod. 40, 1087-1093.

Lobb, D.K. and Dotxington, J.H. (1993) Reprod. Fertil. Dev. 5, 523- 529.

McFarland, K.C., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1989) Science 245, P494-499.

May, J.V., Bridge, A.J., Gotcher, E.D. and Gangrade, B.K. (1992) Endocrinology 131.689697.

May, J.V., Frost, J.P. and Schomberg, D.W. (1988) Endocrinology 123, 168-179.

Morley, P., Armstrong, D.T. and Gore-Langton, R. (1987) J. Cell Physiol. 132, 226-236.

Oberg, K.C., Brown, A. and Carpenter, G. (1990) in Growth Factors, Differentiation Factors and Cytokines (Habenicht, A., ed.), pp. 3- 17, Springer-Verlag. New York.

Prigent, S.A. and Lemoine. N.R. (1992) Prog. Growth Factor Res. 4, I- 24.

Richards, J.-A., Jahsen, T., Hedin, L., Litka, J., Ratoosh, S., Durica, J.M. and Goldring, N.B. (1987) Recent Prog. Harm. Res. 43, 231- 267.

Roy, S.K. and Greenwald, G.S. (1991) Biol. Reprod. 44.889-896. Schreiber, A.B., Winkler, M.E. and Derynck, R. (1986) Science 232,

1250-1253. Singh, B. (1993) Biol. Reprod. 48 (suppl. l), 102 (abstract 175). Singh, B., Rutledge, J.M. and Armstrong, D.T. (1995). Mol. Reprod.

Dev., in press. Skinner, M.K. and Coffey, Jr., R.J. (1988) Endocrinology 123, 2638-

2638. Sordoillet, C., Chauvin, M.A., deperetti, E., Morera, M. and Benahmed,

M. (1991) Endocrinology 128,2160-2168. Steel, R.E.D. and Torrie, J.H. (1960) Principles and Procedures for

Statistics, McGraw-Hill, New York. Teitelbaum, I. (1990) J. Biol. Chem. 265.42184222. Tekpetey, F.R. and Armstrong, D.T. (1991) Biol. Reprod. 45,498-505. Tekpetey, F.R. and Armstrong, D.T. (1994) Mol. Cell. Endocrinol. 101,

49-57. Tekpetey, F.R., Daniel, S. and Armstrong, D.T. (1994) Proc. American

Fertility Society Meeting, San Antonio, TX, abstract O-141. Tosbach, J., Poff, J.P. and Tsang, P.C.W. (1992) Biol. Reprod. 46

(suppl. 1). abstract 202. Trzeciak, W.H., Duda, T., Waterman, M.R. and Simpson, E.R. (1987)

Mol. Cell. Endocrinol. 52,43-50. Vlodavsky, I., Brown, K.D. and Gossodarowicz, D. (1978) J. Biol.

Chem. 253.3744-3750. Yeh, J., Lee, G.Y. and Anderson, E. (1993) Biol. Reprod. 48, 1071-

1081.

localization of epidermal growth factor (egf) receptor in the rat corpus luteum, and egf and...

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