0021-972x/92/7505~1362$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 75, No. 5 Printed in U.S.A

Gestational Age-Dependent Growth Factor on Human TAKESHI MARUO, HIROYA AND MATSUTO MOCHIZUKI Department

of

MATSUO,

KAZUO

Obstetrics and Gynecology, Kobe University

Dual Action of Epidermal Placenta Early in Gestation* MURATA, School of Medicine,

Kobe 650, Japan

ABSTRACT In order to better understand the role of epidermal growth factor (EGF) in the regulation of placental growth and function, effects of EGF on proliferative activity and differentiated function of trophoblast were examined. Explants from very early (4-5 week) placentas and early (6-7 week, 8-9 week, lo-12 week) placentas were respectively cultured under a serum-free condition in the absence or presence of EGF (100 pg/L) for the first 48 h, and the cultures were continued for subsequent 72 h without EGF. The proliferative potential and differentiated function of trophoblast were assessed by immunohistochemical Ki-67 staining and by determining the ability to secrete human CG (hCG) and human placental lactogen (hPL), respectively. Quantitative estimates of proliferative activity based on mean percentage of Ki-67

positive nuclei showed that EGF stimulated proliferative potential of cytotrophoblast in very early (4-5 week) placental explants. The EGF stimulation of trophoblast proliferation was apparent at a 12-h EGFtreated period. By contrast, early (6-12 week) placental explants did not respond to EGF with increase in trophoblast proliferation. Instead, in early placental explant culture EGF stimulated hCG and hPL secretion with a lag period of 72 h, whereas very early placental explants did not respond to EGF with increase in hCG and hPL secretion. These results suggest that EGF exerts gestational age-dependent dual action on the first-trimester placenta: one is to stimulate trophoblast proliferation in 4-5 week placenta and the other is to stimulate differentiated trophoblast function in 6-12 week placenta. (J Clin Endocrinol Metub 75: 1362-1367, 1992)

T

EGF receptors in very early placenta are primarily localized to cytotrophoblast suggests that EGF in very early placenta may be linked to the proliferation rather than to the differentiated function of trophoblast. Recently, a monoclonal antibody, Ki-67, has been produced that labels the nuclei of proliferating cells. The Ki-67 antibody specifically recognizes an as yet undefined human nuclear antigen that is present only in proliferating cells, but absent in resting cells (5, 6). Thus, immunohistochemical Ki67 labeling is useful for evaluation of the proportion of proliferating cells in normal and neoplastic cell populations (7). In the present study, effects of EGF on trophoblast proliferation and differentiated trophoblast function were assessed by determining the percentage of Ki-67 positive nuclei of cultured trophoblasts and by determining the ability to secrete hCG and hPL, respectively. Then, attention was directed to a comparative assessment of the possible effects of EGF on very early placenta and early placenta in order to better understand the role of EGF in the regulation of placental growth and function during the course of early gestation. The data presented demonstrate that EGF exerts gestational age-dependent dual action on the first-trimester placenta: one is to stimulate trophoblast proliferation in 4-5 week placenta and the other is to stimulate differentiated trophoblast function in 6-12 week placenta.

HE VILLOUS trophoblast in early human placenta is composed of two layers of cytotrophoblast and syncytiotrophoblast. It is evident that cytotrophoblast displays highly proliferative and invasive properties early in the first trimester, while syncytiotrophoblast displays little potential for proliferation throughout gestation (1, 2). We have recently demonstrated that in very early (4-5 week) placenta epidermal growth factor (EGF) and EGF receptor are primarily localized to cytotrophoblast, whereas EGF and EGF receptor in early (6-12 week) placenta are predominantly localized to syncytiotrophoblast (3). The simultaneous expression of EGF and EGF receptor in cytotrophoblast of 4- to 5-week placenta and in syncytiotrophoblast of 6- to 12-week placenta raises a possibility that EGF in the first-trimester placenta may act in an autocrine manner. In keeping with the observation that EGF receptors in 6to 12-week placenta are almost exclusively localized to syncytiotrophoblast which contains abundant human CG (hCG) and human placental lactogen (hPL), we have reported that EGF exerts stimulatory effect on hCG and hPL secretion by cultured early placental tissues and that the stimulatory effect of EGF closely correlates with the cellular levels of EGF receptor demonstrable on trophoblast (4). This suggests that EGF in early placenta affects differentiated trophoblast function via its own receptors. On the other hand, the fact that Received February 3, 1992. Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Department of Obstetrics and Gynecology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan. * This work was supported in part by Grants in Aid for Scientific Research 63480368 and 0267074 from the Japanese Ministry of Education, Science and Culture, and by the Ogyaa-Donation Foundation of Japan Association of Maternal Welfare.

Subjects

and Methods

Materials First-trimester placentas were obtained from 12 patients who underwent therapeutic abortion at 4-5 weeks (3 cases), 6-7 weeks (3 cases), 8-9 weeks (3 cases), and lo-12 weeks (3 cases) of gestation. The gestational age of the placenta was determined by estimation of the

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DUAL

ACTION

OF EGF ON EARLY

duration of pregnancy from the date of patient’s last menstrual period. In all cases, gestational age was further evaluated by ultrasound examination. Informed consent was obtained before operation for the use of placental tissues for culture experiments.

Tissue culture An in vifro culture system similar to that previously described was used (8). Briefly, explants of placental villous tissues (total wet wt, 50 mg) were placed on filter paper within plastic multiwell plates, to which 2 mL serum-free medium comprising McCoy’s 5a medium (GIBCO, Grand Island, NY) supplemented with 1 X lo5 U/L penicillin and 50 mg/L streptomycin were added. The placental explants were cultured at 37 C in an atmosphere of 95% air-5% CO? in the presence or absence of EGF (Collaborative Research, Waltham, MA) for the first 2 days, and the cultures were continued for subsequent 3 days in the absence of EGF. In this experiment, 100 *g/L EGF was used as the appropriate dose of EGF as described in our previous study (4). The medium was changed every day. Each set of culture was performed in triplicate. When the cultures were terminated, the medium was collected and stored at -20 C until analyzed.

Immunohistochemical

staining

with Ki-67

Cultured placental explants were frozen in OCT compound (Miles Scientific, Naperville, IL) in liquid nitrogen and stored at -80 C until used. The frozen sections were cut at 5-6 pm, mounted on albumincoated slides, and fixed in acetone at -20 C for 20 min. Slides were removed from the fixative and allowed to dry completely for 30-60 s. The sections were rinsed in cold phosphate-buffered saline thoroughly, exposed to 20% normal goat serum to prevent nonspecific binding of the antibody to the sections after blocking endogenous peroxidase by 0.3% hydrogen peroxide in methanol, and then incubated with a monoclonal antibody Ki-67 (Dako, Glostrup, Denmark) for 1 h at room temperature. The Ki-67 antibody was diluted 1:20 with phosphatebuffered saline containing 0.5% BSA. The avidin/biotin immunoperoxidase method was employed using the polyvalent immunoperoxidase kit (Omnitags, Lipshaw, Ml) (9). Ki-67 labeling was developed with a diaminobenzidine-hydrogen peroxide substrate (DAB, Wako, Osaka, Japan), and sections were lightly counterstained with half-strength hematoxylin. All immunostained sections were analyzed and scored in a blinded fashion without knowledge of the experimental group. Mean percentage of Ki-67 positive nuclei for each group of placental samples was determined by counting the number of trophoblasts for more than 500 nuclei for each group.

Radioautography

of tritiated

PLACENTA

1363

Results Effect of EGF on proliferatiue trophoblast

activity of cultured

placental

Immunostaining with Ki-67 on frozen sectionsof cultured placental explants showed that a substantial number of cytotrophoblast nuclei were positively stained, whereas syncytiotrophoblast nuclei were totally negative for the staining. Figure 1 represents the Ki-67 staining features of frozen sections of very early (4-5 week) placental explants cultured in the presence or absenceof 100 pg/L EGF for 12 h. In the explants cultured with EGF, a greater number of Ki-67 positive cytotrophoblast nuclei were observed relative to the control explants cultured without EGF. By contrast, in cultures of early (8-9 week) placental explants there was no apparent difference in the number of Ki-67 positive cytotrophoblast nuclei between EGF-treated and untreated explants (Fig. 2). Table 1 shows mean percentage of Ki-67 positive nuclei of cytotrophoblasts in sections of very early (4-5 week) placental explants and early (6-12 week) placental explants cultured in the presenceor absenceof 100 pg/L EGF for 12, 24, and 48 h. In cultures of very early 4- to 5-week placental explants, EGF treatment augmented mean percentage of Ki67 positive nuclei of cytotrophoblasts compared to the control explants (P < 0.02). The increase in mean percentage of Ki-67 positive nuclei of cytotrophoblasts in responseto EGF treatment was apparent at 12-h EGF-treated culture period and maintained for up to 24-h EGF-treated culture period, and returned to control level by 48-h EGF-treated culture period. By contrast, in the case of 6- to 7-week placental

thymidine

EGF-treated and -untreated early placental explants cultured for 120 h were incubated with 1 mCi/L tritiated thymidine (New England Nuclear, Boston, MA) at 37 C for 2 h in an atmosphere of 95% air and 5% CO*. After the pulse incubation, the placental explants were subjected to radioautography as previously described (9).

RIA lmmunoreactive hCG and hPL were determined by homologous double antibody RIA for hCG (10) and hPL RIA Kit (Daiichi Radioisotope Lab., Tokyo), respectively.

Statistical

analysis

All experiments were repeated three times with similar results, and the reported results are representative. The statistical significance of the difference between sample mean was determined by Student’s t test. A statistically significant difference was considered to be present at P less than 0.05.

FIG. 1. Ki-67 immunohistochemical staining of very early first-trimester (4-5 week) placental explants cultured in the absence or presence of EGF (100 pg/L) for 24 h. Frozen sections of 4- to 5-week placental explants cultured in the absence (A) or presence (B) for 24 h were stained with Ki-67 using the avidin/biotin-immunoperoxidase method. Arrows indicate Ki-67 positive nuclei. The lighter staining of nuclei is the hematoxylin counterstain. A substantial number of cytotrophoblast nuclei were positively stained, whereas syncytiotrophoblast nuclei were negative for the Ki-67 immunostaining. Original magnification, x200.

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JCE & M .1992 Voll5.No5

contrast, in cultures of early 6- to 7-week placental explants, EGF treatment significantly augmented hCG and hPL secretion compared to EGF-untreated cultures (hCG: P < 0.05, hPL: P < 0.05) (Fig. 5). The stimulatory effects of EGF on hCG and hPL secretion became apparent only 48 h after removal of EGF. Similar stimulatory effects of EGF on hCG and hPL secretion were also attained with the use of early placental explants obtained at 8-9 week gestation (Fig. 5) and lo-12 week gestation (data not shown).

Comment

FIG. 2. Ki-67 immunohistochemical staining of early first-trimester (8-9 week) placental explants cultured in the absence or presence of EGF (100 rg/L) for 24 h. Frozen sections of 8- to g-week placental explants cultured in the absence (A) or presence (B) for 24 h were stained with Ki-67 using the avidin/biotin immunoperoxidase methods. Arrows indicate Ki-67 positive nuclei. The lighter staining of nuclei is the hematoxylin counterstain. Original magnification, x200.

explants, no significant effects of EGF treatment on mean percentage of Ki-67 positive nuclei of cytotrophoblasts were observed. Similar results were attained with the use of early placental explants obtained at 8-9 week gestation or lo-12 week gestation (Table 1). Figure 3 shows radioautographic uptakes of tritiated thymidine by early (8-9 week) placental explants cultured for 120 h. Radioautographic feature of tritiated thymidine uptake by placental tissues cultured for 120 h was similar between EGF-treated and -untreated explants. Effect of EGF on hCG and hPL secretion by cultured placental trophoblasts In cultures of very early 4- to 5-week placental explants, there were no significant differences in hCG and hPL secretion into the medium throughout the whole culture period between EGF-treated and untreated cultures (Fig. 4). By TABLE week,

EGF has been shown to be mitogenic for a variety of cell types (11, 12). Furthermore, it is evident that EGF also affects differentiated function of normal and neoplastic cells (4, 13, 14). Early placental tissues cultured under a serum-free condition have been shown to maintain their viability for a 7day culture period when determined by radioautographic uptakes of 3H-labeled thymidine and uridine (15). New synthesis of hCG has also been demonstrated by measuring incorporation of 3H-labeled proline into the synthesized hormone (16, 17). Thus, the in vitro culture system of placental explants provides a suitable model for investigating the regulation of placental growth and endocrine function. The data obtained in the present study demonstrate that EGF enhances cytotrophoblast proliferation in culture of very early (4-5 week) placental tissues, without affecting the ability to secrete hCG and hPL. The mitogenic effect of EGF observed on very early placenta was great contrast to the effect of EGF observed on early (6-12 week) placenta. Namely, in culture of 6- to 12-week placental tissues, EGF did not affect cytotrophoblast proliferation. Instead, EGF stimulated hCG and hPL secretion by cultured placental tissues. These findings suggest that EGF has gestational agedependent dual action on first-trimester placenta: one is to stimulate the proliferative potential of cytotrophoblasts in very early placenta before 6 weeks of gestation and the other is to stimulate hCG and hPL secretion by early placenta between 6 and 12 weeks of gestation. Thus, EGF appears to exert the two different actions independently in a stepwise manner on the proliferation and differentiated function of trophoblast during the course of first-trimester gestation.

1. Mean percentage of Ki-67 positive nuclei of cytotrophoblasts in frozen sections of very early (4-5 week) and early and lo-12 week) placental explants cultured in the absence or presence of EGF (100 pg/L) for 12, 24, and 48 h Gestational ace

Treatment

(6-7

12-h culture

24-h culture

48-h culture

4-5 week

EGF-untreated EGF-treated

25.2 f 3.4% 40.2 + 5.1%”

28.8 + 2.5% 47.7 f 2.0%”

18.3 * 3.5% 21.7 f 3.1%

6-7 week

EGF-untreated EGF-treated

26.8 + 2.8% 30.1 + 3.3%

28.0 zk 2.6% 32.3 k 3.1%

17.2 3~ 2.4% 19.6 + 2.0%

8-9 week

EGF-untreated EGF-treated

27.9 + 4.2% 24.4 + 3.6%

25.0 It 2.9% 28.6 + 3.1%

16.3 zk 3.0% 12.0 + 2.6%

lo-12

EGF-untreated EGF-treated

28.2 zk 3.0% 27.5 + 2.7%

29.1 + 2.5% 28.8 + 2.4%

15.8 + 2.0% 14.0 + 2.2%

week

The results represent ’ P < 0.02 (us. control

the mean + SD of triplicate cultures. cultures without EGF treatment).

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week,

8-9

DUAL

ACTION

OF EGF

ON EARLY

1365

PLACENTA [ 4 - 5 week

placenta

1

5

(days)

5

(days)

FIG. 3. Radioautographic uptake of tritiated thymidine by EGFtreated and -untreated early (8-9 week) placental tissues cultured for 120 h. Cultured placental tissues were exposed to tritiated thymidine for 2 h. The formalin-fixed, paraffin-embedded sections were subjected to autoradiography. A, EGF-treated explants; B, EGF-untreated explants. Original magnification, X 200.

Although the role of EGF in the induction of differentiated trophoblast function has been described by several investigators (18-21) including us (4), this is the first to describe the mitogenic effect of EGF on cytotrophoblasts in very early human placenta. The stimulatory effect of EGF on proliferative activity of cultured very early placental trophoblast was apparent at 12h EGF treatment. By contrast, the stimulatory effect of EGF on hCG and hPL secretion by cultured early placental explants became apparent only 72 h after termination of EGF treatment. It is of interest to note that EGF stimulation of proliferative potential of very early placental trophoblast takes place as a rapid effect during EGF treatment, whereas EGF stimulation of hCG and hPL secretion by early placental explants occurs as a delayed effect with a lag period of 72 h after removal of EGF. The delayed stimulatory effect of EGF on hCG and hPL secretion is not likely to be due to improved viability of cultured placental explants, since there were no differences in mean percentage of Ki-67 positive nuclei of cytotrophoblasts between EGF-treated and -untreated early placental explants. The mean percentage of Ki-67 positive cytotrophoblast nuclei in early placental explants cultured for 120 h was 4.1 f 2.0% in EGF-treated culture and 3.8 + 1.8% in EGF-untreated culture. Similar results concerning viability of the cultured placental tissueswere also obtained by radioautographic labeling of tritiated thymidine (Fig. 3). The first step in the biological actions of EGF is represented by its binding to specific receptors present in the target cells (22). The binding process is thought to lead to receptor phosphorylation, internalization of the ligand-receptor complex, and generation of signals that mediate the actions of EGF (23). Consistent with the present observation that EGF exerts the dual action in a stepwise manner on cytotrophoblast proliferation in very early placenta and on hCG and hPL secretion by early placenta, we have shown that EGF

1

2

3

4

4. Human CG and hPL secretion into the medium from very early first-trimester (4-5 week) placental explants cultured in the absence or presence of EGF. Very early (4-5 week) placental explants were cultured in the absence (op& lx&) or presence (solid bars) of 100 pg/L EGF during the first 48 h of culture, and the cultures were continued for subsequent 72 h in the absence of EGF. The results represent the mean f SD of triplicate cuhures. FIG.

receptors in very early (4-5 week) placenta are primarily localized to cytotrophoblast, whereas EGF receptors in early (6-12 week) placenta are almost exclusively localized to syncytiotrophoblast. Thus, the change in cytologic localization of EGF receptors from cytotrophoblast to syncytiotrophoblast observed around 6 weeks of gestation is of great importance for the stepwise expressionof the gestational age dependent dual action of EGF on placental trophoblasts during the course of first-trimester gestation. In support of the above mentioned hypothesis that EGF may act as an autocrine signal to regulate placental growth and function in first-trimester gestation, an EGF-like substance measurableby human EGF-RIA has been found to be produced and secreted by cultured early placental trophoblasts. The similarity of this placental EGF-like substanceto authentic human EGF is supported by parallel displacement in a specific human EGF-RIA and coelution during Sephadex G-75 gel filtration of serum-free media conditioned by early placental explants (Maruo, T., K. Murata, H. Matsuo, and M. Mochizuki, unpublished data). Additional evidence for autocrine control of placental growth has been reported. Goustin et al. (24) have demonstrated that the first-timester placenta expressesboth c-myc

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1366

FIG. 5. Human CG and hPL secretion into the medium from early first-trimester (6-7 week, 8-9 week) placental explants cultured in the absence or presence of EGF. Six- to 7-week placental explants (A) and 8- to g-week placental explants (B) were, respectively, cultured in the absence (open bars) or presence (solid bars) of 100 pg/L EGF during the first 48 h of culture, and the cultures were continued for subsequent 72 h in the absence of EGF. The results represent the mean & SD of triplicate cultures. (*), P < 0.05; (**), P < 0.01 (us. control cultures without EGF treatment).

-

MARUO

ET

[ 6-7 week

placenta

(, 1

1 8 - 9 week

I

*

:~ 2

JCE & M. 1992 Vol75.No5

AL.

and c-sis, suggesting the ability to produce platelet derived growth factor (PDGF) and that first-trimester placenta has specific receptors for PDGF and responds to exogenous PDGF by activating the c-myc gene and DNA synthesis. It is therefore likely that EGF, along with other growth factors intrinsic to placental trophoblasts, act together to regulate placental growth via autocrine/paracrine mechanism in firsttrimester gestation. Further studies to characterize factors responsible for the regulation of the synthesis of these growth factors in the placenta should provide an important insight into the mechanism regulating human placental growth and function. References 1. Gerbie AB, Hathaway HH, Brewen JL 1968 Autoradiographic analysis of normal trophoblastic proliferation. Am J Obstet Gynecol. 100:640-650. 2. Kliman HJ, Nestber JE, Sermasi E, Sanger JM, Strauss III JF. 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placenta. Endocrinology. 118:15671582. 3. Ladines-Llave CA, Maruo T, Manalo AS, Mochizuki M. 1991 Cytologic localization of epidermal growth factor and its receptor in developing human placenta varies over the course of pregnancy. Am J Obstet Gynecol. 165:1377-1382. 4. Maruo T, Matsuo H, Oishi T, Hayashi M, Nishino R, Mochizuki M. 1987 Induction of differentiated trophoblast function by epiderma1 growth factor: relation of immunohistochemicallv detected cellular epidermal growth factor receptor levels. J C&n Endocrinol Metab. 64:744-750. 5. Gerdes J, Schwab U, Lemke H, Stein H. 1983 Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 31:13-20. 6. Gerdes J, Lemke H, Baisch H, Wacker H, Schwab U, Stein H. 1984 Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 133:1710-1715.

1

*

~ 3

placenta

4

5

(days)

4

5

(W's)

1

2

3

4

5

(W's)

7. Kamel OW, Franklin WA, Ringus JC, Meyer JS. 1989 Thymidine labeling index and Ki-67 growth fraction in lesions of the breast. Am J Pathol. 134:107-113. 8. Maruo T, Matsuo H, Ohtani T, Hoshina M, Mochizuki M. 1986 Differential modulation of chorionic gonadotropin subunit mRNA levels and chorionic gonadotropin secretion by progesterone in normal placenta and choriocarcinoma cultured in vitro. Endocrinology. 119:855-864. 9. Maruo T, Mochizuki M. 1987 Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation. Am J Obstet Gynecol. 156:721-727. 10. Maruo T, Segal SJ, Koide SS. 1979 Studies on the apparent human chorionic gonadotropin-like factor in the crab ovalipes ocellatus. Endocrinology. 104:932-939. 11. Carpenter G, Cohen S. 1979 Epidermal growth factor. Annu Rev Biochem. 48:193-216. 12. Gospodarowicz D. 1981 Epidermal and nerve growth factors in mammalian development. Annu Rev Physiol. 43:251-265. 13. Hsueh AJW, Welsh TH, Jones PBC. 1981 Inhibition of ovarian and testicular steroidogenesis by epidermal growth factor. Endocrinology. 108:2002-2004. 14. Futamura K, Maruo T, Mochizuki M. 1987 Differential effects of dibutyryl cyclic AMP and epidermal growth factor on the synthesis and secretion of human chorionic gonadotropin and its subunits by trophoblastic and nontrophoblastic cells. Acta Obstet Gynecol Jpn. 39:1641-1648. 15. Maruo T, Ashitaka Y, Mochizuki M, Tojo S. 1974 Chorionic gonadotropin synthesized in cultured trophoblast. Endocrinol Jpn. 21:499-505. 16. Maruo T. 1976 Studies on in vitro synthesis and secretion of human chorionic gonadotropin and its subunits. Endocrinol Jpn. 23:119128. 17. Maruo T, Koide SS. 1980 Large molecular species of chorionic gonadotropin from human placental tissues: biosynthesis and physice-chemical properties. Acta Endocrinol (Copenh). 94:259-267. 18. Huot R, Foidart J, Nardone R, Stromberg K. 1981 Differential modulation of human chorionic gonadotropin secretion by epiderma1 growth factor in normal and malignant placental cultures. J Clin Endocrinol Metab. 53:1059-1063. 19. Lai WA, Guyda HJ. 1984 Characterization and regulation of EGF

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DUAL

ACTION

OF EGF

receptors in human placental cultures, J Clin Endocrinol Metab. 58:“44-352. 20. Morrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy 0.1987 Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab. 65:1282-1290. 21. Barnea ER, Feldman D, Kaplan M, Morrish DW. 1990. The dual effect of epidermal growth factor upon human chorionic gonadotropin secretion by the first trimester placenta in vitro. J Clin Endocrinol Metab. 71:923-928.

ON EARLY

PLACENTA

1367

22. Cohen S, Carpenter G, King Jr L. 1980 Epidermal growth factor receptor interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 255:4834-4839. 23. Schreiber AB, Libermann TA, Lax I, Yarden Y, Schlessinger J. 1983 Biological role of epidermal growth factor-receptor clustering. J Biol Chem. 258:846-850. 24. Goustin AS, Betsholtz C, Pfeiffer-Ohlsson S, et al. 1985 Coexpression of the sis and myc proto-oncogenes in developing human placenta suggests autocrine control of trophoblast growth. Cell. 41:301-312.

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Gestational age-dependent dual action of epidermal growth factor on human placenta early in gestation.

In order to better understand the role of epidermal growth factor (EGF) in the regulation of placental growth and function, effects of EGF on prolifer...
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