0013-7227/92/1313-1202$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 131, No. 3 Printed in U.S.A.

Expression of the Insulin-Like Growth Factor (IGF)-I and -11 and the IGF-I and -11 Receptor Genes during Postnatal Development of the Rat Ovary M. D.

J. LEVY, LEROITH

E.

R.

HERNANDEZ,

E.

Y.

ADASHI,

R.

J. STILLMAN,

C. T.

ROBERTS,

JR.,

AND

Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (M.J.L., C.T.R., D.L.), Bethesda, Maryland20892; University of Maryland School of Medicine (E.R.H., E. Y.A.), Baltimore, Maryland 21201; and the Department of Obstetrics and Gynecology, George Washington University School of Medicine (M. J.L., R. J.S.), Washinggton, D.C. 20037 ABSTRACT

levels after day 10. IGF-I receptor mRNA levels increased lo-fold to a maximum in the 20- to 25-day postnatal period. This pattern was similar to the developmental pattern of [‘Z61]IGF-I binding in the ovary. Two apparent peaks of IGF-II/mannose-6-phosphate receptor mRNA levels were seen, on day 20 and between days 50-80. These specific and significant changes in the expression of the genes encoding the IGFs and their receptors suggest a role for the IGF system in postnatal ovarian development. (Endocrinology 131: 1202-1206,1992)

Solution hybridization/RNase protection assays were used to study the developmental expression of the insulin-like growth factor-I (IGFI), IGF-II, IGF-I receptor, and IGF-II/mannose-6-phosphate receptor genes in the rat ovary between postnatal days l-80. Maximal IGF-I mRNA levels occurred during the 15- to 25-day postnatal period, and the level on day 20 represented a O-fold increase over the baseline at earlier and later stages. IGF-II mRNA levels were maximal during the l- to B-day postnatal period and subsequently declined to undetectable

T

HE insulin-like growth factor (IGF) family encompasses a group of related polypeptides, including insulin, IGFI, and IGF-II (1). They have mitogenic activity (2) and promote the expression of differentiated function through the activation of specific cell surface receptors. The IGF-I receptor is homologous to the insulin receptor and consists of two (Ysubunits and two p-subunits (3). The IGF-II receptor consists of a single-polypeptide chain and is identical to the mannose6-phosphate (M6P) receptor (4). IGF-I (somatomedin-C) is a major regulator of growth and development (5). Over 90% of circulating IGF-I is produced by the liver as a result of GH stimulation (6). A dramatic increase in liver IGF-I mRNA levels between fetal and adult stages suggests that circulating IGF-I has an important role in postnatal growth and development (7-10). Multiple extrahepatic tissues, including the ovary, are also capable of synthesizing IGF-I and IGF-II, and these locally produced factors may also be important in the postnatal development of specific tissues (7, 11-16). Maximal levels of plasma IGFII and mRNA expression have been demonstrated in the fetal and early neonatal stages, with markedly decreased levels in older animals (17). A role for IGF-II in fetal tissue maturation, therefore, has been suggested. It has been demonstrated that a complete intraovarian IGF system, including ligands (15-17), receptors (18), and binding proteins (19), is present. The roles of the IGF system in the Received January 8, 1992. Address all correspondence and requests for reprints to: Dr. Derek LeRoith, Diabetes Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 88243, 9000 Rockville Pike, Bethesda, Maryland 20892.

ovary may include regulation of the formation of the follicular apparatus during the late fetal/early neonatal period as well as mediation of the puberty-promoting effects of GH (20). The overall action of the ligand is primarily dependent upon the presence and concentration of the receptors with which it may interact. While the IGF-I and insulin receptors are transmembrane tyrosine kinases, the IGF-II/M6P receptor, by virtue of its involvement with the uptake and intracellular trafficking of lysosomal enzymes, has been implicated in tissue remodelling and regenerative processes. There has recently been a great deal of interest in the identification of intraovarian regulators. To clarify the potential role of the IGF system during ovarian development, we have examined the patterns of expression of the IGF and IGF receptor genes during postnatal development, and have correlated these with changes in IGF binding to the major mediator of IGF function, the IGF-I receptor. Materials

and Methods

Animals Sprague-Dawley rats were obtained from Taconic Laboratories (Germantown, PA). They were fed ad fibifum, and on postnatal days 1, 5, 10, 15, 20, 25, 35, 50, and 80, groups of rats were killed by cervical dislocation. Pregnant rats were obtained so that delivery of their offspring could be monitored to accurately sample the earlv postnatal &ou{. The ovaries were immediately rekoved and placed* iri dry ice, then stored at -70 C. Ovaries were pooled from 20 rats for postnatal days l-10, from 10 rats for postnatal days 15-20, and from 5 rats for postnatal days 25-80.

RNA isolation Total tissue RNA isothiocyanate-CsCl

was prepared precipitation

by a modification technique (21).

of the guanidinium RNA concentrations

1202

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EXPRESSION

OF IGF AND RECEPTOR

were calculated on the basis of absorbance at 260 nm. Ethidium bromidestained 28s and 18s ribosomal RNA bands were visually inspected after electrophoresis of 5 pg total RNA through 1.5% agarose-2.2 M formaldehyde gels. Solution

hybridization/RNase

protection

assay

The antisense RNA probes used to determine IGF-I, IGF-II, IGF-I receptor, and IGF-II/M6P receptor mRNA levels have been previously described (14, 15,22). The solution hybridizations were performed using a modification of previously described procedures (14). Solution hybridization/RNase protection assays were repeated at least twice, using 20 pg total RNA for all samples and all probes. The only exception was a single assay for days 1, 5, and 10 using the IGF-I receptor and IGF-II/M6P receptor probes due to insufficient RNA. The numerical values generated by the scanning densitometry of multiple autoradiographs were converted to arbitrary optical density units that were expressed as a percentage of the maximum. The average of the two values obtained from the separate experiments was used to plot each point in Fig. 2. Membrane

preparations

and binding

Results

1203

the presence of two protected bands, 322 and 241 bases in length, corresponding to exon 2- and exon l-containing IGFI mRNAs, respectively (14). At all developmental stages, these exons were regulated coordinately, with exon 1 mRNA being the major transcript (>90%). The peak level on day 20 was 9-fold higher (based on the exon 1 signal) than the average level at the earlier and later developmental stages measured (Fig. 2A). As illustrated in Figs. 2B and 3, maximal IGF-II levels occurred during the 1- to 5-day postnatal period. The levels subsequently declined rapidly to a level that was undetectable beyond day 10 at the RNA concentrations analyzed in these experiments. Maximal IGF-I receptor mRNA levels were seen during

studies

In a similar set of experiments, ovaries were removed and pooled from rats between postnatal days l-32. Membranes were prepared for ligand binding studies as previously described (23). [‘251]IGF-I binding was performed, and specific binding was calculated by subtracting binding in the presence of excess unlabeled IGF-I from total ‘*sI]IGF-I binding (18).

GENES

100

-

90

-

60

-

70

-

5

60

!I?

50-

%

‘lo

-

30

-

20

-

10

-

0-

The levels of expression of the IGF-I, IGF-I I, IGF-I receptor, and IGF-II/M6P receptor genes in the rat ovary were determined by solution hybridization/RNase protection assays, using RNA prepared from pooled ovaries from postnatal days l-80. As shown in Fig. 1, maximal IGF-I mRNA levels occurred during the 15- to 25-day postnatal period. The probe used in this solution hybridization/RNase protection assay allowed us to distinguish, in rat ovarian RNA, two of the variant IGF-I mRNAs that diverge in sequence in the 5’-untranslated/prepeptide-coding region. Due to this divergence, hybridization of the 32P-labeled antisense RNA, followed by RNase digestion of unhybridized probe, results in Probe 151015m2535!B938l+-

-20 0

10

20

30

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0

102030405060706090

0

10 20

100 90 60 70 5 60 : so40 30 20 10 01111111'111 0 102030405060708090

30

DAYS Exon 2 -I

Exon 1

-+I

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90

FIG. 2. Composite graph depicting the results of scanning densitometry of duplicate experiments of the type shown in Figs. 1, 3, 4, and 6. A, IGF-I probe; B, IGF-II probe; C, IGF-I receptor probe; D, IGF-II/ M6P receptor probe.

s‘

FIG. 1. Solution hybridization/RNase protection assay with an IGF-I antisense RNA probe. Aliquots of total RNA isolated from ovaries on the indicated postnatal days were assayed as described in Materials and Methods. The arrows on the left indicate the positions of protected probe bands corresponding to IGF-I mRNAs containing leader exon 2 (upper) or 1 (lower) sequences. The arrow on the right indicates the position of the full-length probe carried through the assay in the absence of target RNA and subsequently incubated with (+) or without (-) RNases.

40

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1

5

lo

15

20

253590

66

FIG. 3. Solution hybridization/RNase protection in Fig. 1, using an IGF-II antisense RNA probe.

80

Probe + -

assay, as described

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EXPRESSION

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OF IGF AND RECEPTOR

the 20- to 25-day postnatal period (Fig. 4). The peak level represented an approximately 9-fold increase over the levels seen through day 10 (Fig. 2C), and this was significantly higher than those at all other developmental stages. The initial peak of IGF-I receptor mRNA levels was similar to the developmental pattern of [‘251]IGF-I binding in the ovary (Fig. 5). That the increased IGF-I binding is associated with increased receptor concentration was confirmed by Scatchard analysis (data not shown). The transient decrease in specific [‘251]IGF-I binding seen in the presence of sustained IGF-I receptor mRNA levels may reflect a down-regulation of cell surface receptors, which follows the peak of IGF-I gene expression (and, presumably, ligand binding). IGF-II/M6P receptor gene expression exhibited a postnatal upward trend with two peaks, on day 20 and then again during the 50- to 80-day period (Figs. 2D and 6). Discussion A complete intraovarian IGF system that includes the ligands, receptors, and binding proteins has been demonstrated (24). We have previously shown that the granulosa cell is the site of IGF-I production in the rat ovary (16). Its biosynthesis is increased by FSH and estrogen. The content of rat ovarian IGF-I also appears to be GH dependent (20). 1

5

10

15

20

25

35 go

c6

al

Probe + -

FIG. 4. Solution hybridization/RNase protection assay, as described in Fig. 1, using an IGF-I receptor antisense RNA probe.

20

30

POSTNATAL DAY FIG. 5. [‘Z51]IGF-I specific binding to ovarian membranes as a function of postnatal development.

1

5

10

GENES

15

xl

Endo. 1992 Voll31. No 3

25

35 SIC6

al

Probe + -

FIG. 6. Solution hybridization/RNase protection assay, as described in Fig. 1, using an IGF-II/MGP receptor antisense RNA probe.

A direct effect of GH on IGF-I production by the rat granulosa cell has not yet been demonstrated. A variety of actions have been demonstrated for granulosa cell IGF-I in the rat. These include FSH-supported progesterone and estrogen biosynthesis, acquisition of LH receptors, and proteoglycan synthesis (25, 26). These local actions of IGF-I may be mediated through either autocrine or paracrine mechanisms. This study demonstrates that significant changes in the expression of the IGF-I, IGF-II, and IGF receptor genes occur during postnatal development of the rat ovary. Maximal expression of the IGF-I gene was seen on day 20, the time at which a significant increase in the expression of the IGFI and IGF-II receptor genes was also seen. A different pattern was found for IGF-II, with maximal expression in early postnatal life and greatly decreased expression after day 10. The peak expression of IGF-I and the IGF-I and IGF-II receptor genes occurs at a highly significant time in the development of the rat ovary. Specifically, this coincides with the early development of the follicles destined to be the first to ovulate approximately 19 days later. No large ovarian follicles (type 5b and larger) are present in the rat ovary before the end of the infantile period of development (days 7-21) (27). In view of the known mitogenic effects of IGF-I, it is attractive to propose a role for IGF-I in the development of these follicles. The plasma levels of FSH, a recognized signal for IGF production, increase dramatically between postnatal days 1 and 12 and then decrease steadily until a nadir on the morning of the first estrus (28, 29). This peak immediately precedes the increased IGF-I gene expression seen in this study. Serum levels of estrogen (another regulator of IGF production) in the female rat are also elevated during the infantile period, but this is mostly of adrenal origin and may not be biologically active (30-32). The estrogen then decreases due to the loss of a-fetoprotein that binds estrogen and a consequent increase in the MCR. These low levels are maintained until the preovulatory gonadotropin surge. The potential effects of estrogen on ovarian IGF-I gene expression in viva, therefore, may be indirect and somewhat slow-acting. GH may also be involved in the regulation of rat ovarian function (33-37). It has been shown that the suppression of GH results in a delay in pubertal development. This is potentially due to the direct action of GH in the ovary and not the failure of general body growth or the failure to reach a critical body weight (33-37). We have previously demonstrated specific high affinity IGF-I-binding sites on granulosa cell membranes (38). This binding capacity is up-regulated by FSH, LH, and GH, but not PRL (39). FSH may play a central role in IGF-I respon-

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EXPRESSION

OF IGF AND RECEPTOR

siveness, with CAMP playing an intermediary role in the process. Since FSH may increase IGF-I production by the ovary as well as increase the IGF-I receptor concentration, it seems likely that the peak levels of serum FSH between postnatal days lo-14 (data not shown) (28,29) may stimulate these developmentally associated IGF-I and IGF-I receptor changes in the ovary. Maximal IGF-II gene expression was demonstrated in early postnatal life. This is consistent with the elevated levels found in several other rat tissues in fetal or early postnatal life (17). Corresponding levels of immunoreactive IGF-II have been found in serum (40). This stage of high local IGFII gene expression coincides with the development of the fetal ovary and its follicular apparatus. The overall correlation of the developmental regulatory pattern of rat ovarian IGF-I and IGF-I receptor gene expression is not seen with IGF-II. Indeed, in early postnatal life, when the level of IGFII gene expression is maximal, IGF-II/M6P receptor gene expression is low. An initial peak of IGF-II/M6P receptor gene expression was identified on day 20, followed by a second sustained peak of expression in the adult rat. Due to low IGF-II/M6P receptor levels in the early postnatal period, most of the mitogenic effects of IGF-II may be mediated by the low but detectable levels of IGF-I receptor. The initial peak of IGF-II/M6P receptor gene expression coincides with the peak expression of the IGF-I and IGF-I receptor genes. This may, in fact, result from the tissue events generated by this surge in IGF-I activity. In the adult rat ovary, the cycle of follicular development and atresia may require high levels of the IGF-II/M6P receptor. Given that IGF-II gene expression is extremely low in the adult rat ovary, the function of the IGF-II/MBP receptor during this period may reflect its M6P-binding capacity more than its mediation of IGF action. This role would be similar to its previously documented roles in tissue remodelling and regeneration. References 1. Zapf J, Schmid C, Froesch ER 1984 Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 13:3-10 2. Froesch ER, Schmid C, Schwander J, Zapf J 1985 Actions of insulin-like growth factors. Annu Rev Physiol 47:443-449 3. Ullrich A, Gay A, Tam AW, Yang-Feng?, Tsubokawa M, Collins C. Henzel W. LeBon T. Kathuria S, Chen E, lacobs S, Franke U, RamachandrHn J, Fujka-Yamaguchi Y 1986-Insulin-iike growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-2510 4. Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WJ 1987 Insulin-like growth factor II receptor as a multifunctional binding protein. Nature 329:301-303 5. Zapf J, Schmid CH, Froesch ER 1984 Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 13:3-10 6. Isaksson OGP, Lindahl A, Nilsson A, Isgaard J 1987 Mechanism of the stimulating effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426-431 7. Lund PK, Moats-Staats BM, Hyner MA, Simmons JG, Jansen M, D’Ercole AJ, Van Wyk JJ 1986 Somatomedin C/insulin-like growth factor I and insulin-like growth factor II mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-14546 8. Rotwein P, Pollack KM, Watson M, Milbrandt JD 1987 Insulin-

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10. 11.

12 13.

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like growth factor gene expression during rat embryonic development. Endocrinology 121:2141-2150 Norstedt G, Levinovitz A, Moller C, Eriksson LC, Andersson G 1988 Expression of insulin-like growth factor I (IGF-I) and IGF-II mRNA during hepatic development, proliferation and carcinogenesis in the rat. Carcinogenesis 9:209-216 Han VKM, D’Ercole AJ, Lund PK 1987 Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-195 D’Ercole AJ, Stiles AD, Underwood LE 1984 Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanism of action. Proc Nat1 Acad Sci USA 81:935-939 Murphy LJ, Bell GI, Friesen HG 1987 Tissue distribution of insulinlike growth factor I and II messenger ribonucleic acid in the adult rat. Endocrinology 120:1279-1284 Roberts Jr CT, Lasky SR, Lowe Jr WL, Seaman WT, LeRoith D 1987 Molecular cloning of rat insulin-like growth factor I complementary ribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues. Mol Endocrinol 1:243-249 Lowe Jr WL, Roberts Jr CT, Lasky SR, LeRoith D 1987 Differential expression of alternative 5’-untranslated regions in mRNAs encodinn:r$ insulin-like growth factor I. Proc Nat1 Acad Sci USA 84:8946-

15. Hernandez ER, Roberts Jr CT, Hurwitz A, LeRoith D, Adashi EY 1990 Rat ovarian IGF-II gene expression is theta-interstitial cellspecific: hormonal regulation and receptor distribution. Endocrinology 127:3219-3226 16. Hernandez ER, Roberts Jr CT, LeRoith D, Adashi EY 1989 Rat ovarian insulin-Iike growth factor (IGF-I) gene expression is granulosa cell-selective: 5’-untranslated mRNA variant representation and hormonal regulation. Endocrinology 125:572-578 17. Brown AL, Graham DE, Nissley SP, Hill DJ, Stain AJ, Rechler MM 1986 Developmental regulation of insulin-like growth factor II mRNA in different rat tissues. J Biol Chem 261:13144-13150 18. Poretsky L, Grigorescu F, Seibel M, Moses AC, Flier JS 1985 Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary. J Clin Endocrinol Metab 61:728-734 19. Adashi EY, Resnick CE, Hernandez ER, Hurwitz A, Rosenfeld RG 1990 Follicle-stimulating hormone inhibits the constitutive release of insulin-like growth jactor binding proteins by cultured rat ovarian eranulosa cells. Endocrinoloav 126:1305-1307 20. Davore; JB, Hsueh AJW 1987 GroGth hormone increases ovarian levels of immunoreactive somatomedin-C/insulin-like growth factor I in viva. Endocrinology 120:198-206 21. Lowe Jr WL, Shaffner AE, Roberts Jr CT, LeRoith D 1987 Developmental regulation of somatostatin gene expression in the brain is region-specific. Mol Endocrinol 1:181-187 22. Burguera B, Werner H, Sklar M, Shen-Orr Z, Stannard B, Roberts Jr CT, Nissley SP, Vore SJ, Caro JF, LeRoith D 1990 Liver regeneration is associated with increased expression of the insulinlike growth factor II/mannose-6-phosphate receptor. Mol Endocrino1 4:1549-1554 23. Lowe Jr WL, Boyd FT, Clarke DW, Raizada MK, Hart C, LeRoith D 1986 Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures. Endocrinology 119:25-35 24. Adashi EY, Resnick CE, Hernandez ER, Hurwitz A, Roberts Jr CT, LeRoith D, Rosenfeld R 1991 The intraovarian IGF-I system. In: Spencer EM (ed) Modern Concepts of Insulin-Like Growth Factors. Elsevier, Amsterdam, p 267 25. Adashi EY, Resnick CE, D’Ercole AJ, Svoboda ME, Van Wyk JJ 1985 Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev 6:400-418 26. Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ, Hascall VC, Yanagishita M 1986 Independent and synergistic actions of somatomedin-C in the stimulation of proteoglycan biosynthesis by cultured rat granulosa cells. Endocrinology 118 27. Ojeda SR, Andrews WW, Advis JP, Smith-White S 1980 Recent advances in the endocrinology of puberty. Endocr Rev 1:228

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RECEPTOR

Ojeda SR, Ramiriz VD 1972 Plasma

LH and F5H in maturing rats: Endocrinolgy 90:466-472 Germain BJ, Campbell PS, Anderson JN 1978 Role of the serum estrogen-binding protein in the control of tissue estradiol levels during postnatal developmental of the female rat. Endocrinology 103:1401-1410 Dohler KD, Wittke W 1975 Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. Endocrinology 97898-906 Levina SE 1970 Regulation of the secretion of hypophysical gonadotropins during embryogenesis in man. Probl Endokrinol (Mosk) 16:53-60 Root AW, Shapiro BH, Duckett GE, Godman AS 1975 Effect of synthetic luteinizing hormone-releasing hormone in newborn rats. Proc Sot Exp Biol Med 148:631-635 Ramaley JA, Phares CK 1980 Delay of puberty onset in females due to suppression of growth hormone. Endocrinology 106:1989 Glass AR, Dahms WT, Swerdloff RS 1979 Body fat at puberty rats: alterations by changes in diet. Pediatr Res 13:7-12 Wilen R, Naftolin F 1978 Pubertal food intake and body weight response

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and composition well fed animals.

to hemigonadectomy.

36. 37.

in the feed-restricted female rat: comparison with Pediatr Res 12:263-266 Ronnekleiv 0, Ojeda SR, McCann SM 1978 Under-nutrition puberty and development of the estrogen positive feedback female rat. Biol Reprod 149:414-419 Advis P, Smith-White S, Ojeda SR 1981 Activation of growth hormone short loop negative feedback delays puberty in the female rat. Endocrinology 108:1343-1350

38. Adashi EY, Resnick CE, Hernandez

39.

40.

ER, Svoboda ME, Van Wyk

JJ 1987 Characterization and regulation of a specific cell membrane receptor for somatomedin-C/insulin-like growth factor I in cultured rat granulosa cells. Endocrinology 122:1940-1948 Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1986 Folliclestimulating hormone enhances somatomedin-C binding to cultured rat granulosa cells: evidence of CAMP-dependence. J Biol Chem 261:3923-3928

Moses AC, Nissley SP, Short PA, Rechler MM, White RM, Knight AB, Higa OZ 1980 Increased levels of multiplication-stimulating activity, an insulin-like growth Acad Sci USA 77:3649-3653

factor,

in fetal

rat serum.

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Proc

Nat1

Expression of the insulin-like growth factor (IGF)-I and -II and the IGF-I and -II receptor genes during postnatal development of the rat ovary.

Solution hybridization/RNase protection assays were used to study the developmental expression of the insulin-like growth factor-I (IGF-I), IGF-II, IG...
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