J. Endocrinol. Invest. 15.689-707,1992
REVIEW ARTICLE
Growth factors in the ovary G. Giordano*, A. Barreca*, and F. Minuto** *Cattedra di Endocrinologia, and **Fisiopatologia Endocrina, DISEM, University of Genova, Genova, Italy INTRODUCTION
somatomedin peptides in the local control of the ovarian function have been weil demonstrated. The somatomedins or "insulin-like growth factors" (IGFs) are a family of peptides wh ich possess insulin-like, sulfating and mitogenic activities in many tissues and cell systems (4), and which appear to be the link between the classical endocrine hormones and the autocrine and paracrine GFs, Two different but chemically very similar forms have been purified from human plasma and characterized both at the level of amino acid (5, 6) and nucleotide sequences (7-10), Among the IGFs, IGF-I is a basic peptide (pi 8,5 ± 0,5) composed of a single chain of 70 amino acids, with molecular weight of 7649 Da, showing 43% homology with human proinsulin. It has a potent growth-promoting action both in vitra and in vivo and its plasma concentration is greatly dependent on GH, though it is also influenced by other factors, in particular nutritional ones (11). The IGFI plasma levels are low at birth and increase during childhood until they reach adult values, with a maximum increase during puberty, in concomitance with the growth "spurt" typical of this period of life. IGF-II is a neutral peptide (pi 6,2 ± 0.5) having 50% sequence homology with IGF-I, and eomposed of 67 amino aeids with a mol wt of 7471 Da; its serum concentration appears to be much less GH-dependent and its growth effects are less relevant than those of IGF-1. In contrast, it has much more insulin-like activity than IGF-I, and is considered to be the most important somatomedin for fetal growth, The remarkable structural resemblance between IGFs and insulin shows that all these peptides belong to a family which originates from a common ancestral gene (12,13). The high degree of homology between the primary structures of insulin and IGFs, and the similarities between their tertiary structures allow the IGFs to interact with the insulin receptor and insulin to bind to the "type I" somatomedin receptor (14). This fact would explain, at least in part, the common biological actions of the three peptides. Indeed, it has been demonstrated, in a wide range of tissue and cell systems, that the IGFs exert their metabolie activity through the insulin receptor, while insulin expresses its mitogenic activity through the "type I"
The primary endocrine regulators of gonadal differentiation and function are the pituitary folliclestimulating hormone (FSH) and luteinizing hormone (LH) (1). Recently, however, many experimental data have shown that differentiation of gonadal cells is also modulated by several factors acting in a paracrine and/or autocrine manner. In particular, some growth factors, initially evaluated for their capacity to stimulate ovarian cell proliferation, have been shown to be capable of inducing, directly or by regulating gonadotrophin (Gn) activity, some differentiated functions in follicular cells. It is weil known that the follicle represents the functional unit of the ovary and consists of an external layer of theca interna cells (TC) and internallayers of granulosa cells (GC), which surround the oocyte. It can be hypothesized that, besides the synthesis and actions of oestradiol (E2) and of Gn-induced cAMP increase (2, 3), other factors are of the utmost importance for the development of a preovulatory follicle. A large number of factors involved in the regulation of the differentiation of the follicular cells are the growth factors (GFs). The physico-chemical characteristics, site of synthesis and effects on target cells of some of these GFs will be hereby considered (Table 1). INSULlN-LlKE GROWTH FACTORS AND INSULIN The ascription to GFs of an autocrine and/or paracrine role at the ovarian level arises from the demonstration of their synthesis by follicular cells which, in turn, possess specific receptors and are sensitive to the biological actions of GF. Among these GFs, the role and physiological relevance of
Key-words: Growth lactors. Insulin-Ilke. somatomedin. binding protein. inhibin. prostaglandins. interleukin, ovary, granulosa, theca, gonads, endorphins, vasopreSSin, LHRH, TNF-alpha. Correspondence Dotl G. Giordano, eattedra di Endocrinologla, DISEM, Viale Benedetto xv 6, 1-16132 Genova, Italy.
689
G. Giordano, A. Barreca, and F. Minuto
Table 1 - Biochemica! and bio!ogica! properties of the most characterized tissue growth factors iso!ated from the ovary PEPTIDE IGF-I
MOL. WT. (kDa)
STRUCTURE
ORIGIN
TARGET
ACTION
GCTC
> LH-R E2 PG A
7.6
single chain
GC
IGF-II
7.5
single chain
GC
Insulin
5.7
single chain
EGF
6.1
single chain
TC
GC
< LH-R E2
TGFa.
5.6
single chain
TC
GC
< E2
TC GC (ß2)
TC GC
> LH-R E2 PG. A
TGFß
25
homodimer
Inhibln
32
a.-ß heterodimer
Activin
28
ß-ß homodimer
FGFb
15
single chain
Interleukin-1
17.5
single chain
> LH-R E2 PG A > LH-R E2 PG A
> E2. < PG A GC
< E2. > PG GC
< LH-R E2 PG
Legend GC=granulosa cell. TC=theca cell. LH-R=LH receptor. E2=Estradiol. PG=progesterone. A=androgens. =lncreases
been further confirmed by the presence of high levels of IGF-I mRNA, which imply its gene expression, in Ge (29-31), notably Ge of developing follicles (31). Ovarian IGF-I synthesis appears to be dependent on GH (32, 33), Gn and E2 (34), EGF and TGFalpha (35). As for GH, it is weil known that treatment with GH induces an increase in the intraovarian content of IGF-I in hypophysectomized estrogen-treated rats, increases ovarian progesterone (PG) production in response to Gn, and enhances the rat Ge response to Gn stimulation in vitra (32, 36, 37). Besides IGF-I, IGFBP are also produced by murine and porcine ovarian cells (15-17,38). In this regard, a protein has been recently purified from porcine FF which has a high degree of homology with the amino terminal sequence of the GH-dependent hIGFBP-3, and wh ich seems to have an inhibitory effect on the FSH-induced production of E and PG by rat Ge (39). The inhibitory effect of IGFBP-3, as weil as of IGFBP2 purified from porcine follicular fluids, seems to be exerted by the binding of endogenously produced IGF-I and, possibly, bya direct interaction with the cell to elicit inhibition (40). The fact that FSH in turn exerts an inhibitory effect on the production 01 IGFBP by Ge (41) suggests that the IGFBP exerts a buffer effect on the IGF amplification mechanisms (Fig. 3). More recently, the effect of FSH on the production of IGFBP by Ge from immature estrogenprimed rats has been shown to be dose- and timedependent, i.e. stimulatory at early low doses and inhibitory at late high doses; this biphasic effect of FSH on IGFBP synthesis may regulate the time of follicular growth and the induction of follicular dominance (42). The fact that, using an in situ hybridization technique, the mRNA for IGFBP-2, -3 and -4 have been 10calized in the rat interstitial cells, corpora lutea, and
somatomedin receptor. The IGFs, however, unlike insulin are synthesized by several tissues and cell lines (15) and circulate almost exclusively bound to specific carrier proteins (IGFBP) in the form of high molecular weight complexes (16-18). Of the two classes of IGF-BP complexes identified in adult humans, one (calied 150 K complex) is GH-dependent and includes most of the circulating IGF activity; the other seems to be the only one present in subjects affected by GH-dependent dwarfism, and has a mol wt of 30-45 kDa (19-21). The 150 K complex easily dissociates in acid conditions in a 53 kDa subunit (hIGFBP-3), able to specifically bind the IGF peptide, and in an acid labile subunit which, while not binding the IGF, is necessary to reconstitute the complex in its natural form (21-24). Recently, it has become possible to deduce the amino acid sequence of hIGFBP-3 from complementary DNA (25) and therefore confirm that this protein is different from hIGFBP-1, although they present a considerable amino acid homology (26, 27). A) In vivo and in vitro studies in animals A novel role attributed to somatomedin peptides is related to their capacity to act, on some occasions, more as differentiating rather than mitogenic factors. Regarding the ovary, several experimental data have indicated that the IGF-I can act by paracrine or autocrine mechanisms. Indeed, its synthesis and the presence of its specific receptors have been demonstrated in the follicular cells which, in turn, appear to be sensitive to the actions of IGFcl in a dose-dependent manner. In particular, immunoreactive and bioactive IGF-I is present in medium conditioned by porcine granulosa cells (28). Its production in the rat ovary has
690
Growth factors in the ovary
THEGA GELL
BASEMENT MEMBRANE
GRANULOSA GELL ~
androstenedione
INSULIN
testosterone
androstened ione
Fig. 1 - Some effeets of IGF-I and insulin on ovarian theea and granulosa eells.IGF-1 exerts a stimulatory effeet on the synthesis by Ge of progesterone, oxytoem, proteoglyeans and inhibin as weil as on aromatase aetivity IGF-I also appears to be involved in the synthesis of androgen by TC and, in this way, it is able to inerease the androgenie substrate and thus further enhanee E synthesis in the Ge. Insulin also earry out similar effeets on the proliferation and differentiation of ovarian follieles probably through the type I reeeptor of IGFs .
SHBG
. . = IGF-I reeeptor
!Gf-BP1 STIMULATORY ACTION
lIIIIiIIIIII
INHIBITORY ACTION
atretic Ge, respectively, hints at a different regulation for each of these IGFBP, which may have a role as autocrine/paracrine factors in regulating the local actions of the IGFs in the ovary (43). High-affinity, low-capacity receptors for IGF-I have been shown in porcine (44, 45) and murine (46-49) Ge , and in human ovarian tissues (50) ; in vivo and in vitro administration of FSH and, to a lesser extent LH, increases the number of ovarian IGF-I receptors (46-48). Although its mechanism of action in the follicular cells has not been completely clarified, it has been demonstrated that, at the Ge level, IGF-I induces the activation of adenyl cyclase, and therefore an increase in Gn-induced cAMP (51), and that it also acts at site(s) distal to cAMP generation (52). Besides a potent stimulatory effect on Ge replication in several species (44), IGF-I also exerts a stimulatory effect on the synthesis of progesterone, oxytocin (53, 54), proteoglycans (55) and inhibin (56) as weil as on aromatase activity (57) (Fig. 1). Moreover, it has a synergic role with FSH both on steroidogenesis and on LH receptor induction in Ge (52, 58-60). IGF-I also appears to be involved in the synthesis of androgen by Te (61), and, in this way, it is able to increase the androgenic substrate and thus further enhance E synthesis in the Ge (Figs. 1, 2). IGF-I actions on steroidogenesis include the stimulation of cholesterol side-chain cleavage at the mitochondrial level (62) as weil as
= Insulin reeeptor
the induction of lipoprotein-processing capabilities of Ge , i.e. their LDL-binding capacity, uptake and utilization (63). Recently it has been demonstrated that IGF-I alone causes an increase in P450 scc activity by stimulating increases in P450 scc enzyme
GRANULOSA CELL FSH
l
STEROIDOGENESIS
IGF-I ---+
CELL REPLICATION
t
LH Fig. 2 - Potentiating effeets of IGF-I on FSH funetion. Besides a potent stimulatory effeet on Ge replieation in several speeies, IGF-I has asynergie role with FSH both on steroidogenesis and on LH reeeptor induetion in Ge.
W
691
~
IGF-I receptor
...... =
FSH reeeptor
. . . = LH reeeptor
G. Giordano, A Barreca, and F. Minuto
GRANULOSA CELL FSH
STEROIDO· GENESIS
REPLICATION
STIMULATORY ACTION INHIBITORY ACTION
----+
---+
Fig 3 - Possible effeels of IGF-I and IGF binding proteins on granulosa eel/s. Besides IGF-I. IGFBP are also produeed by ovarian eel/s. A protein purified from porcine FF. whieh has a high degree of homology with Ihe amino terminal sequenee of the GH-dependen t hIGFBP-3, has been fo und to have an inhlbilory erfeeion Ihe FSH-indueed produelion of E and PG by rat Ge. The inhibitory effeet of IGFBP-3. as wel/ as of IGFBP-2 purified from poreine FF. seems 10 be exerled by Ihe bin ding of endogenous/y produeed IGFland, possibly, bya direet interaelion with Ihe eell to elieit inhibition. The fact thai FSH in turn exerts an inhibitory effeel on Ihe produelion of IGFBP by Ge suggests Ihal the IGFBP exerls a buffer effeet on the IGF amplifiealion meehanisms.
W
= IGF-I reeeplor .... = FSH reeep tor
LH
content and mRNA levels, and that IGF-I augments the ability of LH to increase the content of P450 scc and P45017alpha in the same cells (64). All these effects lead to an increased intracellular cholesterol pool which can be used, particularly in the final stages of ovarian follicle maturation, as a substrate in steroid synthesis. Within this family of peptides, IGF-II and insulin also carry out similar effects on the proliferation and differentiation of ovarian follicles. Indeed , both insulin and IGF-II (type 11) receptors have been demonstrated in human (65-68) and rat (69, 70) ovaries. However, as insulin and IGF-II appear to be less powerful than IGF-I (71), it seems likely that both express their actions in this organ through the type I receptor of IGFs (44, 72, 73). Indeed, the maximal effective dose of insulin in inducing functional differentiation of porcine GC is 1000-fold higher than the physiological level , while IGF-I is effective at physiological concentration (100 ng/ml) (74). More recently, the use of antibodies specific for the rat type II IGF receptor (R-II-PAB 1) has confirmed that IGF-I and II hormonal actions at the GC level are exerted via the type I IGF receptor (75) .
111 = LH reeeplor
regard , some of the well-known actions of growth hormone (GH) on the gonads could also be mediated by IGF-I. The connections between gonadal differentiation and GH arise from clinical and experimental data. Indeed, it has been established that the circulating levels of GH are increased during puberty (79) , and that a lack of GH delays th e on set of puberty (80) as weil as reduces the number of ovarian LH receptors and Gn -induced steroidogenesis. On the other hand , replacement of GH in GH-deficiency restores to normal the tim e of puberty onset (81 , 82). Growth hormone therapy also augments the ovarian response to Gn stimulation in women presenting with ovaries that are relatively resistant to conventional Gn therapy (83, 84). It is not completely ascertained, however , whether GH modulates the actions of FSH on GC directly or via IGF produc tion. These two possibilities are not mutually exclusive, as demonstrated by a study we performed on follicular fluids (FF) from patients in whom the in duction of super-ovulation was obtained only when GH was added to the gonadotrophin therapy (85). In these fluids we investigated the presence of GH, IGF-I, Epidermal Growth Factor (EGF), E2 and PG , while the FF of patients responsive to treatment with gonadotrophin alone served as a co ntrol. Characterization of the IGF-l-Iike immunoreactivity found in the FF revealed the presence of both IGFland IGF-I binding capacity. When reacted with radioiodinated IGF-I , this binding material is able to generate a 35 kDa complex similar to that found in
B) In vive and in vitro studies in humans In the last few years , the concept of the IGFs as modulators of other hormonal actions has been expanded with the demonstration that, besides the effects of Gn, and in particular of FSH, on ovarian and testicular tissues , IGF-I is also able to amplify the effects of TSH on thyroid cells (76-78). In this
692
Growth tactars in the avary
nadotrophin-resistant anovulation. The finding of a highly significant correlation between EGF and IGFI concentrations in the FF suggests a role for this growth factor in inducing IGF-I production also in human ovarian tissue in vivo. This is in agreement with what has already been found in porcine Ge in vitra (35) . With regard to the possible effects of IGFI on E2 and PG concentrations, it is noteworthy that in the FF a relationship between IGF-I and E2 or PG concentrations , was evident only when considering the GH-treated patients (Fig. 4). These data suggest a direct GH action, besides the IGF-I mediated GH effects on the differentiation of follicular cells. Alternatively, the GH effects may be explained by the "Dual Effector" theory of Zezulak and Green (97 , 98). This theory emerges from experiments on 3T3 preadipocytes and epiphyseal prechondrocytes, in which GH not only promotes the initial differentiation and stimulates IGF-I produc ti on but also induces a sensibilization to the IGF-I actions, including the mitogenic action . This results in a preferential expansion of the more differentiated cell clones. It is possible, therefore, to assume that both GH and IGF-I, through reciprocal poten tiation , play an important role in the control of follicular growth and development in humans. We have also investigated the stimulatory effect of GH on E2 and PG release by human Ge in vitra (99). During the first 24h of incubation, the Ge obtained from patients treated with Gn and GH released into the medium higher amounts of E2 than did Ge obtained from patients treated with Gn alone. When the in vitra Ge release of steroid due to the in vivo
the amniotic fluid (86-88). Based on the amino acid sequence homology of the N-terminal segment, it appears that the amniotic fluid BP, which behaves similarly to the non-GH-dependent BP in different clinical conditions (89, 90), is the placental protein 12 (91); recently, it has been proposed for the sake of uniformity that all these forms be called hIGFBP1 (18). It has been demonstrated that this BP can inhibit (86, 92) or potentiate (93) the IGF peptide action , depending on the cell systems tested . Therefore, the identification of this BP in the FF could be relevant for the regulation of ovarian cell responsiveness to the IGFs. In patients treated with combined therapy, the results demonstrated the presence in the FF of GH , E2 , and PG in significantly higher concentrations than in control follicles. Acid -extractable IGF-I and EGF concentrations were similar to those of control fluids. Our data demonstrate that GH administration also modifies the intrafollicular levels of the hormone and therefore the extent of its actions on the Ge specific receptors. We have indirectly confirmed the presence of the GH receptors in the ovarian follicles by demonstrating the presence in the FF of high concentrations of a specific GH-binding protein (94), whose amino terminal sequence appears to be identical to the extracellular domain of the GH receptor (95 , 96). On the other hand, the significant correlation found between GH and IGF-I only in the FF of GH-treated patients shows that the local IGFI concentration can be influenced by the administration of GH in patients presenting with go-
350
Estradiol (ng/ml) ------
Progesterone (J,Jg/mJ)
:-j
300
"
.,~
0 0
250
,
0
....
u l!!
200
00 00 0
'50
0
o
tb
50 0
','
I
0 0
50
'00
,so
200
IGF-I o
o
I)
n
urY
o
[fl
o
o
3
0
'b
0
o
o
8
':J
U
' 00
0
0
o '
o
~J
250
300
control
350
h.
l
"
lJ
o o
o
1--1 50
'00
'50
200
250
IGF-I (ng/ml) GH treated 693
300
350
Fi g. 4 - Re lationship between in trafo/licular fluid IGF-I and estradiol or progesterone conce ntration in subjects treated with gonadotropin alone (control; vs E2: r = -0.06; vs PG . r = 0.25) or with gonadotrop in and GH (GH treated; vs E2. r = 0.55, vs PG. r = 0.77; p
REVIEW ARTICLE
Growth factors in the ovary G. Giordano*, A. Barreca*, and F. Minuto** *Cattedra di Endocrinologia, and **Fisiopatologia Endocrina, DISEM, University of Genova, Genova, Italy INTRODUCTION
somatomedin peptides in the local control of the ovarian function have been weil demonstrated. The somatomedins or "insulin-like growth factors" (IGFs) are a family of peptides wh ich possess insulin-like, sulfating and mitogenic activities in many tissues and cell systems (4), and which appear to be the link between the classical endocrine hormones and the autocrine and paracrine GFs, Two different but chemically very similar forms have been purified from human plasma and characterized both at the level of amino acid (5, 6) and nucleotide sequences (7-10), Among the IGFs, IGF-I is a basic peptide (pi 8,5 ± 0,5) composed of a single chain of 70 amino acids, with molecular weight of 7649 Da, showing 43% homology with human proinsulin. It has a potent growth-promoting action both in vitra and in vivo and its plasma concentration is greatly dependent on GH, though it is also influenced by other factors, in particular nutritional ones (11). The IGFI plasma levels are low at birth and increase during childhood until they reach adult values, with a maximum increase during puberty, in concomitance with the growth "spurt" typical of this period of life. IGF-II is a neutral peptide (pi 6,2 ± 0.5) having 50% sequence homology with IGF-I, and eomposed of 67 amino aeids with a mol wt of 7471 Da; its serum concentration appears to be much less GH-dependent and its growth effects are less relevant than those of IGF-1. In contrast, it has much more insulin-like activity than IGF-I, and is considered to be the most important somatomedin for fetal growth, The remarkable structural resemblance between IGFs and insulin shows that all these peptides belong to a family which originates from a common ancestral gene (12,13). The high degree of homology between the primary structures of insulin and IGFs, and the similarities between their tertiary structures allow the IGFs to interact with the insulin receptor and insulin to bind to the "type I" somatomedin receptor (14). This fact would explain, at least in part, the common biological actions of the three peptides. Indeed, it has been demonstrated, in a wide range of tissue and cell systems, that the IGFs exert their metabolie activity through the insulin receptor, while insulin expresses its mitogenic activity through the "type I"
The primary endocrine regulators of gonadal differentiation and function are the pituitary folliclestimulating hormone (FSH) and luteinizing hormone (LH) (1). Recently, however, many experimental data have shown that differentiation of gonadal cells is also modulated by several factors acting in a paracrine and/or autocrine manner. In particular, some growth factors, initially evaluated for their capacity to stimulate ovarian cell proliferation, have been shown to be capable of inducing, directly or by regulating gonadotrophin (Gn) activity, some differentiated functions in follicular cells. It is weil known that the follicle represents the functional unit of the ovary and consists of an external layer of theca interna cells (TC) and internallayers of granulosa cells (GC), which surround the oocyte. It can be hypothesized that, besides the synthesis and actions of oestradiol (E2) and of Gn-induced cAMP increase (2, 3), other factors are of the utmost importance for the development of a preovulatory follicle. A large number of factors involved in the regulation of the differentiation of the follicular cells are the growth factors (GFs). The physico-chemical characteristics, site of synthesis and effects on target cells of some of these GFs will be hereby considered (Table 1). INSULlN-LlKE GROWTH FACTORS AND INSULIN The ascription to GFs of an autocrine and/or paracrine role at the ovarian level arises from the demonstration of their synthesis by follicular cells which, in turn, possess specific receptors and are sensitive to the biological actions of GF. Among these GFs, the role and physiological relevance of
Key-words: Growth lactors. Insulin-Ilke. somatomedin. binding protein. inhibin. prostaglandins. interleukin, ovary, granulosa, theca, gonads, endorphins, vasopreSSin, LHRH, TNF-alpha. Correspondence Dotl G. Giordano, eattedra di Endocrinologla, DISEM, Viale Benedetto xv 6, 1-16132 Genova, Italy.
689
G. Giordano, A. Barreca, and F. Minuto
Table 1 - Biochemica! and bio!ogica! properties of the most characterized tissue growth factors iso!ated from the ovary PEPTIDE IGF-I
MOL. WT. (kDa)
STRUCTURE
ORIGIN
TARGET
ACTION
GCTC
> LH-R E2 PG A
7.6
single chain
GC
IGF-II
7.5
single chain
GC
Insulin
5.7
single chain
EGF
6.1
single chain
TC
GC
< LH-R E2
TGFa.
5.6
single chain
TC
GC
< E2
TC GC (ß2)
TC GC
> LH-R E2 PG. A
TGFß
25
homodimer
Inhibln
32
a.-ß heterodimer
Activin
28
ß-ß homodimer
FGFb
15
single chain
Interleukin-1
17.5
single chain
> LH-R E2 PG A > LH-R E2 PG A
> E2. < PG A GC
< E2. > PG GC
< LH-R E2 PG
Legend GC=granulosa cell. TC=theca cell. LH-R=LH receptor. E2=Estradiol. PG=progesterone. A=androgens. =lncreases
been further confirmed by the presence of high levels of IGF-I mRNA, which imply its gene expression, in Ge (29-31), notably Ge of developing follicles (31). Ovarian IGF-I synthesis appears to be dependent on GH (32, 33), Gn and E2 (34), EGF and TGFalpha (35). As for GH, it is weil known that treatment with GH induces an increase in the intraovarian content of IGF-I in hypophysectomized estrogen-treated rats, increases ovarian progesterone (PG) production in response to Gn, and enhances the rat Ge response to Gn stimulation in vitra (32, 36, 37). Besides IGF-I, IGFBP are also produced by murine and porcine ovarian cells (15-17,38). In this regard, a protein has been recently purified from porcine FF which has a high degree of homology with the amino terminal sequence of the GH-dependent hIGFBP-3, and wh ich seems to have an inhibitory effect on the FSH-induced production of E and PG by rat Ge (39). The inhibitory effect of IGFBP-3, as weil as of IGFBP2 purified from porcine follicular fluids, seems to be exerted by the binding of endogenously produced IGF-I and, possibly, bya direct interaction with the cell to elicit inhibition (40). The fact that FSH in turn exerts an inhibitory effect on the production 01 IGFBP by Ge (41) suggests that the IGFBP exerts a buffer effect on the IGF amplification mechanisms (Fig. 3). More recently, the effect of FSH on the production of IGFBP by Ge from immature estrogenprimed rats has been shown to be dose- and timedependent, i.e. stimulatory at early low doses and inhibitory at late high doses; this biphasic effect of FSH on IGFBP synthesis may regulate the time of follicular growth and the induction of follicular dominance (42). The fact that, using an in situ hybridization technique, the mRNA for IGFBP-2, -3 and -4 have been 10calized in the rat interstitial cells, corpora lutea, and
somatomedin receptor. The IGFs, however, unlike insulin are synthesized by several tissues and cell lines (15) and circulate almost exclusively bound to specific carrier proteins (IGFBP) in the form of high molecular weight complexes (16-18). Of the two classes of IGF-BP complexes identified in adult humans, one (calied 150 K complex) is GH-dependent and includes most of the circulating IGF activity; the other seems to be the only one present in subjects affected by GH-dependent dwarfism, and has a mol wt of 30-45 kDa (19-21). The 150 K complex easily dissociates in acid conditions in a 53 kDa subunit (hIGFBP-3), able to specifically bind the IGF peptide, and in an acid labile subunit which, while not binding the IGF, is necessary to reconstitute the complex in its natural form (21-24). Recently, it has become possible to deduce the amino acid sequence of hIGFBP-3 from complementary DNA (25) and therefore confirm that this protein is different from hIGFBP-1, although they present a considerable amino acid homology (26, 27). A) In vivo and in vitro studies in animals A novel role attributed to somatomedin peptides is related to their capacity to act, on some occasions, more as differentiating rather than mitogenic factors. Regarding the ovary, several experimental data have indicated that the IGF-I can act by paracrine or autocrine mechanisms. Indeed, its synthesis and the presence of its specific receptors have been demonstrated in the follicular cells which, in turn, appear to be sensitive to the actions of IGFcl in a dose-dependent manner. In particular, immunoreactive and bioactive IGF-I is present in medium conditioned by porcine granulosa cells (28). Its production in the rat ovary has
690
Growth factors in the ovary
THEGA GELL
BASEMENT MEMBRANE
GRANULOSA GELL ~
androstenedione
INSULIN
testosterone
androstened ione
Fig. 1 - Some effeets of IGF-I and insulin on ovarian theea and granulosa eells.IGF-1 exerts a stimulatory effeet on the synthesis by Ge of progesterone, oxytoem, proteoglyeans and inhibin as weil as on aromatase aetivity IGF-I also appears to be involved in the synthesis of androgen by TC and, in this way, it is able to inerease the androgenie substrate and thus further enhanee E synthesis in the Ge. Insulin also earry out similar effeets on the proliferation and differentiation of ovarian follieles probably through the type I reeeptor of IGFs .
SHBG
. . = IGF-I reeeptor
!Gf-BP1 STIMULATORY ACTION
lIIIIiIIIIII
INHIBITORY ACTION
atretic Ge, respectively, hints at a different regulation for each of these IGFBP, which may have a role as autocrine/paracrine factors in regulating the local actions of the IGFs in the ovary (43). High-affinity, low-capacity receptors for IGF-I have been shown in porcine (44, 45) and murine (46-49) Ge , and in human ovarian tissues (50) ; in vivo and in vitro administration of FSH and, to a lesser extent LH, increases the number of ovarian IGF-I receptors (46-48). Although its mechanism of action in the follicular cells has not been completely clarified, it has been demonstrated that, at the Ge level, IGF-I induces the activation of adenyl cyclase, and therefore an increase in Gn-induced cAMP (51), and that it also acts at site(s) distal to cAMP generation (52). Besides a potent stimulatory effect on Ge replication in several species (44), IGF-I also exerts a stimulatory effect on the synthesis of progesterone, oxytocin (53, 54), proteoglycans (55) and inhibin (56) as weil as on aromatase activity (57) (Fig. 1). Moreover, it has a synergic role with FSH both on steroidogenesis and on LH receptor induction in Ge (52, 58-60). IGF-I also appears to be involved in the synthesis of androgen by Te (61), and, in this way, it is able to increase the androgenic substrate and thus further enhance E synthesis in the Ge (Figs. 1, 2). IGF-I actions on steroidogenesis include the stimulation of cholesterol side-chain cleavage at the mitochondrial level (62) as weil as
= Insulin reeeptor
the induction of lipoprotein-processing capabilities of Ge , i.e. their LDL-binding capacity, uptake and utilization (63). Recently it has been demonstrated that IGF-I alone causes an increase in P450 scc activity by stimulating increases in P450 scc enzyme
GRANULOSA CELL FSH
l
STEROIDOGENESIS
IGF-I ---+
CELL REPLICATION
t
LH Fig. 2 - Potentiating effeets of IGF-I on FSH funetion. Besides a potent stimulatory effeet on Ge replieation in several speeies, IGF-I has asynergie role with FSH both on steroidogenesis and on LH reeeptor induetion in Ge.
W
691
~
IGF-I receptor
...... =
FSH reeeptor
. . . = LH reeeptor
G. Giordano, A Barreca, and F. Minuto
GRANULOSA CELL FSH
STEROIDO· GENESIS
REPLICATION
STIMULATORY ACTION INHIBITORY ACTION
----+
---+
Fig 3 - Possible effeels of IGF-I and IGF binding proteins on granulosa eel/s. Besides IGF-I. IGFBP are also produeed by ovarian eel/s. A protein purified from porcine FF. whieh has a high degree of homology with Ihe amino terminal sequenee of the GH-dependen t hIGFBP-3, has been fo und to have an inhlbilory erfeeion Ihe FSH-indueed produelion of E and PG by rat Ge. The inhibitory effeet of IGFBP-3. as wel/ as of IGFBP-2 purified from poreine FF. seems 10 be exerled by Ihe bin ding of endogenous/y produeed IGFland, possibly, bya direet interaelion with Ihe eell to elieit inhibition. The fact thai FSH in turn exerts an inhibitory effeel on Ihe produelion of IGFBP by Ge suggests Ihal the IGFBP exerls a buffer effeet on the IGF amplifiealion meehanisms.
W
= IGF-I reeeplor .... = FSH reeep tor
LH
content and mRNA levels, and that IGF-I augments the ability of LH to increase the content of P450 scc and P45017alpha in the same cells (64). All these effects lead to an increased intracellular cholesterol pool which can be used, particularly in the final stages of ovarian follicle maturation, as a substrate in steroid synthesis. Within this family of peptides, IGF-II and insulin also carry out similar effects on the proliferation and differentiation of ovarian follicles. Indeed , both insulin and IGF-II (type 11) receptors have been demonstrated in human (65-68) and rat (69, 70) ovaries. However, as insulin and IGF-II appear to be less powerful than IGF-I (71), it seems likely that both express their actions in this organ through the type I receptor of IGFs (44, 72, 73). Indeed, the maximal effective dose of insulin in inducing functional differentiation of porcine GC is 1000-fold higher than the physiological level , while IGF-I is effective at physiological concentration (100 ng/ml) (74). More recently, the use of antibodies specific for the rat type II IGF receptor (R-II-PAB 1) has confirmed that IGF-I and II hormonal actions at the GC level are exerted via the type I IGF receptor (75) .
111 = LH reeeplor
regard , some of the well-known actions of growth hormone (GH) on the gonads could also be mediated by IGF-I. The connections between gonadal differentiation and GH arise from clinical and experimental data. Indeed, it has been established that the circulating levels of GH are increased during puberty (79) , and that a lack of GH delays th e on set of puberty (80) as weil as reduces the number of ovarian LH receptors and Gn -induced steroidogenesis. On the other hand , replacement of GH in GH-deficiency restores to normal the tim e of puberty onset (81 , 82). Growth hormone therapy also augments the ovarian response to Gn stimulation in women presenting with ovaries that are relatively resistant to conventional Gn therapy (83, 84). It is not completely ascertained, however , whether GH modulates the actions of FSH on GC directly or via IGF produc tion. These two possibilities are not mutually exclusive, as demonstrated by a study we performed on follicular fluids (FF) from patients in whom the in duction of super-ovulation was obtained only when GH was added to the gonadotrophin therapy (85). In these fluids we investigated the presence of GH, IGF-I, Epidermal Growth Factor (EGF), E2 and PG , while the FF of patients responsive to treatment with gonadotrophin alone served as a co ntrol. Characterization of the IGF-l-Iike immunoreactivity found in the FF revealed the presence of both IGFland IGF-I binding capacity. When reacted with radioiodinated IGF-I , this binding material is able to generate a 35 kDa complex similar to that found in
B) In vive and in vitro studies in humans In the last few years , the concept of the IGFs as modulators of other hormonal actions has been expanded with the demonstration that, besides the effects of Gn, and in particular of FSH, on ovarian and testicular tissues , IGF-I is also able to amplify the effects of TSH on thyroid cells (76-78). In this
692
Growth tactars in the avary
nadotrophin-resistant anovulation. The finding of a highly significant correlation between EGF and IGFI concentrations in the FF suggests a role for this growth factor in inducing IGF-I production also in human ovarian tissue in vivo. This is in agreement with what has already been found in porcine Ge in vitra (35) . With regard to the possible effects of IGFI on E2 and PG concentrations, it is noteworthy that in the FF a relationship between IGF-I and E2 or PG concentrations , was evident only when considering the GH-treated patients (Fig. 4). These data suggest a direct GH action, besides the IGF-I mediated GH effects on the differentiation of follicular cells. Alternatively, the GH effects may be explained by the "Dual Effector" theory of Zezulak and Green (97 , 98). This theory emerges from experiments on 3T3 preadipocytes and epiphyseal prechondrocytes, in which GH not only promotes the initial differentiation and stimulates IGF-I produc ti on but also induces a sensibilization to the IGF-I actions, including the mitogenic action . This results in a preferential expansion of the more differentiated cell clones. It is possible, therefore, to assume that both GH and IGF-I, through reciprocal poten tiation , play an important role in the control of follicular growth and development in humans. We have also investigated the stimulatory effect of GH on E2 and PG release by human Ge in vitra (99). During the first 24h of incubation, the Ge obtained from patients treated with Gn and GH released into the medium higher amounts of E2 than did Ge obtained from patients treated with Gn alone. When the in vitra Ge release of steroid due to the in vivo
the amniotic fluid (86-88). Based on the amino acid sequence homology of the N-terminal segment, it appears that the amniotic fluid BP, which behaves similarly to the non-GH-dependent BP in different clinical conditions (89, 90), is the placental protein 12 (91); recently, it has been proposed for the sake of uniformity that all these forms be called hIGFBP1 (18). It has been demonstrated that this BP can inhibit (86, 92) or potentiate (93) the IGF peptide action , depending on the cell systems tested . Therefore, the identification of this BP in the FF could be relevant for the regulation of ovarian cell responsiveness to the IGFs. In patients treated with combined therapy, the results demonstrated the presence in the FF of GH , E2 , and PG in significantly higher concentrations than in control follicles. Acid -extractable IGF-I and EGF concentrations were similar to those of control fluids. Our data demonstrate that GH administration also modifies the intrafollicular levels of the hormone and therefore the extent of its actions on the Ge specific receptors. We have indirectly confirmed the presence of the GH receptors in the ovarian follicles by demonstrating the presence in the FF of high concentrations of a specific GH-binding protein (94), whose amino terminal sequence appears to be identical to the extracellular domain of the GH receptor (95 , 96). On the other hand, the significant correlation found between GH and IGF-I only in the FF of GH-treated patients shows that the local IGFI concentration can be influenced by the administration of GH in patients presenting with go-
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Fi g. 4 - Re lationship between in trafo/licular fluid IGF-I and estradiol or progesterone conce ntration in subjects treated with gonadotropin alone (control; vs E2: r = -0.06; vs PG . r = 0.25) or with gonadotrop in and GH (GH treated; vs E2. r = 0.55, vs PG. r = 0.77; p
