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The role of insulin-like growth factors and epidermal growth factor-relatedpeptides in intraovarianregulation in the pig ovary

The autocrine and paracrine role of the insulin-like growth factors (IGFs) and epiderrnal growth factor (EGF)-related peptides in pig ovary are reviewed. For convenience, each of these regulatory systems is divided into several interactive components: regulated expression of the growth factors, growth factor reception at the cell surface and intracellular action of the growth factors. In addition, the concept of regulated bioavailability and targeting of growth factors in the extracellular space is developed as an important control locus and area for future study. With regard to the 1GF system, these components include two ligands — IGF-1 and -II, both expressed in the porcine ovary —and the possibility of three receptors. IGF-I and the type I IGF receptor appear to be the most important in stimulating ovarian function and amplifying hormone action. In addition, the 'set-point' of the ovarian 1GF system may be determined by the activity of several IGF-binding proteins (IGFBPs). At least four of these proteins are expressed in the pig ovary. Studies of their regulation and action in ovarian cells indicate that they can function as antagonists to FSH and the IGFs. However, preliminary evidence suggests a more dynamic model in which these proteins may direct the site and timing of IGF effects. There are fewer data on the EGF system. At least four EGF-related peptides are expressed in pig ovaries, but insufficient information is available to predict their physiological regulation. These peptides are potent mitogens for ovarian cells. Their steroidogenic effects are more variable, apparently depending on the level of differentiation of target cells as well as the presence or absence of critical co-factors. Differential targeting and activation of these peptides can be predicted from their primary structure. However, few data are yet available to substantiate these mechanisms in the ovary. The data in each of these areas suggest a pivotal role for these systems in ovarian function. However, control of these systems in vivo will be required to confirm this hypothesis.

The autocrine and paracrine role of the insulin-like growth factors (IGFs) and epiderrnal growth factor (EGF)-related peptides in pig ovary are reviewed. For convenience, each of these regulatory systems is divided into several interactive components: regulated expression of the growth factors, growth factor reception at the cell surface and intracellular action of the growth factors. In addition, the concept of regulated bioavailability and targeting of growth factors in the extracellular space is developed as an important control locus and area for future study. With regard to the 1GF system, these components include two ligands -IGF-1 and -II, both expressed in the porcine ovary -and the possibility of three receptors. IGF-I and the type I IGF receptor appear to be the most important in stimulating ovarian function and amplifying hormone action. In addition, the 'set-point' of the ovarian 1GF system may be determined by the activity of several IGF-binding proteins (IGFBPs). At least four of these proteins are expressed in the pig ovary. Studies of their regulation and action in ovarian cells indicate that they can function as antagonists to FSH and the IGFs. However, preliminary evidence suggests a more dynamic model in which these proteins may direct the site and timing of IGF effects. There are fewer data on the EGF system. At least four EGF-related peptides are expressed in pig ovaries, but insufficient information is available to predict their physiological regulation. These peptides are potent mitogens for ovarian cells. Their steroidogenic effects are more variable, apparently depending on the level of differentiation of target cells as well as the presence or absence of critical co-factors. Differential targeting and activation of these peptides can be predicted from their primary structure. However, few data are yet available to substantiate these mechanisms in the ovary. The data in each of these areas suggest a pivotal role for these systems in ovarian function. However, control of these systems in vivo will be required to confirm this hypothesis.

Introduction/Historical Overview
The need to invoke intraovarian regulatory principles has been obvious for several decades and derives mainly from the selectivity of ovarian growth and differentiation during the reproductive cycle. Steroid hormones were early candidates for this regulation and are now generally accepted as important infraovarian regulators.
However, in this review, attention will be directed toward the regulatory peptides that have been implicated in these processes. Initial efforts to define such peptide regulators emphasized  , 1988b;Adashi, 1992). This will be a selective review emphasizing data collected in porcine ovarian systems and that derived from our own laboratory in particular. These data concern the ovarian IGF system and the epidermal growth factor-related peptides. New data on these growth factors suggest reconsideration of their function. In addition, these two growth factor families are among the most intensely investigated of the autocrine/paracrine systems in the ovary, and provide convenient examples of the current state-of-the-art in the field, the techniques that can be brought to bear on such physiological questions, and the additional evidence that will be required to establish their role definitively.
The classical components of these systems include the ligands or growth factors themselves, their receptors and an intracellular signal transduction pathway controlling either cell replication or steroidogenic differentiation of ovarian cells. In the last few years, it has become clear that this framework omits a significant point for regulation of these systems. This step determines growth factor bioavailability, targeting and activation -processes that seem crucial to the operation of autocrine and paracrine mechanisms (Flaumenhaft and Rifkin, 1992;Logan and Hill, 1992). Data accumulated through investigations of each of these components have resulted in a relatively comprehensive picture of the ovarian IGF system. Data on the EGF family of peptides is less complete, but recent studies suggest that a better understanding will soon be obtained.

IGF expression
The ability of pig ovarian cells to secrete IGEs has been surmised from studies measuring such factors in ovarian follicular fluid, ovarian cell conditioned media, and by analysis of the expression of mRNA for such peptides. Our laboratory has focused on this area in particular and our results have been recently reinforced and extended by others.
IGFs can easily be measured in pig follicular fluid (Hammond et al., 1982(Hammond et al., , 1985. IGF-I and -II are both present (Hammond et al., 1985;Spicer et al., 1992;Samaras et al., 19936).  , 19936), which showed that IGF-I is expressed predominantly in the membrana granulosa of medium size follicles and, to a lesser extent, in functioning corpora lutea (Fig. 1). These studies indicated that the expression of IGF-I in ovarian follicles in vivo was quite variable with some follicles being virtually devoid of mRNA.
In an attempt to relate the expression of IGF-I more clearly to recognized parameters of ovarian follicular development, we examined a well-characterized collection of ovarian follicles during the period of synchronized follicular growth triggered by progestin withdrawal (Samaras et al., 1993a). These studies showed statistically significant correlations of IGF-I with days of the follicular phase, follicular size, and oestradiol concentration.
In this model, IGF-I expression reached peak values on day 5 after altrenogest withdrawal and then declined (after the LH surge) on day 7. Collectively, these data indicate that IGF-I is expressed m the pig ovary and regulated in a physiologically meaningful fashion_ These studies are consistent with the notion that IGF-I may be involved in, or at least linked to, the process of follicular growth and selection. Although IGF-II peptide and mRNA (Hammond et al., 1993b) can also be readily detected, we did not find a relationship of this peptide to hormone action or follicle development. There is less mRNA for IGF-I in the porcine corpus luteum than in the follicle. However, mRNA for IGF-I can easily be detected in the corpus luteum; our initial studies suggest that the peptide is expressed in both large and small luteal cells (J. Gadsby, J. Lovdal, J. Barber and J. M. Hammond, unpublished). Evidence concerning the secretion of IGE-1 by porcine ovarian cells and its hormonal regulation has been provided directly by studies with cultured pig granulosa cells. Cells from 1-3 mm follicles of immature gilts secrete IGE-I in serum-free cultures for 7 -10 days and respond to treatment with FSH, oestradiol, LH and growth hormone (Hsu and Hammond, 1987a,b) and EGE (Mondschein and Hammond, 1988). Recently, Hatey et al. (1992) found that granulosa cells from medium sized (3-5 mm) follicles expressed ICE-I mRNA in culture and that the amount of mRNA was increased by gonadotrophic hormones. We have recently shown a threefold increase in steady-state IGF-I mRNA with forskolin as a cAMP inducer (S. Samaras, unpublished). Since IGE-I has been shown to increase the steroidogenic effectiveness of gonadotrophins and cAMP (see below), these data suggest that ICE-I can serve as an autocrine amplifier of gonadotrophin action in cells or follicles that develop the ability to express this peptide.
This possibility was directly tested with antibodies that bind IGE-1 (Mondschein et al, 1989) and these antibodies caused a 50% reduction in FSH-dependent steroidogenesis.

Expressionof EGT-hke peptidesin the porcine ovary
There are relatively few data on the expression of EGF-like peptides in the porcine ovary. However, these studies reflect substantial current interest in this problem. Several years ago, we found that the concentrations of receptor-active EGF-like peptides in pig follicular fluid were substantially higher than in pig plasma (Hsu et al., 1987) and that this activity declined in the preovulatory follicle. On the basis of these data and complementary studies in rodents (Kudlow et al., 1987) and cows (Skinner et al., 1987), we   et al., 1990, 1991Grimes and Hammond, 1992;Grimes et al., 1992Grimes et al., , 1993a  -3, and particularly -5, have been shown to be stimulated by treatment with IGET (Grimes and Hammond 1992). In contrast, gonadotrophins and cAMP generally inhibit these binding proteins (Mondschein et al., 1990;Grimes el al., 1992). However, concentrations of IGFBP-4 are increased at submaximal concentrations of gonadotrophins, and IGF-I treatment has insignificant effects (Grimes et al., 1993a). Interestingly, PGE2 inhibits while PGF," stimulates expression of IGFBP-3 in cultures of partially luteinized granulosa cells (Grimes et al., 19936), suggesting that the expression of this protein could be involved in the antagonistic luteotrophic and luteolytic effects of these hormones. Collectively, these data support the notion that the IGFs and their binding proteins function antagonistically in the regulation of the growth of the ovarian follicle and maintenance of the corpus luteum in pigs. This concept is supported by the inhibitory action of exogenous IGFBPs on ovarian cellular function and by the opposing effects of gonadotrophins and follicular growth on the secretion of the IGFBPs versus IGF-I and steroids. However, this concept fails to account for the complexity of the system, including the divergent expression of the several members of the IGFBP family and stimulatory actions of the proteins in other systems (Clemmons, T991). Undoubtedly a more complex relationship between the expression of the IGFs the IGFBPs, and the presentation of the IGFs to their receptors on ovarian target cells will eventually be developed.  (Fig. 2). Central to this model is the existence of an extracellular pool of IGFs complexcd to the IGFBPs, which serves as a reservoir of potentially active 1GFs. With processing of the complex, the IGFs can be presented to receptors on target cells. This model has not been fully substantiated in any system, but data for the existence of activity at each of the control loci depicted have been developed in one or more systems (Clemmons, 1991;Rechler, 1993): these include binding of thc IGFBPs to attachment sites on the cell glycocalyx (integrins or related attachment sites) or to mucopolysaccharides in the extracellular matrix; post-translational modification of the IGFBPs by phosphorylation, glycosylation or proteolytic processing could modify the affinity of these proteins for the IGFs and, hence, delivery of the IGE's to the receptors. Our own studies (Grimes and Hammond, in press) suggest that IGFBP-3 is proteolytically processed by porcine granulosa cells, probably by a serine protease, and that this action is inhibited by IGF-I. Similar effects can also be shown for 1GFBP-5 (Fig. 3) for which a striking induction of IGH3P-5 by IGHI is associated with a diminution in its low molecular weight processing variant. Further studies of this sort as well as of the critical steps in interaction of IGF-1-1GFBP complexes at the cell surface need to he performed before the full importance of these mechanisms can be understood. Current data suggest that these effects may be important in controlling the overall set point of the ovarian 1GF system_

Bioavailability and targeting of EGI-like peptides
Little or no information is available regarding post-translational processing of the EGF-like peptides in the pig ovary. It is not known why the pig ovary expresses four discrete growth factors which apparently bind to the same receptor, but the answer may require a better understanding of differential processing and targeting of these peptides within the ovary. Data from other systems suggest that these processes occur and are potential sites for regulation. For example, EGF and TGF-o are often secreted as a large precursor that spans the membrane of the secretory cell and requires proteolytic cleavage to be released into the extracellular space (Prigent and Lemoine, 1992). Site-directed mutagenesis of this cleavage site has shown that TGF -u can be active without full secretion, but the targeting of such actions may be restricted to the cell of origin and immediately adjacent cells (Lee, 1994 It is also possible that there are targeting mechanisms for heparin binding EGE-like peptide based on the demonstrated attachment of this molecule to mucopolysaccharides similar to the extracellular matrix, This peptide could then be released proteolytically or displaced by sulfated molecules in steps parallel to those outlined in our model for the IGEs (Fig. 2). Such mechanisms occur for many if not most growth factor-based autocrine and paracrine systems (reviewed in Logan and Hill, 1992;Flaumenhaft and Rifkin, 1992)

Actions of the ICEs
The actions of the IGFs on the pig ovary have now been studied for more than 10 years and have been extensively reviewed in other publications (Hammond et at, 1988b(Hammond et at, , 1991Giudihe, 1992) al., 1993). The preliminary data suggest that a similar phosphorylation cascade operates in the bovine ovary (Chakravorty et al., 1992), and that related processes occur in the pig ovary (Romero et al.;1993). More detailed information is available concerning the effects of the IGFs on cell specific end points; and these can be divided into growth or differentiative pathways which predominate depending on the presence or absence of other extracellular stimulators and on the level of differentiation of the ovarian cell. Although 1GF-1 is a mitogen for ovarian cells, it is not particularly potent when used alone. However, in the presence of EGF or related peptides, it causes a synergism in DNA synthesis and cell replication (Hammond and English, 1987;May et al., 1988). In the presence of gonadotrophins, the steroidogenic or differentiative actions of the 1GFs predominate. 1GF-I has the potential for synergizing with the gonadotrophins at several points in the steroidogenic pathway (see Giudice, 1992, for review). These interactions, coupled with the previously described effects of gonadotrophins to increase 1GF-1 secretion and decrease inhibitory 1GFBPs represent an integrated, mutually facilitatory set of mechanisms whereby gonadotrophin effects may be amplified through the ovarian 1GF system.

Conclusions
From the foregoing discussion a case can be made for a pivotal role for 1GFs and EGF-like peptides in the growth and development of the pig ovary. The level of understanding of the several components of these systems is becoming increasingly detailed. However, studies to date have relied heavily on in vitro techniques. It will be necessary to gain control over ovarian growth factors in vivo to confirm the growth factor hypothesis and to manipulate such systems to our advantage. Studies to date that bear on this question include hormonal manipulation of the 1GF system in the ovary (and throughout the body) by the administration of growth hormone. In women, considerable success has been reported in achieving an increase in gonadotrophin sensitivity (reviewed in Giudice, 1992) as predicted by the in vitro results reviewed above. However, comparable studies in pigs (Bryan et al., 1989;Guthrie et al., 1992;Spicer et at, 1992) do not clearly demonstrate the predicted increase in ovarian development and gonadotrophin responsivity.
In fact, these studies suggest an inhibition of several critical aspects of ovarian function (despite a dramatic increase in ovarian IGF-I concentrations).
These data indicate that intraovarian IGF-I cannot be viewed as the final common pathway for ovarian cellular growth and differentiation. Critical timing of activation of the IGF system and the presence or absence of other supportive hormonal and intraovarian factors, for example gonadotrophins and 1GFBPs, may be crucial.
In theory, ovarian growth factors could also be modulated by genetic manipulation of growth factor expression. A number of laboratories have now begun 'knock out' experiments to disrupt growth factor gene function systematically in transgenic mice. The IGE-11 gene has been disrupted by these techniques with modest effects (if any) on reproductive function in the preliminary analysis which has been performed (DeChiara et al., 1991). Once again, these studies indicate that the ICE system may not be the final common pathway for ovarian function, but a facilitatory mechanism. In addition, it seems likely that the body has compensatory mechanisms to avoid potentially lethal mutations in critical gene families. In the j M. Hammond et al.
ICF system, in particular, this is manifest by a multiplicity of ligands and receptors that have discrete but overlapping functions.
In the EGF family of peptides, the overlap in ligands is even greater. An ideal experiment might entail transgenic animals with growth factor genes driven by ovary-specific, hormoneresponsive promoters, for example FSH receptor or aromatase. To our knowledge, such experiments have not been completed in any species.