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In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mumol/(min.mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein.(ABSTRACT TRUNCATED AT 250 WORDS)

A peptide that is recognized by antibodies to human somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) has been partially purified from cultured Sertoli cells prepared from sexually immature rats. The mol wt of this peptide is about 25,000, as determined by gel filtration chromatography and immunoblot analysis of samples resolved by polyacrylamide gel electrophoresis. Isoelectric focusing indicated that the isoelectric point of this peptide was near neutrality. However, a smaller peptide of mol wt 8,000 that cross-reacted with antibodies to Sm-C/IGF-I, was released after gel filtration in acetic acid. Similarly, reverse phase HPLC on a C18 column under acidic conditions released a Sm-C/IGF-I immunoreactive peptide of 8,000 mol wt. This smaller species apparently resulted from the dissociation of this peptide from a binding protein. Unlike the larger neutral form, the isoelectric point of the smaller peptide was 9.8. This pI is similar to the GH-dependent Sm-C/IGF-I peptide isolated from rat serum. The small peptide, unlike the larger form, reacted in a parallel manner to human Sm-C/IGF-I in the Sm-C/IGF-I RIA and radioreceptor assays. In addition, the 8,000 mol wt peptide behaved as a progression factor in the BALB/c-3T3 assay and competed with [125I]Sm-C/IGF-I for binding to the type I Sm-C/IGF-I receptor from cultured rat Sertoli cells. In summary, results of this study demonstrate that rat Sertoli cells in culture secrete a peptide that is the rat equivalent of human Sm-C/IGF-I.

Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polypeptide required for the proliferation of many cell types. Since several attempts in our laboratory to recover monoclonal antibody-secreting hybrids to this peptide by the direct fusion of hyperimmunized splenocytes with myeloma cells had been unsuccessful, we modified our approach by coculturing hyperimmunized BALB/c splenocytes and a small amount of the antigen for 5 days prior to fusion with the P3X63Ag.8.653 myeloma cell line. Of 88 microcultures at risk, specific antibody was detected in 24. Two clones were expanded in ascites fluid and characterized as to isotype, affinity, and specificity. Both were IgG1,kappa and bound human Sm-C/IGF-I with affinity constants of 1.09 and 1.01 X 10(10) liter/mol, respectively. Both clones were quite specific for Sm-C/IGF-I with inconsequential binding to insulin-like growth factor II, multiplication-stimulating activity, any of the chymotryptic fragments of Sm-C/IGF-I, insulin preparations, hGH, hTSH, mEGF, or mouse albumin. In vitro boosting after primary in vivo immunization appears to provide monoclones of an IgG isotype in contrast to primary in vitro immunization, which reportedly favors an IgM isotype. The antibodies produced in this study have proved to be extraordinarily useful in defining the physiologic role of Sm-I/IGF-I with immunoneutralization techniques and in the purification of human Sm-C/IGF-I by affinity chromatography.

Somatomedin-C (SM-C) and insulin-like growth factor I (IGF-I) produced identical curves of competition in a RIA for SM-C, and in placental cell membrane receptor assays for SM-C and insulin. Somatomedin-A (SM-A), insulin-like growth factor II (IGF-II) and two forms of multiplication stimulating activity (MSA) were less than 5% as potent as SM-C/IGF-I in the RIA and less than 50% that of SM-C/IGFi in the SM-C receptor assay. In the insulin receptor assay, IGF-II and MSA III-2 were much more potent than SM-C/IGF-I. The present data suggest that SM-C and IGF-I are functionally identical substances.

Recent studies have documented the ability of somatomedin-C (Sm-C) to synergize with FSH in the induction of rat granulosa cell progesterone biosynthesis. This direct cytodifferentiative action of Sm-C proved distinct from its established mitogenic property. To determine whether Sm-C partakes in the differentiation of granulosa cell functions other than progesterone biosynthesis, the role of Sm-C in the acquisition of LH receptors was investigated in vitro. Granulosa cells were obtained from immature, hypophysectomized diethylstilbestrol-treated rats and cultured under serum-free conditions for up to 72 h. Specific granulosa cell LH/hCG binding under basal conditions was relatively low and was not significantly affected by treatment with highly purified Sm-C (50 ng/ml). In contrast, treatment with FSH (oFSH; NIH FSH S14; 20 ng/ml) produced a 3.8-fold increase in LH/hCG binding to 3167 +/- 660 (+/- SE) cpm/10(6) cells. However, concurrent treatment with Sm-C (50 ng/ml) resulted in a 6.1-fold enhancement of the FSH effect. The ability of Sm-C to synergize with FSH in the induction of LH receptors was dose and time dependent, with an apparent median effective dose (ED50) of 6.2 +/- 0.6 (+/-SE) ng/ml, and a minimal time requirement of 24 h or less. Examination of the binding parameters of the LH/hCG receptor revealed that the Sm-C-mediated increase in LH/hCG binding was due to enhanced binding capacity, but not affinity (Kd = 4.4 X 10(-11) M). Decreasing dilutions (1:128,000-1:8,000) of a monoclonal antibody raised against Sm-C (sm 1.2), produced progressive and virtually complete immunoneutralization of the synergistic interaction between Sm-C and FSH, suggesting specificity of action. The ability of Sm-C to enhance LH receptor induction was associated with increased hCG-stimulated progesterone biosynthesis, suggesting that the newly acquired receptors are functional in nature. Our present findings indicate that nanomolar concentrations of Sm-C are capable of enhancing FSH-stimulated LH/hCG-binding capacity, but not affinity, in a time- and dose-dependent fashion. These observations suggest that the direct cytodifferentiative effect of Sm-C is not limited to the induction of progesterone biosynthesis, but other key granulosa cell functions may also be subject to modulation by this regulatory peptide.

alpha IR-3, a monoclonal antibody that interacts with the somatomedin-C receptor, inhibited the binding of somatomedin-C, but not of insulin, to human placental membranes and intact IM-9 cells. alpha IR-1, a monoclonal antibody that interacts with the insulin receptor, did not inhibit the binding of either hormone. Inhibition of somatomedin-C binding by alpha IR-3 was mainly due to a decrease in its affinity. 125I-Labeled alpha IR-3 bound specifically to placental membranes and intact IM-9 cells and was inhibited by concentrations of unlabeled alpha IR-3 that were lower than those required to inhibit somatomedin-C binding. [125I]alpha IR-3 binding was also inhibited by somatomedin-C and insulin, but only at very high concentrations. A410, a rabbit antiserum that reacts with both receptors for insulin and somatomedin-C, also inhibited labeled alpha IR-3 binding. alpha IR-I did not. These results help to define the epitopes with which these antibodies interact.

Somatomedin-C (SM-C) was purified from Cohn fraction IV of human plasma by a series of steps which included cation-exchange chromatography, gel filtration, isoelectric focusing, and reverse-phase high-pressure liquid chromatography. The peptide isolated contained 10,142 units/mg, as judged by a radioimmunoassay, representing a 780,000-fold purification from native plasma. The isolated peptide was basic (pI 8.1-8.5) and was judged to be no less than 90% pure. The best fit for integral amino acids was obtained with 78 residues. Limited sequence data were obtained on the N terminus and on five fragments obtained by tryptic digestion after blocking the lysine residues. Three of these fragments and the N terminus could be aligned with portions of insulin-like growth factor I (IGF-I). Of the 25 residues so aligned, 22 were identical with IGF-I. In addition, two tryptic fragments were obtained which are not present in the sequence of IGF-I. SM-C and IGF-I produced identical curves of displacement in radioimmunoassay and radioreceptor assays for SM-C and in an insulin radioreceptor assay. The potencies of SM-C and IGF-I in these assays differed significantly from IGF-II, somatomedin-A, and several preparations of multiplication stimulating activity. At a concentration of 1 ng/mL, SM-C stimulated the progression of Balb/c 3T3 cells into DNA synthesis and when injected in vivo restored mitosis in lens epithelium of hypophysectomized frogs.

A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatomedin-C (Sm-C) has recently been found to amplify the FSH-mediated acquisition of granulosa cell progestin biosynthetic capacity, aromatase activity, and LH receptors, an effect distinct from its established replicative property. To further characterize the cellular mechanism(s) underlying the synergistic interaction of Sm-C with FSH, we have set out to evaluate the intermediary role of cAMP in this regard. Isolated granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for up to 3 days under serum-free conditions. The basal extracellular accumulation of cAMP remained unchanged in response to treatment with highly purified Sm-C (50 ng/ml). However, concurrent treatment with increasing concentrations (0.3-50 ng/ml) of Sm-C, produced dose- and time-dependent increments in the FSH-stimulated accumulation of cAMP, with an apparent median effective dose (ED50; mean +/- SE) of 5.1 +/- 0.6 ng/ml, a maximal response 8.8-fold greater than that induced by FSH alone, and a minimal time requirement of 1-2 days. Given increasing concentrations of FSH, treatment with a constant concentration (50 ng/ml) of Sm-C resulted in 1.7-, 5.8-, and 4.3-fold increases in cAMP accumulation for 10, 30, and 100 ng/ml FSH, respectively. The ability of Sm-C to augment FSH-stimulated cAMP accumulation was evident and, in fact, enhanced by ZK62711 (Rolipram; 3 X 10(-6) M)-induced blockade of cAMP-phosphodiesterase activity. Decreasing dilutions (1:64,000 to 1:1,000) of a monoclonal antibody raised against Sm-C (sm 1.2) produced progressive and complete immunoneutralization of the synergistic interaction of Sm-C with FSH, suggesting specificity of action. Taken together, these findings suggest that Sm-C, acting at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (0.6-2.0 nM), is capable of amplifying FSH-stimulated cAMP accumulation in a time- and dose-dependent manner. These observations suggest that the synergistic action of Sm-C is exerted, at least in part, at a site(s) proximal to cAMP generation.

Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH-selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently observed that nanomolar concentrations of exogenously added somatomedin-C (Sm-C) are capable of synergizing with FSH in the induction of cultured rat granulosa cell progesterone biosynthesis and LH receptors without altering granulosa cell survival or replication. To further characterize the cytodifferentiative properties of Sm-C, we have undertaken to investigate whether the acquisition of granulosa cell aromatase activity is also subject to modulation by this intraovarian peptide. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were initially cultured for up to 3 days in an androstenedione-free medium, during which time aromatase activity was induced by FSH in the absence or presence of Sm-C (treatment interval). At the conclusion of this period, the cells were washed and reincubated for an additional 8-h test interval, during which time aromatase activity was estimated. Basal aromatase activity, as assessed by the conversion of unlabeled androstenedione (10(-7) M) to radioimmunoassayable estrogen, was negligible, remaining unaffected by treatment with highly purified Sm-C (50 ng/ml) alone. However, concurrent treatment with Sm-C (50 ng/ml) produced a 7.0-fold increase in the FSH (100 ng/ml; NIH FSH S14)-stimulated accumulation of estrogen. Similarly, Sm-C produced a 6.1-fold increase in FSH-induced aromatase activity, as assessed by the stereospecific generation of tritiated water from [1 beta-3H]androstenedione substrate. Sm-C-potentiated aromatase activity was dose and time dependent, with an apparent median effective dose of 5.0 +/- 1.9 (+/- SE) ng/ml and a minimal time requirement of 24 h or less, but was independent of the FSH dose employed. Although bovine insulin and multiplication-stimulating activity, like Sm-C, proved capable of augmenting aromatase activity (albeit at a substantially reduced potency), little or no effect was observed for either porcine or rat relaxin, a distantly related member of the insulin-like growth factor family. Examination of the apparent kinetic parameters of the aromatase enzyme revealed that the Sm-C-mediated potentiation of aromatase activity was due to enhancement of the apparent maximal reaction velocity, but not substrate affinity (Km = 2.8 X 10(-8) M). Our findings indicate that nanomolar concentrations of exogenously added Sm-C synergize with FSH in the enhancement of the maximal reaction velocity, but not Km, of granulosa cell aromatase in a dose- and time-dependent fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

The ovarian granulosa cell has recently been shown to be a site of insulin-like growth factor-I (IGF-I) production, reception, and action. In large measure, IGF-I action (in the rat) appears contingent upon its ability to synergize with FSH, a major promoter of granulosa cell differentiation. It is the objective of the in vitro studies reported herein to elucidate the cellular mechanism(s) whereby IGF-I amplifies FSH hormonal action, placing special emphasis on the potential role of the putative intracellular second messenger cAMP in this regard. Basal FSH binding (115.7 +/- 2.1 fmol/mg cell protein) to rat granulosa cells cultured under serum-free conditions remained unchanged after 72 h of treatment with IGF-I (50 ng/ml) by itself (107.1 +/- 1.0 fmol/mg cell protein). In contrast, treatment with FSH (20 ng/ml) resulted in a significant (P less than 0.05) decrease in FSH binding capacity (but not affinity) relative to controls in either the absence or presence of IGF-I. Whereas treatment with FSH resulted in a substantial increase in forskolin-stimulatable adenylate cyclase activity (10 +/- 1.7% conversion of [3H] ATP to [3H]cAMP), concurrent treatment with IGF-I resulted in 2.2-fold enhancement of FSH action. This IGF-I effect proved dose dependent with an apparent median effective dose of 3.6 +/- 0.8 ng/ml, a concentration in keeping with its granulosa cell receptor binding affinity. Significantly, however, IGF-I proved capable of enhancing Bt2cAMP-stimulated progesterone accumulation suggesting that IGF-I may be also acting at site(s) distal to cAMP generation. Taken together, these and previous studies indicate that nanomolar concentrations of exogenously added IGF-I may be interacting with the FSH transduction signal at multiple cellular site(s) to effect amplification of FSH action.

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.

The characteristics and regulation of the murine granulosa cell type I insulin-like growth factor (IGF) receptor under in vivo conditions were studied. In vivo treatment of immature hypophysectomized diethylstilbestrol-treated rats with increasing doses (0.3-30 microgram/rat, twice daily) of FSH for 72 h resulted in dose-dependent increments in specific granulosa cell somatomedin-C (Sm-C)/IGF-I binding, peaking (5150 +/- 350 cpm/3 x 10(5) cells) at the 10 micrograms/rat (twice daily) dose level to yield a 2.6-fold increase relative to that in untreated controls. This FSH (10 micrograms/rat, twice daily) effect proved time dependent; the first significant (P less than 0.05) increase in binding (3670 +/- 150 cpm/3 x 10(5) cells) was noted after 48 h of treatment (1.6-fold increase). Significantly, little or no variation was observed for basal Sm-C/IGF-I binding over the course of the experiment, suggesting that this component of Sm-C/IGF-I receptor complement may be independent of the trophic influence(s) of the pituitary gland. Equilibrium competition studies carried out with granulosa cells derived from both control and FSH-treated rats revealed linear Scatchard plots consistent with a single class of noninteracting binding sites, a 2.8-fold increase in FSH-associated Sm-C/IGF-I-binding capacity, but not affinity (Kd control, 1.9 +/- 0.3 nM; kd FSH, 2.6 +/- 0.9 nM). Limited specificity studies of the FSH-induced receptor revealed related peptides to compete for Sm-C/IGF-I binding with a relative rank order of potency of Sm-C/IGF-I much greater than multiplication-stimulating activity greater than insulin, a pattern compatible with a type I IGF receptor. A series of other polypeptides, including porcine relaxin, porcine proinsulin, epidermal growth factor, basic fibroblast growth factor as well as transforming growth factor-alpha and -beta (TGF beta) were nonreactive. Significantly, the induced type I IGF receptor proved functionally coupled to granulosa cell proteoglycan biosynthesis. The ability of FSH (10 micrograms/rat, twice daily) to enhance granulosa cell Sm-C/IGF-I binding was significantly (P less than 0.05) up-regulated (1.53-fold amplification) by ovine GH (100 micrograms/rat, twice daily); a down-regulatory effect (64% inhibition) was observed for a potent GnRH agonist [( D-Ala6,Des-Gly10]GnRH ethyl amide; 25 micrograms/rat, twice daily). Once induced, the Sm-C/IGF-I receptor of the granulosa cell required the continued presence of either FSH or LH for its maintenance; the lactogenic receptor agonist PRL had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The ovarian granulosa cell has recently been shown to be a site of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) production, reception, and action. These observations have generally been interpreted to suggest the existence of an autocrine loop concerned with granulosa cell physiology. It is the objective of the in vitro studies reported herein to extend these observations by evaluating the interaction of Sm-C/IGF-I with the adjacent thecal-interstitial cell. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with Sm-C/IGF-I (50 ng/ml) or hCG (1 ng/ml), resulted in 2.1- and 4.0-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstane-17-one), the main androgenic steroid identified in culture media. However, combined treatment with both agents unmasked a synergistic interaction producing a 3.3-fold increase in the hCG-stimulated accumulation of androsterone, an effect consequent to enhanced androgen biosynthesis rather than diminished degradation. Unaccounted for by an increase in viable ovarian cell numbers and independent of the hCG dose (0.1-10 ng/ml) used, the Sm-C/IGF-I effect proved time and dose dependent, with a projected minimal effective dose of 3 ng/ml and a minimal time requirement of 72 h. [125I]Iodo-Sm-C/IGF-I binding to untreated highly enriched thecal-interstitial cells proved saturable, with a single class (Hill coefficient = 0.98 +/- 0.01) of high affinity (Kd = 3.0 nM), low capacity (maximum binding = 10,840 +/- 2,108 sites/cell) binding sites. Limited specificity studies using related peptides produced a rank order of competitive potency of: Sm-C/IGF-I greater than multiplication stimulating activity greater than insulin, a pattern compatible with the presence of type I IGF receptors. Other related peptides, such as porcine proinsulin and porcine desoctapeptide insulin, proved weakly effective in inhibiting Sm-C/IGF-I binding to its receptor; unrelated peptides such as porcine relaxin and erythropoietin were without effect. Taken together, these findings suggest that 1) the thecal-interstitial cell, like the granulosa cell, may be a site of Sm-C/IGF-I reception and action, and 2) the ability of high dose insulin to stimulate ovarian androgen biosynthesis may be due to its capacity to act as a Sm-C/IGF-I surrogate, its high dose requirements reflecting cross-interaction with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently shown that nanomolar concentrations of somatomedin-C (Sm-C), are capable of enhancing the FSH-mediated (but not basal) accumulation of progesterone (Po) by cultured rat granulosa cells. To further characterize this direct cytodifferentiative effect of Sm-C, granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats were cultured under serum-free conditions for up to 96 h. Concurrent treatment with highly purified Sm-C (50 ng/ml) produced 10.2- and 3.6-fold increments in the FSH (20 ng/ml)-stimulated accumulation of Po and 20 alpha-hydroxy-4-pregnen-3-one, respectively. Sm-C-augmented Po biosynthesis was dose- and time dependent, but was independent of the FSH dose employed. Significantly, this effect of Sm-C could not be accounted for by enhancement of cellular viability or plating efficiency, nor by an increase in the number of cells, or their DNA synthesis. Furthermore, specific inhibition of DNA synthesis with cytosine-1-beta-D-arabinofuranoside was without significant effect on the ability of SM-C to enhance FSH-supported Po biosynthesis. Insulin, like Sm-C, also synergized with FSH in the induction of Po biosynthesis. However, insulin [ED50 = 19.2 +/- 1.6 (SE) micrograms/ml] was approximately 4800-fold less potent than Sm-C [ED50 = 4.0 +/- 0.3 (SE) ng/ml] in this regard, and exerted little or no effect at concentrations presumed to saturate the putative high affinity granulosa cell insulin receptor. Although maximal stimulatory doses of Sm-C (75 ng/ml) or insulin (100 micrograms/ml) produced comparable increments in FSH-supported Po biosynthesis, combined treatment with maximal doses of both peptides did not prove additive. Pertinently, the direct cytodifferentiative effect of Sm-C is exerted at (nanomolar) concentrations compatible with its receptor-binding affinity as observed in all other cell types studied. Thus, Sm-C is not likely to be acting through the putative high affinity insulin receptor but rather through its own high affinity recognition sites. Similarly, the cytodifferentiative action of high dose insulin may reflect the consequences of its cross-interaction with the putative Sm-C, rather than the insulin receptor. These findings are in keeping with the suggestion that the granulosa cell may be the site of Sm-C reception and action and that Sm-C of intraovarian or circulatory origin may participate in the differentiation, as well as replication, of the developing granulosa cell.

Somatomedin-C (Sm-C) was purified from Cohn fraction IV of human plasma by previously published methods. Purity was established by SDS polyacrylamide electrophoresis followed by silver staining of the gel. Amino acid analysis of an acid hydrolysate revealed no significant discrepancies from the amino acid composition of insulin-like growth factor I (IGF-I). The first 24 residues beginning at the amino terminal glycine were identical to the corresponding residues in IGF-I. Tryptic and chymotryptic degradation followed by determination of the amino acid composition and sequence of the resultant peptides was used to complete the primary structure of Sm-C. The results of these studies document that Sm-C and IGF-I are identical peptides, thus supporting previous observations that Sm-C and IGF-I are qualitatively and quantitatively indistinguishable in radioligand and biological assay systems.

The role of somatomedin-C (Sm-C) in the acquisition of granulosa cell progesterone biosynthesis was investigated in vitro in a primary culture of rat granulosa cells cultured for 72 h under serum-free conditions. Basal progesterone accumulation was negligible and remained unaffected by treatment with highly purified Sm-C (50 ng/ml). Whereas treatment with FSH (20 ng/ml) produced a 9-fold increase in progesterone accumulation, the concurrent application of increasing concentrations (0.3-50 ng/ml) of Sm-C brought about dose-dependent increments in the FSH-stimulated accumulation of progesterone with a median effective dose of 4.0 +/- (SE) 0.3 ng/ml and a maximal response 9.6-fold greater than that induced by FSH alone. A monoclonal antibody raised against Sm-C (sm 1.2) produced complete immunoneutralization of the synergistic interaction between FSH and Sm-C, supporting the specificity of the Sm-C effect and arguing against the possible involvement of copurified contaminant(s) in the preparation used. Treatment of granulosa cells with the highest dose of Sm-C tested (50 ng/ml), in the absence or presence of FSH, did not result in significant alterations in cell number, DNA content, plating efficiency or viability. Taken together, our findings indicate that Sm-C is capable of synergizing with FSH in the induction of granulosa cell progesterone biosynthesis. Significantly, this ability of Sm-C to augment differentiated phenotypic expression of the developing granulosa cell is distinct from its well established growth-promoting property and may thus represent a novel biologic effect of this polypeptide.

The ovarian granulosa cell has recently been found to be a site of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) production, reception, and action, thereby raising the prospect of a novel autocrine control mechanism concerned with granulosa cell ontogeny. It is the objective of the in vitro studies reported herein to explore the characteristics of the murine granulosa cell membrane Sm-C/IGF-I receptor and its regulation by gonadotropic, lactogenic, and beta 2-adrenergic signalling. Provision of FSH (150 ng/ml) to granulosa cells from immature rats cultured for 72 h under serum-free conditions resulted in a 3.1-fold increase over control values in specific cell-bound [125I]iodo-Sm-C/IGF-I. Binding to FSH-primed cells proved time, temperature, and pH dependent; optimal steady state conditions were achieved after an 8-h incubation at 15 C and a pH of 8.0. Although subject to regulation by the cellular density of plating, the binding of [125I]iodo-Sm-C/IGF-I to its receptor proved saturable (apparent Kd = 3.3 X 10(-9) M) as well as reversible; complete or partial tracer displacement was effected by competitive inhibition and dilution, respectively. Specificity studies revealed the competition for [125I]iodo-Sm-C/IGF-I binding to follow a relative rank order of potency of Sm-C/IGF-I much greater than multiplication-stimulating activity greater than insulin, but disclosed limited or no displacement by a series of chemically related and unrelated polypeptides. By Scatchard and Hill analysis, both control and FSH-treated cells displayed a single class of noninteracting binding sites; the FSH-enhanced binding represented largely increased binding capacity, rather than affinity. Significantly, up-regulation of granulosa cell Sm-C/IGF-I binding was not limited to FSH; qualitatively comparable increments in [125I]iodo-Sm-C/IGF-I binding were obtained after treatment with luteotropic, and beta 2-adrenergic (but not lactogenic) granulosa cell agonists. Taken together, these studies provide further evidence for the existence of high affinity, low capacity, specific cell membrane receptors for Sm-C/IGF-I in cultured rat granulosa cells. Our findings further indicate that the ability of FSH to enhance granulosa cell Sm-C/IGF-I binding largely reflects increased binding capacity rather than affinity and that this heterologous up-regulatory phenomenon may not be limited to FSH. As such, our observations of comparable up-regulation after luteotropic and beta 2-adrenergic (but not lactogenic) stimulation are in keeping with the view that cAMP may play an intermediary role in the regulation of granulosa cell type I IGF receptors.

The role of somatomedin-C (Sm-C) in the regulation of granulosa cell proteoglycan biosynthesis was investigated in vitro in a primary culture of rat granulosa cells labeled with [35S]sulfate. Basal [35S]sulfate incorporation into extracellular proteoglycans was increased by 93 percent in response to treatment with highly purified Sm-C (50 ng/ml) by itself. Whereas treatment with a minimally effective dose of FSH (20 ng/ml) alone produced a 43 percent increase over basal levels in extracellular [35S]sulfate-labeled proteoglycans, concurrent treatment with Sm-C yielded a 2.7-fold amplification of the FSH effect. Qualitatively similar results were obtained when [35S]sulfate incorporation into cellular proteoglycans was determined, the latter accounting for approximately one half of the total radioactivity incorporated. Significantly, fractionation of the major extracellular proteoglycan species revealed FSH to favor the exclusive production of dermatan sulfate (1.6-fold increase), whereas Sm-C supported the simultaneous biosynthesis of both heparan and dermatan sulfate (2.5- and 1.8-fold increments, respectively). Moreover, Sm-C proved capable of diverting FSH-driven proteoglycan biosynthesis from the exclusive stimulation of dermatan sulfate towards the enhanced production of heparan sulfate over dermatan sulfate. These findings suggest that while Sm-C may synergize with FSH in stimulating granulosa cell proteoglycan biosynthesis, it is also able to act in tis own right to effect marked quantitative as well as qualitative alterations in proteoglycan economy. Given the possible role of proteoglycans in follicular antrum formation and follicular atresia, our findings raise the possibility that Sm-C of granulosa cell origin may partake in the growth as well as the demise of the developing ovarian follicle.

A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein. The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E. coli tryptophan promoter. The fusion protein, termed L0-[Thr59]-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay. Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells. After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed [Thr59]-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays. It was also mitogenic in Balb/c 3T3 cells. These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.