Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Effects of epidermal growth factor, fibroblast growth factor and bovine serum albumin on ornithine decarboxylase activity of procine granulosa cells.

Epidermal growth factor, a potent mitrogen for granulosa cells produced a three-fold stimulation of ornithine decarboxylase activity in porcine granulose cells in vitro. Fibroblast growth factor, another compound with mitogenic activity for granulose cells, did not stimulate ornithine decarboxylase. Maximally effective concentrations of a commercial preparation of bovine serum albumin equalled the maximal effect of epidermal growth factor on this enzyme activity. The dominant stimulator(s) in the albumin preparation eluted after bovine serum albumin in gel filtration. At maximally effective concentrations, luteinizing hormone produced substantially greater stimulation than either epidermal growth factor or the bovine albumin preparation. Combinations of saturating doses of any two of these stimulators produced additive effects on enzyme activity.     

c-fos reduces growth factor requirements for mitogenic stimulation of L6 rat myoblasts

Addition of fetal calf serum (FCS) to serum-deprived L6J1 rat myoblasts increases fos-like immunoreactivity. The nuclear immunoreactivity reached a maximum 2 h after serum addition. Effects of the c-fos protein on myoblast proliferation were analyzed in L6J1 rat myoblasts transfected with the murine c-fos gene under control of a metallothionein promoter. L6J1 myoblasts with elevated expression of transfected c-fos reached higher cell densities than neo transfected control myoblasts when approaching a stationary phase in normal culture conditions (5% FCS). The differences in cell densities were even more pronounced at low serum concentrations (0.5% FCS). c-fos transfected cells also had a faster growth rate than did control cells in serum-free medium supplemented with calcium chloride, lithium chloride, sodium selenite, hydrocortisone, and insulin. The cell morphology of c-fos transfected L6J1 myoblasts was not affected compared to control myoblasts. These results suggest that c-fos protein expression in L6J1 myoblasts is activated by serum and that mitogenic stimulation of L6J1 myoblasts is facilitated by the presence of elevated amounts of c-fos protein.     

Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.     

Effect of dihydrocytochalasin B on the induction of DNA synthesis by epidermal growth factor, insulin and serum in Swiss 3T3 cells

The addition of a microfilament-disorganizing agent--dihydrocytochalasin B B (5-10 micrograms/ml)--to to quiescent confluent or sparse (in 0.5% serum) Swiss 3T3 cells, 1-2 hours prior to stimulation, inhibited the initiation of DNA synthesis induced by an epidermal growth factor (7.5-10 ng/ml) and insulin (0.5-1.0 micrograms/ml), but exerted a low effect on serum stimulation. DNA synthesis was measured 21-23 hours after the growth factor administration by 14C-thymidine incorporation in acid-insoluble material and the ratio of this fraction to exogenous thymidine uptake. Moreover, the polar solvent dimethylsulfoxide, present in culture medium at low concentration (0.1-0.5%), also caused a decrease in the basal level of 14C-thymidine incorporation in resting cells, and a less decrease in the induced incorporation.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8

To study the fine specificity of the HLA-D region, a panel of human T-lymphocyte clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DRw8. HLA-DR-homozygous peripheral blood lymphocytes (PBLs) were stimulated with DR-heterozygous PBLs in primary mixed lymphocyte cultures for 4 days. Blasts were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T-cell growth factor and irradiated stimulator cells. Viable clones were subsequently tested in proliferation assays against the original stimulator and a limited panel of stimulators bearing relevant DR specificities. Initial primings produced approximately 800 clones; some recognized DR-associated antigens, 70 recognized only their original stimulator, and approximately 50% were nonresponsive. Analysis on extended stimulator panels revealed alloantigenic complexity within similar DR-associated antigens as recognized by TLCs. The data are consistent with evidence that extreme heterogeneity exists within the HLA-D region.Abbreviations used in this paper cpm counts per minute - DNV double normalized value - EBV Epstein-Barr virus - FCS fetal calf serum - HLA human major histocompatibility complex - HTC homozygous typing cell - LCL lymphoblastoid cell line - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PLT primed lymphocyte typing - TCGF T-cell growth factor - TLC T-lymphocyte clone - T-max maximized T-test analysis     

The effect of recombinant erythropoietin on murine megakaryocyte colony formation

We investigated the effect of a recombinant human erythropoietin preparation (recombinant Epo) on murine megakaryocyte (MK) colony formation in serum-free and serum-containing culture systems, in order to study the relationship between Epo and megakaryopoiesis. Pokeweed mitogen spleen-conditioned medium (PWM-SCM), a standard source of MK colony stimulator, dose-dependently stimulated MK colony formation in the two culture systems. The plating efficiency of serum-free cultures was almost equal to that of cultures containing serum. Recombinant Epo also dose dependently stimulated MK colony formation in serum-containing cultures. However, in serum-free cultures recombinant Epo alone did not stimulate the growth of MK colonies; with the addition of fetal calf serum (FCS) to the serum-free cultures, recombinant Epo induced the growth of MK colonies. Furthermore, recombinant Epo enhanced MK colony formation through the stimulation of PWM-SCM or murine interleukin 3 (IL-3) in serum-free cultures. Our data show that Epo can act as a stimulator of megakaryopoiesis in collaboration with a factor in serum, or with an MK colony stimulator such as IL-3.     

Insulin-like growth factor I/somatomedin-C (IGF-I/SM-C) and glucocorticoids synergistically regulate mitosis in competent human fibroblasts

In serum-free medium, insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) was weakly mitogenic for adult human fibroblasts in culture. However, in the presence of 0.5% human hypopituitary serum (HHS), which by itself had little effect, there was a marked dose-dependent response to IGF-I/SM-C with a 10- to 20-fold increase in [3H]thymidine incorporation at 25 ng/ml IFG-I/SM-C. With the further addition of dexamethasone or hydrocortisone to the combination of IGF-I/SM-C + 0.5% HHS, there was a dramatic synergistic effect resulting in a 60- to 70-fold increase in [3H]thymidine incorporation. This stimulation was two times greater than that seen with 20% FCS. In contrast, glucocorticoids had no effect in serum-free medium or with HHS alone. These [3H]thymidine incorporation results were clearly supported by cell replication studies. Dose-response curves for 125I IGF-I/SM-C binding and IGF-I/SM-C stimulation of [3H]thymidine incorporation were similar with 1/2 maximal effects for both at 5 ng/ml. However, the striking synergism seen with glucocorticoids occurred in the absence of any glucocorticoid-induced change in IGF-I/SM-C binding, indicating that the interaction of IGF-I/SM-C and glucocorticoids occurs at a postreceptor level. These data demonstrate that in the presence of a low concentration of HHS, IGF-I/SM-C and glucocorticoids stimulate complete cell cycle traverse and replication of human fibroblasts.     

Mitogenic activity of glia maturation factor: Interaction with insulin and insulin-like growth factor-II

The mitogenic activity of glia maturation factor (GMF) was tested on sparse-cultured cells. GMF stimulates the growth rate of normal astroblasts and fibroblasts grown in the presence of fetal calf serum (FCS), and raises the saturation density of the cells over what is imposed by the corresponding serum concentrations. GMF has no mitogenic effect in the complete absence of serum. The mitogenicity of GMF is also demonstrable in defined media where certain serum components are present. In particular, GMF in combination with the defined medium N2 partially mimics the proliferative effect of serum alone. Insulin, an ingredient of N2, can substitute for the complete N2 formula. Insulin-like growth factor-II (IGF-II), in turn, can substitute for insulin. The interaction of GMF with insulin or IGF-II can be demonstrated in a sequential manner, suggesting that GMF is a competence factor. Since insulin is required at a concentration well above the physiologic serum level, and must be used at a dose 1000 times higher than IGF-II, we suspected that insulin acts on IGF-II receptors. This was substantiated by the demonstration of IGF-II receptors and the absence of detectable insulin receptors on the astroblasts. The combined effect of IGF-II and GMF mimics the combined effect of 10% FCS and GMF, in both growth rate and saturation density.     

The growth of mouse melanoma cells in hormone-supplemented, serum-free medium.

A mouse melanoma line, M2R, derived from the B₁₆ transplantable tumor can be grown in a hormone-supplemented serum-free medium. Cells grown in medium supplemented with insulin, transferrin, testosterone (or progesterone), follicle-stimulating hormone, nerve growth factor (NGF) and leutinizing releasing hormone show growth rates equivalent to that seen in medium supplemented with 5% fetal calf serum (FCS). This hormone-supplemented medium will support the growth of cells indefinitely. In cells grown in hormone-supplemented medium, insulin is shown to increase incorporation of glucose into glycogen and fatty acids. Transferrin is shown to act in part as an iron transport protein, although another role in growth stimulation cannot be eliminated. It is suggested that progesterone stimulates growth via an androgenic breakdown product and thus acts in a manner similar to testosterone.     

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.     

Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes

Polyvinyl alcohol (PVA) was used as a substitute for serum in a vitrification solution for in vitro matured bovine oocytes. In vitro matured bovine oocytes were cryopreserved in various vitrification solutions (VS) supplemented with different concentrations (0.05, 0.1, 0.5, and 1%) of PVA, 20% fetal calf serum (FCS) or without macromolecule supplementation in a gel-loading tip (GL-tip). After warming, vitrified oocytes were examined for effects on survivability, fertilizability, and embryonic development in vitro. At 18 h in vitro fertilization after vitrifying and warming, the number of surviving mature oocytes vitrified in VS without macromolecule supplementation was significantly (P < 0.05) lower than those with macromolecule supplementation. For fertilizability after vitrification, there was no significant difference in the penetration rate of oocytes among fresh oocytes (98.7%); oocytes vitrified in VS supplemented with 0.1 (76.8%), 0.5 (70.2%), or 1% (80.3%) PVA; 20% (84.1%) FCS; or without supplementation (61.7%). Also, the normal fertilization rate was not significantly different in oocytes vitrified with 0.1 (56.5%), 0.5 (43.5%), or 1% (49.7%) PVA and 20% (60.6%) FCS, compared with fresh oocytes (84.0%). Subsequently, vitrified oocytes were examined for embryonic development effects in vitro. The highest proportion of cleaved oocytes after vitrification was obtained in VS supplemented with 0.1% (18.8%) PVA. Additionally, the proportion of development to morula stage (7.7%) in the oocytes vitrified in a VS supplemented with 0.1% PVA was significantly (P < 0.05) superior to that of the 0, 0.5, and 1% PVA-vitrified groups. However, the beneficial effect of PVA addition was not found in blastocyst development. Embryonic development of vitrified oocytes was significantly lower than that of fresh oocytes. In conclusion, the present results indicate that 0.1% PVA supplementation in VS results in a significantly higher rate of morula stage embryos than 0, 0.5, and 1% PVA supplementation, and could replace FCS in VS for vitrification of in vitro matured bovine oocytes.     

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.0%) did not support viability or maturation of the cumulus-oocyte complex as well as higher doses of FCS (5, 10, or 20%) for either species. BSA failed to support cumulus expansion for bovine or hamster cumulus-oocyte complexes. All doses of FCS examined supported cumulus expansion in bovine cumulus-oocyte complexes, whereas the hamster complexes required at least 1.0% FCS to induce cumulus expansion. The addition of a serum filtrate, Solcoseryl, with BSA improved viability of the cumulus in the bovine but did not support cumulus expansion or completion of Meiosis I in bovine complexes. In vitro fertilization could be accomplished in media containing FCS by increasing the heparin concentration in the bovine system or reducing FCS for the hamster system. Polyspermy was increased when FCS was the protein supplement. It is not known whether this is an interaction of FCS with the sperm or oocyte. In conclusion, FCS was found necessary for follicle-stimulating-hormone (FSH)-induced cumulus expansion. It also improved cumulus cell viability and completion of the first meiotic division in complexes of both species compared with BSA.     

Dihydrotestosterone (DHT) regulation of insulin-like growth factor II mRNA in neonatal rats

Insulin-like growth factors (IGFs) are well known as peptide mitogens and important growth factors in fetal as well as in early postnatal development. In particular, IGF II is strongly expressed during fetal life and in neonatal animals. Very little is known about the regulation of IGF II gene expression. In order to study in detail the regulation of IGF II mRNA levels in the liver by the potent nonaromatizable androgen dihydrotestosterone (DHT), we have used quantitative in situ hybridization to detect the mRNA encoding for this growth factor. Pups were separated into 4 groups and injected twice a day immediately after birth with 3 different doses of DHT: 0.1 mg DHT/day, 0.25 mg DHT/day, 0.5 mg DHT/day for 4 and 7 days, and the control groups were injected with the vehicle alone. Animals were perfused with 4% paraformaldehyde and sections from the liver, heart, kidneys and brain were cut with a cryostat. A [³⁵S]-labeled cDNA probe was used to detect IGF II mRNA levels. After hybridization, sections were autoradiographed with X-ray films and then coated with liquid photographic emulsion. Densitometric measurement revealed that, at 4 days of age, IGF II mRNA levels were lower in DHT-treated rats than in control animals. No statistically significant differences in IGF II mRNA levels were observed among the three groups treated with the different doses of DHT, thus revealing that even the lowest dose of DHT (0.1 mg/day) used was sufficient to inhibit IGF II gene expression in neonatal rats. Moreover, at 7 days of age, DHT-treated rats showed the same levels of IGF II mRNA as those observed in rats treated with DHT for 4 days. These results suggest that DHT may play an important role in the regulation of IGF II gene expression in the rat liver during the neonatal period.     

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Growth hormone (GH) plays an important role in regulation of animal growth, metabolism and lactation[1]. Numerous studies have shown that exogenous somatotropin (ST) can increase average daily weight gain, improve feed efficiency, stimulate protein deposition and muscle growth and decrease lipid accretion rate[1]. The original somatomedin hypothesis suggested that the effect of GH on postnatal growth was mediated by insulin-like growth hormone factor 1 (IGF-I) which was thought to be deriv…