Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.     

Intrapituitary regulatory system of mammotrophs in the mouse

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor alpha (TGF-alpha) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17beta (E2, 10(-9) M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-alpha antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-alpha mRNA and growth hormone by in situ hybridization and fluorescence-immunocytochemistry demonstrated that TGF-alpha mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-alpha-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-alpha produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.     

Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha-(difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF-stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.     

Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.

Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.     

5-Hydroxytryptamine stimulates fluid secretion in locust malpighian tubules independently of cAMP

5-Hydroxytryptamine (5-HT) stimulates fluid secretion by semi-isolated Malpighian tubules of Locusta in a dose-dependent manner. The threshold of stimulation is between 10(-8) and 10(-7) M 5-HT; maximal activation occurs at doses greater than 10(-6) M. Relative to the activation induced by diuretic hormone (storage lobe extracts), 5-HT increases the rate of fluid secretion by only 65%. Phentolamine, the alpha-adrenergic blocker, failed to inhibit either DH or 5-HT stimulated secretion. Diuretic hormone raises the levels of intracellular of cAMP, and activates adenylate cyclase in plasma membrane preparations of Locusta Malpighian tubules. 5-HT (10(-4) M) has no effect in either assay system. Thus 5-HT can stimulate fluid secretion independently of cAMP. A hypothetical model for hormone stimulated fluid secretion by Locusta Malpighian tubules, involving dual-receptor activation, is proposed. Other biogenic amines, including octopamine, adrenalin, dopamine, synephrine and the formamidine chlordimeform were tested for their ability to stimulate fluid secretion. Only dopamine showed a weakly stimulatory effect.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

Aging alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha

Administration of pharmacological doses of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) in young rats stimulates gastric mucosal proliferation, but, in aged rats, the same treatment inhibits proliferation. This may be due to enhanced ligand-induced internalization of EGF receptor (EGFR). In support of this, we demonstrated that although a single injection of EGF (10 microg/kg) or TGF-alpha (5 microg/kg) in young (4-6 mo old) rats greatly increased membrane-associated EGFR tyrosine kinase activity, the same treatment slightly inhibited the enzyme activity in aged (24 mo old) rats. This treatment also produced a greater abundance of punctate cytoplasmic EGFR staining in gastric epithelium of aged rats, consistent with EGFR internalization. In vitro analyses demonstrated that exposure of isolated gastric mucosal cells from aged but not young rats to 100 pM TGF-alpha resulted in marked increases in intracellular EGFR tyrosine kinase activity and that induction of EGFR tyrosine kinase activity in mucosal membranes from aged rats occurred at doses 1,000-fold less than those required in young rats. Our data suggest that aging enhances sensitivity of the gastric mucosa to EGFR ligands. This may partly explain EGFR-mediated inhibition of gastric mucosal proliferation in aged rats.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor and transforming growth factor-alpha stimulate the proliferation of mouse uterine stromal cells

Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E(2)) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E(2) (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-alpha and the combination of E(2) and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha-, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Blastocoel expansion in the preimplantation mouse embryo: stimulation of sodium uptake by cAMP and possible involvement of cAMP-dependent protein kinase

Elevating cAMP levels in mouse blastocysts increases the rate of blastocoel expansion (F. Manejwala, E. Kaji, and R. M. Schultz, 1986, Cell, 46, 95-103), which requires extracellular sodium (F. Manejwala, E. J. Crago, Jr., and R. M. Schultz, 1989, Dev. Biol. 133, 210-220). We report that cAMP analogs that can activate the cAMP-dependent protein kinase stimulate 22Na+ uptake by cavitating mouse blastocysts and that inhibitors of cAMP-dependent protein kinase activity inhibit the cAMP-stimulated increase in both the rate of blastocoel expansion and 22Na+ uptake.     

New actions of melatonin on tumor metabolism and growth

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.     

Transforming growth factor-alpha binds to the epidermal growth factor receptor in gastric mucosa.

The ability of transforming growth factor-alpha (TGF-alpha) to interact with the gastric mucosal epidermal growth factor (EGF) receptor was investigated using a mucosal membrane preparation. TGF-alpha inhibited specific binding of [125I]EGF to its receptor, but the IC50 for TGF-alpha was at least 100 fold greater than that observed for unlabeled EGF. Cross-linking studies revealed no attachment of [125I]TGF-alpha to EGF-receptor size components, and the unlabeled TGF-alpha was only weakly effective in inhibiting cross-linking of [125I]EGF to the 170 kDa receptor. However, when the cytosolic fraction was reconstituted with the membrane preparation, an enhancement in binding of [125I]TGF-alpha to the EGF receptor occurred in a manner dependent on the concentration of cytosolic protein. Hence the binding characteristics of TGF-alpha to the EGF receptor in gastric mucosa are different from those for EGF.     

FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte

Although it has been shown that granulosa cells regulate the growth and meiotic maturation of mammalian oocytes, there is little evidence of a role for the oocyte in the differentiation or function of granulosa cells. To test the hypothesis that the oocyte participates in the regulation of granulosa cell function, oocytes were removed from isolated oocyte-cumulus cell complexes by a microsurgical procedure and oocytectomized complexes were tested for their ability to undergo expansion in response to follicle-stimulating hormone (FSH). FSH increased the levels of intracellular cAMP, the activity of the hyaluronic acid-synthesizing enzyme system, and induced cumulus expansion in intact complexes. In contrast, FSH did not induce increased hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized complexes. Therefore, the participation of the oocyte is necessary for the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in vitro in response to stimulation with FSH. FSH induced the elevation of intracellular cAMP to the same extent in both intact and oocytectomized complexes and the cAMP analog 8-bromo cyclic adenosine monophosphate (8Br-cAMP) did not stimulate expansion in oocytectomized complexes. Therefore, the influence of the oocyte on cumulus expansion occurs downstream from the elevation of cAMP levels in the cumulus cells. Epidermal growth factor (EGF), a potent stimulator of cumulus expansion in intact complexes, which probably acts by a mechanism at least initially different from FSH, failed to stimulate cumulus expansion after oocytectomy. Next, oocytectomized complexes were either cocultured with germinal vesicle stage denuded oocytes or cultured in medium conditioned by denuded oocytes. In both cases, FSH or EGF stimulated expansion by oocytectomized complexes. The degree of expansion was directly correlated to the number of oocytes used to condition the medium. Contact between the oocyte and the cumulus cells is not necessary for cumulus expansion. Rather, a factor(s) secreted by the oocyte is necessary for the cumulus cells to undergo expansion in response to either FSH or EGF. FSH did not induce expansion of oocytectomized complexes in media conditioned by various somatic cells such as granulosa cells, fibroblasts, and Sertoli cells; by a mixed population of male germ cells; or by spermatozoa. This suggests that the expansion enabling activity is specific to the oocyte. These results demonstrate that the oocyte participates in the regulation of cumulus cell function.