Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

Binding of native, glucosamine-labeled, luteinizing hormone to Leydig cells.

A method for obtaining pituitary glycoprotein hormones labeled either in the protein backbone or in the carbohydrate moiety, by incubation of pituitary slices in the presence of radioactive leucine or glucosamine, has been developed. This report describes the subsequent purification of the “native” labeled luteinizing hormone from the incubation medium and its use in binding studies with testicular tissue. The purified radioactive luteinizing hormone specifically bound to Leydig cells could be displaced with excess human chorionic gonadotropin, but was unaffected by excess follicle stimulating hormone. Binding data indicated that $\text{K}{}_{\mathrm{d}}\text{= 5.2 × 10}{}^{\mathrm{?9}}\text{m}$ and approximately 6 × 10⁴ binding sites per cell.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

Phospholipid methylation by intact rat Leydig cells

Phospholipid methylation by intact Leydig cells was investigated by determining the incorporation of radioactivity from [3H-methyl] methionine into phospholipids. Leydig cells incorporated significantly more radioactivity into phospholipids than did unpurified testicular cells, non-Leydig testicular cells, or red blood cells. Approximately 40% of the radioactivity was found in phosphatidylcholine, indicating that the methyltransferase pathway for the synthesis of this phospholipid is highly active in rat Leydig cells. Addition of luteinizing hormone to cells preloaded with [3H-methyl] methionine did not alter the rate of phospholipid methylation. However, phospholipid methylation by Leydig cells desensitized by the injection of human chorionic gonadotropin 1 to 7 days previously was reduced by approximately 60%. Inhibition of phospholipid methylation to 75% of normal with homocysteine thiolactone did not affect luteinizing hormone-stimulated androgen production. Further inhibition of phospholipid (and protein) methylation by treatment with homocysteine thiolactone and 3-deazaadenosine significantly reduced luteinizing hormone-stimulated androgen production. The results of this study demonstrate that the methyltransferase pathway for the synthesis of phosphatidylcholine is highly active in intact Leydig cells but is reduced in desensitized Leydig cells. There does not appear to be a close association between the activity of this pathway and the ability of luteinizing hormone to acutely stimulate androgen production.     

Optimizing testosterone production by purified adult rat Leydig cells in vitro

Summary We sought to establish conditions that increased the duration of testosterone production by fully differentiated adult rat Leydig cells in primary culture. A freshly isolated suspension of highly purified adult rat Leydig cells produced 83 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated in Medium 199 in a 1.5 ml microfuge tube with shaking for 3 h with a maximally stimulating concentration of ovine luteinizing hormone (LH). Unfortunately, adult rat Leydig cells that were allowed to attach only to a plastic culture dish flattened out, and testosterone production diminished rapidly. Leydig cells in Dulbecco's modified Eagles' medium-Ham's F12 (1∶1; vol/vol) containing Cytodex 3 beads pre-equilibrated in culture medium containing fetal bovine serum attached to the beads and remained viable, but produced only 30 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated for 24 h with similar stimulation. Leydig cells similarly cultured and maximally stimulated with LH, responded to bovine lipoproteins (<1.222 g/ml) producing 105 ng of testosterone/10⁶ Leydig cells·h⁻¹ when incubated with 1 mg/ml bovine lipoprotein. Therefore, lipoproteins maintain the steroidogenic capacity of purified adult rat Leydig cells in primary culture for 24 h. Paper presented at the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation. This research was supported in part by the National Institutes of Health (grant HD-07204), The Population Center (grant HD-06268), and EPA cooperative agreement (CR81-2765), an NSF equipment grant, and a Mellon Foundation Postdoctoral Fellowship for Gary Klinefelter. Although the research described herein has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement (CR81-2765) to the Division of Reproductive Biology at Johns Hopkins University, it has not been subjected to the agency's peer and policy review, and therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

Regulation of testicular LH receptors by homologous hormone: in vitro studies on receptor occupancy and receptor loss.

A method is described which makes use of 4M MgCl₂ to dissociate the testicular luteinizing hormone-receptor complex without altering either the binding capacity or binding affinity of the receptor. Using this method, it was demonstrated that $\text{in vitro}$ incubation at 4° of decapsulated rat testes with various concentrations of luteinizing hormone or with human chorionic gonadotropin resulted in a reduction in binding capacity. This reduction of binding capacity could not be completely accounted for by occupation of receptors by homologous hormone, suggesting that receptors were lost. Thus negative regulation of LH receptors by LH and hCG was observed. The reduction in LH binding capacity was specific for LH and hCG, dose dependent and time related. FSH, prolactin and growth hormone did not exert the same effect.     

Pharmacological Approaches to the Identification and Classification of Myometrial Oxytocin Receptors

Abstract

Three binding sites have been recently reported on the rat, calf and sheep myometrial cells, with dissociation constants (K_d) roughly 10^?9, 10^?7 and 10^?5 mol/1. Oxytocin receptor for the uterotonic response in vitro was identified pharmacologically: 1) The analysis of dose-response curves has been based on a partial irreversible inhibition of the receptor on isolated rat uterus by the method of Furchgott and Bursztyn, and by the newly suggested plotting of K_d vs. maximal response for an increasing degree of irreversible inhibition. 2) pA₂-values (reflecting K_d) of structural analogues of oxytocin acting as competitive inhibitors of the parent hormone have been analysed according to Free and Wilson. Contribution of side chains in individual positions of the nonapeptide chain were computed, tested on additivity and then used for back-computation of a K_d for oxytocin. Results of all experiments reveal a K_d for oxytocin receptor (rat uterus in vitro) of 2 × 10^?7 mol/l. Possible endocrine functions of the high and low affinity sites have not been clarified as yet.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.     

Tumor necrosis factor-alpha binding in porcine primary stromal-vascular cell cultures

Summary The binding characteristics of tumor necrosis factor-α receptors (TNFRs) in primary stromal-vascular cultures from fat tissue of 7-d-old pigs were analyzed. Cells were plated and maintained in 10% fetal bovine serum from day 0 to day 3 and then switched to serum-free medium from day 3 to day 6 to induce lipid filling. On days 3 and 6 of culture, some of the cells were lysed for ligand and immunoblotting and the remainder subjected to competitive and inhibitory-binding assays. Media from day 6 of culture were subjected to ligand and immunoblotting. Competitive binding analysis showed one-site bindings, with IC₅₀s in the nanomolar andK _ds in the picomolar ranges, that were not signficantly different at both time-points of measurement. However, the B_max decreased significantly with differentiation. Preincubation with antibody against TNF receptor type 1 (TNFR1) or TNF receptor type 2 reduced the specific binding by 95 and 15%, respectively, suggesting a dominating role of TNFR1 in¹²⁵I-labeled TNFα (¹²⁵I-TNFα) binding. This was further supported by ligand blotting of cell lysates. Ligand and immunoblotting of cell lysates indicated that TNFα utilizes both types of surface receptors and their isoforms which were not modified during differentiation. Ligand blotting of media revealed soluble receptors with high M_r implying the formation of multimers. Immunoblotting suggested the presence of both types of TNFRs, but a greater abundance of soluble TNFR1. Also, it indicated the additional formation of smaller oligomers from both types of soluble receptors suggesting higher affinity of larger multimers for¹²⁵I-TNFα.     

The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells

Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

Prolactin modulates steroidogenesis by rat granulosa cells: II. Effects on estradiol

The effects of prolactin on secretion of estradiol by rat granulosa cells obtained at two stages of differentiation and cultured under various conditions were determined. Relatively undifferentiated granulosa cells were obtained from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats and cultured in serum-free medium or with 10% serum. More highly differentiated granulosa cells were obtained on the morning of proestrous from the preovulatory follicles of immature rats in which an estrous cycle had been induced with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% serum. Cells were cultured for 3 days with graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with follicle-stimulating hormone (FSH; 300 ng/ml), testosterone (0.5 microM), or FSH + testosterone. In control cultures (no prolactin) the relatively undifferentiated granulosa cells from HPX rats secreted negligible quantities of estradiol except when both FSH and testosterone were supplied. Prolactin alone or in combination with FSH or testosterone had no effect on estradiol secretion, but prolactin in combination with FSH + testosterone significantly decreased secretion in a dose-dependent fashion. This set of prolactin treatments was applied in both serum-free medium and medium containing 10% serum, with similar results under both culture conditions. The inhibitory effects of prolactin appeared to be reversible if cells were cultured with prolactin for only 1 day, but were not reversed if cells were cultured with prolactin for 2 or 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)