Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED(50) (dose that produces a response that is 50% of the maximum response) 60+/-5.7ng/ml and 8+/-1.8ng/ml (mean+/-s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/10(6) cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific (125)I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4 degrees C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5'-[beta,gamma-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50-60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.     

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.     

Immunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation.

Specific anti-(lutropin receptor) antibodies were produced by immunizing rabbits with lutropin receptor purified from pseudopregnant rat ovary. The anti-receptor serum at 1:100 dilution together with anti-(rabbit gamma-globulin) serum immunoprecipitated 70% of 3H-labelled, purified lutropin receptor and 42% of 125I-chorio-gonadotropin-receptor complex. The antiserum inhibited hormone binding to rat ovarian particles. Pseudopregnant rat ovarian particles were labelled with periodate/NaB3H4 and solubilized with Triton X-100. The Triton X-100 extract was subjected to immunoprecipitation using the anti-receptor serum. When the immunoprecipitate was dissolved and analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate under reducing conditions followed by fluorography, a receptor polypeptide with an apparent Mr 95000 was detected. A receptor down-regulating dose of choriogonadotropin was injected into pseudopregnant rats and their ovaries were removed and homogenized 4 days later, and analysed for immunoprecipitable receptors as above. No receptor molecules were found. Accordingly, the lutropin receptor molecules actually disappear rather than merely become masked from hormone during homologous down-regulation.     

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

Lutropin receptors from male and female tissues: different responses to a lutropin receptor binding inhibitor.

An inhibitor for lutropin receptor site binding (LH-RBI), which strongly inhibited the binding of 125I-labeled ovine lutropin ([125I]oLH) to ovarian LH receptors, did not inhibit the [125I]oLH binding to testicular LH receptors. Preincubation of the LH-RBI with [125I]oLH did not affect the binding of preincubated ]125I]oLH to ovarian LH receptors. No inhibition of [125I]oLH binding to testicular LH receptors was observed even uhen the concentration of LH-RBI was significantly increased or when the testicular LH receptors uere first incubated with LH-RBI prior to the addition of [125I]oLH and a second incubation. Scatchard analysis revealed that the dissociation constant of [125I]oLH binding was essentially the same in the presence or absence of LH-RBI. The results suggest that: (i) the lutropin receptor of ovaries, but not of testes, has a specific LH-RBI binding site in addition to the lutropin binding site, and (ii) the binding of the LH-RBI produces an "allosteric" type of inhibition to the binding of lutropin at the lutropin binding site.     

Comparison of the thermal stability characteristics of native and deglycosylated ovine pituitary lutropin

The receptor binding, immunological and biological activities of native ovine lutropin were almost completely eliminated when aqueous solutions of the hormone were kept in a boiling water bath for 30 or 60 min. Similar exposure of chemically deglycosylated lutropin revealed that this preparation was relatively more stable to heat treatment. The conformational features of deglycosylated lutropin required for receptor binding and immunological activity were significantly retained after thermal treatment. The heated deglycosylated lutropin solution still retained its ability to antagonize cyclic AMP accumulation stimulated by the native hormone in rat testicular interstitial cells. Specificity of receptor (lutropin) binding or inhibitory activity was not lost by heating of deglycosylated lutropin as revealed by lack of an effect in follitropin radio-receptor assays.     

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.     

A Leydig cell tumour: a model for the study of lutropin action.

The properties of cells isolated from a Leydig cell tumour have been compared with normal rat testis Leydig cells. These cells were found to be similar in the following respects: 1. Lutropin-stimulated cyclic AMP and testosterone production. 2. Lutropin-activated protein kinase activity followed by phosphorylation of endogenous proteins of mol. wts. 57,000and 14,000. 3. Parallel lutropin dose vs. response curves for phosphorylation of the endogenous proteins and for testosterone production. 4. Two forms of isoenzyme, cyclic AMP dependent protein kinase, present. They differed mainly with respect to the lutropin-stimulated testosterone production, which was much lower in the tumour cells compared with the normal adult testis Leydig cells (4.6 +/- 1.1 and 114 +/- 16 ng testosterone/10(6) cells per 2 h, respectively). However, the lutropin-stimulated steroid production in the tumour cells was quantitatively comparable with the normal rat Leydig cell when the metabolism of pregnenolone in intact cells and mitochondria was inhibited by addition of SU-10603 and/or cyanoketone. It is concluded that the Leydig cell tumour used in this study can be used to investigate certain aspects of lutropin action where large quantities of cells are required.     

Estradiol-17 beta inhibition of lutropin and dibutyryl cyclic AMP induced steroidogenesis in intact bovine luteal cells. Absence of effect on induced protein phosphorylations

Estradiol-17 beta is known to inhibit in a dose dependent manner the lutropin-induced stimulation of progesterone synthesis in luteal cells without affecting the intracellular cyclic AMP increase produced by the hormone. The hypothesis that this inhibitory action could involve an inhibition of the cyclic AMP dependent phosphorylation of cytosolic proteins was investigated by using incubations of selected small bovine luteal cells. Doses of 10 and 100 micrograms/ml of estradiol-17 beta inhibited respectively 60 and 90% of the progesterone synthesis induced by lutropin as well as by dibutyryl cyclic AMP in small bovine luteal cells. At the concentrations of 10 and 100 micrograms/ml, estradiol-17 beta was unable to affect the cyclic AMP dependent protein kinase activation induced by lutropin. At the concentration of 10 micrograms/ml the steroid was without effect on the lutropin or dibutyryl cyclic AMP induced protein phosphorylations. However 100 micrograms/ml of estradiol-17 beta seemed to produce a slight inhibition of the induced protein phosphorylations.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Modulation of testicular lutropin receptors in the developing male rat

In the developing male rat around 40 days of age, the testis appears to contain the maximum amount of lutropin receptors per unit weight. During this period, circulating levels of testosterone markedly increase without the concomitant major surges in lutropin levels. The increased sensitivity and responsiveness of tests to basal levels of circulating lutropin during this period is accompanied by enhanced serum prolactin levels suggesting that this hormone may be involved in this process. The finding that prolactin treatment of pubertal rats for 3 days induced the formation of more testicular lutropin receptors supports the above premise. However, shortterm immunoneutralisation of endogenous prolactin did not significantly alter the specific binding of [ 125 I ]-labelled lutropin to testicular membranes. Interestingly, during development, a close correction exists between receptor occupancy and capacity of the tissue to bind labelled lutropin. The apparent dissociation between serum lutropin levels, on the one hand and tissue occupancy and free receptor contents on the other, suggests that factors other than lutropin (presumably prolactin) are involved in the modulation of the sensitivity and the responsiveness of the testis to lutropin during early development.     

Effects of beta subunit acylation on lutropin receptor site binding.

The effects of various modifications on the beta subunit of lutropin have been studied using the binding characteristics of the reconstituted hormone in the rat testicular radioligand assay. Conditions for iodinating lutropin and lutropin derivatives were determined which resulted in 15 per cent specific binding when tested immediately and retention of 6 to 7 per cent specific binding even after storage for 6 months. Acetimidinyl, acetyl, and carbamyl derivatives of the beta subunit were prepared and combined with unmodified alpha subunit to form reconstituted lutropin. Modification of the beta subunit was shown to have no effect on the time course of binding to testicular receptors or, with one exception, on the extent of receptor saturation. Very high concentrations of lutropin reconstituted with acetylated beta subunit showed an anomalous binding behavior. Scatchard plots of the binding data support the view that the native hormone has a unique receptor affinity which is irreversibly disrupted by separation of subunits and that derivatization of the beta subunit does not alter this parameter further. These data also suggest that there are no significant differences in the amino groups modified on the beta subunit. Competition and preincubation tests for receptor sites that reacted only with modified lutropin and not with the native hormone were negative.     

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of rat lutropin

To elucidate a possible role of sialic acid moiety in the electrical heterogeneity of rat pituitary lutropin, seven components separated were individually treated with neuraminidase. Some intermediates with isoelectric points corresponding to the native components were concomitantly seen at the serial stages of the enzyme treatment. All the treated components showed an isoelectric point of about 10.0 which was the same to the isoelectric point of one of the seven components. Desialylation of the components with less biological activity caused enhancement of the in vitro cyclic AMP producing- and testosterone producing-activities as well as the binding activity to the receptor. It is concluded that the number of sialic acid moiety in lutropin is responsible for the charge heterogeneity and the biological potency of the hormone.     

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP.

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Dissociation of lutropin-induced loss of testicular lutropin receptors and lutropin-induced desensitization of testosterone synthesis.

The relationship between changes in testicular lutropin receptors, as measured by specific binding of 125I-labeled human chorionic gonadotropin, and testosterone synthesis in response to lutropin (testicular responsiveness) was studied in intact and hypophysectomized rats. Administration of a single 200-microgram dose of ovine lutropin to intact rats results at 3 days in a 58% decrease in lutropin receptors associated with a parallel decrease in testicular responsiveness. A single 30-microgram dose of lutropin to intact rats resulted in a comparable decrease in lutropin receptors with a transient increase in testicular responsiveness. Rats receiving twice-daily injections of 15 microgram lutropin for 10 days exhibited a 48% decrease in lutropin receptors by day 3 which persisted during the 10-day treatment period, but was accompanied by a progressive increase in testicular responsiveness to lutropin. Hypophysectomy resulted in an 80% loss of receptors and a 72% loss in responsiveness 7 days after surgery. Daily treatment with lutropin initiated immediately following surgery resulted in a further dose-dependent decrease in lutropin receptors and a dose-dependent increase in testicular responsiveness. Loss of lutropin receptors was not due to occupancy of the receptor by exogenous lutropin. These studies demonstrate a dissociation between the negative regulation of lutropin receptors and testicular responsiveness to lutropin. Furthermore, the studies in hypophysectomized rats indicate that lutropin is the only hormone essential for maintenance of steroidogenesis and that this is independent of lutropin receptor concentration.     

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion and cellular cyclic AMP levels. Cultured ovine and rat anterior pituitary cells show markedly different responses

Human pancreatic growth hormone-releasing factor (GRF-44-NH2) stimulated growth hormone (GH) secretion and intracellular cyclic AMP levels in cultured pituitary cells from both sheep and rat. Somatostatin (SRIF), over a wide range of doses and time, showed no significant effect on the elevated cyclic AMP levels in sheep cells, but did block the GH release in a dose-dependent manner. In rat cells, however, SRIF inhibited GRF-stimulated cyclic AMP levels by 75% maximum (still 8-fold greater than the basal levels) and GH release to almost half the basal value. We conclude that somatostatin inhibits GRF-elevated cyclic AMP levels in rat pituitary cells but not in sheep cells.     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.