Possible role for arachidonic acid in the control of steroidogenesis in hen theca

Studies were conducted to evaluate if arachidonic acid (C20:4) could function as a second messenger within theca cells from the second largest preovulatory (F2) follicle from the ovary of the domestic hen. Arachidonic acid stimulated basal progesterone and androstenedione production, but inhibited LH-induced androstenedione production. The stimulatory effects of arachidonic acid were not altered by either cyclooxygenase or lipoxygenase pathway inhibitors (indomethacin and nordihydroguaiaretic acid, respectively), but were blocked by agents that prevented mobilization and/or efflux of calcium (TMB-8 and verapamil). The inhibitory effects of arachidonic acid on LH-stimulated steroidogenesis were determined to occur both prior and subsequent to cAMP formation. Fifty and 100 microM arachidonic acid attenuated LH- (10 ng) and forskolin- (0.2 microM) induced cAMP levels, and decreased androstenedione and estradiol production following treatment with 8-bromo-cAMP. Phospholipase A2 (PLA2) and the calcium ionophore, A23187, stimulated the release of 3H from theca cells prelabeled with [3H]arachidonic acid, and both PLA2 and the closely related fatty acid, eicosatrienoic acid (C20:3), could replicate the inhibitory effects of arachidonic acid on LH-stimulated androstenedione production. Finally, neither indomethacin nor nordihydroguaiaretic acid blocked the inhibitory effects of arachidonic acid on LH-promoted androstenedione production. We conclude that arachidonic acid can be released within theca cells in response to physiologic (PLA2) and pharmacologic agents (A23187), and accordingly, that it may act directly as a second messenger to modulate both basal and LH-stimulated steroid production.     

Decreased androstenedione production with increased follicular maturation in theca cells from the domestic hen (Gallus domesticus)

Collagenase-dispersed theca cells from the 3rd and 4th largest ovarian follicles (T3) were responsive to LH stimulation of both oestrogen and androstenedione production, whereas theca cells from the largest follicle (T1) failed to respond to the gonadotrophin stimulation. Similarly, 8-bromo cAMP and forskolin were more effective in stimulating oestrogen and androstenedione production in T3 than in T1 cells, indicating that post-receptor events were involved in the decreased LH responsiveness of T1 cells. The C17-20-lyase activity, as measured by conversion of [3H]17-hydroxyprogesterone to androstenedione, was greatly reduced in T1 cells as compared to T3 cells. The results demonstrate that a decrease in C17-20-lyase activity, in addition to a decrease in aromatase activity, contributes to the loss of LH-stimulated steroidogenesis in mature theca cells.     

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation

LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.     

Calcium,calmodulin, and the production of prostacyclin by cultured vascular endothelial cells

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell lysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.     

Inhibition of androgen and oestrogen production by clomiphene citrate in avian theca cells

Isolated theca cells (2 X 10(5)/ml) were pre-incubated for 1 h in the presence or absence of clomiphene citrate (10(-12)-10(-4) M). Ovine LH (50 ng/ml) was added and cells were incubated for an additional 3 h. A 50% inhibition of LH-stimulated androstenedione and oestrogen production was obtained with doses of 10(-8) M and 2 X 10(-7) M clomiphene, respectively. Furthermore, the effect of clomiphene on LH-stimulated androstenedione production was reversed by washing clomiphene from the cells before stimulation with LH. In subsequent experiments, the effects of clomiphene on C17-20-lyase and aromatase activities were examined. Conversion of [3H]17-hydroxyprogesterone to androstenedione was inhibited by 50% when theca cells were pretreated with 10(-5) M-clomiphene. In addition, conversion of testosterone to oestrogen by theca cells was inhibited in a dose-dependent manner by clomiphene, with 50% inhibition occurring at a dose of 5 X 10(-6) M. The results show that clomiphene treatment in vitro inhibits androgen and oestrogen production in theca cells by inhibitory effects on the activities of C17-20-lyase and aromatase. In addition to the widely-accepted effects of clomiphene on the hypothalamic-pituitary axis, the present findings add further support to the suggestion that clomiphene exerts direct effects on ovarian steroidogenesis.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma. Role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B4 (LTB4), leukotriene D4 (LTD4) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15 microM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB4, LTD4 and histamine. An antagonist of calmodulin, trifluoperazine (1-200 microM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC50 of this compound for inhibition of histamine was much lower (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Effects of calcium channel activators BAY-K8644 and CGP-28392 on steroidogenesis in granulosa cells of the domestic hen

The effects of calcium agonists BAY-K8644 and CGP-28392 on steroidogenesis was examined in chicken granulosa cells in short term incubation. BAY-K8644 (5-500 nM) and low doses of CGP-28392 (1-10 microM) failed to appreciably affect basal and LH-stimulated progesterone production whether tested in calcium free, low (0.05 mM) or high (3 mM) calcium containing medium. However, higher concentrations of CGP-28392 (50-250 microM) inhibited significantly (P less than 0.01) both basal and LH-stimulated steroidogenesis in a dose-related manner independently of extracellular calcium availability. The suppressive effect of CGP-28392 was manifest with submaximally and maximally stimulating LH doses. In additional experiments with non-hormonal agonists such as forskolin, dibutyryl cyclic AMP and kaurenol, BAY-K8644 and low CGP-28392 concentrations were again without effect on steroidogenesis. By comparison, higher CGP-28392 doses suppressed the stimulatory effects of all three agonists dose-dependently. These results demonstrate that, the calcium channel agonists are incapable of inducing a steroidogenic response in chicken granulosa cells. Since BAY-K8644 and CGP-28392 (low dose, 1-10 microM) failed to influence steroidogenesis in the dose range that induced maximal physiologic responses and calcium influx in a variety of cells, it is concluded that chicken granulosa cells lack the type(s) of channels specific for them. Hence the usefulness of BAY-K8644 and CGP-28392 as Ca2+ probes may be tissue-specific. The inhibitory effects of CGP-28392 appear to be non-specific.     

Second messenger pathways mediating chicken luteinizing hormone secretion from dispersed pituitary cells.

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Effect of calmidazolium (R 24571) on the electrical and contractile properties of smooth muscle cells in the guinea pig ureter

Calmidazolium in macromolecular concentrations inhibited the electric and contractile activity of smooth muscle cells (SMC). The concentrations causing a 50% inhibition of oscillations on the action potential (AP) plate were equal to 1 X 10(-6) microM, AP amplitude was 3 X 10(-6) microM and contraction amplitude was 1 X 10(-6) microM. Calcium ionophore A 23187/8 X 10(-7) microM, added to the normal Krebs solution, decreased rapid AP components amplitude and increased the contraction power of the isolated SMC strip by 62 +/- 9%. A 23187, though to a lesser extent, increased smooth muscle contractions during the action of calmidazolium. With combined use of A 23187 and calmidazolium, rapid AP components were depressed to a greater extent than each of them taken separately. The data obtained point to the presence of calmodulin or similar protein in SMC of the calcium channels.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.