Demonstration of follicle stimulating hormone (FSH) activity in hypophyseal extracts of various vertebrates by the response of the Sertoli cells of the chick.

In newly hatched cockerels, administration of ovine FSH (NIH-FSH-S10) caused hypertrophy of the Sertoli cells of the testis and consequently the increase in the diameter of the seminiferous tubule. A linear log-dose response relation was observed in a dose range between about 90 and 1000 μg of NIH-FSH-S10. Administration of ovine LH (NIH-LH-S16) did not induce significant increase in the diameter of the tubule. Simultaneous administration of LH with FSH neither potentiated nor inhibited the effect of FSH on the tubule diameter. Either chicken adenohypophyseal glycoprotein and a saline extract of acetonedried bullfrog adenohypophyses caused a dose-dependent widening of the seminiferous tubule but a crude extract of salmon hypophyses did not. Mean relative FSH potency was 0.76 × NIH-FSH-S10 in the chicken extract and 0.024 × NIH-FSH-S10 in the bullfrog extract.     

Onset of the response to chorionic gonadotropin in the chick embryo testis

The stage of development of the chick embryo testis when it begins to respond to gonadotropin stimulation was investigated. The testosterone secretion in vitro, measured by radioimmunoassay, was employed to evaluate the response to hCG in testis from 8 to 16 days of incubation. At 8 to 10 days of the chick embryo development, the testis secreted testosterone, but no increment in the steroid production has been observed after hCG treatment. On the contrary, at 12, 14, and 16 days a clear increase in testosterone secretion has been demonstrated when hCG was added to the culture medium. The absence of hCG response before 12 days of incubation agrees with the hypothesis of an early independence period between testis and adenohypophysis during embryonic development.     

Intratesticular secretion of a factor(s) with major stimulatory effects on Leydig cell testosterone secretion in vitro

As intratesticular communication between the Sertoli and Leydig cells must take place via testicular interstitial fluid (IF), we have tested whether this fluid contains a factor(s) which has effect on testosterone secretion by Percoll-purified Leydig cells in vitro. Addition of increasing concentrations of charcoal-stripped IF to Leydig cells caused dose-dependent stimulation of basal testosterone secretion and greatly enhanced the response to a maximally stimulating dose of hCG. These effects were first evident after 2 h of incubation and were progressive up to 24 h. Charcoal-stripped serum from the same donor animals also had some stimulatory effect on basal and, occasionally, on hCG-stimulated testosterone secretion, but the magnitude and pattern of this effect and its limited dose-dependence were very different from the effects seen with IF. The effects of IF and serum were not altered by the addition of an antiserum to LH. It is therefore concluded that testicular IF contains a factor(s), not derived from serum, which can alter Leydig cell testosterone responsiveness. This factor(s) was heat-sensitive with a MW of greater than 10 000 daltons. IF from the abdominal testes of adult rats which had been made unilaterally cryptorchid contained much higher levels of the stimulatory factor(s) compared to the contralateral scrotal testes, and this is of interest because of the well-documented alteration in Sertoli-Leydig cell interaction that occurs in this situation. These results provide the first direct evidence that one or more factors capable of altering testosterone secretion are produced within the testis of the normal adult rat, and raises the possibility that this factors(s) may be involved in the local regulation of the intratesticular testosterone levels.     

The dynamics of the steroidogenic response of perfused Xenopus testis explants to gonadotropins

The dynamics of androgen production by perfused Xenopus testis explants has been investigated under various gonadotropic stimulations. Androgens were measured in perfusate by radioimmunoassay. The behavior of hormones in the perfusion system was assessed. Pieces of testis were capable of producing androgens after more than 20 hr in perifusion. Amplitude and duration of the response to a gonadotropic stimulation were the same 3 and 10 hr after the beginning of the experiment. After stimulation by pituitary extract, hCG, or carp GTH, there was a rapid tenfold increase in androgen secretion with a maximum after 1.5 hr and then a slow decrease to a basal line level. A typical log dose-response curve was obtained with hCG and cGTH. After 6 hr continuous stimulation by pituitary extract, steroid secretion increased and reached a plateau 4 hr later. The decrease occurred only after the end of stimulation. The same amount of pituitary extract injected into the column at the rate of 4 min stimulations every hr gave the same type of response. As far as is known, the present data are the first on perifusion systems on the testes of lower vertebrates.     

Fibroblast growth factor enhances human chorionic gonadotropin synthesis independent of mitogenic stimulation in Jar choriocarcinoma cells

The Jar choriocarcinoma cell line was used as an in vitro placental cell model to determine the effects of polypeptide growth factors on hCG beta secretion. Epidermal and fibroblast growth factor (FGF) treatment of serum-free cultures stimulated hCG beta secretion 2.5- and 4.0-fold over basal serum-free control levels within a 15-h incubation period. Insulin-like growth factor I, nerve growth factor, and transforming growth factor-beta had no significant effect on hCG beta secretion. FGF at concentrations as low as 0.125 ng/ml significantly elevated medium hCG beta levels without increasing cell number or total cellular protein. FGF stimulation of secretion was not detectable until 2 h of treatment. Intracellular hCG beta remained constant (23%) relative to total hCG beta (cell plus medium) as total hCG beta increased 3-fold, suggesting that FGF stimulated de novo hCG beta synthesis. Insulin significantly augmented the FGF-induced hCG beta stimulation without stimulating hCG beta production itself.     

LH/hCG receptors and stimulation of testosterone biosynthesis in the rat testis: changes during foetal development in vivo and in vitro

The development of gonadotropin receptors to LH/hCG in the foetal rat testis from 14 to 20 days of gestation was monitored by quantitative binding assays using [125I]hCG and compared to testosterone secretion under basal and stimulated conditions in vitro. Specific hCG binding was first detected on day 15. Thereafter the binding increased gradually with advancement in gestational age and correlated with LH-stimulated secretion of testosterone in vitro. On day 18 of gestation the KA was 0.82 x 10(10) M-1 and the binding capacity was 0.57 fmoles per testis. No binding was detectable in the female gonads at this age. The differentiation of hCG receptors obtained in vitro was very low, although it was sufficient to give a full response to LH with testosterone biosynthesis. The results of the present study suggest that functional receptors do not appear before the capacity to synthesize testosterone is expressed and that their appearance is not dependent on factors extrinsic to the testis. However, additional factors could be necessary for a full development of the receptors.     

True hermaphroditism: from female to male endocrine status

True hermaphroditism was revealed by monthly intrascrotal bleeding in a 21-yr-old subject of male phenotype who had undergone surgical treatment for gonadal ectopy at the age of 7 yr. The presence of an ovary was demonstrated by the endocrine profile of an ovulatory menstrual cycle. Evidence for the presence of a testis was provided by a plasma testosterone increase after hCG administration (5000 IU/day for 3 days) and its spontaneous response to an endogenous preovulatory LH peak. Further endocrine studies revealed that both gonads were stimulated by endogenous gonadotropins. At surgery, a hemiuterus and an ovary with corpus luteum were found in the left hemiscrotum, and a testis and epididymis were found in the right hemiscrotum. After removal of the ovary, the subject passed from a predominantly female to a male endocrine status, which suggests that the endocrine secretion of the testis was inhibited by the negative feedback effect of ovarian steroids on gonadotropin secretion.     

Regulation of inhibin secretion by Sertoli cell-enriched cultures

Sertoli cells secrete a factor which has the same bioactivity as ovine testicular lymph inhibin: it selectively suppresses the secretion of FSH by cultured pituitary cells. We investigated the factors that acutely modulate the secretion of this inhibin by cultured Sertoli cells derived from immature rats. The secretion of inhibin was studied on day 7 of culture after a 24 h period of incubation in the presence or absence of steroids, gonadotrophins and foetal bovine serum, added alone or in various combinations. It could be demonstrated that aromatisable as well as non-aromatisable natural and synthetic androgens promote the secretion of inhibin in a dose-dependent way. FSH and pregnant mare serum gonadotrophin--at concentrations that clearly stimulate Sertoli cell aromatase activity--did not affect basal or androgen-stimulated production of inhibin. hCG was equally uneffective. The effect of androgens was not modified by the addition of an aromatase inhibitor but it was neutralized by the antiandrogen cyproterone acetate. Oestradiol-17 beta did not influence the secretion of inhibin whereas progesterone decreased it. Serum enhanced basal as well as androgen stimulated secretion of inhibin. It is concluded that androgens are the major factor which acutely stimulates the production of Sertoli cell inhibin.     

Localization and secretion of inhibin/activin subunits in the human and subhuman primate fetal gonads

Little is known about the ability of the fetal primate gonads to produce inhibin/activin. We investigated the presence of the alpha-, beta A-, and beta B-subunits of inhibin/activin in fetal human (16-23 weeks gestational age) and rhesus monkey (days 150-157 of gestation; term = 165 days) testes and ovaries by immunocytochemistry. The regulation of alpha-inhibin secretion by gonadotropins was studied in fetal testicular cultures. In the human fetal testis, alpha-subunit immunostaining was found in interstitial and intratubular cells, while beta A- and beta B-subunit immunostaining occurred in clusters of Leydig cells that were clearly demarcated from groups of Leydig cells that were immunonegative. In the late gestational monkey testis, the alpha-subunit was localized in tubular cells, and the beta B-subunit was present in the tubules and interstitium. Testicular cells from midgestation human testes secreted detectable immunoreactive alpha-inhibin in response to FSH and hCG stimulation; alpha-inhibin levels were significantly higher after hCG than FSH. In contrast, levels of alpha-inhibin secreted by rhesus monkey testicular cells were significantly increased by FSH, but not hCG. In the ovary, only weak beta B-subunit immunoreactivity was detected in granulosa cells of a few primary follicles from midgestational human fetal ovaries. In contrast, all three subunits were found in granulosa cells of numerous primary and secondary follicles in the late gestation rhesus monkey ovary. In light of recent evidence that inhibins/activins have actions on gonadal differentiation and growth modulation in vitro, as well as endocrine effects on the fetal pituitary, we propose that these proteins may have intragonadal and endocrine roles in human and subhuman intrauterine gonadal development.     

Studies on human chorionic gonadotropin secretion: effects of EGTA, lanthanum, and the ionophore A23187.

Human malignant trophoblast cells that secrete human chorionic gonadotropin (hCG) in culture were employed to assess the calcium requirement for hormone production. Cellular and secreted hCG was measured by radioimmunoassay. Cells cultured for 7 h in Ca2+- and Mg2-free medium or in Ca2+-free medium, secreted less hCG than cells cultured in medium containing Ca2+. Addition of ethylene glycol bis (beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), ethylenediamine tetraacetic acid (EDTA), or La2+ inhibited hCG release from the cells, but did not affect the amount of hCG in the cells. Inhibition of hCG release was dependent on time of incubation and on concentration of agent added. Inhibition of hCG secretion by EGTA was reversed upon removal of the EGTA from the culture fluid or by addition of equimolar Ca2+ to the fluid. These results demonstrated that divalent cations, probably Ca2+, are required for release and further synthesis of hCG. Addition of the divalent cation ionophore A23187 (0.1 to 10 muM) failed to increase hCG secretion by the malignant trophoblast, in contrast to the stimulatory effect of this agent in other secretory systems. Incubation of the cells for 15 to 60 min with 10 muM A23187 reduced hCG secretion, and this inhibition was reversed upon removal of the ionophore from the culture fluid. The studies with the ionophore supported other evidence indicating basic differences between hormone secretory mechanisms in the trophoblast compared to other endocrine tissues.     

Emergence of beta-adrenergic sensitivity in the developing chicken heart.

Muscle cells dissociated from 5-day embryonic chicken hearts showed dose-dependent increases in both chronotropic rates of contraction and cyclic AMP (cAMP) levels in response to epinephrine (EPI), an effect that could be blocked by beta-adrenergic antagonists. However, 2- to 2.5-day embryonic chicken myocardial cells, although similar to 5-day heart cells with respect to the organization of myofibrils, failed to respond to EPI either by increased rates of contraction or by elevated levels of intracellular cAMP. Development of beta-adrenergic sensitivity in 2- to 2.5-day cells did not occur even after several days of growth in culture. However, addition of an extract prepared from 11-day chicken embryos to 2- to 2.5-day muscle cell cultures at any point during in vitro growth resulted in the development of sensitivity to EPI as measured by increases in both the beating frequency and cAMP levels after a 48-hr incubation in embryo extract (EE). The basal level of cAMP in cells unresponsive to EPI is 5 times that in EPI-sensitive muscle cells from older hearts (5 days). The high basal level of cAMP in these cells is reduced to a level characteristic of cells from older hearts when treated with the EE. Once sensitivity was acquired, it was retained as a stable trait of the muscle cells in culture. Furthermore, EE-treated 2- to 2.5-day cells showed less reduction of positive chronotropy in response to multiple doses of EPI than did cells prepared from 5-day hearts. Fractionation of EE on Sephadex G-200 showed that the activity was present in the large-molecule fractions. It is concluded that the development of beta-adrenergic sensitivity can be induced in unresponsive heart cells cultured from 2- to 2.5-day embryos by a factor(s) in the EE that is not adsorbed on Sephadex G-200.     

Decreased Leydig cell responsiveness in the testicular feminized male rat.

The Stanley-Gumbreck pseudohermaphrodite or testicular feminized male (tfm) rat exhibits a decreased Leydig cell sensitivity to human CG (hCG) measured by androgen and cyclic-3',5',-adenosine monophosphate production in vitro. These changes were associated with an 80% reduction in the number of LH receptors in the tfm testis, when compared on the basis of equivalent amounts of testis particle protein or per 10(6) isolated Leydig cells. Androstenedione and not testosterone is the major androgen secreted by the tfm Leydig cell and androstenedione secretion is, therefore, a more appropriate end point than testosterone secretion for Leydig cell function in tfm animals. A dose of hCG (3 ng/2 ml) which elicited a near maximal response in androgen production from the decapsulated testes and Leydig cell suspensions of normals rats, did not significantly stimulate androgen production from Leydig cells of the tfm animals. A much higher dose of hCG (200 ng/2 ml) gave a response from the tfm Leydig cells which was comparable to that obtained with 3 ng from Leydig cells of normal littermates. This indicates that the small number of LH receptors on the tfm Leydig cell membrane are functional and that the reduction in receptor number results in a decrease in the sensitivity of response to LH rather than a reduction in the maximum steroid response.     

Influence of the thymus on steroidogenesis by rat ovarian cells in vitro

Thymic hormones and factors have been shown to modulate the function of other endocrine glands including the gonads. Absence of the thymus during development results in ovarian dysgenesis characterized by a decrease in the number of follicles and corpora lutea, bringing about severe changes in reproductive function. To examine whether thymic secretions might affect ovarian activity, whole dispersed ovarian cells obtained from immature rats pretreated with pregnant mare serum gonadotrophin were exposed to a thymus fraction of approximately 28 kDa and also to the media from incubated thymuses (TIM) and the conditioned media from cultured thymic reticuloepithelial cells (TCM). The thymic fraction caused a dose-dependent decrease in human chorionic gonadotrophin (hCG)-stimulated production of progesterone, oestradiol and testosterone, but had no effect on their synthesis in the absence of hCG. Similarly, hCG-induced production of these steroids was decreased by TIM and TCM. Progesterone secretion was the most markedly affected. These results suggest: (1) that the thymus contains a factor with a molecular weight of approximately 28 kDa which interacts with hCG in ovarian cells, (2) that the thymus can release active substances which modify steroid secretion by the ovary in vitro and (3) that the reticuloepithelial cells of the thymus are involved in the secretion of factors which modulate the stimulation by hCG of steroidogenesis in ovarian cells.     

Ethanol Modulates the Hormone Secretory Responses Induced by Epidermal Growth Factor in Choriocarcinoma Cells

Analysis of clinical data has implicated ethanol (EtOH) as an embryotoxic agent and as an agent that disrupts normal placental structure and function. Because epidermal growth factor (EGF) is an important regulator of placental function, we have studied the effects of EtOH on EGF-induced hormone secretion using JEG-3 choriocarcinoma cells that serve as a model for trophoblast cells. EtOH at physiological (5-100 m m ) concentrations modulated effects of EGF in a time and dose-dependent manner. EGF-induced P₄ secretion was increased by 20–100 mM EtOH after a 2-day pretreatment of cells with EtOH, but not after a 6-day pretreatment. Preincubation with 50 mM EtOH doubled the P₄ responses to 50 and 100 ng/ml EGF. Although a 2- or 4-day preincubation of cells with 10–50 mM EtOH increased the secretion of E₂ in response to 20 ng/ml EGF, a 6-day preincubation inhibited the secretory response to EGF. Pretreatment of cells with 10–50 m m , but not 100 m m EtOH for 2 to 6 days enhanced the human chorionic gonadotropin (hCG) secretory response to EGF. At 50 mm EtOH, the secretion of hCG in response to EGF was increased 2-fold. EtOH also increased basal hCG secretion in a dose-dependent manner between 10–50 m m EtOH. These results suggest that EtOH may modulate EGF-stimulated hormone secretion from cells of placental origin. Such alterations, if they occur in vivo, may impact on the function of the placenta and could potentially explain the pathophysiology of alcohol toxicity during pregnancy.     

Regulation of testosterone production in Leydig cells from fetal mice under dynamic conditions: effect of human chorionic gonadotrophin and 8-bromo-cyclic AMP

The temporal release of testosterone by Leydig cells from 18-day-old mouse fetuses in response to human chorionic gonadotrophin (hCG) and to 8-bromocyclic AMP (8-bromo-cAMP) was investigated under short-term incubation (180 min) conditions. A rapid and large increase in testosterone release was induced by a 5-min exposure to hCG (20 i.u./l) or 8-bromo-cAMP (10 mmol/l). The testosterone response of fetal Leydig cells to the two gonadotrophic stimuli was Gaussian in distribution with a peak value of testosterone by 15-20 min. Repeated exposure to hCG resulted in a reduced testosterone response but an increased accumulation of cAMP. The apparent resistance of fetal Leydig cells to hCG could not be overcome either by increasing the hCG concentration (to 2000 i.u./l) or by exposing the cells to 8-bromo-cAMP (10 mmol/l). Continuous exposure to hCG (200 i.u./l) divided into multiple small doses (each 8 i.u./l) induced testosterone secretion with different kinetic characteristics: a three-fold longer time-lag between hormone exposure and the peak value; a twofold greater testosterone response (P less than 0.001) and a gradual decrease of testosterone secretion. Oestradiol significantly reduced basal and hCG-stimulated testosterone production only at a high concentration (10 mumol/l). These results indicate that continuous or pulsatile exposure to hCG can induce refractoriness of fetal Leydig cells. The similarity between the actions of hCG and 8-bromo-cAMP on fetal steroidogenesis suggests that this rapid defect is not primarily due to a depletion of gonadotrophin receptors but results from disruption of regulatory mechanisms at the post-receptor level.     

Biphasic effect of gonadotropin-releasing hormone and its agonist analog (HOE766) on in vitro testosterone production by purified rat Leydig cells

GnRH and GnRH agonists have stimulatory and inhibitory effects on testicular testosterone secretion both in vivo and in vitro. To determine whether they are exerted directly on the Leydig cells and to explore the temporal relationships, we examined the effects of acute (3 h) and chronic (24-72 h) exposure of purified (greater than or equal to 80%) rat Leydig cells to GnRH and its agonist analog HOE766 (D-Ser-t-BU6,des-Gly-NH2 10LHRH ethylamide; Hoechst, Frankfurt, Germany) on testosterone production. GnRH and HOE766 enhanced basal testosterone secretion by freshly isolated or cultured Leydig cells. HOE766 was at least 100 times more potent than GnRH. However, exposure of Leydig cells to HOE766 for 24 h or longer lead to a significant reduction in hCG responsiveness without altering basal testosterone secretion. Both the stimulatory and inhibitory effects were dose related, with a maximal response elicited by 10(-9) M HOE766. HOE766 reduced Leydig cell sensitivity to hCG (ED50) stimulation, but did not alter the slope of the dose-response curves. Thus, GnRH and its agonist appear to have a dual and biphasic effect on the Leydig cells. Acute exposure stimulates basal testosterone secretion (and occasionally the hCG response), whereas chronic exposure decreases the response to hCG stimulation. These data provide additional evidence that GnRH has a direct effect on Leydig cell steroidogenesis.     

Endocrine testicular function in vivo and in vitro in infertile men.

In order to detect any possible Leydig cell dysfunction associated with male infertility, the endocrine capacity of the tests was investigated in vivo and in vitro in 21 infertile men. Plasma testosterone was determined before and after 3 days of hCG stimulation. Testicular tissue obtained by bilateral biopsies was subjected to (1) histological examination, (2) determination of basal testosterone concentration and (3) incubation with hCG. Patients were grouped according to histology. In vitro basal and stimulated testicular testosterone was similar in patients with normal histology, Sertoli-cell-only syndrome and spermatogenic arrest. Tissue from patients with Leydig cell hyperplasia showed 3-fold higher basal testosterone levels and a greater response to hCG. All patients had plasma testosterone levels and responses to hCG in the normal range. There was no significant correlation between the data obtained in vivo and in vitro, indicating that testosterone determinations in peripheral blood do not necessarily reflect the intratesticular situation. There was no evidence for gross abnormality in Leydig cell function accompanying disturbed spermatogenesis.     

8-Bromo-3',5'-adenosine monophosphate stimulates the endocrine activity of human cytotrophoblasts in culture

Cytotrophoblasts, purified from human term placentae, were cultured in the absence or presence of 8-bromo-cAMP or 8-bromo-cGMP. 8-Bromo-cAMP provoked a dose-dependent increase in the secretion of hCG and progesterone within 24 h. After 48 h, hCG secretion increased by more than 200-fold, and progesterone secretion increased nearly 5-fold. 8-Bromo-cGMP had no effect on hCG secretion. In culture in serum-supplemented medium, the mononuclear cytotrophoblasts aggregated and fused to form syncytia. This morphological transformation was not affected by 8-bromo-cAMP. Immunocytochemical studies of the alpha- and beta-subunits of hCG in control and 8-bromo-cAMP-stimulated cultures demonstrated that the cyclic nucleotide analog promoted the synthesis of both subunits in all cellular forms, including single mononuclear cells, cell aggregates, and syncytia. In serum-free medium, the cytotrophoblasts did not aggregate or form syncytia, yet they responded to 8-bromo-cAMP with an increase in hCG secretion. We conclude that the endocrine function of cytotrophoblasts can be stimulated by a cAMP-dependent mechanism which can be initiated independently of the formation of a syncytium.     

In vitro model of the first phase of testicular descent: identification of a low molecular weight factor from fetal testis involved in proliferation of gubernaculum testis cells and distinct from specified polypeptide growth factors and fetal gonadal hormones

The gubernaculum testis is the connective tissue organ that causes the testis to descend. How the process of testicular descent is regulated is not fully understood. Current hypotheses postulate that a nonandrogenic fetal testicular factor controls the first phase of descent, that is characterized by growth of the gubernaculum and transabdominal migration of the testis. When gonadal extracts from fetuses with ages corresponding to the first phase of testicular descent (50, 60, and 75 days) were tested on gubernacular cells, the growth stimulatory effect of testicular extracts exceeded the effect of both ovarian extract and fetal calf serum. Gonadal extracts from 80-, 90-, and 100-day-old fetuses showed only a minor sex difference. No sex difference or age-dependent changes were detected when fetal gonadal extracts were tested on murine 3T3 cells. Polypeptide growth factors (epidermal growth factor, insulin, fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-beta) were tested for growth stimulatory activity and had only minor effects on gubernaculum cells. Fetal testicular hormones (anti-Müllerian hormone, inhibin, and androgenic steroids) did not induce initiation of DNA synthesis at concentrations that are highly bioactive in typical target systems. When testicular samples were dialyzed, the high mol wt fraction (greater than 3500) had lower growth stimulatory activity in gubernaculum cells, but not 3T3 cells. Bioactivity of ovarian extracts and fetal calf serum was not diminished after dialysis. The low mol wt fraction (less than 3500) of testicular extract was distinctly stimulatory to gubernaculum cells but not 3T3 cells, and the low mol wt fraction of ovarian extracts did not stimulate growth in either cell type. It was concluded that the fetal porcine testis during the first phase of testicular descent contains low mol wt factor(s) to which gubernaculum cells and not 3T3 cells are responsive. The bioactive fraction probably contains the factor(s) that initiate testicular descent. We suggest the name descendin for this new activity.     

Effect of lipoxygenase products on choriogonadotropin secretion by cultured human choriocarcinoma JEG-3 cells pre- and post-stimulation with epidermal growth factor and a phorbol ester

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.