An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Effect of retinol and retinoic acid on testosterone production by rat Leydig cells in primary culture

Adult rat Leydig cells, purified by Percoll density gradient centrifugation, were used to determine the effect of retinol and retinoic acid on steroidogenesis. It was found that both retinoic acid and retinol stimulated testosterone production. Although retinol was less potent than retinoic acid, retinol had the greater efficacy. When these retinoids were tested in the presence of a maximal dose of LH, it was found that retinol inhibited LH-stimulated testosterone synthesis whereas retinoic acid had no similar effect. These results demonstrate for the first time that retinol and retinoic acid have a direct effect on Leydig cell steroidogenesis in culture suggesting that retinoids play a role in the maintenance and regulation of Leydig cell function.     

Comparative role of retinol, retinoic acid and beta-carotene on progesterone secretion by pig corpus luteum in vitro

Quadruplicate wells of pig luteal cells were incubated for 24 h in the presence of different concentrations of retinol, beta-carotene (0, 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M) or retinoic acid (0, 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M). In addition, the responsiveness of luteal cells to LH challenge was also evaluated. Progesterone was assayed in the media. Cell viability was estimated using trypan blue exclusion and showed over 95% viability. In the presence of LH, progesterone content in the medium was increased by 7-fold. As compared to their respective controls, all concentrations of retinoic acid and beta-carotene increased progesterone content in the media. The highest level of stimulation was observed with 1 x 10(-6) M-retinoic acid (5-fold increase) and 1 x 10(-7) M-beta-carotene (10-fold increase). Only 1 x 10(-5) M-retinol stimulated progesterone secretion (over 3-fold). Therefore, retinol, retinoic acid and beta-carotene stimulate progesterone secretion by pig luteal cells in vitro.     

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Retinoids and cultured human fibroblasts : Effects on cell growth and presence of cellular retinoic acid-binding protein

We have examined the effects of retinoids on growth of cultured human skin fibroblasts from four individuals. Retinoic acid and retinol both produce a dose-dependent inhibition of growth in the four strains examined; retinoic acid was more potent than retinol in this respect. The growth inhibitory effect of retinoic acid is characterized by a decrease in the exponential growth rate, which is reversible upon removal of retinoic acid from the growth medium; the final saturation density, however, is not modified by retinoic acid treatment. No alterations of cell morphology, viability, or adhesiveness to substratum are induced by the retinoid concentrations utilized. The inhibitory effect of 10⁻⁶ M retinoic acid on cell growth is not affected by the concentration of fetal calf serum (FCS) in the medium. In all four human fibroblast strains examined, specific binding of [³H]retinoic acid to cytosol is present as determined by sucrose-density gradient centrifugation. Despite the effects of retinol on fibroblast growth, no cytoplasmic binding of [³H]retinol could be demonstrated in these cells.     

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10^?8 to 3 × 10^?6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10^?7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Further evidence in support of a short-loop feedback action of estrogen on ovarian androgen production

We have demonstrated previously an ability of estrogen to inhibit ovarian androgen production. We report here further evidence in support of this intraovarian short-loop feedback mechanism. Thecal cells from ovarian follicles of estradiol-17β (E)-treated rats demonstrated an enhanced capability of producing progesterone in response to LH $\text{in vitro}$. In contrast, testosterone production by the same thecal preparations was markedly inhibited by pretreatment with E, suggesting a selective inhibitory action of E at the level of the androgen-producing cells in the ovarian follicle. In a somewhat contrasting experiment in hypophysectomized rats, while simultaneous administration of purified follicle-stimulating hormone (FSH) antagonized an inhibitory action of E on ovarian progesterone production, treatment of the hypophysectomized rats with either E alone or concomitantly with E plus FSH still attenuated ovarian testosterone production by these animals in response to acute LH stimulation. These results are consistent with a direct inhibitory action of estrogen at the level of the ovarian C_17α-hydroxylase /C_17,20-lyase enzyme system.     

Steroid production in vitro by granulosa, theca, and luteal cells from goat ovaries

Basal progesterone (P4) production by isolated goat ovarian cells in vitro was in the order corpus luteum (CL) greater than granulosa (G) greater than theca (TH), while estradiol (E2) production was in the order TH greater than G greater than CL. In G cells, various concentrations (0.01 to 100 micrograms/ml) of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) increased P4 and E2 secretion. Testosterone (T, 10(-9) to 10(-5) M) produced dose-dependent increases in P4 and E2 secretion. Testosterone and LH together had an additive effect on E2 secretion. The combined effect of the lower (less than 10(-6) M) concentrations of T and LH on P4 production was marginally higher than either agent alone, but the increase was statistically insignificant; at higher concentrations of T (10(-6) and 10(-5) M) in combination with LH, P4 secretion was similar to that with LH alone, but was significantly (p less than 0.01 and less than 0.001, respectively) less compared to that with T alone. Follicle-stimulating hormone and T together produced a synergistic effect on E2 and an additive effect on P4 production. In TH cells, a dose-dependent increase in P4 and E2 production was observed with LH and hCG, but the effect of FSH was not significant. Testosterone produced a dose-dependent increase in P4 and E2 secretion. Testosterone and LH together induced higher steroid production than either agent alone. However, the increase was not statistically significant compared to T alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K (retinol-) (β) (-LG?)=?6.4 (±?.6)?×?10(6)?M(-1) and K (retinoic acid-) (β) (-LG?)=?3.3 (±?.5)?×?10(6)?M(-1). The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid-protein complexes with the free binding energy of -8.11?kcal/mol for retinol and -7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52-51% and a major increase in turn structure from 13 (free protein) to 24-22%, in the retinoid-β-LG complexes, indicating a partial protein destabilization.     

Effects of retinoid beta-glucuronides and N-retinoyl amines on the differentiation of HL-60 cells in vitro

Retinoyl beta-glucuronide and retinyl beta-glucuronide, which are naturally occurring water-soluble metabolites of vitamin A, induce the granulocytic differentiation of HL-60 cells in vitro, as evidenced by an increased reduction of nitroblue tetrazolium. The relative effectiveness of various retinoids in differentiation is retinoic acid greater than retinoyl beta-glucuronide greater than retinyl beta-glucuronide. Under the selected assay conditions, retinol, hydroxyphenyl-retinamide, retinamide, and N-retinoyl-phenylalanine are essentially inactive in differentiation. At concentrations of retinoids from 10(-9) to 10(-5) M, cell viability was best with the retinoid beta-glucuronides and retinamide, less with retinoic acid and retinol, and poorest with the N-retinoyl aromatic amines. Cellular growth was depressed only slightly by retinyl beta-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl beta-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl beta-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid beta-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.     

Effect of cyanoketone on follicle-stimulating hormone (FSH) induction of receptors for FSH in granulosa cells of the rat

Follicle-stimulating hormone (FSH) enhances the conversion of testosterone or androstenedione into estradiol by stimulating the aromatase enzyme system. Estradiol also enhances FSH action. Thus, a synergistic action of FSH and estradiol may be required for maturation of ovarian follicles. We hypothesized that estradiol may be required for FSH action. Thus, blocking estrogen synthesis should prevent FSH-induced increases in FSH receptors. Hypophysectomized rats were divided into five groups and injected subcutaneously with: 1) saline, 2) cyanoketone (0.05 mg, blocks the conversion of pregnenolone to progesterone), 3) ovine FSH (oFSH, 200 micrograms), 4) cyanoketone then oFSH 24 h later, or 5) cyanoketone plus estradiol [or progesterone, testosterone, promegestrone (R5020), dihydrotestosterone (DHT), 2 mg], then FSH 24 h later. Animals were decapitated at 0, 12 or 24 h after an injection of oFSH, and membrane receptors for FSH and luteinizing hormone (LH), plus nuclear receptors for estradiol from granulosa cells, were measured. LH receptor levels were increased only after administration of FSH and estradiol. At 0 and 24 h, numbers of FSH or estradiol receptors were similar in saline- and cyanoketone-treated animals. FSH alone increased (P less than 0.01) FSH and estradiol receptors 3-fold and 4-fold, respectively, over controls by 12 and 24 h. Cyanoketone prevented these increases in FSH and estradiol receptors. Estradiol replacement fully reversed the effects of cyanoketone on FSH action. Replacement with progesterone and testosterone was able to only partially restore levels of FSH receptors; however, estradiol receptor numbers were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

Interactions between inhibin, oestradiol and progesterone in the control of gonadotrophin secretion in the ewe

Experiments were carried out to test the hypothesis that inhibin and oestradiol act synergistically to inhibit the secretion of FSH, to test for effects of progesterone, and to compare the FSH and LH responses to ovarian feedback. In Exp. 1, with 11 ovariectomized and 12 intact Romanov ewes during the anoestrous season, doses of oestradiol (administered by means of subcutaneous implants) that restored normal LH pulse frequencies were insufficient to restore normal concentrations of FSH. In Exp. 2, with 48 ovariectomized Welsh Mountain ewes during the breeding season, a factorial design with 4 ewes per cell was used to assess the responses in LH and FSH to 3 doses of oestradiol (s.c. implants) and 4 doses of bovine follicular fluid ('inhibin', 0.2-1.6 ml s.c. every 8 h). This was done initially in the absence of progesterone and then after 7 days of treatment with progesterone (s.c. implants). Analysis of variance revealed a significant synergistic interaction between oestradiol and inhibin on the plasma concentrations of FSH. Progesterone had little effect. In contrast, there was a significant synergistic interaction between oestradiol and progesterone on the concentrations of LH. 'Inhibin' also inhibited LH secretion but this effect was independent of the two steroids. We conclude that there are basic differences in the way that ovarian feedback acts to control the secretion of LH and FSH in the ewe. FSH secretion appears to be primarily controlled by the synergistic action of oestradiol and inhibin on the anterior pituitary gland, while the secretion of LH is inhibited during the follicular phase by an effect of oestrogen at pituitary level and during the luteal phase by the synergistic action of oestradiol and progesterone at the hypothalamic level. Inhibin, or another non-steroidal factor in follicular fluid, may also play a minor role in the control of LH secretion.     

Identification of LH/hCG receptors in rabbit uterus

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.