Morphology of the release of vitamin A-containing lipid droplets by perisinusoidal lipocytes in the rat liver

Vitamin A-containing lipid droplets in perisinsoidal lipocytes (or fat-storing cells) of rat liver were found to be exocytotically released from the cells in the form of a 'lipid droplet-protein complex' following intraportal injection of retinol (17, 33, 67 or 100 micrograms). Intraportal injection of retinol produced varied numbers and sizes of vacuoles with a limiting membrane in perisinusoidal lipocytes. The substance within the vacuoles exhibited a meshwork-like configuration in sections from slices incubated in a medium for revealing acid phosphatase activity or the corresponding control medium, and was immunoreactive to retinol-binding protein and proteinaceous in nature. The occurrence and number of the vacuoles depended on the dosage of injected retinol, being greatest at a dosage of 100 micrograms of retinol and becoming progressively less at dosages of 67, 33 and 17 micrograms. The vacuoles appeared to be formed by vacuolization of cisternae of the rough endoplasmic reticulum. Little or no esterase activity was found in lipid droplets in perisinusoidal lipocytes before the intraportal injection of retinol, but after the injection lipid droplets which had fused with the vacuoles became strongly positive for this enzyme activity. This suggests that hydrolysis of retinyl esters may occur in the process of complex formation in perisinusoidal lipocytes.     

The mechanism of uptake of retinol by plasma-membrane vesicles.

The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.     

Treatment of the rat hepatic stellate cell line,PAV-1, by retinol and palmitic acid leads to a convenient model to study retinoids metabolism

The main site of vitamin A storage in the liver is the hepatic stellate cells (HSC). Involvement of HSC in vitamin A metabolism has mainly been studied using primary culture, which represents the most physiological model but technically suffers several drawbacks (yield, low reproducibility, etc.). To circumvent these problems, we have previously established and characterised an immortalised rat HSC line named PAV-1. This study aimed to investigate in PAV-1 and in primary HSC (i) the incorporation of retinol and its esterification, (ii) the cellular retinol-binding protein (CRBP) content, (iii) the acid retinyl ester hydrolase activity (aREH), (iv) the thermal susceptibility and (v) the lipid composition of the membranes, which may play a crucial role in retinol transport across cellular membrane. In routine conditions of culture, the rate of retinol esterification in PAV-1 was low (5.2%) compared to that obtained with primary HSC (69.9%). Retinol pre-treatment doubled this esterification rate (10.7%) and the CRBP content in PAV-1. The co-incubation with retinol and palmitic acid enabled PAV-1 to esterify retinol with a rate close to that of primary HSC (66.2% vs. 69.9%) and with similar retinyl ester profiles. aREH activity was higher in primary HSC than in PAV-1. Thermal susceptibility and phospholipid composition of membranes in PAV-1 treated cells were similar to those of primary HSC. In conclusion, our study shows that PAV-1 cells treated with retinol and palmitic acid is a sound and convenient model for studying vitamin A mobilisation, a fundamental physiological event occurring in HSC.     

Immunohistochemical localization of retinoid binding proteins at the materno-fetal interface of the porcine epitheliochorial placenta

Retinol and retinoic acid that are potent modulators of gene expression are vital for development and growth of the conceptus. Apart from being transported across the placenta, retinol and retinoic acid may also be active in the placenta per se. Three proteins involved in 1) serum transport of retinol (retinol binding protein [RBP]), 2) cellular transport and metabolism of retinol (cellular RBP [CRBP] I), and 3) retinoic acid (cellular retinoic acid binding protein [CRABP] I), respectively, have been located by immunohistochemistry during gestation in the porcine placenta. This is a diffuse epitheliochorial placenta composed of areolar-gland subunits, where transport of larger molecules takes place, and interareolar regions, where gas-exchange and trophoblast absorption of hemotroph occur. Immunoreactive-RBP (ir-RBP) as well as CRBP I (ir-CRBP) was detected in uterine glands and in areolar trophoblasts, suggesting that RBP-retinol is secreted by the glands and absorbed by the trophoblasts. Both proteins were present also at the interareolar regions, with ir-CRBP in both the uterine epithelium and the apposing trophoblasts, but ir-RBP only in the former. The localization of ir-CRABP was, in contrast, strictly limited to interareolar trophoblasts. Together these findings suggest that 1) the areolar gland subunits are important for transport of retinol and retinol-RBP, and 2) retinoid binding proteins are involved in the development and growth of the porcine placenta.     

Retinol to retinol-binding protein (RBP) is low in obese adults due to elevated apo-RBP

Elevated serum retinol-binding protein (RBP) concentration has been associated with obesity and insulin resistance, but accompanying retinol values have not been reported. Assessment of retinol is required to discriminate between apo-RBP, which may act as an adipokine, and holo-RBP, which transports vitamin A. The relations between serum RBP, retinol, retinyl esters, BMI, and measures of insulin resistance were determined in obese adults. Fasting blood (> or =8 h) was collected from obese men and women (n = 76) and blood chemistries were obtained. Retinol and retinyl esters were quantified by HPLC and RBP by ELISA. RBP and retinol were determined in age and sex-matched, nonobese individuals (n = 41) for comparison. Serum apo-RBP was two-fold higher in obese (0.90 +/- 0.62 microM) than nonobese subjects (0.44 +/- 0.56 microM) (P < 0.001). The retinol to RBP ratio (retinol:RBP) was significantly lower in obese (0.73 +/- 0.13) than nonobese subjects (0.90 +/- 0.22) (P < 0.001) and RBP was strongly associated with retinol in both groups (r = 0.71 and 0.90, respectively, P < 0.0001). In obese subjects, RBP was associated with insulin (r = 0.26, P < 0.05), homeostatic model assessment of insulin resistance (r = 0.29, P < 0.05), and quantitative insulin sensitivity check index (r = -0.27, P < 0.05). RBP was associated with BMI only when obese and nonobese subjects were combined (r = 0.25, P < 0.01). Elevated serum RBP, derived in part from apo-RBP, was more strongly associated with retinol than with BMI or measures of insulin resistance in obese adults. Investigations into the role of RBP in obesity and insulin resistance should include retinol to facilitate the measurement of apo-RBP and retinol:RBP. When evaluating the therapeutic potential of lowering serum RBP, consideration of the consequences of vitamin A metabolism is paramount.     

Extraction and recombination studies of the interaction of retinol with human plasma retinol-binding protein

Methods have been developed for the removal of retinol from human plasma retinol-binding protein (RBP), so as to form the retinol-free apoprotein, and for the recombination of apo-RBP with retinol to again form the holoprotein. Retinol is removed from RBP by gently shaking a solution of RBP with heptane under controlled conditions. During the shaking, retinol is gradually extracted from the RBP and into the heptane phase. The reassociation of apo-RBP with retinol is achieved by exposing a solution of apo-RBP to Celite coated with a thin film of retinol, followed by isolation of the RBP by gel filtration on Sephadex G-100. This procedure results in the recombination of apo-RBP with an amount of retinol almost identical with that previously removed by extraction. The two-phase extraction procedure was used to explore some of the factors which affect the interaction of retinol with RBP. The retinol-RBP complex was most stable in the lower portion of the pH range 5.6 to 10. The rate of removal of retinol from the RBP-prealbumin complex (the form in which RBP normally circulates in plasma) was markedly less than the rate of its removal from RBP alone. The interaction of retinol with RBP appears to be stabilized by the formation of the RBP-prealbumin complex. The recombination procedure was employed to examine the specificity of the binding of retinol to RBP, by determining whether compounds other than all-trans-retinol would effectively bind to apo-RBP. Apo-RBP did not bind cholesterol, but displayed a slight affinity for phytol. The affinity of RBP for beta-carotene was minimal, whereas both retinyl acetate and retinal were bound about one-third as effectively as all-trans-retinol. In contrast, retinoic acid bound to apo-RBP almost as effectively as did retinol. Each of two isomers of retinol, 13-cis and 11,13-di-cis-retinol, bound to apo-RBP to some extent. The 13-cis isomer appeared to bind somewhat less effectively than did the 11,13-di-cis isomer. The binding of retinol to RBP is highly but not absolutely specific.     

Effect of pentoxifylline on arachidonic acid metabolism,neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A₂ (PLA₂), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 M), it increased the quantity of stored lipids. Retinol and PTF (5 M and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA₂ activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE₂, but not of PGF₂. However, when in presence of retinol, we observed a slightly higher increase in PGE₂ and PGF₂ production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.     

Regulation of plasma retinol binding protein secretion in human HepG2 cells

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.     

Effect of retinol and retinoic acid on permeability,electrical resistance and phase transition of lipid bilayers

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K⁺, I^? and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.     

Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Retinoids and retinoid-metabolic gene expression in mouse adipose tissues

Vitamin A and its analogs (retinoids) regulate adipocyte differentiation. Recent investigations have demonstrated a relationship among retinoids, retinoid-binding-protein 4 (RBP4) synthesized in adipose tissues, and insulin-resistance status. In this study, we measured retinoid levels and analyzed the expression of retinoid homeostatic genes associated with retinol uptake, esterification, oxidation, and catabolism in subcutaneous (Sc) and visceral (Vis) mouse fat tissues. Both Sc and Vis depots were found to contain similar levels of all-trans retinol. A metabolite of retinol with characteristic ultraviolet absorption maxima for 9-cis retinol was observed in these 2 adipose depots, and its level was 2-fold higher in Sc than in Vis tissues. Vis adipose tissue expressed significantly higher levels of RBP4, CRBP1 (intracellular retinol-binding protein 1), RDH10 (retinol dehydrogenase), as well as CYP26A1 and B1 (retinoic acid (RA) hydroxylases). No differences in STRA6 (RBP4 receptor), LRAT (retinol esterification), CRABP1 and 2 (intracellular RA-binding proteins), and RALDH1 (retinal dehydrogenase) mRNA expressions were discerned in both fat depots. RALDH1 was identified as the only RALDH expressed in both Sc and Vis adipose tissues. These results indicate that Vis is more actively involved in retinoid metabolism than Sc adipose tissue.     

Uptake of retinol by cultured fibroblasts

Cultured fibroblasts of adult rats were used to determine whether they could take in retinol administered to the culture medium at physiological concentration. After the administration of retinol, cells were observed with a phase-contrast fluorescence light microscope (LM) and a transmission electron microscope (TEM). Retinol and retinyl fatty acyl esters (RFAE) stored in the cells were analyzed with high-performance liquid chromatography (HPLC). It was revealed that these fibroblasts could take in retinol in the medium at a concentration of 1 x 10(-7) M and store it in lipid droplets in the cytoplasm as retinyl palmitate and other RFAE.     

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol-RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.     

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.