Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period.

Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.

Localization of GATA2 in the nuclear and cytoplasmic regions of ovine conceptuses.

GATA transcription factors are emerging as critical regulators in trophoblast development and its gene regulation. The purpose of this study was to examine the expression and cellular localization of GATA2 in ovine conceptuses during the peri-implantation period. In Western blot analyses, GATA2 proteins were found in days 15, 17 and 21 ovine conceptuses (day 0=day of estrus). Using immunohistochemistry and immunofluorescence analyses, we found that GATA2 was localized in days 15, 17 and 21 ovine conceptuses, and more importantly, GATA2 protein was detected in both nuclear and cytoplasmic regions of the trophectoderm. To our knowledge, the present study is the first to demonstrate that GATA2 is localized in two cellular compartments of the trophectoderm in ovine and many other mammalian species, and suggests that the difference in GATA2 location plays a role in the regulation of down-stream genes during the early pregnancy period.

Expression and in situ localization of GATA4, 5 and 6†mRNAs in ovine conceptuses and uterine endometria during the peri-implantation period.

In vertebrates, six GATA transcription factors, GATA1 through GATA6, have been identified and GATA1-3 is known to be involved in hematopoietic developments, while GATA4-6 play roles in cardiac and endoderm developments. Recently, we and others have found that GATA2 and GATA3 found in the trophectoderm plays a role in gene expression specific to this cell type, but GATA4-6 have not been well characterized in early embryonic developments. Using quantitative polymerase chain reaction (qPCR) and in situ hybridization, we examined the expression of GATA4, 5 and 6 messenger RNAs (mRNAs) in ovine conceptuses and uteri during the peri-implantation period. In ovine conceptuses, GATA4, 5 and 6 transcripts were present on days 15, 17 and 21 (day 0 = day of mating), and high GATA5 and 6 mRNAs were found on day 21, most of which were localized in the trophectoderm and endoderm. Moreover, minute and substantial GATA4 and 5 mRNAs were found in days 15 and 21 uterine endometria, respectively. Increase in GATA4-6 transcripts in day 21 uteri indicates that in addition to GATA1-3, GATA4-6 may also play a potentially novel role in the development of ovine trophectoderm, endoderm and/or uterine endometria following conceptus attachment to the uterine epithelium.

Down-regulation of interferon tau gene transcription with a transcription factor, EOMES.

Interferon tau (IFNT), produced for a short interval during early pregnancy by the ruminant embryonic trophectoderm, is essential for the maintenance of early pregnancy, but the mechanism by which it is transcriptionally regulated has not been fully determined. To identify a transcription factor(s) involved in the down-regulation of IFNT genes, mRNAs for various known transcription factors were investigated by reverse-transcriptase and real-time PCR in conceptus tissues collected on Days 15, 17, and 21, or Days 17, 20, and 22 of ovine or bovine pregnancy, respectively. In particular, the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in Day 17 or 22 ovine or bovine conceptuses. Interaction between EOMES and the identified transcription factors was studied using transient transfection, revealing that ovine/bovine IFNT-reporter transactivation was down-regulated by EOMES. Transcription factor interactions with EOMES were further studied through immunoprecipitation, demonstrating an association between EOMES and cAMP-response element binding protein (CREB)-binding protein (CREBBP). Uterine flushing media collected from cyclic or early pregnancy animals were added to bovine trophoblast CT-1 cells cultured on type-I collagen (monoculture) or bovine uterine epithelial cells (coculture) in an attempt to regulate EOMES expression. In the coculture, but not the monoculture, addition of uterine flushing from Day 17 pregnant animals resulted in increased EOMES expression in CT-1 cells. These results suggest that as conceptuses attach to the uterine epithelium, IFNT gene transcription is down-regulated by an increase in EOMES expression and EOMES-CREBBP binding in the attached trophoblast cells.

Expression and potential role of GATA factors in trophoblast development.

Despite exhaustive studies, molecular mechanisms governing blastocyst formation, implantation to the uterine endometrium and placentation have not been definitively characterized. GATA family proteins are a group of zinc finger transcription factors, for which gene ablations eventually result in embryonic death later in pregnancy. These findings suggested that GATA factors are not essential for early embryonic development. However, recent studies from our laboratory and others have revealed that GATA proteins are involved in the regulation of key genes expressed by the trophectoderm that underpin the transition from the morula to trophoblast, and trophectoderm maintenance. Consequently, it is important to consider the current understanding how GATA factors govern early trophectoderm development.

Presence of Transcription Factor OCT4 Limits Interferon-tau Expression during the Pre-attachment Period in Sheep.

Interferon-tau (IFNT) is thought to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. We and others have observed that OCT4 expression persists in the trophectoderm of ruminants; thus, both CDX2 and OCT4 coexist during the early stages of conceptus development. The aim of this study was to examine the effect of CDX2 and OCT4 on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG-3 cells were cotransfected with an ovine IFNT (-654-bp)-luciferase reporter (-654-IFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with Cdx2, Ets2 and Jun increased transcription of -654-IFNT-Luc by about 12-fold compared with transfection of the construct alone. When cells were initially transfected with Oct4 (0 h) followed by transfection with Cdx2, Ets2 and/or Jun 24 h later, the expression of -654-IFNT-Luc was reduced to control levels. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Thus, when combined with the other transcription factors, OCT4 exhibited little inhibitory activity towards CDX2. An inhibitor of the transcriptional coactivator CREB binding protein (CREBBP), 12S E1A, reduced CDX2/ETS2/JUN stimulated -654-IFNT-Luc expression by about 40%, indicating that the formation of an appropriate transcription factor complex is required for maximum expression. In conclusion, the presence of OCT4 may initially minimize IFNT expression; however, as elongation proceeds, the increasing expression of CDX2 and formation of the transcription complex leads to greatly increased IFNT expression, resulting in pregnancy establishment in ruminants.

Coculture system that mimics in vivo attachment processes in bovine trophoblast cells.

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.

Functions of interferon tau as an immunological regulator for establishment of pregnancy.

The establishment of a successful pregnancy requires a "fine quality embryo", "maternal recognition of pregnancy", and a "receptive uterus" during the period of conceptus implantation to the uterine endometrium. In ruminants, a conceptus cytokine, interferon tau (IFNT), a major cytokine produced by the peri-implantation trophectoderm, is known as a key factor for maternal recognition of pregnancy. IFNT can be considered one of the main factors in conceptus-uterus cross-talk, resulting in the rescue of ovarian corpus luteum (CL), induction of endometrial gene expressions, activation of residual immune cells, and recruitment of immune cells. Much research on IFNT has focused on the CL life-span (pregnancy recognition) and uterine gene expression through IFNT and related genes; however, immunological acceptance of the conceptus by the mother has not been well characterized. In this review, we will discuss the progress in IFNT and implantation research made by us and others for over 10 years, and relate this progress to pregnancy in mammalian species other than ruminants.

Expression of GATA1 in the ovine conceptus and endometrium during the peri-attachment period.

The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes.

Function of a transcription factor CDX2 beyond its trophectoderm lineage specification.

The transcription factor caudal-related homeobox 2 (CDX2) regulates trophectoderm differentiation, but its function beyond trophectoderm differentiation is not well characterized. CDX2 was shown to regulate a trophoblast-specific gene, interferon τ (IFNT), in the ruminants. However, its regulatory mechanism has not been determined. Here, we report a new role of CDX2 in histone modifications of the IFNT gene. Chromatin immunoprecipitation assays using ovine conceptuses obtained from d 14, 16, 16.5, or 20 of pregnancy (d 0, day of mating) revealed that H3K18 acetylation was highly detectable at the upstream and open reading frame regions of the IFNT gene on d 14 and 16, when CDX2 reached its peak expression. From d 16.5, when the conceptus initiates attachment to uterine epithelial cells, histone acetylation along with CDX2 expression declines. Two candidate CDX2 binding sites (-300 to -294 bp and -293 to -287 bp) of the bovine IFNT gene promoter region were detected from chromatin immunoprecipitation and luciferase assay. When Cdx2 constructs were transfected into bovine ear-derived fibroblast cells, histone acetylation was increased, concurrent with the recruitment of cAMP response element binding protein-binding protein, which has histone acetyltransferase activity. H3K18 acetylation was seen in the proximity of the CDX2 binding region located at the IFNT gene's upstream region in CT-1 cells, but when these cells were treated with specific CDX2 small interfering RNA, H3K18 acetylation was decreased. These findings suggest that CDX2 regulates its targeted gene through cAMP response element binding protein-binding protein recruitment, which correlates with greater histone acetylation.

Retinol-binding protein (RBP), retinol and ß-carotene in the bovine uterus and plasma during the oestrous cycle and the relationship between systemic progesterone and RBP on day 7.

In the dairy cow, low systemic concentrations of progesterone are known to be a major factor associated with early embryo loss. Endometrial expression of the gene encoding retinol-binding protein (RBP) is sensitive to small changes in progesterone on day 7 of the oestrous cycle. The objectives of the present study were to measure RBP concentrations in bovine uterine flushings and plasma across different days of the oestrous cycle and to examine the relationship between uterine RBP and systemic concentrations of progesterone. Uterine flushings and plasma were collected from cows on days 3, 7, 11 and 15 of the oestrous cycle. Uterine RBP concentrations were five- to 15-fold higher (P < 0.001) on day 15 compared with the other days and twofold higher (P < 0.001) in the uterine horn ipsilateral to the corpus luteum on day 15. RBP concentrations were similar in flushings and plasma across days 3-11; however, day 15 RBP concentrations were six- to 15-fold higher (P < 0.001) in uterine flushings. No significant relationship was found between the concentration of systemic progesterone and RBP concentrations on day 7. Overall, the results of the present study indicate a local controlling mechanism operating at the level of the endometrium to regulate RBP secretion, most likely progesterone.

Glutathione content and antioxidant enzyme expression of in vivo matured sheep oocytes.

Retinoids play important roles in many diverse biological functions such as cell growth, morphogenesis, differentiation, and reproduction. Previous studies demonstrated that retinol administration to ewes, followed by natural service, resulted in embryos with improved competence to develop under standard in vitro conditions (5% CO(2) in air). Additional studies provided evidence that retinol may have some antioxidant effect by improving blastocyst development in cattle under atmospheric conditions (5% CO(2) in air). Glutathione is an important non-protein, sulphydryl compound found in oocytes and embryos, which acts to decrease oxidative stress. The purpose of the present study was to evaluate the effects of retinol administration to ewes on the content of glutathione and glutathione-related and antioxidant enzymes in in vivo matured sheep oocytes. Briefly, ewes were administered retinol or vehicle during superovulation, and after 60h the oviducts were removed and mature oocytes collected. Glutathione content did not differ significantly between oocytes collected from retinol-treated ewes (6.78+/-3.81pmol/oocyte) and control ewes (6.38+/-1.58pmol/oocyte). Transcripts encoding for manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu-Zn SOD), glutathione synthetase (GS), and glutathione transferase pi (GSTp) were detected in single ovine oocytes; however, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not reveal any significant differences in transcripts between oocytes from retinol-treated ewes and those from control ewes.

Induction of endogenous interferon tau gene transcription by CDX2 and high acetylation in bovine nontrophoblast cells.

Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production.

BACKGROUND: Prostaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production. METHODS: Cells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates. RESULTS: BEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity. CONCLUSION: Results from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

Distinct phospholipase A2 enzymes regulate prostaglandin E2 and F2alpha production by bovine endometrial epithelial cells.

BACKGROUND: The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production. METHODS: Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed. RESULTS: Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production. CONCLUSION: Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.

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.