Requirement of the Fas ligand-expressing luteal immune cells for regression of corpus luteum

Apoptosis in corpus luteum (CL) is induced by prolactin (PRL) in female rats. PRL-induced apoptosis in CL is mediated by the Fas/Fas ligand (FasL) system. The CL consists of steroidogenic and non-steroidogenic cells, including immunocytes. Fas mRNA was detected only in the luteal steroidogenic cells, and FasL mRNA was expressed only by the non-steroidogenic CD3-positive luteal immunocytes. Removing the luteal immune cells from the luteal cells inhibited PRL-induced luteal cell apoptosis effectively. Thus, FasL-expressing non-steroidogenic luteal immunocytes are required for PRL-induced luteal cell apoptosis and heterogeneous induction of apoptosis by Fas/FasL in CL.     

Immune cell-endothelial cell interactions in the bovine corpus luteum

Early embryonic mortality accounts for a substantial portionof reproductive failure in agriculturally important livestock,including the dairy cow. The maintenance of early pregnancyrequires a fully functional corpus luteum (CL) that is not susceptibleto regression following fertilization, yet the cellular mechanismsof luteal regression are not clearly understood. Immune-cellaccumulation within the CL at the time of regression is a well-documentedphenomenon in a variety of species. In the dairy cow, immune-cellaccumulation precedes luteal regression by several days andcoincides with an increase in expression of the chemokine monocytechemoattractant protein 1 (CCL2), suggesting that immune-mediatedevents promote tissue destruction. Recent studies indicate thatendothelial cells comprising the CL are a primary source ofCCL2 secretion. Moreover, although uterine-derived prostaglandinF₂ (PGF) initiates luteal regression in the cow, PGF does notdirectly provoke CCL2 secretion by luteal endothelial cells.Instead, PGF-induced luteal regression is thought to requirecooperative interaction among immune cells, endothelial cells,and steroidogenic cells of the CL to further promote CCL2 secretion,enhance immune-cell recruitment, and eliminate luteal tissue.This brief review focuses on putative interactions between immunecells and endothelial cells derived from the bovine CL thatresult in enhanced CCL2 expression and the elaboration of otherinflammatory mediators (for example, cytokines), which perpetuateluteal regression. Fundamental knowledge of immune-endocrineinteractions within the reproductive system of cows has relevanceto other CL-bearing mammals, including humans and endangeredanimals, particularly in the development of methods to controland/or improve fertility. Thus, it is a timely topic for thissymposium concerning ecological immunology and public health.     

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.     

Demonstration of oxytocin release by bovine luteal cells utilizing the reverse hemolytic plaque assay.

Corpora lutea (CL) of a number of species produce oxytocin (OXT). In the present experiments we studied basal, prostaglandin (PG) F2 alpha-stimulated and ascorbate-stimulated OXT release from individual bovine luteal cells utilizing the reverse hemolytic plaque assay (RHPA). Using a mixture of C- and N-terminus-specific antisera against OXT, we were able to demonstrate OXT plaque formation by individual luteal cells. CL consist of two steroidogenic cell types: large luteal cells (LLC), believed to derive from granulosa cells and to produce and secrete OXT, and small luteal cells (SLC), thought to derive from theca cells. To distinguish between these two cell types, we designated cells greater than 20 microns as LLC and those less than 20 microns as SLC. On the basis of this morphological parameter, OXT release from both LLC and SLC was demonstrable. After an incubation period of 15 h, 7% of both cell types formed OXT plaques. PGF 2 alpha and ascorbate increased the size of plaques surrounding both LLC and SLC to more than 200% and 240%, respectively (basal plaque size = 100%). The number of plaque-forming cells increased only slightly in the presence of either PGF 2 alpha or ascorbate in comparison to basal conditions. We suggest that the RHPA can be used to demonstrate peptide release from luteal cells. It is concluded that LLC may be subdivided into functional subclasses because less than 10% of bovine luteal cells release OXT. Known OXT secretagogues increased the amount of OXT released. It appears that not only LLC but also SLC secrete this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size distribution of luteal cells from pregnant and non-pregnant marmoset monkeys and a comparison of the morphology of marmoset luteal cells with those from the human corpus luteum

The size distribution of marmoset luteal cells was determined on Days 6, 14 and 20 after ovulation in non-pregnant cycles and in early pregnancy. Image analysis was used to estimate the cell diameter of dispersed cells prepared from the marmoset corpus luteum (CL). Steroidogenic cells showed a size distribution consistent with one population of cells. There was a significant increase in mean cell diameter (P less than 0.05) from Day 6 to Day 14 in pregnant and non-pregnant animals with no further increase on Day 20. Micrographs of marmoset luteal tissue showed cells of greater than 10 micron containing the organelles typical of steroid-producing cells, and smaller non-steroidogenic cells surrounding the steroid-producing cells. On the basis of microscopy, there were no areas within the CL where cell composition was noticeably different. In contrast, micrographs of human luteal tissue showed two types of steroidogenic cell; most cells were similar to those in the marmoset CL but a smaller population of smaller cells could be distinguished around the periphery and along vascular septa. It is likely that these smaller and larger types of steroidogenic cells are of theca and granulosa cell origin respectively, the two cell populations differing in the degree of electron density and amount of rough endoplasmic reticulum. A distinguishing feature between marmoset and human luteal cells was the shape of the mitochondrian which were considerably rounder in marmoset luteal cells. The origin of steroidogenic cells in the marmoset CL is unclear, although in marmosets and man the luteal cell types display morphological characteristics distinct from the large and small luteal cells described for CL of the domestic ungulates.     

Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites

Background  

The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.     

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Hypoxia is an important physiological process which ensures corpus luteum (CL) formation and development, thus playing an important role in steroidogenesis. Recent studies have shown that CL develops in an analogous to tumorigenesis by accumulation of hypoxia-inducible factor-1 alpha subunit (HIF1A) in response to hypoxia. To investigate the relationship among hypoxia, steroidogenesis, and cell proliferation during CL lifespan, histological and steroidogenic analyses of CL were performed at various CL stages in non-pregnant Holstein. Also, the hypoxia-mediated steroidogenesis and cell proliferation were studied in vitro with both primary luteal and luteinized granulosa cells. Our results showed that progesterone (P(4)) concentration increased with the upregulation of steroidogenic protein including steroidogenic acute regulatory protein (STAR) and CYP11A1 (P450scc) in the middle luteal stage. On the other hand, the cell proliferation- or hypoxia-associated proteins were upregulated in the early stage, including the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), HIF1A, and aryl hydrocarbon receptor nuclear translocator (ARNT). In primary culture, phospho-protein kinase A (p-PKA) was downregulated, as were P(4) secretion and steroidogenic proteins both under oxygen-conditioned hypoxia in luteal cells and cobalt chloride-induced hypoxia in luteinized granulosa cells. However, under the treatment of hypoxia, PCNA, which was downregulated in luteal cells, was upregulated together with HIF1A and VEGFA in luteinized granulosa cells. Taken together, present study suggested that hypoxia downregulated steroidogenesis through PKA signaling and that the hypoxia-regulated cell proliferation could be activated during CL formation.     

Effects of prostaglandin F2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum

Receptors for prostaglandin (PG) F2 alpha in the ovine corpus luteum are localized on large steroidogenic luteal cells. Therefore, it was hypothesized that during luteolysis, the first demonstrable effects of PGF2 alpha would occur in the population of large luteal cells. To test this hypothesis, the numbers and sizes of large and small luteal cells, fibroblasts, capillary endothelial cells, and pericytes were determined in corpora lutea collected 12, 24, or 36 h (6 animals/group) following administration of PGF2 alpha on Day 10 postestrus and from untreated ewes on Days 10 and 12 postestrus. The numbers and sizes of luteal cells were determined after enzymatic dissociation of the luteal tissue into single cell suspensions and by morphometric analysis of luteal slices. Serum levels of progesterone decreased (p less than 0.05) within 12 h of treatment, indicating that luteolysis was induced. Recovery of the two types of steroidogenic luteal cells following enzymatic dissociation was different (p less than 0.05). Recovery of both steroidogenic cell types decreased with time after PGF2 alpha treatment, suggesting that they had become more fragile. As determined by morphometry, the number of large luteal cells was not different at any time point examined; however, by 36 h after treatment, the average diameter of large luteal cells had decreased (p less than 0.05). In contrast, by 24 h after treatment, there was a decrease in the number of small luteal cells (p less than 0.05) but no change in their diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine corpus luteum is an extrapituitary site of prolactin production

Prolactin (PRL) is known to be synthesized not only in the anterior pituitary, but also in other organs including the ovary. Among its various functions, PRL is regarded as the most important constituent of the luteotropic complex in rodents and pigs. The purpose of the present study was to determine whether PRL is produced locally in bovine corpus luteum (CL) and to determine its possible roles in CL. In the present study, we examined changes during the luteal phase in (1) the expressions of PRL and PRL receptors (long form: l-PRLR, short form: s-PRLR) in CL and (2) the localization of PRL in CL. We also measured the levels of PRL mRNA in cultured luteal cells and luteal endothelial cells. Furthermore, the effect of PRL on progesterone (P4) and prostaglandin (PG) F2alpha production by cultured bovine luteal cells was examined. Semiquantitative RT-PCR analysis revealed that the mRNAs for PRL and its two receptors, l- and s-PRLR, were expressed in all luteal stages examined. PRL mRNA expression was less in the regressed stage (days 19-21 after ovulation) than in the other stages. Both l-PRLR and s-PRLR mRNA expressions were higher in the late luteal stage (days 15-17) than in the other stages, while the ratio of l-PRLR to s-PRLR was less in the regressed stage than in the other stages. PRL mRNA was also detected in cultured luteal cells and luteal endothelial cells. PRL protein was immunohistochemically detected only in CL of the mid- and regressed stages. It was detected in smooth muscle cells of the intraluteal arterioles and endothelial cells but not in luteal cells and other cell types of CL. Exposure of cultured luteal cells obtained from mid-stage CL (days 8-12) to bovine PRL (100, 200 ng/ml) for 24 hr did not affect P4 and PGF2alpha production by the cells. The present study demonstrates for the first time the expressions of PRL and PRLR mRNA in bovine CL throughout the luteal phase. The overall results strongly suggest that the bovine CL is an extrapituitary site of PRL production.     

The expression of prolactin and its cathepsin D-mediated cleavage in the bovine corpus luteum vary with the estrous cycle

In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.     

Microvascular endothelial cells of the corpus luteum

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).     

Differences of microvascular endothelium in the bovine corpus luterum of pregnancy and the corpus luteum of the estrous cycle

Summary— The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells (‘cobblestone growth pattern’ and ‘arcuate growth pattern’) were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to be status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.     

Developmental sensitivity of the bovine corpus luteum to prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1): is ET-1 a mediator of the luteolytic actions of PGF2alpha or a tonic inhibitor of progesterone secretion?

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.     

Regression of the corpus luteum of pregnancy following parturition in the ewe

Corpora lutea (CL) of pregnancy from single-lambing ewes were examined by light and electron microscopy within 24 h and at 8, 15, 23, 31 and 41 days after parturition (2 ewes per stage). Within 24 h of parturition the structure of the CL was well preserved and both large and small luteal cells, characteristic of this species, were present in substantial numbers. However, both types of luteal cell contained numerous cytoplasmic lipid droplets, and smooth endoplasmic reticulum and secretory granules in large luteal cells were less prominent than in normal functional CL of cyclic ewes. Leucocytic infiltration, and death of some luteal and endothelial cells, were also observed at this stage. Further regression of the CL progressed slowly, and lipid-rich large luteal cells were still readily recognisable 15 days after parturition. The size of the CL declined progressively, and the proportion of tissue occupied by intercellular substances increased. Corpora albicantia approximately equal to 3-4 mm in diameter were still recognisable 41 days after parturition. It was concluded that luteal regression post partum progresses much less rapidly than at the end of the oestrous cycle.     

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.     

Tumor necrosis factor alpha receptors in microvascular endothelial cells from bovine corpus luteum.

There is sufficient evidence to prove that tumor necrosis factor alpha (TNFalpha) modulates bovine corpus luteum (CL) function. Our previous study demonstrated that functional TNFalpha receptors are present on luteal cells in bovine CL throughout the estrous cycle. The purpose of the present study was to identify the presence of functional TNFalpha receptors on the microvascular endothelial cells derived from developing bovine CL. TNFalpha receptors were analyzed by a radioreceptor assay using (125)I-labeled TNFalpha on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). (125)I-Labeled TNFalpha binding was maximal after incubation for 30 h at 37 degrees C, and the specificity of binding was confirmed. A Scatchard analysis showed the presence of two binding sites (high- and low-affinity) for TNFalpha receptors on both CS and TS cells. The dissociation constant (K(d)) values and concentrations of the high-affinity binding sites for TNF receptors were similar for CS and TS cells. However, K(d) values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells (P < 0.05 or lower). The expression of TNF receptor type 1 (TNF-RI) mRNA was determined in both cell types. Furthermore, TNFalpha significantly stimulated prostaglandin E(2) and endothelin-1 secretion by both CS and TS cells (P < 0.05 or lower). These results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, and suggest that TNFalpha plays two or more roles in regulating the secretory function of the endothelial cells.     

Hormonal regulation and cell-specific expression of endothelin-converting enzyme 1 isoforms in bovine ovarian endothelial and steroidogenic cells

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.