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Ovary |
a Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel
b Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, follicle, growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by pretreatment with type A ET-1 (ETA) receptor antagonists [5, 7, 9] suggest that this peptide functions as an important component of the luteolytic cascade. In addition, ET-1 may prevent premature luteinization of follicular cells [10]. These functions of ET-1 are possible because of its ability to inhibit steroidogenesis in follicular and in luteal cells and through other mechanisms, such as the induction of tumor necrosis factor [11] and the interplay with nitric oxide [12]. ET-1, a 21-amino acid (aa) peptide, is synthesized from a pre pro (pp) protein of approximately 200 aa, which is proteolytically cleaved into the inactive 38-aa big ET-1 and is further processed to the mature active form of ET-1 by zinc-binding metalloendopeptidase ET-converting enzyme 1 (ECE-1) [13]. ECE-1 null mice exhibit a phenotype similar to that of ET-1- or ETA-deficient mice, demonstrating the significance of ECE-1 in generating bioavailable ET-1 [14]. ECE-1 may play a role in ET-1 overproduction that characterizes some pathophysiological conditions, when ET-1 is elevated in parallel to ppET-1 [15, 16]. ECE-1 can also be regulated independently of the ppET-1 gene, and alterations in the enzyme contributed directly to the changes in ET-1 levels in fetal lung [17], the cyclic contraction of the seminiferous tubules [18], and the development of the corpus luteum (CL) [19].
Regulation of ET-1 synthesis is even more complex when considering the presence of different isoforms of ECE-1. These isoforms have comparable biological activity; however, by virtue of the different N-terminal cytoplasmic tails they differ in their intracellular localization [20, 21]. Despite their importance, little is known of the cellular distribution of the various ECE-1 isoforms in vivo, and the localization of these isoforms to different ovarian cells has not yet been studied. Additionally, we and others [19, 22] have noted that ovarian ECE-1 levels vary throughout the cycle, but the factors controlling ECE-1 expression remain unknown. Clarification of these regulatory mechanisms and of the site at which ECE-1 functions is necessary to have a better understanding of the reproductive roles of ET-1. This study was therefore undertaken to investigate the hormonal regulation of ECE-1 in follicles and CLs and to identify the cell-specific expression of ECE-1 isoforms in endothelial and steroiodogenic cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Super Script II RNase H- Reverse Transcriptase and Ultra pure electrophoresis agarose gel were obtained from Gibco BRL Life Technologies (Gaithersburg, MD). Deoxynucleotide triphosphates, random hexamer oligodeoxynucleotides, and Taq DNA polymerase were from Farmentas (Vilnius, Lithuania). Oigonucleotide primers were synthesized by Biotechnology General (Rehovot, Israel). Penicillin, streptomycin, neomycin, and fetal calf serum (FCS) were from Biological Industries (Beit HaEmek, Israel). Dulbecco modified Eagle medium:Ham F12 medium (1:1, v/v), bovine big ET-1, porcine ET-1, protease inhibitor cocktail for mammalian cell extracts, insulin, and horseradish peroxidase (HRP)-conjugated goat anti rabbit IgG (H+L) were from Sigma (St. Louis, MO). Bandeiraea simplicifolia lectin 1 (BS-1) was from Vector Laboratories (Burlingame, CA). Collagenase type IV was from Worthington Biochemical Corporation (Freehold, NJ). Uncoated magnetic beads Dynabeads M-450 were from Dynal (Oslo, Norway). Bovine LH (USDA bLH-B-5) was kindly provided by the USDA Animal Hormone Program (Beltsville, MD). Insulin-like growth factor I (IGF-I) was kindly provided by Prof. Arie Gertler (Hebrew University of Jerusalem, Rehovot, Israel).
CL Collection, Luteal Cell Dispersion, and Enrichment of Subpopulations
CLs were collected at a local slaughterhouse, and luteal stage was determined by macroscopic examination, as described by Fields and Fields [23]. CLs were dispersed and luteal cell subpopulations were enriched as previously described [19]. CLs were sliced and dispersed in M-199 containing 0.5% BSA and collagenase (420 U/ml). Dispersed luteal cells were suspended in 1% BSA in PBS and mixed with magnetic tosylactivated beads coated with BS-1 (the endothelial cell-specific lectin) at a bead:endothelial cell ratio of 1:3. The adherent cells were washed and concentrated using a magnet until the supernatant was free of cells. Both BS-1-positive cells (endothelial cells [19]) and nonadherent cells (enriched luteal steroidogenic cells) were collected for RNA extraction.
Follicular Cell Isolation and Cultures
Granulosa and theca cells were isolated from healthy bovine follicles (estradiol in follicular fluids >150 ng/ml) as previously described [24]. Enriched theca interna cells were obtained as follows. Dissociated theca layer cells were incubated with BS-1-coated magnetic beads, and BS-1-adherent cells were then discarded. BS-1-negative cells (enriched theca interna cells) were collected for RNA extraction.
Granulosa cells were either incubated for 24 h in the presence of 1% FCS, LH (100 ng/ml), or ET-1 (10-7 and 10-8 M) or were incubated for 5 days in the presence of insulin (20, 200, or 2000 ng/ml) or IGF-I (1, 10, or 100 ng/ml). At the end of the incubation period, cells were collected for total protein or RNA extraction.
RNA Extraction and Semiquantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from cells using the guanidinium thiocyanate method [25]. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed as previously described, with glyceraldehyde 3-phosphate dehydrogenase (G3PDH) as an internal standard [26]. Sequence analysis or restriction mapping ascertained the identity of the PCR products. Specific forward primers for the bovine ECE-1 isoform were designed according to the 5' end of each isoform (accessions U27342 and Z35306 for primers ECE-1a and ECE-1b, respectively). Primers at the 3' end of the cDNA, common to all isoforms (1277–1906), were used to detect total ECE-1 expression. Sequence analysis showed that these primers do not amplify ECE-2. Computer searches and sequence alignments were performed by using software from Genetics Computer Group (Madison, WI). Primers are listed in Table 1.
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In Situ Hybridization
Procedures for in situ hybridization were as previously described [19]. A 550-base pair cDNA encoding bovine ECE-1 was generated using RT-PCR. The cDNA was ligated into the pGEM-T-Easy vector and sequenced. Both antisense and sense [35S]UTP-labeled cRNA probes 107 cpm/ml were transcribed from linearized cDNA templates using a transcription kit (Promega Corp., Madison, WI). Hybridization was performed in a humidified oven at 55°C for 20 h. After hybridization, slides were washed, treated with RNase, dehydrated, dipped in NTB-2 emulsion (Kodak, Rochester, NY), and exposed for 21 days at 4°C. Slides were developed, lightly counterstained with hematoxylin and eosin, and mounted for microscopic examination. For each follicle, two sections were hybridized with the antisense probe, and one section was hybridized to the sense probe. Hybridization intensity was measured using the Bioquant image analyses system (R&M Biometrics, Nashville, TN). The image analysis system determined the number of graphic pixels occupied by the silver grains.
Western Blot Analyses
Proteins were extracted in lysis buffer (20 mM Tris HCl pH 8.6, 1% SDS, 1 mmol/L PMSF, and protease inhibitor cocktail). Homogenates were centrifuged for 15 min at 2000 x g, and protein concentrations of the supernatant were determined using DC reagents (BioRad, Hercules, CA). Samples containing 10 µg protein were separated by 7.5% SDS-PAGE under reducing conditions and electrically transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH). After 2 h of blocking in Tris-buffered saline with Tween-20 containing 5% BSA, membranes were incubated for 2 h with anti-ECE-1 antiserum (kindly provided by M. Yanagisawa) [27] directed against ECE-1 C-terminal peptide. The membranes reacted with primary antibodies (diluted 1:4000) were washed and then incubated with HRP-conjugated goat anti-rabbit IgG for 1 h at room temperature, and binding was detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Western blots were exposed to x-ray films and subsequently scanned and quantified using NIH Image (v. 1.61).
Statistical Analyses
Data are presented as means ± SEM. The expression of the various genes was quantified using the densitometric analysis relative to an internal standard (G3PDH). Protein levels were quantified using densitometric analysis relative to total protein as determined with a Comassie stain. Statistical analysis was carried out using the JMP package (v. 3.2) [28]. A one-way ANOVA was used to determine the significance of differences between individual groups. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cellular localization of ECE-1 mRNA in developing bovine antral follicles was examined using in situ hybridization with an antisense probe (Fig. 1). In small and mid-size follicles, ECE-1 mRNA was present in antral and less in basal granulosa cells, whereas in large follicles the granulosa layer was uniformly labeled. The intensity of granulosa cell labeling was highest in large follicles; intensities (in arbitrary units) were 19.3 ± 0.9, 33.1 ± 1.1, and 56.1 ± 2.0 for small, medium, and large follicles, respectively. ECE-1 mRNA appeared in the theca cell layer of mid-size and large follicles, with hybridization intensity (in arbitrary units) lower than that in the corresponding granulosa layer (e.g., 43.2 ± 2.9 vs. 56.1 ± 2.0, respectively, in the large follicle; Fig. 1E). In addition to follicular cells, ECE-1 mRNA was also detected in the endothelial cells lining the blood vessels in the theca layer (Fig. 1C). No specific hybridization was observed with the sense probe.
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We next quantified ppET-1 and ECE-1 gene expression in granulosa and theca cells separated from large bovine follicles (Table 2). In agreement with the in situ hybridization data, granulosa and theca cells expressed ECE-1, and its levels in granulosa cells were higher than those measured in theca cells (Table 2). RT-PCR for ppET-1 showed negligible levels in the avascular granulosa cells and high levels of expression in the theca cell layer. The theca interna layer was peeled along with the basement membrane; therefore, dispersed theca cells were found with endothelial cells. To distinguish ppET-1 gene expression between these two cell types, theca interna cells were enriched by the use of magnetic beads coated with the endothelial cell-specific lectin BS-1. Partial elimination of endothelial cells was confirmed by reduced expression of the endothelial cell marker CD31 (Table 2). Reducing endothelial cell number from the dissociated theca layer commensurately reduced ppET-1 levels in the remaining theca interna cell fraction (Table 2). It therefore appears that ET-1 expression is mainly localized to the endothelial cells of the bovine follicle. In contrast, ECE-1 expression was not concomitantly reduced, suggesting that both endothelial and theca interna cells express the ECE-1 mRNA (Table 2) in agreement with the in situ hybridization data (Fig. 1).
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Identification of ECE-1 Isoforms in Ovarian Endothelial and Steroidogenic Cells
Two isoforms of bovine ECE-1 capable of cleaving intra- or extracellular big ET-1 were identified [21]. Therefore, we sought to identify which ECE-1 isoforms are present in endothelial and steroidogenic cells in the follicle (Fig. 2a) and CL (Fig. 2b). Luteal cells were enzymatically dispersed and separated into endothelial and steroidogenic cell fractions, as previously described [19]. PCR amplification was carried out using specific primers for the a and b isoforms. The intracellular ECE-1a isoform was exclusively expressed in the resident luteal endothelial cells. Conversely, the membrane-bound ECE-1b isoform was expressed in both steroidogenic and endothelial cells of the preovualtory follicle and the CL (Fig. 2). In follicles, the intracellular ECE-1a isoform was present in theca layer cells (containing endothelial cells) and absent in the theca interna-enriched cell fraction (Fig. 2a), suggesting that ECE-1a is only expressed in follicular endothelial cells similar to the endothelium in the CL.
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Hormonal Regulation of ECE-1 Expression in Luteal Cells
The pattern of ECE-1 mRNA levels in bovine CL tissue collected during various stages of the cycle is illustrated in Figure 3. Lowest levels were found in early stages of CL development (Days 2–4). As the CL matured (Days 7–12), ECE-1 levels increased and remained elevated in the late CL (Days 13–16). ECE-1 levels declined only in the regressed gland (Day 20+; Fig. 3).
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To gain better insight into the regulation of ECE-1 expression during the luteal phase, the direct effects of several luteotrophic and luteolytic factors were studied. A 24-h incubation of granulosa cells with LH (100 ng/ml) markedly downregulated (6-fold) ECE-1 levels (Fig. 4). Incubation in medium alone did not affect ECE-1 levels, which remained similar to those measured in freshly isolated cells before culture (Fig. 4). We then examined the effects of other luteotrophic factors, IGF-I and insulin, on ECE-1 expression. Culture of granulosa cells in the presence of various doses of IGF-I or insulin for 5 days induced a dose-dependent upregulation of ECE-1 protein levels (Fig. 5). A similar result was observed for ECE-1 mRNA levels (data not shown). The effects of insulin were detected over a wide range of concentrations (Fig. 5; 20–2000 ng/ml), indicating that this hormone may have acted via both the insulin and type 1 receptors. ECE-1 expression in IGF-I/insulin-treated cells increased with time of culture along with the ability of the cells to produce progesterone (data not shown). When incubated with ET-1 for 24 h (at either 10 or 100 nM), a significant decrease in ECE-1 levels was observed in dispersed luteal cells (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ECE-1 is a critical enzyme in the biosynthesis of ET-1, a peptide that has diverse functions. This enzyme may therefore be a target for pharmacological intervention to control numerous physiological or pathological conditions involving the ET-1 system [16, 29].
ECE-1 may also be involved in the processing or degradation of other substrates. This enzyme efficiently hydrolyzes a number of peptides other than big ET-1, including bradykinin, substance P, and neurotensin [30] and the beta-amyloid peptides involved in Alzheimer's disease [31].
ECE-1 mRNA expression is modulated in many pathological proceses involving the cardiovascular system. These changes take place at the level of the endothelial cell and are mediated by ET-1 itself or other vasoactive substances [32, 33]. The present study is the first to document the regulation of ECE-1 and its isoforms in ovarian parenchymal cells. IGF-I and insulin markedly induced ECE-1 in cultured granulosa cells, and this increase was dose and time dependent. In addition to effects measured when µg/ml amounts of insulin were added, a significant effect was also detected at low insulin concentrations (20 ng/ml), suggesting that insulin may affect ECE-1 by acting via its own receptor. Insulin or IGF-I plays an important role in ovarian physiology and has been associated with normal ovarian follicular growth and the acquisition of steroidogenic capacity [34–36]. Insulinopenic animals and IGF-I mutant mice exhibit low ovarian weight, reduced follicular size, and impaired steroidogenesis [37, 38].
IGF-I, insulin, and IGF-I receptors were detected by various methods in bovine follicles and CLs [39, 40] and could therefore be involved in regulating ECE-1 in vivo. Although the presence of the various components of the IGF-I system in ovaries of domestic species is well documented, the exact roles of IGF-I and its receptor mRNA during either follicular or luteal development remain controversial [39–41]. Nevertheless, recent studies showed that because of IGF binding protein-degrading proteases, IGF bioavailability is increased during development of the selected follicle to the preovulatory stage [42]. Elevated free IGF-I could be responsible for augmenting ECE-1 expression in growing follicles, as suggested by this study. These findings are consistent with ET-1 levels being higher in large than in small follicles [10]. The cellular source of ET-1 in follicles differs among species. In bovine follicles, ET-1 is produced by basement membrane endothelium (this study) [43], whereas granulosa cells do not express ppET-1 and similarly bovine luteal steroidogenic cells also lack ET-1 mRNA [2, 19]. These findings, however, contrast with those in pigs and humans in which ET-1 mRNA was detected in granulosa cells by in situ hybridization. Regardless of whether bovine granulosa cells transcribe the ET-1 gene, they can produce the active ET-1 peptide by cleaving big ET-1. There is abundant evidence demonstrating the presence of ET-1 in ovarian follicles of various species [10, 43, 44]. The prevailing notion is that this peptide acts to prevent premature luteinization of follicular cells. However, ET-1 does not selectively inhibit progesterone production; ET-1 inhibited gonadotropin-induced changes in granulosa cell morphology [45] and cAMP and estradiol production by these cells [46, 47]. Therefore, the physiological role of ET-1 in follicles needs further research.
Exposure of preovulatory granulosa cells to LH markedly reduced their ECE-1 expression. This finding corroborates our previous findings of low ECE-1 levels during early stages of luteal development [19]. The decline in ECE-1 levels in response to LH may also account for the reduction in ET-1 peptide levels demonstrated in post-ovulatory bovine follicles and the young CL [19, 48]. The inhibitory effects of ET-1 on luteal progesterone biosynthesis both in vitro and in vivo are well established [5–9]. Therefore, negligible amounts of ET-1 peptide in the young CL and lack of its induction by PGF2 [4] may confer resistance to luteolytic signals. As a result, locally produced factors such as IGF-I or PGF2 can act to support luteotrophic processes [39, 49]. LH also inhibits ETA receptors in luteal cells [50]. These receptors are present on luteal cells and promote the antisteroidogenic effects of ET-1 [5, 7]. Therefore, the LH surge that triggers luteal cell differentiation also lowers ET-1 and ETA to ensure optimal luteal development.
During luteal regression when high levels of ET-1 are present, ECE-1 levels decrease (this study) [22]. This decline may be accomplished by ET-1 itself as implied by findings reported here. Yet, this observation may appear puzzling, because it is unclear how the CL could synthesize large amounts of ET-1 with reduced levels of ECE-1. The temporal sequence of events induced after PGF2 can clarify this ambiguity. Stimulation of ET-1 is a rapid event, and elevation of ppET-1 mRNA and peptide concentration are detected as soon as 4 h after PGF2 injection [3, 4]; however, ECE-1 levels decline 44 h later [22]. This time frame is more than sufficient for ET-1 to inhibit steroidogenesis (2–4 h) and cause vasoconstriction, which are the initial steps in the process of luteolysis.
In humans, four ECE-1 isoforms, 1a–1d, have been identified [51, 52]. ECE-1 isoforms are identical in most of their aa sequence and differ in their cytoplasmic N-terminal tail as a result of different promoters a single gene [20, 53]. When expressed in Chinese hamster ovary (CHO) cells, ECE-1 isoforms displayed distinct subcellular localization. Human (h) ECE-1a was strongly expressed at the cell surface, hECE-1b was exclusively intracellular, and hECE-1c and -1d were also expressed on plasma membrane, although less strongly than hECE-1a [51]. In cows, two ECE-1 isoforms have been identified [21]. When overexpressed in CHO cells, bovine (b) ECE-1a (<60% aa identity with the sequence of hECE-1a) is localized to an intracellular compartment (possibly the Golgi apparatus). The form designated bECE-1b (the bovine orthologue of hECE1c; 88% identity in aa) is expressed mostly on the cell surface, similar to expression of the human form [21].
Identification of the cellular and subcellular distribution of ECE-1 isoforms in ovarian cells would allow determination of the site where big ET-1 is activated. We found that steroidogenic cells enriched from midcycle CL and follicular granulosa and theca cells exclusively express ECE-1b. This finding is supported by our previous findings demonstrating that luteal granulosa cells can cleave exogenously added big ET-1 [19]. In contrast, endothelial cells lining blood vessels in the CL or the follicle express both ECE-1a and ECE-1b mRNA. Based on these findings, a model describing ET-1 biosynthesis in the ovaries is proposed (Fig. 7). According to this model, endothelial cells expressing ppET-1 and the two ECE-1 isoforms can secrete both the precursor, big ET-1, and mature ET-1. The exact relationship between these products would require more detailed knowledge of the expression of each isoform; however, it is tempting to speculate that differential regulation of ECE-1a versus ECE-1b could result in a varible big ET-1:ET-1 ratio. Steroidogenic cells however only express the cell surface form of ECE-1, ECE-1b, without ppET-1. To produce ET-1, these cells are dependent on the extracellular supply of big ET-1. Nonetheless, they can regulate the amount of ET-1 produced in a manner independent of endothelial cells by modulating ECE-1 levels. Emoto et al. [21] suggested an analogous model for the formation of ET-1 in vascular endothelium and underlining smooth muscle cells. Similar ET-1 activation in different organs (coronary arteries and CL or follicle) indicates the physiological significance of this cellular arrangement, wherein endothelial cells provide the precursor (big ET-1) and the underlying tissue, either smooth muscle or steroid-secreting epithelial cells, and convert it into mature ET-1. Thus, ET-1 is generated near its site of action, i.e., the ETA receptor, to ensure that short-lived ET-1 is active.
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The hormonal regulation and intracellular localization of bovine ECE-1 isoforms as they were revealed in this study may provide insight into ET-1 biosynthesis and mode of action in various ovarian compartments.
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FOOTNOTES |
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2 Correspondence: Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel. FAX: 972 89465763; rina.meidan{at}huji.ac.il
Received: 11 July 2002.
First decision: 7 August 2002.
Accepted: 30 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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