Nitzan Levy

Administration of Prostaglandin F2α During the Early Bovine Luteal Phase Does Not Alter the Expression of ET-1 and of Its Type A Receptor: A Possible Cause for Corpus Luteum Refractoriness

Abstract Luteal regression is initiated by prostaglandin F2α (PGF2α). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF2α. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF2α-induced luteolysis. Therefore, in this study, we compared the effects of PGF2α administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF2α receptors (PGF2α-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF2α analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF2α analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF2α injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450scc) was only affected when PGF2α was administered during midcycle. Nevertheless, PGF2α elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF2α-R was transiently affected. Such effects probably result from PGF2α acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF2α, possibly the endothelium, could yet be nonresponsive during the early luteal phase.

The ovarian endothelin network: an evolving story

The endothelin (ET) system consists of three ET isopeptides, several converting enzyme isoforms and two G-protein-coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Less than 20 years after the initial detection of ET-1 in granulosa cells, the ovarian ET network continues to expand with the discovery of new members and functions. ETs influence a broad range of essential reproductive processes, such as ovulation, steroidogenesis and luteolysis. Therefore, a more comprehensive understanding of the ovarian ET network might provide new strategies for controlling reproduction. This review presents up-to-date findings on the ET network in the ovary.

Endothelin-1 receptors and biosynthesis in the corpus luteum: Molecular and physiological implications

Endothelin-1 (ET-1), a 21-amino acid peptide was initially identified as a potent vasoconstrictor, ET-1 plays an important role in the female reproductive cycle: its quick ascent during luteal regression, ability to inhibit steroidogenesis in vitro and in vivo, combined with the observation that the luteolytic effects of prostaglandin F2alpha (PGF2alpha) were delayed by pretreatment with ET-1 receptors type A (ETA) antagonists suggest that this peptide functions as an important element of the luteolytic cascade. The observation that ETA receptor expression was inversely correlated with steroidogenesis in luteal cells; namely factors which stimulated steroidogenesis inhibited ETA receptor levels is also in accord with the inhibitory role of ET-1 in corpus luteum (CL) function. Contrary to the mature mid cycle CL, the CL of early cycle is refractory to PGF2alpha-induced luteolysis. PGF2alpha administered at early luteal phase (day 4 of the cycle) failed to increase luteal ET-1 gene expression or its ETA receptors. In contrast, both genes were markedly induced in mid cycle CL exposed to PGF2alpha. ET-1 gene is transcribed as prepro ET-1 (ppET-1) and the active form of peptide is derived from the inactive intermediate big ET-1, by endothelin-converting enzyme-1 (ECE-1), therefore alterations in mature ET-1 levels can be achieved by modulating the expression of ppET-1 and/or ECE-1. Analysis using in situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA. The ppET-1 mRNA, on the other hand, was only expressed by resident endothelial cells, suggesting that luteal parenchymal and endothelial cells cooperate in the biosynthesis of mature bioactive ET-1. A significant, four-fold elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to mid luteal phase. This increase was accompanied by a significant rise in ET-1 peptide. Surprisingly however, ppET-1 mRNA levels remained similar during early and mid luteal phase. Collectively, these studies demonstrate that: (a) the various components of ET-1 system (ET-1/ECE-1/ETA) are dynamically and independently regulated during bovine luteal life span. (b) The CL becomes PGF2alpha-responsive only when both ppET-1 and ECE-1 genes are expressed at a level which enable an uninterrupted ET-1 biosynthesis.

Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain.

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and β-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH2 terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3′, which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH2-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Hormonal Regulation and Cell-Specific Expression of Endothelin-Converting Enzyme 1 Isoforms in Bovine Ovarian Endothelial and Steroidogenic Cells

Abstract 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.

Luteolysis in Ruminants: Past Concepts, New Insights, and Persisting Challenges

It is well established that in ruminants, and in other species with estrous cycles, luteal regression is stimulated by the episodic release of prostaglandin F2α (PGF2α) from the uterus, which reaches the corpus luteum (CL) through a countercurrent system between the uterine vein and the ovarian artery. Because of their luteolytic properties, PGF2α and its analogues are routinely administered to induce CL regression and synchronization of estrus, and as such, it is the basis of protocols for synchronizing ovulation. Luteal regression is defined as the loss of steroidogenic function (functional luteolysis) and the subsequent involution of the CL (structural luteolysis). During luteolysis, the CL undergoes dramatic changes in its steroidogenic capacity, vascularization, immune cell activation, ECM composition, and cell viability. Functional genomics and many other studies during the past 20 years elucidated the mechanism underlying PGF2α actions, substantially revising old concepts. PGF2α acts directly on luteal steroidogenic and endothelial cells, which express PGF2α receptors (PTGFR), or indirectly on immune cells lacking PTGFR, which can be activated by other cells within the CL. Accumulating evidence now indicates that the diverse processes initiated by uterine or exogenous PGF2α, ranging from reduction of steroid production to apoptotic cell death, are mediated by locally produced factors. Data summarized here show that PGF2α stimulates luteal steroidogenic and endothelial cells to produce factors such as endothelin-1, angiopoietins, nitric oxide, fibroblast growth factor 2, thrombospondins, transforming growth factor-B1, and plasminogen activator inhibitor-B1, which act sequentially to inhibit progesterone production, angiogenic support, cell survival, and ECM remodeling to accomplish CL regression.