Background
The ovulatory surge of luteinizing hormone (LH) stimulates events within the dominant ovarian follicle which lead to ovulation. One such action of LH is to increase PGE2 levels within the follicle [
1]. PGE2 stimulates ovulatory events such as expansion of cumulus granulosa cells and enhanced expression of proteases associated with follicle rupture [
2]. Blockade of PGE2 production within the follicle [
3,
4] or genetic manipulation which disrupts PGE2 production or PGE2 receptor expression [
5,
6] can prevent ovulation, further highlighting the essential role of receptor-mediated actions of PGE2 in mammalian ovulation.
PGE2 acts via G-protein coupled EP receptors [
7]. Four distinct EP receptors (EP1, EP2, EP3, and EP4) have been identified. To date, examination of follicular EP receptor expression has focused on granulosa cells, with both cumulus and mural granulosa cells shown to express multiple types of EP receptors [
8,
9]. While PGE2 acts at both cumulus and mural granulosa cells of ovulatory follicles to promote periovulatory events [
10,
11], comparatively little is known about the oocyte as a target for PGE2 action. Detection of EP2 mRNA in bovine oocytes has been reported [
12]. In contrast, a recent report suggests that mouse oocytes do not express EP receptor proteins [
10]. The purpose of this study is to determine if the mammalian oocytes express EP receptors capable of signal transduction and modulation of oocyte function.
Discussion
The present study demonstrates that mammalian oocytes express functional receptors for PGE2. Multiple EP receptor mRNAs were detected in monkey and mouse oocytes, but only EP2 and EP4 receptor proteins were consistently detected by immunofluorescence. These discrepancies are reminiscent of our studies of EP expression in monkey mural granulosa cells before administration of the ovulatory gonadotropin stimulus, when EP1 and EP4 mRNA were detected, but these EP proteins were not detected by immunofluorescence [
8]. EP3 and EP4 mRNA were detected in all mouse oocytes examined, while only 1 of 3 oocyte preparations had detectable EP2 mRNA. A previous report suggested that EP2 and EP4 receptors were present in the cumulus, but not oocyte, of the mouse cumulus-oocyte complex [
10]. Our detection of EP receptor proteins within the oocyte, with inconsistent detection of EP mRNAs, might suggest that cumulus cells are the source of these EP receptor proteins. However, use of confocal microscopy in the present study permitted localization of EP receptors specifically to oocytes of mice and monkeys, suggesting that mammalian oocytes express receptors capable of direct response to PGE2.
Previous studies by this laboratory and others focused on granulosa cells as the primary target for PGE2 action within the periovulatory follicle. Mural cells are well established as expressing multiple EP receptors. Granulosa cells from monkey periovulatory follicles expressed mRNA for all 4 EP receptors, with EP1, EP2, and EP3 receptor proteins detected by immunofluorescence [
8]. Granulosa cells from human follicular aspirates were reported to express mRNA for EP2 and EP4, while EP1 and EP3 mRNA were not detected [
31]. The present study confirmed that monkey and mouse cumulus granulosa cells expressed EP2 and EP4 receptor proteins. Previously, Segi and colleagues used in situ hybridization to successfully localize mRNA for EP2 and EP4 (but not EP1 and EP3) to cumulus granulosa cells within mouse periovulatory follicles [
9]. More recently, EP2, EP3 and EP4 mRNA and proteins were localized to mouse cumulus cells [
10]. While the present study shows that EP2 and EP4 receptor proteins are expressed by mouse cumulus, further studies will be needed to confirm expression of EP3 receptor proteins. It is possible that cumulus cell expression of EP receptors changes in response to the midcycle LH surge, which may explain discrepancies between these studies. Recently, theca cells have also been suggested as a target for PGE2 action, with detection of mRNA for EP2, EP3, and EP4 in bovine theca [
32] and EP2 mRNA in mouse theca cells [
9]. Taken together, these findings support the concept that multiple cell types within the periovulatory follicle, including cumulus granulosa cells, mural granulosa cells, theca cells, and now oocytes, are potential targets for PGE2 action.
EP receptors utilize G proteins to mediate the effect of PGE2 binding to these transmembrane receptors [
7]. EP2 and EP4 are reported to couple exclusively with Gαs to activate adenylyl cyclase and increase intracellular cAMP. In both monkey and mouse oocytes, stimulation of EP2 and EP4 receptors increased cAMP as anticipated. The distribution of EP2 receptors at the surface of monkey oocytes was patchy, while EP4 receptors were distributed more uniformly around the surface of monkey oocytes. Microdomains have been reported in the vicinity of plasma membranes, wherein a receptor associates with specific G proteins, membrane-associated enzymes, and other components of the intracellular signaling apparatus [
33]. Microdomains present the possibility that EP2 and EP4 receptors may preferentially associate with different isoforms of adenylyl cyclase or cAMP phosphodiesterases in oocyte plasma membranes. In mouse oocytes, EP2 was localized throughout the cytoplasm while EP4 was primarily perinuclear in location. Although prostaglandin receptors are often located in plasma membranes, presence of prostaglandin receptors, including EP4, in perinuclear membranes has been reported [
34]. Since EP2 and EP4 receptors were differentially distributed within monkey and mouse oocytes, cAMP generated via each receptor may have a unique function. Furthermore, species differences in EP2 and EP4 distribution suggests that PGE2 may regulate different functions in oocytes obtained from different mammalian species.
Stimulation of EP2 and EP4 receptors increased oocyte cAMP levels. Levels of cAMP measured in this study were similar to previous reports of basal, PGE1-, and PGE2-stimulated cAMP in mouse and bovine oocytes [
35,
36]. Many pathways have been suggested to elevate oocyte cAMP levels, including ligand-independent receptors coupled to Gαs and adenylyl cyclase [
37,
38], suppression of cAMP phosphodiesterase activity to limit cAMP breakdown [
39], and transfer from granulosa cells [
40]. In vivo, cumulus cells surrounding oocytes also express EP2 and EP4 receptors, and cAMP generated within cumulus cells can move to the adjacent oocyte through junctional complexes connecting these cells. The actions of PGE2 on individual cell types within the oocyte-cumulus complex are difficult to discern in vivo. However, these studies do support the hypothesis that oocytes can respond directly to PGE2 via stimulation of EP2 and EP4 receptors to contribute to oocyte cAMP concentrations.
Levels of cAMP within the oocyte are critical in the control of resumption of meiosis. The present study shows that PGE2 treatment delayed progression of meiosis in mouse oocytes as well as in monkey oocytes without and with surrounding cumulus. This observation is consistent with the concept that high cytoplasmic cAMP within the oocyte prevents resumption of meiosis; low cAMP permits meiosis to continue [
41]. Several agents which increased adenylyl cyclase-generated cAMP levels also delayed resumption of meiosis in bovine oocytes without and with surrounding cumulus [
36]. When mouse oocytes were treated in vitro with cholera toxin or PGE1, trends towards increased cAMP and delayed resumption of meiosis have been reported [
35]. These findings, taken together with data from the present study, suggest that elevated PGE2 within the follicle may delay resumption of meiosis in vivo, with more pronounced effects in species with longer intervals between the midcycle LH surge and ovulation. This concept is consistent with previous studies showing delayed induction of COX-2 expression and prostaglandin synthesis in follicles of species with long periovulatory intervals [
1,
42‐
44].
Fertilization and subsequent embryonic development of denuded mouse oocytes was not altered by PGE2 treatment. This finding is consistent with previous studies showing that in vitro fertilization rates for oocytes stripped of surrounding cumulus cells were similar between wild type mice and mice lacking EP2 receptor expression [
5]. Overall, elevated follicular PGE2 may delay nuclear maturation somewhat, but PGE2 exposure does not alter fertilization of mouse oocytes.
In contrast, PGE2 treatment of monkey oocytes without surrounding cumulus had a negative impact on the fertilization rate. Interestingly, PGE2 treatment of cumulus-intact oocytes did not alter subsequent fertilization. PGE2 treatment reduced oocyte nuclear maturation in the absence and presence of surrounding cumulus, suggesting that progression of meiosis is modulated by PGE2 action directly at oocytes. In the context of the periovulatory follicle, PGE2 action at EP receptors on cumulus cells and elsewhere in the follicle may directly or indirectly regulate critical maturational changes within the oocyte which promote successful fertilization.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DMD conceived of the monkey oocyte/embryo studies, conducted monkey oocyte/embryo studies, performed cyclic AMP assays, and drafted the manuscript. LKMG conducted mouse oocyte/embryo studies. CAVV provided monkey sperm and advised on optimation of monkey IVF and oocyte/embryo culture. LKC conceived of the mouse oocyte/embryo studies, performed RNA amplification/RT-PCR for mouse and monkey oocytes, and helped to draft the manuscript. All authors read and approved the final manuscript.