We report here on the generation of mice deficient in the expression of the PGE2 EP2 receptor. Although these mice are generally healthy, female Ep2–/– mice have significantly reduced pregnancy rates, and when these animals do become pregnant, they deliver smaller litters than Ep2+/–and wild-type controls. We show that this decrease in fertility is primarily due to a significantly lower fertilization rate in the Ep2–/– females. This reduction in fertilization is not due to an intrinsic defect of the ovum, because the in vitro fertilization rates of the Ep2–/– and wild-type ova are identical. In addition to this decreased fertility, we show that Ep2–/– animals have reduced blood pressure on normal diets and that this hypotension can be partially ameliorated by increased dietary salt.
Although all 4 EP receptors are expressed in the uterus, reduced fertility has not been reported for either the EP3- or EP4-deficient mice (13
). Although the fertility of the EP2-deficient mouse is decreased, implantation and decidualization is identical to that seen in wild-type animals. Decreased implantation or decidualization was not reported for the FP-, IP-, or TP-receptor–deficient mice (11
). These observations are somewhat surprising given the abnormalities of these reproductive functions in the COX-2–deficient mice. A number of explanations for these findings can be put forth. First, it is possible that PGD2
activation of EP1 is important for implantation. It is also possible that, despite the seemingly unique pattern of expression of the various prostanoid receptors, their functions overlap enough that implantation is not affected by the loss of a single receptor. Alternatively, a yet to be identified COX-2 product might be important for implantation.
Although ovulation is primarily triggered by a surge of luteinizing hormone, a role for PGs in this process has also been suggested. For example, inhibitors of PG synthesis, such as indomethacin, can prevent follicular rupture, and this effect can be overcome by administration of PGE2
). Similarly, mice deficient in COX-2 demonstrated impaired ovulation (10
). Our results indicate that these effects of PGs on ovulation are not mediated through the EP2 receptor, because both natural and hormonally induced ovulation in Ep2–/–
females were no different from wild-type controls.
Although PGs are produced throughout the female reproductive tract and by ovulated cumulus cell-oocyte complexes, their precise role in the fertilization process remains unclear (23
). Again, direct evidence for a role of prostanoids in fertilization came from studies of the COX-2–deficient mice. The results presented here are consistent with the hypothesis that at least one function of prostanoids in fertilization is mediated by PGE2
through the EP2 receptor. To define the mechanism by which loss of the EP2 receptor leads to decreased fertilization, a number of experiments were carried out. To determine if decreased fertilization was due to delayed or premature release of the ovum, ovulation was induced by administration of hCG, and the appearance of the eggs in the oviduct, as well as the rate of fertilization, was examined. Although the appearance of the ampulla in the oviduct was similar, the ova released after the animals were treated with hCG still failed to become fertilized. In addition, ova and their cumulus complexes from Ep2–/–
animals were morphologically indistinguishable from those from wild-type controls. Therefore, it seems unlikely that the decreased fertilization of eggs from Ep2–/–
mice results from an alteration in the timing of ovulation.
The fertilization process requires successful transport of sperm to the oviducts, where they can be sequestered for interaction with the ova. PGs have been shown to facilitate sperm transport through the female reproductive tract, not only by their actions on sperm but also by their effects on female reproductive tissue, including oviductal smooth muscle (24
). Numerous sperm were found in the oviducts of Ep2–/–
females within 12 hours of coitus, making it unlikely that alteration in sperm transport is the cause of fertilization failure in the EP2-deficient animals.
Together, these findings suggest that the reduced fertilization seen in these animals could be a function of PGs intrinsic to the ovum and cumulus cells, or that EP2-mediated PGE2
functions are essential for establishing an oviductal microenvironment conducive to successful fertilization. To distinguish between these 2 possibilities, we compared in vitro and in vivo fertilization of the Ep2–/–
ovum. Successful in vitro fertilization of ova from Ep2–/–
animals does not support the former hypothesis. These results are consistent with studies showing that maturation of the ovum is independent of PGs (25
). Previous studies have also suggested that ovum viability is unaffected by inhibitors of PG synthesis in vitro (5
Our studies are consistent with the hypothesis that PGE2
acts through the EP2 receptor to define a microenvironment in the oviduct conducive to fertilization. Although such a function in reproductive physiology has not previously been ascribed to these lipids, it is consistent with the diverse actions of PGs in numerous organ systems. For example, PGs have been shown to modify ion transport in a number of epithelia (27
). It is possible that the ionic composition of the oviductal fluid is altered in the Ep2–/–
animals and that this, in turn, leads to reduced fertilization.
also has potent effects on the cardiovascular system. A role for PGE2
in blood pressure homeostasis has been recognized for years, but these actions are complex, involving regulation of vascular tone and sodium balance (17
). To examine the role of the EP2 receptor in blood pressure regulation, we measured systolic blood pressure in conscious Ep2–/–
animals. On a normal diet (0.4% NaCl), systolic blood pressure was reduced by 13 mmHg in the EP2-deficient mice compared with controls. To determine whether this difference in blood pressure could be overcome by providing excess sodium in the diet, Ep2–/–
mice were fed high-salt diets containing 6% NaCl (wt/wt). This increase in dietary NaCl content had no effect on blood pressures in the wild-type mice. In contrast, on the high-salt diet, blood pressure increased significantly in Ep2–/–
mice to levels that were not significantly different than controls. Thus, reduced blood pressure in the Ep2–/–
mice is sodium sensitive and can be overcome with dietary sodium loading. This suggests that the absence of EP2 receptors produces an abnormality in sodium handling by the kidney. EP2 receptor expression in the kidney has been demonstrated by both RT-PCR and Northern analysis (29
), although its cellular functions in the kidney have not been clearly elucidated.
Our findings of reduced blood pressure in the Ep2–/–
animals may seem somewhat paradoxical given the known vasorelaxant actions of PGE2
). However, whereas acute infusions of PGE2
cause vasodilation, chronic infusions of PGE2
increase blood pressure through direct stimulation of renin secretion (33
). Furthermore, inhibitors of PG synthesis have been shown to lower blood pressure and renin levels in patients with renovascular hypertension (34
). It is well established that PGs can stimulate renin release through actions on the juxtaglomerular apparatus; hormones that increase intracellular cAMP levels stimulate renin release by these specialized cells (35
). Because 2 EP receptors, EP2 and EP4, are Gs
protein–coupled, it has been suggested that one or both of these receptors may mediate this action of PGE2
To determine whether abnormal regulation of the renin-angiotensin system might contribute to altered blood pressure regulation and salt sensitivity in Ep2–/–
mice, we measured PRA in these animals. Despite their lower blood pressures while on a normal diet, neither PRA nor renin mRNA was elevated in the Ep2–/–
animals (Figures and ). This differs from other lines of mice harboring targeted mutations that lower blood pressure, where PRA and renin mRNA are consistently elevated as a compensatory mechanism (20
). These findings suggest that renin responses are significantly impaired in EP2-deficient mice. Failure to stimulate PRA may contribute to the reduced blood pressure seen in these animals while they are on a normal diet.
The contribution of PGs to many important biologic processes has been recognized for years. Recently, mice carrying homozygous null mutations for genes encoding enzymes in the PG biosynthetic pathway and in PG receptors themselves have furthered our understanding of the role that these lipid mediators play in various aspects of reproduction (10
). The role of PGs in maintaining circulatory homeostasis has been more controversial and less understood. Here we define critical roles for the EP2 PGE2
receptor in blood pressure regulation and female reproduction. Specifically, we present evidence supporting the hypothesis that PGE2
, acting though the EP2 receptor, is important for establishment of the microenvironment required for successful fertilization of the released ovum. We also show that the EP2 receptor is essential for normal blood pressure homeostasis. Mice lacking this receptor are relatively hypotensive, and this hypotension can be partially reversed by salt loading. Finally, compensatory increases in renin are not seen in these animals, suggesting that the EP2 receptor may be involved in PG-stimulated renin release. Continued investigations into the specific roles of the receptors that mediate the actions of PGs will further our understanding of these important lipid mediators and potentially may lead to the development of more specific and less toxic therapies for a variety of disease processes.
Note added in proof.
During the review process of this manuscript, an analysis of EP2 receptor–deficient mice was independently reported in the February 1999 issue of Nature Medicine (5:217–220).