Prostaglandin E2 involvement in mammalian female fertility: ovulation, fertilization, embryo development and early implantation

Ovulation requires adequate cumulus cell expansion to enable cumulus-oocyte complex (COC) detachment from the follicle wall and follicle rupture to release the oocyte into the oviduct. It was found that the LH surge increases PGE2 levels in the dominant follicle to modulate the action of gonadotropin for cumulus cell expansion and the expression of proteases associated with follicle rupture [15‐18]. Female mice deficient in EP2, a PGE2 receptor, presented with abnormal cumulus cell expansion and unruptured follicles, which resulted in infertility or sub-fertility [7, 19].

The essential genes that are induced in COCs and are responsible for expansion include the following: oocyte maturation genes of epidermal growth factor (EGF)-like factors, such as amphiregulin (Areg), epiregulin (Ereg) and betacellulin (Btc) [20, 21]; and matrix-forming and stabilizing elements, such as hyaluronan synthase 2 (Has2) and tumor necrosis factor α-induced protein 6 (Tnfaip6) [20, 22]. Studies have shown that PGE2 up-regulates the expression of many of these genes, including Areg, Ereg, Has2 and Tnfaip6, in granulosa and cumulus cells via its receptor EP2 [21, 23]. Moreover, there is evidence that the cAMP pathway induces the expression of the cumulus expansion-related genes Has2 and Tnfaip6 in cumulus cells [4] and that PGE2 increases cAMP concentrations in cumulus cells during ovulation [23]; these findings suggest a direct role of PGE2 in cumulus expansion via these growth factors. The role of the protein kinase B and mitogen-activated protein kinases3/1 (PKB-MAPK3/1) pathway in cumulus expansion has also been documented [24, 25], and PGE2 was found to activate this pathway for cumulus cell expansion and meiosis resumption [26].

PGE2 was found to be involved in not only cumulus expansion but also in meiotic maturation [27]. Cyclic adenosine monophosphate (cAMP) is a well-known mediator of meiotic maturation. PGE2 increases cAMP production in follicles, resulting in the maturation and cumulus expansion of oocytes [23, 28]. The PGE2 receptors EP2 and EP4, which are predominant in cumulus and granulosa cells [29], can increase intracellular cAMP levels when they are coupled to adenylate cyclase [30, 31]. In an in vitro study using mouse oocytes, treatment with an agonist selective for EP2 and EP4 increased cAMP production and subsequently increased ovulation rates [32], whereas the genetic manipulation of genes encoding EP2 and EP4 resulted in the inhibition of meiotic maturation and cumulus expansion [10, 33]. Several factors are responsible for maintaining spindle integrity during meiotic maturation. MAPK regulates spindle integrity during the meiotic maturation of oocytes [34, 35]. MAPK activity depends on phosphorylation. PGE2 was found to be responsible for the phosphorylation of MAPK [36], suggesting that PGE2 activates MAPK and indirectly induces the meiotic maturation of oocytes.

PGE2 was thought to mediate LH signals for meiotic maturation. Angiotensin II stimulation by LH has been reported to promote the meiotic maturation of oocytes by blocking the inhibitory effect of theca cells [37, 38]. It was demonstrated that the effects of angiotensin II in this process are mediated by PGE2 [39‐41]. In an in vitro bovine oocyte study, indomethacin supplementation blocked the meiotic maturation of bovine oocytes induced by angiotensin II, whereas PGE2 treatment restored meiotic maturation to levels comparable to those induced by angiotensin II [39]. Human chorionic gonadotropin (hCG), a substitute for LH that stimulates oocyte maturation and ovulation in assisted reproduction, was reported to increase PGE2 and ovulatory gene expression through prostaglandin transport (PGT) in human granulosa cells [42].

Even though LH and PGE2 were shown to trigger cumulus expansion and meiotic maturation separately [11‐13], according to the above findings, we suggest similarity and synergetic effects between LH and the PGE2 pathways in regulating cumulus expansion and meiotic maturation. First, LH and PGE2 receptors are members of the G protein-coupled receptor (GPCR) family, and they trigger the ovulation process via activating adenylate cyclases to increase cAMP concentrations [23, 43, 44] for the synthesis of EGF-like factors, including Areg and Ereg, in granulosa and cumulus cells [23, 44‐46]. Because the PGE2/cAMP pathway has been studied in meiotic maturation [23, 28], but not in the stimulation of Areg and Ereg secretion, we hypothesize that PGE2 induces cAMP pathways to stimulate EGF-like growth factors in cumulus and granulosa cells via its receptors (the same as LH); we propose this since these growth factors are positively involved in both cumulus expansion and oocyte meiotic maturation (Fig. 2).

Cumulus cells synthesize and secrete extracellular matrices (ECMs) composed of mostly hyaluronan deposited in the intercellular space [47, 48]. ECM deposition in the inter-cumulus cell space leads to cumulus expansion, increases ECM viscosity and confers resistance capacity against biochemical and mechanical stress to the cumulus ECM. Chemokines were reported to modulate cumulus ECM consistency [49‐51]. It has been demonstrated that upon ovulation, cumulus cells secrete various factors, including chemokine receptors and chemokines, such as CCL7, CCL2, and CCL9, which can increase the ECM viscosity to protect the oocyte from mechanical stress [49, 51]. The increased ECM consistency in cumulus cells by CCL-CCR signals impedes the passage of sperm and prevents fertilization [3, 49]. PGE2-induced activation of the cAMP pathway [50] disassembles and attenuates the ECM consistency [7, 50] by inhibiting the secretion of the chemokines CCL7 and CCL2 [50, 52]; these effects create a free space for sperm penetration.

In addition, interleukin-1β (IL-1β) stimulates the secretion of CCL2 in different cells [53, 54] and granulosa cells [55]. Given that cumulus cells are derived from granulosa cells, we hypothesize that IL-1β also stimulates CCL2 secretion in cumulus cells. It was found that PGE2 inhibits IL-1β in the myometrial cells of pregnant women [56], suggesting that PGE2 also inhibits the expression of CCL2 stimulated by IL-1β. Increased IL-1β expression was identified in cumulus cells lacking the PGE2 receptor EP2 [49], and we suspect a subsequent increase in CCL2 expression in these cells lacking PGE2 receptors. Taken together, we suggest that PGE2 acts via EP2 as a negative regulator of the chemokines CCL7 and CCL2 in the cumulus ECM to enhance ECM disassembly for sperm penetration and subsequent fertilization (Fig. 3a, b).

Neutrophils are mobilized in oviduct in response to the presence of sperm, a process resembling classical inflammation [57‐59]. When protective signals become ineffective, the neutrophils fight against the sperms and reduce their motility and fertilization potential [60‐62].

PGE2 exerts immunosuppressive activity [63], which inhibits the phagocytosis of sperm by neutrophils and macrophages [64, 65]. In the oviduct, in addition to the PGE2 secreted after epithelial cell stimulation by LH [66], it was found that sperm binding to the epithelial cells also induces PGE2 release [67], suggesting an auto-defense mechanism exerted by sperm in the oviduct. Via its receptor EP2, PGE2 may protect sperm from the phagocytic activity of polymorphonuclear neutrophils (PMNs) in the oviduct [68‐70]. Neutrophils phagocytize spermatozoa either through cell attachment or by entrapping them in neutrophil extracellular traps (NETs) [71, 72]. A previous study demonstrated that PGE2 inhibits NET formation [73]. We suggest that the immunosuppressive potential of PGE2 and its inhibition of NET formation may result in enhanced sperm survival and higher motility and binding capacity of sperm in the oviduct, which subsequently increase the fertilization rate (Fig. 3c).

After ovulation, mechanisms that protect the corpus luteum from regression should be initiated to stimulate the continuous production of factors protecting and promoting embryo development and early implantation. An increase in PGE2 biosynthesis and signaling in the uterus during the luteal phase was noted in sheep [79] and pig models [80], suggesting the role of this PG during early pregnancy. Through its receptor EP2, PGE2 induces the expression of LH receptors on the corpus luteum, resulting in an increase in progesterone (P4) synthesis [81, 82]. Previous studies reported that decreased blood flow to the luteal-containing ovary and decreased progesterone production result in luteolysis [83]. Blood flow has to be maintained for incoming bio-chemical factors, including LH, E2 and PGE2, to stimulate the corpus luteum to synthesize and secrete various factors, mostly P4, as well as to disseminate the secreted factors in the systemic system for embryo development and early implantation [84]. Via its receptor EP4, PGE2 was shown to enhance utero-ovarian blood circulation by increasing adenylate cyclase (AC) activity, which in turn increases nitric oxide synthase (OS) activity to increase synthesis and nitric oxide (NO) release, a vasodilator [85].

Interferon tau (IFN-t) was reported to reverse the PG secretion profile in endometrial cells during early pregnancy, thus resulting in increased PGE2 secretion and decreased PGF2a secretion [86, 87]. The increased PGE2/PGF2α ratio during early pregnancy is thought to prevent luteolysis and to support the luteal function for embryo development and successful implantation. It was reported that embryos secrete 17-β estradiol (E2) to stimulate continuous P4 production by the corpus luteum [88, 89]. The roles of embryo-secreted estradiol would be anti-luteolytic and lyteoprotectant, which are properties of PGE2. Previous studies demonstrated a simultaneous increase in E2 secretion by the embryo [90] and a shift from PGF2α to PGE2 secretion with a subsequent increase in the PGE2/PGF2α ratio during early pregnancy [91, 92]. Given the spatio-temporal pattern of E2 and PGE2 secretion and their localization, we hypothesize that there is an interaction between E2 and PGE2 to inhibit luteolysis. Moreover, E2 was shown to increase PGE2 production by stimulating PTGS2 secretion, which potentiates its activity through increasing the expression of EP2 [93]; E2 also decreases PGF2α production by inhibiting the secretion of PGFS (prostaglandin F2alpha synthase) and carbonyl reductase 1 (CBR1) [93], enzymes involved in PGF2α secretion [94‐96].

We hypothesize that PGE2 exerts its anti-luteolytic properties to protect and enhance embryo development and early implantation by inducing LH receptors on the corpus luteum for continuous progesterone secretion, by increasing nitric oxide (NO) synthesis and production to maintain adequate utero-ovarian blood flow and by mediating the effects of E2 and IFN-t to stimulate P4 secretion (Fig. 5a).

Different signals, including chemokines and chemokine receptors, produced in the endometrium are essential for embryo development and early implantation. Previous studies found that PGE2 modulates the expression of CXCR4, which is a receptor for the chemokine CXCL12, in hematopoietic stem cells [97] and stromal cells of the endometrium [98]. Embryonic signals were shown to regulate the expression of CXCR4 receptors in the endometrium [99], and the chemokine CXCL12 was identified in blastocysts [100].

PGE2 secreted by the embryo [101] binds to EP2 receptors in the endometrium to stimulate CXCR4 via the epidermal growth factor receptor (EGFR)-phosphatidyl inositol-3 kinase (PI3K) and extracellular signal-regulated kinase (ERK1/2) pathways [102]. The EGFR, PI3K and ERK1/2 pathways were shown to mediate early embryo implantation by inducing the expression of genes involved in cell growth, differentiation and uterine receptivity [103‐105]. Moreover, high levels of CXCR4 expression were found in endometrium stromal cells at the apposition site of the embryo during the implantation window [106]. Furthermore, the PI3K/ERK1/2 pathways are known as pro-survival pathways that enhance the growth, proliferation, differentiation and survival of embryonic and endometrial cells [107]. We hypothesize that PGE2 secreted by embryos influences the apposition, adhesion and invasion of trophoblastic cells to the decidua by stimulating the expression of the chemokine CXCR4 receptor at the implantation site via the EP2, EGFR, PI3K and ERK1/2 pathways and/or by stimulating the synthesis of the chemokine CXCL12, which in turn will bind to CXCR4 receptors. In addition, we suggest that PGE2 uses the PI3K/ERK1/2 pathways to promote embryonic growth, proliferation, differentiation and survival, as well as endometrial cell growth, proliferation and differentiation in decidual cells.

The above findings indicate that PGE2 is essential for enhancing pre-implantation embryo development and early implantation. However, other mechanisms need to be further investigated (Fig. 5b).