Role of Candidate Genes Regulating Uterine Prostaglandins Biosynthesis for Maternal Recognition of Pregnancy in Domestic Animals

1 Sam Higginbottom Institute of Agriculture, Technology & Sciences, Allahabad 211007, India 2 Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India 3 Division of Surgery, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India 4Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, DUVASU, Mathura 281001, India 5 Animal Nutrition Division, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India


Introduction
Successful embryo development and survival include formation of blastocyst, implantation into the uterus, formation of placenta, development of the heart, and vascularisation of both embryo and fetus to assist nutrient deliverance [1].Among these developmental events, implantation of embryo is a crucial step and its success mostly depends on the efficiency with which the maternal recognition of pregnancy (MRP) is established [2,3].The MRP includes series of events that are synchronized by the endocrine interaction between the mother and the embryo.
The majority of studies examining the molecular mechanisms of conceptus-endometrial interactions carried out during the peri-implantation period of pregnancy have focused on the maternal side, describing changes in the transcriptome of the endometrium [4,5].Major factor affecting gene expression in the endometrium is day of the estrous cycle/early pregnancy.In other words, irrespective of pregnancy status, the temporal changes in gene expression in the endometrium are similar in pregnant and cyclic animals up to the time of MRP [5,6].
The survivability and opportunity of successful development of an embryo are influenced directly and indirectly by Step (i) Step (ii) Step (iii) Prostaglandin E2 (PGE2) Prostaglandin F2  ) (PGF2 various paracrine and autocrine factors (steroid hormones, growth factors, and cytokines), controlling uterine microenvironment [7].Out of these factors, hormones such as PGF2, PGE2 released during the preimplantation period influence molecular interactions involved in maintenance of pregnancy through reciprocal interactions between the conceptus and endometrium [8].Proteome and transcriptome studies have revealed quantitative and qualitative changes in the genes at different stages of the estrous cycle and pregnancy [8][9][10][11][12].These spatial and temporal changes were mainly due to the expression pattern of sets of genes in the uterine environment, where the endometrium is especially important for the embryo-maternal interaction and successful establishment of pregnancy.A number of important candidate genes have been identified, which regulate estrous cycle through PGF2 and PGE2 production, namely, AKR1B5, PGFS, PGFR, PGES, and COX-2.
In ruminants, endometrial prostaglandins (PGs) are important regulators of estrous cycles, uterine receptivity, implantation, and parturition [13].Two types of PGs, namely, PGF2 and PGE2 are known to induce opposite effects in tissues such as kidney (human), vascular system (human), uterine myometrium (pig, cow), and corpus luteum (CL) (pig) [13].In ruminants, PGF2 and PGE2 are the primary PGs produced in the uterine endometrium, but their secretory patterns are different [14,15].In ruminants, PGF2 of uterine origin is responsible for luteolysis, and high levels of PGF2 can disturb pregnancy at any time in several species (namely human horse pig, sheep, and cow) [16].Luteolysis, defined as functional and/or structural regression of CL, is a key process in ovarian cycle [17,18].In domestic species, luteolysis must be initiated to permit a new ovarian cycle to begin when conception does not occur.Conversely, the function and integrity of CL are required to establish and maintain pregnancy.The conceptus must therefore somehow abrogate luteolysis, a process commonly referred to as the MRP [17,18].In contrast to PGES, PGE2 induces systematic effects and acts as a luteotrophic or antiluteolytic agent [19].PGE2 also exerts an immunomodulation that helps prevent rejection of the conceptus [20].The PGE2/PGF2 ratio affects CL function, endometrial cell growth and differentiation, blood flow, vascular permeability, embryo migration, and implantation [21].
PGs are produced in response to a variety of physiological and pathological stimuli that activate phospholipase A2, resulting in the hydrolytic release of arachidonic acid (AA) from membrane phospholipids [22][23][24][25].AA is then metabolized to the unstable cyclic endoperoxide PGH2 by the cyclooxygenase enzymes COX-1 and COX-2 (also known as prostaglandin G/H synthases PGHS-1 and PGHS-2).In cows and sheep, endometrial COX-2 expression was found to be transiently induced during late diestrus around the expected time of luteolysis, whereas COX-1 expression was found to be invariant (in sheep) or undetectable (in cows) throughout the estrous cycle [26,27].Therefore, cyclooxygenase (COX) is the rate-limiting enzyme that catalyses the initial step in prostaglandins (PGs) production [28].

Biosynthetic Pathway of PGF2𝛼 and PGE2
PGs are synthesized in the cell from the essential fatty acids.PGH2, the common precursor of all PGs, is generated from AA by prostaglandin synthase/cyclooxygenase (PGHS/COX).However, AA is created from membrane phospholipids (phospholipase-A2) in the presence of cytosolic phospholipase A2 (cPLA2) [29].AA is then metabolized to the unstable cyclic endoperoxide PGH2 by the COX-1 and COX-2 enzymes (Figure 1).Therefore, COX is an enzyme that is responsible for the formation of PGs.There are two isoforms encoded by distinct genes [30]: the constitutive isoform, COX-1, is widely expressed in a variety of tissues and cells, whereas the inducible form, COX-2, is regulated by factors such as cytokines or tumor promoters [31].COX-1 is constitutively expressed in most tissues and responsible for housekeeping functions and immediate response to levels of AA above 10 M.COX-2 is regulated by factors such as cytokines or tumour promoters and supports sustained production of PGs from relatively low levels of AA (below 2.5 M) [32].PGH2 produced by COXs is the common precursor for generation of primary PGs including PGE2, PGF2, by cell-specific isomerases and synthases (such as PGES for PGE2 and PGFS for PGF2).The downstream enzymes, PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH2 to PGE2 and PGF2, respectively.
Among the different PGs, PGE2 and PGF2 are the main prostanoids produced in the human [33,34] and bovine [35] endometrium.The physiological importance of PGs in reproduction has been confirmed in the mouse, where targeted disruption of COX-1 or COX-2 genes reduced reproductive efficiency [36][37][38].Null mutation for cPLA2, a PG biosynthesis enzyme upstream of COX-2, also leads to an infertile phenotype [39].COX is the rate-limiting enzyme that catalyses the initial step in prostaglandins production [28].At the receptor level, deletion of the PGF2 receptor (PGFR) showed that it is necessary for parturition in the mouse [40].Here, we review the genes responsible for regulation and maintenance of estrus cycle through PGs production.

Prostaglandin F2𝛼 (AKR1C3 and AKR1B5
).Among different classes of PGs, PGF2 is one of the main prostanoids produced by bovine endometrium [35].In ruminants, it regulates ovarian cycle through initiating the regression (luteolysis) of CL and functions as major luteolytic agent [17,41].In particular, PGF2 is involved in labor and luteolysis to initiate a new estrous cycle in many species.Apart from sex steroids, prostaglandins are probably the most important regulators of female reproductive function (ovulation, uterine receptivity, implantation, and parturition) and associated pathologies [42].PGF2 can be produced from three distinct pathways (Figure 2).The major route in the formation PGF2 is via reduction of PGH2 by 9, 11-endoperoxide reductase activity, referred to as PGF2-synthetase (PGSF).PGFS, a monomeric enzyme, catalyzes the conversion of PGH2 to PGF2.PGFS expression has been demonstrated in several tissues, namely, uterus [43], placenta [44], CL [41], lungs [45], and liver [46].
All recognized mammalian PGFSs belong to the aldoketoreductase (AKR) 1C family and are generally associated with hydroxysteroid dehydrogenase (HSD) activity [29].The members of AKR1C3 play an important role in biosynthesis of PGs, catalyzing the formation of PGF2 and 9-, 11-PGF2 from PGH2 and PGD2, respectively [63].However, none of known functional PGFSfrom AKR1Cfamily was detected in bovine endometrium, while the expression of PGF2 was very high [35].
Aldoketoreductase 1B5 (AKR1B5) was the most likely PGFS involved in the production of PGF2 in bovine endometrium at the time of luteolysis [35].Interestingly, with its 20HSD activity, this enzyme can also inactivate progesterone, another factor regulating endometrial function.Therefore, AKR1B5, an old enzyme, established with a new function, as a functional PGFS [35].The human equivalent of the bovine AKR1B5 is AKR1B1 belonging to the AKR superfamily [64].AKR1B1 also known as the aldose reductase is highly expressed in the placenta for glucose metabolism and in the eye and kidney for osmotic regulation [65].Recently, Chapdelaine et al. [66] accumulated several lines of evidence supporting the hypothesis that AKR1B1 is a functional PGFS in the human endometrium.Kang et al. [67] demonstrated AKR1B1 association with PGF2 production in human endometrial cell lines and in decidualized stromal cells.In a cell free system, purified AKR1B1 recombinant protein is able to produce PGF2 from PGH2.Endometrial cell lines transiently transfected with an expression vector coding for AKR1B1 exhibit increased ability to release PGF2.In contrast, when AKR1B1 expression is knockdown with specific siRNAs, PGF2 production is decreased.They have found that the other potential PGF synthase (AKR1C3) is also expressed in endometrial cell cultures, but its contribution to PGF2 production remains to be determined.
The endometrial release of PGF2 in response to oxytocin is the initial signal triggering luteolysis in animals, and ovarian PGF2 contributes to the luteolytic process in primates including humans [68].In presence of a viable embryo, the default luteolytic signal is counteracted by an antiluteolytic or a luteotrophic signal or a combination of both to maintain the production of progesterone.PGF2 is also a potent constrictor of the myometrium and uterine blood vessels [69].In humans, PGs interact with cytokines and prolactin (PRL) to regulate decidualization and with angiogenic and coagulation factors to regulate menstruation [70].During the menstrual cycle, the concentration of PGF2 is apparently higher than PGE2 during the secretory phase, whereas levels of both PGs are low during the proliferative phase.The concentrations of PGE2 remain low, whereas PGF2 goes higher during menstruation and lower during the implantation [71].Recent reviews concur to state that across species (namely human horse pig, sheep, and cow), PGF2 and PGE2 are universally important in the regulation of endometrial function [70,72].

Prostaglandin F2 Receptor (PGFR).
Many of uterine effects are associated with the expression and activation of prostanoid receptors, which have been derived from in vitro or in vivo studies involving the genetic or pharmacological manipulation of prostanoid receptors.Some of the known effects of prostanoid signalling include angiogenesis, proliferation, adhesion, alterations in cellular morphology, motility, invasion [33,73], and pain perception [74,75].
PGF2 exerts its biological function through interactions with FP prostanoid receptors (PGFR).Reproductive tissues express different classes of prostaglandin receptors [76].PGFR is a receptor for PGF2.It is a member of the Gprotein coupled receptor family.Main effects of prostaglandin binding to the receptor are uterine (smooth muscle) contraction [77][78][79][80][81]. PGF2 mediates luteolysis via activation of this receptor [40] and is also involved in modulating intraocular pressure and smooth muscle contraction in uterus and gastrointestinal tract sphincters.Knockout studies in mice suggest that the interaction of PGF2 with this receptor in ovarian luteal cells initiates luteolysis and thus induces parturition [40].
There is evidence demonstrating that PGFR may be regulated both during pregnancy labour, implicating its potential role in the parturition process.In rats, PGFR expression in the uterus remained low through 15-20 days of gestation, whereas at day 22 and during parturition (day 23), PGFR expression significantly increased [82][83][84].In addition, mice lacking the PGFR gene remain pregnant post term [40], and administration of THG113, a PGFR antagonist, can delay preterm birth in both mice and sheep [85,86].This led to the suggestion that PGFR antagonists may prove to be of benefit for the treatment of preterm labour [87].

Prostaglandin E2 Synthase (PGES).
PGES is a terminal prostanoid synthase that can enzymatically convert COX product PGH2 to PGE2 [62].PGE2 induces systemic effects and may act as a luteotrophic or antiluteolytic agent [19].PGE2 also exerts an immunomodulation that helps to prevent rejection of the conceptus [20].The accepted pathway for the production of PGE2 involves first the generation of PG endoperoxide H2 primarily through COX-2 and then conversion into PGE2 via PGES.It has been observed that PGES may be an important rate-limiting enzyme for PGE2 biosynthesis in bovine endometrium [88].
It has been reported that recognition and establishment of pregnancy depends on the regulation of the balance between PGF2 as the luteolytic signal and PGE2 as the antiluteolytic or luteotrophic signal.In this respect, the relative production of PGE2 and PGF2 may be more important than the absolute production of each PG.The PGE2/PGF2 ratio can be modulated in favour of PGE2 and establishment of pregnancy by three different pathways: (1) the well-accepted hypothesis of a decrease in the production of PGF2; conversion of PGE2 into PGF2 by a putative 9-keto-prostaglandin E2 reductase and (2) an increase in PGE2 through the stimulation of PGE synthase.Asselin et al. [89,90] reported that PGE2 production is increased in both types of endometrial cells following treatment with recombinant ovine interferon tau (IFNT), a key factor for establishment of pregnancy recognition signal.In addition, IFNT silences expression of estrogen receptor alpha which prevents upregulation of oxytocin receptor (OXTR) expression, which is required for the endometrium to generate oxytocin-dependent luteolytic pulses of prostaglandin F2 (PGF2) in response to oxytocin from the CL and/or posterior pituitary in nonpregnant ewes.In this way, IFNT maintains CL function for continued production of progesterone, the unequivocal hormone of pregnancy that stimulates and maintains endometrial functions necessary for conceptus survival and growth in the uterus [91].Dorniak et al. [92] supported the hypothesis that PGs synthesized and secreted by the conceptus act in paracrine alone or in concert with IFNT and progesterone to coordinately regulate endometrial functions that govern conceptus growth and elongation during the peri-implantation period of pregnancy in sheep.Therefore, PGs and IFNT from the conceptus coordinately regulate endometrial functions important.In epithelial cells, the alteration in PG production was such that the primary PG produced was changed from PGF2 to PGE2.Arosh et al. [27] have shown that the expression of PGES mRNA closely followed that of COX-2 during the bovine estrous cycle.Presence of PGES and its modulation under conditions known to influence PG production in bovine endometrial was reported for the first time by Parent et al. [88].

Cyclooxygenase (COX) 2.
PGs are generated via the COX pathway, and COX is the rate-limiting enzyme for conversion of arachidonic acid into PGH2 [30,93].COX exists in to two isoforms that are encoded by two separate genes, COX1 and COX-2, which are also known as prostaglandin endoperoxide H synthases (PGHS)-1 and (PGHS)-2 [30].These enzymes are responsible for the conversion of arachidonic acid into PGH2.Although COX1 is a constitutively expressed enzyme in a variety of cell types, COX-2 is the inducible enzyme Table 1: In our knowledge, status of study for mRNA expression pattern of some important genes regulating PG synthesis in different species.

Name of genes
Expression reported in endometrium of different mammalian species, across estrous/menstruation cycle (in vivo) Symbol represents, reports, ✓: available, X: not available.
that plays role in various pathological and physiological conditions in animal tissues.Although COX1 deficient female mice are fertile, they have specific defects in parturition, whereas COX-2 deficient female mice are infertile with abnormalities in ovulation, fertilization, implantation, and decidualization [36,38,94].The requirement of COX-2 for normal blastocyst implantation and decidualization in mice is due to the role of COX-2-derived PGs in regulation of vascular endothelial growth factor and angiopoietin signaling that influence uterine vascular permeability and angiogenesis [61,88,95].Parent et al. [32] have correlated the expression of PGES with that of COX-2, which is an important enzyme for the production of PGE2 in bovine endometrium.Increasing this production will modulate the PGE2/PGF2 ratio and contribute to establishment of pregnancy.

Spatiotemporal Expression of AKR1B5, AKR1C3, PGFR, PGES, and COX-2 Genes in Endometrium, across Estrus/Menstruation Cycle
Although the ovine model has greatly advanced our understanding of how the fate of the corpus luteum is controlled, it is clear that the mechanisms involved in the maternal recognition of pregnancy vary considerably from one species to another [17,18,96].Therefore, spatiotemporal expression of these genes (at transcript level) in endometrium, across estrus/menstruation cycle, was studied extensively in different species, namely, human, monkey, horse, pig, and sheep (Table 1).The reproductive pattern of the goat is rather similar to the sheep in many respects, but there are two important differences between these two species.First, the oestrous cycle length in the goat is longer (19-21 days) as compared with the sheep (16-17 days).Second, the goat relies exclusively on progesterone secreted by the CL for the maintenance of pregnancy, since the goat placenta, unlike the sheep, does not produce progesterone [17].Despite having their significant role for the regulation of estrus cycle and maintenance of pregnancy, the above review suggested that the goat is one of the few domestic species in which these genes are still to be studied.

Conclusions
Endometrial PGs (PGF2 and PGE2) synthesis, secretion and associated genes for their function, is being discussed in this review.Both PGF2 and PGE2 function to establish and maintain the pregnancy by their luteolysis and luteotropic nature.The expression of these genes has been studied in various species, namely, human, monkey, horse, pig, cow, and sheep.However, in goat these genes and their expression are still to be studied.So, there is the need to explore the expression pattern of these genes in this important domestic species too.Along with these, the particular ratio of PGE2/PGF2 is also unclear, which ultimately decides the microenvironment of uterus.In conclusion, the answer of these questions we left can solve the problems in reproductive performance.

Figure 1 :
Figure 1: Selective representation for production of PGE2 and PGF2.Steps involved (i) release of arachidonic acid (AA) from plasma membrane phospholipids through cytosolic phospholipase A2 (cPLA2).(ii) Conversion of AA into PGH2, the common precursor for all PGs, occurs through PGH synthase/COX, for which there are two isoforms COX-1 and COX-2.(iii) Conversion of PGH2 into active PG by terminal synthase PGES and PGFS.

Figure 2 :
Figure 2: Known biosynthetic pathways leading to the formation of PGF2.