lit review endo

Abigail Martin 07360932 4BY2

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Genes involved in embryo- endometrium interactions in mares during Maternal

Recognition of Pregnancy (MRP)

Abstract

Introduction

There are many important reasons for studying equine reproduction and improving its

efficiency for its own sake [1]. 16% of thoroughbreds experience pregnancy failure, with

60% happening within the first three weeks of gestation [2]. Economically, the equine

industry is substantial in many countries and poor reproductive results are very costly.

Although equine reproductive studies are still in their early stages, it is known that genes

expressed in the endometrium (the lining of the uterus) of mares play a necessary role in the

establishment and maintenance of maternal pregnancy. The actions of the conceptus (the

spherical shaped sac containing the embryo proper, its associated membranes and fluid) in the

uterus must be considered as it is also important in MRP [3].

Using biochemical pathways, the conceptus signals its presence causing arrest of the

oestrous cyclic process and maintains primary Corpus Luteum (CL) function. The events

controlling these processes are known as “maternal recognition of pregnancy” (MRP) [4]. For

all ruminants it is therefore understood that for MRP to transpire, communication between the

conceptus and the endometrium is necessary, thus creating a synchronous uterine

environment for embryonic development. Very little is known about the molecular crosstalk

between the embryo and endometrium in equines because their MRP mechanisms are

different to other ruminants [5].

Progesterone is a principal hormone involved in maintaining pregnancy in all

mammals. In the presence of an embryo in the uterus, the life span of the CL (corpus luteum)

is prolonged and endometrial release of prostaglandin F2α (PGF2α) into the bloodstream is

blocked, preventing luteolysis (regression of the CL) [3]. MRP depends on species- specific

signals produced by the embryo and recognised by the uterine lining and vice versa.

The aim of this review is to first, give an overview of the first 3 weeks of pregnancy

in the mare and its embryonic development. Secondly, to highlight key prostaglandins and

genes that have a strong influence on MRP involved in the conceptus- endometrium

interactions, especially those leading up to implantation in equids and in ruminants. Finally,

to discuss the values of studying equine embryology.


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Early stage development of the equine embryo

After fertilisation, the zygote cleaves via first mitosis to give two cells called

blastomeres surrounded by the zona pellucida. Cleavages are divisions of cells in the early

embryo. The cleavage ends with the formation of the blastula. These begin during the

transport of the embryo through the oviduct into the uterus but, this process is a species-

specific stage during development. Mares are unusual to other animals with regard to passage

through the oviduct, whereby only developing embryos can pass through unlike unfertilised

oocytes which cannot [3,6].

The equine blastocyst enters the oviduct around day 6.5 after ovulation has occurred.

The blastocyst contains an inner cell mass and a blastocyst cavity surrounded by a monolayer

of trophectoderm. By day 7 the zona pellucida has shed and the embryo is surrounded by a

tough acellular mucin-like glycoprotein capsule [7]. The capsule acts to protect, and prevents

the trophoblast from elongating, unlike other domestic animals such as cattle and pigs, and

gives it its spherical structure. It facilitates rapid growth between days 11 and 16, nutrition

and migration of the conceptus in the uterine lumen via peristaltic myometrial forces [5].

During the migration from one uterine horn to the other, the conceptus has surface

contact with the endometrium. The conceptus signals its presence in the uterus via anti-

luteolytic signals thus, the embryo prevents luteolysis by first, placing itself near to the

endometrium then attaching to it [3], between day 17 and 21 [8]. This event is known as

fixation, where the conceptus containing the embryo proper becomes attached to the

endometrial surface. Fixation is the termination of the extended mobility stage in the uterus,

positioning and fixing the embryo at the base of one of the uterine horns. This termination is

because of an increase in uterine tone and embryo growth, predominantly in the right horn of

mares [8]. In rodents and primates the term implantation is used here but, in these animals the

embryo penetrates the epithelium and advances into the endometrial connective tissue where

it is implanted [3]. Fig.1 outlines the stages of development of an equine embryo during MRP

from day 0 to day 28.

In mares luteolysis can occur as early as days 11 and 13 [5,9]. Luteolysis can be

defined as the functional and/ or structural regression of the CL [10]. It is a crucial process in

the ovarian cycle whereby, post-ovulation, luteolysis is initiated by an Oxytocin-dependant


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Fig. 1: The approximate timing of key developmental stages of growth in the equine embryo during the first 4

weeks of gestation .Taken from [1]

high- frequency burst release of PGF2α (prostaglandin F2α) from the posterior pituitary and the

CL [1]. Simultaneously, the oxytocin receptor is upregulated in the endometrium, this is

similar for cows and sheep [10]. Luteolysis is abrogated by the conceptus for the pregnancy to

continue, usually from day 14 [5].

The relationship between endometrium and embryo

For the establishment of pregnancy, a number of complex interactions are necessary.

This is during the pre-fixation period. These interactions take place between the ovary,

endometrium and embryo. The ovaries produce oestradiol and progesterone, these hormones

act on the endometrium to produce various proteins and prostaglandins. When an embryo is

present in the uterus, proteins nourish the developing embryo. As already mentioned,

prostaglandin acts on the CL causing luteolysis if a viable embryo is not present in the animal

[11]. Depending on the species, the conceptus secretes steroids, prostaglandins, growth

factors and cytokines. These factors influence the endometrium: preventing prostaglandin


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secretion and stimulating protein secretion, or by directly acting on the ovary to stimulate

progesterone secretion [12].

The pig, horse and guinea pig are oxytocin dependant species. During MPR the CL

produces progesterone and oxytocin stimulating endometrial synthesis of prostaglandin thus,

causing luteolysis. Interferon- tau (IFN-τ) is the MRP factor in ruminants such as sheep,

cows, goats [13, 14]. IFN-τ genes are not present in horses and equine conceptuses do not

produce interferon like proteins during early pregnancy but, it is an important factor in other

ruminants and therefore must be acknowledged [15]. IFN-τ silences expression of the

estrogen receptor (ESR1) preventing expression of endometrial oxytocin receptors. If an

embryo is present, oxytocin cannot induce PGF2α synthesis preventing luteolysis. It is

released from the endometrial glands and stimulates histotrophe production, i.e. secretions

that nourish the embryo in the uterine cavity. Pigs have a different mechanism to halt

luteolysis. Oxytocin is produced in the CL and promotes synthesis of PGF2α like ruminants.

The trophectoderm of a pig trophoblast produces oestradiol, thus preventing luteolysis. PGF2α

is secreted into the uterine cavity where it is degraded by rate limiting enzymes instead of the

maternal bloodstream as in equids and ruminants [3].

Conceptus associated proteins involved in endometrium communication

The components of systems for the exchanging of materials between the conceptus

and the mare are often found in the cellular yolk sac wall called the capsule. The proteins

β2M, uterocalin, uteroglobulin, ganglioslide activator protein (GM2AP) and phospholipase

A2 (PLA2) are mostly expressed in large amounts approaching the time before fixation.

A major luteolysis- associated protein of the conceptus capsule is β2 Microglobulin

(β2M). It is the most abundant protein within the capsule. β2M undergoes proteolysis in the

capsule and its sequence is shortened around the time of fixation [16]. The removal of amino

acids is due to the conversion of the intact form of β2M to its cleaving form to the

endometrium around day 17, when the embryo becomes fixed to the uterine lumen. The

origin of the β2M has not been determined but Quinn et al. state that it is likely to stem from

the endometrium or yolk sac wall tissue. Its function is the light chain complex of various

MHC class 1 complexes. It is believed to also have an earlier function in the capsule before

degradation occurs [17].

Uterocalin/ P19 is a characterised lipocalin which act as a small hydrophilic carrier

protein. It is secreted by the endometrial glands in late diestrus and in early pregnancy in

equines. The cationic protein binds to capsule and its main function is to transport and deliver


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lipophillic substances such as nutrients into the yolk sac [16]. It is only present in the yolk sac

fluid before fixation. Uterocalin has only been demonstrated in equines so far. Similar to β2M

it lessens as the capsule degrades, therefore uterocalin and β2M only appear to be essential

during early embryonic growth [17].

The uteroglobin gene is expressed in the equine uterus. First identified in the uterine

of rabbits, it is a member of the large secretoglobulin super family of proteins. Like

Uterocalin it binds small lipophillic molecules and also, is present in ample amounts in the

uterine lavage fluids but, only in the yolk sac before fixation [16, 17]. Uteroglobulin is found

in various mammals including some without encapsulate conceptuses.

The ganglioslide activator protein (GM2AP) is expressed in large amounts by the

equine capsule up until around day 18.5 of pregnancy. This protein is involved in the

transport of glycolipids. It has only been found to be expressed by the capsule so far [18].

Phospholipase A2 (PLA2) are a complex group of enzymes that cleave glycerol based

phospholipids and a fatty acid, usually arachidonic acid, a cyclooxygenase substrate from the

eicosanoid family (a group of signalling molecules constructed by the oxidation of twenty-

carbon essential fatty acids) [16, 17, 19]. The PLA2 enzymes play a valuable role in

regulating ovarian function, pregnancy and delivery, they are also important in inflammation

and haemostasis. PLA2 synthesis in the equine uterus was found to bind to the capsule and

increase its concentration when PGF2α was administered to block fixation [16, 17, 19]. It is

also believable that synthesis of PLA2 assists in the degradation and removal of the

conceptus capsule.

http://en.wikipedia.org/wiki/Lipid_signaling

http://en.wikipedia.org/wiki/Oxidation

http://en.wikipedia.org/wiki/Essential_fatty_acid


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Membrane phospholipids

PLA2

Arachidonic acid

COX-1 COX-2

PGH2

PGE2 PGF2α

PGE synthesis

PGF synthesis

Fig. 2 Systematic mechanisms of the expression of prostaglandin E2 and prostaglandin F2α in the equine uterine

lining. Endometrial membrane phospholipids are converted to arachidonic acid by active phospholipase A2

(PLA2). Arachidonic acid is metabolised to an intermediate prostaglandin H2 (PGH2) by two cyclooxygenase

enzymes (COX-1 and COX-2). Prostaglandin E (PGE) is synthesised to prostaglandin E2 (PGE2) and

prostaglandin F is synthesised to prostaglandin F2α (PGF2α).

Prostaglandin synthase expression in the equine endometrium

Boerboom et al. hypothesise that the conceptus could inhibit luteolysis by altering

expression of enzymes involved in prostaglandin (PG) synthesis [10]. The effects of

prostaglandins are mediated by membrane bound receptors in the uterus. Prostaglandin F

receptor (PGFR) is a contractile receptor that binds PGF2α and prostaglandin E2 receptor

(PGER) acts as a relaxant and may play an important role in uterine receptivity. The PGER

(prostaglandin E2 receptor) has four subtypes [20]. Prostaglandins are synthesised in response

to many pathological and physiological stimuli that activate phospholipase A2 (PLA2)

converting membrane phospholipids to arachidonic acid then metabolises to cyclic

endoperoxide prostaglandin H2 (PGH2) by two cyclooxygenase isoform enzymes COX-1 and

COX-2 [9, 10]. This is shown in fig 2. COX-1 and COX-2 catalyse arachidonic acid and they

are considered to be the rate-limiting enzymes in prostaglandin production. The receptors


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PGER and PGFR along with the cyclooxygenase enzymes play an important role in

degrading prostaglandin during MRP [20].

COX-1 expression was shown to remain low in the uterus during the oestrus cycle and

become upregulated during MRP. It is understood that its expression may be influenced by

oestradiol [20]. COX-2 is a key modifier of PG metabolism during early pregnancy in mares.

It can be stimulated in different cell types by different factors, i.e. Follicle stimulating

hormone and luteinising hormone in ovarian follicles or by cytokines influencing epithelial

cells [20]. PGH2 is the intermediate prostaglandin formed from the prostaglandin synthesis

reaction, it is then converted to prostaglandin E2 (PGE2) and PGF2α by prostaglandin E

synthesis (PGEs) and prostaglandin F synthesis (PGFs) [15]. Balances between these two

biosynthesis are considered important in ruminants for successful pregnancy to take place. It

is not fully understood whether the relative expression of enzymes PGE2 and PGF2α changes

throughout the oestrous cycle and pregnancy in equid endometrium [21]. Ealy et al. state that

in equids, the primary target for conceptus secretions is COX-2, which leads to believe that

endometrial COX-2 is blocked by the presence of the conceptus but, the amount of PGEs and

PGFs is not affected [10, 13].

In relation to differences in the biological roles in the reproductive tracts interactions

in equid verses ruminant, Boerboom el al’s findings were that there was a lack of endometrial

expression of PGE2 in equines and also, that it does not have an effect on the lifespan of the

CL or in luteolysis. In bovine endometrium PGE2 regulation plays an important role in

parallel with COX-2 in the establishment of pregnancy. PGE2 is produced in large quantities

by the conceptus, Boerboom et al. hypothesise that it may have the responsibility of acting as

a supplement for the endometrium rather than serving to stimulate myometrial contractions

therefore, moving the conceptus around the uterus distributing antiluteotic signal molecules

which is a process common in ruminants. By targeting the expression of the COX-2 enzyme

the equine conceptus can regulate endometrial prostaglandin synthesis [10, 15].

In cows and sheep endometrial COX-1 expression was almost undetectable and COX-

2 was found to increase and be temporarily expressed around the time of luteolysis (i.e., day

15) [10]. COX-2 expression levels in these ruminants is prolonged by pregnancy, of which

does not affect the amount of PGF2α released by the uterus of the ewe but affects the

pulsatility of its secretion [10].


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There are fundamental differences in the molecular mechanisms underlying MRP in

mares, cows and ewes. Oxytocin stimulation of PGF2α secretion is reduced when pregnant in

these animals but in mares the receptor number is higher at time of luteolysis resulting in

prevention of luteolysis being more dependent on inhibiting the up-regulation of COX-2

rather than oxytocin receptor [22].

Gene regulation in the equine endometrium during MRP

Equine gene homologue Gene symbol Function

Up regulated genes

Homo sapiens GM2 ganglioside activator GM2A Lipid transport

Heat shock proteins

Equus caballus similar to heatshock 22kDa protein 8 HSPB8 Response to stress

Homo sapiens heat shock 27kDa protein family member 7 HSPB7 Response to stress; cardiovascular

Equus caballus similar to crystallin Alpha B CRYAB Protein tyrosine kinase signalling; anti apoptosis

Cell- cell signalling

Equus caballus siminlar to interferon stimulated gen 15 ISG15 Cell-cell signalling; protein modification process

Equus caballus similar to ATPase, H+ transporting ATP6V0A4 Ion/proton transport

Equus caballus siminlar to stanniocalin STC1 Cell-cell signalling: calcium ion homeostasis

Transport

Homo sapiens proton/ amino acid transporter 2 (PAT2) SLC36A2 Amino acid transporter

Equus caballus similar to solute carrier family 46 member 2 SLC46A2 Transport

Putative Secretory Proteins

Equus caballus similar to fibroblast growth factor 9 FGF9 Growth factor activity

Equus caballus TIMP metallopeptidase inhibitor 1 TIMP1 metallopeptidase inhibitor

Equus caballus similar to fibroblast growth factor binding protein FGFBP Growth factor binding

Down regulated genes

Equus caballus insulin-like growth factor binding protein 1 IGFBP1 Regulation of cell growth

Equus caballus similar to connective tissue growth factor CTGF Cell adhesion; regulation of cell growth; angiogenesis

Equus callabus estrogen receptor 1 ESR1 Estrogen receptor signalling

Table 1. A gene profile of genes which are up and down-regulated within the endometrium during MRP.

Adapted from [9, 23].

Regulation of genes within the endometrium in response to a conceptus

Many of the identified genes that are regulated in the endometrium are involved in the

endometrial remodelling in response to a conceptus. After looking at the conceptus response to

the endometrium it is necessary to study the endometrial response with respect to the conceptus. It

is important the conceptus- endometrium interactions remain synchronous for MRP to occur and


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for embryo growth and development to be successful. Table 1 shows a summary of commonly

upregulated and down regulated genes that are expressed in the endometrium during early

pregnancy in response to the conceptus.

Merkl et al. determined differential expression of genes in the endometrium on day 8

and 12 of pregnant and cyclic mares, taking endometrial biopsies on the relevant days and using

qPCR and microarrays to show total mRNA expression in response to a conceptus. By day 12,

they noticed a significant difference in the expressions recorded. They found the higher mRNA

levels were due to a response to the conceptus that prevented the down regulation of the genes

they analysed, except for FGF9 gene, a fibroblast growth factor, which was additionally

upregulated. Merkl et al. stated that the regulation of steroid hormones may be responsible for

these interactions [9].

Heat shock proteins are proteins whose expression is upregulated when introduced to

a stressful environment. The three main proteins regulated by oestrogens and involved in the

equine endometrium are heat shock proteins B7 and B8 (HSPB7 and HSPB8) and Crystalin, alpha

B (CRYAB). The endometrium is sensitive and receptive towards the conceptus. Klein et al.

hypothesised that these proteins prepare the endometrium for fixation in the conceptus [23].

Cell-cell signalling mediates the transfer of information from one cell to another [24].

This process and its upregulation of genes are essential for the successful establishment of

pregnancy. Stanniocalcin 1 (STC1) is a pregnant-specific uterine gene which is upregulated in the

endometrium across species. In the endometrium of equids STC1 expression has not been

reported but, its expression has been recorded in sheep, pigs, humans and mice. In sheep, STC1

mRNA and protein are required for regulating growth and differentiation of the embryo and its

placenta, and act as a marker for implantation in pigs. In humans and mice STC1 expression is

highest during embryo implantation in the endometrium. It therefore plays a species- independent

role in early pregnancy [9, 23].

During MRP, amino acid transport is necessary for embryo development. Transport

mediator protein, solute carrier 36 member 2 (SLC36A2) is a proton/ amino acid symporter. This

simply means that two molecules can be transported by this protein in the same direction through

the conceptus capsule using a common carrier mechanism. Klein et al. assume that its

upregulation is due to the demands of rapidly growing conceptus for nutrients [23].


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Secretory proteins make up a considerable amount of gene material upregulated in the

uterine cavity. When secretory protein production is poor, pregnancies often show detrimental

effects [25]. Commonly upregulated secretory proteins were luteinizing hormone (LH), tissue

inhibitor of metalloproteinase 1 (TIMP1) and insulin growth factors (IGFs). LHB has been

observed in women and is believed to prepare the uterus for implantation. It interrupts cellular

apoptosis, encourages angiogenesis and employs a particular immune responses [11, 25]. This is

understood to be similar in equids in preparing the endometrium for fixation. TIMPs regulate the

extracellular matrix production and tissue reconstruction during implantation and fixation thus, it

is a contributing factor to endometrial reconstruction in various species and equine pregnancies

[23]. As described earlier, FGF9 a fibroblast growth factor is also potentially involved in

endometrial reconstruction [9, 23]. IGFBP1 and IGF1 are both found to be upregulated and

expressed in Klein et al. and Merkl et al. studies on expressions in the endometrium. It is

suggested IGFBP1 and IGF1 have a function as transporters of IGFs between the endometrium

and uterine lumen of the horse [9, 23].

A gene of interest is estrogen receptor 1 (ESR1), involved in the initiation of

luteolysis in cyclic mares. Pre-fixation period ESR1 is down regulated. This is due to the intrusion

of the conceptus [23]

The value of equines in reproductive studies

When it comes to looking at maternal recognition of pregnancy, equines exhibit many

differences in comparison to other domestic ruminants (goats, sheep, pigs, cattle). But there

are also similarities. For example in cattle 70%- 80% of total embryonic loss occurs between

days 8-16 after insemination, a similar time-frame like equines in embryonic loss. Equines

have many advantages in using them as models over other ruminants for the study of

pregnancy in the first month, and for the total embryonic loss that occurs during this time.

There is no other domestic species of which a conceptus can be collected intact i.e within the

first four weeks of pregnancy while undergoing advanced organogenesis. It can be obtained

from the uterus repeatedly, atraumatically and easily with no maternal tissues attached [1].

Although methods and techniques for this research have developed greatly over the past

decade, there are still many unanswered questions in embryonic development and its

interactions within the uterus. Fortunately, this acts as an incentive to undertake this research

and combat the problem of early embryonic loss and extend our knowledge of endometrium-


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embryo cross-talk. Betteridge states that “research will quite certainly also pay dividends in

unanticipated ways in the broader field of reproductive biology” [1].

A disadvantage is that the embryo cannot be accessed easily until around day 6.5

after the blastocyst has been released from the oviduct. At present, surgery or slaughter is the

necessary procedure to collect a blastocyst from the oviduct. Once the blastocyst enters the

uterus it can be accessed easily using harmful techniques. Surgery or slaughter is most

certainly not efficient. It is possible to successfully culture cleavage stage embryos in vitro.

There is not a system that can meet the requirements of a growing blastocyst and provide it

with the correct environment to allow it to produce a capsule as it does in the uterus [26].

Therefore, there is a need to improve methods to produce embryos in vitro.

From an immunological point of view, the embryo- maternal interactions has

highlighted events that struggle to be detected in other species [25].

Conclusion

It is clear that embryo- endometrial interactions are not detected by a single gene, but

by a series of individual genes and pathways regulating specific phases of endometrial

function, conceptus development, and the influences the conceptus has on endometrial

function during maternal recognition of pregnancy. In only very recent years has there been

much understanding of the embryo- maternal cross talk. Transcriptional profiling of the equid

endometrium during MRP has only been published this year, of which are the first few of

their kind to be reported. Common features have been found in equine and human

pregnancies [11, 25, 27]. Equines have proven to be a positive addition in the studies of

early reproductive research. Many questions remain in this area of research, such as further

investigations into communication between the embryo and endometrium via gene

expression, the physical relationship between the capsule and the yolk sac and its signalling

efforts towards the endometrium and the conceptus synchronisation in the uterine cavity. By

focusing on particular genes that are expressed during early pregnancy it will be possible to

improve methods for diagnosis and treatment of mares that are prone to losing the conceptus

during the first three weeks of pregnancy. There is the need for rigour in the study of

embryonic development and its products presumed to play an important role in

communication between embryo and mother in any species.


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