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BIOLOGY OF REPRODUCTION 54, 294-302 (1996)
Maternal Recognition of Pregnancy'
R. Michael Roberts, 2 Sancai Xie, and Nagappan Mathialagan
Departments of Animal Sciences and Biochemistry, University of Missouri-Columbia
Columbia, Missouri 65211
ABSTRACT
Maternal recognition of pregnancy reflects the various ways in which the mother responds to the presence of aconceptus within her
reproductive tract. A part of the biochemical information she senses may be irrelevant to pregnancy outcome, but some reflects the
attempts by the conceptus to gain some measure of control over corpus luteum function, uterine blood supply, the mother's immune
system, and other aspects of maternal physiology. Most probably as a result of ongoing genetic conflict between the mother and the
conceptus, a bewildering range of placental structures and trophoblast signaling mechanisms are encountered in eutherian mammals
despite the fact that the uterus and conceptus share acommon interest, which isthe successful outcome of the pregnancy. Here we review
some of the ways that such mammals maintain luteal function inearly pregnancy and briefly discuss the related topics of embryonic loss
and maternal monitoring of conceptus fitness. We next address the view that the conceptus isan intruder, recognized as foreign by the
mother, that likely survives by using strategies analogous to those employed by successful parasites. Inthis context, we describe the
pregnancy-associated glycoproteins, multiple isoforms of which are released at the trophoblast-endometrial interface during pregnancy
of ungulate species. These molecules, which are structurally related to pepsin, are proposed to bind and sequester antigenic peptides,
thereby serving an immunoprotective role.
INTRODUCTION
allograft-it does not become vascularized by the host's
blood supply as would a true tissue graft-it must nevertheless take steps to avoid a losing confrontation with the
maternal immune system.
Considering their importance, it is surprising how little
of these physiological processes is understood. In a few
species, a coherent picture is beginning to emerge as to how
the early embryo intervenes to extend the life span of the
CL, but the basis for long-term CL maintenance generally
remains obscure. In many species, ovarian progesterone
production is required throughout gestation and not simply
as a stop-gap measure until the placenta can take over [7].
There is little known about how the embryo manipulates
maternal blood supply and endometrial structure in the
early stages of pregnancy. Possibly some of the already
identified growth factors are implicated, but unique systems
could also be at play. During implantation, some insight into
the proteinases, proteinase inhibitors, and cell adhesive molecules that regulate trophoblast invasion is being realized.
There are many species, however, in which access to maternal resources through endometrial invasion is not an option. In the pig, for example, the uterine epithelium is never
broached by the trophoblast, but maternal and placental
capillaries do reach within a few microns of each other by
penetrating deeply between the epithelial cells on the two
opposing surfaces of the uterus and chorion [8]. How the
conceptuses attract maternal capillaries remains unknown.
Finally, the topics of immune recognition and fetal allograft" escape have been discussed in many thousands of
papers, but with little resolution of the main issues.
In this symposium paper, we first briefly review what is
known about the ways in which the early embryo intervenes to rescue the CL, thereby concentrating on a narrow
Although there is evidence that the mother quickly becomes cognizant of the cleavage-stage embryo within her
reproductive tract and reacts to its presence [1-4], her early
responses do not generally appear to be essential for the
pregnancy to proceed. For example, embryo transfer to
nonmated females can be accomplished successfully in cattle 2 wk after conception provided that the recipient is suitably synchronized with the donor [5]. Even in the human,
transfer can be delayed almost until the blastocyst would
begin to implant [6]. There comes a time in all species, however, when the presence of a conceptus (the embryo plus
its membranes) or several conceptuses in the case of litterbearing animals, is necessary to keep the uterus in a continued receptive state. The normal cyclic regression of the
corpus luteum (CL) in all species where the length of the
pregnancy exceeds that of the ovarian cycle must, for example, be prevented in order to maintain progesterone production. The conceptus has also to ensure that an adequate
supply of maternal blood reaches the sites of placentation
so that gas and nutrient exchange are facilitated. As a result,
the endometrium may become considerably modified as the
placenta gains its foothold. Finally, there is little doubt that
expression of paternally inherited and fetal genes ensures
that the conceptus is recognized as foreign by the mother.
Although it may be misleading to regard the placenta as an
'Contribution from the Missouri Agricultural Experiment Station Journal Series Number
12,375). This research was supported by grants HD29483 and HD21893 from the National
Institutes of Health.
2
Correspondence: R. Michael Roberts, Departments of Animal Sciences and Biochemistry, 158 Animal Sciences Center, University of Missouri-Columbia, Columbia, MO 65211.
FAX: (314) 882-6827; e-mail: ansc0135mizzoul.missouri.edu
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MATERNAL RECOGNITION OF PREGNANCY
aspect of maternal recognition of pregnancy. We shall then
present our own slant on some related topics: early embryonic loss and maternal monitoring of embryo fitness. Next,
we address the concept of the conceptus as an intruder,
antigenically foreign to the mother, that likely survives by
avoidance of direct immune confrontation. Finally, we close
the review by describing some work from this laboratory
on an unusual group of molecules, the pregnancy-associated glycoproteins, that are released at the trophoblast-uterine interface in a surprisingly broad collection of mammals.
Their function remains unknown, but their predicted ability
to bind basic, hydrophobic peptides has suggested to us
that they may play a role in minimizing maternal recognition
of paternal and fetal immune epitopes. Throughout the review we have taken the position that genetic conflict [9] has
played a dominant role in influencing interactions between
the mother and the conceptus, but it should not be overlooked that both have common genetic interests in that half
their genes are shared. Pregnancy is probably neither a
completely harmonious relationship, in which the conceptus and uterus function together for the entire benefit of the
former, nor one that is one-sidedly parasitic.
Before proceeding into these topics, a brief overview of
placental morphology is provided. The placenta is the
bridgehead between the fetus and the mother, central to the
evolutionary success of eutherian mammals and the main
source of endocrine signals from the conceptus, yet its
structure is disconcertingly variable between taxonomic
groups. Such variability has profound implications with regard to all manner of maternal-conceptus communication
during pregnancy.
PLACENTAL ORGANIZATION
Whereas the progression of the one-celled zygote to the
multicellular blastocyst and the development of the inner
cell mass into the fetus are processes in which easy comparisons can be made across species, the ways in which a
mature placenta can form are bewilderingly diverse. This
variability in placental structure has always been a source
of confusion to the evolutionary biologist. Why would an
organ apparently not exposed directly to external evolutionary pressure and so pivotal for reproductive success
show such diversity? Haig [9] has suggested that the evolution of the mammalian placenta has been driven by the
powerful forces of genetic conflict between the mother and
her developing offspring. Rather than pregnancy being a
cooperative, nurturing relationship, Haig argues that the
genes expressed by the mother and conceptus often have
quite different immediate interests and goals and are therefore frequently at odds. For example, what might be the
optimal amount of nutrients for the fetus could be so costly
to the mother that her ability to care for other offspring
could be compromised. There is insufficient space in this
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review to develop Haig's argument at length, but its theme
of conflict will reappear several times.
Blastocysts, once they have escaped from the zona pellucida, develop in markedly different ways. Mouse and human blastocysts almost immediately attach to the uterine
surface and begin to invade, although the manners in which
the two trophoblasts breach the epithelium and colonize
the stromal layer below are quite distinct [10]. Nevertheless,
in both species trophoblast tissue gains access to the maternal blood supply and placentation is of the hemochorial
type. In the less invasive endotheliochorial placentation noted
in dogs and cats, for example, blood vessels of the mother
become surrounded by trophoblast but are not penetrated.
The uterine epithelium is, however, completely eroded at the
zone of attachment and invasion. At the other extreme, pigs
and related ungulates such as camels and hippopotamuses
demonstrate an epitheliochorial placentation in which both
the uterine epithelium and the outer placental cell layer of
chorion (trophectoderm) remain intact throughout pregnancy [11]. As a result, fetal and maternal blood supplies
remain separated by six cell layers. Horses and related species exhibit a variation on this theme. A band of chorionic
cells (the girdle) migrates from the chorion and lodges in
the endometrium to form transient islands of trophoblast
cells called endometrial cups. The trophoblast of ruminant
ungulate species also shows limited invasiveness. In this
type of placentation, which was originally classified as syndesmochorial but is now more appropriately termed synepitheliochorial [12], binucleate cells migrate from the chorion and fuse with uterine epithelial cells. As a result of
additional cell-to-cell fusions, an extensive syncytium develops between the maternal and fetal tissues in the sheep.
In cattle, cell fusion is less extreme, and the barrier layer
becomes partially populated with trinucleate cells. Goats,
deer, and other ruminant ungulates all present slight variations on this synepitheliochorial scheme.
Beyond this basic organization of cell layers, there are
additional levels of complexity in the gross forms adopted
by mammalian placentas [13]. A placenta may be diffuse or
cotyledonary, discoid or zonary, for example. These features will not be discussed here, but they undoubtedly influence the interchange of biochemical information between mother and fetus.
From this brief discussion it should not be assumed that
the structures of the placental membranes are completely reliable as taxonomic guides to mammalian classification (but
see [14]). For example, the ungulates and near ungulates (including elephants, manatees, and hyraxes) do not all possess
the superficial types of placentation described above [11]. The
hyraxes, for example, have the hemochorial type. Similarly,
lemurs and related prosimians have epitheliochorial placentae, much like those of equids and quite unlike those of higher
primates [15]. It remains debatable whether noninvasive placentation should be regarded necessarily as primitive. Mam-
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mals with epitheliochorial or synepitheliochorial placentae
range from the tropics to the arctic and are found on both
land and in the sea. They include the largest and many of the
most successful terrestrial mammals.
Whatever evolutionary pressures led to the extraordinary
diversity of placental types, the consequences for maternal
fetal interchange of information have been profound. For
example, the syncytial trophoblast layer of the hemochorial
placenta of the human has immediate access to nutrients in
maternal blood and can release placental hormones directly
into the maternal bloodstream. As a result, the mother probably has to take strong countermeasures to avoid direct exploitation of her nutritional resources by the fetus [9]. On
the other extreme, the pig fetus must obtain nutrients such
as iron or vitamin A, which are transported on macromolecules, in the form of uterine secretions (or histotrophe). Special absorptive structures called areolae, develop opposite
the openings of each uterine gland to take up maternal secretory products [16]. Conversely, trophoblast signals must
negotiate an intact uterine epithelium in order to gain access
to the maternal circulation. It is not surprising, therefore,
that the biochemical signals utilized by the placentas of the
pig and human are quite different. Rapid evolution of placental structure has not only had an impact on placental
nutrient exchange but also on the way that the mother and
conceptus exchange information.
RESCUE OF THE CORPUS LUTEUM: DIVERGENT
EXAMPLES OF TROPHOBLAST SIGNALING
The ways in which different species counteract the maternal drive towards luteolysis during early pregnancy are
remarkably diverse. In man and the anthropoid apes, luteolysis is initiated by an intraovarian mechanism whose basis
remains unclear, although many believe it requires local production of PGF2 a [7, 17]. Luteolysis in these species is avoided
by the intervention of blood-borne chorionic gonadotrophin
(CG) produced by implanting trophoblast cells [18]. The CG
probably binds to LH receptors, hence stimulating progesterone production, but also exerts a protective action against
PGF 2 ,. Its action is, therefore, luteotrophic and luteoprotective [19, 20]. Ever increasing doses of CG are required to
maintain the CL of haplorhine primates such as Rhesus, and
it remains unclear whether functionality can be prolonged
indefinitely by continued CG injection [19]. It seems possible,
therefore, that additional factors produced by the conceptus
contribute to the long-term viability of the CL of pregnancy.
It is not known whether the strepsirhine primates, such as
the lemurs, which have noninvasive placentae of the diffuse,
epitheliochorial kind [201, even produce a CG. Without early
direct access of the trophoblast to the maternal bloodstream,
direct luteotrophic support of the CL might not be possible.
The idiosyncratically high concentrations of CG attained during the first trimester of human pregnancies, which are orders
of magnitude higher than required to saturate binding to the
LH receptor, are another curious feature of this hormone.
They have been said to reflect an immunoprotective role [21].
Haig [9] on the other hand, argues that the high concentrations of both CG and placental lactogen are probably a result
of the continued escalation of the conflict between the
mother and her fetus. He argues that placental hormones are
endocrine signals primarily serving the interests of the fetus.
The mother most likely has mechanisms to buffer the effects
of these high concentrations of fetal hormones but may also
monitor their concentrations to gauge the size and competence of her fetus (see embryonic loss). The evolutionary
response of the placenta could have been to increase the
production of these hormones, in part to gain better control
of maternal physiology but possibly also in an attempt to
relay false information that may enhance the possibility of
acceptance by the mother. Alternatively, the placental hormones may not be subject to normal feedback regulation and
are produced until some limit of the biochemical pathway is
reached.
Rodents, including the rat and mouse, do not produce a
chorionic gonadotrophin at all. Mature females have incomplete estrous cycles and fail to develop a full luteal phase.
During pseudopregnancy in the rat, which can be induced
by sterile mating or simply by stimulating the cervix of a
receptive female, the cycle is lengthened to 12 days before
the CL regress. This extension of CL life span is the result
of surges of pituitary prolactin release caused by stimulation
of the pelvic nerve and activation of a neural reflex arc (see
[22]). Prolactin has the ability to maintain an active population of LH receptors on luteal cells and may also, by a
manner unknown, provide some protective action against
PGF2 ,. If the rat is pregnant, a series of placental lactogens
and prolactin-like hormones produced by the placenta and
deciduum replace pituitary prolactin at about mid-gestation.
As far as is known, in all ungulates the CL regresses as
the result of release of the luteolytic hormone, PGF2,,, from
the uterine endometrium late in the estrous cycle [23]. Ewes,
cows, gilts, and mares that have had their uteri removed
early in the cycle, for example, can often maintain functional CL for weeks to months and often as long as a normal
pregnancy. If an ungulate is to remain pregnant after mating, this luteolytic signal must be negated. The action of
conceptuses in this instance is to prevent luteolysis, i.e.,
antiluteolytic rather than luteotrophic. Unfortunately, despite what appears to be a common underlying mechanism
for luteolysis, the antiluteolytic devices employed to maintain the CL by different ungulates differ considerably. In the
case of the pig, estrogen released by the trophoblast as it
begins to elongate is probably the initial signal to the
mother that she is pregnant, and injections of large amounts
of estrogen between Days 11 and 15 of the estrous cycle
are usually sufficient to cause a pseudopregnancy that can
last for several months. Introduction of estrogen into the
MATERNAL RECOGNITION OF PREGNANCY
uterine lumen in amounts likely to mimic those produced
by conceptuses during pregnancy, however, usually extends the estrous cycle for only a few days if at all (see [24]).
Therefore, production of other factors is probably necessary
to allow continuation of pregnancy in the pig. The interferon-y and the unusual Type I interferon, expressed just
subsequent to the initial burst of estrogen, have not been
implicated, however [25].
The equine conceptus, unlike that of the pig, does not
elongate. Instead, it forms an encapsulated spherical structure that between Days 12 and 14, the period critical for CL
maintenance, migrates throughout the length of the uterine
lumen many times per day [26, 271. This constant patrolling
may be the key to the mechanism that inhibits PGF2Q release. Whether local paracrine signals are responsible or
whether there is direct contact stimulation of luminal epithelial cells is unclear. What seems likely is that the antiluteolytic factors are neither estrogen, as in swine, nor Type
I interferons, as in cattle and sheep [27, 28]. Curiously, the
horse is the only known species outside the higher primates
that produces a chorionic gonadotrophin, but it does so
transiently and only after pregnancy is well established. In
reality, eCG is misnamed and is a placental form of LH,
unique only in the manner it is processed posttranslationally
[29, 30]. As a product of the invading chorionic girdle cells,
it first appears in maternal blood at about Day 35 [31].
Equine CG does enhance serum progesterone concentrations, largely as a result of stimulating the luteinization of
additional ovarian follicles [31, 32], but its necessity during
pregnancy remains equivocal. Because no endometrial
cups are formed, donkey fetuses carried in mares produce
no CG, but the majority abort between Days 85 and 100
[32]. By contrast, horse embryo transferred to donkey recipients form large endometrial cups and generally go to term.
In cattle and sheep the conceptus begins to intervene in
the luteolytic process three to four days before the CL actually become dysfunctional (see [33]). As in swine and
equids, this period precedes definitive attachment of the
trophoblast to the uterine wall. In these species, the antiluteolytic substance, an unusual Type I interferon (interferon (IFN)-t, has been reviewed on numerous occasions
in the literature (see [33-35]) and will not be discussed in
detail here. It is released in large quantities by the mononucleate cells of the trophectoderm as the blastocyst begins
to elongate at about Days 12-13 in sheep and Days 14-16
in cattle. Despite the fact that IFNT shares most, if not all,
of the biological activities of related Type I IFNs, such as
IFNa, and competes for binding to the same receptor complex, there is some evidence that it may be a more potent
reproductive hormone [34]. Exactly how it acts on maternal
endometrium is unclear, but its presence in the lumen
clearly suppresses the normal pattern of pulsatile release of
PGF2 , in the late estrous cycle, possibly by a mechanism
that involves down regulation of estrogen receptors in the
297
uterine epithelium, which in turn prevents a rise in oxytocin
receptor number [28, 35, 36]. IFNT provision to nonpregnant ewes or cows in the critical period when the CL would
normally regress extends the estrous cycle in most animals
but only occasionally results in a prolonged pseudopregnancy. Again, other factors, possibly placental lactogens [30]
that are produced subsequently to the initial rescuing surge
of IFNT, seem to be necessary to ensure that continued CL
function is preserved.
These examples are good illustrations of the diverse
mechanisms used by different species to intervene in maternal control of CL function. As interest broadens to cover
other groups such as carnivores and insectivores, until now
largely ignored, other strategies will doubtlessly emerge.
Nature has not chosen to present the scientific community
with an easy set of rules to follow.
MATERNAL RECOGNITION OF PREGNANCY
AND EMBRYONIC LOSS
Much prenatal mortality occurs in all mammals. In the
human, it has been estimated that as many as two-thirds of
pregnancies fail in healthy women attempting to conceive
[37]. An even higher amount of embryonic wastage occurs
following in vitro fertilization and embryo transfer [38]. The
majority of these losses occur prior to or during implantation. Consequently, menstrual cycle length is not extended
and the failure of the pregnancy goes unnoticed. In sheep
and cattle, embryonic losses are also relatively high, with
most occurring in the first 3 wk of pregnancy [39, 40]. In
litter-bearing species, such as the pig, between 30% and
40% of conceptuses are lost during the 114-day gestation,
but wastage is again highest in the early stages before placentation is fully established [41].
Embryo transfer experiments carried out in domestic
farm species and rodents have indicated that the uterus
must be approximately in phase with the embryo if the
transfer is to be successful [39-41]. While the pig is particularly poor in accepting out-of-phase embryos, the human
seems rather more tolerant in this regard. Nevertheless, a
receptive window, presumably programmed by maternal
hormones, still exists, and it limits when a blastocyst can
implant [42, 43].
As a result of such observations, natural asynchrony can
probably be regarded as one cause of embryonic loss. Unfortunately, even though embryo transfer and human IVF
studies have given insight into the limits of maternal tolerance
to an out-of-phase embryo, they have provided little information on the physiological causes of embryonic death.
Natural asynchrony between the embryo and the mother
can arise from several causes. The late onset of the first meiotic
division may lead to some oocytes being delayed in their maturation. In pigs and other multiparous species, for example,
ovulation occurs over several hours, and the variability in em-
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ROBERTS ET AL.
bryonic morphology seen at Day 12 has been shown to be
the result of late-ovulated eggs giving rise to less developed
embryos [41, 44]. It is these smaller embryos that subsequently
appear to be lost. A second natural cause of asynchrony may
be due to delayed fertilization [41, 45]. Finally, embryos are
known to cleave at different rates [46].
Why should asynchronous and particularly embryos delayed in their development die? One explanation is that the
uterine milieu is only narrowly permissive, even hostile to
out-of-phase embryos, and places severe restrictions on
which embryos will survive and which will die. A second
is that embryos lagging in development fail to signal to the
mother in sufficient time or in a suitably robust manner to
trigger responses in the mother that may be necessary for
the pregnancy to continue. In both cases the mother provides barriers that the embryo must surmount. Resources
are not to be risked on progeny that are perceived rightly
or wrongly to be genetically flawed or otherwise unfit. The
ability of injected interferons to improve pregnancy success
in ewes may be due to the rescue of embryos delayed, but
otherwise normal in development, that would be otherwise
incapable of rescuing the CL (see [341).
In litter-bearing species there is also likely to be genetic
conflict between conceptuses. Because there are several
conceptuses within the confines of the uterus, there will
ultimately be competition for space and nutritional resources. In the pig, and most probably other species, conflict becomes evident well before space becomes limiting
[24, 411. As discussed earlier, pig conceptuses attain control
over maternal progesterone production at about Days 1112 of pregnancy by releasing estrogen and probably other
factors just prior to the time the CL would normally regress,
but a second consequence of estrogen production is that it
induces the massive release of uterine secretions from the
uterine glandular and surface epithelium [47]. This material--sometimes known as histotrophe-undoubtedly
serves a nutritional role and bathes the rapidly elongating
conceptuses [8]. Curiously, if estrogen is given as a single
i.m. injection prior to Day 10.5 of pregnancy [48] or if the
pigs eat moldy corn containing zeralenone early in their
pregnancy [491, the conceptuses continue to progress for a
while but their development is fatally compromised and the
pig returns to heat ten days or so later than the normal time.
An argument has been made that it is retinol, the precursor
of several potential teratogens including retinoic acid,
which is present at concentrations orders of magnitude
higher in these secretions than in serum, that is embryocidal
[47]. Before Day 10, conceptuses may not have progressed
sufficiently to develop mechanisms to avoid vitamin A toxicity [24], while at Days 11 and 12, advanced conceptuses,
by producing estrogen before their smaller siblings, selectively purge the latter from the litter. Sibling competition in
some species is more extreme than in the pig where embryonic loss is relatively low. For example, the pronghorn
antelope produces about 20 embryos, but only one survives
in each horn. In this instance, embryos have special structures for killing their littermates [50].
MATERNAL IMMUNE RECOGNITION OF PREGNANCY: IS
THERE ANYTHING TO BE LEARNED FROM PATHOGENS?
The goal of pregnancy is for the conceptus to develop
to such a point that survival outside the uterus is possible,
but because the fetal/placental unit is a product of its paternal as well as its maternal genes, it is potentially at risk
as a target for the immune system from conception until
birth. Despite the fact that the uterus can mount an effective
immune attack against infection by bacteria and that antibodies directed against fetal transplantation (MHC) antigens
as well as embryo-specific antigens can be detected in maternal blood [50], the "foreign" component does not usually
become a target for cytotoxic T-lymphocytes. How the histoincompatible conceptus survives in an immunocompetent uterus continues to be a mystery.
It has been emphasized that trophoblast cells may not
express a normal complement of histocompatibility antigens on their surfaces [51], thereby minimizing tissue incompatibility and also reducing the presentation of paternal
antigens in the form of peptides to maternal T-cells. The
mouse trophoblast, for example, is largely MHC-free, while
the human possesses a truncated HLA-G molecule with limited polymorphism and possibly no ability to present antigen, but which creates an illusion of self [52]. In both these
examples the trophoblast has features that are likely to minimize either a vigorous T-cell or natural killer (NK) cell response. A lack of normal MHC display may not be universal,
however. The invading chorionic girdle cells of the horse
trophoblast possesses MHC antigens on their surfaces and
elicit a vigorous immune reaction by the mother [53]. In any
case, the idea that tissues are rejected largely on the basis
of direct presentation of foreign MHC antigens may be exaggerated in the light of recent experiments performed with
donor tissue from mice lacking both class I and class II
MHC. Rejection here was only slightly slower than observed
in mice with MHC histoincompatibility [54]. Indirect responses to nonclassical antigens undoubtedly must play a
part in the rejection process [55]. The fetus, therefore, whatever its MHC status, is still foreign and unlikely to avoid the
scrutiny of immune cells that continually infiltrate the endometrium and scavenge the uterine lumen. Ewes carrying
chimeric sheep-goat conceptuses also produce a strong antibody reaction to goat antigens yet do not reject the fetus
[56]. The conceptus, therefore, is not unlike a multicellular
parasite and, like a successful parasite, has been obliged to
perfect mechanisms that minimize confrontations with a potentially hostile immune system. These mechanisms must
be local and subtle and should not compromise the overall
defense mechanism of the host. If this were to happen, e.g.,
MATERNAL RECOGNITION OF PREGNANCY
by systemic immunosuppression, the host would be constantly under threat from pathogens.
Because the goals of a fetus and an invading parasite are
somewhat alike, it is instructional to examine the strategies
used by the latter to form an extended association with the
host (Table 1). There have been several recent reviews on
this topic [57-60] and so only a selective overview will be
presented.
Defense mechanisms used against an invading pathogen
can be empirically divided into two types: those that are relatively nonspecific and generally rapid and those that are
more precisely targeted and require time to be mobilized [60].
The first kind is likely to be elicited by substances shed or
secreted by the invader. Such chemicals may act as attractants
for macrophages and granulocytes, activate the hydrolytic enzymes of the complement pathways, or induce local cytokine
production. The invaders may become direct targets for NK
cells that are responding to abnormal surface components.
The presence of a preexisting antibody to a surface antigen
may also trigger an immediate response. If the invader survives immediate scrutiny, it must avoid activating cytotoxic Tlymphocytes and any antibody-directed lytic mechanisms that
it might have initiated. Local cytokine production must be
down-regulated or redirected in some manner to avoid massive inflammatory responses. Cytokines and lymphokines,
such as interferons, are agents that inform lymphoid cells
when to divide, where to concentrate, and how to act, but
they may also be immunosuppressive. Therefore, diversionary tactics, often involving molecular mimicry, such as the
production of cytokine-binding proteins or cytokine analogues that put out misinformation, are popular among pathogens and are probably within the capabilities of the placenta,
whose synthetic range is extraordinarily broad. The production of large amounts of IFNy by the pig trophoblast in the
early attachment period [25] and of IFNT by preimplantation
sheep and cow conceptuses [34] cannot be expected to go
unnoticed by the mother. Although the local immune system
may be activated in some manner, the overall effects could
be disruptive. Many pathogens secrete antiinflammatory prostanoids, such as PGE 2 and steroids, while others produce antioxidant enzymes that protect against reactive oxygen species
released by eosinophils and other types of leukocytes. Is it
possible that the production of similar compounds by conceptuses has a similar immunosuppressive intent?
One particularly favored tactic used by pathogens is the
production of proteinase inhibitors, which can be used to
block steps in the complement cascade of reactions and to
neutralize the trypsin-like enzymes and granzymes released
from various granulocytic leukocytes and cytotoxic lymphocytes. One particularly intriguing strategy utilized by virulent strains of Vaccinia virus is to inhibit a cysteine proteinase of the host known as interleukin-1l3 converting
enzyme (often abbreviated ICE), which cleaves the IL-1if
precursor [61]. The inhibitor is one of several members of
299
TABLE 1. Some of the tactics used by invading pathogens to avoid immune destruction.a
*
*
*
*
*
*
*
*
*
*
*
Protective coats and immunoneutral surfaces
Genetic switch mechanisms
Proteolytic destruction of host "effector" proteins
Synthesis of antiinflammatory agents
Production of enzymes that protect against or eliminate destructive chemicals
released by host attack cells
Inducing production of antibody types that do not direct a cellular response
Subverting the action of cytokines by molecular mimicry
Interference with leukocyte adhesion
Inhibition of T- and B-cell proliferation
Inhibition of proteinases produced by attack cells
Interference with antigen presentation
aFor more specific information, reader is referred to references 57-60.
the serpin superfamily of proteins encoded by Vaccinia and
other viral genomes. Some others may block antigen presentation by the cells they infect [62]. Indeed, inhibiting proteolytic events in antigen-presenting cells may be a key to
several evasive strategies practiced by chronic parasites.
The hookworm Ascaris lumbricoides, for example, produces a polypeptide that inhibits several aspartyl proteinases [63], including cathepsin E [64], while certain filarial
worms release a cystatin-like molecule, which can probably
inhibit cathepsin B [65]. Both cysteinyl and aspartyl proteinases are regarded as key components of the steps that lead
to antigen presentation at the cell surface. The inhibitors
probably block the intracellular hydrolysis of polypeptides
released by an invading pathogen and taken up by antigenpresenting cells. The end result would be a failure of cytotoxic T-lymphocytes to respond to alloantigen.
It is unclear which, if any, of the tactics listed in Table 1
are pursued during pregnancy to protect the fetus, but they
offer attractive possibilities. Like any parasite, the trophoblast is likely to possess a broadly based, flexible system of
immunoprotection. It would be most unlikely to rely upon
a single mechanism.
PREGNANCY-ASSOCIATED GLYCOPROTEINS (PAG):
POTENTIAL INVOLVEMENT IN MATERNAL RECOGNITION
OF PREGNANCY
The properties of these curious molecules are summarized in Table 2. A more detailed review is presented below.
a) Discovery of PAG
In 1982 the partial purification and characterization of a
pregnancy-specific protein (PSP-B) was reported from cattle
[66]. Over the next decade PSP-B was never thoroughly
characterized, although a range of molecular weights was
published. More recently, Zoli et al. [67] isolated several isoforms of a pregnancy-associated glycoprotein (PAG) from
bovine placental tissue. The methodology was similar to
that of Butler et al. [66] in that an antiserum was first generated against placental extracts from which antibodies
300
ROBERTS ET AL.
TABLE 2. Summary of properties of pregnancy-associated glycoproteins.
*
*
*
*
*
Members of aspartic proteinase gene family
Exhibit typical bilobed structure of pepsin
Multiple gene family
Majority appear not to be proteolytically active
Expressed abundantly introphectoderm, including trophoblast binucleate cells,
from preimplantation until term
* Some can be detected in maternal serum following implantation
* Exhibit a range of molecular sizes, but majority have apparent mass of
-70 kDa
* Occur inall ungulate species so far examined
against common maternal antigens had been removed by
immunoadsorption. This reagent was then used to track one
antigenic form of PAG (here called PAG-1, because there
are many kinds) through a series of purification steps that
led to the isolation of an acidic glycoprotein product of Mr
-67 000. It is now clear that PSP-B and PAG-1 are identical
in sequence [68].
The presence of PAG-1 (or PSP-B) in blood serum has
provided the basis of a potentially useful pregnancy test in
cattle. The antigen generally becomes detectable by about
Day 20 postbreeding; most pregnant cows appear to have
measurable levels by Day 24 [69-71]. This period coincides
with the time that the trophoblast attaches itself firmly to
the uterine wall and when placentation begins. In cattle,
concentrations of the antigen rise gradually during gestation
and peak just prior to parturition. The antiserum raised
against PSP-B (PAG-1) has since been used to monitor pregnancy in a variety of domestic and wild species [72, 73].
b) The cDNA Cloning of PAG
The antiserum generated by Zoli et al. [67] was used to
screen cDNA libraries prepared from mid-pregnant placental
tissues of cattle and sheep [74]. The cDNA so isolated were
approximately 1.3 kbp in length and coded for polypeptides
of 380 and 382 amino acids in cattle and sheep, respectively.
Each polypeptide had a predicted signal sequence of 15 residues and, in the case of the bovine PAG, where an amino
terminal sequence of the purified glycoprotein had already
been obtained [71], it was clear that a pro-peptide of 38 residues had also been removed. Most surprisingly, the PAG
clearly belonged to the aspartic proteinase gene family and
especially resembled pepsin, where the extent of sequence
identity was close to 50%. Despite the resemblance to pepsin,
the purified bPAG-1 product had no detectable proteolytic
activity towards denatured hemoglobin, yet it bound tightly
to immobilized pepstatin and so presumably had an intact
substrate-binding cleft.
Examination of the normally conserved regions in the
amino terminal and carboxyl terminal lobes, which together
constitute the catalytic centers of aspartic proteinases, revealed yet further surprises. Ovine PAG-1 (oPAG-1) lacked
one of the invariant aspartyl residues considered essential
for catalysis and could not, therefore, be active, while
bPAG-1 had an alanine substituted for a normally invariant
glycine residue. A molecular modeling study performed in
Dr. Jordan Tang's laboratory at the University of Oklahoma
has indicated that the presence of this relatively bulky
methyl side chain on alanine would also cause loss of enzymatic activity [741. Thus, it seems unlikely that either ovine
or bovine PAG-1 are proteinases. Consequently, the removal of the pro-peptide from bPAG-1 is probably not
achieved by autocatalytic mechanisms. More likely some
other proteinase is responsible.
c) Expression of PAG-1 during Pregnancy
Immunocytochemical studies have shown that PAG-1 in
both cattle and sheep is localized to granules within binucleate cells of the outer layer of the placenta [74, 75]. These
cells start to appear just prior to the time that the preplacenta (trophoblast) attaches to the uterine wall (around
Day 17 in cattle) and constitute the invasive components of
the placenta. After they fuse with uterine epithelial cells, the
dense secretory granules discharge their contents from the
basolateral face of the uterine epithelium [12].
The PAG-1 mRNA is expressed abundantly from the time
binucleate cells first form, just prior to implantation, until
term (-145 days in sheep, -280 days in cattle) [74]. In this
regard their expression differs markedly from that of the
other trophoblast-specific product of ruminant species discussed earlier, the IFNz. The latter is localized to the mononucleate cells of the trophectoderm and is expressed for
only a few days prior to the beginning of implantation [34].
d) The PAG Are a Highly Polymorphic Group of Molecules
Nucleic acid screening of the mid-pregnant bovine cDNA
libraries under low stringency has identified cDNA for additional PAG molecules. The first to be characterized encoded a protein with 87% amino acid sequence identity to
bPAG-1. It has been named bPAG-1,,, [76]. A second highly
abundant cDNA, which represents a 376 amino acid polypeptide (PAG-2) with only 58% amino acid sequence identity to bPAG-1, was isolated by a similar screening strategy
[77]. More recently, screening of a Day 25 library has revealed yet additional cDNA representing PAG-related molecules that are distinct from PAG-1 and PAG-2 (S. Xie and
R.M. Roberts, unpublished results). A similar wide range of
cDNA have been identified from ovine and porcine cDNA
libraries ([78]; S. Xie, B. Szafranska and R. Nagel, unpublished results).
A high degree of polymorphism among oPAG has been
demonstrated by purifying individual variants from culture
medium of explant tissue from Day 100 placenta (S. Xie and
R.M. Roberts, unpublished results). Sequential use of ammonium sulfate precipitation, Cibacron blue dye adsorption
chromatography, and several high performance ion-exchange and gel filtration steps gave pure products varying
MATERNAL RECOGNITION OF PREGNANCY
in size, charge, and ability to be recognized by some of the
antisera in our possession. When these purified fractions
were microsequenced, it was clear that their amino termini
differed from PAG sequences inferred from cloned cDNA
as well as from sequences reported by Atkinson et al. [791
for PAG-like molecules that bound the monoclonal antibody SBU-3. There seems little doubt that many different
forms of PAG exist, differing both at their amino-termini as
well as elsewhere in their sequences.
An analysis of the genes for bPAG-1 [76] and bPAG-2 (J.
Green and S. Xie, unpublished results) has confirmed the
9-exon structure typical of all aspartic proteinase genes. By
using a probe encompassing exons 7 and 8, which represents one of the most conserved regions of PAG relative to
others in the gene family, Southern genomic blotting of bovine genomic DNA has revealed the presence of many PAGrelated genes [76].
e) A Proposed Functionfor PAG
Data obtained by molecular modeling the structures of
b- and oPAG-1 relative to pepsin and related aspartic proteinases have shown that the PAG have a well-defined peptide-binding cleft with a preference for basic, relatively
hydrophobic polypeptides and confirmed that they are unlikely to have enzymatic activity (K. Guruprasad, T. Blundell, and R.M. Roberts, unpublished results). A less detailed
comparison of three different bovine PAG, two ovine PAG,
two recently described porcine PAG, and an equine PAG
has suggested that the peptide-binding specificities of the
molecules are unalike.
Two possible functions for PAG suggest themselves [741.
The first is that they could be hormones, which, by virtue
of their binding clefts, are able to bind specific cell surface
receptors on maternal target cells. The discovery of many
different PAG and the realization that each of them is likely
to present a distinct specificity seems not to favor such an
endocrine role. The second suggestion is that PAG sequestered or transported peptides, but again the lack of specificity seems inconsistent with such a theory. On the other
hand, PAG variability may be a key to function. A hypothesis we are presently testing is that PAG are able to compete
with the MHC for peptides processed for antigen presentation. In such a manner, they could interfere with activation
of T-cells.
The PAG are widely distributed. So far they have been
discovered in Artiodactyla and Perissodactyla,mammalian
orders whose ancestors diverged at least 55 million years
ago. It seems unlikely that they are merely inconsequential
curiosities. Instead, we predict they have an important function in pregnancy.
The rapid proliferation of PAG subtypes within a species
might also be yet another reflection of genetic conflict. If so,
it would seem likely that the mother would take steps to coun-
301
teract effects of PAG on her system. She apparently does. We
have recently shown that the uterine serpins, which are produced by the glandular epithelium of the uteri of sheep, cattle
and pigs in response to progesterone and which are weak
inhibitors of pepsin, are able to bind PAG [80].
ACKNOWLEDGMENTS
We thank our coworkers, particularly Jonathan Green, Bozena Szafranska, and Robert
Nagel, whose unpublished work is quoted in this paper, and Gail Foristal who typed the
paper. The critical reading of the manuscript by Dr. David Haig and three anonymous reviewers was invaluable.
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