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Comparative Aspects of Conceptus Signals for Maternal Recognition of Pregnancy“ FULLER W. BAZER, ROSALIA C. M. SIMMEN, AND FRANK A. SIMMENb Departments of Animal Science and b Dairy Science University of Florida Gainesville, Florida 32611

INTRODUCTION Pregnancy is established and maintained in response to interactions between the conceptus (embryo/fetus and associated membranes and fluids) and endometrium. This chapter describes events associated with establishment of intimate contact between the conceptus and uterine endometrium and the conceptus signals affecting the maternal system which allow establishment of pregnancy and maintenance of an intrauterine environment supportive of pregnancy.

ANTILUTEOLYTIC FACTORS FROM CONCEPTUSES OF PIGS AND SHEEP Conceptus “signals” must be produced at precise times and in adequate amounts for maintenance of morphological and functional integrity of the corpus luteum (CL) and continued production of progesterone by CL, i.e., maternal recognition of pregnancy. I Maintenance of an intrauterine environment that will support conceptus development is dependent on progesterone. Endometria of nonpregnant sheep and pigs secrete Prostaglandin F2, (PGF), the uterine luteolytic factor, in a pattern necessary to induce regression of CL (luteolysis). Subprimate species have a uterine-dependent ovarian cycle; therefore, conceptusesof sheep and pigs produce proteins, steroids and/or prostaglandins which inhibit uterine production of luteolytic amounts of PGF. Primates, however, have a uterine-independent ovarian cycle. Luteolysis appears to be dependent on intra-ovarian mechanisms which are inhibited by a luteotrophic signal, i.e., human chorionic gonadotropin (hCG). Sheep conceptuses secrete an antiluteolytic protein called ovine trophoblast protein1 (oTP-1) between Days 10 and 21 of pregnancy. oTP-I secreted by mononuclear cells of the trophectoderm, has a molecular weight of about 19 OOO and PI values of 5.3 to 5.7.2oTP-1 has high amino acid sequence homology with interferons of the alpha-I1 (IFNU,,) class;3 potent antiviral a ~ t i v i t yantiproliferative ,~ activity (C. Pontzer, F. W. Bazer, and H.M. Johnson, unpublished results), and immunosuppressive a ~ t i v i t y . ~ a

This paper is published as University of Florida Experiment Station Journal Series No. R-Oo760. 202

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oTP-1 is the only antiluteolytic protein secreted by sheep conceptuses. oTP-I inhibits endometrial secretion of luteolytic pulses of PGE2 Endometrium of cyclic ewes releases PGF in a pulsatile manner between Days 14 and 17 of the cycle, which results in luteolysis.2 In a progesterone-dominated uterus, progesterone and estradiol from ovarian follicles interact to induce endometrial receptors for oxytocin. Oxytocin from CL and/or posterior pituitary stimulates uterine secretion of luteolytic pulses of PGF.2 The pattern of oxytocin release is not different between cyclic and pregnant ewes; however, endometrial oxytocin receptor numbers are few or absent in pregnant ewes.6 In the total array of ovine conceptus secretory proteins (oCSP), oTP-I alone has antiluteolytic activity.2 oTP-1 does not interfere with binding of oxytocin to its receptor, oxytocin stimulation of its second messenger system, or oxytocin stimulation of endometrial secretion of PGF when endometrial oxytocin receptors are present.2 Therefore, synthesis of endometrial oxytocin receptors and oxytocin-induced PI turnover and PGF secretion are inhibited when endometrium of cyclic ewes is exposed to oTP-1 on Days 12 through 14.7 Concentrations of prostaglandin EZ(PGE) in utero-ovarian vein blood of pregnant ewes increase markedly on Days 13 and 14 and PGE may play a luteal protective role.2 The PGE may interfere with the luteolytic activity of PGF through a competitive mechanism or alternatively, PGE may accelerate depletion of luteal oxytocin prior to formation of endometrial oxytocin receptors on Days 14 to 16. The theory of maternal recognition of pregnancy in pigs has been reviewed extensively.2 This theory states that PGF is secreted in an endocrine direction (toward the uterine vasculature) in cyclic pigs and transported to the CL to exert its luteolytic effect. However, in pregnant pigs, the direction of secretion of PGF is exocrine (into the uterine lumen) where it is sequestered to exert its biological effects in urero. Available results suggest that estradiol induces endometrial receptors for prolactin in pigs? which may allow prolactin to induce calcium cycling across the epithelium and redirect secretion of PGF into the uterine lumen. Pig conceptuses secrete two major classes of proteins (pCSP) between Days 10.5 and 18 of gestations which include a protein(s) with antiviral activity.9 However, available results indicate that pCSP are not antiluteolytic. lo

RETINOID BINDING PROTEINS Retinol binding protein(s), a major component of pCSP and oCSP, is secreted by pig conceptuses from Day 10 of pregnancy and by endometrial epithelium of pigs in response to progesterone. Retinoids affect gene transcription, cellular differentiation and proliferation, epithelial cell integrity and function, steroidogenesis, hematopoiesis and immune cell function, and interferon production.11 Each of these functions is related to the establishment and maintenance of pregnancy.

PLASMINOGEN ACTIVATOR AND PLASMIN/TRYPSIN INHIBITOR Plasminogen activatorsof both the tissue (tPA) and urokinase (uPA) type are present in uterine secretions of pigs and may be involved in tissue remodeling, e.g., tropho-

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blast outgrowth, implantation and uterine development, including angiogenesis.12 Progesterone-induced inhibitors of plasmin and possibly tPA and uPA are present in uterine secretions of pigs and prevent invasive implantation of pig blastocysts, as is the case for PA from trophectoderrn of rodent blastocysts. Pig blastocysts are invasive when transferred to ectopic sites, but not in the uterine lumen where protease inhibitors secreted by the endometrial surface epithelium are adsorbed to the trophectoderm. This may restrict effects of PA to remodeling of conceptus tissues. Protease inhibitors may also protect secreted proteins from degradation in the uterine lumen or during transplacental transport into the fetal-placental circulation.

ENDOMETRIAL SECRETIONS IN PIGS AND SHEEP Conceptuses of pigs and sheep exhibit noninvasive implantation and depend upon secretions of endometrial epithelium for most, if not all of pregnancy.'*. Peptides and proteins which serve as nutrients, enzymes, regulatory factors, and transport molecules are present in uterine secretions. Enzymes include lysozyme, cathepsins B, D and E, aminoacylpeptidase, plasminogen activator, glucose phosphate isomerase, p-Nacetylglucosaminidase, hyaluronidase, oxytocinase, acid phosphatase (uteroferrin), and a number of glycosidases in pig uterine secretions.12 Glucose, fructose, riboflavin, ascorbic acid, sodium, potassium, calcium, amino acids, retinol, retinoic acid, prostaglandins, and an array of free and conjugated steroids are also found in uterine luminal fluid.13 Uterine secretory activity is greatest when the uterus is under the influence of progesterone. However, estrogens stimulate release of progesterone-induced proteins from secretory granules, as well as synthesis and secretion of a number of other proteins. Estrogens and catecholestrogens secreted by pig blastocysts beginning on Days 10.5- 11 act directly or indirectly to cause vasodilation of arterioles, increase uterine blood flow, stimulate uterine growth and endometrial secretory activity, stimulate uterine production of prostaglandins, increase endometrial receptors for prolactin, and possibly alter effects of cholinergic and adrenergic agents on the uterine endometrium and myometrium.14 In pregnant gilts, there is an increase in calcium on Days 11-12 followed by an increase in total protein, PGF, PGE, sodium, potassium, and selected amino acids between Days 12 and 14. Alanine, glycine, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, and valine are higher in uterine flushings from pregnant than cyclic gilts. 15. Glucose accumulates in the uterine lumen after Day 12, but only in pregnant pigs. Androgens and progestins increase in uterine flushing of pregnant, but not cyclic gilts between Days 9 and 15 after onset of estrus and can be converted to free estrogens by pig conceptuses.16 Pig endometrium and uterine secretions also contain P-endorphin17 and methionine-enkephalin;l*however, their function(s) is not known. There is also an increase in the turnover of norepinephrine and dopamine, as well as an increase in CAMPand cGMP in endometrium of pigs in early pregnancy.19 These results suggest that interactions between the conceptus and endometrium alter uterine and conceptus functions during pregnancy. Proteins secreted by endometrial epithelium of sheep during early gestation have received little attention. Total protein and activities of the enzymes succinic dehydrogenase, glutamic-oxaloacetic transaminase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, P-glucuronidase and glycogen phosphoryl-

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ase are highest in uterine flushings from diestrus ewes and ovariectomized ewes treated with progesterone.20 Endometrial secretory proteins have regulatory roles, e.g. , regulation of the maternal immune system to allow survival of the conceptus allograft. Uterine secretions of the cow, sheep and pig contain proteins that inhibit in vitro proliferation of lymphocytes in response to mitogens. In addition, skin allografts placed into the uterine lumen are protected from rejection in ewes injected with progesterone for 30 days, the time required to stimulate secretion of two major proteins (Mr = 57 and 59k) which are referred to as ovine uterine milk proteins (oUTMP).~'Endometria and conceptuses of sheep, cows and pigs also secrete high molecular weight glycoproteins which have immunosuppressive activity in vitro.22 The mechanism by which these immunosuppressive proteins block a maternal cytotoxic attack on the conceptus is not known. Lack of expression of polymorphic transplantation antigens by trophoblastkhorion may also be critical to survival of the conceptus allograft.23 In pigs, uterine secretory activity is greatest between Days 12 and 16 and Days 35 and 75 of gestation when the progester0ne:estrogen ratio in plasma is greatest. Numbers of endometrial receptors for prolactin are highest (Days 35 to 75) when uterine secretory activity and transport of nutrients into the fetal fluids are greatest.24 In sheep, induction of secretion of oUTMP by progesterone requires about 30 d a ~ s . 2In~pregnant ewes, the major increase in uterine secretory activity occurs after 90 days of gestation and is temporally associated with increased production of ovine placental lactogen (oPL) and increasing concentrations of progesterone in maternal plasma. It has been suggested that oPL and/or prolactin, as well as estrogen, are necessary for induction of progesterone receptors and, in turn, induction of uterine secretory activity by progesterone in ewes.25

UTEROFERRIN, A HEMATOPOIETIC GROWTH FACTOR

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Uteroferrin (UF) is a progesterone-induced glycoprotein secreted by the glandular epithelium of pig endometrium. UF has a Mr of about 35 OOO, a PI of about 9.7, acid phosphatase enzymatic activity and 2 molecules of iron per molecule of UF. The major role of UF has been presumed to be transport of iron from uterine endometrium to conceptus;26 however, UF has recently been shown to have hematopoietic growth factor activity as Localization of UF is in fetal liver, the major hematopoietic organ of fetal pigs, and in yolk sac during early pregnancy.26Iron from UF may be released to ferritin and transferred to erythroblasts by a process called ropheocytosis for synthesis of hemoglobin. Transcriptional activity of the UF gene is low, but distinct between Days 10 and 15 of pregnancy, declines to undetectable levels between Days 16 and 30 and then increases to maximal levels between Days 45 and term (115 days).2* However, translation of mRNA for UF decreases after Day 60 of pregnancy in pigs, but factors inhibiting translation of UF mRNA are not known. UF exists as a purple Mr 35 OOO monomer and a rose-colored heterodimer (Rose) when bound to one of three UF-associated proteins.29The UF-associated proteins have high amino acid sequence homology with serine protease inhibitors; however, the UF-associated proteins are not known to inhibit proteases.M Both UF and Rose have colony forming unit (CFU) and burst forming unit (BFU) activities for erythroid (CFU-

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E) cells, as well as CFU-granulocyte-monocyte/macrophage (CFU-GM) activities for myeloid cells.” Human placenta contains a UF-like protein, the tartrate-resistant Type 5 acid phosphatase, which also has hematopoietic growth factor activity (F. W. Bazer, D. Worthington-White, M. A. Davis, and S. Gross, unpublished results) and 82% amino acid sequence homology with porcine UF.31.32

GROWTH FACTORS OF PREGNANCY Growth factors exhibit diverse modes of action with respect to uterine growth, differentiation and function. At various stages of uterine development, uterine-derived growth factors may, through paracrine and autocrine mechanisms stimulate cellular DNA synthesis, mediate epithelial-stromal interactions, facilitate blastocyst implantation, increase placental vascular circulation, accelerate fetal growth and development and induce extracellular matrix remodeling.33.34 Recent studies to elucidate the roles of endometrial growth factors in maternal-fetal interactions have derived from use of immunologicaland molecular probes to monitor the expression levels of mRNAs and corresponding protein(s) in uterine and fetal (placental) tissues. Understandably, studies have been more extensive with rodent models, but sufficient data have emerged to indicate possible involvement of peptide growth factors in maternal-fetal communication in primates and other mammals. The most extensively characterized peptide growth factors in primate reproduction are epidermal growth factor (EGF) and the insulin-like growth factors (IGFs). EGF actions are targeted to all layers of human uterus including stroma, glandular epithelium and myometrium, based upon detection of specific high affinity EGF receptors in each of these tissues.35 EGF mRNAs are synthesized primarily in uterine epithelia in the m0use;3~however, no studies to date have localized production of EGF in any human uterine cell types. The role(s) of EGF in the uterus may be related to creating a favorable environment for the developing conceptuses, as well as a mitogen for endometrial epithelial cells. The role of EGF in myometrium is less clear, although results of recent studies indicated that it stimulates uterine contractions in vitro.37 Componentsof the IGF pathway have been elucidated in both the human and baboon. In humans, the placenta is a major site of IGF-I and -11 production during pregnancy.38 Highest expression of placental IGF-I mRNAs occur during the first trimester, whereas IGF-I1 mRNA levels peak during the second trimester of pregnancy.39 These developmental changes correspond closely to uterine endometrial steady-state levels of IGF mRNAs in pregnant pigs, in which highest expression of IGF-I and -11 mRNAs are detected during the preimplantation period and mid-pregnancy, respectively.40-4’Similar results have now been demonstrated in sheep endometrium where expression of IGF-I and -11 are temporally correlated with the peri-implantation period.42 The autocrine/paracrine mechanism of IGF action in the placenta likely mediates aspects of placental metabolism, growth, and development. Placental IGFs may also stimulate growth and development of the conceptus, since these tissues contain IGF receptors and respond physiologically to I G F s . ~IGF ~ binding proteins (IGFBPs) are synthesized in endometrial and decidual tissues and may mediate the biological actions of IGFs.4 In humans and baboons, IGFBP-1 is the predominant secretory component during gestation and has been localized in decidual stromal cells,4s amniotic fluid,& and endometrial glandular epitheli~m.~’ It has been shown that IGFBP-1 inhibits

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binding of IGF-I to its receptor in human endometrial membranes in vitro.48A role for IGFBP-I has been postulated in implantation of baboon and human concept use^.^^.^^ Human placenta also contains factor(s) which stimulate endothelial cell proliferation, migration and angiogenesis in vivo and in vitro.49 This factor was found to be homologous to basic fibroblast growth factor (bFGF), which was also localized in conditioned media and homogenates of human fetal membranes and cotyledons from term placentae.49 FGFs (both acidic and basic forms) have been isolated from uterine tissues .of early pregnant gilts,% and recent data suggest the presence of these growth factors in sheep uteri Are specific growth factors responsible for initiation of morphological development of conceptuses from spherical to tubular to filamentous forms and initiation of events leading to the secretion of steroids and/or proteins responsible for maternal recognition of pregnancy? Just prior to initiation of estrogen secretion and morphological transition from spherical to tubular to filamentous forms, there is mesodermal outgrowth from the embryonic disc of pig conceptuses, i.e., Days 10 to 12 of pregnancy, which coincides with peak values for IGF-I in uterine secretions of both cyclic and pregnant pigs. Furthermore, conceptuses of prolific Chinese Meishan pigs develop more rapidly, secrete more estrogens and are exposed to higher amounts of IGF-I in uterine secretions than conceptuses of less prolific Large White pigs.51 Secretion of significant amounts of IFNa-like proteins by pig conceptuses does not begin until Days 13 to 14 or well after mesodermal outgrowth is e~tablished.~ The growth factor associated with inducing mesoderm in amphibians is PFGFS2 and both aFGF and PFGF stimulate proliferation of mesoderm-derived cells. Fibroblast growth factors are present in pig endometrium and uterine secretions,M but temporal associations between secretion of FGFs, morphological changes in conceptuses and initiation of secretion of estrogens by pig conceptuses have not been established. The most dramatic increase in rate of secretion of oTP-1 by sheep conceptuses occurs on Day 13.53Both IGF-I and IGF-I1 are present in Day 13 uterine secretions of sheep;42however, their effects on conceptus development and secretion of oTP-1 are not known. Co-culture of sheep endometrium and conceptuses from Day 16 of pregnancy results in a two- to threefold increase in secretion of oTP-1 which suggests that endometrial factors can influence secretion of oTP-1.2 Rates of development of bovine conceptuses are accelerated by injecting cows with exogenous progesterone (100 mg/day) on Days 1-4 after estrus.” Bovine trophoblast protein-I (bTP-I), an IFNaIl, responsible for maternal recognition of pregnancy in cattle was secreted by conceptuses with accelerated development, but not by conceptuses from control cows. Platelet-derived growth factor (PDGF) mRNA and protein are expressed in human placenta throughout pregnancy, but highest levels are at midtrimester.55 This period coincides with maximum proliferation of cytotrophoblast cells, peak levels of IGF-II and accelerated fetal growth. An autocrine mechanism of control is involved in PDGF action in placenta since coordinate expression of mRNAs (and corresponding protein) for PDGF receptor occur at this time. Human and murine decidua and placenta also synthesize colony-stimulating factor-1 (CSF-1),%957 coincident with expression of its specific receptor (c-fms) in placenta.58Elevated levels of CSF are also found in amniotic fluid throughout gestation, suggesting a role in placental growth regulation. The presence of the transforming growth factor (TGF) family of peptides has not been examined extensively in human uterine tissues. Expression of three forms of murine

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TGF-P (PI, P 2 and 0 3 ) is predominant in the placenta and developing embryos,sg@J although the relative levels in tissues vary. The TGF-Ps may influence mesoderm induction. In frog embryos, a protein called Vgl exhibits sequence relatedness to TGF-P and is localized in the vegetal hemisphere where it may influence mesoderm formation.61-62 Uterine production of TGF-P has not been demonstrated in primates and domestic animals, but potential functions are predicted. In sheep, TGF-P may mediate maternal signaling by inducing trophoblast outgrowth and influencing secretion of oTP-I. In pigs, estrogens produced by the conceptus may induce uterine production of TGF-P, which could then facilitate conceptus development. Conceptus estrogens have been shown to induce endometrial secretion of IGF-I in the pig (unpublished observations from our laboratories); and, similarly estrogen increased levels of three isoforms of TGF-P in rat uterus.63 Within the uterine microenvironment during pregnancy, the developing conceptus is also a source of growth factors. Using the highly sensitive method of polymerase chain reaction (PCR), synthesis of TGFs, IGF-11, PDGF, FGF and interleukin-6 (IL-6) by developing mouse embryos have been demonstrated.-65 Sheep conceptuses also secrete IGF-I and IGF-II,4*and preimplantation pig conceptuses exhibit low, but detectable levels of IGF-I mRNAs.4 Although studies on fetal production of growth factors in primates have not been extensive, existing data from studies of rodents and domestic animals suggest similar responses.

SUMMARY Maintenance of corpus luteum (CL) function is essential for establishment of pregnancy in mammals. Estrogens from pig conceptuses (embryo and associated membranes) initiate events that, with prolactin, redirect secretion of the uterine luteolytic hormone prostaglandin F2a (PGF) from an endocrine (to uterine veins) to an exocrine (to uterine lumen) direction to prevent luteolysis. Ovine conceptuses secrete ovine trophoblast protein-1 (oTP-l), which exhibits high amino acid sequence relatedness with alpha I1 interferons (IFNarr)and inhibits synthesis of endometrial receptors for oxytocin and uterine production of luteolytic pulses of PGF. Estrogens and oTP-1 are local antiluteolytic signals to endometrium, whereas human chorionic gonadotrophin (hCG) appears to have a direct luteotrophic effect on CL. A progestational endometrium secretes proteins that serve as growth factors, transport proteins, regulatory proteins and enzymes, as well as transporting nutrients into the uterine lumen to support conceptus development.

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