Potential roles for endometrial inhibins, activins and follistatin during human embryo implantation and early pregnancy

Potential roles for endometrial inhibins, activins and follistatin during human embryo implantation and early pregnancy
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  TRENDSin Endocrinology & Metabolism Vol.13 No.4 May/June 2002 1043-2760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(01)00559-8 144 Review Members of the transforming growth factor- β (TGF- β) superfamily are produced by the humanendometrium, and have been implicated in regulating angiogenesis, decidualization, implantation andimmunomodulation at the materno–fetal interface [1].Here, we focus on the synthesis and possible functionsof the closely related inhibins and activins in theendometrium. Inhibins and activins are dimericglycoproteins of the TGF- β superfamily, whichshare common β subunits and are functionalantagonists (Fig. 1). Recently, two novel mammalian β subunits were identified, β C and β E [2,3]. Little isknown about the patterns of synthesis or functions of these subunits in human tissues, although they donot appear to play major roles in reproduction ordevelopment in mice [4].Initially identified as endocrine regulators of pituitary follicle stimulating hormone (FSH) release[5],evidence is now accumulating for the important localactions of inhibins and activins, particularly asparacrine regulators of reproductive function [6]. Activin A, in particular, has been attributed with rolesin modulating cellular proliferation, differentiation,apoptosis, tissue remodeling and inflammation[7–11]. Accordingly, activin β subunits are found in all reproductive tissues, in addition to manynonreproductive tissues [12]. The local functions of inhibins are less well known, and synthesis of   subunits is generally limited to endocrine organs. Activin receptors  Activins elicit cellular responses through interactionwith serine/threonine kinase receptors [13]. There aretwo known subtypes of activin receptors – I and II.Ligand binding occurs with the type II receptor,which then recruits and activates a type I receptor. Activated type I receptors phosphorylate intracellularmediators Smad 2 or Smad 3, which then interactwith Smad 4, and this complex translocates to thenucleus, where it promotes gene expression [14].Two type II receptors have been identified, ActRIIAand ActRIIB. Distinct phenotypes are seen inmice deficient in either ActRIIAor ActRIIB, related toeither as yet unidentified differences in mode of actionor non-overlapping patterns of synthesis [15,16]. ActRIIAis essential for folliculogenesis and thereforefertility, whereas mice deficient in ActRIIB canreproduce normally. Similarly, two subtypes of typeIreceptors have been described, ActRIA(ALK2; activinreceptor-like kinase 2) and ActRIB (ALK4). However, ActRIAmight be a receptor for the closely relatedbone morphogenetic proteins (BMPs) and Müllerianinhibitory substance, indicating that ActRIB is themain activin signaling receptor [17–19]. Follistatin:an activin-binding protein The local bioactivity of activin is tightly regulated bythe coordinate synthesis of its binding protein,follistatin, which binds and neutralizes activin withhigh affinity, by preventing interaction with the typeIIreceptors [20]. Two isoforms of follistatin exist (FS-288and FS-315). Circulating activin is commonly detectedin complex with the long-form FS-315 [21], whereasthe truncated FS-288 exhibits high affinity forproteoglycans, and thus is commonly associated withcell membranes [22]. Cell surface-bound FS-288 hashigher neutralizing ability than does FS-315, probablybecause of its additional actions as a clearance factorfor excess activin, by the internalization anddegradation of the follistatin–activin complex [23]. Potential roles for endometrialinhibins,activins and follistatinduring human embryo implantationand early pregnancy Rebecca L.Jones,Lois A.Salamonsen and Jock K.Findlay Rebecca L.Jones*Lois A.SalamonsenJock K.Findlay Prince Henry’s Instituteof Medical Research,POBox 5152, Clayton,VIC3168, Australia.*e-mail: The human endometrium is a remarkably dynamic tissue,undergoing cyclesof proliferation,differentiation and breakdown every 28 days.In preparationfor embryo implantation,the endometrium differentiates or decidualizes,involving widespread morphological and functional differentiation ofendometrial stromal cells.If pregnancy occurs,the decidua regulatestrophoblast invasion and forms the maternal component of the placenta.Uterine remodeling has long been known to be regulated by the ovarian steroidhormones 17 β   -estradiol and progesterone;however,only recently has theimportance of paracrine factors in mediating the cellular and biochemicalchanges been recognized.Many growth factors and cytokines,such as inhibinsand activins,whose expression is generally limited to developmental andpathological states,are produced by actively remodeling endometrial cells,and play crucial roles in regulating endometrial cell function.Here we presentevidence for integral roles for the inhibin and activin family in the paracrineregulation of endometrial receptivity,decidualization and implantation. Published online: 7 March 2002  Inhibin receptors/binding proteins No signaling inhibin receptor has been identified todate; however, most actions of inhibins can beexplained by their antagonism of activin action [24]. Accordingly, inhibin can compete with activin forbinding to ActRIIs, but does not stimulate typeIreceptor phosphorylation, and thus has no signaling capacity. This simple competition model is flawedbecause compared with activin, inhibin exhibits verylow affinity for ActRII. Several high-affinity, inhibin-specific binding proteins have been identified thatare potential receptors, including inhibin-binding protein (INhBP/p120) [25], betaglycan [26], and twonovel binding proteins [27,28]. INhBPmightselectively enhance the interaction of inhibinB with activin type I receptors [29], although binding affinities have not been published. Betaglycan canbind inhibin through the  subunit and enhancethe interaction of inhibin with ActRII, showing thatinhibin can antagonize activin action even at lowconcentrations [30]. Inhibin and activin subunit synthesis in humanendometrium The synthesis of inhibin and activin subunits in the endometrium varies with the different stages of the menstrual cycle and in early pregnantendometrium, as the uterus remodels anddifferentiates to form the decidua [31–34]. Innonpregnant endometrium, inhibin  subunit isfound heterogeneously at low levels in glandularand surface epithelium [32,33] (Fig. 2a). During decidualization at the end of the menstrual cycle and in early pregnancy, immunostaining for  subunit changes from epithelial to stromal cells, and both protein and mRNAare detectable indecidualized stroma [33,34] (Fig. 2a). Inhibin andactivin β  Aand β B subunits show a similar patternof synthesis, with the epithelial cells being thepredominant source in nonpregnant endometrium,and stromal cells beginning to produce β subunitswith the onset of decidualization [31,33] (Fig.2a).Importantly however, β  Aand β B subunits aremaintained in glandular and surface epitheliumduring early pregnancy. Quantitative mRNA expression studies by northern analysis indicatethat the production of all three subunits increasesthrough pregnancy, with maximal synthesis in third trimester decidua [34]. Further evidence forupregulation of inhibin and activin subunitsynthesis with decidualization was obtained fromstudies where the endometrium was extensivelydecidualized by the intrauterine delivery of progestin [33]. TRENDSin Endocrinology & Metabolism Vol.13 No.4 May/June 2002 145 Review TRENDS in Endocrinology & Metabolism α C α Npropre α  subunit β Apropre β A subunit β Bpropre β B subunitInhibin AInhibin BActivin ABActivin AActivin B α    β A  α    β B  β A  β A  β A  β B  β B  β B Fig. 1. Structure ofinhibins and activins.Allinhibin and activinsubunits are synthesizedas preproproteins, whichrequire proteolyticdigestion to yield themature subunit. Themature  subunit (  C)can form dimers with β Aor β B subunits to forminhibin A and inhibin B,respectively. Dimerizationof β subunits alone yieldsactivin A, activin AB andactivin B. NondecidualizedDecidualized (a)(b) (i)(i)(ii)(iii)(iv)(iii)(ii) Fig. 2. Immunohistochemical localization of inhibin and activinsubunits, activin receptors and inhibin- and activin-binding proteins inhuman endometrium. (a) Immunohistochemical localization of (i)  ,(ii) β A and (iii) β B subunits of inhibin and activin in nondecidualizedcycling endometrium and in the decidua of early pregnancy. Positivelocalization is shown by brown immunostaining, whereas nuclei arestained blue with a nonspecific counterstain. (b) Representativeactivin receptor subtypes localized by immunohistochemistry tonondecidualized (i and ii) and decidualized (i, inset) endometrialstromal cells, with additional staining in vascular endothelial cells(ii,inset). Follistatin (FS-288) protein is present in glandular epithelialcells and especially prominent in intensely stained ‘vesicles’ in thesecretory phase (iii), whereas betaglycan was localized byimmunohistochemistry to glandular epithelial cells and stromal cells(iv). Scale bar in (ai) = 100 µ m and applies to all photographs, except(bi) scale bar = 100 µ m and (bi) inset scale bar = 25 µ m. Reproducedfrom [33], by permission of Oxford University Press.  Inhibin–activin dimer formation in humanendometrium The capacity of a particular cell type to producebioactive inhibin and activin dimers depends on thecomplement of subunits that it synthesizes. Theavailability of  subunit probably regulates whetherinhibins or activins are produced. Epithelial cells innonpregnant human endometrium synthesize  and β subunits and therefore have the potential toproduce both inhibin and activin dimers, whereas inearly pregnancy they produce only β subunits and soonly activins will be formed (Table 1). Stromal cellscan produce both inhibins and activins only during and after decidualization. These extrapolations from the synthesis patternsof subunits are supported by the measurement of dimer production from endometrial cells invitro .Isolated epithelial cells can secrete both dimericinhibins and activins, although activin Awasdetectable at farhigher concentrations (200pg per10 5 cells) than inhibin A(6 pg per 10 5 cells) [35].Nondecidualized stromal cells secrete very lowlevels of inhibin–activin dimers in vitro . Recentexperiments indicate that high concentrations of activin A(5000pg per 10 5 cells) are secreted bystromal cells after in vitro decidualization [36],equivalent to levels detectable in maternal serumduring the third trimester of pregnancy [37]. This is consistent with the dramatic upregulation of activin subunit synthesis seen with the onset of decidualization [33]. Activin Aproduction by theearly pregnancy decidua was verified byimmunohistochemical analysis using an antibodyspecifically raised against the dimeric form [31].Overall, the human endometrium appears to be asignificant source of activins and inhibins,especially in the preparation for and during theearly stages of pregnancy. Regulation of inhibin and activin bioactivity within the endometrium Follistatin is produced by endometrial epithelialglands and decidualized stromal cells [38] (Fig.2b).Its synthesis is significantly increased in earlypregnancy, in concert with increased production of activin subunits by decidualized stromal cells [31,33].The colocalization of follistatin with activin subunitsand receptors in decidualized stromal cells is evidencefor the tight local regulation of activin action in theperi-implantation endometrium. Secretion of follistatin from epithelial cells might be important forrestricting the bioavailability of activin within theuterine lumen. Follistatin might also have activin-independent effects, suggested by the distinctphenotypes observed when the genes encoding follistatin and the activin β  Asubunit are deleted oroverexpressed [39,40]. Follistatin can also bind othermembers of the TGF- β superfamily, including inhibin[20] and BMPs 2, 4 and 7 [41,42]. BMPs 2, 4 and 7 areproduced in large amounts in the decidualized mouseuterus [43,44], but have not yet been reported in thehuman endometrium.Inhibin can also regulate activin bioactivity bycompeting with activin for binding to ActRII incomplex with betaglycan. Betaglycan is synthesizedby the human endometrium, by the same cells thatproduce activin receptors and inhibin and activinsubunits [38] (Fig. 2b). Synthesis of betaglycan isupregulated in the decidua of early pregnancy,correlating with increased synthesis of the inhibin  subunit by decidualized stromal cells. This impliesthat inhibin action is important during earlyimplantation and placentation. Betaglycan mightalso be important for presenting TGF- β s(particularly TGF- β 2) to their type II receptor [45],enhancing TGF- β signaling and actions in theendometrium and placenta. Potential endometrial actions of inhibins and activins The roles of endometrially derived inhibins andactivins are not yet known; however, theendometrium is a potential target for activin action.Expression of mRNAfor individual activin receptorsubtypes has previously been shown in isolatedendometrial cells in culture by RT-PCR [35]. Recentresearch describes the detection of mRNAand proteinfor all receptor subtypes (ActRIA, IB, IIAand IIB) inthe endometrium, and their specific localization onlyto stromal cells and vascular endothelium [38](Fig.2b). Furthermore, ActR mRNAexpression ismaximal in the early secretory phase and earlypregnancy, that is, immediately before and during decidualization and implantation. Activin A during stromal cell decidualization  In the secretory phase, the human endometriumundergoes decidualization, in preparation forinvasion by trophoblast if pregnancy occurs.Decidualization involves widespread remodeling of the endometrium, with the differentiation of stromalcells to enlarged, rounded decidual cells. Many paracrinefactors, including prostaglandin E 2 (PGE 2 ) [46],interleukin 11 [47] and corticotrophin-releasing hormone [48], promote decidualization, facilitating the progression of stromal cell differentiation TRENDSin Endocrinology & Metabolism Vol.13 No.4 May/June 2002 146 Review Table 1. Expression of inhibin/activin subunits andactivin receptors in human endometrial cells a,b Female status c ProteinsEpithelial cellsStromal cells Nonpregnant     A /  B    ActR    Pregnant     A /  B    ActR    a Abbreviations: ActR, activin receptors;  , inhibin/activin   subunit;  A, inhibin/activin  A subunit;  B, inhibin/activin  B subunit. b Reproduced from [33], by permission of Oxford University Press. c Expression analyzed in nonpregnant endometrium during themenstrual cycle and in the decidua of early pregnancy.  throughout the endometrium if pregnancy isachieved. In both the rodent and human uterus, theexpression patterns of the genes encoding activin,follistatin and activin receptors are consistent witha role in decidualization [33,38,49]. Activin is apotent cytodifferentiation factor [7,8], and alsoplays an active role in repairing and remodeling tissues [10]. Utilizing an established model of stromal cell decidualization in vitro , it was recentlydemonstrated that activin Apromotesdecidualization [36]. This effect can be inhibited byco-treatment with follistatin, verifying the specificityof the response, and providing strong evidence forautocrine and paracrine actions of activin Ain theprogression of decidualization (Fig. 3). Thedownstream targets of activin Ain decidual cellshave not been explored. However, in other cell types,activin Astimulates the production of many factorsassociated with decidualization, such as PGE 2 ,MMP-2 and fibronectin [50,51]. Further studies arerequired to confirm the role of activin Aindecidualization invivo . Immunomodulatory actions of activin A  Activin Ais also an immunomodulatory factor, similarto the closely related TGF- β s [52]. It is present inelevated concentrations in synovial fluid of patientswith rheumatoid arthritis [53] and inflammatorybowel disease [54]. The role of activin Aduring theinflammatory response is unclear, because it appearsto have both pro- and anti-inflammatory actions, asdefined by its effects on cytokine production [55].Studies in sheep have shown that systemic levels of activin Aincrease very rapidly in response to aninflammatory stimulus, and concentrations peakbefore those of the early-response proinflammatorymediator tumor necrosis factor  [56].This is of relevance to the endometrium, becausemenstruation, and implantation to some extent,share many features with an inflammatory response. At menstruation, there is a large infiltration of polymorphonuclear leukocytes and monocytes, whichproduce a wide array of inflammatory cytokines andenzymes and probably contribute to the focalbreakdown of the endometrium [57]. Asubset of endometrial macrophages and neutrophils synthesizeactivin β  Asubunits premenstrually [32], whereas adistinct subset of macrophages and mast cells presentin proliferating and basal endometrium synthesize β B subunits [33] (Fig. 4). Asimilar pattern of differential synthesis of β  Aand β B subunits is alsoseen in infiltrating leukocytes during wound healing,with the rapid and transient appearance of  β  Asubunit-producing leukocytes, whereas β B subunitproduction appears later and persists during re-epithelization [10]. This suggests that, in somesituations, activins Aand B fulfil different roles, withactivin Aassociated with acute inflammation anddifferentiation, and activin B involved predominantlyin tissue repair. Endometrial angiogenesis  Endometrial endothelial cells synthesize activin β  Aand β B subunits [33], activin receptors and follistatin[38]. Activin Aacts as an autocrine and paracrine inhibitorof angiogenesis, and activin subunits and receptorsare produced by steady-state endothelial cells [58].Conversely, follistatin induces angiogenesis, and isupregulated during endothelial cell proliferation andmigration [59]. Three peaks of angiogenesis occur inthe endometrium: (1) during repair following menstruation; (2) during proliferation underestrogenic control; and (3) during the formation of thespecialized spiral arterioles [60]. Whether activin isinvolved in regulating endometrial angiogenesis isunknown, although no specific correlation of activinor follistatin synthesis with angiogenic incidence hasbeen reported. Potential functions for endometrially derived inhibinsand activins during early implantation In addition to its proposed role in facilitating decidualization, endometrial activin Amight havepotent effects on embryonic and placentaldevelopment during the early stages of humanimplantation. In many species, including  Xenopus and mice, activin Ais involved in embryogenesis[61,62] and is synthesized dynamically with itsreceptors and binding proteins during earlyembryonic development [63,64]. Asimilar role hasnot been verified in humans, although activin βΑ subunits and type I and II receptors are expressed byhuman blastocysts [65]. In later pregnancy, activin βΑ and βΒ subunits are synthesized by many developing gonadal and nongonadal organs [66]. The placenta is an established target for andsource of activins [67]. Activin receptor subtypes are TRENDSin Endocrinology & Metabolism Vol.13 No.4 May/June 2002 147 Review(a)(b) TRENDS in Endocrinology & Metabolism ActRActivin ADecidualizedstromal cellSpiralarterioleActivin Apromotesdecidualizationof neighboringstromal cellsNondecidualizedstromal cell Fig. 3. Potential autocrine/paracrine actions of activin A in the decidualizing human endometrium.Decidualization is initiated in stromal cells surrounding the spiral arterioles, and progresses throughthe endometrium if pregnancy is achieved. During decidualization, stromal cells enlarge and becomerounded, and begin to synthesize and secrete dimeric activin A. Both nondecidualized anddecidualized stromal cells express activin receptors (ActR). Activin A promotes decidualization ofstromal cells in vitro  . It is therefore proposed that decidualized stromal cells surrounding spiralarterioles produce activin A, which can act in a paracrine manner on neighboring nondecidualizedstromal cells to promote their differentiation. In this manner, activin A might facilitate the progressionof decidualization through the endometrium.  expressed by first trimester placenta [68], localizedspecifically to the syncytiotrophoblast [69]. Later inpregnancy, activin receptor expression changes fromsyncytial to vascular, indicating a shift in activinfunction as pregnancy advances. In early pregnancy,activin Amight facilitate implantation, because itpromotes cytotrophoblast differentiation towards aninvasive phenotype, and stimulates the productionof paracrine agents involved in invasion [51], and theplacental hormones human chorionic gonadotropin,17 β -estradiol and progesterone [70,71]. Therefore, activin Awas thought to be animportant mediator of early embryonic and placentaldevelopment, until the surprising finding thatdeletion of the gene encoding the β  Asubunit in miceresulted in apparently normal implantation and onlyminor embryonic developmental defects [40].However, these studies do not explore a maternalcontribution to intrauterine levels of activin A. βΑ subunit-deficient mice do not survive beyondbirth, because of a feeding defect, so it is not possibleto examine their fertility. However, there is substantial evidence that thehuman endometrial epithelium produces inhibin andactivin subunits and dimeric activin A[31–33,35].During the secretory phase of the menstrual cycle(at the time of blastocyst implantation), epithelialcell products are secreted predominantly into theuterine lumen and, accordingly, activin Ais presentin uterine fluid of cycling women [35]. It is probablethat the uterine fluid supporting thepreimplantation embryo contains maternallyderived activin A, and hence potential actions viaactivin receptors expressed on the blastocyst couldbe envisaged (Fig. 5a). Following the initialimplantation events, trophoblast cells invade thematernal decidua and therefore are in intimatecell–cell contact with decidualized stromal cells. Activin Aproduced by decidualized stromal cells [36]might play a further role in augmenting invasion andsupporting placental function (Fig. 5b). ActivinA produced by both maternal endometrial epithelialand decidual cells at the time of fertilization andimplantation could therefore have an important rolein maternal–fetal communication during embryoand trophoblast development, and thus could beimportant for the establishment of pregnancy. Maternal serum levels of inhibins and activins  The endometrium might also contribute to theincreased concentrations of inhibins and activin inmaternal serum during pregnancy [37]. Although theplacenta has been assumed to be the major source[72],other components of the maternal–fetal unit mightcontribute, including the fetal membranes anddecidua [36,73]. Activin Alevels rise dramaticallythroughout pregnancy. Follistatin levels risecoordinately with activin Ain the first half of pregnancy, but in the later stages of pregnancy, theyremain stable, indicating that there is an overallexcess of activin A[37], which might have endocrineactions at this time. The reason for elevatedcirculating inhibins and activins is unknown.Regardless, maternal serum concentrations of inhibins and activins are useful clinical indicators of fetal and maternal health during pregnancy [74,75]. TRENDSin Endocrinology & Metabolism Vol.13 No.4 May/June 2002 148 Review TRENDS in Endocrinology & Metabolism 0123MPESMSLS4 ∗ (a)(b)(g)(h)(c)(c) (i)(ii) (e)(e) (i)(ii) (d)(d) (i)(ii) (f)(f) (i)(ii) lele          β    A  -  p  o  s   i   t   i  v  e   l  e  u   k  o  c  y   t  e  s 0123MPESMSLS ∗          β    B  -  p  o  s   i   t   i  v  e   l  e  u   k  o  c  y   t  e  s Stage of menstrual cycle Fig. 4. The presence of activin β A and β B subunits in endometrial leukocytes. (a) β A subunits in asubset of leukocytes (le) within the endometrial stroma in the mid-secretory phase of the menstrualcycle. (b) β B-positive leukocytes in proliferative-phase endometrium. Serial tissue sections werestained with leukocyte subtype markers to determine the identity of β subunit-positive migratory cells(arrows). (c) A macrophage (i) immunostained for CD68 and (ii) staining positively for the β A subunit.(d) A neutrophil (i) identified by neutrophil-elastase immunostaining and (ii) also positive for the β Asubunit. (e) Macrophages positive for (i) CD68 and (ii) the β B subunit. (f) Mast cell (i) identified bymast-cell-tryptase staining and (ii) also expressing the β B subunit. The histograms show semi-quantitative scoring for leukocytes positively stained for (g) β A and (h) β B subunits at the differentstages of the menstrual cycle. Abbreviations: ES, early secretory phase; LS, late secretory phase;M,menstrual; MS, mid-secretory phase; P, proliferative. Significantly higher numbers (* P  < 0.05) of β A-positive leukocytes were detected in the late-secretory phase of the cycle, whereas maximalnumbers of β B-positive leukocytes were identified in the proliferative phase of the menstrual cycle.Scale bars = 100 µ m (a,b); 20 µ m (c,d); and 50 µ m (e,f).Reproduced from [32,33], by permission ofOxford University Press.


Apr 29, 2018
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