The TGF-β Superfamily and Its Roles in the Human Ovary and Placenta

SCIENTIFIC BASIS OF RE.PRODUCTIVE MEDICINE

THE TGF-p SUPERFAMILY AND ITS ROLES IN THE HUMAN OVARY AND PLACENTA Chun Peng, PhD Department of Biology, York University, Toronto ON

Abstract: The transforming growth factor-~ (TGF-~) superfamily consists of a large group of growth and differentiation factors, such as TGF-~s, activins, inhibins, growth and differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). These molecules act through specific receptor complexes that are composed of type I and type II serinelthreonine receptor kinases. The receptor kinases subsequently activate Smad proteins, which then propagate the signals into the nucleus to regulate target gene expression. Several ligands in this family, such as TGF-~s, activins, inhibins, BMP-IS, and GDF-9, play important roles in regulating human ovarian functions, includ, ing follicle development and maturation.Activin and TGF-~ are also involved in regulating placental development and functions. Abnormal expression or function of these ligands has been found in several pathological conditions. This review summarizes the role of the TGF-~ superfamily in human ovarian and placental regulation and function, and the potential clinical implications. Resume : La superfamille du facteur de croissance transformant beta (TGF-~) est composee d'un groupe considerable de facteurs de croissance et de differenciation, notamment les TGF-~, les activines, les inhibines, les facteurs de croissance et de differenciation (FCD) et les proteines morphogenetiques osseuses (PMO). Ces molecules agissent par I'intermediaire de complexes de recepteurs specifiques composes du recepteur de la serine-kinase et de la threonine-kinase de type I et de type II. Le recepteur de ces kinases active ensuite les proteines Smad, qui propagent alors les signaux dans Ie noyau de maniere it reguler I'expression du gene cible. Plusieurs ligands de cette famille, notamment les TGF-~, les activines, les inhibines, les PMO-IS et les FCD-9, jouent des roles importants dans la regulation des fonctions ovariennes humaines, particulierement dans Ie developpement et la maturation des follicules. L'activine et les TGF-~ participent aussi it la regulation du developpement et des fonctions placentaires. On a decouvert une expression ou un fonctionnement anormal de ces ligands dans plusieurs etats pathologiques. Cette analyse

KeyWords Activins; bone morphogenetic proteins; transforming growth factor-~; ovary, human; placenta, human Competing interests: None declared. Received on March 21. 2003 Revised and accepted on June 6. 2003 JOGe

resume Ie role de la superfamille du TGF-~ dans la regulation et Ie fonctionnement ovariens et placentaires humains et ses implications cliniques possibles.

J Obstet

Gynaecol Can 2003;2S( I0):834-44.

INTRODUCTION

The transforming growth factor-/3 (TGF-/3) superfamily consists of a large group of growth and differentiation factors that are involved in regulating many cellular activities, including proliferation, differentiation, apoptosis, migration, and adhesion. 1-4 These growth factors are generally produced by a variety of cells and tissues throughout the body. 1-4 Many of the TGF-/3s are important regulators of reproductive processes, such as hormone production by the hypothalamus-pituitary-gonadal axis4-7 and by the placenta4-6; germ cell development4,8 and maturation4; and normal progression of pregnancy.4.6This review summarizes the structure and signalling of the TGF-/3 superfamily and its roles in human ovary and placenta, focusing on TGF-/3s, activins, inhibins, GOF-9, and BMP-15 (GOF-9B). THE

TGF-~

SUPERFAMILY

LIGANDS

The TGF-/3 superfamily consists of more than 35 peptide growth factors. 5 Based on their sequence similarities, these growth factors are divided into several subfamilies (Table 1), including TGF-/3s, activins/inhibins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GOFs), and glial cell line-derived neurotrophic factors (GONFs).I,5,9 Peptide growth factors in the TGF-/3 superfamily are all produced as prepropeptides, which are then cleaved into a signal peptide, an N-terminal peptide, and a C-terminal mature peptide. I.5 Characteristic of this superfamily, the mature peptides have 6 to 9 conserved cysteine residues, which form intra- and intermolecular disulfide bonds. 5The biologically active forms of the ligands are homo- or heterodimers linked by an intermolecular disulfide bond. I,5 In the cases of GOF-9, BMP-15, and GOF-3, which do not have the cysteine involved in forming intermolecular disulfide bond, the dimers are thought to be held together through noncovalent association. 1.5 OCTOBER 2003

In mammals, the TGF-~ subfamily has 3 members: and TGF-~3, each containing 2 identical chains of the mature peptide.I,J The TGF-~s are usually produced and secreted in an inactive latent form in which the N-terminal peptide, or latency-associated peptide (LAP), remains noncovalently associated with the dimer of the mature peptide. This LAP-TGF-~ complex can also form larger latent complexes with other proteins, such as latent TG F-~ binding protein (LTBP).I,IO Upon dissociation from the latent complexes, TGF-~ becomes activated. Activins and inhibins were initially identified as gonadal peptides that regulate pituitary follicle-stimulating hormone (FSH) secretion. 4, 11 Homo- or heterodimerization of 3 subTGF-~l, TGF-~2,

units, a, ~A, and ~B, generate 5 proteins in the activin/inhibin subfamily: activin-A (~A~A), activin-AB (~A~B), activin-~ (~B~B), inhibin-A (a~A), and inhibin-B (a~B). Additional ~ subunits (~C, ~D, and ~E) have also been discovered in animals and humans. 12-14 Both GDF-9 and BMP-15 were discovered using polymerase chain reaction (peR) with degenerate primers specific for conserved regions of the TGF-~ superfamily. 15,16 Unlike most members of the TGF-~ superfamily that have widespread tissue distribution patterns, these proteins are expressed predominantly in oocytes. 16 ,17 Formation ofhomodimers of BMP-15 and GDF-9 through noncovalent association has recently been demonstrated. 18

TABLE I THE MAMMALIAN TGF-13 SUPERFAMILY: LIGANDS, RECEPTORS, AND SMADS* Subfamily

Ligands Type"

Receptors Type I

Smads Co-R

R-Smads

Smad2/3 Smadl/S

I-Smads

TGF-13

TGF-131/2/3

TI3RII

ALKS ALKI

Activinllnhibin

ActivinAlBIAB InhibinA InhibinB

ActRIIAlB ActRIIA ActRIlB

ALK4

BMP2

BMP2/4

BMPRII

ALK3/6

Smadl/S/8

Smad6/7

BMPS

BMPS/6/7/8

BMPRII ActRIl

ALK2/3/6

Smad 1/518

Smad6/7

BMP3

BMP3 GDFIO

? ?

ALK2? ?

? ?

? ?

BMP9

BMP9/10

?

?

?

?

BMPII

BMPII GDF8

? ActRIlB

? ?

? Smad3

? ?

GDFS

GDFS/6/7

BMPRII ActRIIAlB

ALK6

Smad 1/5/8

Smad6/7

GDFI

GDFI/3

?

?

?

?

GDF9

GDF9 BMPIS

BMPRII BMPRII

? ALK6

Smad2 SmadllS/8

? Smad6/7

Other members

Nodal AMH GDFIS

ActRIlB AMHRII ?

ALK7/4 ALK6 ?

Smad2/3 Smadl ?

Smad7 ? ?

GDNF

GDNF Neuturin Artemin Persephin

Betaglycan PI20

ALK4

Smad7 Smad6/7

Smad2/3 Smad7 Blocks activin signalling Blocks activin signalling

RetlGFRal RetlGFRa2 Ret/GFRa3 Ret/GFRa4

*'rGF-I3: transforming growth factor-l3; TI3RII: TGF-13 type II receptor; ALK: activin receptor-like kinases; ActRIIA: activin type IIA receptor; ActRIIB: activin type liB receptor; BMP: bone morphogenetic protein; BMPRII: BMP type II receptor; GDF: growth and differentiation factor; AMH: anti-Mullerian hormone; AMHRII: AMH type II receptor; GDNF: glial cell line-derived neurotrophic factor; GFR: GDNF family receptor.

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OCTOBER 2003

RECEPTORS

With the exception of the GDNF subfamily, which interacts with Ret tyrosine kinase and a GDNF family receptor a (GFRa), 19,20 ligands in the TGF-f3 superfamily interact with cell surface receptors that have serinelthreonine kinase activities (Table 1).1-5,9,10,21,22 A signalling receptor complex is composed of 2 distinct receptor proteins, type I and type II, of which seven type I receptors and 5 type II receptors have been identified in mammals. I,5,9 The seven type I receptors are referred to as activin receptor-like kinases (ALKs): ALK4 is involved in activin signalling; ALK5, and less certainly ALK1, is a specificTGF-f3 receptor; ALIG and ALK6 mediate the actions of several BMPs; and ALK7 and ALK4 can bind with Nodap,10,22 The five type II receptors are activin type lIA (ActRIIA) and type lIB (ActRIIB) , TGF-f3 type II (Tf3RII), BMP type II receptor (BMPRII), and anti-Miillerian hormone (AMH) type II receptor (AMHRII). Although the type II receptors were named based on their first ligand identified, they can interact with different ligands when associated with appropriate type I receptors (Table 1).5,9,10,22 Both type I and type II receptors have a single transmembrane domain, an extracellular ligand binding domain that is rich in cysteine residues (also known as an activin receptor domain), and an intracellular serine/threonine kinase domain. Type I receptors also have a segment of glycine and serine residues (GS box) preceding the kinase domain. l -4 Both type I and type II receptors must be phosphorylated in order to become active kinases. I -4 Autophosphorylation of type II receptors has been demonstrated, and it is believed that they exist as constitutively active kinases. I ,9 Activins and TGF-f3s first bind to their type II receptors and the type I receptors are subsequently recruited into the complex,I-4,9,1O,21 whereas BMPs interact directly with both type II and type I receptors. 1-4,9,10,22 Following receptor binding, tetrameric receptor complexes composed of two type I and two type II receptor molecules are formed. 1,9 The type II receptors then phosphorylate the type I receptors at their GS domain, 1-4,9 whereupon the phosphorylated rype I receptors can then transmit signals to downstream signalling molecules, Smads (Figure).9,10,21,22 Inhibins are functional antagonists of activins and neutralize activin actions in many systems. 23 ,24It has been suggested that inhibins abolish activin signalling by disrupting the assembly of activin receptor complexes. 23 ,24 Betag1ycan, a proteoglycan known as TGF-f3 type III receptor, can form a complex with inhibin A and ActRIIA, thereby blocking activin signalling. 23 This provides a potential mechanism by which inhibin-A neutralizes activin actions. Inhibin-B, on the other hand, inhibits activin signalling by binding to an inhibin binding protein, formerly known as P120, and forming complexes with ALK4 and ActRIIB.24 BINDING PROTEINS AND ACCESSORY RECEPTORS

In addition to signalling receptors, other proteins can also bind to TGF-f3ligands and modulate their actions. Binding proteills bind JOGC

to ligands, making them unavailable for their signalling receptors, 1,10,25 whereas accessory receptors facilitate the binding of ligands to their receptors, enhancing the receptors' activities.I,lo As stated above, TGF-f3 is synthesized and released ill a latent form by associating with LAP, and in this association, it is not recognized by its receptors and is biologically inactive. 1,10 Follistatins were initially identified as activin binding proteins, but it is now known that they can also interact with other members oftheTGF-f3 family, including BMP-15. 26 Two isoforms offollistatin, follistatin-288 and follistatin-315, both bind to activins with high affinity and prevent the interaction between activins and their receptors, thereby neutralizing activin actions. 25 Follistatin-288 can also enhance endocytotic degradation of activins. 25 Several secreted proteins, such as Noggin, Chordin, DAN, Cerberus, and Gremlin, which play critical roles during embryonic development, have also been identified to antagonize BMP action by binding to BMPs and thereby preventing interaction between BMPs and their receptors. 10 When betaglycan, which has high affinity for all isoforms ofTGF-f3s, binds to TGF-f3s, it increases TGF-f3 activities by increasing the affinity ofTGF-f3s, especially TGF-f32, to the type I and type II signalling receptors. I ,1O Endoglin, a glycoprotein with some structural similarity to betaglycan, has also been shown to act as an accessory receptor for TGF-f3.I,1O INTRACELLULAR SIGNAL TRANSDUCERS

Smad proteins are the only family of intracellular signalling molecules whose role in TGF-f3 signalling is well characterized. In vertebrates, this family is composed of 8 members, which are grouped into 3 subfamilies based on their functional characteristics. I,9,10,21 The first subfamily is receptor-regulated Smads (R-Smads, including Smads 1,2,3,5, and 8) that are activated through phosphorylation by type I receptors. The second subfamily, common Smad (Smad4), forms complexes with phosphorylated R-Smads. The inhibitory Smads (I-Smads, Smads 6 and 7) form the third subfamily and inhibit signalling by the TGF-f3 superfamily. The R-Smads can be further grouped into 2 subclasses: activin- orTGF-f3-activated Smad (AR-Smads) and BMP-activated Smads (BR-Smads).9 The AR-Smads include Srnad2 and Smad3 and are activated by ALK4, ALK5, and ALK7. The BR-Smads, Smad1, 5, and 8, can be activated by ALIG and ALK6. ALK1 and ALK2 also activate Smad1 and Smad5 (Table 1).9 All R-Smads have 2 conserved domains, MH1 and MH2, located at the N- and C-terminals, respectively. 1,10 They also have a SSXS phosphorylation motif at the C-terminals.I,1O Once phosphorylated, all the R-Smads can form oligomeric complexes with Smad4 and become translocated into the nucleus. I,9,1O,27 Smad4 also has the MH 1 and MH2 domains but lacks the C-terminal phosphorylation motif found in R-Smads. The I-Smads have only the MH2 domain and they inhibit signalling by the TGF-f3 superfamily by preventing the OCTOBER 2003

phosphorylation of R-Smads by type I receptors or the association between R-Smads and Smad4. 1,9,10,27 Both Smad6 and Smad7 can inhibit the activation of Smad 1, Smad5, and Smad8, whereas only Smad7 has been proven to inhibit Smad2 and Smad3 signalling (Figure).1,9,10,27

with transcription coactivators or corepressors, respectively (Figure). 1,9,10,27

R-SmadlSmad4 complexes are transcriptional active, in that they have been shown to regulate the transcription of a variety of genes. Although most R-Smad/Smad4 complexes can bind to a specific DNA sequence known as Smad Binding Element (SBE), such binding usually has relatively low affinity and specificity, thus Smads generally bind to DNA together with cell-specific DNA binding cofactors. 1,9,10,27 Smad signalling can lead to gene induction or repression, through its interaction

Studies in animals have shown that the TGF-~ superfamily is involved in regulating ovarian functions, such as follicular cell proliferation,4.7 differentiation,4,7 follicle development,4,7 and atresia,4,7 as well as oocyte maturation. 4,7 Results from limited studies carried out in humans suggest that the TGF-~ superfamily also plays important regulatory roles in the human ovary, 4,7,28-48 although its precise involvement in human ovarian functions is still not clear and remains to be further defined.

THE TGF-~ SUPERFAMILY IN THE HUMAN OVAJff

BM P2/4151 617181 I 5 GDF5/6/7

ActivinA/AB/B TGF- ~1/2/3

AMH

Nodal GDF8

RII

target gene

Signal transduction pathways for the TGF-~ superfamily. Activins, TGF-~s, Nodal, and GDF8 first bind to their specific type II receptors, which then recruit and phosphorylate corresponding type I receptors. The type I receptors (ALK4, ALK5, and ALK7) phosphorylate and thereby activate AR-Smads (Smad2 and Smad3). Most BMPs and GDFs interact with their specific type I and type II receptors directly. The type I receptors (ALK2, ALK3, and ALK6), upon phosphorylation by their type II receptors, activate BR-Smads (Smad I, SmadS, and Smad8). Following activation by their type I receptors, both AR-Smads and BR-Smads interact with Smad4 to transduce the signals to the nucleus. Within the nucleus, the R-Smad/Smad4 complex interacts with DNA-binding cofactors, transcription coactivators, or corepressers to regulate target gene expression.

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OCTOBER 2003

ACTIVINS AND INHIBINS

Production of activins and inhibins has been demonstrated in the human ovary.28-37 Activin and inhibin subunits are expressed in cultured human granulosa-luteal cells and in intact ovarian follicles,28-31 and can be detected in human follicular fluid, as well as in the culture medium of granulosa cells or cumulusoocyte complex. 32-37 Activin receptor and follistatin mRNAs are expressed in human granulosa-luteal cells and cumulus cells 28 ,38,39 and activin type II receptors (ActRIIA and lIB) immunoreactivities have also been detected in ovarian follicles 40 along with Smad2 and Smad4 proteins. 4o Many studies conducted in animal models have shown that activins and inhibins are important autocrine and paracrine regulators of follicular functions. 4,41,42 In general, act ivins induce FSH receptors and promote proliferation of granulosa cells, follicle development, and oocyte maturation. 4,41,42 Activins also inhibit androgen production,4 but have stimulatory or inhibitory effects on progesterone and estradiol production, depending on the degree of granulosa cell differentiation. 4 Inhibins generally have antagonist effects as activins. Similar findings have been reported in the human ovary. In cultured human thecal cells, basal and LH-induced androgen production was inhibited by activin-A, but enhanced by inhibin-A.43,44 Activin-A suppressed basal and human chorionic gonadotropin (hCG)-induced progesterone production in luteal or granulosa-luteal cells. 45 ,46 Similarly, it also inhibited basal as well as inhibin-A and FSH-induced progesterone and estradiol production in cultured granulosa cells taken from antral folliclesY Furthermore, activin-A promoted granulosa cell proliferation. 48 Finally, activin-A stimulated meiotic maturation of human oocytes. 47 TGF-~s

All isoforms ofTGF-~ are expressed in the human ovary.49,50 TGF-~ 1 is expressed in granulosa, theca, and luteal cells, as well as in oocytes, whereas TGF-~2 is primarily expressed in granulosa and small luteal cells. 49 Although expression of TGF-~3 has not been examined for adult human ovary, it has been found to be expressed in oocytes of fetal ovary.50 Both TGF-~151 and TGF-~252 have been detected in follicular fluid obtained from women undergoing in vitro fertilization. In addition to the ligands, TGF-~ signalling components, including receptors and Smads, have been identified in follicular cells and oocytes. 40 ,50,53,54 Although studies in mammals have implicated TGF-~ in various aspects of ovarian development, including steroidogenesis, maturation, ovulation, and follicular atresia, 55 inconsistent findings are often reported, 55 due likely to different animal models or varying degrees of cell differentiation. In a human ovarian theca-like tumour cell line, TGF-~ 1 inhibited forskolin-stimulated androstenedione production, probably by inhibiting expression of steroidogenic acute regulatory protein lOGC

(StAR)56 and 17a-hydroxylase.57 In granulosa-luteal cells, both and TGF-~2 potently induced activin/inhibin ~B subunit mRNA expression. 58 In addition, TGF-~l inhibited apoptosis ofluteinized granulosa cells and may therefore have a luteotropic effect. 59 Furthermore, a role forTGF-~l in promoting oocyte maturation and fertilization has been suggested by the finding that the level ofTGF-~ 1 in follicular fluid at the time of oocyte retrieval correlated positively with the number of fertilized oocytes and subsequent pregnancy.60 Taken together, these findings suggest that TGF-~ acts as an autocrine and paracrine regulator in the human ovary.

TGF-~ 1

GDF-9 AND BMP-IS

Both GDF-9 and BMP-15 are found in human oocytes, 16,17 and GDF-9 mRNA expression begins earlier than that ofBMP-15 during follicle development. 17 GDF-9 mRNA is also expressed in granulosa-luteal cells. 61 Messenger RNAs of several receptors and Smads known to mediate BMP-15 action,62 including BMP type II receptor, ALK6, Smadl, and Smad5, have . recently been found in cultured human granulosa-luteal cells. 63 These findings suggest that GDF-9 and BMP-15 can regulate follicular functions in the human ovary. The critical roles of GDF-9 and BMP-15 in maintaining normal folliculogenesis and fertility have been established in sheep and mice. 64-66 Sheep heterozygous for a point mutation in BMP-15 gene have an increased ovulation rate, but animals homozygous for the same mutation are infertile because follicle growth is arrested at the primary preantral stage. 64 GDF-9 knockout mice are infertile due to the arrest of folliculogenesis in the primary follicle stage,65 whereas BMP-15 knockout mice are only subfertile with decreased ovulation and fertilization rates. 66 Interestingly, there appears to be an interaction between GDF-9 and BMP-15, as BMP-15(-I-)/GDF-9(+I-) females have severe defects in folliculogenesis, cumulus cell expansion, and fertilization. 66 Several studies conducted in humans have shown that GDF-9 can regulate human ovarian functions. 61 ,67 In cultured human granulosa and theca cells, GDF-9 inhibited cAMP-induced steroid production and expression of several key proteins involved in steroidogenesis, including StAR, cholesterol side chain cleavage enzymes, and aromatase. 61 Also, in ovarian follicle cultures, GDF-9 stimulated the development of primordial follicles into secondary follicles. 67 The role of BMP-15 in the human ovary has not been reported. TGF-~

SUPERFAMILY IN THE HUMAN PLACENTA

Evidence suggests that several members of the TGF-~ superfamily, especially activin-A and TGF-~s, regulate human placental development and functions and thereby play important regulatory roles during pregnancy.4-7,68

OCTOBER 2003

TGF-~2 are expressed throughout gestation, TGF-~3 levels are

ACTIVINSIINHIBINS

The production of activins/inhibins by human placenta is well documented. 4,68-70 Activin and inhibin subunit mRNAs and proteins have been detected in the placenta at both mRNA and protein levels. 68 Activin-A is the major form of activins produced by the placenta,32,69 while inhibin-A is the principal form of bioactive inhibins in maternal circulation during pregnancy,68 being mainly produced by the placenta during mid to late pregnancy.68,70 Both type I and type II activin receptor mRNAs are expressed in trophoblast cells from early to late stages of pregnancy.38,71-73 Recently, activin receptor proteins have been found in the syncytium of first and second trimester placentae, and in vascular endothelial cells of villous blood vessels in third trimester.74 Betaglycan, which is now known to interact with both TGF-~ and inhibin, has also been identified in the placenta.75 Finally, Smad2, Smad3, and Smad4 mRNAs have been detectf;d in human trophoblast cells.76,77 Activin-A may play several important roles during pregnancy, from implantation, maintenance of pregnancy, to parturition. 78-83 During early pregnancy, activin-A may promote implantation78 because it has been shown to induce cytotrophoblast outgrowth,79 promote the differentiation of villous cytotrophoblasts into invasive extravillous cytotrophoblasts,79 and stimulate the production of hormones important for the implantation process, such as hCG,4,80 progesterone,4,76,79,80 and estradiol?6,81 Since progesterone is critical in the maintenance of pregnancy,4,78 activin-A, by stimulating its production, may also play an important role in supporting pregnancy. Evidence supporting a role of activin-A in parturition includes the observations that maternal serum activin-A levels increased in women undergoing spontaneous labour 4,80,82 and that activin-A stimulated oxytocin and prostaglandin E2 release,4,80,83 but this notion remains controversial. More recent studies have found that activin-A levels in maternal circulation did not increase during the last 3 weeks of pregnancy or during labour. 84 Moreover, activin receptors were not found in myometrium,85 and there were no differences in either activin-A or activin receptor protein levels between placentae collected after active labour and those from Caesarean births. 85 Thus, whether or not activin-A is involved in parturition requires further clarification. The role of inhibin-A in the placenta, in general, is to antagonize the effect of activin-A. It has been reported that inhibin-A blocked activin-A-induced GnRH, hCG, and progesterone release from cultured placental cells. 86 Similarly, in perifused trophoblast cells, the stimulatory effect of activin-A on hCG secretion was inhibited by inhibin-A.87 TGF.~S

All isoforms ofTGF-~ mRNAs and proteins are expressed in human placenta. 88 TGF-~ 1 is predominantly expressed in syncytiotrophoblast cells, whereas extravillous cytotrophoblast has the highest level ofTGF-~2 expression. 89 While TGF-~l and JOGC

significantly decreased in the third trimester. 9o The differential expression pattern ofTGF-~ isoforms in the placenta suggests that they have different functions during pregnancy. Type I and type II TGF-~ receptors are also expressed in the placenta. 89,91,92 These receptors can be detected in first trimester92 and term placentae,89 and are abundantly expressed in syncytiotrophoblast and extravillous trophoblast cells. 89 TGF-~ is thought to regulate trophoblast invasion but the exact form ofTGF-~s responsible for this action is unclear. Earlier studies demonstrated TGF-~l to inhibit cytotrophoblast proliferation,93 differentiation, migration, and invasiveness of extravillous cytotrophoblasts. 94 However, recent studies using antisense oligonucleotides demonstrated TGF-~3 to be the endogenous form ofTGF-~ that inhibited trophoblast invasiveness. 95 Other actions ofTGF-~ in the placenta include regulation of syncytiotrophoblast cell differentiation and hormone production. TGF-~ has been reported to inhibit the differentiation of villous syncytiotrophoblast% and the secretion ofhCG and human placental lactogen. 81,96 Recently, it was reported that TGF-~ 1 .can inhibit progesterone and estradiol production, most likely through the inhibition of cholesterol transport and aromatase, respectively. 97 Taken together, the evidence suggests that both activin-A and TGF-~s play important regulatory roles in the human placenta. Many effects of activin-A and TGF-~s appear to be antagonistic in nature (Table 2), an interesting observation

TABLE 2 SUMMARY OF ACTIVIN AND TGF.~ ACTIONS IN HUMAN PLACENTA" Activin

TGF.~

NE

L

i

L

NO

L

hCG

i

L

GnRH

i

NO

Cytotrophoblast proliferation Invasiveness Differentiation into syncytiotrophoblast Hormone production

NE

L

Progesterone

i

L

Estradiol

i

L

hPL

*NE: no effect; NO: not determined; hCG: human chorionic gonadotropin; GnRH: gonadotropin-releasing hormone; hPL: human placental lactogen.

OCTOBER 2003

considering both act through a common Smad-signalling pathway. The mechanisms underlying their actions in human placenta remain to be investigated. CLINICAL ASPECTS

Abnormal secretion or function of activins, inhibins, and TGF-~s have been found in several reproductive disorders, such as polycystic ovary syndrome (PCOS) ,98-1 0I ovarian cancers, I02-114 and pregnancy-related diseases. I15-123 Table 3 summarizes reported data on abnormal levels of activins, inhibins, and TGF-~s in reproductive diseases related to the ovaries and placenta. Their potential diagnostic applications in several reproductive disorders are discussed below. POLYCYSTIC OVARY SYNDROME

It has been suggested that a higher ratio of follistatin/activinA may contribute to the pathophysiology of PCOS, since serum follistatin levels were significantly elevated while activinA concentrations were lower in women with PCOS compared to normal controls. 98 ,99 Several studies have reported elevated levels of inhibin a subunit, inhibin-A and inhibin-B in women with PCOS,100 while others have found no difference in either inhibin-Aor inhibin-B levels between women with PCOS and those without. 100 It has been suggested that the dissimilar findings of these studies may be related to different sample sizes, differences in the presence or absence of dominant follicles in the inclusion of women as having PCOS, and differences in body mass index (BMI).100 A significant correlation between BMI and inhibin-B levels in women with PCOS has been demonstrated. 100,101

TABLE 3

ALTERATION OF SERUM LEVELS OF ACTIVINS, INHIBINS, AND TGF-/31 IN REPRODUCTIVE DISEASES· Disorders

pcos

Inhibin-A Inhibin-B Activin-A TGF-/31

i I NC

i INC

Granulosa tumour

i

i

Preeclampsia

i

NC

J.

i

i

i INC

Preterm labour Down syndrome pregnancies

i

i

Hydatidiform mole

i

i

'PCOS: polycystic ovary syndrome; NC: no change.

lOGC

OVARIAN CANCER

The 3 major types of ovarian cancers are epithelial, stromal, and germ cells. 102 Cancer antigen (CA) 125 is a tumour marker effective in the detection of ovarian epithelial cancer, but less effective in detecting stromal cancer. 103 Inhibins, particularly the a-subunit, have been found to be very effective in monitoring stromal cancers.IOI-103 Elevated inhibin-B and inhibin-A levels were observed in patients with granulosa cell tumours. 103-106 Inhibin a-subunit was detected in all granulosa cell tumours, and its level was greatly increased. 103-106 All granulosa cell tumours can be detected using inhibin assays, compared to a 30% detection rate of granulosa cell tumours with CA 125.103 The inhibin assays are also effective in detecting mucinous carcinomas.103 Since epithelial ovarian cancers can be effectively detected by CA 125, and the granulosa cell tumours and mucinous carcinomas are readily detectable by inhibin assays, a combination of CA 125 and inhibin assays may provide an effective tool to monitor the majority of ovarian cancers. 100 TGF-~ may be involved in ovarian tumourigenesis, especially in ovarian epithelial cancer. 107-110 Both tumour suppressing and oncogenic activities of TGF-~ have been reported. I 10 In normal ovarian surface epithelium and early stages of ovarian carcinomas, TGF-~ exerts both antiproliferative and proapoptotic actions. 107-110 However, in later stages, ovarian cancer cells become resistant to the growth inhibitory effect ofTGF-~.1 10,111 TGF-~ also promotes neoplastic behaviours, such as invasiveness. I 10,112 Mutations ofTGF-~ receptors have been found in ovarian cancers. I13,1 14 PREECLAMPSIA

In preeclampsia, cytotrophoblast invasion is restricted and uteroplacental perfusion is reduced. 115 Both activin-A and inhibinA levels in maternal circulation were significantly higher in women with preeclampsia than in women with healthy pregnancies, 116,1l8,120 whereas inhibin-B levels were similar. I 17,120 The marked increase in serum activin-A and inhibin-A levels in preeclampsia appears to be due to increased placental production. 119,121 It has therefore been suggested that activin-A and inhibin-A could be used as markers of preeclampsia. 122,123 A possible association between preeclampsia and TGF-~ has also been suggested from the finding that plasma concentrations ofTGF-~l were significantly higher in women with preeclampsia compared to women with healthy pregnancies. 124 ,125 Placental contents ofTGF-~1125 and TGF-~3126 have also been reported to be increased in preeclamptic pregnancies. Placental explants from preeclamptic pregnancies failed to exhibit spontaneous invasion, but trophoblast invasive capability was restored ifTGF-~3 production was inhibited, 126 suggesting that the overexpression ofTGF-~3 may be responsible for failure of trophoblast invasion in preeclamptic pregnancies. In contrast to these reports, it has also been found that expression ofTGF-~l, TGF-~2, and TGF-~3 in the OCTOBER 2003

placenta remains unchanged in preeclampsia compared with normal pregnancy.90

REFERENCES I.

OTHER POTENTIAL USES OF

2.

ACTIVIN-A AND INHIBIN-A

The possibility of using activin-A and/or inhibin-A as potential diagnostic markers for prediction of pregnancy outcomes has been evaluated. 122,123,127·130 Activin-A and inhibin-A may be useful markers of molar pregnancy as their levels in maternal plasma are elevated in hydatidiform mole. 127,128 In women using assisted reproductive techniques, higher activin-A levels were associated with multiple pregnancies, 129 and lower inhibin-A levels were associated with lack of embryo development. 129,13o Therefore, serum inhibin-A and activin-A concentrations may be useful for early prediction of pregnancy outcome. 122,123 Higher activin-A and inhibin-A levels in maternal serum and in placental extracts have been detected in Down syndrome pregnancies. 131·133 Although activin-A concentrations in the maternal circulation had been found to be higher in women with preterm labour,116 recent studies have suggested that activin-A is not a useful marker for the prediction of preterm delivery.134,135

3. 4. 5.

2002;23:787~23.

6.

7. 8. 9.

10. II.

12.

CONCLUSION 13.

Increasing evidence has indicated that members of the TGF-~ superfamily are important regulators of human ovarian and placental functions. Activins, inhibins, and TGF-~s, as well as BMP-15 and GDF-9, regulate a variety of ovarian functions, such as follicular development and maturation, modulation of gonadotropin activities, and hormone production. Activins, inhibins, and TGF-~s also have important regulatory functions in the human placenta, including trophoblast proliferation and differentiation and hormone production, and may therefore be involved in the initiation, maintenance, and termination of pregnancy. However, many of the physiological roles of these regulators in human ovary and placenta and the mechanisms underlying their action in human reproductive tissues are not yet known. Finally, although activins, inhibins, and TGF-~s have been implicated in the pathophysiology of several conditions related to pregnancy or the reproductive tract, more clinical studies are needed to fully evaluate their usefulness in diagnosis, monitoring, and treatment of these diseases.

14.

15.

16.

17.

18.

19.

20.

ACKNOWLEDGEMENTS

21.

Research in my laboratory was supported by grants from Canadian Institutes of Health Research.

22. 23.

24.

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