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Activin A selectively represses expression of the membrane-bound isoform of Kit ligand in human fetal ovary

2009; Elsevier BV; Volume: 92; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2009.03.095

1556-5653

Andrew J. Childs, Richard A. Anderson,

Pluripotent Stem Cells Research

Germ cell–expressed activin A promotes germ cell survival and proliferation indirectly, possibly by maintaining ovarian pregranulosa cells in an undifferentiated state and preventing primordial follicle formation until oocytes have matured sufficiently. We have previously demonstrated that recombinant activin A can repress the expression of Kit ligand (KITLG) in cultures of human fetal ovaries, and here we extend this finding to show that this repression acts selectively on the membrane-bound isoform of Kit ligand without affecting the soluble form, providing a potential mechanism for suppressing direct interaction between somatic and germ cells and thus inhibiting precocious primordial follicle formation. Germ cell–expressed activin A promotes germ cell survival and proliferation indirectly, possibly by maintaining ovarian pregranulosa cells in an undifferentiated state and preventing primordial follicle formation until oocytes have matured sufficiently. We have previously demonstrated that recombinant activin A can repress the expression of Kit ligand (KITLG) in cultures of human fetal ovaries, and here we extend this finding to show that this repression acts selectively on the membrane-bound isoform of Kit ligand without affecting the soluble form, providing a potential mechanism for suppressing direct interaction between somatic and germ cells and thus inhibiting precocious primordial follicle formation. The formation of the primordial follicle pool during fetal life is a critical determinant of reproductive lifespan in the adult female. Follicle formation involves breakdown of syncitial "nests" of germ cells, resulting in increased association of germ cells and somatic cells, a process which begins around 18 weeks gestation in the human ovary. Although a number of germ cell–intrinsic and –extrinsic factors have been implicated in regulating the timing and extent of primordial follicle formation, the mechanisms of the process remain poorly understood (1Skinner M.K. Regulation of primordial follicle assembly and development.Hum Reprod Update. 2005; 11: 461-471Crossref PubMed Scopus (367) Google Scholar). Activin, a member of the transforming growth factor β superfamily of growth factors with pleiotropic roles in both male and female gonado- and gametogenesis, is a key regulator of follicle formation in human and mouse (2Martins da Silva S.J. Bayne R.A. Cambray N. Hartley P.S. McNeilly A.S. Anderson R.A. Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation.Dev Biol. 2004; 266: 334-345Crossref PubMed Scopus (96) Google Scholar, 3Bristol-Gould S.K. Kreeger P.K. Selkirk C.G. Kilen S.M. Cook R.W. Kipp J.L. et al.Postnatal regulation of germ cells by activin: the establishment of the initial follicle pool.Dev Biol. 2006; 298: 132-148Crossref PubMed Scopus (161) Google Scholar). In vitro culture of human fetal ovaries in the presence of activin A results in an increase in germ cell proliferation (2Martins da Silva S.J. Bayne R.A. Cambray N. Hartley P.S. McNeilly A.S. Anderson R.A. Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation.Dev Biol. 2004; 266: 334-345Crossref PubMed Scopus (96) Google Scholar). In vivo, systemic administration of activin to neonatal mice before primordial follicle formation results in a substantial increase in prepubertal primordial follicle number (3Bristol-Gould S.K. Kreeger P.K. Selkirk C.G. Kilen S.M. Cook R.W. Kipp J.L. et al.Postnatal regulation of germ cells by activin: the establishment of the initial follicle pool.Dev Biol. 2006; 298: 132-148Crossref PubMed Scopus (161) Google Scholar). In the human fetal ovary, activin is produced by germ cells in syncitial clusters, but it is down-regulated as these break down and is undetectable in oocytes in primordial follicles (2Martins da Silva S.J. Bayne R.A. Cambray N. Hartley P.S. McNeilly A.S. Anderson R.A. Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation.Dev Biol. 2004; 266: 334-345Crossref PubMed Scopus (96) Google Scholar, 4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). Although activin receptors are expressed by both germ and somatic cells in the human fetal ovary (2Martins da Silva S.J. Bayne R.A. Cambray N. Hartley P.S. McNeilly A.S. Anderson R.A. Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation.Dev Biol. 2004; 266: 334-345Crossref PubMed Scopus (96) Google Scholar), the phosphorylated (active) forms of their downstream transcriptional regulators SMAD2 and SMAD3 are detectable only in the somatic cells, indicating that these cells are the targets of activin signaling (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). The effects of activin on germ cell survival and/or proliferation must therefore occur indirectly through juxtacrine/paracrine interactions with neighboring somatic cells rather than autocrine interactions with germ cells. Kit signaling has diverse roles in numerous developmental processes, including melanogenesis, hematopoiesis, gametogenesis, and promoting cell proliferation, migration, and survival. Work from our laboratory and others has recently identified the Kit ligand (KL)/c-Kit receptor system as a potential activin-regulated somatic-to-germ cell signaling pathway in human fetal and mouse ovaries (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar, 5Pangas S.A. Jorgez C.J. Tran M. Agno J. Li X. Brown C.W. et al.Intraovarian activins are required for female fertility.Mol Endocrinol. 2007; 21: 2458-2471Crossref PubMed Scopus (101) Google Scholar). In cultures of disaggregated human fetal ovaries, recombinant human activin A (rhActA) repressed expression of the KITLG gene (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar), consistent with increased Kitlg expression in the ovaries of mice with targeted disruptions of activin subunits, and repression of Kitlg in rhActA-treated murine granulosa cells (5Pangas S.A. Jorgez C.J. Tran M. Agno J. Li X. Brown C.W. et al.Intraovarian activins are required for female fertility.Mol Endocrinol. 2007; 21: 2458-2471Crossref PubMed Scopus (101) Google Scholar). In human fetal ovaries, c-Kit is expressed in undifferentiated proliferating germ cells and in oocytes in primordial follicles, but is absent from germ cells in intermediate stages of maturation, i.e., in clusters (6Robinson L.L. Gaskell T.L. Saunders P.T. Anderson R.A. Germ cell specific expression of c-Kit in the human fetal gonad.Mol Hum Reprod. 2001; 7: 845-852Crossref PubMed Scopus (95) Google Scholar, 7Hoyer P.E. Byskov A.G. Mollgard K. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries.Mol Cell Endocrinol. 2005; 234: 1-10Crossref PubMed Scopus (91) Google Scholar). This pattern of Kit down-regulation as ovarian germ cells enter meiosis until oocytes are assembled into primordial follicles is conserved in several mammalian species, indicating that repression of Kit signaling may be required for normal entry into and progression through meiosis (8Manova K. Nocka K. Besmer P. Bachvarova R.F. Gonadal expression of c-Kit encoded at the W locus of the mouse.Development. 1990; 110: 1057-1069PubMed Google Scholar, 9Clark D.E. Tisdall D.J. Fidler A.E. McNatty K.P. Localization of mRNA encoding c-Kit during the initiation of folliculogenesis in ovine fetal ovaries.J Reprod Fertil. 1996; 106: 329-335Crossref PubMed Scopus (40) Google Scholar). Strikingly, the expression pattern of the activin βA subunit is the direct opposite, being detectable only in germ cells in nests and down-regulated before follicle formation, so that the expression of c-Kit and the activin βA subunit are mutually exclusive (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). Kit ligand expression is detectable in both germ and somatic cells in the human fetal ovary before meiosis (∼7 weeks gestation), but is restricted to somatic cells around germ cell clusters or forming primordial follicles at later gestational ages (∼18 weeks) (7Hoyer P.E. Byskov A.G. Mollgard K. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries.Mol Cell Endocrinol. 2005; 234: 1-10Crossref PubMed Scopus (91) Google Scholar). As a result of alternative splicing of the KITLG gene transcript, two protein isoforms of KL are produced. Inclusion of exon 6, which encodes a proteolytic cleavage site that is efficiently cleaved by membrane-associated proteases, produces a soluble extracellular form known as KL-1, and exclusion of this exon produces the predominantly membrane-associated KL-2 isoform (10Huang E.J. Nocka K.H. Buck J. Besmer P. Differential expression and processing of two cell associated forms of the Kit-ligand: KL-1 and KL-2.Mol Biol Cell. 1992; 3: 349-362Crossref PubMed Scopus (273) Google Scholar). Kit ligand 1 therefore can act as a paracrine factor over distance, whereas KL-2 acts in a juxtacrine fashion. Expression of the two isoforms is developmentally regulated in many tissues, including the ovary, suggesting that changes in the KL-1:KL-2 ratio during development may have functional significance (10Huang E.J. Nocka K.H. Buck J. Besmer P. Differential expression and processing of two cell associated forms of the Kit-ligand: KL-1 and KL-2.Mol Biol Cell. 1992; 3: 349-362Crossref PubMed Scopus (273) Google Scholar, 11Manova K. Huang E.J. Angeles M. De Leon V. Sanchez S. Pronovost S.M. et al.The expression pattern of the c-Kit ligand in gonads of mice supports a role for the c-Kit receptor in oocyte growth and in proliferation of spermatogonia.Dev Biol. 1993; 157: 85-99Crossref PubMed Scopus (266) Google Scholar). Kit ligand 2 is considered to be the more potent isoform, because it triggers more sustained tyrosine kinase activation and greater receptor stability on binding c-Kit than occurs with KL-1 (12Miyazawa K. Williams D.A. Gotoh A. Nishimaki J. Broxmeyer H.E. Toyama K. Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-Kit gene–encoded protein than its soluble form.Blood. 1995; 85: 641-649Crossref PubMed Google Scholar). In the absence of soluble KL, female germline development proceeds normally (13Tajima Y. Moore M.A. Soares V. Ono M. Kissel H. Besmer P. Consequences of exclusive expression in vivo of Kit-ligand lacking the major proteolytic cleavage site.Proc Natl Acad Sci U S A. 1998; 95: 11903-11908Crossref PubMed Scopus (72) Google Scholar), but it is compromised at various stages between primordial germ cell migration to primary follicle development in KL-2–deficient mutants depending on the severity of the genetic lesion (14Flanagan J.G. Chan D.C. Leder P. Transmembrane form of the Kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant.Cell. 1991; 64: 1025-1035Abstract Full Text PDF PubMed Scopus (610) Google Scholar, 15Kuroda H. Terada N. Nakayama H. Matsumoto K. Kitamura Y. Infertility due to growth arrest of ovarian follicles in Sl/Slt mice.Dev Biol. 1988; 126: 71-79Crossref PubMed Scopus (109) Google Scholar, 16Huang E.J. Manova K. Packer A.I. Sanchez S. Bachvarova R.F. Besmer P. The murine Steel panda mutation affects Kit ligand expression and growth of early ovarian follicles.Dev Biol. 1993; 157: 100-109Crossref PubMed Scopus (189) Google Scholar). Kit ligand 2, but not KL-1, has also been reported to induce the up-regulation of Kit expression by oocytes cultured on feeder cells expressing either one of the isoforms (17Thomas F.H. Ismail R.S. Jiang J.Y. Vanderhyden B.C. Kit ligand 2 promotes murine oocyte growth in vitro.Biol Reprod. 2008; 78: 167-175Crossref PubMed Scopus (52) Google Scholar). Because KL can induce Kit expression, and activin A represses KL expression, we hypothesised that a possible role of activin may be to repress KL in somatic cells during meiosis until oocytes have reached a sufficient stage of maturation to form primordial follicles. Given the comparative importance of the two KL isoforms in ovarian development, we speculated that the repressive effect of activin may be exerted differentially on the two KL isoforms, with repression of the membrane-associated form of potentially greater functional significance in inhibiting oocyte maturation and follicle formation. To test this, we investigated the effect of activin treatment on the expression of transcripts encoding the soluble and membrane-associated isoforms of KL in cultures of disaggregated human fetal ovaries. Ovaries were obtained from five morphologically normal human fetuses after medical termination of pregnancy. Consent was obtained in accordance with national guidelines, and the study was approved by the Lothian Research Ethics Committee. Pregnancies were terminated by treatment with mifepristone (200 mg orally) followed 48 hours later by misoprostol (800 μg, three hourly per vaginam). Gestational age was determined by ultrasound before termination and confirmed subsequently by direct measurement of foot length. Ovaries were dissected into Hank balanced salt solution (Sigma-Aldrich, Poole, U.K.) and disaggregated and cultured as described previously (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar) in the presence or absence of 100 ng/mL rhActA (R&D Systems, Abingdon, U.K.). After culture, RNA was extracted and cDNA prepared as described previously (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). Because primers that only amplify the KL-2 isoform cannot be obtained, we measured the expression of total KL (KL-1 and KL-2) and soluble KL-1 alone by conventional 20 μL reverse-transcription polymerase chain reaction (PCR) reactions using primers amplifying either both KL spliceforms (total KL: forward 5′- CATTGTTGGA TAAGCGAGATGG-3′; reverse 5′- CACTCCACAAGGT CATCG-3′), specific to the soluble isoform (KL-1: forward 5′- CATTGTTGGATAAGCGAGATGG-3′; reverse 5′- GCCTTCCTATTACTGCTACTGC-3′), or to the housekeeping gene RPL32 (primers to which have been reported previously [4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar]). A single PCR reaction was performed for each amplicon on each specimen. Reaction concentrations and PCR cycling conditions used were as described previously (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). The PCR products were separated on 2% Tris-acetate-EDTA agarose gels and photographed under ultraviolet light. Images were imported into ImageJ (National Institutes of Health, Bethesda, MA) for densitometric analysis (Fig. 1A ). Pixel intensities of total KL and KL-1 PCR products were corrected to that of the housekeeping gene RPL32. Data are the mean ± SEM of five independent experiments. Significance was determined by t test. Culture of disaggregated human fetal ovaries for 24 hours in the presence of rhActA resulted in a significant repression of total KL expression (55 ± 12% of untreated control; P=.025), but had no effect on the level of KL-1 expression (107 ± 49% of control; ns) (Figs.1A and 1B). The reduction in total KL expression in response to activin A reported here is similar to what we have reported previously (∼40% reduction in total KL [4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar] vs. ∼45% reduction here). For this reduction in total KL to occur in the absence of a change in KL-1 indicates that the change in total KL transcript levels detected in response to activin A–treated cultures results almost entirely by specific repression of KL-2 expression, indicating that activin A selectively represses the expression of the membrane-bound isoform of KL. The data presented here provide direct evidence in support of our earlier hypothesis that one role of germ cell–expressed activin during human fetal ovarian development may be to repress membrane-associated KL expression in somatic/pregranulosa cells, thus preventing the induction of c-Kit receptor expression in oocytes and possibly subsequent promotion of meiotic arrest and primordial follicle assembly (Fig. 1C) (4Coutts S.M. Childs A.J. Fulton N. Collins C. Bayne R.A. McNeilly A.S. et al.Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression.Dev Biol. 2008; 314: 189-199Crossref PubMed Scopus (54) Google Scholar). A role for KL as a suppressor of meiotic progression has been previously proposed, because treatment of rat oocytes with soluble KL can inhibit germinal vesicle breakdown and prevent extrusion of the first polar body (18Ismail R.S. Dube M. Vanderhyden B.C. Hormonally regulated expression and alternative splicing of kit ligand may regulate Kit-induced inhibition of meiosis in rat oocytes.Dev Biol. 1997; 184: 333-342Crossref PubMed Scopus (44) Google Scholar). The data presented here may support a similar role for KL in the fetal ovary and provide a mechanism for the promotion of primordial follicle number induced by activin administration to neonatal mice (3Bristol-Gould S.K. Kreeger P.K. Selkirk C.G. Kilen S.M. Cook R.W. Kipp J.L. et al.Postnatal regulation of germ cells by activin: the establishment of the initial follicle pool.Dev Biol. 2006; 298: 132-148Crossref PubMed Scopus (161) Google Scholar). These results further underscore the relative importance of the membrane-associated isoform of KL in the oogenic process, in support of data from mouse mutants indicating that KL-2, but not KL-1, is essential for oogenesis (13Tajima Y. Moore M.A. Soares V. Ono M. Kissel H. Besmer P. Consequences of exclusive expression in vivo of Kit-ligand lacking the major proteolytic cleavage site.Proc Natl Acad Sci U S A. 1998; 95: 11903-11908Crossref PubMed Scopus (72) Google Scholar, 14Flanagan J.G. Chan D.C. Leder P. Transmembrane form of the Kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant.Cell. 1991; 64: 1025-1035Abstract Full Text PDF PubMed Scopus (610) Google Scholar). Presently, no data exist on the relative levels or sites of expression of the two KL isoforms during human fetal ovary development, e.g., whether, as might be predicted by our model, KL-2 is preferentially expressed by somatic cells in close association with clustered germ cells and, more importantly, whether KL expression is altered during the period of activin production by germ cells in vivo. Furthermore, they provide a starting point for future studies into the presently unknown molecular mechanisms by which activin signaling can modulate the production/degradation of specific KL mRNA spliceforms. Although changes in splicing patterns in response to a wide variety of extracellular stimuli have been reported in numerous systems (19Stamm S. Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome.Hum Mol Genet. 2002; 11: 2409-2416Crossref PubMed Scopus (174) Google Scholar), we believe this to be the first report of selective changes in the expression of mRNA spliceforms in response to growth factor signaling within the human fetal ovary. The authors are grateful to Anne Saunderson, Joan Crieger, and the staff of the Bruntsfield Suite, Royal Infirmary of Edinburgh, for patient recruitment.