EXI’ERIMENTAL
CELL
RESEARCI-I
202,
199-202
(19%)
SHORT NOTE -1 and Ets-2 Protooncogene Expression in Th of the Adult Mouse Ovar ALEXANDRA Ludwig
Institute
fm
Cancer
Research,
St. Mary’s
ROWE’ Hospital
AND FRIEDRICH Medical
We have investigated the mRNA expression of the E&-I and Ets-2 genes in murine gonads and found expression in adult ovaries. In situ hybridization experiments show that the Ets genes are predominantly expressed in theta cells and cells of ovarian interstitium. By gel retardation experiments we detected DNA binding proteins in ovaries that specifically bind to the ETS motif, suggestimg the expression of Ets or Et.+related proteins. Our results raise the possibility of Ets-2 involvement in ovarian pathology seen in patients with own’s syndrome. 62 1992 Academic Press, Inc.
INTRODUCTION The members of the Ets family of protooncogenes are related to the viral oncogene v-E@ one of the transforming genes of the avian retrovirus E26 which causes erythroblastosis and myeloblastosis in chickens [l-3]. .Members of the Ets family, which include Ets-1, Ets-2, Erg, Elk-I, and Ets-2, have been identified in many species including buman, mouse, Xenopus, and Drosophila [3,4]. The protooncogenes Ets-1 and Ets-2 are localized on human chromosomes I1 and 21[5], respectively, and are believed to participate in the regulation of cell growth. Ets and Ets-related genes share a highly conserved -85-amino acid region, the ETS domain, which mediates sequence-specific DNA binding. It has recently been shown that Ets-1 and Ets-2 are transcription factors which activate transcription through binding to the ETS sequence motif in viral and cellular promoters [6-B]. In the mouse9&s-J and Ets-2 show distinct patterns of tissue-specific expression. Et.+1 is expressed at high levels mainly in the thymus, whereas Ets-2 is expressed in a wide range of tissues, particularly developing and proliferating tissues, in addition to the thymus [9]. Here we show that Ets-1 and Ets-2 mRNAs are expressed at high levels in the adult mouse ovary. In situ hybridization studies demonstrate that expression occurs in * Present address: Molecular ular Medicine, John Radcliffe ‘To whom correspondence dressed.
School,
Norfolk
PROPST' Place,
London
WZ lPG,
United
Kingdom
theta cells and corpora lutea. We also show that protein extracts from adult mouse ovaries ex binding to tbe ETS motif, consistent of functionally active Ets proteins o tion factors in theta cells.
RNA
expression analysis. Tissues were obtained from adult mice in accordance with the guidelines for the care and use of experimental animals. Total RNA from tissues was prepared using RNAzol B (CINNABIOTECX) as described [IQ] and analyzed by Northern blot hybridization after fractionation on 1% agarose-formaldehyde gels [ll]. Following electrophoresis the gels were stained with ethidium bromide to visualize the l8S and 26s ribosomal RNA bands to ascertain that equal amounts of intact RNA were loaded in each lane. Probes used were a I.3kb EcoRI fragment from the plasmid pAl1 containing part of exon 8, the entire exon 9, 1.6 kb of 3’ untranslated sequence of the mouse Ets-1 cDNA, and a 1.6 kb EcoRU Aval fragment from plasmid pA3/I.6 containing the entire open reading frame of the mouse Ets-2 cDN.4 ]12] ??-Iabeled by random priming as described [ll]. Plasmids pAl1 and pA3/1.6 were kindly provided by Dr. D. Watson. In situ hybridizations were carried out using [“S]UTP-labeled RNA probes generated from the above Ets-1 or Em-2 templates [ll] on sections ofpa~afo~maldehyde-fixed tissues as described 1131. Gel retardation assay. Whole cell extracts from ovary, testis, and thymus were prepared by homogenization of fresh tissue in a Dounce homogenizer in a buffer containing 20 mMHepes (pIl7.9), 25% (v/v) glycerol, 0.42 MNaCl, 1.5 mMMgQ, 0.2 mMEDTA, 0.5 mMPMSF, 0.5 mM DTT, 1.5 mM benzamidine, I.5 pg/ml leupeptin, 1 pg/ml pepstatin, 100 pg/ml aprotinin, and 1 pg/ml chymostatin followed by centrifugation at 300,OOOg for 30 min at 2’C. Supernatants (termed whole cell extracts) were stored at -7O’C prior to use in binding assays. Protein concentration was determined using the Bio-Rad protein assay system. The oligonucleotides used were the ETS oligonucleotide containing the ETS motif (underlined), 5’-GTCGAGCCGGAAGTTCGA-3’ [4, 6, 141, and tha mutant oligonucleotide containing two mutations in the ETS motif (underlined), 5’. TCGAGCAAGAAGTTCGAG-3’. In each case complementary oiigonucleotides designed to yield a 5’-protruding guanosine after annealing were annealed and end-labeled with [3zP]dCTP using the KIenow fragment of DNA polymerase I Ill]. DNA binding assays and subsequent gel electrophoresis of the DNA-protein complexes were performed as described 1151.
BALB/c
Expression of Ets-I and .Ets-2 in Addt Ets-1 and Ets-2 mRNA levels in adult mouse gonadal tissue were examined by Northern blot hybridization. High levels of both Em-1 and Ets-2 mRNA were
Parasitology Unit, Institute of MolecIIospital, Oxford OX3 9DU, UK. and reprint requests should be ad-
199
0014-4827/92
$5.00
200
SHORT
Ets-
4.7
kb-
1.9
kb-
1
123
Et&-2
456
FIG. 1. Northern blot analysis of RNA expression of Ets-1 and Ets-2 in murine gonadal tissue. Twenty-five micrograms of total RNA from adult BALB/c ovary (lanes 1 and 4), testis (lanes 2 and 5), or thymus (lanes 3 and 6) was analyzed using “P-labeled Et.s-I-specific (lanes l-3) or Ets-Z-specific (lanes 4-6) probes. The 4.7- and 1.9.kb markers indicate the position of the 28s and 18s ribosomal RNA, respectively.
tected in ovary but not in testis (Fig. 1). For Ets-1 we detect a major 5.3-kb transcript and minor transcripts ranging in size from 2 to 4 kb. These transcripts appear to be identical in size to those seen in the thymus (Fig. 1) [9]. For Ets-2 we detect 3.5-kb transcripts in ovaries, again corresponding in size to the transcripts seen in thymus (Fig. 1) [9]. We did not detect the very low level of Ets-2 RNA reported to be expressed in testis of 5 to 6-week-old mice [9] probably due to the use of total rather than poly(A)+ RNA in our experiments. In situ hybridization was performed on sections of mouse ovaries to determine the cell types expressing Ets-1 and Ets-2. After hybridization with an Ets-l-specific probe, silver grains were detected over cells of the theta interna which surrounds developing and atretic follicles (Fig. 2). Hybridization was also seen in a patchy distribution throughout corpora lutea and scattered throughout the interstitial tissue but not over granulosa cells (Fig. 2) or oocytes (not shown). Similar results were obtained with an Ets-2-specific probe. Ets-2 was found to be specifically expressed in theta cells although at lower levels than those of Ets-1 (not shown). Higher levels of Ets-2 expression were detected in specific cells in interstitium and corpus luteum (Fig. 2E). As a control, further ovarian sections were hybridized to Ets-1 and Ets-2 sense riboprobes, and these showed no specific hybridization (Fig. 2). Moreover, sections of adult testis were also hybridized with Ets-1 and Ets-2 antisense riboprobes and no specific hybridization was detected (not shown). Specific Ets DNA Binding Activity
in Ovary Extracts
To demonstrate the presence of Ets or Ets-related DNA binding proteins in extracts from adult mouse
NOTE
ovary, gel retardation assays were performed. When ovary extracts were incubated with 32P-labeled oligonucleotide containing the preferred ETS DNA binding motif [4,6,14] specific retarded complexes were formed (Fig. 3). The detection of these complexes was prevented by an excess of identical, unlabeled competitor oligonucleotides. Excess unlabeled mutant competitor, differing by two nucleotides in the ETS motif, did not prevent formation of the complexes, showing the specificity of the binding proteins for the ETS motif. Similar complexes were obtained with thymus extracts (Fig. 3). The presence of multiple retarded complexes of distinct size could result from different monomeric and/or hetero- or homo-oligomeric Ets or Ets-related proteins binding specifically to the ETS motif. Whereas these results are consistent with the presence of Ets-1 and/or Ets-2 proteins in ovary and thymus, the observed complexes could be the result of Ets-related proteins such as Elk [16]. Indeed, extracts from testis, where we were unable to detect Ets-1 and Ets-2 mRNA expression, gave similar results in the gel retardation assays (not shown) and it has been demonstrated that the Ets-related Elk genes are expressed at high levels in mouse testis [17]. DISCUSSION In this report we show for the first time that two members of the Ets protooncogene family, Ets-1 and Ets-2, are expressed in the adult mouse ovary, predominantly in cells of the theta interna. Two previous reports showed that Ets-2 is expressed in oocytes of Xenopus [18] and sea urchin [19], and it was suggested that Ets-2 was necessary for meiotic maturation of Xenopus oocytes [18]. In the present study we were unable to detect Ets-1 or Ets-2 expression in murine oocytes, which could be due to insufficient sensitivity of the in situ hybridization technique or to species differences. Our results raise the possibility that the transcription factors Ets-1 and Ets-2 play an important role in theta cell function. Theta cells produce androgens and progestins [20] and probably take part in the extensive remodeling of ovarian tissue in association with the continuous development and atresia of follicles [21,22]. Interestingly, the ETS binding motif is found in the promoter regions of three genes whose products are involved in the remodeling and restructuring of tissuestromelysin [8], collagenase [23], and urokinase plasminogen activator [24]. Thus, one possible function for the Ets genes in theta cells could be the transcriptional control of genes required for tissue reorganization. In support of this hypothesis, it is relevant to note that we were unable to detect the expression of Ets-1 or Ets-2 in Leydig cells. In the testis, Leydig cells are the androgenproducing analogues of theta cells. This suggests that the Ets genes are involved in the growth regulatory aspect of theta cell function, rather than the steroidogenie function, which is shared by Leydig cells. More-
SHORT
F ‘WI&. 2. Cellular localization of 32s -1 specitic (A-C) or Ets-Z-specific and silver grains is seen over the layers tcu ~ (R) Dark field photomicrograph silv tior RN !&is
NOTE
201
Ets-1 and Ets-2 mRNA in adult mouse ovary. Sections from adult mouse ovary were hybridized to (E) antisense RNA probes or to Ets-1 (D) or Ets-2 (F) sense RNA control probes. (A) Deposition of black of theta ceils surrounding three growing follicles (f) and over cells of the ~~~e~~t~ti~~ and corpus Meurn of the same section as that in A with enhanced visibihty of silver gram deposition. Note the absence of er grains over granulosa cells in the center of the follicles. (C) High-magnification view of a layer oftheca cells displaymg strong bybridizaL (D) Absence of specific silver grain deposition over tbeca cells (arrowhead) and an atretic follicle (a) after bybridizstion to the Ets-1 sense A control probe. (E) Patchy distribution of silver grains over cells of ovarian mterstitium and corpus luteum foIlowmg hybridization to the -Z-specific probe. (Fj Control byb~idizatio~ to the Ets-2 sense RNA probe. ~a~ni~~atio~ is 60X in A, R, and D-F and 130X m C.
202
SHORT
extract:
thymus -
ComDetitor:
E
ovary M
-
E
M
NOTE 2.
Leprince, D., Gegonne, A., Coll, J., de Taisne, A., Lagrou, C., and Stehelin, D. (1983) Nuture
3.
Watson, Oncogs
over, since the ETS motif is also found in retroviral promoter regions, expression of E&-l and Ets-2 in theca cells could contribute to the high level of ecotropic provirus expression seen in theta cells of certain hybrid mice [25]. In humans, ETS-2 is located on chromosome 21 [5], and it has been suggested that altered expression of ETS-2 may be responsible for some of the pathology seen in Down’s syndrome patients with trisomy of chromosome 21 [5,26]. Assuming that theta cell expression of ETS-2 is conserved in humans, our hypothesis for a role of .ETS-2 in the regulation of ovarian function could explain the fact that Down’s syndrome patients show abnormal ovarian histology. In these patients the ovaries contain fewer and smaller follicles than normal and growth and atresia of follicles are reduced, although fertility may be retained [27]. We are grateful to Dr. D. Watson for the kind gift Ets-2 plasmids and for valuable suggestions throughout tion of this manuscript.
of Ets-1 and the prepara-
REFERENCES 1.
Nunn, (1983)
Received Revised
M. F., Seeburg, P. H., Moscovici, Nuture 306, 391-395.
February 21,lSSZ version received May
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C., and Duesberg,
P. H.
T. S. (1990)
Crit.
Reu.
Karim, F. D., Urness, L. D., Thummel, C. S., Klemsz, M. J,, McKercher, S. R., Celada, A., Van Beveren, C., Maki, R. A., Gunther, C. V., Nye, J. A., and Graves, B. J. (1990) Gene Deu. 4, 1451-1453.
5.
Watson, D. K., McWilliams-Smith, -M. J., Kozak, C., Reeves, R., Gearhart, J., Nunn, M. F., Nash, W., Fowle, J. R., III, Duesberg, P., Papas, T. S., and O’Brien, S. J. (1986) hoc. N&l. Acud. Sci. USA 83,1792-1796.
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Wasylyk, B., Wasylyk, and Stehelin, D. (1990)
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Wasylyk, EMBOJ
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Bhat, N. K., Fisher, R. J., Fujiwara, S., Ascione, R., and Papas, T. S. (1987)Proc. Ned. Acad. Sci. USA 84,3161-3165. P., and
Sacchi,
Chomczynsk~, 156-1159.
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Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Watson, D. K., McWilliams, M. J., Lapis, P., Lautenberger, J. A., Schweinfest, C. W., and Papas, T. S. (1988) Proc. NatL Acad. Sci. USA 85, 7862-7866.
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Hogan, B., Constantini, F., and Lacy, the Mouse Embryo, Cold Spring Harbor Spring Harbor, NY.
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O’Hare, P., and Goding, Rao, V. N., and Reddy,
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Rao, V. N., Huebner, K., IsobeT M., Ar-Rushdi, and Reddy, E. S. P. (1989) Science 244,66-‘70.
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Chen, Z.-Q., Burdett, Papas, T. S. (1990)
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Chen, Z*-Q., Kan N. C., Pribyl, L., Lautenberger, drianakis, Es, and Papas, T. S. (1988) Dev. BioZ.
R. J., Mavrothalassitis, Oncogene 6,2249-2254.
N. {1987)
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Manipulating Press, Cold
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C. R. (1988) Cell 52,435-445. E. S. P. (1992) Oncogene 7,65-70. A., Croce,
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Bogovich> 1201-1208.
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R. J., Jr. (1988)
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Skinner,
J., Osteen,
K., and
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FIG. 3. Specific Ets DNA binding activity in tissue extracts. Twenty micrograms of whole cell extracts from adult thymus (lanes l-3) and ovary (lanes 4-6} was used in a DNA binding assay with a double-stranded “P-labeled ETS oligonucleotide containing the ETS motif. Lanes 1 and 4, no competitor (-); lanes 2 and 5, IOO-fold excess of unlabeled ETS oligonucleotide (I?,); lanes 3 and 6, lOO-fold excess of unlabeled mutant oligonucleotide (M).
R., and Papas,
1,409-436.
4.
9.
1
D. K., Ascione,
C., Schneeberger, 306,395-397.
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Lock, L. F., Keshet, E., Gilbert, D, G., Jenkins, land, N. G. (1988) EMBO J. 7,4169-4177.
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N. A., and Cope-
M. (1978)
CELL
RESEARCI-I
202,
199-202
(19%)
SHORT NOTE -1 and Ets-2 Protooncogene Expression in Th of the Adult Mouse Ovar ALEXANDRA Ludwig
Institute
fm
Cancer
Research,
St. Mary’s
ROWE’ Hospital
AND FRIEDRICH Medical
We have investigated the mRNA expression of the E&-I and Ets-2 genes in murine gonads and found expression in adult ovaries. In situ hybridization experiments show that the Ets genes are predominantly expressed in theta cells and cells of ovarian interstitium. By gel retardation experiments we detected DNA binding proteins in ovaries that specifically bind to the ETS motif, suggestimg the expression of Ets or Et.+related proteins. Our results raise the possibility of Ets-2 involvement in ovarian pathology seen in patients with own’s syndrome. 62 1992 Academic Press, Inc.
INTRODUCTION The members of the Ets family of protooncogenes are related to the viral oncogene v-E@ one of the transforming genes of the avian retrovirus E26 which causes erythroblastosis and myeloblastosis in chickens [l-3]. .Members of the Ets family, which include Ets-1, Ets-2, Erg, Elk-I, and Ets-2, have been identified in many species including buman, mouse, Xenopus, and Drosophila [3,4]. The protooncogenes Ets-1 and Ets-2 are localized on human chromosomes I1 and 21[5], respectively, and are believed to participate in the regulation of cell growth. Ets and Ets-related genes share a highly conserved -85-amino acid region, the ETS domain, which mediates sequence-specific DNA binding. It has recently been shown that Ets-1 and Ets-2 are transcription factors which activate transcription through binding to the ETS sequence motif in viral and cellular promoters [6-B]. In the mouse9&s-J and Ets-2 show distinct patterns of tissue-specific expression. Et.+1 is expressed at high levels mainly in the thymus, whereas Ets-2 is expressed in a wide range of tissues, particularly developing and proliferating tissues, in addition to the thymus [9]. Here we show that Ets-1 and Ets-2 mRNAs are expressed at high levels in the adult mouse ovary. In situ hybridization studies demonstrate that expression occurs in * Present address: Molecular ular Medicine, John Radcliffe ‘To whom correspondence dressed.
School,
Norfolk
PROPST' Place,
London
WZ lPG,
United
Kingdom
theta cells and corpora lutea. We also show that protein extracts from adult mouse ovaries ex binding to tbe ETS motif, consistent of functionally active Ets proteins o tion factors in theta cells.
RNA
expression analysis. Tissues were obtained from adult mice in accordance with the guidelines for the care and use of experimental animals. Total RNA from tissues was prepared using RNAzol B (CINNABIOTECX) as described [IQ] and analyzed by Northern blot hybridization after fractionation on 1% agarose-formaldehyde gels [ll]. Following electrophoresis the gels were stained with ethidium bromide to visualize the l8S and 26s ribosomal RNA bands to ascertain that equal amounts of intact RNA were loaded in each lane. Probes used were a I.3kb EcoRI fragment from the plasmid pAl1 containing part of exon 8, the entire exon 9, 1.6 kb of 3’ untranslated sequence of the mouse Ets-1 cDNA, and a 1.6 kb EcoRU Aval fragment from plasmid pA3/I.6 containing the entire open reading frame of the mouse Ets-2 cDN.4 ]12] ??-Iabeled by random priming as described [ll]. Plasmids pAl1 and pA3/1.6 were kindly provided by Dr. D. Watson. In situ hybridizations were carried out using [“S]UTP-labeled RNA probes generated from the above Ets-1 or Em-2 templates [ll] on sections ofpa~afo~maldehyde-fixed tissues as described 1131. Gel retardation assay. Whole cell extracts from ovary, testis, and thymus were prepared by homogenization of fresh tissue in a Dounce homogenizer in a buffer containing 20 mMHepes (pIl7.9), 25% (v/v) glycerol, 0.42 MNaCl, 1.5 mMMgQ, 0.2 mMEDTA, 0.5 mMPMSF, 0.5 mM DTT, 1.5 mM benzamidine, I.5 pg/ml leupeptin, 1 pg/ml pepstatin, 100 pg/ml aprotinin, and 1 pg/ml chymostatin followed by centrifugation at 300,OOOg for 30 min at 2’C. Supernatants (termed whole cell extracts) were stored at -7O’C prior to use in binding assays. Protein concentration was determined using the Bio-Rad protein assay system. The oligonucleotides used were the ETS oligonucleotide containing the ETS motif (underlined), 5’-GTCGAGCCGGAAGTTCGA-3’ [4, 6, 141, and tha mutant oligonucleotide containing two mutations in the ETS motif (underlined), 5’. TCGAGCAAGAAGTTCGAG-3’. In each case complementary oiigonucleotides designed to yield a 5’-protruding guanosine after annealing were annealed and end-labeled with [3zP]dCTP using the KIenow fragment of DNA polymerase I Ill]. DNA binding assays and subsequent gel electrophoresis of the DNA-protein complexes were performed as described 1151.
BALB/c
Expression of Ets-I and .Ets-2 in Addt Ets-1 and Ets-2 mRNA levels in adult mouse gonadal tissue were examined by Northern blot hybridization. High levels of both Em-1 and Ets-2 mRNA were
Parasitology Unit, Institute of MolecIIospital, Oxford OX3 9DU, UK. and reprint requests should be ad-
199
0014-4827/92
$5.00
200
SHORT
Ets-
4.7
kb-
1.9
kb-
1
123
Et&-2
456
FIG. 1. Northern blot analysis of RNA expression of Ets-1 and Ets-2 in murine gonadal tissue. Twenty-five micrograms of total RNA from adult BALB/c ovary (lanes 1 and 4), testis (lanes 2 and 5), or thymus (lanes 3 and 6) was analyzed using “P-labeled Et.s-I-specific (lanes l-3) or Ets-Z-specific (lanes 4-6) probes. The 4.7- and 1.9.kb markers indicate the position of the 28s and 18s ribosomal RNA, respectively.
tected in ovary but not in testis (Fig. 1). For Ets-1 we detect a major 5.3-kb transcript and minor transcripts ranging in size from 2 to 4 kb. These transcripts appear to be identical in size to those seen in the thymus (Fig. 1) [9]. For Ets-2 we detect 3.5-kb transcripts in ovaries, again corresponding in size to the transcripts seen in thymus (Fig. 1) [9]. We did not detect the very low level of Ets-2 RNA reported to be expressed in testis of 5 to 6-week-old mice [9] probably due to the use of total rather than poly(A)+ RNA in our experiments. In situ hybridization was performed on sections of mouse ovaries to determine the cell types expressing Ets-1 and Ets-2. After hybridization with an Ets-l-specific probe, silver grains were detected over cells of the theta interna which surrounds developing and atretic follicles (Fig. 2). Hybridization was also seen in a patchy distribution throughout corpora lutea and scattered throughout the interstitial tissue but not over granulosa cells (Fig. 2) or oocytes (not shown). Similar results were obtained with an Ets-2-specific probe. Ets-2 was found to be specifically expressed in theta cells although at lower levels than those of Ets-1 (not shown). Higher levels of Ets-2 expression were detected in specific cells in interstitium and corpus luteum (Fig. 2E). As a control, further ovarian sections were hybridized to Ets-1 and Ets-2 sense riboprobes, and these showed no specific hybridization (Fig. 2). Moreover, sections of adult testis were also hybridized with Ets-1 and Ets-2 antisense riboprobes and no specific hybridization was detected (not shown). Specific Ets DNA Binding Activity
in Ovary Extracts
To demonstrate the presence of Ets or Ets-related DNA binding proteins in extracts from adult mouse
NOTE
ovary, gel retardation assays were performed. When ovary extracts were incubated with 32P-labeled oligonucleotide containing the preferred ETS DNA binding motif [4,6,14] specific retarded complexes were formed (Fig. 3). The detection of these complexes was prevented by an excess of identical, unlabeled competitor oligonucleotides. Excess unlabeled mutant competitor, differing by two nucleotides in the ETS motif, did not prevent formation of the complexes, showing the specificity of the binding proteins for the ETS motif. Similar complexes were obtained with thymus extracts (Fig. 3). The presence of multiple retarded complexes of distinct size could result from different monomeric and/or hetero- or homo-oligomeric Ets or Ets-related proteins binding specifically to the ETS motif. Whereas these results are consistent with the presence of Ets-1 and/or Ets-2 proteins in ovary and thymus, the observed complexes could be the result of Ets-related proteins such as Elk [16]. Indeed, extracts from testis, where we were unable to detect Ets-1 and Ets-2 mRNA expression, gave similar results in the gel retardation assays (not shown) and it has been demonstrated that the Ets-related Elk genes are expressed at high levels in mouse testis [17]. DISCUSSION In this report we show for the first time that two members of the Ets protooncogene family, Ets-1 and Ets-2, are expressed in the adult mouse ovary, predominantly in cells of the theta interna. Two previous reports showed that Ets-2 is expressed in oocytes of Xenopus [18] and sea urchin [19], and it was suggested that Ets-2 was necessary for meiotic maturation of Xenopus oocytes [18]. In the present study we were unable to detect Ets-1 or Ets-2 expression in murine oocytes, which could be due to insufficient sensitivity of the in situ hybridization technique or to species differences. Our results raise the possibility that the transcription factors Ets-1 and Ets-2 play an important role in theta cell function. Theta cells produce androgens and progestins [20] and probably take part in the extensive remodeling of ovarian tissue in association with the continuous development and atresia of follicles [21,22]. Interestingly, the ETS binding motif is found in the promoter regions of three genes whose products are involved in the remodeling and restructuring of tissuestromelysin [8], collagenase [23], and urokinase plasminogen activator [24]. Thus, one possible function for the Ets genes in theta cells could be the transcriptional control of genes required for tissue reorganization. In support of this hypothesis, it is relevant to note that we were unable to detect the expression of Ets-1 or Ets-2 in Leydig cells. In the testis, Leydig cells are the androgenproducing analogues of theta cells. This suggests that the Ets genes are involved in the growth regulatory aspect of theta cell function, rather than the steroidogenie function, which is shared by Leydig cells. More-
SHORT
F ‘WI&. 2. Cellular localization of 32s -1 specitic (A-C) or Ets-Z-specific and silver grains is seen over the layers tcu ~ (R) Dark field photomicrograph silv tior RN !&is
NOTE
201
Ets-1 and Ets-2 mRNA in adult mouse ovary. Sections from adult mouse ovary were hybridized to (E) antisense RNA probes or to Ets-1 (D) or Ets-2 (F) sense RNA control probes. (A) Deposition of black of theta ceils surrounding three growing follicles (f) and over cells of the ~~~e~~t~ti~~ and corpus Meurn of the same section as that in A with enhanced visibihty of silver gram deposition. Note the absence of er grains over granulosa cells in the center of the follicles. (C) High-magnification view of a layer oftheca cells displaymg strong bybridizaL (D) Absence of specific silver grain deposition over tbeca cells (arrowhead) and an atretic follicle (a) after bybridizstion to the Ets-1 sense A control probe. (E) Patchy distribution of silver grains over cells of ovarian mterstitium and corpus luteum foIlowmg hybridization to the -Z-specific probe. (Fj Control byb~idizatio~ to the Ets-2 sense RNA probe. ~a~ni~~atio~ is 60X in A, R, and D-F and 130X m C.
202
SHORT
extract:
thymus -
ComDetitor:
E
ovary M
-
E
M
NOTE 2.
Leprince, D., Gegonne, A., Coll, J., de Taisne, A., Lagrou, C., and Stehelin, D. (1983) Nuture
3.
Watson, Oncogs
over, since the ETS motif is also found in retroviral promoter regions, expression of E&-l and Ets-2 in theca cells could contribute to the high level of ecotropic provirus expression seen in theta cells of certain hybrid mice [25]. In humans, ETS-2 is located on chromosome 21 [5], and it has been suggested that altered expression of ETS-2 may be responsible for some of the pathology seen in Down’s syndrome patients with trisomy of chromosome 21 [5,26]. Assuming that theta cell expression of ETS-2 is conserved in humans, our hypothesis for a role of .ETS-2 in the regulation of ovarian function could explain the fact that Down’s syndrome patients show abnormal ovarian histology. In these patients the ovaries contain fewer and smaller follicles than normal and growth and atresia of follicles are reduced, although fertility may be retained [27]. We are grateful to Dr. D. Watson for the kind gift Ets-2 plasmids and for valuable suggestions throughout tion of this manuscript.
of Ets-1 and the prepara-
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