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Women’s Health Research Institute, Wyeth Ayerst Research, Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Susan Fitzpatrick, Women’s Health Research Institute, Wyeth Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: fitzpas2{at}war.wyeth.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several of these TGFß family members, including inhibin, activin, and
Mullerian inhibiting substance (MIS), are known to play an important
role in reproduction. Recently, gene targeting has been applied to
investigate the precise role of several members in reproduction
(reviewed in Ref.7). Inhibin and activin are synthesized primarily in
the gonads (8) and regulate FSH levels from the pituitary, thereby
modulating fertility in males and females (9). Female mice deficient in
-inhibin appeared to have a block in folliculogenesis and/or
oogenesis, although deficient male mice do not display gross
reproductive defects (10). Furthermore, these mice developed gonadal
sex cord stromal tumors (granulosa/Sertoli cell tumors), suggesting
that -inhibin has tumor suppresser activity (10). Mice deficient in
activin receptor type II displayed reproductive defects in both females
and males (11). MIS is important for regression of the Mullerian duct
in males, and male mice deficient in MIS developed uteri and oviducts
and had Leydig cell hyperplasia (12). Clearly, members of the TGFß
superfamily are essential regulators of growth and development in the
gonads.
Growth/differentiation factor-9 (GDF-9) is a novel member of the TGFß superfamily that was identified using degenerate primers corresponding to conserved regions of the known family members (13). GDF-9 protein shares 21–34% homology to other family members in the -COOH terminal portion that encodes the mature protein and no homology in the proregion. Furthermore, GDF-9 messenger RNA (mRNA) has been reported to have a highly specific and restrictive cellular and tissue distribution. In particular, GDF-9 mRNA has been reported to be exclusively expressed in the ovary, specifically within the oocyte (14). GDF-9 mRNA is transcribed in follicles beginning at the primary stage, but is not present at the primordial stage. The highly specific localization of GDF-9 mRNA within the ovarian follicles suggested that this potential growth factor is necessary for folliculogenesis and fertility. Indeed, disruption of the GDF-9 mouse gene led to an abnormal organization of granulosa cells within the follicle and infertility in females, indicating an essential function for GDF-9 in follicular development (15). The follicles were arrested in the primary stage and contained only one layer of granulosa cells with no thecal cell components. Therefore, GDF-9 is an oocyte-derived factor required for normal folliculogenesis, perhaps by regulating granulosa and thecal cell functions.
Curiously, despite the essential role of GDF-9 in the ovary and the similarity of gene expression between the ovary and testis, GDF-9 mRNA was reported to be absent from the testis (13). We were, therefore, surprised to discover during our routine RT-PCR analyses that GDF-9 mRNA was present in the testis as well as the hypothalamus of the brain. We investigated the presence of GDF-9 mRNA in nonovarian tissues of mouse, rat, and human. Indeed, GDF-9 mRNA is expressed in the testis and hypothalamus of mouse and rat and, surprisingly, in human tissues, including several nonreproductive tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern blot analysis
Total RNA was extracted from various rodent tissues using TRIzol
reagent as described by the manufacturer (Life Technologies,
Gaithersburg, MD). Polyadenylated [poly(A)+] mRNA was
either isolated from total RNA using Fast Track 2.0 (Invitrogen, San
Diego, CA) or Oligotex mRNA (Qiagen, Chatsworth, CA). Additional
poly(A)+ mRNAs were purchased from Clontech (Palo Alto, CA)
and Ambion (Austin, TX). Five micrograms of poly(A)+ RNA
were size-separated on a denaturing 1% agarose formaldehyde gel and
blotted onto Hybond-N+ nylon membrane (Amersham Life
Science, Arlington Heights, IL). The entire mouse GDF-9 complementary
DNA (cDNA) or a fragment of the rat GDF-9 pro region cDNA corresponding
to nucleotides 280–770 of the published mouse cDNA sequence (13, 16)
was generated by PCR and radiolabeled with [-32P]deoxy
(d)-CTP by random priming. Blots were hybridized in Rapid-Hyb buffer
(Amersham) at 65 C using 2 x 106 cpm/ml denatured
probe. The membranes were washed at high stringency [0.1 x SSC
(0.015 M NaCl and 0.0015 M sodium citrate, pH
7.0) and 0.1% SDS for 30 min at 65 C] and exposed to X-Omat AR film
(Eastman Kodak, Rochester, NY) at -80 C. The mouse Northern blot and
the rat Northern blot were exposed for 7 and 4 days, respectively, at
-80 C. The human Northern blots were either obtained from Clontech
(Human Multiple Tissue Northern blot II containing 2 µg mRNA/lane) or
prepared using poly(A)+ mRNA (5 µg/lane) obtained from
Clontech. Both human blots were hybridized with an
[-32P]dCTP-labeled fragment of the human GDF-9 cDNA
generated by PCR and corresponding to DNA encoding for amino acids
12–355 (14), which encodes primarily the pro region. The MTN blot was
exposed for 7 days at -80 C, whereas the other human Northern blot was
exposed for 4.5 days. The membranes were stripped in boiling 0.05% SDS
and reprobed with the pTRI-GAPDH-Mouse or -Human plasmid (Ambion) to
measure glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Southern blot analysis
A mouse GENO-BLOT (Clontech), containing 4 µg genomic DNA
digested with different restriction enzymes, was probed with the entire
mouse GDF-9 cDNA as described for the Northern blots. A human GENO-BLOT
(Clontech), containing 4 µg genomic DNA digested with different
restriction enzymes, was probed with the human GDF-9 partial cDNA as
described above with the final wash in 0.2 x SSC and 0.1% SDS.
The mouse and human Southern blots were exposed for 2.5 days at -80
C.
In situ hybridization histochemistry
A plasmid containing the entire mouse GDF-9 cDNA was linearized
with either SpeI (antisense) or ApaI (sense,
control), and RNA polymerase was used to generate
[35S]UTP-labeled complementary RNA probes for in
situ hybridization.
The in situ hybridization methodology used for these studies has been described previously (17, 18). Briefly, commercial paraffin-embedded tissue sections were deparaffinized, postfixed in paraformaldehyde, treated with acetic anhydride, and then delipidated and dehydrated. Processed section-mounted slides were hybridized with 200 µl antisense or sense (control) riboprobe (6 x 106 dpm/slide) in a 50% formamide hybridization mix and incubated overnight at 55 C in an open air humidified slide chamber. The next day, the slides were immersed in 2 x SSC containing 10 mM dithiothreitol, treated with ribonuclease A (20 µg/ml, 30 min, 37 C), and washed (twice for 30 min) at 65 C in 0.1 x SSC to remove nonspecific label. After dehydration in a graded series of alcohol-ammonium acetate (70%, 95%, and 100%), the slides were exposed to BioMax (BMR-1, Kodak) x-ray film for 3 days and then dipped in NTB2 nuclear emulsion (Kodak; diluted 1:1 with 600 mM ammonium acetate). The slides were exposed for 2–12 weeks in light-tight black desiccated boxes, photographically processed, stained in cresyl violet, and protected with coverslips. The experiment was performed twice. The ovarian sections were exposed for 2 weeks at -20 C, and the testicular sections were exposed for 12 weeks.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The expression and mRNA size in the
ovary were similar to those originally reported (13, 14). However, in
contrast to these reports, a similarly size mRNA and minor longer
transcripts were also seen in one testicular sample. It is unclear why
GDF-9 mRNA was not detected previously in the testis (13). GDF-9 mRNA
was also detected in the hypothalamus (a tissue not previously
examined). The mouse probe was able to detect GDF-9 mRNA in rat ovary,
although to a lesser extent due to differences in homology sequence.
The blot was reprobed with GAPDH as an internal control. To determine
that these signals were GDF-9, RT-PCR was used to amplify mRNA products
from mouse ovary, testis, and hypothalamus. Subsequent sequencing of
these products revealed that they were indeed GDF-9 (data not
shown).
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, GDF-9 mRNA was detected in the ovary,
testis, and hypothalamus, but not in the uterus or heart. The major
transcript was approximately 1.6 kb and additional minor transcripts
were observed in the ovary (
3.4 and 6.3 kb) and testis (8.4
and 10.5 kb). Rat GDF-9 mRNA expression in the ovary was confirmed
by sequencing products obtained by RT-PCR.
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, GDF-9 mRNA was detected in the mouse
I-10 Leydig cell line, but not in the mouse TM3 Leydig or rat R2C
Leydig (data not shown) cell lines. Four transcripts were seen in the
I-10 cells (4.6, 4.0, 2.0, and 1.55 kb). RT-PCR and sequence
analysis confirmed the presence of GDF-9 mRNA in I-10 cells. However,
we were not able to relate the multiple mRNA transcripts with the
single RT-PCR product. As the PCR product was within the translated
region, it is possible that different 5'- and 3'-untranslated regions
account for the size differences between GDF-9 mRNA transcripts in I-10
cells.
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), silver grains representing
GDF-9 mRNA were detected in oocytes from a secondary follicle.
Expression was also observed in oocytes from primary, secondary, and
tertiary follicles, but not in primordial follicles (data not shown).
The present results agree with those previously reported (13). GDF-9
mRNA expression was not observed in somatic granulosa, thecal, or
interstitial cells. Occasionally, silver grains were seen over cumulus
granulosa cells, which may result from the close proximity to the
oocyte. Background grain levels were uniformly low in all samples
hybridized to sense probe (Fig. 4B).
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, A and B) and darkfield (Fig. 5C)
photomicrographs, silver grains indicating GDF-9 mRNA were concentrated
over seminiferous tubules in two areas: those containing large
pachytene primary spermatocytes (e.g. stages VII–X of the
cycle) and those containing many early round spermatids
(e.g. stages I–III). In contrast, there was an obvious lack
of signal over 1) adluminal areas of tubules containing more mature,
elongating spermatids, 2) more basal portions of tubules containing
spermatogonia, and 3) stage IX–XII, leptotene or zygotene, early
primary spermatocytes. Although the close physical relationship between
Sertoli and germ cells made it difficult to rule out expression of
GDF-9 mRNA by Sertoli cells, there were obviously areas of Sertoli cell
cytoplasm that lacked signal, particularly in those regions where
Sertoli cells surrounded maturing spermatids in adluminal areas and in
basal regions where Sertoli cells surrounded spermatogonia and early
primary spermatocytes. The level of background signal was uniformly low
in all samples, and signal above background was seen only in samples
exposed to antisense probes, with no specific signal in those sections
hybridized to sense probes (Fig. 5C).
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) or prepared in-house from
commercial human mRNA (Fig. 6B) were probed with an
[-32P]dCTP-labeled partial human GDF-9 cDNA probe (see
Materials and Methods). GDF-9 mRNA was detected
predominantly in the testis, and several transcript sizes were
observed. Major GDF-9 mRNA transcripts of about 4.3 and 2.1 kb were
also detected in the ovary, but at a lower level than in the testis.
The ovarian mRNA on the MTN blot II was obtained from 6 individuals,
aged 31–57 yr, and the testicular mRNA was obtained from 12 males,
aged 22–70 yr. The attenuated expression of GDF-9 mRNA in human ovary
compared with testis may be due to dilution of the ovarian sample with
mRNA from postmenopausal females containing few oocytes. Unexpectedly,
low levels of GDF-9 mRNA were also detected in nonreproductive human
tissues, including the pituitary, uterus, and other tissues. The
nonreproductive tissue samples were obtained from multiple individuals,
including both sexes, and covered a wide age range. GDF-9 mRNA
expression in the testis and pituitary was confirmed by RT-PCR and
sequence analysis. However, we were not able to relate the multiple
mRNA transcripts in testis with the single RT-PCR product. As the PCR
product was within the translated region, it is possible that different
5'- and 3'-untranslated regions account for the size differences of
GDF-9 mRNA transcripts in testis.
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) or human (Fig. 7B) genomic DNA,
digested with several restriction enzymes, were probed with the
appropriate [-32P]dCTP-labeled cDNA. The banding
pattern obtained with mouse DNA was consistent with the known map of
the mouse GDF-9 gene (16). Furthermore, as in the mouse, the banding
pattern obtained with human DNA was simplistic and did not indicate the
presence of a second gene. Indeed, manipulation of the GDF-9 gene by
homologous recombination did not reveal the presence of a second gene
(15). Therefore, it appears that a single GDF-9 gene is present in mice
and humans, and it is expressed in both ovarian and nonovarian
tissues.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were surprised to discover that GDF-9 mRNA was also expressed in the mouse, rat, and human testis using Northern blot analysis, sequence analysis of RT-PCR products, and in situ hybridization histochemistry. It is not clear why expression in the mouse testis, using Northern blot analysis of mRNA, was not detected previously (13). Here, using in situ hybridization histochemistry, GDF-9 mRNA was detected specifically in two types of germ cells: large spermatocytes that are in the later, pachytene stage of prophase of meiosis I, and postmeiotic, immature, round spermatids. Although the close physical relationship between Sertoli and germ cells makes it difficult to rule out expression of this gene by Sertoli cells in samples subjected to autoradiographic analysis, there were obvious areas of Sertoli cell cytoplasm that lacked signal. These included regions where Sertoli cells surrounded maturing spermatids in adluminal areas and basal regions where Sertoli cells surrounded spermatogonia and early primary spermatocytes.
With this approach, we cannot entirely rule out the possibility that the GDF-9 mRNA transcript is also located, in a compartmentalized fashion, in regions of Sertoli cell cytoplasm enveloping these germ cells. Regardless, GDF-9 mRNA is expressed in pachytene primary spermatocytes and round spermatids in the testis.
Based on the expression pattern of GDF-9 mRNA by in situ hybridization histochemistry, we did not expect to detect GDF-9 mRNA in the mouse I-10 cell line, a line derived from a Leydig cell tumor. The dedifferentiation of these cells by immortalization may have led to inappropriate expression of GDF-9 mRNA, although GDF-9 mRNA was not expressed in TM3 cells, another mouse Leydig cell line. Although multiple transcripts were observed in I-10 cells, including two transcripts not seen in mouse testis tissue, sequence analysis of RT-PCR-derived products confirmed that GDF-9 mRNA transcripts were present in I-10 cells.
The discovery of GDF-9 mRNA in the testis is not totally unexpected, as
certain members of the TGFß family are expressed in the testis and
regulate testicular function. For example, immunoreactive inhibin
(composed of the subunits ßA or ßB) and activin (composed of
the subunits ßAßA, ßAßB, or ßBßB) are detected in Sertoli
cells (primarily), Leydig cells, immature germ cells (fetal and
prepubertal testis), and Leydig cell lines (reviewed in Ref. 20; 21).
Expression of the -, ßA-, and ßB-subunits is hormonally and
developmentally regulated (reviewed in Ref. 20; 22). There is also
cross-regulation between family members. For example, TGFß1 and
activin A can enhance inhibin gene expression (23), whereas ßA
expression is enhanced 200-fold in inhibin -deficient mice (24).
Interaction between germ and Sertoli cells by paracrine-acting factors
can also regulate the expression of activin/inhibin. Inhibin secretion
from Sertoli cells is stimulated by the presence of germ cells
(25, 26, 27, 28), and ßA expression in Sertoli cells during development
fluctuates with the stage of the spermatogenesis (20). Conversely,
activins A and B, but not inhibin A, stimulate germ cell proliferation
(29), and receptors for activin have been identified in spermatocytes
and spermatids (30, 31, 32, 33). Thus, members of the TGFß family regulate
spermatogenesis and are regulated by factors from germ cells.
A physiological role for activin/inhibin in the testis has been
demonstrated in humans and rodents. Inhibin B levels correlate with
Sertoli cell function and are low in some forms of male infertility
(34). Surprisingly, the absence of inhibin (-subunit knock-out) did
not alter spermatogenesis and fertility in young male mice, but older
mice developed gonadal sex cord stromal tumors and consequently became
infertile (reviewed in Ref. 7; 10). Activin RII knock-out male mice had
normal stages of spermatogenesis but reduced fertility due to a
decreased number of spermatozoa as a consequence of a reduction in
seminiferous tubule volume (11).
The role of GDF-9 in the testis remains to be elucidated. Extrapolating from the roles of other family members in the testis and ovarian data from GDF-9 knock-out mice, GDF-9 may act as a paracrine growth factor and regulate the growth and differentiation of other germ cells or Sertoli cells. Importantly, the GDF-9 knock-out male mice were fertile, although no data were presented regarding testicular function (e.g. sperm count, maturation, mobility, or viability) or testosterone levels in these animals. Although activin has been shown to be important for spermatogenesis, ßB knock-out animals were fertile (7, 35). It is possible that in the testis, GDF-9 activity, like activin activity, may be vestigial or compensated for by other TGFß family members. A reexamination of the GDF-9 knock-out mice and immunohistological localization of the protein may help elucidate the role of GDF-9 in the testis.
GDF-9 mRNA was also detected in the hypothalamus of mice and rats using Northern blots and sequence analysis of PCR products. However, we were unable to demonstrate expression in the hypothalamus using in situ hybridization histochemistry. It is possible that a distinct gene that is homologous to GDF-9 was detected by Northern blot but not by in situ hybridization despite the use of a probe that is specific to the pro region. However, the size of the mRNA is consistent with GDF-9, and most importantly, sequence analyses of RT-PCR products confirmed the presence of authentic GDF-9 RNA transcripts. Furthermore, Southern blot results are consistent with the presence of a single GDF-9 gene. The discrepant in situ hybridization histochemistry results remain unresolved. However, there is evidence for the localization and physiological action of TGFß family members in the hypothalamus. For example, activin ßA-subunit mRNA (36) and immunoreactivity (37, 38) as well as activin RI and RII immunoreactivities (39) have been detected in the hypothalamus. Furthermore, infusions of activin A into the dorsal hypothalamus resulted in increased water consumption and urine volume (40), whereas injection of activin A into the paraventricular nucleus of the hypothalamus enhanced CRF release (41).
We also demonstrate here using Northern blot analyses that GDF-9 mRNA is expressed in multiple tissues in humans. The expression was greatest in the testis, and we were surprised at the low level of expression in the ovary. Because the ovarian mRNA is from premenopausal and postmenopausal women, the lower expression of GDF-9 mRNA in human ovary compared with testis is most likely due to a dilution of the mRNA sample with mRNA from women with few oocytes. To confirm that mRNA expression in the other human tissues was indeed GDF-9, RT-PCR products from pituitary were sequenced and determined to be authentic GDF-9. As in the mouse, Southern blot analysis was consistent with the presence of a single GDF-9 gene. Therefore, detection of GDF-9 in RNA from ovary and testis as well as other human tissues suggests that GDF-9 may play an important role in reproductive tissues and perhaps have a more ubiquitous role in other tissues in humans.
Together, the present studies demonstrated that GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Like that in the ovary, mRNA expression in the testis is primarily confined to germ cells. In addition, GDF-9 mRNA was detected in the human ovary and testis as well a wide variety of nonreproductive tissues. Although the identification and function of GDF-9 protein in nonovarian tissues remain to be determined, these results suggest that GDF-9 may have physiological roles in addition to those in the ovary.
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Acknowledgments |
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Footnotes |
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Received November 14, 1997.
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inhibin
deficient mice. Biochem Biophys Res Commun 203:105–112[CrossRef][Medline]
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N. Kaivo-Oja, J. Bondestam, M. Kamarainen, J. Koskimies, U. Vitt, M. Cranfield, K. Vuojolainen, J. P. Kallio, V. M. Olkkonen, M. Hayashi, et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 755 - 762. [Abstract] [Full Text] [PDF]
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J. L. Juengel, N. L. Hudson, D. A. Heath, P. Smith, K. L. Reader, S. B. Lawrence, A. R. O'Connell, M. P.E. Laitinen, M. Cranfield, N. P. Groome, et al. Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Are Essential for Ovarian Follicular Development in Sheep Biol Reprod, December 1, 2002; 67(6): 1777 - 1789. [Abstract] [Full Text] [PDF]
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N. Yamamoto, L. K. Christenson, J. M. MCAllister, and J. F. Strauss III Growth Differentiation Factor-9 Inhibits 3'5'-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2849 - 2856. [Abstract] [Full Text] [PDF]
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F. L. Teixeira Filho, E. C. Baracat, T. H. Lee, C. S. Suh, M. Matsui, R. J. Chang, S. Shimasaki, and G. F. Erickson Aberrant Expression of Growth Differentiation Factor-9 in Oocytes of Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1337 - 1344. [Abstract] [Full Text] [PDF]
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E. V. Solovyeva, M. Hayashi, K. Margi, C. Barkats, C. Klein, A. Amsterdam, A. J.W. Hsueh, and A. Tsafriri Growth Differentiation Factor-9 Stimulates Rat Theca-Interstitial Cell Androgen Biosynthesis Biol Reprod, October 1, 2000; 63(4): 1214 - 1218. [Abstract] [Full Text]
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K.J. Bodensteiner, K.P. McNatty, C.M. Clay, C.L. Moeller, and H.R. Sawyer Expression of Growth and Differentiation Factor-9 in the Ovaries of Fetal Sheep Homozygous or Heterozygous for the Inverdale Prolificacy Gene (FecXI) Biol Reprod, June 1, 2000; 62(6): 1479 - 1485. [Abstract] [Full Text]
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J. Aaltonen, M. P. Laitinen, K. Vuojolainen, R. Jaatinen, N. Horelli-Kuitunen, L. Seppä, H. Louhio, T. Tuuri, J. Sjöberg, R. Bützow, et al. Human Growth Differentiation Factor 9 (GDF-9) and Its Novel Homolog GDF-9B Are Expressed in Oocytes during Early Folliculogenesis J. Clin. Endocrinol. Metab., August 1, 1999; 84(8): 2744 - 2750. [Abstract] [Full Text]
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M. Hayashi, E. A. McGee, G. Min, C. Klein, U. M. Rose, M. v. Duin, and A. J. W. Hsueh Recombinant Growth Differentiation Factor-9 (GDF-9) Enhances Growth and Differentiation of Cultured Early Ovarian Follicles Endocrinology, March 1, 1999; 140(3): 1236 - 1244. [Abstract] [Full Text]
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K.J. Bodensteiner, C.M. Clay, C.L. Moeller, and H.R. Sawyer Molecular Cloning of the Ovine Growth/Differentiation Factor-9 Gene and Expression of Growth/Differentiation Factor-9 in Ovine and Bovine Ovaries Biol Reprod, February 1, 1999; 60(2): 381 - 386. [Abstract] [Full Text] [PDF]
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