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Division of Reproductive Biology, Department of Gynecology and
Obstetrics, Stanford University School of Medicine (M.H., E.A.M., G.M.,
C.K., A.J.W.H.), Stanford, California 94305-5317 and Scientific
Development Group
1 (U.M.R., M.v.D.), N.V.
Organon, Oss,
The Netherlands
Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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content in explants of neonatal ovaries. In contrast, the stimulatory
effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that
was apparently not bioactive. Thus, the present study demonstrates the
important role of GDF-9 in early follicle growth and differentiation.
The availability of recombinant bioactive GDF-9 allows future studies
on the physiological role of GDF-9 in ovarian development in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During initial recruitment of ovarian follicles, intraovarian factors may stimulate some primordial follicles to initiate growth, whereas the rest of the follicles remain quiescent for months or years. Alternately, initial recruitment of follicles may be due to a release from inhibitory stimuli that maintains the resting follicles in stasis. GDF-9 is a protein of the TGF-ß/activin family originally identified as an oocyte product (11, 12). Subsequent studies using GDF-9 mutant mice indicated this protein is important for the initial development of ovarian follicles. Animals deficient in GDF-9 showed an arrest of follicle development beyond the primary stage with one layer of granulosa cells (13). However, the exact role of GDF-9 in follicle growth and differentiation has not been elucidated due to the unavailability of bioactive GDF-9.
Using a serum-free culture system of preantral follicles from rats, we have demonstrated the ability of cGMP analogs to suppress follicle cell apoptosis and the stimulatory effect of FSH on preantral follicle growth (14). Here, we isolated rat GDF-9 complementary DNA (cDNA), generated recombinant GDF-9 proteins and produced specific GDF-9 antibodies. Using the serum-free cultures of preantral follicles and neonatal ovarian explants, it was demonstrated that GDF-9 is a growth and differentiation factor for early ovarian follicles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of antibodies specific for rat mature GDF-9
For antibody production, the mature region of rat GDF-9 (amino
acids 307 to 440) was subcloned into the pGEX-4T-2 vector
(Pharmicia). After transformation into Escherichia
coli strain TOP10 (Invitrogen, San Diego, CA),
expression of a fusion protein consisting of glutathione S-transferase
(GST) and mature GDF-9 was induced following treatment with
isopropyl-1-thio-ß-D-galactoside (IPTG). The fusion
protein in bacterial lysate was purified using a glutathione-Sepharose
4B-affinity column (Pharmicia), emulsified in Freund’s
adjuvant and injected into rabbits. The titer of antiserum against
GDF-9-GST protein was checked by ELISA using the fusion protein as the
antigen and compared with that of the preimmune serum. IgG was purified
using the Protein G Sepharose 4 Fast Flow column
(Pharmicia). In immunoblots, the GDF-9 antibodies do not
bind related recombinant activin A and inhibin A (data not shown).
Expression of recombinant rat GDF-9 protein in 293T cells
Expression vectors for wild-type and epitope-tagged GDF-9 were
constructed using pcDNA3.1 Zeo (Invitrogen). N-tagged
GDF-9 encoded a Flag epitope for M1 antibody followed by six histidine
residues fused to the amino-terminus of mature GDF-9. The resulting
junctions encoded the following sequences; — RHRR (cleavage site of
GDF-9)/DYKDDDDK (M1)/HHHHHH/GQKTLS—. These constructs were generated
using PCR and confirmed by DNA sequencing.
Human embryonic kidney 293T cells were transfected with the expression vector using the calcium phosphate precipitation method (16). Clonal cell lines stably expressing wild-type and tagged GDF-9 were selected under 1 mg/ml of Zeocin (Invitrogen) and maintained in DMEM/F12 (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 120 µg/ml Zeocin. When the cells reached 90–100% confluence, the medium was replaced with DMEM/F12 without Zeocin. After 4 days of serum-free culture, the condition media were harvested, and cell debris was cleared. For some experiments, the supernatant was concentrated 20- to 30-fold using Biomax-10 (Millipore Corp., Bedford, MA).
Characterization of recombinant rat GDF-9 protein
Conditioned media from 293T cells expressing GDF-9 were
electrophoresed in 14% SDS-PAGE gels before transfer of proteins to
nitrocellulose (Hybond-ECL) or polyvinylidene difluoride membranes
(Hybond-P, Amersham, Buckinghamshire, UK). Immunoblots
were performed using antirat GDF-9 antisera (1:8000 dilution), M1
antibody (10 µg/ml; Kodak, New Haven, CT) as the primary antibody.
The horseradish peroxidase-conjugated sheep antimouse IgG (1:6250
dilution for M1 antibodies) or donkey antirabbit IgG (1:6250 dilution
for GDF-9 antibodies) were used as the secondary antibody. Signals were
detected following immunofluorescent imaging using the ECL Plus System
(Amersham).
To remove N-linked carbohydrate side chains, conditioned media were diluted in the deglycosylation buffer (50 mM sodium phosphate buffer, pH 7.4, 1% SDS, 1%-mercaptoethanol, 0.5% Nonidet-P40, and 25 mM EDTA) and incubated with 1U/30 µl endoglycosidase F (Boehringer Mannheim, Indianapolis, IN) at room temperature for 1 h (17). Samples were mixed with Laemmli buffer under reducing conditions (100 mM dithiothreitol and 5% mercaptoethanol) for immunoblots. N-tagged GDF-9 was purified to homogeneity using Nickel column (Pharmicia) and verified by Coomassie blue staining. Levels of wild-type GDF-9 were estimated in immunoblots using the purified N-tagged GDF-9 as standard.
Immunohistochemical analysis
Ovaries were collected from 5- and 20-day-old Sprague Dawley
rats (Simonsen, Gilroy, CA) and fixed in 3% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4) for 24 h at 4 C. After
cryoprotection in 17% sucrose/PBS for 18 h at 4 C, tissues were
frozen in tissue-Tek O.C.T. embedding medium (Miles Scientific,
Elkhart, IN) under liquid nitrogen at -70 C. Frozen sections (6 µm)
were cut using a Leica Corp. CM 1800 cryostat (Leica Corp. Microsystems, Deerfield, IL) at -20 C and thaw-mounted on
microscope slides coated with 0.1% poly-L-lysine
(Sigma Chemical Co., St. Louis, MO). Slides were incubated
at room temperature for 2 h in blocking buffer 1 (2% normal
rabbit serum, 1% BSA, 100 mM glycine, and 2 mM
NaN3 in PBS, pH 7.4), followed by 4 h in incubation buffer (1%
BSA, 100 mM glycine in PBS, pH 7.4) containing either
rabbit antirat GDF-9 antiserum (1:200) or normal rabbit serum (1%).
After four washes of 30 min each, tissues were then incubated for
1 h in blocking buffer 2 (2% donkey serum, 1% BSA, 100
mM glycine in PBS, pH 7.4), and for 45 min in incubation
buffer containing donkey antirabbit IgG conjugated with horseradish
peroxidase (1:2000). After washing five times for 30 min each, tissues
were incubated for 5 min with the peroxidase substrate solution
(3,3'-diaminobenzidine tetrahydrochloride), and the reaction was
stopped with copious amounts of distilled water for 30 min before
dehydration and microscopic examination.
Cultures of preantral follicles and ovarian explants
Ovaries were collected from 14-day-old rats, and preantral
follicles (130–150 µm in diameter) were dissected microscopically
using a fine needle (14). Follicles were cultured individually in
96-well dishes lined with polycarbonate membranes in 100-µl medium
overlaid with 50-µl sterile mineral oil at 37 C in a moist atmosphere
of 5% CO2 and 95% air. Basal medium consisted of -MEM supplemented
with 1% ITS (insulin, 10 ng/ml; transferrin, 5.5 ng/ml; selenium, 5
ng/ml), 2.5 mM 8-bromo-cGMP, penicillin 100 U/ml,
streptomycin 100 µg/ml. Follicles were cultured in 100 µl of the
basal medium in the presence or absence of serum-free conditioned media
containing 20 ng recombinant GDF-9 and/or 5 IU/ml of human FSH
(ISIAFP-1; 6150 IU/mg from the National Hormone and Pituitary
Distribution Program, NIDDK, NIH). For control groups, conditioned
media were harvested from nontransfected 293T cells or cells expressing
N-tagged GDF-9. Follicle diameter was measured daily as the average
distance between the outer edges of the basement membrane in two
perpendicular planes. At the end of the 72 h incubation, follicles
were collected for protein content determination by the bicinchoninic
acid protein assay, using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL) (18).
For ovarian explant cultures, ovaries from 6-day-old rats were collected. As previously described (19), dissected ovaries were placed on sterile lens paper layered on glass beads in 24-well dishes, and cultured for 2 days under an atmosphere of 60% oxygen, 5% CO2 and 35% nitrogen at 37 C. The basal medium (900 µl/well) consisted of DMEM/F-12 containing 4.5 mg/ml glucose, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Individual ovaries were incubated in the serum-free medium with or without 50 µM forskolin and/or conditioned media containing recombinant GDF-9. Statistical differences between different groups were determined using ANOVA followed by Student’s t test.
Immunoblot analysis of inhibin-
Neonatal ovarian explants from each treatment group were
collected and kept frozen. They were later thawed in lysis buffer (50
mM Tris-HCl, 150 mM NaCl, 1% SDS, 5
mM EGTA, 0.5 mM MgCl2, 0.5 mM
MnCl2, and 0.2 mM phenylmethyl-sulfonylfluoride) at 50 µl
buffer per ovary and homogenized. Inhibin- levels were determined
using immunoblotting as previously described (20). Briefly, samples
were fractionated using SDS-PAGE in 10% polyacrylamide gels and
transferred to polyvinylidene difluoride membranes. The membranes were
then incubated with a mouse monoclonal antibody to human inhibin-
(1:2000 dilution, Serotec, Oxford, UK), followed by
incubation with horseradish peroxidase-conjugated sheep antimouse IgG
and immunofluorescent imaging with the ECL System.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Similar to the cloned
GDF-9 cDNAs, the rat cDNA (440 amino acids in length) encodes a protein
with a hydrophobic signal peptide and the pro-region (306 residues),
followed by the mature protein (134 residues). Rat GDF-9 shows an
overall homology of 91% and 74% with its mouse and human
counterparts, respectively (11, 12). In the mature protein, the
homology reaches 96% and 92%, respectively. A cluster of basic amino
acids in the putative cleavage site (RHRR; boxed in Fig. 1) between
pro-region and the mature protein is conserved. In addition, multiple
cysteines presumably important for forming the disulfide bonds to
maintain the secondary structure of GDF-9 are also conserved. Similar
to the mouse and human GDF-9, but distinct from most other
TGF-ß/activin family members (11), the rat GDF-9 has a Cys to Ser
substitution immediately before the fourth cysteine in the mature
protein. The lack of this Cys could prevent GDF-9 homodimerization. In
the mature protein, a single putative N-linked glycosylation site was
identified in all three species (Fig. 1, triangles) whereas
three conserved glycosylation sites were found in the pro-region. Also,
an additional putative glycosylation site unique for the rat protein
was found in the pro-region between the second and third consensus
sites.
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, Coomassie blue staining revealed the induction of fusion protein
production in the bacteria lysate following IPTG treatment. After
purification using a glutathione affinity column, a distinct band
of GDF-9-GST fusion proteins could be identified. These proteins
were used to generate GDF-9 antibodies in rabbits. As shown in Fig. 2B, specific antibodies were found to bind the original antigen in solid
phase, whereas the preimmune serum was only effective at 1,000-fold or
higher concentrations.
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, A and B),
many follicles were at the primordial stage with some advanced to
primary and a few to the secondary stage. GDF-9 staining was detected
in the oocyte of primary (1F) and secondary but not primordial
follicles (PMF). In ovaries from rats at 20 days of age (Fig. 3, C and
D), follicle development had advanced further. In addition to primary
and secondary follicles (2F), GDF-9 staining was found in the oocyte of
preantral follicles (PAF). Of interest, GDF-9 staining in preantral
follicles was not uniform with the oocytes from some follicles showing
lower signals. In contrast, no staining was found in sections incubated
with the normal rabbit serum for ovaries from both 5- and 20-day-old
rats (Fig. 3, E and F).
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) were used to transfect human 293T
cells. Cells permanently expressing GDF-9 proteins were selected and
conditioned media used to analyze GDF-9 secretion. As shown in Fig. 4B, immunoblot analysis under reducing conditions indicated that media from
cells transfected with wild-type GDF-9 cDNA contained two bands of
mature GDF-9 with apparent mol wt of 15 and 19 kDa, respectively. In
addition, a diffused band of 60–80 kDa presumably representing
uncleaved GDF-9 was also apparent. Likewise, secretion of N-tagged
GDF-9 could be found in media from transfected cells using antibodies
against either GDF-9 (Fig. 4B) or specific tags (data not shown). No
staining was found in the conditioned media of nontransfected
cells.
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, the migration pattern of immunoreactive bands from
media of cells expressing wild-type GDF-9 was different before (20, 24,
70–95 kDa) and after (15, 19, 60–80 kDa) treatment with reducing
reagents, suggesting the presence of intramolecular disulfide bonds and
conformational changes induced by reducing agents. Due to the absence
of bands corresponding to GDF-9 dimers, these data suggest the
secretion of monomeric GDF-9 as predicated from its unique Cys to Ser
substitution in the mature protein (11). The double bands for mature
GDF-9 were likely the result of varying glycosylation because only one
lower band of 15 kDa was detected after removal of carbohydrate side
chains with N-glycosidase F treatment (Fig. 4D). Furthermore, the
apparent size of the high MW uncleaved GDF-9 was also decreased
following enzyme treatment, suggesting its glycoprotein nature. The
N-tagged GDF-9 was further purified using the Nickel column (Fig. 4E).
Coomassie blue staining of purified N-tagged GDF-9 was found as two
major bands of 22 and 18 kDa that corresponded to immunoblot results.
The purified tagged GDF-9 was used as standards in immunoblots to
estimate the amount of nontagged GDF-9 in the conditioned media of
transfected 293T cells.
GDF-9 stimulation of preantral follicle growth and
differentiation
Using a serum-free culture of preantral follicles in which
follicle cell apoptosis was suppressed by a cGMP analog (14), we tested
the effect of recombinant GDF-9 on follicle growth. As shown in Fig. 5A, treatment with wild-type GDF-9 (200
ng/ml) increased the diameter of preantral follicles in a
time-dependent manner (P < 0.01). Consistent with
earlier findings (14), treatment with FSH also stimulated follicle
growth and combined treatment with both GDF-9 and FSH led to an
additive increase in follicle diameter. Furthermore, treatment with
N-tagged GDF-9, with or without FSH, did not alter follicle diameter,
suggesting the N-tagged GDF-9 is not bioactive. The potent stimulatory
effect of GDF-9 on follicle growth was further confirmed by
morphological analysis (Fig. 5B). Although no major increase in oocyte
size was found after treatment with GDF-9 or FSH, the diameter of these
follicles were clearly increased, mainly as the result of changes in
granulosa cells. The protein content of cultured follicles was also
estimated (Fig. 5C). Treatment with GDF-9 alone increased the protein
content by 1.9-fold as compared with the 2.0-fold increase induced by
FSH. Combined treatment with both hormones led to an additive
(3.4-fold) increase.
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, a differentiation marker
for early follicles. As shown in Fig. 6A, treatment of neonatal ovaries with GDF-9 led to a dose-dependent
increase in inhibin- levels based on the detection of a 41-kDa band
in immunoblots. At 5 µg/ml GDF-9, a 3.8 ± 0.6-fold increase
(mean ± SD; n = 3) in inhibin- levels was
detected. In contrast, treatment with N-tagged GDF-9 at the same
concentrations was ineffective (P > 0.01). Because
both VIP and ß-adrenergic agents are known stimulators of the protein
kinase A pathway (22), the ovarian explants were further treated with
forskolin to increase endogenous cAMP production. Forskolin treatment
also increased inhibin- production (4.6 ± 2.9-fold, n =
3) and the combined treatment of GDF-9 and forskolin led an additive
increase (11.4 ± 8.2 fold as compared with controls, n = 3).
These data suggested that GDF-9 is a differentiation factor for early
follicles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by early follicles. GDF-9 is composed of pro- and mature regions separated by a tetrabasic cleavage site. Comparison of the amino acid sequence of rat GDF-9 with its human and mouse counterparts indicated that the mature GDF-9 protein is highly conserved (>92%) in these species, consistent with its important role in reproduction. Multiple cysteines are conserved in all three species, presumably essential for the formation of disulfide bonds to stabilize the unique "hand-shaped" protein (23). However, rat GDF-9, like GDF-3 and GDF-9 from mouse and human, lacks the conserved cysteine residue found in other TGF-ß/activin family members believed to be important for their homodimerization through intermolecular disulfide bonds (24). In addition, one and four N-linked glycosylation sites were found in the mature and pro-region of rat GDF-9, respectively. Consistent with these structural predictions, immunoblot analysis indicated that recombinant GDF-9 produced by mammalian cells are likely glycosylated monomers with varying amounts of carbohydrate side chains in both pro- and mature regions.
Using a bacterially derived fusion protein between GST and mature GDF-9, specific GDF-9 antibodies were generated. In contrast to a study using antibodies against the pro-region of GDF-9 (12), this is the first report of antibodies against the mature GDF-9 protein, shown to be useful for immunoblot and immunohistochemical analyzes. Consistent with earlier in situ hybridization studies of GDF-9 mRNA (12, 21), the mature GDF-9 protein was found in the oocyte of primary, secondary and larger follicles, indicating that the GDF-9 transcripts, unlike many other oocyte transcripts (25), are indeed translated into proteins. Of interest, GDF-9 expression was uniform in primary and secondary follicles but heterogeneous in the larger preantral follicles with a few of them showing lower signals.
During development, granulosa cells of primordial follicles start to
divide (26, 27), followed by morphological changes to the cuboidal
shape characteristic of primary follicles. The exact mechanism by which
the majority of primordial follicles remain dormant and a few of them
initiate growth is unclear. The role of the oocyte in the initial
recruitment of follicles has been considered. GDF-9 is produced by
growing oocytes in primary and larger follicles but is absent in
primordial follicles; disruption of the GDF-9 gene in mice prevents
follicle development beyond the primary/early secondary stage (13). The
present findings of a growth-promoting role of GDF-9 on early follicles
are consistent with the observed phenotype in GDF-9 mutant mice. In
addition, the observed stimulatory role of GDF-9 on inhibin-
production suggests this growth factor can also enhance early follicle
differentiation. Although wild-type GDF-9 was bioactive, N-tagged GDF-9
was not, suggesting the importance of an intact N terminus for GDF-9
function as has been found for the related activins (28).
It is apparent that, in addition to its potential role in the initiation of primordial follicle growth (13), GDF-9 also plays an important role in the continuing growth and differentiation of early follicles. Thus, GDF-9, like FSH (14), is a growth and differentiation factor for early follicles. However, analysis of ovarian phenotypes in mutant mice with deletion of GDF-9 (13) or FSH-ß gene (29) indicated that GDF-9 is required for the initial recruitment of primordial follicles to enter the growing pool whereas FSH likely plays a facilitatory role in early follicle development because follicle development could progress to the early antral stage in the absence of circulating FSH. In addition to GDF-9 and FSH, activins, ovarian paracrine factors with limited sequence homology to GDF-9, also promote early follicle development (30, 31).
It has been proposed that endogenous activators of cAMP may play a role in initial follicle recruitment and treatment of ovarian explants from neonatal rats with VIP or norepinephrine increases cAMP production and accelerates early follicle development (19). Because the first follicles that grow in the rat ovary are in the highly innervated corticomedullary junction, the first wave of follicle growth may be facilitated by these local neurotransmitters. Our findings using neonatal ovarian explants further suggests that, in addition to neurotransmitters and VIP of neuronal origin, the oocyte-derived GDF-9 also plays an important role in early follicle development. The demonstration of GDF-9 actions in the present model systems also allows convenient in vitro bioassays for GDF-9.
A gap junction protein, connexin 37, is expressed at the oocyte-granulosa cell junction by the time follicles have developed to the secondary stage whereas follicles of mice that lack connexin 37 do not progress normally (32). These findings underscore the importance of oocyte-granulosa communication. Several in vitro studies further demonstrated that oocyte factors have an effect upon granulosa cell differentiation and steroidogenesis (33, 34, 35). Among the known factors capable of regulating preantral follicle growth, GDF-9 is the only one of oocyte origin. The oocyte plays an essential role in transmitting the correct genetic material to the offspring and GDF-9 secreted by oocyte might play a potential role in regulating the development of somatic cells in a given follicle, thus determining the follicle fate. Based on the finding that GDF-9 is important for early follicle development, it is interesting to investigate the genetic makeup of oocyte in individual follicles exhibiting differential GDF-9 secretion and to use GDF-9 as a functional marker for oocytes.
The mechanism by which GDF-9 regulates follicle growth and differentiation remains to be elucidated. Because granulosa cells constitute the majority of cells in the present preantral follicles, they are likely to be the major target for GDF-9. However, one cannot rule out theca cells as the GDF-9 targets because mutant mice showed the absence of theca cells in ovarian follicles (13). GDF-9 likely binds to specific plasma membrane receptors present in granulosa and/or theca cells. Like other members of the TGF-ß/activin family proteins, GDF-9 could signal through serine-threonine kinase receptors (36, 37, 38, 39, 40). The availability of recombinant bioactive GDF-9 and findings of the growth and differentiation actions of GDF-9 on follicles allow future characterization of specific GDF-9 receptors in the ovary and intracellular signaling pathways. Elucidation of factors involved in the initial recruitment of follicles could provide new treatments for patients with premature ovarian failure. The possibility of suppressing initial follicle recruitment and preventing the growth initiation of resting follicles could also be the basis for designing treatments that would preserve the resting follicle pool, thus extending the female fertile period and delaying menopause.
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Acknowledgments |
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Footnotes |
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Received September 15, 1998.
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I Demeestere, J Centner, C Gervy, Y Englert, and A Delbaere Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents Reproduction, August 1, 2005; 130(2): 147 - 156. [Abstract] [Full Text] [PDF]
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F. Otsuka, R. K. Moore, X. Wang, S. Sharma, T. Miyoshi, and S. Shimasaki Essential Role of the Oocyte in Estrogen Amplification of Follicle-Stimulating Hormone Signaling in Granulosa Cells Endocrinology, August 1, 2005; 146(8): 3362 - 3367. [Abstract] [Full Text] [PDF]
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P.A. Johnson, M.J. Dickens, T.R. Kent, and J.R. Giles Expression and Function of Growth Differentiation Factor-9 in an Oviparous Species, Gallus domesticus Biol Reprod, May 1, 2005; 72(5): 1095 - 1100. [Abstract] |
140 µm in diameter) were dissected from rats at day 14 of age and
cultured in serum-free media with 2.5 mM bromo-cGMP in the
presence or absence of GDF-9 (200 ng/ml) and/or FSH (5 IU/ml). Some
cultures were treated with N-tagged GDF-9 (200 ng/ml) with or without
FSH. The number of follicles used for each group is shown in
parentheses.