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Department of Pathology (J.A.E., C.Y., P.W., M.M.M.) Department
of Molecular and Human Genetics (J.A.E., M.M.M.), and Department of
Cell Biology (M.M.M.) Baylor College of Medicine Houston, Texas
77030
Department of Applied Biochemistry (K.N.) Tohoku
University Sendai, Miyagi, Japan 981-8555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase, LH receptor
(LHR), and c-kit, the receptor for kit ligand]. This
demonstrates that in the absence of GDF-9, the follicles are
incompetent to emit a signal that recruits theca cell precursors to
surround the follicle; 2) The primary follicles of GDF-9-deficient mice
demonstrate an up-regulation of kit ligand and inhibin-. This
suggests that these two important secreted growth factors, expressed in
the granulosa cells, may be directly regulated in a paracrine fashion
by GDF-9. Up-regulation of kit ligand, via signaling through
c-kit on the oocyte, may be directly involved in the
increased size of GDF-9-deficient oocytes and the eventual demise of
the oocyte; 3) After loss of the oocyte, the cells of the
GDF-9-deficient follicles remain in a steroidogenic cluster that
histologically resembles small corpora lutea. However, at the molecular
level, these cells are positive for both luteal markers
(e.g. LHR and P-450 side chain cleavage) and nonluteal
markers (e.g. inhibin and P-450 aromatase). This
demonstrates that initially the presence of the oocyte prevents the
expression of luteinized markers, but that the absence of GDF-9 at an
early timepoint alters the differentiation program of the granulosa
cells; and 4) As demonstrated by staining with either proliferating
cell nuclear antigen (PCNA) or Ki-67 and TUNEL
(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)
labeling, the granulosa cells of GDF-9-deficient type 3b primary
follicles fail to proliferate but also fail to undergo cell death. This
suggests that granulosa cells of type 3b follicles require GDF-9 for
continued growth and also to become competent to undergo apoptosis,
possibly through a differentiation event. Thus, these studies have
enlightened us as to the paracrine roles of GDF-9 as well as the normal
steps of granulosa cell and theca cell growth and differentiation
within ovarian follicles. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The mammalian transforming growth factor-ß superfamily, the largest
family of secreted proteins, consists of more than 30 members (4).
In vivo studies have demonstrated that individual members of
this family exhibit a wide range of key biological functions, including
cellular differentiation during early development of the embryo
(e.g. nodal, BMP-2, and BMP-4), development of the eye and
kidney (e.g. BMP-7), craniofacial development
[e.g. activin ßA and transforming growth factor-ß3
(TGF-ß3)], suppression of the immune system (e.g.
TGF-ß1), and regulation of muscle mass [e.g. growth
differentiation factor-8 (GDF-8)] (5). Members of this family have
also been shown to play essential roles during mammalian sexual
differentiation and in gonadal function (5, 6). For example, absence of
Müllerian inhibiting substance results in male
pseudohermaphroditism, absence of inhibin- leads to ovarian and
testicular tumors, and absence of BMP-8a and BMP-8b, important
testicular proteins, results in male infertility due to defects in
spermatogenesis. GDF-9, BMP-15, and BMP-6 are TGF-ß superfamily
members expressed in the mammalian oocyte beginning at the type 3a
follicle (one-layer) stage and expressed through ovulation (4, 7, 8).
Using the GDF-9 knockout mice (9), we have previously shown that
absence of GDF-9 results in a block in folliculogenesis at the type 3b
stage (late, one-layer primary follicle stage). In addition, we have
demonstrated that there are other secondary effects of absence of
GDF-9, including apparent failure of the thecal layer to form and
defects in the oocyte (9, 10). Among the changes that occur in the
oocyte are defects in oocyte meiotic competence, including abnormal
germinal vesicle breakdown and spontaneous parthenogenetic activation,
and an increased rate of growth of the oocyte. In the present study, we
have examined the molecular changes within the ovary that result from
the absence of GDF-9. Our findings are important in defining the key
role of GDF-9 in follicle development as well as contibuting to the
understanding of normal ovarian physiology.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and cyclin-cdk complexes, is expressed during G1,
increases through the G1/S transition, is high in
G2, and declines sharply in M phases of the cell cycle
(11). Similarly, Ki-67, a component of the granular nucleolus, is
expressed in all cell cycle phases except G0 (12). The most
highly proliferative granulosa cells are found in the wild-type antral
follicle, in which the majority of the granulosa cells are PCNA (Fig. 1A
) and Ki-67 positive (data not shown).
In type 3b (large one-layer) and type 4 (two-layer) follicles of
wild-type ovaries, immunohistochemical analysis of PCNA (Fig. 1, B and
C) or Ki-67 (data not shown) showed that more than 50% of the
granulosa cells in the cross-sections are positive (i.e.
positive defined as intense red staining of the nucleus; negative
defined as light or diffuse staining of the nucleus and cytoplasm). In
contrast, the type 3b follicles with intact oocytes in the
GDF-9-deficient ovary demonstrated less than 10% positive staining
granulosa cells per cross-section (Fig. 1E), suggesting that nearly all
of the granulosa cells are blocked at G0. However, soon
after oocyte degeneration, granulosa cell differentiation begins to
occur in the follicles of the GDF-9-deficient ovary, and more cell
nuclei stain positively for both PCNA (Fig. 1, D–F) and Ki-67 (data
not shown). Oocyte nuclear staining with the PCNA antibody is detected
in growing and full-grown oocytes of GDF-9-deficient type 3a and 3b
follicles, consistent with previous studies demonstrating the presence
of PCNA in nuclei of oocytes of primary through antral follicles in
wild-type ovaries (11).
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, G–J). In contrast, sections of GDF-9-deficient ovaries
contained 2–10 positively labeled cells per ovarian section (Fig. 1, K
and L). No positively staining cells were found in the one-layer
follicles, but an occasional apoptotic cell was seen in follicles with
degenerating oocytes, within the steroidogenic follicular nests, or
within the interstitial tissue. Thus, in the absence of GDF-9, the
majority of granulosa cells of the type 3b follicle appear to remain
dormant and fail to either proliferate or die.
Analysis of Cell Cycle Inhibitors
p21 and p27 are well documented inhibitors of the cell cycle
and are correlated with cell cycle arrest upon luteinization in the
ovary (13). Based on the relative lack of proliferation of the
granulosa cells in the GDF-9 knockout ovaries, we examined expression
of both p21 and p27 mRNA by in situ hybridization and p27
protein by immunohistochemistry. p21 mRNA was detected at low levels
ubiquitously in both wild-type and GDF-9-deficient ovaries with higher
levels in wild-type atretic follicles and scattered cells in the
corpora lutea, and in the luteinized follicular nests of the
GDF-9-deficient ovary (data not shown). In wild-type ovaries, p27 mRNA
is also expressed ubiquitously at low levels throughout the ovary but
is more abundant in the corpora lutea of wild-type ovaries (Fig. 1, M
and N). In the GDF-9-deficient ovary, granulosa cells of the one-layer
follicles express detectable levels of p27 message, while small groups
of cells in the center of the GDF-9-deficient ovary express higher
levels (Fig. 1, O and P). Similarly, nuclear p27 immunoreactivity is
clearly detectable in the majority of luteinized granulosa cells within
the wild-type corpus luteum (Fig. 1Q), and within the luteinized
follicular nests of the GDF-9-deficient ovary (Fig. 1R). Reduced p27
nuclear staining is also present in granulosa cells of both wild-type
and GDF-9-deficient one-layer follicles, which is clearly higher than
the staining in the negative control or in the interstitial cells (Fig. 1, Q and R). Although it is impossible to estimate protein levels by
immunohistochemistry, there does not appear to be a dramatic difference
in p27 immunoreactivity between the one-layer follicles of the GDF-9
knockout and wild-type ovaries. This suggests that p27 protein
overexpression is not the reason for the block in folliculogenesis at
the primary follicle stage in the GDF-9-deficient ovaries.
Thecal Layer Development
We have previously reported that a morphologically distinct thecal
layer could not be detected by light and electron microscopic analysis
in GDF-9-deficient ovaries (9). However, a flattened layer of
fibroblastic cells outside of the granulosa cell basement membrane
rings the type-3b follicles in the GDF-9-deficient ovaries. To confirm
the absence of a true thecal layer, in situ hybridization
was carried out with a probe for cytochrome P-450 17-hydroxylase
(17-OH), a theca cell-specific enzyme necessary for androgen
production. In the wild-type ovary, cells expressing 17-OH begin to
associate with type 3b and type 4 follicles and form complete rings
just outside the granulosa cell basement membranes by the multilayer
preantral follicle stage (Fig. 2, A and
B). In contrast, in the GDF-9-deficient ovaries, only a few cells
expressing 17-OH are scattered in the interstitium and not
associated with follicles (Fig. 2, C and D). These 17-OH-positive
cells may represent a theca cell precursor population that is
responding to the elevated serum LH (9). Similarly, absence of LHR and
c-kit mRNA around the follicle (see below) confirms that a
theca cell layer fails to form. These data suggest that GDF-9 signaling
is required either directly or indirectly to recruit theca cell
precursors to the early preantral follicles.
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). By in situ hybridization,
c-kit mRNA is localized to the oocyte and theca-interstitial
cells of the wild-type ovary, but is excluded from granulosa cells as
previously demonstrated (Fig. 4, A and B
and Ref. 17). In the GDF-9-deficient ovaries, c-kit mRNA
localizes only to oocytes, with only background levels of silver grains
present over other cell types (Fig. 4, C–F).
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). Insulin-like
growth factor-I (IGF-I), which is also expressed in the granulosa cells
of early preantral follicles, however, does not show a similar increase
in expression in the GDF-9-deficient ovary (Fig. 3D), suggesting that
the increase in kit ligand represents a specific regulatory
interaction, rather than a tissue composition effect. Although in
situ hybridization is not a reliable method for quantitating mRNA
expression, we can compare relative expression levels between wild-type
and GDF-9-deficient ovaries by positioning sections from both types of
ovaries close together on the same slide to minimize interslide
variability. Under these conditions, we can barely detect kit ligand
expression in the wild-type ovary (Fig. 4, G and H), with a faint
signal above background apparent in granulosa cells of preantral
follicles. In contrast, in the GDF-9-deficient ovaries, granulosa cell
expression of kit ligand is abundant (Fig. 4, I and J). Type 3a and
early type 3b follicles have detectable levels of kit ligand, while the
largest one-layered follicles show more intense staining (Fig. 4, K and
L). In the event that paracrine factors produced by multilayered
follicles of adult ovaries repressed kit ligand expression, we examined
ovaries from 10-, 17-, and 28-day-old wild-type mice. Although kit
ligand was somewhat easier to detect in these immature ovaries because
of the increased number of preantral follicles, the relative expression
level per follicle was never comparable to the level seen in the
GDF-9-deficient ovaries at similar ages (data not shown). Kit ligand
expression is increased further in the granulosa cells of asymmetric
follicles, which are presumably destined to undergo oocyte degeneration
(Fig. 4, K and L). However, soon after the oocyte degenerates, kit
ligand expression disappears (Fig. 4, I and J) and is also absent in
the follicular nests.
There are two alternatively-spliced forms of kit ligand, KL-1 and KL-2,
which differ by 84 bp. This alternative splicing results in an
additional 28 amino acids in KL-1, which includes a proteolytic
cleavage site. Since membrane-bound kit ligand is more active than free
kit ligand, KL-2, the more stable, cell-associated form, is
consequently more potent (14). By nonquantitative RT-PCR using primers
that can distinguish KL-1 from KL-2, we detected both forms of kit
ligand in both the wild-type and GDF-9-deficient ovaries (Fig. 3B).
Furthermore, immunohistochemical analysis of the KL protein in the
GDF-9-deficient ovaries demonstrates the same pattern of expression as
the KL mRNA, with the most intense staining occurring in asymmetric
follicles (data not shown). Taken together, these results suggest that
GDF-9 negatively regulates kit ligand expression in granulosa cells in
a paracrine manner and that active, KL protein is synthesized.
TGF-ß Superfamily Members (Activins, Inhibins,
Follistatin)
Activins and inhibins have been implicated in the regulation of
granulosa cell proliferation and follicle growth both in
vivo and in vitro (18, 19). Hence, we
compared the expression levels and pattern of expression
of inhibin-, activin-ßA, activin-ßB, and the activin-binding
protein, follistatin, in wild-type and GDF-9-deficient ovaries.
Surprisingly, by Northern blot analysis, inhibin- is expressed at
similar levels in GDF-9-deficient vs. wild-type ovaries
(Fig. 3C). In wild-type ovaries, inhibin is expressed in granulosa
cells of all growing follicles (type 3a through the preovulatory
stage), but is excluded from corpora lutea (Fig. 5, A and B). In GDF-9-deficient ovaries,
inhibin- is expressed highly in the one-layer follicles, in the
follicles with degenerating oocytes, and in the central steroidogenic
follicular nests (Fig. 5, C and D). This indicates that the cells of
the follicular nests, although similar to corpora lutea in many
respects, are developmentally different than granulosa cells in
wild-type ovaries that have proliferated, formed into multilayer
follicles, and associated with an active thecal layer before
luteinizing.
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, E and F, and Fig. 6, A, B, I, and J). Interestingly, expression of ßB in atretic
follicles is low and localized specifically to the perioocyte cells,
while in healthy follicles, all granulosa cells express high levels
(Fig. 6, E and F). GDF-9-deficient
ovaries have almost undetectable ßB-mRNA by Northern blot analysis
and very low levels of follistatin (Fig. 3C). These genes are expressed
in the granulosa cells of the one-layer follicles, more robustly in
follicles with degenerating oocytes, and at highest levels in
oocyte-deficient follicular nests (Fig. 6, C, D, G, and H).
ßA-message is not detectable in the one-layered follicles, weakly
localizes to granulosa cells of follicles with degenerating oocytes,
and is expressed at high levels in oocyte-deficient follicular nests.
However, by Northern blot analysis the levels of ßA mRNA are
equivalent between GDF-9-deficient and wild-type ovaries (Fig. 3C).
These results indicate that the GDF-9-deficient ovaries have retained
the ability to make both activin-ß and inhibin- subunits. However,
the relative amounts of each subunit and thus the net production of
each type activin (A, B, or AB) or inhibin (A or B) remains to be
determined.
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, E
and F). FSHR is expressed in multilayer preantral and antral follicles
(Fig. 7, A and B). Aromatase, which is normally induced by FSH
stimulation of the granulosa cells, is expressed at high levels
specifically in the preovulatory follicle (Fig. 7, I and J). In the
GDF-9-deficient ovary, ERß is expressed at low levels in the
one-layer follicles, and at somewhat higher levels in the follicles
with degenerating oocytes (Fig. 7, G and H). FSHR is also expressed in
the follicles with degenerating oocytes (Fig. 7, C and D), while
aromatase is expressed at high levels only in the follicular nests with
completely degenerated oocytes (Fig. 7, K and L). Aromatase expression
in these follicular nests in the GDF-9-deficient ovaries suggests that
a functional FSH signaling pathway is present similar to granulosa
cells of preovulatory follicles of a wild-type ovary.
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and data not shown). Consistent
with the Northern blot data, in situ hybridization shows
that in wild-type ovaries stimulated with PMSG and hCG, COX-2 is
expressed by the granulosa cells of preovulatory follicles (Fig. 8, A and B and Ref. 23). The highest
expression at 5 h occurs in the cumulus cells of the wild-type
ovary (Fig. 8, A and B), while COX-2 expression is completely
undetectable in the GDF-9-deficient ovary (Fig. 8, C and D).
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, E and F) (21). In the GDF-9-deficient ovary, granulosa
cells of nonluteinized and luteinized follicular nests express LHR at
very high levels (Fig. 8, G and H). Stimulation of theca and luteinized
granulosa cells by LH stimulates production of the steroidogenic enzyme
P-450 scc and subsequent synthesis of progesterone (3). In wild-type
ovaries, P-450 scc protein is present in theca cells, corpora lutea,
and secondary interstitial tissue (Fig. 8, I and J). In GDF-9-deficient
ovaries, P-450 scc protein is detected at low levels in nonluteinized
follicular nests and at much higher levels in the steroidogenic
luteinized follicular nests (Fig. 8, K and L). Female 6-week-old
GDF-9-deficient mice have average serum progesterone levels of 3.4
ng/ml, compared with 2.6 ng/ml in wild-type mice, indicating that these
nests are not only capable of expressing markers but also functioning
like miniature corpora lutea. However, as mentioned earlier, these
follicular nests also express inhibin-, a marker that is normally
never observed at significant levels in corpora lutea (see below). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). GDF-9 mRNA (8) and protein (24) are
absent in primordial follicles and are first observed in type 3a
primary follicles. Ovaries from the GDF-9 knockout mice show a block at
the type 3b primary follicle stage. Our studies in this manuscript
demonstrate that the granulosa cells of the type 3b follicle
essentially lay dormant; neither cell division nor apoptosis is
observed in the granulosa cells of the follicles until the oocyte is
lost. Thus, although, GDF-9 protein is synthesized at the type 3a
stage, the GDF-9 signal transduction cascade must only become essential
at the type 3b stage for further follicular growth. Clearly, these
studies suggest that recruitment of primordial follicles and growth of
the granulosa cells from the primordial follicle stage (<20 granulosa
cells) to the type 3b stage (90 granulosa cells) (25) are not dependent
on GDF-9. Possibly another oocyte-secreted growth factor is required
for these approximately two rounds of cell divisions from the
primordial to the type 3b primary follicle stage. One factor that may
be instrumental in many of these processes is a recently identified
transcription factor, FIG. FIG mRNA is present in primordial
follicles, and FIG protein has been shown to regulate the
transcription of zona pellucida genes. It is likely, in combination
with additional factor(s), to regulate the transcription of many other
oocyte genes at this stage (26), potentially including a growth factor
that signals the pregranulosa cells to initiate replication.
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It has previously been hypothesized that an oocyte-derived factor regulates kit ligand expression by a paracrine mechanism (27). In gonadotropin-stimulated mice, there is a gradient of kit ligand expression whereby granulosa cells farthest from the oocyte (i.e. mural granulosa cells) express the highest levels, while those closest to the oocyte (i.e. cumulus cells) express very low or undetectable levels (28). Our observations of dramatically elevated kit ligand in GDF-9-deficient follicles suggest that GDF-9 is one of the oocyte-secreted paracrine factors that negatively regulates kit ligand expression. This hypothesis is supported by evidence from our in vitro studies of GDF-9 action (see Ref. 24) demonstrating that GDF-9 can regulate other genes (i.e. hyaluronan synthase 2, COX-2, steroidogenic acute regulator protein, urokinase plasminogen activator, and LHR) that are differentially expressed with respect to oocyte proximity in antral follicles. Thus, action of other oocyte-produced and extrafollicular factors unopposed by GDF-9 may contribute to the increased kit ligand expression that we observe. The kit ligand that is produced in the GDF-9-deficient ovaries also appears to be active. GDF-9-deficient ovaries contain significantly more mast cells per section (data not shown), possibly due to kit ligand-stimulated increased recruitment and proliferation, as has previously been reported in other systems. In addition, kit ligand has also been shown to stimulate oocyte growth in vitro (27). The oocytes in the GDF-9-knockout ovary grow more rapidly and to a 15% greater maximum size compared with the controls (10), before ulitmately degenerating, providing further evidence of functional granulosa cell-derived kit ligand signaling through c-kit on the oocyte. By Northern blot analysis, we show that other members of the TGF-ß superfamily continue to be expressed in the GDF-9-deficient ovary.
By Northern blot analysis, inhibin- and activin-ßA subunits are
expressed in GDF-9-deficient ovaries at similar levels to controls,
whereas activin-ßB and follistatin are dramatically decreased.
In vitro activin A has been shown to stimulate follicular
growth (18) and to promote FSH stimulation of granulosa cell DNA
synthesis (29). Additionally, activin A plus FSH stimulates
granulosa cells from immature follicles to produce progesterone, but
decreases progesterone synthesis by granulosa cells from differentiated
follicles cultured with or without FSH (30). In the GDF-9-deficient
ovary, locally produced activin A in the follicular nests may stimulate
limited granulosa cell proliferation and enhance the response of these
cells to the elevated serum gonadotropins to express P-450 aromatase
and P-450 scc. The activin effect in the GDF-9-deficient ovaries may be
enhanced by the reduced level of follistatin, an activin-binding
protein and antagonist. However, since the follicular nests express
both the inhibin- subunit and activin-ßA, it is unclear how much
activin A (vs. inhibin A) is being produced.
It has been suggested that once a follicle is recruited from the quiescent pool of primordial follicles into the growing pool, there are only two developmental endpoints: cell death or terminal differentiation (1, 25, 31). Follicles normally continue to grow until they either undergo atresia or luteinize after ovulation; under normal circumstances follicles cannot arrest at any intermediate stage. In the GDF-9-deficient ovary, we observe progressive granulosa cell differentiation, albeit uncoupled from granulosa cell proliferation and normal follicular morphogenesis. Evidence suggesting that the oocyte plays a key role in regulating gene expression within the follicle is accumulating. For example, removal of the oocyte from a rabbit follicle in vivo triggers luteinization of the follicle (32). In culture, oocytes have also been shown to inhibit expression of LHR (33), a marker of preovulatory follicles and corpora lutea.
There are at least three possible interpretations of our data regarding
the functional differentiation of the granulosa cells in the
GDF-9-deficient ovary. The first is that the GDF-9-deficient oocytes
still produce a factor other than GDF-9 that inhibits granulosa cell
differentiation, but when the oocyte degenerates the follicles
luteinize. The second possibility is that GDF-9 itself is the
luteinization-suppressing agent. The third possibility is that
luteinization is one endpoint of a default pathway that every granulosa
cell follows in a cell-autonomous manner after awakening from
quiescence, and that regulation of the process occurs by inducing cell
death before the granulosa cells reach that endpoint. Whatever the
reason for luteinization, the progression to a luteinized phenotype is
not simply turning on the genes that we see in the fully differentiated
corpus luteum, but a progressive process in which markers of
intermediate stages of follicles are expressed. For example, serial
sections (data not shown) hybridized with probes for aromatase, LHR,
and P-45017OH, showed distinct aromatase-positive, LHR-negative;
aromatase-positive, LHR-positive; and aromatase-negative, LHR-positive
follicular nests (all were 17OH negative). Additionally, aromatase
and FSHR, both markers of antral follicles, appear preferentially
expressed in degenerating oocyte- containing or nonluteinized
follicular nests (Fig. 9, ovary panel C) rather than the more
steroidogenic-appearing, fully luteinized nests (Fig. 9, ovary panel
D). In the wild-type ovary, the morphology of the follicle
(i.e. number of granulosa cell layers, antrum, presence or
absence of an oocyte) has allowed us to classify granulosa cell
markers. However, in the GDF-9-deficient ovary, follicular nests that
are at different stages of differentiation may histologically look very
similar. For this reason, it may appear that both luteinized and
nonluteinized markers are being abnormally coexpressed, especially for
markers such as aromatase and FSHR in which there is some variability
in the prevalence of expressing clusters. However, the widespread
expression of inhibin-, a marker clearly excluded from the CL in the
wild-type ovary in the majority of the central clusters, suggests that
the luteal clusters are not identical to normal CL. This suggests that
some luteal cell markers are actively suppressed, and this inhibitory
factor is not present or the cells are not responsive to it.
In the GDF-9-knockout, the thecal layer fails to form as determined by
light and electron microscopy and absence of the thecal layer markers
P-450 17-hydroxylase, LHR, and c-kit around the one-layer
type 3b follicles. However, 17-hydroxylase-positive cells, presumed
theca cell precursors, are still present throughout the interstitium.
These data suggest that GDF-9, either directly or indirectly, regulates
thecal layer development (Fig. 9). Previous studies have suggested that
a preantral follicle-derived factor is necessary for thecal layer
formation and that follicles with more than two layers of granulosa
cells, but not one-layer follicles, appear to be more competent at
stimulating thecal layer formation (34). Thus, absence of a thecal
layer could be due to the failure of GDF-9 to stimulate the formation
of a type 4 follicle and/or the secretion of the theca cell recruitment
factor. Based on its expression in granulosa cells of preantral
follicles, kit ligand is a candidate thecal layer recruitment factor.
However, our findings that kit ligand is highly expressed in the
GDF-9-knockout ovary makes this possibility unlikely. The finding that
theca precursor cells are still present suggests that intragonadal and
extragonadal factors continue to normally regulate differentiation and
gene expression (i.e. 17-hydroxylase expression) in these
cells.The present studies open up new avenues of research for
understanding ovarian function and the intraovarian role of GDF-9.
Future studies will include determining the factors that are required
for the regulation of primordial follicle recruitment and the
development of the thecal layer during ovarian folliculogenesis and the
interactions of GDF-9 in these processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RNA Isolation and Northern Blot Analysis
Total RNA was extracted from various tissues of wild-type and
GDF-9–deficient C57BL/6/129SvEv hybrid mice using RNA STAT-60 (Leedo
Medical Laboratories, Houston, TX) as described by the manufacturer and
quantitated on a spectrophotometer. Fifteen micrograms of total RNA of
each sample were electrophoresed on a 1.2% agarose/7.6% formaldehyde
gel and transferred to Hybond N nylon membrane (Amersham,
Arlington Heights, IL). Table 1 includes
a summary of all the specific cDNA fragments used to make probes in
these studies. Probes were generated by random priming with
[32P]dATP using the Strip-EZ probe synthesis kit
(Ambion, Inc., Austin, TX). The membrane was hybridized,
washed, and subjected to autoradiography as previously described (35).
The probe was removed from the membrane using the Strip-EZ removal
reagents (Ambion, Inc.) following the manufacturer’s
protocol. The same blots were then reprobed with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA
as a loading control. Signals for each probe were quantitated on a
Molecular Dynamics, Inc. (Sunnyvale, CA)
photodensitometer.
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includes a summary of the specific
probe information. [-35S]UTP-labeled antisense
and sense probes were generated using the Riboprobe T7/T3 or Riboprobe
T7/SP6 Combination System (Promega Corp., Madison, WI).
Hybridization was carried out at 55 C with 5 x 106
cpm of each riboprobe per slide for 16–18 h in 50% deionized
formamide/0.3 M NaCl/20 mM Tris-HCl (pH 8.0)/5
mM EDTA/10 mM NaPO4 (pH 8.0)/10%
Dextran sulfate/1x Denhardt’s/0.5 mg/ml yeast RNA. High-stringency
washes of 2x SSC/50% formamide and 0.1x SSC at 65 C were carried
out. Dehydrated sections were dipped in NTB-2 autoradiographic
emulsion (Eastman Kodak Co., Rochester, NY) and exposed
2–14 days, depending on the probe, at 4 C. After developing, the
slides were counterstained with hematoxylin and mounted for
photography.
Immunohistochemistry
Ovaries were fixed in 20% neutral buffered formalin for 3
h, processed, embedded in paraffin, and sectioned at 4 µm thickness.
Detection of PCNA was conducted as previously described (38) using a
mouse anti-PCNA monoclonal antibody (Novocastra Laboratories, Newcastle
upon Tyne, UK). The rabbit antimouse P-450 scc polyclonal antiserum was
a kind gift from Michael J. Soares at the University of Kansas Medical
Center, and used at a 1:625 dilution in 1x PBS, 0.05% Tween-20
(PBST), with 2% normal mouse serum (Sigma Chemical Co.)
and 2% normal goat serum (Vector Laboratories, Inc.,
Burlingame, GA). Rabbit anti-Ki-67 polyclonal antiserum (Novocastra)
was diluted 1:300 and rabbit anti-p27 polyclonal antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was diluted 1:125 in
1% BSA, 0.1% NaN3 in PBS, with 2% normal mouse serum and
2% normal goat serum. Successful staining for both Ki-67 and p27
required antigen retrieval methods. For Ki-67 staining, sections were
steamed for 35 min in 0.1 M citrate buffer, pH 6.0. For p27
staining, sections were steamed for 35 min in pH 8.0 Tris-EDTA antigen
retrieval solution. For Ki-67, p27, and P-450 scc, all sections were
blocked for 30 min in 1x PBS with 0.05% Tween-20, 2% normal mouse
serum (Sigma Chemical Co.), and 2% normal goat serum and
incubated with the primary antibody for 1 h at room temperature.
PCNA detection was accomplished using the Super Sensitive Mouse
Antibody Animal Detection kit (BioGenex Laboratories, Inc., San Ramon, CA) containing antimouse IgG-biotinylated
secondary antibody preabsorbed with rat tissue. P-450 scc, p27, and
Ki-67 antibodies were detected using the Super Sensitive Rabbit
Antibody Detection kit (Biogenex Laboratories, Inc.)
containing antirabbit IgG-biotinylated secondary antibody preabsorbed
with mouse tissue. PCNA and P-450 scc were detected using
streptavidin-conjugated alkaline phosphatase label and New Fuschin
substrate (BioGenex Laboratories, Inc.) while p27 was
detected with streptavidin-conjugated horseradish peroxidase
label (BioGenex Laboratories, Inc.) and
3,3'-diaminobenzidine tetrahydrochloride substrate (Vector Laboratories, Inc.).
TUNEL Assay
Ovaries were stained for apoptotic cells by a modified TUNEL
method using the Apoptag Plus Complete Apoptosis Detection kit (Oncor
Laboratories, Gaithersburg, MD) following the manufacturer’s
instructions. Nuclei were counterstained with Propidium
iodide/Antifade mounting media (Oncor Laboratories).
RT-PCR Analysis
Oligo-dT-primed cDNA from 1 µg of either control or
GDF-9-deficient ovarian RNA was synthesized using Superscript reverse
transcriptase (Gibco BRL, Gaithersburg, MD) following the
manufacturer’s protocol. One microliter of each RT reaction (1/20
of total) was used in each 25 µl PCR reaction primed with kit
ligand-specific oligonucleotides:
5'-CCAGAAACTAGATCCTTTACTCCT-3'(sense, nucleotides 493–517 of
S40364) and 5'-CTGTTGCAGCCAGCTCCCTTAG-3' (antisense, nucleotides
943–919 of S40364) primers which span introns and an 84-bp
alternatively spliced exon. Amplification of the KL-1 form yields a
product of 450 bp, and amplification of KL-2 form yields a product of
366 bp. Products were separated on a 2% agarose gel and visualized by
ethidium bromide staining.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These studies were supported in part by sponsored research grants from Genetics Institute (Cambridge, MA) and Metamorphix (Baltimore, MD) and NIH Grant HD-33438 (to M.M.M.). Julia A. Elvin is a student in the Medical Scientist Training Program supported in part by NIH Grants GM-07330 and GM-08307 and the Baylor Research Advocates for Student Scientists (BRASS) organization.
Received for publication February 1, 1999. Revision received March 22, 1999. Accepted for publication March 24, 1999.
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J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
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F. H. Thomas, J.-F. Ethier, S. Shimasaki, and B. C. Vanderhyden Follicle-Stimulating Hormone Regulates Oocyte Growth by Modulation of Expression of Oocyte and Granulosa Cell Factors Endocrinology, February 1, 2005; 146(2): 941 - 949. [Abstract] [Full Text] [PDF]
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D. Schmidt, C. E. Ovitt, K. Anlag, S. Fehsenfeld, L. Gredsted, A.-C. Treier, and M. Treier The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance Development, February 15, 2004; 131(4): 933 - 942. [Abstract] [Full Text] [PDF]
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S. Shimasaki, R. K. Moore, F. Otsuka, and G. F. Erickson The Bone Morphogenetic Protein System In Mammalian Reproduction Endocr. Rev., February 1, 2004; 25(1): 72 - 101. [Abstract] [Full Text] [PDF]
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 R. L. Krisher The effect of oocyte quality on development J Anim Sci, January 1, 2004; 82(13_suppl): E14 - 23. [Abstract] [Full Text] [PDF]
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K. H. Burns, J. E. Agno, L. Chen, B. Haupt, S. C. Ogbonna, K. S. Korach, and M. M. Matzuk Sexually Dimorphic Roles of Steroid Hormone Receptor Signaling in Gonadal Tumorigenesis Mol. Endocrinol., October 1, 2003; 17(10): 2039 - 2052. [Abstract] [Full Text] [PDF]
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K. H. Burns, G. E. Owens, S. C. Ogbonna, J. H. Nilson, and M. M. Matzuk Expression Profiling Analyses of Gonadotropin Responses and Tumor Development in the Absence of Inhibins Endocrinology, October 1, 2003; 144(10): 4492 - 4507. [Abstract] [Full Text] [PDF]
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M. Hsieh, S. M. Mulders, R. R. Friis, A. Dharmarajan, and J. S. Richards Expression and Localization of Secreted Frizzled-Related Protein-4 in the Rodent Ovary: Evidence for Selective Up-Regulation in Luteinized Granulosa Cells Endocrinology, October 1, 2003; 144(10): 4597 - 4606. [Abstract] [Full Text] [PDF]
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Y. Yang, A. Balla, N. Danilovich, and M. R. Sairam Developmental and Molecular Aberrations Associated with Deterioration of Oogenesis During Complete or Partial Follicle-Stimulating Hormone Receptor Deficiency in Mice Biol Reprod, October 1, 2003; 69(4): 1294 - 1302. [Abstract] [Full Text] [PDF]
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K. L. Loveland, M. Bakker, T. Meehan, E. Christy, V. von Schonfeldt, A. Drummond, and D. de Kretser Expression of Bambi Is Widespread in Juvenile and Adult Rat Tissues and Is Regulated in Male Germ Cells Endocrinology, September 1, 2003; 144(9): 4180 - 4186. [Abstract] [Full Text] [PDF]
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X. Wu, P. Wang, C. A. Brown, C. A. Zilinski, and M. M. Matzuk Zygote Arrest 1 (Zar1) Is an Evolutionarily Conserved Gene Expressed in Vertebrate Ovaries Biol Reprod, September 1, 2003; 69(3): 861 - 867. [Abstract] [Full Text] [PDF]
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D. M. Duffy Growth Differentiation Factor-9 Is Expressed by the Primate Follicle Throughout the Periovulatory Interval Biol Reprod, August 1, 2003; 69(2): 725 - 732. [Abstract] [Full Text] [PDF]
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 D. H. Castrillon, L. Miao, R. Kollipara, J. W. Horner, and R. A. DePinho Suppression of Ovarian Follicle Activation in Mice by the Transcription Factor Foxo3a Science, July 11, 2003; 301(5630): 215 - 218. [Abstract] [Full Text] [PDF]
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J. F. Couse, M. M. Yates, V. R. Walker, and K. S. Korach Characterization of the Hypothalamic-Pituitary-Gonadal Axis in Estrogen Receptor (ER) Null Mice Reveals Hypergonadism and Endocrine Sex Reversal in Females Lacking ER{alpha} But Not ER{beta} Mol. Endocrinol., June 1, 2003; 17(6): 1039 - 1053. [Abstract] [Full Text] [PDF]
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C. M. Milner and A. J. Day TSG-6: a multifunctional protein associated with inflammation J. Cell Sci., May 15, 2003; 116(10): 1863 - 1873. [Abstract] [Full Text] [PDF]
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C. R. Harlow, M. Rae, L. Davidson, P. C. Trackman, and S. G. Hillier Lysyl Oxidase Gene Expression and Enzyme Activity in the Rat Ovary: Regulation by Follicle-Stimulating Hormone, Androgen, and Transforming Growth Factor-{beta} Superfamily Members in Vitro Endocrinology, January 1, 2003; 144(1): 154 - 162. [Abstract] [Full Text] [PDF]
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J.-S. Roh, J. Bondestam, S. Mazerbourg, N. Kaivo-Oja, N. Groome, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells Endocrinology, January 1, 2003; 144(1): 172 - 178. [Abstract] [Full Text] [PDF]
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H. Chang, C. W. Brown, and M. M. Matzuk Genetic Analysis of the Mammalian Transforming Growth Factor-{beta} Superfamily Endocr. Rev., December 1, 2002; 23(6): 787 - 823. [Abstract] [Full Text] [PDF]
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A. Bachelot, P. Monget, P. Imbert-Bollore, K. Coshigano, J. J. Kopchick, P. A. Kelly, and N. Binart Growth Hormone Is Required for Ovarian Follicular Growth Endocrinology, October 1, 2002; 143(10): 4104 - 4112. [Abstract] [Full Text] [PDF]
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C. R. Harlow, L. Davidson, K. H. Burns, C. Yan, M. M. Matzuk, and S. G. Hillier FSH and TGF-{beta} Superfamily Members Regulate Granulosa Cell Connective Tissue Growth Factor Gene Expression in Vitro and in Vivo Endocrinology, September 1, 2002; 143(9): 3316 - 3325. [Abstract] [Full Text] [PDF]
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K. H. Burns, G. E. Owens, J. M. Fernandez, J. H. Nilson, and M. M. Matzuk Characterization of Integrin Expression in the Mouse Ovary Biol Reprod, September 1, 2002; 67(3): 743 - 751. [Abstract] [Full Text] [PDF]
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E. E. Nilsson and M. K. Skinner Growth and Differentiation Factor-9 Stimulates Progression of Early Primary but Not Primordial Rat Ovarian Follicle Development Biol Reprod, September 1, 2002; 67(3): 1018 - 1024. [Abstract] [Full Text] [PDF]
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S. Varani, J. A. Elvin, C. Yan, J. DeMayo, F. J. DeMayo, H. F. Horton, M. C. Byrne, and M. M. Matzuk Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility Mol. Endocrinol., June 1, 2002; 16(6): 1154 - 1167. [Abstract] [Full Text] [PDF]
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M. Hsieh, M. A. Johnson, N. M. Greenberg, and J. S. Richards Regulated Expression of Wnts and Frizzleds at Specific Stages of Follicular Development in the Rodent Ovary Endocrinology, March 1, 2002; 143(3): 898 - 908. [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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A. L. L. Durlinger, M. J. G. Gruijters, P. Kramer, B. Karels, H. A. Ingraham, M. W. Nachtigal, J. Th. J. Uilenbroek, J. A. Grootegoed, and A. P. N. Themmen Anti-Mullerian Hormone Inhibits Initiation of Primordial Follicle Growth in the Mouse Ovary Endocrinology, March 1, 2002; 143(3): 1076 - 1084. [Abstract] [Full Text] [PDF]
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J. J. Eppig, K. Wigglesworth, and F. L. Pendola The mammalian oocyte orchestrates the rate of ovarian follicular development PNAS, February 20, 2002; (2002) 52658699. [Abstract] [Full Text] [PDF]
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T. El-Hefnawy and A. J. Zeleznik Synergism Between FSH and Activin in the Regulation of Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D2 Expression in Rat Granulosa Cells Endocrinology, October 1, 2001; 142(10): 4357 - 4362. [Abstract] [Full Text] [PDF]
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K. H. Burns, C. Yan, T. R. Kumar, and M. M. Matzuk Analysis of Ovarian Gene Expression in Follicle-Stimulating Hormone {beta} Knockout Mice Endocrinology, July 1, 2001; 142(7): 2742 - 2751. [Abstract] [Full Text] [PDF]
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