Molecular Characterization of the Follicle Defects in the Growth Differentiation Factor 9-Deficient Ovary
Julia A. Elvin,
Changning Yan,
Pei Wang,
Katsuhiko Nishimori and
Martin M. Matzuk
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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Growth differentiation factor-9 (GDF-9), a
secreted member of the transforming growth factor-ß superfamily, is
expressed at high levels in the mammalian oocyte beginning at the type
3a primary follicle stage. We have previously demonstrated that
GDF-9-deficient female mice are infertile because of an early block in
folliculogenesis at the type 3b primary follicle stage. To address the
molecular defects that result from the absence of GDF-9, we have
analyzed the expression of several important ovarian marker genes. The
major findings of our studies are as follows: 1) There are no
detectable signals around GDF-9-deficient follicles for several theca
cell layer markers [i.e. 17
-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.
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INTRODUCTION
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The ovarian follicle is the functional unit of the female
reproductive system, consisting of an oocyte surrounded by granulosa
and theca cells. During normal folliculogenesis, oocyte growth and
maturation are coordinated with granulosa and theca cell proliferation
and differentiation within the follicular unit (reviewed in Ref. 1).
Follicular recruitment of a quiescent primordial follicle into the
growing pool is morphologically characterized by an increase in oocyte
size and a squamous-to-cuboidal transition of the associated granulosa
cells, followed by secretion of the glycoprotein-rich zona pellucida.
Normally, once follicular growth is initiated, the oocyte grows to full
size, and the granulosa cells proliferate to form multiple layers of
granulosa cells in response to intrafollicular signals (2).
Preantral follicle development encompasses approximately seven
doublings of the original granulosa cells and is regulated primarily by
paracrine and autocrine mechanisms. In contrast, large follicles are
responsive to both intraovarian and extraovarian regulation and
actively participate in the hypothalamic-pituitary-gonadal axis. Unless
rescued by elevated FSH, most multilayer preantral and early antral
follicles undergo atresia, a degenerative process characterized by
widespread apoptotic death of the granulosa cells. In surviving
follicles, LH stimulates theca cell androgen production, while FSH
stimulates granulosa cell proliferation, aromatization of androgens to
estrogens, and LH receptor (LHR) expression (3). Follicular estrogen
feeds back on both the hypothalamus and pituitary to trigger the LH
surge, while granulosa cell-derived inhibin decreases pituitary FSH
secretion. The ability of the mammalian ovary to produce and respond to
this complex milieu of regulatory factors is essential for efficient
reproductive function.
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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Cell Cycle Progression
GDF-9-deficient follicles with intact oocytes contain only one
layer of granulosa cells and fail to form follicles consisting of
multiple layers of granulosa cells. We hypothesized that this lack of
increase in granulosa cell number is due to either a lack of granulosa
cell proliferation or to an increase in granulosa cell apoptosis.
Proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase
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
, DF) 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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Figure 1. Immunohistochemical and in Situ
Hybridization Analysis of Ovarian Cell Cycle Markers
AF, Ovaries from 9-week-old control (AC) and GDF-9-deficient (DF)
mice were stained with an anti-PCNA monoclonal antibody. A, A large
antral follicle (AnF) with positively staining (dark
red) granulosa cells (GC) and cumulus cells (CC) (low power). B
and C, One-layer (type 3b) and two-layer (type 4) follicles contain
multiple positive nuclei and positive germinal vesicle (GV). The
adjacent corpus lutea (CL) contains a few elongated, darkly staining
cells (true positive) as well as diffusely staining luteinized granulosa cells (nonproliferating) (B, low power; C,
high power). In panel C a cluster of small follicles in the wild-type
ovary contains a high proportion of positively staining nuclei in a
type 4 follicle, a single stained nuclei in the type 3b follicle, and
no staining of the primordial (type 2) follicle. D, Low power (D) and
high power (E and F) views of a GDF-9-deficient ovary demonstrates
normal and abnormal follicles including oocyte-containing one-layer
follicles (type 2, 3a, and 3b), which occupy the periphery, and
follicles with degenerating oocytes (DO) and follicular nests, which
predominate in the center of the section. In panels E and F, large type
3b follicles have few PCNA-positive granulosa cells, but oocytes (O)
demonstrate intense PCNA-positive staining of the GV. Type 3a follicles
and follicles with degenerating oocytes (DO) have multiple positive
granulosa cells. GL, Apoptosis detection in control (GJ) and
GDF-9-deficient (K and L) follicles. All nuclei stained with propidium
iodide (red), and apoptotic nuclei were labeled
green by modified TUNEL with fluorescein detection (low
power). G and H, A healthy antral follicle (AnF) and an atretic
follicle (AtF) showing apoptotic nuclei in the atretic follicle in
panel H. I and J, Scattered apoptotic nuclei in a corpus luteum are
also seen in panel J but are absent in an adjacent healthy type 4
follicle. K and L, Several apoptotic nuclei are present in the
follicular nests (FN) as seen in panel L, but elsewhere the apoptotic
cells are absent. MR, p27 expression in control (M, N, and Q) and
GDF-9-deficient (O, P, and R) ovaries. MP, p27 mRNA expression was
analyzed by in situ hybridization using a specific cDNA
antisense probe. Hybridization signal is visualized by darkfield
illumination (N and P), and can be compared with tissue morphology in
brightfield panel (M and O, low power). Q and R, p27 protein is
detected by immunohistochemistry (brown), and
nuclei are counterstained with hematoxylin (blue). In
control ovaries, p27 is expressed at high levels in most cells of the
corpus luteum (CL) and primordial follicles (type 2) and at low levels
in preantral follicles (e.g. type 3a, 3b, and 4). In
GDF-9-deficient ovaries, high levels are detected in some cells of the
luteinized follicular nests (FN) and primordial follicles (type 2),
while lower levels are seen in granulosa cells of type 3b follicles.
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TUNEL (Terminal Deoxynucleotidyl
Transferase-Mediated dUTP Nick End Labeling) Labeling
Although normal follicular development in the GDF-9-deficient
ovaries arrests at the type 3b stage and oocyte loss eventually
results in granulosa cell differentiation, histological examination of
the GDF-9-deficient ovaries surprisingly failed to detect any
apoptotic-appearing cells. To confirm the relative absence of apoptotic
cells in the GDF-9-deficient ovaries, we used a TUNEL assay to
fluorescently label DNA ends. Granulosa cells of wild-type atretic
antral follicles and corpora lutea contained many positively labeled
cells (Fig. 1
, GJ). In contrast, sections of GDF-9-deficient ovaries
contained 210 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.
Analysis of c-kit and kit Ligand Expression
The c-kit/kit ligand-signaling pathway has been shown
to be important for germ cell proliferation and folliculogenesis
(14, 15, 16). By Northern blot analysis, we show that c-kit mRNA
is expressed in GDF-9-deficient ovaries and that levels are comparable
to or slightly higher than wild-type ovaries (Fig. 3B
). 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
, CF).

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Figure 3. Analysis of Gene Expression in Control (WT)
vs. GDF-9-Deficient (-/-) Ovaries
A, Northern blot analysis of c-kit and KL gene
expression in control and GDF-9-deficient ovaries. The lower
panels are the GAPDH loading controls. Band intensities
(normalized to GAPDH) for GDF-9-deficient: control yields a ratio of
1.4:1 for c-kit and 32:1 for KL (E). B, RT-PCR products
of control or GDF-9-deficient ovarian RNA amplified for 27 or 30 cycles
with primers spanning the alternatively spliced exon, which
distinguishes KL-1 from KL-2. Both samples amplify two products of 366
bp and 450 bp, as compared with the 100-bp DNA ladder. C, Northern blot
analysis of inhibin , activin ßA, activin ßB, and follistatin
gene expression in control and GDF-9-deficient ovaries. (GAPDH not
shown.) Comparison of band intensities normalized to GAPDH for -/-:
control yields a ratio of 1.1:1 for inhibin- , 1:1.2 for
activin-ßA, 1:5.9 for activin-ßB, and 1:3 for follistatin (E). D,
Northern blot analysis of COX-2 in PMSG (48 h)/hCG (5 h)-treated
control and GDF-9-deficient ovaries, and IGF-I in unstimulated control
and GDF-9-deficient ovaries (18S not shown). The band intensities were
normalized to 18S for -/-: control yields a ratio of 1:56 for COX-2
and 1.4:1 for the larger ( 7 kb) and 1:1.6 for the lower ( 1 kb)
forms of IGF-I (E).
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Figure 4. Localization of c-kit and Kit
Ligand mRNA
Expression of c-kit mRNA in adult control (A and B) and
GDF-9-deficient (CF) ovaries analysis by in situ
hybridization using specific cDNA antisense probes. (AD)
c-kit expression is localized to oocytes (O) in both
ovaries, and theca/interstitial cells (TI) in the control (low power).
E and F, At higher magnification in the GDF-9-deficient ovary, it is
clear that only intact oocytes (O) express c-kit, and that degenerating
oocytes (DO) no longer express c-kit (high power). GL, Expression of
kit ligand mRNA in control (G and H) and GDF-9-deficient (IL)
ovaries. G and H, Low levels of KL are expressed in granulosa cells of
preantral follicles (i.e. type 3b and 4 follicles),
scattered cells in the corpus luteum, and in interstitial cells (low
power). IL, In GDF-9-deficient ovaries, the largest type 3b follicles
express high levels of KL, and asymmetric follicles (AsF) express even
higher levels. The follicular nests (FN) no longer express KL (I and J,
low power; K and L, high power).
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In contrast to the c-kit expression results, kit
ligand (KL) expression in GDF-9-deficient ovaries is increased 32-fold
compared with expression in wild-type ovaries (Fig. 3B
). 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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Figure 5. Localization of Inhibin- and Activin-ßA mRNA
Expression of inhibin- and activin-ßA mRNA in adult control (A, B,
E, F, I, and J) and GDF-9-deficient (C, D, G, H, K, and L) ovaries was
analyzed by in situ hybridization using specific cDNA
antisense probes. A and B, Control ovaries show expression of
inhibin- in granulosa cells (GC) of preantral and antral follicles,
while cells of the corpora lutea (CL) are negative (low power). C and
D, In GDF-9-deficient ovaries, granulosa cells are positive, with
follicles with degenerating oocytes (DO) and the follicular nests (FN)
having the highest levels of expression (low power). E and F,
Expression of activin-ßA localizes to granulosa cells of healthy
antral follicles (AnF), is less in atretic follicles (AtF), and is
absent in corpora lutea of control ovaries (E and F) (low power). At
higher magnification (I and J), activin-ßA cannot be detected in type
4 follicles. G, H, K, and L, In GDF-9-deficient ovaries, activin-ßA
is not expressed in primordial (type 2) or one-layer (types 3a and 3b)
follicles, but is expressed at low levels in the follicles with
degenerating oocytes (DO) and at very high levels in the follicular
nests (FN). (G and H, low power; K and L, high power).
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The two inhibin/activin-ß subunits, ßA and ßB, and the activin
binding protein, follistatin, are expressed in overlapping patterns in
the wild-type ovary. All three genes are expressed in granulosa cells
of multilayer preantral and antral follicles (Fig. 5
, 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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Figure 6. Localization of Activin-ßB and Follistatin mRNA
Expression in adult control (A, B, E, F, I, and J) and GDF-9-deficient
(C, D, G, H, K, and L) ovaries was analyzed by in situ
hybridization using specific cDNA antisense probes. A and B, Control
ovaries show high level expression of activin ßB in granulosa cells
of antral follicles (AnF), while cells of the corpora lutea (CL) and
most cells of the atretic follicles (AtF) are negative (low power). E
and F, At higher magnification, low levels of activin-ßB are detected
in two-layer (type 4) follicles. In GDF-9-deficient ovaries, granulosa
cells of one- layer (type 3a and 3b) follicles have detectable
expression of activin-ßB (C and D, low power), while follicles with
degenerating oocytes (DO) and follicular nests (FN) have higher levels
of expression (G and H, high power). Expression of follistatin
localizes to granulosa cells of both preantral follicles (PF) and
antral follicles (AnF), but is excluded from the corpus luteum (CL) in
control ovaries (I and J). K and L, In GDF-9-deficient ovaries
expression localizes to follicles with degenerating oocytes and
follicular nests (FN) (low power).
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Characterization of Antral Follicle Markers (ERß, FSHR,
Cytochrome P-450 Aromatase)
To further characterize the follicles of the GDF-9-deficient
ovary, we analyzed the expression of other markers associated with
antral granulosa cell functional differentiation: FSH receptor (FSHR),
cytochrome P-450 aromatase (aromatase), and estrogen receptor-ß
(ERß). We previously showed by Northern blot analysis that FSHR is
expressed similarly in both GDF-9-deficient and wild-type ovaries, and
that aromatase expression is 50% of the wild-type level (9). ERß is
also expressed at low but similar levels in the knockout vs.
wild-type ovaries (data not shown). By in situ hybridization
in the wild-type ovary, we detected FSHR, ERß, and aromatase
specifically in the granulosa cells as previously reported (3, 20, 21).
ERß is expressed at low levels in one-layer follicles and at higher
levels in multilayer follicles (Fig. 7
, 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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Figure 7. Localization of Antral Follicle Markers: Estrogen
Receptor-ß, FSH Receptor, and P-450 Aromatase
Expression in adult control (A, B, E, F, I, and J) and GDF-9-deficient
(C, D, G, H, K, and L) ovaries was analyzed by in situ
hybridization using specific cDNA antisense probes. ERß expression
localizes at low levels to granulosa cells of preantral follicles (PF)
including type 3 and type 4 follicles and to antral follicles (AnF) at
higher levels (A and B), while FSHR is only detectable in antral
follicles (E and F). In GDF-9-deficient ovaries, follicles with
degenerating oocytes (DO) and follicular nests (FN) have detectable
levels of ERß (C and D) and FSHR (G and H). I and J, P-450 aromatase
is barely detectable in one antral follicle (AnF) of the control ovary,
while a larger preovulatory follicle (POF) shows abundant expression. K
and L, In the GDF-9-deficient ovary, follicles with degenerating
oocytes (DO) show low-level expression of aromatase, but the follicular
nests (FN) demonstrate highest expression (low power).
|
|
Characterization of Periovulatory and Luteal Markers
[Cyclooxygenase 2 (COX-2), LHR, Cholesterol Side Chain Cleavage
Cytochrome P-450]
As mentioned previously, GDF-9-deficient ovaries contain multiple,
centrally located nests of cells that have the appearance of luteinized
granulosa cells. By electron microscopic analysis, the cells of these
nests contain multiple lipid droplets and mitochondria with tubular
cristae typical of highly steroidogenic cells (9). To confirm their
steroidogenic nature and similarity to luteal cells, we analyzed the
expression of COX-2 and LHR message, and cholesterol side chain
cleavage cytochrome P-450 protein (P-450 scc). As COX-2 is expressed in
the wild-type ovary only within a discrete window of time after the LH
surge (22, 23), we used ovaries from 3-week-old mice stimulated for
48 h with PMSG, and then hCG for 5 h for both Northern blot
analysis and in situ hybridization. By Northern blot
analysis, RNA from superovulated ovaries showed three distinct bands,
while no bands could be detected in the unstimulated wild-type or
GDF-9-deficient ovary lanes (Fig. 3D
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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Figure 8. Localization of Ovulatory and Luteal Markers:
COX-2, LHR, and P-450 Cholesterol Side Chain Cleavage
Expression of COX-2 in adult control stimulated with PMSG for
48 h, followed by hCG for 5 h (A, B, E, and F) and
unstimulated GDF-9-deficient (C, D, G, and H) ovaries was analyzed by
in situ hybridization using specific cDNA antisense
probes. COX-2 expression localizes to granulosa cells of preovulatory
follicles (POF) only, with cumulus cells (CC) surrounding the oocyte
(O) expressing higher levels than mural granulosa cells (MGC) (A and
B). Sections of GDF-9-deficient ovaries (C and D) failed to demonstrate
COX-2 expression and were indistinguishable from sections hybridized
with the sense probe (data not shown). EH, LHR expression is high in
corpora lutea (CL) of control ovaries (E and F) and in follicles with
degenerating oocytes and in the follicular nests (FN) in the
GDF-9-deficient ovaries (G and H) (low power). IL, P-450 scc protein
was detected immunohistochemically (dark staining), and
sections were not counterstained. In control ovaries, P-450 scc is
produced by corpora lutea (CL), theca (T), and interstitial (IC) cells
(I, J), but not by preantral or antral granulosa cells. In
GDF-9-deficient ovaries, P-450 scc is detectable at low levels in
follicles with degenerating oocytes (DO) and at higher levels in the
follicular nests (FN) (K and L, low power).
|
|
In contrast to the above-mentioned COX-2 expression data, we have
previously shown that LHR is expressed in the GDF-9-deficient ovary at
levels comparable to the wild-type ovary by Northern blot analysis (9).
LHR is expressed by theca cells, granulosa cells of preovulatory
follicles (data not shown), and luteinized granulosa cells of corpora
lutea (Fig. 8
, 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
|
---|
We have previously demonstrated that absence of GDF-9 in the
mammalian oocyte leads to infertility due to a block in follicular
development. In the present studies, we attempted to further
characterize the defects at the molecular level (summarized in Fig. 9
). 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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Figure 9. Summary of the GDF-9 Knockout Ovary Studies
High-power views of the follicles (A and B) and follicular nests (C and
D) from PAS-stained sections of GDF-9-deficient ovary. To the
right of each box is a list of markers expressed at that
stage. Boxes in low-power image indicate typical
location where each follicle type can be found in the GDF-9-deficient
ovary (top, low power). A, Normal primordial (type 2)
and primary follicles (types 3a and 3b). B, A follicle containing an
overgrown oocyte with an uneven distribution of granulosa cells. C, A
follicular nest with an empty zona pellucida remnant
(magenta) after oocyte degeneration (nonluteinized). D,
A luteinized follicular nest containing hypertrophied, foamy-appearing,
highly steroidogenic granulosa cells. Model of GDF-9 action in early
folliculogenesis (left). GDF-9 mRNA and protein are
present in type 3a follicles. GDF-9 signaling to the granulosa cells is
required for granulosa cell proliferation and growth from the type 3b
to type 4 follicle and negatively regulates kit ligand (KL) and
inhibin- expression. Thecal layer recruitment begins at the type 4
follicle stage and may be a direct effect of GDF-9 (purple
arrow) or alternatively GDF-9 may induce the granulosa cells to
produce a recruitment factor (blue arrow). The role of
GDF-9 in larger preantral and antral follicles is still unknown. In the
preovulatory follicle (type 8), GDF-9 stimulates hyaluronan synthase 2
(HAS2), steroidogenic acute regulatory protein (StAR), and COX-2 and
suppresses LHR and urokinase plasminogen activator (uPA) (24 ).
|
|
Unlike the dramatic apoptosis observed in atretic follicles of
wild-type ovaries, there is minimal apoptosis observed in the GDF-9
knockout ovary. Normally, apoptosis occurs in antral follicles at a
point after they become responsive to and dependent on FSH. Even though
granulosa cells of the GDF-9-deficient ovary express antral markers, we
rarely see apoptotic cells. These observations lead to several
alternative explanations. GDF-9 may be required to induce competence to
respond to proapoptotic stimuli. Alternatively, although the elevated
serum FSH (9) cannot overcome the type 3b block, it may promote
granulosa cell survival. Finally, the granulosa cells in the
GDF-9-deficient ovaries may bypass this apoptosis-competent state by
differentiating after the oocyte degenerates to form the steroidogenic
follicular nests. Thus, GDF-9 is an important factor for the
differentiation of the granulosa cells, allowing granulosa cells of
follicles beyond the type 3b stage to acquire specific characteristics
such as the capability to undergo apoptosis.
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-45017
OH, showed distinct aromatase-positive, LHR-negative;
aromatase-positive, LHR-positive; and aromatase-negative, LHR-positive
follicular nests (all were 17
OH 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.
 |
MATERIALS AND METHODS
|
---|
Experimental Animals
All experimental mice were maintained in accordance with the NIH
Guide for the Care and Use of Laboratory Animals. Unless otherwise
indicated, ovaries from adult C57Bl/6/129SvEv hybrid mice 612
weeks of age were used for both RNA isolation and specimens for
in situ hybridization and immunohistochemistry. For the
studies of COX-2, 3-week-old mice received ip injections of 7.5 IU
Gestyl (Diosynth B.V., Oss, Holland), and then with 5 IU of hCG
(Sigma Chemical Co., St. Louis, MO) 48 h later.
Ovaries were collected 5 h after hCG injection.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from various tissues of wild-type and
GDF-9deficient 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 manufacturers
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.
In Situ Hybridization
In situ hybridization was performed essentially as
described previously (36) with the following modifications. Freshly
dissected ovaries from wild-type or GDF-9-deficient C57Bl/6/129SvEv
hybrid mice were fixed in 4% paraformaldehyde-PBS overnight,
processed, and embedded in paraffin. Sections (5 µm thick) were cut
and pretreated as described. Table 1
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 1618 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 Denhardts/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
214 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 manufacturers
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
manufacturers 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 493517 of
S40364) and 5'-CTGTTGCAGCCAGCTCCCTTAG-3' (antisense, nucleotides
943919 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.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Carolyn Bondy, S. K. Dey, Wade Harper, Anita
Payne, and Jian Zhou for the gifts of the cDNA probes; Dr. Michael
Soares for the anti-P-450 scc antibody; Rebecca Robker and Dr. Joanne
Richards for teaching us the in situ hybridization
technique; Kim Paes for help with figures; Shirley Baker for aid in
manuscript preparation; and Drs. T. Rajendra Kumar and Joanne Richards
for critical review of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: mmatzuk{at}bcm.tmc.edu
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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