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Departments of Medicine (S.-A.W., J.C., J.M.H.), Cellular and Molecular Physiology (J.M.H.), and Neuroscience and Anatomy (T.L.W., J.C., S.W.L.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: James M. Hammond, Section of Endocrinology, Diabetes and Metabolism, H044, Hershey Medical Center, Hershey, Pennsylvania 17033.
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Abstract |
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PMSG/hCG treatment induced the appearance of IGFBP-2 transcripts in the ovarian interstitium. Following PMSG/hCG-induced ovulation, IGFBP-2 and -4 and IGF type-I receptor mRNAs were strongly expressed in virtually all luteal cells, whereas IGFBP-3 and -5 transcripts were selectively localized to some cell types in the corpus luteum. Conversely, IGF-I mRNA was essentially undetectable in the corpus luteum.
This study represents the most comprehensive and detailed analysis of the physiology and anatomy of the mouse ovarian IGF system, and shows that 1) IGFBP-5 is linked to the survival of the slow growing and immature preantral follicles; 2) IGF-I is associated with the growth and survival of the rapidly growing large preantral and antral follicles; 3) IGFBP-4 is an atretogenic candidate for mouse ovarian follicles; 4) ovulatory doses of PMSG/hCG up-regulate IGFBP-2 mRNA expression in the ovarian interstitium; and 5) transcripts of IGF type-I receptor and IGFBP-2 through -5, but not those of IGF-I are highly expressed in the mouse corpus luteum.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A more definitive characterization of the murine ovarian IGF system is warranted for several reasons. First, the relevance of the rat intraovarian IGF system to mouse physiology is uncertain; for instance, IGFBP-2 messenger RNA (mRNA) is expressed in the rat thecal-interstitial compartment, but apparently restricted to granulosa cells in the mouse ovary (16). It has been suggested that IGF-I mRNA expression in mouse ovaries is restricted to healthy follicles (16), but the histological assessment of follicular health or atresia was not documented in this study. Thirdly, to the best of our knowledge, the existence of a correlation between specific IGFBPs and follicular growth or atresia in the murine ovary has never been addressed. Follicular atresia is associated with the appearance of IGFBP-4 and -5 transcripts in rat granulosa cells (12, 13, 14) and increases in IGFBP-2, -4, and -5 in sheep antral follicles (18). In the pig, IGFBP-2 seems most tightly correlated with atresia (19). Finally, previous studies in the mouse were conducted in immature animals; thus, nothing is known about gonadotropin-induced changes in the expression of IGF system components or levels in preovulatory follicles and luteal cells. A more extensive characterization of murine intraovarian IGFs and their receptor and binding proteins also would provide a firm basis for interpreting ongoing studies assessing reproductive effects of mutations in the IGF system in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization
35S-labeled antisense complementary RNA (cRNA)
probes were synthesized from rat complementary DNA sequences for IGF-I
(20), -II (21), and -IR (22) and IGFBP-2 (23), -3 (24), -4 (25), and -5
(from Dr. Peter Rotwein, Washington University (St. Louis, MO) by
either T3 (IGFBP-3), T7 (IGF-I and -II and IGFBP-2 and -4), or Sp6
(IGFBP-5 or IGF-IR) RNA polymerase-mediated transcription of linearized
plasmids using a transcription reaction kit (Promega Corp.
Co., Madison, WI). For controls, IGF-II and IGFBP-5 sense cRNAs were
synthesized by Sp6 and T7 transcription, respectively. Unincorporated
35S-nucleotides were removed by filtration on Sephadex G50
Quick Spin columns (Boehringer-Mannheim, Indianapolis,
IN).
Sections were transferred directly from -70 C into 4% paraformaldehyde/PBS, rinsed in PBS, dehydrated in a series of alcohols, acetylated in 0.25% acetic anhydride (vol/vol), 0.05 M triethanolamine, 0.3% acetic acid for 10 min, washed in 0.2x SSC (1xSSC contains 0.15 M sodium chloride, 0.015 M sodium citrate), and dehydrated. Sections were prehybridized 1–3 h at room temperature (RT) in 50% deionized formamide; 0.6 M NaCl; 10 mM Tris HCl, pH 7.5; 0.02% Ficoll; 0.02% BSA; 0.02% polyvinylpyrrolidone; 1 mM EDTA; 0.1% sheared herring sperm DNA; 0.5 mg/ml yeast total RNA; and 0.05 g/ml transfer RNA. Next, hybridization was carried out overnight at 50 C in prehybridization buffer containing 35S-labeled cRNA probe (4 x 107 cpm/ml), 0.1 mg/ml sheared herring sperm DNA, 10% dextran sulfate, 0.1% SDS, and 10 mM dithiothreitol. Sections were rinsed subsequently in 2x SSC and washed in 50% formamide, 1x SSC, and 10 mM dithiothreitol at 45 C and O.5x SSC at RT. Unhybridized probe was then degraded by ribonuclease A [100 mg/mL in 0.5 M NaCl; 10 mM Tris, pH 7.5; and 1 mM EDTA] for 30 min at RT, and sections were washed in 0.2x SSC at 55–60 C for 2 h and dehydrated through graded alcohols. Sections were exposed to autoradiographic film (Kodak Biomax MR, Eastman Kodak Co., Rochester, NY) overnight to evaluate the intensity of hybridization signals, then dipped in Kodak NTB2 liquid emulsion and exposed for 1–3 weeks depending on the probe at 4 C. For a given probe, exposure times were the same for all animals. This were 3 weeks for IGF-I, -II, -IR, IGFBP-2, and -3 and 1 week for IGFBP-4 and -5. Slides were developed in Kodak-D19 developer and counterstained with Mayer’s hematoxylin and examined with both bright-field and dark-field microscopy.
Quantitative analysis of in situ hybridization
Quantitative analysis of hybridization signals was performed
using a video-NIH Image Analysis program (Scion Corp., Frederick, MD)
linked to a microscope. Each probe was tested simultaneously on three
different mouse strains, and three to four animals were examined per
strain and per probe. For each animal and for each probe, one
representative section was chosen in the largest cross-section of the
ovary. Grain density was determined in a constant area of 200
µm2 using a x20 objective and dark-field illumination.
Under these conditions, there was excellent correlation between the
number of silver grains determined by visual count and image analysis.
Grain density was determined in four nonoverlapping constant (200
µm2) areas of primary or preantral follicles. Six such
areas were considered for antral follicles and corpora lutea. Only
microscopic fields completely occupied with cells were measured, except
for the background for which grain density was determined in four large
random areas (5000 µm2) devoid of tissues. For each
slide, specific labeling was determined by subtracting average
background value from the values obtained for each follicular category.
For sense probes, the specific hybridization signal determined using
these methods was negligible.
Morphological characterization of ovarian follicles
Stages of follicular development were defined as follows:
primary (a single layer of cuboidal granulosa cells), large preantral
(at least two layers of cuboidal granulosa cells and no antrum), small
antral (several layers of granulosa cells and one or several small
antral cavities but no cumulus oophorus stalk), and large antral
(several layers of granulosa cells, a single large antral cavity, and a
well-defined cumulus oophorus stalk). In preliminary studies, IGFBP-5
mRNA expression was heterogenous between subclasses of the preantral
stage. Consequently, for IGFBP-5, the large preantral stage was
further divided into secondary (two layers of granulosa cells) and
tertiary (three or more layer of granulosa cells and no antral
cavity).
Follicles were classified as healthy on the following basis: zero (primary follicles) to no more than three pyknotic nuclei (preantral and antral follicles), granulosa cells regularly apposed on an intact basement membrane, no fibroblastic morphology in the granulosa cell compartment, and no evidence of infiltration of the oocyte by somatic cells. Follicles were classified as early atretic if they contained at least one pyknotic nucleus in primary and four in larger follicles and an irregular basal lamina. In addition to these criteria, late atretic follicles also contained fibroblast cell in the granulosa cell compartment and oocyte infiltration by somatic cells. Other criteria for assessing follicular atresia such as dissolution of germinal vesicle, chromatin condensation, or cytoplasmic fragmentation of the oocyte were not easy to apply in these studies because the integrity of these cellular compartments is not well preserved in frozen sections.
Only follicles with a visible oocyte were considered to ensure proper follicular counting and classification. A total of 33–35 primary, 30–51 healthy preantral, 18–49 early atretic preantral, 6–40 late atretic preantral, 12–26 small antral, and 0–12 large antral follicles (large antral follicles were not apparent in many sections of immature animals) were analyzed depending on the IGF system component considered.
In situ end labeling of apoptotic nuclei
Two related but distinct procedures were used to detect
apoptosis in ovarian tissues. These procedures used either the enzyme
terminal deoxynucleotidyl transferase (TUNEL) or the Klenow fragment of
DNA polymerase I (KLUNEL) to catalyze the transfer of digoxygenin
(DIG)-labeled nucleotides to the 3'OH end of fragmented DNA. The KLUNEL
procedure has been validated in a variety of tissues (26, 27) but not
in the ovary. Conversely, the use of the TUNEL procedure to detect
apoptosis in ovaries has been previously documented (28, 29, 30). Sections
were processed identically for the two procedures except for some
differences in enzyme buffers. For the TUNEL procedure, sections were
incubated for 1 h in a buffer containing 300 U/ml Tdt
(Boehringer-Mannheim) and 0.01 M DIG DNA mixture
(Boehringer-Mannheim) in 25 mM Tris HCl, 200 mM
potassium cacodylate, and 1 mM CoCl2. For
KLUNEL, sections were incubated for 1 h at RT with 50 U/ml Klenow
polymerase I (Worthington Biochemical Corp., Lakewood, NJ)
and 0.01 M DIG DNA mixture in a buffer containing 50
mM Tris HCl, 5 mM MgCl2, 10
mM ß-mercaptoethanol, and 0.005% BSA (buffer 1).
Incubations were stopped by washing slides in either 300 mM
NaCl, 30 mM sodium citrate (TUNEL), or buffer 1 containing
0.3% Triton X-100 (KLUNEL) at RT. The remaining steps were identical
for both procedures. Sections were incubated with a 1:500 dilution of
an alkaline phosphatase-conjugated sheep anti-DIG antibody for 1 h
at 37 C, and the alkaline phosphatase reaction developed in
BCIP/NBT (Boehringer-Mannheim) for 45 sec, followed by
counterstaining with 1% methyl green.
In one series of experiments, consecutive ovarian sections from five mice were compared sequentially with the TUNEL and KLUNEL procedures. The same population of follicles were labeled on adjacent sections using either DNA fragmentation assay. In a second series of experiments, consecutive ovarian sections from five mice were used sequentially for in situ hybridization and in situ end labeling (KLUNEL) to determine whether or not granulosa cells expressing IGF-I or IGFBP-2 or -4 transcripts were apoptotic.\.
Statistical analyses
The data presented are expressed as the mean ±
SEM. Repeated measures ANOVA models were used to assess
differences between mean densities, adjusted for the average background
density, within and between follicle categories (primary through
antral, healthy, and atretic) and mouse strains for each probe (IGF-I
and IGFBP-2, -4, or -5), and for untreated and hormone-treated mouse
groups. No differences were observed between MF1, C57, and 129 for
IGF-I or IGFBP-2, -4, or -5, and consequently, results were pooled
across the strains. The covariance structure of the models accounted
for both within- and between-mouse correlation (31). Bonferroni
adjustments were made to P values and confidence intervals
for the contrasts of interest to adjust for multiple comparisons such
that the overall probability of a type I error (
) was 0.05. All
analyses were performed using the SAS statistical package (SAS Institute, Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and hormone-treated immature mice
(Fig. 2A). In morphologically healthy
follicles, IGF-I mRNA levels were lower (P < 0.001) in
primary follicles than in larger preantral and antral follicles in
untreated (Fig. 3) and hormone-treated
immature mice (not shown). In granulosa cells of healthy antral
follicles, IGF-I mRNA was expressed as a gradient; the signal was
stronger in the cumulus oophorus and in the vicinity of the antrum than
in the mural compartment (P < 0.001; Fig. 3).
Irrespective of hormonal treatment, IGF-II mRNA signal was mainly
expressed in ovarian blood vessels; in addition, there was minor
diffuse signal in the rest of the ovary (data not shown).
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) and gonadotropin-treated immature mice (Fig. 2A),
IGFBP-2 mRNA was detected in the granulosa cell compartment of most
follicles from the primary to antral stage. IGFBP-2 transcripts were
also present in some primordial follicles (not shown). In healthy
follicles, the level of IGFBP-2 transcripts did not change as a
function of follicular development (Fig. 4). A strong induction of IGFBP-2 mRNA
expression was also observed in the ovarian interstitium of
PMSG/hCG-treated (Fig. 2A) but not of untreated immature mice (Fig. 1).
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and 2A). IGFBP-3 mRNA was strongly
expressed in some interstitial cells and isolated cells—presumably
endothelial—in the thecal compartment of some large preantral and
antral follicles in untreated (Fig. 1) and hormone-treated mice (Fig. 2, A and B). Modest and relatively constant levels of IGFBP-3
transcripts were also detected in peripheral follicular cells at all
stages of follicular development (Fig. 2B). IGFBP-4 transcripts were
constitutively expressed in the thecal and interstitial compartments in
ovaries of untreated (Fig. 1) and hormone-treated mice (Fig. 2A).
However, in granulosa cells of healthy follicles, IGFBP-4 transcripts
were very low throughout follicular development (Figs. 1, 2A, and 4).
Irrespective of hormone treatment, IGFBP-5 transcript levels were
highest in the germinal epithelium of the ovary and in granulosa cells
of healthy follicles during the primary and secondary stages but
decreased sharply (P < 0.001) in late preantral and
subsequent stages (Figs. 1, 2A, and 5). A
closer examination revealed IGFBP-5 mRNA expression as early as the
primordial follicular stage (not shown).
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IGF-I and IGFBP-2, -4, and -5 mRNA expression in relation to
follicular atresia
The levels of IGF-I and IGFBP-2, -4, and -5 mRNA were determined
quantitatively as a function of histological criteria for follicular
health or atresia. There was an inverse relationship between the levels
of IGF-I mRNA expression and the stage of atresia in preantral
follicles of untreated (P < 0.05; Fig. 6) or gonadotropin-treated immature
(P < 0.01; results not shown) mice. There was no
difference in the level of IGFBP-2 mRNA expression between healthy and
early atretic follicles during the preantral stage, but there was a
decrease in IGFBP-2 expression in late atretic stages
(P < 0.05; Fig. 6). In granulosa cells, IGFBP-4
transcripts were very low in healthy preantral (Fig. 6) and antral
(results not shown) follicles but increased proportionally with the
stage of atresia (P < 0.001, Fig. 6). These results
are in contrast with constitutive expression in the theca noted above.
IGFBP-5 mRNA expression was highest in healthy primary and secondary
follicles and decreased with each stage of atresia (P
< 0.001, Fig. 7).
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). The presence of
apoptotic cells clearly preceded morphological signs of atresia. On the
other hand, follicles showing morphological signs of atresia also
contained apoptotic granulosa cells, except for some late stage atretic
follicles that were completely depleted of granulosa cells. When
consecutive sections were used sequentially for in situ
hybridization (IGF-I or IGFBP-2 or -4 mRNA) and in situ end
labeling (KLUNEL) of fragmented DNA, IGF-I mRNA was always expressed in
healthy follicles with at least two layers of granulosa cells, but
occasionally, a positive signal was also detected in apoptotic
follicles (Figs. 9, A and B). Transcripts
of IGFBP-2 mRNA were detected in both apoptotic and nonapoptotic
follicles (Figs. 9, C and D). In contrast, granulosa cell expression of
IGFBP-4 mRNA was restricted to apoptotic follicles (Figs. 9, E and
F).
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) and -II mRNA (not
shown) were low or absent in the corpus luteum, whereas transcripts of
IGF-IR and IGFBP-2 were easily detected in virtually all luteal cells
(Fig. 10, B and C, respectively). In contrast to the absence of IGFBP-4
mRNA expression in granulosa cells of healthy follicles, its transcript
levels increased rapidly in luteinizing cells and were present in some
luteal cells following gonadotropin treatment (Fig. 10E). As mentioned
above, IGFBP-3 mRNA expression during follicular development was
restricted to a few cells of the thecal compartment in antral
follicles. Following ovulation, some cells morphologically identical to
those IGFBP-3-positive cells appeared to infiltrate the forming corpus
luteum (Fig. 10D). Other luteal cell types were negative for IGFBP-3
mRNA expression (Fig. 10D). Similarly, IGFBP-5 transcripts were
expressed in some but not all luteal cells (Fig. 10F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, the involvement of IGF-I in follicular development is suggested by a rapid increase of its transcript levels as follicle growth progresses toward the antral stage. IGF-I was also linked to follicular survival in the current study, because the levels of IGF-I mRNA were inversely correlated with atresia as determined morphologically. It has been shown that apoptotic cell death with accompanying DNA fragmentation is the molecular mechanism underlying the initiation and progression of follicular atresia (32). Our results show that early apoptosis was not always associated with a decrease in IGF-I transcript levels. In fact a decrease in IGF-I mRNA was only detected once morphological signs of atresia had developed. Therefore, a decrease in IGF-I mRNA levels is likely a consequence, rather than a cause of granulosa cell apoptosis. It has been previously reported that follicular development becomes arrested at the early antral stage in IGF-I null mutant mice (1). That study (1) and another (17) indicate that IGF-I regulates antrum formation and FSH action (increased aromatase activity) in granulosa cells of antral follicles. Collectively, these observations clearly demonstrate a requirement for IGF-I in follicular development beyond the antral stage. However, IGF-I is not essential before antrum formation because preantral follicles can develop in IGF-I knockout mice (1). Nonetheless, the presence of IGF-IR transcripts in granulosa cells of preantral follicles suggests earlier development of IGF responsivity. It is noteworthy that early stages of follicular development, e.g. primary follicles, which are characterized by a low growth rate (34, 35), were associated with low IGF-I expression in our study, whereas IGF-I increased to a maximum in rapidly growing large preantral and early antral follicles (34, 35). Together, these observations suggest that IGF-I may be rate limiting for preantral follicle development.
In contrast to the studies with IGF-I, few previous studies link the IGFBPs to follicular development or death in the mouse. Adashi et al. (1997) noted that IGFBP-2 was expressed predominantly in the granulosa compartment in agreement with our current studies, a situation different than in the rat (16). However, these authors failed to note a strong positive or negative correlation between IGFBP expression and follicle health or atresia. In contrast, numerous data have linked IGFBP-2 (19), -4 (13, 14), and -5 (12, 14) with atresia in other species. Surprisingly, granulosa cell IGFBP-2 mRNA expression, which was abundant, failed to correlate with any measure of follicular development or atresia in the current study.
Our current observations that IGFBP-4 mRNA is expressed in granulosa cells of some but not all follicles is consistent with a previous report (16). In our study, we also determined that IGFBP-4 mRNA expression in granulosa cells was restricted to the same follicles in which apoptotic granulosa cells were detected. Consistent with these results, follicles showing histological signs of atresia also expressed IGFBP-4 transcripts in their granulosa cell compartment. Interestingly, the expression of IGFBP-4 transcripts in granulosa cells preceded the appearance of morphological signs of atresia. Together, these data suggest that IGFBP-4 could be a marker of incipient follicular atresia in the mouse ovary. It is possible that IGFBP-4 promotes follicular atresia by sequestrating IGF-I, thereby reducing its availability to interact with IGF-IR in granulosa cells.
Transcripts of IGFBP-5, like those of IGF-I, were inversely correlated with follicular atresia. In contrast, it has been reported that in the rat ovary, IGFBP-5 mRNA is restricted to atretic preantral follicles (12, 14). The confinement of IGFBP-5 transcripts to granulosa cells of healthy primary and secondary follicles is remarkable because these follicles have the slowest growth rate (35) and a limited steroidogenic capacity (36). The capability to synthesize estradiol is acquired by granulosa cells later, during the antral stage, and IGF-I regulates gonadotropin stimulation of aromatase activity (36, 17). Therefore it is possible that IGFBP-5 prevents the premature differentiation, i.e. steroidogenic capability, of granulosa cells in healthy primary and secondary follicles, perhaps by sequestrating IGF-I. Alternatively, the recent identification of IGFBP-5 receptors in other systems raises the possibility of an IGF-independent action of this binding protein in preantral follicles (37, 38, 39).
Our observations suggest that the mouse corpus luteum is a source of all the binding proteins considered (IGFBP-2 through -5) but not of IGF-I or -II. The absence of in situ transcripts of the IGFs and the expression of IGF-IR and IGFBP-2 to -5 to luteal cells suggest that extraovarian IGF-I or -II traveling through the ovarian microvasculature could supply the corpus luteum. In fact, recent studies in humans suggest that extraovarian IGF-IGFBP complexes may reach the ovarian follicular fluid as well (40). Very little is known about the role of IGFBPs in the regulation of luteal function. It is possible that IGFBP-3 and -5, both of which can form a ternary complex with the IGFs and the acid-labile subunit (41), target circulating IGF to luteal cells. IGFBP-4 has generally manifested an inhibitory action on the ovary (42), and in our study, atresia and luteinization were both marked by an increase in IGFBP-4 mRNA and a decrease in IGF-I transcripts in luteal and granulosa cells. Granulosa cells of atretic and luteinizing follicles also produce more progesterone and less estradiol (36). Collectively, it is possible that IGFBP-4 regulates granulosa cell luteinization, perhaps by sequestrating IGF-I, therefore reducing its availability for IGF-IR. Similarly, in the pig ovary, IGFBP-4 mRNA expression has been reported to increase in preovulatory follicles and to be associated with granulosa cell luteinization (43).
Although the actions of the IGFBPs in the ovary are incompletely understood, the data from multiple studies suggests that the effects of IGFBP-4 are the most consistently inhibitory (see Ref. 44 for a review). In contrast, IGFBP-2, -3, and -5 have each been shown to exhibit both positive and negative effects on cellular function in a variety of culture systems (44, 45). Although stimulatory effects of these IGFBPs have not yet been demonstrated in ovarian cells, the correlation of expression of some proteins, e.g. IGFBP-5, with follicular growth and development suggests that this may be plausible in vivo.
In summary, we demonstrated that critical changes occur in the level of expression of IGF-I and IGFBP-4 and -5 genes during follicular growth and atresia in mice. Changes in IGF-I transcripts parallel the ability of preantral follicles to move from a slow to a rapid growth phase. During this period of slow growth, IGFBP-5 is strongly expressed, suggesting that it may regulate IGF effects. A major finding of this study is that IGFBP-4 is an atretogenic candidate for mouse ovarian follicles; the appearance of its transcripts in granulosa cells coincides with the hallmark of apoptosis (DNA fragmentation) and precedes morphological signs of follicular atresia.
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Acknowledgments |
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Footnotes |
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Received June 29, 1998.
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