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Department of Medicine, Section of Endocrinology (S.-A.W., J.A.B., J.M.H.), Diabetes, and Metabolism, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Department of Anatomy, Physiological Sciences and Radiology (J.E.G.), College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
Address all correspondence and requests for reprints to: James M. Hammond, M.D., Head, Section of Endocrinology, Diabetes and Metabolism, Hershey Medical Center, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: jhammond{at}PSGHS.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated
mature pigs and from slaughterhouse sows. CTGF transcripts were induced
during the late preantral stage in granulosa and theca cells
concomitantly with the appearance of endothelial cells in the theca.
CTGF mRNA expression increased in granulosa cells to a maximum
(P < 0.01) in mid-antral follicles but was down
regulated (P < 0.01) in preovulatory follicles. In
contrast, granulosa cell mac25 mRNA expression was undetectable between
the preantral and mid-antral stage but was strongly induced in
terminally differentiated granulosa cells of preovulatory follicles.
CTGF mRNA and peptide were also detected in the theca
externa/interstitium and in vascular endothelial cells of ovarian blood
vessels, whereas mac25 transcripts, which were also abundant in ovarian
blood vessels increased in the theca interna with follicular
development. Transcripts of cyclin D1, a marker of cell proliferation,
appeared during the early antral stage and were moderate in granulosa
cells but abundant in capillary endothelial cells in the theca interna,
underneath the basement membrane. Following ovulation, CTGF and cyclin
D1 mRNAs were associated with the migration of endothelial cells into
the CL. Subsequently, there was a marked up-regulation of CTGF mRNA
expression in granulosa luteins concomitantly with an increase in
endothelial cell proliferation within the CL. We hypothesize that CTGF
may promote ovarian cell growth and blood vessel formation during
follicular and luteal development whereas mac25, a tumor inhibitor, may
promote terminal differentiation of granulosa cells in preovulatory
follicles. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10 mg/animal, Lutalyze, Upjohn Co., Kalamazoo, MI) on day 12 of
the estrous cycle. Animals were ovariectomized 3, 5, 6, 7, or 9 days
after prostaglandin treatment. In this model, ovulation occurs on day 6
or 7 following prostaglandin treatment. All experimental protocols for
animal care and use were performed in accordance with the NIH Guide and
Care of Laboratory Animals and with approval of the North Carolina
State University Institutional Animal Care and Use Committee. Ovarian
pieces approximately 1 cm x 1 cm x 1 cm in size were
embedded in OCT and frozen on dry ice. Ten micrometer-thick frozen
sections were subsequently prepared for in situ
hybridization and immunohistochemistry. Ovaries from both immature and
cyclic pigs were also collected from a local slaughterhouse. Ovaries
from cyclic pigs were classified as either preovulatory, early luteal,
mid-luteal, or late luteal phase. Pieces of such ovaries were fixed in
Bouin’s for 24 h, dehydrated, paraffin-embedded and used for
immunohistochemistry.
Complementary (c)DNAs and cRNA probes
The following RT-PCR primers were designed from sequences
published in GenBank. Porcine mac25 (forward: 5' CGT GTG CAA GTG CCG
CTA C 3'; reverse: 5' TAC CTT GTT CCA GAT GAG GGC AG 3'). Porcine CTGF
(forward: 5' GGG TTA CCA ATG ACA ACG CTT TC 3'; reverse: 5' TGG CAG GCA
CAA GTC TTG ATG 3'). These primers were used to amplify fragments
corresponding to 280-bp (mac25) and 320-bp (CTGF) from porcine
granulosa cell cDNA preparations. The resulting fragments were cloned
into the TA vector, sequenced, and used to generate
35S-labeled cRNA antisense and sense probes for
in situ hybridization as described previously (15).
35S-labeled cRNA probes were also synthesized
from human cyclin D1 (16) and aromatase (17) cDNAs. Aromatase gene
expression along with morphological criteria such as absence of
pyknotic granulosa cell nuclei and integrity of the basal lamina were
used to identify healthy follicles (15). Cyclin D1 mRNA localization
was used as a marker of endothelial cell proliferation in the theca and
the corpus luteum (15).
In situ hybridization
In situ hybridization was conducted as described
previously (16). Consecutive sections of porcine ovaries were
sequentially hybridized with either mac25, CTGF, cyclin D1, or
aromatase cRNA probes. This allowed for direct comparison of their mRNA
distribution within the same ovarian structures. A mac25 mRNA sense
probe, which gave a uniform and nonspecific binding pattern, was used
as a generic sense control. Following the in situ procedure,
sections were exposed to autoradiographic film (Kodak
Biomax MR, Eastman Kodak Co., Rochester, NY) for 1 to 4
days to evaluate the overall intensity of hybridization signals, then
dipped in Kodak NTB2 liquid emulsion and exposed for 1
(CTGF) or 2 weeks (mac25, cyclin D1, and aromatase) at 4 C. For a given
probe, exposure time was the same for all animals. Sections were
developed in Kodak Dektol-19 (Eastman Kodak Co., Rochester, NY) and counter-stained with hematoxylin.
Immunohistochemistry
Immunohistochemical localization of the endothelial cell marker,
factor VIII was conducted on sections consecutive to those used for
mac25, CTGF and cyclin D1 in situ hybridization. Frozen
sections were air-dried at room temperature for at least 2 h,
fixed in cold acetone (10 min), rinsed in PBS (pH 7.4) and the
nonspecific binding was blocked by incubation with CAS-Block
(Zymed Laboratories, Inc., South San Francisco, CA).
Sections were then incubated for 2 h in the presence of factor
VIII antiserum (Zymed Laboratories, Inc.) diluted 1:20.
The subsequent steps were conducted using a nonbiotin amplification kit
(NBA, Zymed Laboratories, Inc.) and according to the
manufacturer’s instructions. Immunohistochemical localization of CTGF
protein was performed on Bouin’s-fixed, paraffin-embedded ovarian
sections. Briefly, sections were deparaffinized, rehydrated and
subjected to a microwave antigen retrieval procedure (3 times 4 min at
1000 Watts). Nonspecific binding was blocked with CAS-Block
(Zymed Laboratories, Inc.) and sections were incubated
overnight at 4 C with rabbit antihuman CTGF antiserum (1:25; generously
provided by Dr. Youngman Ho, Oregon Health Sciences University,
Portland, OR). The rest of the procedure was as described for factor
VIII.
Statistical analyses
Grain density was only quantified for CTGF mRNA, which was
abundantly expressed in granulosa cells. Three to 5 animals were used
for each time point within the follicular phase (Days 3, 5, 6, and 7).
Grain density was measured using NIH image analysis software in random
but constant and nonoverlapping areas of microscopic field at a
magnification of 20x. Four such areas were counted within the
granulosa cell compartment of any follicle considered, and only areas
covered by cells were counted. The background was determined by
counting grain density in four areas of slide devoid of tissue,
providing an internal control for each slide. For each follicle
considered, the background value was subtracted from the density of
grains occupying the granulosa cell compartment. A total number of 42
small preantral, 61 large preantral, 40 healthy small antral, 25
atretic small antral, and 28 healthy large antral follicles were used
to quantify CTGF mRNA.
The data were subjected to two-way ANOVA with follicular phase time points (Days 3, 5, 6, and 7) and stages of follicular development as factors, respectively, to determine the effects of temporal and spatial regulation on CTGF mRNA expression. No differences were seen in CTGF mRNA expression between time points. Consequently, data were pooled across time points and subjected to one-way ANOVA. Differences in mRNA expression between stages of follicular development were determined by the Dunn’s multiple comparison test.
The effects of follicular condition (healthy vs. atretic) on CTGF mRNA expression was examined within a single follicular category i.e. small antral follicles. Treatment comparisons i.e. healthy vs. atretic were conducted using the t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2, and 3
depict the relationship between angiogenic events and mRNA expression
of CTGF, mac25, cyclin D1, and aromatase during various stages of
porcine follicular development. Immunoreactivity for factor VIII shows
that endothelial cells appear in the developing theca compartment
during the end of preantral stage (Fig. 1F). These endothelial cells
increase in density as a function of follicular development, forming a
network of capillaries in the area of the theca interna directly
adjacent to the follicular basement membrane (Fig. 2A and 3A).
As expected, expression of aromatase mRNA, a marker of granulosa cell
differentiation and follicular health, was absent in preantral
follicles (not shown) but increased as a function of follicular
development during the antral stage (Fig. 2F and 3E). As reported
before (15), transcripts of cyclin D1, a marker of endothelial cell
proliferation were low in preantral follicles (Fig. 1I). In antral
follicles, cyclin D1 mRNA levels were moderate in granulosa cells but
enhanced in a subset of cells within the theca interna (Figs. 2E and 3D). A closer examination of this subset of theca cells that strongly
express cyclin D1 mRNA shows that they colocalized with factor
VIII-expressing cells (endothelial cells) in the area directly
underneath the follicular basement membrane (Figs. 2A, 2E, 3A, and 3D).
Moreover, as reported before, cyclin D1 mRNA expression in theca and
granulosa cells was restricted to healthy, aromatase expressing
follicles (Figs. 2E, 2F, 3D, and 3E).
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and 1E; 2C and 3B). Granulosa cell CTGF mRNA levels were
lowest in small preantral follicles (Fig. 4) but increased as a function of
follicular development (P < 0.01) beginning in the
late preantral stage (Figs. 1D, 1E, and 4), to a maximum in small- to
mid-sized antral follicles (Figs. 2C and 4). However, in granulosa
cells of preovulatory follicles, CTGF mRNA expression was greatly
(P < 0.01) reduced (Figs. 3B and 4). The timing and
pattern of follicular CTGF mRNA expression closely paralleled periods
of angiogenesis in growing follicles. In the forming theca compartment
of large preantral follicles, the induction of CTGF mRNA
transcripts coincided with the appearance of endothelial cells (Figs. 1, D–F, and 2, A–C). In the fully differentiated theca compartment of
large antral follicles, CTGF mRNA expression was restricted to discrete
cells in the theca externa/interstitium (Fig. 3B), which also receives
abundant blood supply (Fig. 3A). In contrast, the differentiated theca
interna, which contains cells expressing both factor VII
immunoreactivity and cyclin D1 transcripts, did not express CTGF mRNA
(Fig. 3, A, B, and D). Aromatase mRNA was colocalized with CTGF
transcripts in small- and mid-sized healthy antral follicles (Fig. 2, C
and F), but not in fully differentiated granulosa cells of preovulatory
follicles where aromatase expression was maximum and CTGF/rP-2
transcripts were down-regulated (Fig. 3, B and E). CTGF mRNA was also
detected in pregranulosa cells and oocytes from primordial follicles
(Fig. 1, A and B). Although factor VIII was also detected in a few
discrete somatic cells in the vicinity of primordial follicles (Fig. 1C), it did not appear that CTGF mRNA expressing cells (Fig. 1B) were
endothelial cells.
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) and cyclin D1 (data not shown)
transcript expression were completely abolished. In those follicles,
CTGF transcript levels were greatly reduced (Figs. 5B and 6; P < 0.001)
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, 1H, 2D, and 3C). Mac25 mRNA was
not detected in small preantral follicles (results not shown) but
increased with the appearance of vascular endothelial cells in the
developing theca beginning in late preantral follicles (Figs. 1F, 1G, and 1H). Mac25 transcript levels were further enhanced in the
increasingly vascularized theca of antral follicles (Figs. 2A, 2D, 3A, and 3C). In contrast, mac25 messenger RNA (mRNA) levels were greatly
reduced in theca cells of atretic antral follicles (Fig. 5D).
Granulosa cell mac25 transcripts were undetectable in preantral (Fig. 1G and 1H) and small- to mid-sized antral follicles (Fig. 2D) but
strongly induced in preovulatory follicles (Fig. 3C). A mac25 sense
mRNA probe gave a uniform hybridization pattern (Fig. 3F).
Immunohistochemical localization of CTGF in porcine follicular and
luteal tissues
CTGF protein was strongly detected in the cytoplasma of oocytes of
primordial follicles and their associated somatic cells (Fig. 7A). There was, however, a rapid decrease
in oocyte (OO) CTGF immunoreactivity as follicles started to grow (Fig. 7, B and C). Moderate CTGF immunoreactivity was found in granulosa
cells of growing follicles between the preantral and the mid-antral
stages (Fig. 7, B and C). However, CTGF immunoreactivity was completely
attenuated in granulosa cells of large antral follicles (Fig. 7D). A
moderate signal was also detected in discrete cells within the theca of
antral follicles (Fig. 7, C and D). In early developing luteal tissue,
a strong CTGF immunoreactivity was present in virtually all luteal
cells. No immunoreactivity was detected in parallel control sections
incubated in the absence of primary antibody (Fig. 7F).
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, A and B) and
message (Fig. 8, F and G) were strongly expressed in endothelial cells
associated with various intraovarian blood vessels. Blood
vessels-expressing CTGF proteins were found in both the ovarian
cortex (Fig. 8A) and the more heavily vascularized hilar region of the
ovary (Fig. 8B). Mac25 (angiomodulin) mRNA expression (Fig. 8, H and I)
and, as expected, factor VIII-related antigen (Fig. 8D) were also
detected in endothelial cells associated with blood vessels throughout
the ovary.
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) and CTGF mRNA-expressing cells (Fig. 9C) was very similar to that of endothelial cells (Fig. 9A). Clearly,
cyclin D1 (Fig. 9B) and CTGF (Fig. 9C) mRNA-expressing cells, like
endothelial cells (Fig. 9A) were more concentrated in the theca lutein
compartment and appeared to infiltrate the granulosa lutein compartment
following the rupture of the follicular basal lamina. Moderate mac25
mRNA expression was also detected in thecal-lutein-like cells but not
in granulosa-luteins on Day 7 (Fig. 9D). Two days after ovulation,
cyclin D1 mRNA expressing cells had literally invaded the corpus luteum
(Fig. 9E). Concomitantly, mac25 transcripts became virtually
undetectable (data not shown) whereas CTGF mRNA expression was
enhanced, mainly in granulosa luteins (large luteal cells), although
the possibility that some theca luteins (small luteal cells) were also
positive could not be discounted (Fig. 9F). However, in regressing
luteal cells, CTGF transcripts were clearly restricted to granulosa
luteins (Fig. 9G). At that latter stage, cyclin D1 mRNA expression was
diminished (results not shown) whereas factor VIII was still detected
in endothelial cells throughout the corpus luteum (Fig. 9H).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The absence of follicular CTGF mRNA expression during the small preantral stage and its induction and increase between the large preantral and mid-antral stage is significant because this period coincides with the appearance of vascular endothelial cells in the developing thecal compartment as demonstrated in the current study and in others (19). Moreover, it is during this same period (large preantral to mid-sized antral stage) that maximum follicular growth rate occurs (20, 21). During these growth periods, both the CTGF mRNA and peptide are highly enriched in blood vessels throughout the ovary. Coupled with other data that this peptide is a potent angiogenic factor (4, 11, 12, 18), it is reasonable to propose a role for CTGF in the development of new blood vessels during the rapid growth of antral follicles.
The function of CTGF at other sites of expression is not obvious. Both its mRNA and peptide were abundant in primordial follicles, in oocytes in particular. Further, the function of CTGF mRNA expression in granulosa cells around the time of antrum formation is unknown. However, it could subserve a growth promoting function on these cells as well. The marked decrease in both CTGF immunoreactivity and mRNA expression in preovulatory follicles coincides with the cessation of granulosa cell division (22), and therefore, is consistent with the notion that this peptide is involved in follicular growth.
In contrast to CTGF, mac25 may act as a growth suppressor during follicular development. Its mRNA expression is undetectable in granulosa cells of growing follicles and is induced in the differentiating granulosa cells of preovulatory follicles. Such cells become replicatively quiescent (22). These findings are reminiscent of previous studies in which mac25 mRNA was found to be induced in senescent mammary epithelial cells (6) and down regulated in various tumors (7, 8, 23, 24).
In comparison to CTGF, considerably fewer data exist on the action of this peptide. Mac25 has strong homology with follistatin (FS), an activin-binding protein (9). The carboxyl terminus of FS is not represented in mac25 (9). Interestingly, a carboxyl-truncated form of FS has a stronger activin-binding activity than the native peptide (9). Activin prevents, whereas FS promotes, granulosa cell luteinization in preovulatory follicles (25, 26). Therefore, the induction of mac25 mRNA expression in granulosa cells of preovulatory follicles coincides with a shift away from activin-promoted effects and toward FS-mediated effects. Further studies would be required to test this notion; specifically, the ability of mac25 to actually bind activin remains to be demonstrated. The positive correlation between mac25 mRNA expression and theca cell growth is somewhat surprising in view of its putative growth inhibitory effects in other systems. A role in theca cell differentiation could be invoked on the basis of its homology with follistatin. Thus, activin inhibits, while follistatin potentiates, LH-induced differentiation of theca cells (27).
Interestingly, the localization of cyclin D1 mRNA-expressing cells in the theca and luteal tissues was virtually identical to that of endothelial cells. This finding suggests that the main thecal and luteal cells expressing this cell cycle regulator are likely endothelial cells. Our observation that cyclin D1 transcripts are totally absent in atretic follicles is also consistent with the notion that blood supply to these degenerating follicles is impaired.
CTGF plays a critical role in wound healing (4) and it is not surprising that following ovulation—a wound-like process—-, this peptide mRNA expression was up-regulated in the corpus luteum. The pattern of CTGF mRNA expression during luteogenesis coincides with the migration and subsequent proliferation of vascular endothelial cells. According to our study, CTGF expressing cells migrate into the luteal tissue shortly after ovulation. It is these cells from the theca/interstitium, which are initially responsible for the growth factor expression in the CL. We observed an identical temporal and spatial distribution of CTGF, Factor VIII and cyclin D1 mRNA, consistent with other findings that CTGF promotes the migration of endothelial cells (4). Subsequently, the marked increase in CTGF mRNA and its peptide in granulosa-luteins coincide with increases in the number of cyclin D1 mRNA-expressing cells and vascular endothelial cells in the CL. These observations altogether are also suggestive of a possible role of CTGF in promoting endothelial cell proliferation. In the regressing CL, cyclin D1 transcripts are abolished whereas those of CTGF are still present, perhaps associated with the migration of microphages into the corpus luteum during luteolysis (28).
In summary, induction and increases in CTGF mRNA expression during active periods of follicular and luteal development suggest that it could play a role in porcine follicular growth. CTGF is a potent mitogen for endothelial cells and can stimulate angiogenesis and tissue regeneration in a variety of models. This growth factor may therefore promote ovarian cell growth and/or blood vessel formation during follicular development and later during luteogenesis. It is significant that mac25, a potent tumor inhibitor and a follistatin analog is only detected in porcine granulosa cells during the final stage of follicular development. At this time, granulosa cells stop dividing, terminally differentiate and lose FSH responsiveness. Mac25 could play a role in these events as antagonist of cell replication and a follistatin-like modulator of ovarian cell function.
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Acknowledgments |
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Footnotes |
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Received October 26, 1999.
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References |
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vß2, promotes endothelial cell survival, and
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