Platelets Are Novel Regulators of Neovascularization and Luteinization during Human Corpus Luteum Formation

Article (PDF Available)inEndocrinology 148(7):3056-64 · August 2007with26 Reads
DOI: 10.1210/en.2006-1687 · Source: PubMed
Abstract
The human corpus luteum is a unique endocrine organ that is periodically constructed from the ovulated follicle. During human corpus luteum formation, which is well known as a pathophysiological model for tissue remodeling, the precise mechanisms by which centripetal vascular development is regulated remain unknown. Recently platelets were reported to contain chemoattractive substances with the potential to induce endothelial migration. In this study, we examined the involvement of platelets in the early tissue remodeling process of the human corpus luteum. An immunohistochemical study demonstrated that considerable amounts of red blood cells and CD41-positive platelets were localized at extravascular sites among luteinizing granulosa cells after ovulation. Platelet deposition gradually decreased and became limited near the central cavity toward which microvessels were extending. Platelets were hardly observed in the midluteal phase when the vascular network had already been established. These platelets expressed CD62P/P-selectin and were colocalized with extracellular matrix, suggesting that platelets had been activated by the extracellular matrix. Progesterone production by luteinizing granulosa cells that were isolated from patients undergoing in vitro fertilization therapy was significantly promoted by direct contact with platelets during 4-d culture. Platelet-derived soluble factors induced spreading in granulosa cell morphology. These factors also increased the migration of human umbilical vein endothelial cells, whereas luteinizing granulosa cells attenuated platelet-induced endothelial cell migration. These findings lead us to propose the novel concept that platelets are regulators of endothelial cell migration and granulosa cell luteinization in the remodeling process of the human corpus luteum.

Figures

Platelets Are Novel Regulators of Neovascularization
and Luteinization during Human Corpus Luteum
Formation
Kazumi Furukawa, Hiroshi Fujiwara, Yukiyasu Sato, Bin-Xiang Zeng, Haruko Fujii, Shinya Yoshioka,
Eiichiro Nishi, and Takeshi Nishio
Department of Gynecology and Obstetrics (K.F., H.Fujiw., B.-X.Z., H.Fujii, S.Y.), and Molecular Pathology Unit (E.N.),
Horizontal Medical Research Organization, and Department of Integrative Brain Science (T.N.), Faculty of Medicine, Kyoto
University, Kyoto 606-8507, Japan; and Department of Obstetrics and Gynecology (Y.S.), Osaka National Hospital, Osaka
540-0026, Japan
The human corpus luteum is a unique endocrine organ that is
periodically constructed from the ovulated follicle. During
human corpus luteum formation, which is well known as a
pathophysiological model for tissue remodeling, the precise
mechanisms by which centripetal vascular development is
regulated remain unknown. Recently platelets were reported
to contain chemoattractive substances with the potential to
induce endothelial migration. In this study, we examined the
involvement of platelets in the early tissue remodeling pro-
cess of the human corpus luteum. An immunohistochemical
study demonstrated that considerable amounts of red blood
cells and CD41-positive platelets were localized at extravas-
cular sites among luteinizing granulosa cells after ovulation.
Platelet deposition gradually decreased and became limited
near the central cavity toward which microvessels were ex-
tending. Platelets were hardly observed in the midluteal
phase when the vascular network had already been estab-
lished. These platelets expressed CD62P/P-selectin and were
colocalized with extracellular matrix, suggesting that plate-
lets had been activated by the extracellular matrix. Proges-
terone production by luteinizing granulosa cells that were
isolated from patients undergoing in vitro fertilization ther-
apy was significantly promoted by direct contact with plate-
lets during 4-d culture. Platelet-derived soluble factors in-
duced spreading in granulosa cell morphology. These factors
also increased the migration of human umbilical vein endo-
thelial cells, whereas luteinizing granulosa cells attenuated
platelet-induced endothelial cell migration. These findings
lead us to propose the novel concept that platelets are regu-
lators of endothelial cell migration and granulosa cell lutein-
ization in the remodeling process of the human corpus
luteum. (Endocrinology 148: 3056 –3064, 2007)
T
HE HUMAN CORPUS luteum (CL) is a unique endocrine
organ that is newly constructed from the ovulated follicle
during the menstrual cycle. This process is well known as a
physiological model for tissue remodeling. The CL produces
progesterone, which is essential for inducing and maintaining
embryo implantation in the uterus early in pregnancy. To sup-
ply this hormone to the systemic circulation, two major phe-
nomena are accomplished during CL formation, i.e. granulosa
cell luteinization and neovascularization.
Just before follicular rupture, granulosa cells in the follic-
ular fluid, which contains anticoagulant substances (1), pro-
ceed to luteinization and shift their main products from
estrogen to progesterone. After ovulation, these granulosa
cells undergo hypertrophy to differentiate into large luteal
cells, being in contact with migrating endothelial cells (2) and
producing extracellular matrix (ECM) around the luteal cells
(3–5).
Dramatic centripetal angiogenesis also occurs from the
vascular network surrounding the follicle, although follicu-
lar fluid contains antiangiogenetic factors (6). During ovu-
lation, the follicular basement membrane is destroyed and
endothelial cells just outside the membrane migrate into the
inner granulosa cell layer. In the human CL, it takes several
days to complete mature vascular networks among the luteal
cells (2), finally achieving vascular anastomosis in the central
cavity area, which is the luteal remnant of the antral cavity
of a ruptured follicle.
To induce neovascularization, luteinizing granulosa cells
have been proposed to secrete several soluble angiogenic
factors such as vascular endothelial growth factor (VEGF),
angiogenin, endocrine gland-VEGF, and angiopoietin (7–10).
Luteinizing theca cells were also proposed to play some role
in angiogenesis in human CL (11). In addition, we previously
reported that luteinizing granulosa cells increased the cell
surface expression of ephrin B1 and melanoma cell adhesion
molecule (MCAM), which are reported to regulate endothe-
lial migration and vessel formation by cell-to-cell contact
(12–14). Furthermore, ECM produced by luteinizing granu-
losa cells is considered to modulate the migration and out-
growth of endothelial cells (15). However, there is no definite
evidence demonstrating that there is local dominance of an-
giogenic factors in the central cavity to maintain endothelial
migration toward the area until final the anastomosis is
First Published Online April 19, 2007
Abbreviations: CL, Corpus luteum; ECM, extracellular matrix; FCS,
fetal calf serum; FITC, fluorescein isothiocyanate; HCG, human chori-
onic gonadotropin; HUVEC, human umbilical vein epithelial cell; IVF,
in vitro fertilization; mAb, monoclonal antibody; MCAM, melanoma cell
adhesion molecule; pAb, polyclonal antibody; VEGF, vascular endo-
thelial growth factor.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
0013-7227/07/$15.00/0 Endocrinology 148(7):3056–3064
Printed in U.S.A. Copyright © 2007 by The Endocrine Society
doi: 10.1210/en.2006-1687
3056
achieved. Thus, the precise mechanisms by which centripetal
vascular development is regulated remain unknown.
During ovulation, a decrease of vascular stability beneath
the follicular basement membrane is evident, and conse-
quently blood cells migrate into extravascular spaces around
luteinizing granulosa cells (2, 16). In addition, blood vessels
begin to penetrate into the granulosa cell layer and some
vessels open into the antral cavity, filling it with blood (17).
Accordingly, fresh bleeding toward the central cavity is often
observed for4dinCLintheearly stage (2, 18). Thus, blood
plasma fluid and blood cells, including red cells, normally
flow among luteinizing granulosa cells that are surrounded
by ECM in the extravascular spaces (2, 16), and the CL in this
stage is occasionally called the corpus rubrum. However, the
fibrin net was mainly observed in the central cavity area, and
only sparsely among luteinizing granulosa cells (2), theoret-
ically suggesting that there is some anticoagulant system(s)
operating around granulosa cells. This condition is consid-
ered essential for maintaining the local circulation of tissue
fluid throughout fresh CL and recruiting this progesterone-
containing tissue fluid into the systemic circulation. How-
ever, very little attention has been given to this paradoxical
issue, and it remains unclear how the dynamic kinetics of
tissue fluid are controlled, regulating the coagulation sys-
tems throughout the process of corpus luteum formation
until the establishment of a viable vascular network.
Together with red blood cells, platelets, which are a type
of blood cells that plays an important role in coagulant sys-
tems, are likely to be exuded into extravascular spaces among
luteinizing granulosa cells. Recently platelets were reported
to contain chemoattractive substances potentially capable of
inducing endothelial migration (19) and to play an important
role in pathological processes such as atherosclerosis, wound
healing, and tissue remodeling (20, 21). Therefore, we ex-
amined the precise spatiotemporal distribution of platelets in
human CL and estimated their possible roles in CL forma-
tion, which is one of the dynamic and physiological phe-
nomena involving in tissue remodeling.
Materials and Methods
Reagents
The mouse antihuman integrin
IIb/CD41 (clone M148) and throm-
bomodulin/CD141 (clone 1009) monoclonal antibodies (mAbs) were
obtained from Novocastra Laboratories Ltd. (Newcastle, UK). Fluores-
cein isothiocyanate (FITC)-conjugated mouse antihuman CD41 mAb
(clone M148) was purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). The mouse antihuman P-selectin/CD62P (clone AK-4) mAb
was obtained from BD Biosciences-PharMingen (Tokyo, Japan). The
mouse antihuman collagen type IV (clone IV-3A9) mAb and antihuman
fibronectin (clone 96–21F2) mAb were purchased from Daiichi Fine
Chemical (Takaoka, Japan), and antihuman fibrin mAb (clone E8) was
obtained from Chemicon (Temecula, CA). Antihuman 3
-hydroxys-
teroid dehydrogenase rabbit polyclonal antibody (pAb) was purchased
from Oxygene (Dallas, TX). FITC-conjugated and nonconjugated mouse
IgG1 (clone DAK-GO1) and IgG2b (clone DAK-GO9) mAbs and rabbit
Ig for negative controls were all obtained from Dako (Glostrup, Den-
mark). For the secondary antibody, FITC-conjugated rabbit antimouse
Ig pAb (Dako), FITC-conjugated swine antirabbit Ig pAb (Dako) and
rhodamine-conjugated goat antimouse Ig pAb (Santa Cruz) were used.
The mouse antihuman MCAM (clone S-Endo1; Alexis Biochemicals, San
Diego, CA) and antihuman integrin
5 (clone SAM-1; Chemicon) mAbs
were used for flow cytometry.
Tissues
CL of the early (CL d 2–5, n 17) and midluteal (CL d 7–8, n 5)
phases were obtained from 22 women, aged between 25 and 43 yr. All
women had undergone unilateral ovarian cystectomy or oophorectomy
and contralateral wedge resection to treat benign ovarian tumors. All of
the women had a history of regular menstrual cycles (28 –30 d), and their
ovulatory basal body temperature charts were consistent with normal
luteal phase length. No patient used contraceptives or GnRH analogs
within at least 3 months before the operation. The CL day was reeval-
uated according to histological dating, using hematoxylin and eosin-
stained tissue sections that were fixed with 10% formalin and embedded
in paraffin (2). The migration of endothelial cells and the size of luteal
cells were used for this classification. In the present study, the term CL
day was used according to this definition. For example, CL d 2 was the
day after ovulation, which was confirmed by transvaginal ultrasonog-
raphy and histological dating. Informed consent for the use of these
tissues was obtained from each donor. Use of the materials was also
approved by the Ethics Committee of Kyoto University Hospital.
Immunohistochemistry
Double-immunofluorescence staining was performed as previously
described (22, 23). Frozen tissues were sliced to 7-
m thickness using a
cryostat microtome (Cryocut 1800; Reichert-Jung, Heidelberg, Ger-
many), immediately air dried on Neoplene (Nisshin EM, Tokyo, Japan)-
coated glass slides, and fixed in acetone at 20 C for 5 min. The frozen
sections were incubated with antihuman collagen type IV mAb [5
g/
ml, diluted in culture medium containing 10% fetal calf serum (FCS;
Equitech-Bio, Inc., Kerrville, TX) and 0.1% NaN3], antihuman fibronec-
tin mAb (5
g/ml), antihuman fibrin mAb (5
g/ml), antihuman CD41
mAb (5
g/ml), antihuman CD62p mAb (5
g/ml), or mouse negative
control IgG1 (5
g/ml). After the slides were washed in PBS, they were
incubated with rhodamine-conjugated goat antimouse immunoglobu-
lin. The washed slides were blocked with mouse anti-TNP (trinitrophe-
nyl) mAb (unrelated mAb; 20
g/ml) and then incubated with FITC-
conjugated antihuman integrin
IIb/CD41 mAb (10
g/ml). Otherwise,
for secondary staining, the washed slides were incubated with antihu-
man 3
-hydroxysteroid dehydrogenase rabbit pAb (10
g/ml) or con-
trol rabbit Ig (10
g/ml), followed by FITC-conjugated swine antirabbit
Ig. The slides were washed, mounted with a mounting agent (Perma
Fluor aqueous mounting medium; Immunon, Pittsburgh, PA), and ex-
amined under a confocal laser-scanning microscope (Carl Zeiss Inc.,
Jena, Germany).
Isolation of human luteinizing granulosa cells, platelets,
and umbilical vein epithelial cells (HUVECs)
Fresh human luteinizing granulosa cells were obtained from 38 pa-
tients aged from 25 to 39 yr who had undergone treatment for in vitro
fertilization (IVF) as previously described (3). Human platelets were also
isolated from patients undergoing IVF treatment as described (23, 24).
Whole blood was obtained from patients undergoing IVF treatment,
immediately mixed with 3.8% (vol/wt) trisodium citrate dihydrate (ra-
tio of blood to citrate was 9:1) in polypropylene tubes, and centrifuged
at 200 g for 15 min at 22 C. The platelet-rich plasma was centrifuged
after adding a mixture of 4.5% wt/volcitric acid and 6.6% wt/voldex-
trose at 50
l/ml platelet-rich plasma. The sedimented platelets were
resuspended in RPMI 1640 containing 5.4 mm EDTA, stabilized for 10
min at room temperature, centrifuged at 980 g for 10 min at 22 C, and
then was suspended in RPMI 1640 (2 10
8
/ml).
HUVECs were separated from the umbilical cord as described pre-
viously (25). After washing the inner wall of the umbilical vein with PBS
to remove fetal blood, the lumen was filled with PBS containing Ca
2
and Mg
2
as well as 0.05% collagenase (Wako Pure Chemical Industries
Ltd., Osaka, Japan) and incubated for 30 min at room temperature. After
the detached cells were collected, the remaining endothelial cells in the
inner layer of the umbilical vein were collected by further washing with
RPMI 1640 containing 15% FCS. The isolated HUVECs were cultured
using HuMedia-EB2 (Kurabo, Osaka, Japan) containing 2% FCS, human
epithelial growth factor (10 ng/ml), hydrocortisone (1
g/ml), human
basic fibroblast growth factor (3 ng/ml), and heparin (10
g/ml) as well
as gentamicin (50 mg/ml) and amphotericin B (50
g/ml). Immuno-
Furukawa et al. Platelets Regulate CL Formation Endocrinology, July 2007, 148(7):3056–3064 3057
cytochemical staining using anti-CD141/thrombomodulin mAb con-
firmed the greater than 95% purity of endothelial cells in the above
preparation.
Informed consent for the use of granulosa cells, platelets and HUVECs
in this study was obtained from all donors. Use of the materials was also
approved by the Ethics Committee of Kyoto University Hospital.
Luteinizing granulosa cell culture with platelets
Isolated human granulosa cells were resuspended in culture medium
consisting of RPMI 1640 medium supplemented with 10% FCS. These
cells (1 10
5
cells/ml /well) were cultured in each well of 24-well plates
(Becton Dickinson, Lincoln Park, NJ) in triplicate in the presence or
absence of recombinant human chorionic gonadotropin (HCG) (5 IU/
ml; Rhoto Pharmaceutical Co. Ltd., Osaka, Japan) and isolated platelets
(2 10
7
and 2 10
8
cells/ml). Granulosa cells were also incubated with
platelets (2 10
8
cells/ml) in 0.45-
m-pore culture chambers (Intercell,
Kurabo Co. Ltd., Osaka, Japan), which prevented direct contact between
granulosa cells and platelets. The culture medium was gently replaced
with fresh medium every 2 d, and the collected medium was subjected
to a RIA. Morphological changes were observed under a phase-contrast
microscope and recorded using a digital camera (Camedia C5050; Olym-
pus, Tokyo, Japan). The average length to width ratio of 30 cells in each
well and the mean values of triplicate wells were calculated. The cell
circumference and size (area) were calculated using National Institutes
of Health Image 1.63 (n 5).
Assay of progesterone in culture media
The concentrations of progesterone in the culture medium were mea-
sured using RIA kits (Immunotech, Marseille, France). Inter- and in-
traassay coefficients of variation were 5.7 and 5.3%, respectively.
Matrigel invasion assay
The invasion assay was carried out as previously described (26). A
6.4-mm-diameter culture insert with an 8-
m-pore membrane filter
(Becton Dickinson) was placed in collagen type I-coated 24-well plates
(Asahi Techno Glass, Tokyo, Japan). The upper surface of the membrane
filter was precoated with diluted Matrigel (Becton Dickinson; 300
g/
ml). The lower well was filled with 700
l RPMI 1640 (1% FCS) with or
without (control) platelets (2 10
8
cells/well) and/or granulosa cells
(1 10
5
cells/well) in the presence or absence of HCG (5 U/ml). Then
isolated HUVECs (2.5 10
5
cells per 300
l of RPMI 1640 with 1% FCS)
were inoculated into the upper chamber. After a 3-h incubation at 37 C,
HUVECs that reached the lower surface were fixed with 100% methanol
at 20 C for 5 min and were FITC stained using anti-CD141 mAb. The
stained filters were examined under a confocal laser-scanning micro-
scope and the numbers of CD141-positive cells were counted for quan-
tification using National Institutes of Health Image 1.63 (26). These
experiments were performed in triplicate (n 7), and the average was
defined as the invading cell number. Each result was expressed as the
percentage of invading cell numbers found in the control (without
coculture or additives).
Proliferation assay
HUVECs were cultured in the intercell chambers for 48 h in the
presence or absence of granulosa cells (1 10
5
cells/well), HCG (5
U/ml), and/or platelets (1 10
8
cells/well). The number of HUVECs
in each intercell chamber was assessed using the Premix WST-1 cell
proliferation assay system (Takara, Kusatsu, Japan) and ELISA plate
reader (Molecular Device, Menlo Park, CA) according to the manufac-
turer’s instructions.
Flow cytometry
Flow cytometry was performed as described previously (3). Detached
granulosa cells (n 5) cultured with or without HCG (5 U/ml) or
platelets (1 10
8
cells/ml) were reacted with antihuman MCAM, in
-
tegrin
5, or control mAb (100
g/ml, 10
l) and then with FITC-
conjugated rabbit pAb. Cell surface labeling was analyzed using a FAC-
Scalibur (Becton Dickinson).
Statistics
Data are shown as means sem. The concentration of progesterone
in the culture medium, average length to width ratio, cell circumference
and size of cultured luteinizing granulosa cells, cell numbers of
HUVECs, and the mean intensity in flow cytometry were analyzed by
ANOVA, followed by Scheffe´’s F test for multiple comparison. The
difference was considered significant at P 0.05.
Results
Immunohistochemical localization of CD41-positive
platelets
InCLond2(n 3), edematous changes induced by
inflammatory reaction during ovulation remained evident,
and red blood cells were located among luteinizing granu-
losa cells in all samples (Fig. 1, A–C).
IntheCLond3(n 6), extravascular blood was still
evident among the sparsely lining granulosa cells that were
not yet fully luteinized (Fig. 1D). In another sample, fresh
bleeding into the antral cavity was observed from some
vessels that had started to penetrate into the granulosa cell
layer (Fig. 1, E and F). In this stage, vascular structures were
detected in the luteinizing granulosa cell layer (Fig. 2, A and
B). In the later stage of CL d 3, although intraantral bleeding
was limited to within the peripheral site of the cavity by
fibrin network, extravascular blood was still observed
among the granulosa cells that became more luteinized (Fig.
1, G and H). To coincide with the above observation, CD-
41-positive platelets were diffusely observed among 3
-hy-
droxysteroid dehydrogenase-positive luteinizing granulosa
cells (Fig. 3, A and B).
IntheCLond4(n 4), endothelial cell migration almost
reached the central area, and abundant extravascular blood
was observed among luteinizing granulosa cells (Fig. 2, C
and D). In the later stage of CL d 4, uncoagulated blood red
cells were still observed at extravascular sites among lutein-
izing granulosa cells, which had considerably increased cy-
toplasmic volume and came into contact with each other (Fig.
1, I and J). In this stage, platelet deposition among luteinizing
granulosa cells was mainly observed in the central area of the
CL (Fig. 3, C and D).
IntheCLond5(n 4), granulosa cell luteinization
proceeded further, and small luteal cells could be distin-
guished from large luteal cells (Fig. 1K). Extravascular blood
lakes that contained degenerative materials were sparsely
observed among large luteal cells (Fig. 1L). In this stage,
platelet deposition was almost limited to within the central
cavity (Fig. 3, E and F).
In the CL on d 7–8 (n 5), vascular networks had been
established (Fig. 2E), and vascular anastomosis in the central
cavity was observed (Fig. 2F). In this stage, there were few
extravascular blood lakes and no platelet deposition among
large luteal cells or in extravascular spaces was observed
(Fig. 3G).
In the CL on d 3, CD 41-positive platelets were deposited
in the ECM showing fibronectin and collagen type IV around
luteinizing granulosa cells (Fig. 3H). In the CL on d 4, a fibrin
net was mainly observed in the central cavity but was
sparsely detected among luteinizing granulosa cells, as de-
scribed previously (Fig. 3I) (2). In the CL on d 5, thrombo-
modulin/CD141-positive endothelial cells were observed to
3058 Endocrinology, July 2007, 148(7):3056–3064 Furukawa et al. Platelets Regulate CL Formation
migrate through the luteinizing granulosa cell layer into the
central cavity, forming vascular anastomoses (Fig. 3, J and K).
Platelets that were deposited in the central region expressed
P-selectin/CD62P (Fig. 3, L and M), showing that these plate-
lets were activated.
The effects of platelets on the morphology of luteinizing
granulosa cells
In the presence of HCG, granulosa cells became round
or oval, a change that resembles luteal cell transformation,
whereas in the presence of platelets, spreading of lutein-
izing granulosa cells was enhanced during 48 h of culture
(Fig. 4, A–C). Supporting this observation, both the cal-
culated cell areas and circumferences of cultured granu-
losa cells were significantly reduced by HCG, whereas
these parameters were promoted by platelets (Fig. 4, E and
F), indicating that there are functional differences between
HCG and platelets in the effects on the morphological
changes of luteinizing granulosa cells. Under conditions of
direct contact with platelets, similar microscopic morpho-
FIG. 1. Extravascular blood among luteinizing granulosa cells during human CL formation. A–C, Two samples of CL on the day after ovulation
(CL d 2); D–H, Three samples of CL d 3; I and J, CL d 4; K and L, CL d 5. C, F, H, J, and L are magnified images of the boxed areas in B, E,
G, I, and K. A–C, Inflammatory edema remained in the peripheral stroma (St) and abundant red blood cells were observed around luteinizing
granulosa cells (LGC). D, Extravascular blood was still evident among the sparsely lining granulosa cells that had not yet undergone
luteinization. E and F, Fresh bleeding into the antral cavity was observed. G and H, Intraantral bleeding was limited within the peripheral
site of the cavity. Extravascular blood was still observed among the granulosa cells that became more luteinized. I and J, Uncoagulated blood
red cells were still observed at extravascular sites among luteinizing granulosa cells that were considerably increased in cytoplasmic volume,
and in contact with each other. K and L, Granulosa cell luteinization proceeded further and small luteal cells (SL) could be distinguished from
large luteal cells (LL). Extravascular blood lakes that contained degenerative materials were sparsely observed among large luteal cells. Bar
100
m. CC, Central cavity.
FIG. 2. Neovascularization during human CL forma-
tion. A and B, CL d 3; C and D, CL d 4; E and F, CL
d 7. B, D, and F are magnified images of the boxed
areas in A, C, and E. A and B, Red blood cells at
extravascular sites were observed around luteinizing
granulosa cells (LGL), peripheral stroma (St), and
central cavity (CC). B, Microvessels (arrows) were
observed in the middle area. C and D, Note red blood
cells at extravascular sites around the further lutein-
ized granulosa cells. D, Microvessels (arrow) were
observed in the central area. E and F, Granulosa cells
were already transformed into large luteal cells (LL),
being enlarged and tightly contacting each other.
There were no red cells at extravascular sites and
microvessels (arrows) were observed around the large
luteal cells. Anastomoses of microvessels were estab-
lished in the central cavity (arrowheads). Bar, 100
m. LGC, Luteinizing granulosa cells; SL, small lu-
teal cells.
Furukawa et al. Platelets Regulate CL Formation Endocrinology, July 2007, 148(7):3056–3064 3059
logical changes were induced in luteinizing granulosa
cells (Fig. 4D). Flow cytometry showed that the expression
levels of MCAM and integrin
5 cell surface markers for
luteinization, on granulosa cells were increased by HCG,
as described previously (4, 14), but not by platelets (Fig.
4, G and H).
The effects of platelets on progesterone production by
luteinizing granulosa cells
During2dofculture, although the level of progesterone
production in the groups that were treated with HCG and
platelets seemed higher than that in the control group (with-
out HCG or platelets), the differences were not significant
(Fig. 5A). During an additional 2-d culture, progesterone
production by luteinizing granulosa cells was significantly
elevated under conditions of direct contact with platelets, as
observed in the group treated with HCG (Fig. 5B). Under
conditions of indirect contact using intercell chambers, there
was no significant enhancement of progesterone production
(Fig. 5B).
The effects of platelets and luteinizing granulosa cells on
endothelial cell migration and proliferation
To investigate the in vitro effects of both luteinizing
granulosa cells and platelets on endothelial cell function
we used HUVECs because it is very difficult to obtain a
sufficient amount of migrating endothelial cells from hu-
man CL tissue.
In cocultures with platelets, the number of migrated
HUVECs was significantly enhanced (Fig. 6A). However,
luteinizing granulosa cells, which had been reported to pro-
duce angiogenic factors, showed little effect on HUVEC mi-
gration, even with stimulation by HCG. Unexpectedly, the
enhancing effect of platelets was attenuated by luteinizing
granulosa cells.
Endothelial cell proliferation was promoted by both lu-
FIG. 3. Immunofluorescence staining of human
CL. A–G, Double staining using anti-CD41 mAb
(red stained using rhodamine) and anti-3
-hy-
droxysteroid dehydrogenase pAb (green stained
using FITC). In the CL ond3(AandB),CD-41-
positive platelets were diffusely observed among
3
-hydroxysteroid dehydrogenase-positive lu-
teinizing granulosa cells. Ond4(CandD),plate-
let deposition among luteinizing granulosa cells
was observed only in the central area. Ond5(E
and F), platelets were mainly observed in the cen-
tral cavity. In the CL on d 7 (G), platelets were
hardly observed. On d 3 (H), platelets (green
stained) were deposited in the collagen type IV
(red stained) around luteinizing granulosa cells.
On d 4 (I), the fibrin net (red stained) was mainly
observed in the central cavity but was also
sparsely detected among the luteinizing granu-
losa cells. J–M, Ond5(JandK)thrombomodulin/
CD141-positive endothelial cells (red stained) mi-
grated through the luteinizing granulosa cell
layer into the central cavity, forming vascular
anastomoses. L, Platelets (green stained) depos-
ited in the central region expressed P-selectin/
CD62P (red stained). M is a higher magnification
of the boxed area in L. Bar, 100
m. CC, Central
cavity; LGC, luteinizing granulosa cells; LL, large
luteal cells.
3060 Endocrinology, July 2007, 148(7):3056–3064 Furukawa et al. Platelets Regulate CL Formation
teinizing granulosa cells and platelets. There were no sig-
nificant differences observed among the treated groups (Fig.
6B).
Discussion
In this study, immunohistochemical examination indi-
cated that platelet deposition was highly correlated with the
angiogenic and luteinizing processes in human CL, suggest-
ing that platelets are involved in human CL formation. Un-
coagulated blood was observed at extravascular sites among
luteinizing granulosa cells in all specimens derived from the
early luteal phase, confirming that human CL formation is an
intriguing model of tissue remodeling. The distribution of
fibrin, which reflects the local status of the coagulatory sys-
tem, was also positively correlated with platelet localization.
Platelets deposited in the extravascular spaces were shown
to be in contact with the ECM. In general, direct interaction
with the extracellular matrix induces platelets to release bi-
ologically active substances in cytoplasmic granules (27). In
support of this, the deposited platelets showed coexpression
of CD62P/P-selectin. From these findings, it is speculated
that platelets are activated during CL formation in vivo and
that substances derived from platelets play some roles in
constructing a new endocrine organ.
To investigate the physiological role of platelets in the
luteinization of granulosa cells, we examined the effects of
platelets on progesterone production by cultured luteinizing
granulosa cells. In the presence of HCG, progesterone pro-
duction was significantly enhanced during 4-d culture. No-
tably, coculture with platelets also promoted progesterone
production by granulosa cells, suggesting that platelets fa-
cilitate luteinization of granulosa cells.
This study also showed that coculturing with platelets
induced marked spreading of granulosa cell morphology,
suggesting that platelets enhanced the adhesive property of
granulosa cells. Previously we reported that luteinizing gran-
ulosa cells showed increased expression of MCAM (14),
which was reported to regulate cell attachment to endothelial
cells (13), and integrin
5, which is a receptor for fibronectin
(4). Although HCG enhances the expression of these mole-
cules, there was no significant change in MCAM or integrin
5 expression by coculturing with platelets in this study.
Thus, although the increase in size induced by platelets is
compatible with the morphological change during the lu-
teinization process, the mechanism may differ from that in-
duced by HCG. In contrast to their effects on progesterone
production, morphological changes elicited by platelets did
not require direct contact with granulosa cells, suggesting
that platelets affect granulosa cell luteinization through more
than a single pathway.
For more than a decade, the contribution of white blood
cells to CL function has been proposed (28). However, there
has not been any report concerning the direct effects of plate-
lets on human luteal function. Although this study did not
FIG. 4. Platelet-induced morphological changes in cultured granulosa cells. Human granulosa cells were cultured for 48 h in the absence (A)
or presence of HCG (B), and platelets without (C) or with (D) direct interaction. Granulosa cells became round in the presence of HCG, whereas
platelets induced spreading in granulosa cells with or without direct interaction. Bar,50
m. Both calculated cell areas (E) and circumferences
(F) were significantly reduced by HCG but increased by platelets under indirect contact conditions. As shown by flow cytometry, the expression
levels of MCAM (G) and integrin
5 (H) were enhanced by HCG treatment, whereas these expression levels were not affected by platelets. **,
P 0.01.CTR, Control.
Furukawa et al. Platelets Regulate CL Formation Endocrinology, July 2007, 148(7):3056–3064 3061
provide precise information about the mechanism by which
platelets promote progesterone production, it can be pro-
posed that platelets are novel local regulators of the lutein-
ization of human granulosa cells.
Then we examined the effects of platelets on endothelial
cell migration using HUVECs and a Matrigel invasion assay.
In the human early CL, it was reported that angiogenic fac-
tors such as VEGF and endocrine gland-VEGF were pro-
duced by luteinizing granulosa cells, and their production
was promoted by LH/HCG stimulation (29, 30). Although
these factors were proposed to contribute to endothelial cell
proliferation and maintenance of the vascular structure in
human CL, there is no evidence showing that granulosa cells
enhance endothelial cell migration. In this study, endothelial
cell migration was not enhanced by granulosa cells. Even in
the presence of HCG, there was no significant effect on en-
dothelial migration. However, when platelets were cocul-
tured with endothelial cells, endothelial cell migration was
promoted (31). Although there are some functional differ-
ences between endothelial cells in umbilical veins and ovu-
lating follicles (32), these findings suggest that platelets are
more potent stimulators of endothelial cell migration than
luteinizing granulosa cells.
Platelets contain several factors such as VEGF and sphin-
gosine 1-phosphate, which may contribution to vascular ex-
tension (33). When cocultured with platelets, granulosa cells
unexpectedly showed inhibition of endothelial cell migration
induced by platelets in the presence or absence of HCG.
These findings may be accorded with a recent report that
culturing of HUVECs with conditioned medium from cul-
tured human luteinized granulosa cells lead to the expres-
sion of antiangiogenic factors at the transcript level in en-
dothelial cells (6). To achieve fine and mature vascular
networks among fully luteinized luteal cells, the processes of
vascularization and luteinization as well as the arrangement
of ECM should be synchronized. These findings may reflect
a crucial role of granulosa cells in regulating adequate neo-
vascularization by protecting against excessive stimulatory
effects of platelets on endothelial migration.
Ovulation and subsequent corpus luteum formation are
considered to mimic an inflammatory reaction (34). Accu-
mulating evidence shows that there is a cross-talk between
inflammation and coagulation systems (35), whereby inflam-
mation not only leads to the activation of coagulation, but
coagulation also considerably affects inflammatory activity
(36). This inflammatory reaction is also considered to elicit
cell migration and proliferation in cooperation with the co-
agulation system (35). This study provides further evidence
FIG. 5. Platelet-induced progesterone production in cul-
tured granulosa cells. During the 2-d culture (A), proges-
terone production tended to be increased by HCG and plate-
let treatment. During the additional 2-d culture (B),
progesterone production was significantly promoted in the
groups cultured with HCG and by direct contact with plate-
lets. However, under conditions of indirect contact using
intercell chambers, there was no significant enhancement.
*, P 0.05, **, P 0.01.
FIG. 6. The effects of platelets on endothelial cell migration
and proliferation. In cocultures with platelets, the number
of migrated endothelial cells was enhanced (A). These en-
hancing effects of platelets were attenuated in the presence
of luteinizing granulosa cells (LGC). In contrast, endothe-
lial cell migration was not affected by coculturing with gran-
ulosa cells in the presence or absence of HCG. The prolif-
eration of endothelial cells was slightly promoted by both
luteinizing granulosa cells and platelets in the presence or
absence of HCG (B). There were no significant differences
observed among the treated groups. *, P 0.05, **, P 0.01.
3062 Endocrinology, July 2007, 148(7):3056–3064 Furukawa et al. Platelets Regulate CL Formation
that platelets, which are the main contributors to the coag-
ulation system, induce cell differentiation and migration in
the tissue remodeling process in the adult human ovary.
In conclusion, this study showed that the extravascular
localization of platelets accords with the neovascularization
process during human CL formation. The direct interaction
of platelets with granulosa cells was demonstrated to pro-
mote progesterone production by granulosa cells. In addi-
tion, platelet-derived soluble factors induced morphological
changes in granulosa cells, suggesting that platelets are in-
volved in the process of differentiation of human granulosa
cells toward large luteal cells. Furthermore, platelet-derived
soluble factors were shown to be a greater stimulant of en-
dothelial migration than granulosa cells. Although it remains
unknown how the coagulation system is controlled to main-
tain adequate kinetics of tissue fluid among luteinizing gran-
ulosa cells, the present results lead us to propose a novel
concept whereby platelets regulate spatiotemporal construc-
tion of vascular networks in the early human CL (Fig. 7).
These findings also support the recent concept that platelets
play an important role in wound healing processes and will
contribute to clarifying the mechanism of extravascular cir-
culation in inflammatory lesions.
Acknowledgments
The authors are grateful to Ms. Mizuho Takemura for excellent tech-
nical assistance.
Received December 15, 2006. Accepted April 9, 2007.
Address all correspondence and requests for reprints to: Hiroshi
Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of
Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail:
fuji@kuhp.kyoto-u.ac.jp.
This work was supported by Grants-in-Aid for Scientific Research
(16390474 and 17591731).
Disclosure Statement: The authors have nothing to disclose.
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endocrine community.
3064 Endocrinology, July 2007, 148(7):3056–3064 Furukawa et al. Platelets Regulate CL Formation
    • "Moreover, the blood clot formed during ovulation might stimulate cell migration. Indeed, platelets are a better stimulant for endothelial cells migration than granulosa cells themselves [19] . Examples of proangiogenic cytokines acting on this stage of the cycle include the cytokines fibroblast growth factor 2 (FGF2), VEGF, plateletderived growth factor (PDGF) family, and angiopoietin (Ang). "
    [Show abstract] [Hide abstract] ABSTRACT: In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.
    Full-text · Article · Jun 2013
    • "Additionally, the direct interaction of blood platelets with granulosa cells was demonstrated to promote progesterone production by granulosa cells. Platelets regulate spatiotemporal construction of vascular networks in the early human CL [29]. During folicular development, both types of follicular cells (granulosa and theca cells) simultaneously produce the estradiol, which is a potent mitogen of granulosa cells [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Corpus luteum (CL) is a small, transient endocrine gland formed following ovulation from the secretory cells of the ovarian follicles. The main secretory product of CL is progesterone, which is required for the establishment and maintenance of pregnancy and regulates various reproductive functions. Progesterone plays a key role in the regulation of the length of estrous cycle and in the implantation of the blastocysts. Additionally, progesterone serves as a negative feedback mechanism to the hypothalamus to suppress further follicular development. The inadequate progesterone production is the major cause of infertility and embryonic loss, since progesterone is essential for both endometrial growth and embryo survival. Corpus luteum is formed following ovulation, but the real stimulus for luteinization represents the preovulatory LH surge from hypophysis. Even in case when ovulation does not occur, granulosa cells will differentiate and form CL, while oocyte will be trapped within the non- Correspondence should be addressed to Kaveh Mohammadi Khanghah,; Tel: +98-9189846319. Journal of Biology and today's world 2013, volume 2, issue 3, pages: 153-172 154 | P a g e ovulating structure. The major biologic mechanisms involved in CL development, function, and regression will be discussed in this review.
    Full-text · Article · Jan 2013 · Molecular Human Reproduction
    • "Stimulation of granulosa cells by hCG as well as by IGFs and hypoxia induced up-regulation of VEGF (Hazzard et al., 1999; Tropea et al., 2006; Taylor et al., 2007 ) that is cardinal for generation of healthy ovulatory follicles and CL (Distler et al., 2003). In addition to VEGF, other factors such as angiopoietin (1 and 2; (Sugino et al., 2005), leukocytes (Polec et al., 2011) and platelets (Furukawa et al., 2007; Nurden, 2007) contribute to the remodeling of endothelial cells and luteinized granulosa cells in the process of CL formation. In the current study we found that PEDF regulation is hormonally affected inversely to VEGF, further implying a role for PEDF as a negative regulator of ovarian angiogenesis. "
    [Show abstract] [Hide abstract] ABSTRACT: Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by human chorionic gonadotropin (hCG); namely, PEDF level was nearly undetectable under high doses of hCG while VEGF level was significantly elevated. The anti-angiogenic nature of PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells (HUVECs). Depleting PEDF from primary granulosa cells conditioned-media accelerated endothelial cells proliferation, migration and tube formation.Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.
    Full-text · Article · Oct 2012
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