ARTICLE IN PRESS

Ann Anat 187 (2005) 529—537

www.elsevier.de/aanat

Unique expression and regulatory mechanisms

of EG-VEGF/prokineticin-1 and its receptors
in the corpus luteum$
Tatiana Kisliouk, Helena Podlovni, Rina Meidan

Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew
University of Jerusalem, Rehovot 76100, Israel

Received 8 June 2005; accepted 1 July 2005

KEYWORDS Summary
Endothelial cells;
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) or Prokineticin-
Luteal steroidogenic
1 (PK-1) is a novel cysteine-rich protein that belongs to the AVIT protein family. EG-
cells;
VEGF/PK-1, described as selective angiogenic mitogen, is widely expressed in
VEGF;
different tissues including steroidogenic endocrine glands. This review summarizes
Angiogenesis;
the expression and functions of EG-VEGF/PK-1 in corpus luteum (CL)-derived cells:
Apoptosis;
endothelial and steroidogenic cell types. EG-VEGF/PK-1 mRNA is expressed by luteal
Hypoxia
steroidogenic cells of human, rat and bovine ovaries, but was absent from the luteal
Endothelial cells CLEC. Luteal EC expressed high levels of both PK-receptors PK-R1 and
PK-R2 – the two G protein-coupled PK-1 receptors. Interestingly, expression of EG-
VEGF/PK-1 and VEGF were inversely regulated in human and bovine luteinized
granulosa cells. EG-VEGF/PK-1 elevated [3H]-thymidine incorporation, MAPK activa-
tion and c-jun/fos mRNA expression and enhanced LEC proliferation. EG-VEGF/PK-1
also inhibited serum starvation-induced apoptosis in these cells. Stress conditions such
as serum withdrawal, TNFa and chemical hypoxia markedly increase PK-R2 expression,
whereas mRNA levels of PK-R1 remain unchanged, implying that the anti-apoptotic
effect of PK-1 on LEC may be mediated via PK-R2. Besides its direct mitogenic and
anti-apoptotic effects, EG-VEGF/PK-1 elevated VEGF mRNA expression in bovine
luteal steroidogenic cells, which possesses only PK-R1. Together, these findings suggest
an important role for PK-1 in luteal function by acting as a mitogen and survival factor
in LEC. Nevertheless, the inverse regulation of EG-VEGF/PK1 and VEGF mRNA
expression by ovarian cells and the distribution of its receptors may suggest that in
addition to its angiogenic effects, EG-VEGF/PK-1 may also play other roles in ovary.
& 2005 Elsevier GmbH. All rights reserved.

$
Main Lecture for the 100th Meeting of the Anatomische Gesellschaft in Leipzig, Germany, 11–14 March 2005.
Corresponding author. Tel.: +972 89489394; fax: +972 89465763.
E-mail address: rina.meidan@huji.ac.il (R. Meidan).

0940-9602/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.aanat.2005.07.005
 ARTICLE IN PRESS
530
Introduction
The corpus luteum (CL) is a short-lived endocrine
gland which develops from the granulosa and theca
cells of the preovulatory follicle. The main role of
the CL is the production of progesterone, which is
required for the establishment and maintenance of
pregnancy by acting on the genital tract, most
importantly on the uterus (Smith et al., 1994;
Niswender et al., 2000).
In the absence of embryonic signals the CL
undergoes luteolysis and eventually the tissue is
eliminated by apoptosis (for review see McCracken
et al., 1999). The development, function and
regression of the CL are controlled by a large
number of regulators. In most mammals, luteinizing
hormone (LH) is the main luteotropin and prosta-
glandin F2a (PGF2a) is the main luteolytic agent
(McCracken et al., 1999; Carambula et al., 2003;
Wang et al., 2003). Insulin-like growth factors (IGF-I
and -II), growth hormone (GH), prolactin and
steroid hormones can synergize with LH to regulate
ovarian function and were all shown to have direct
effects on ovarian cells in different species (Zetser
et al., 2001; Schams and Berisha, 2002, 2004;
Schams et al., 2002; Webb et al., 2002).
The rapid cyclical changes in luteal growth and
regression are associated with rapid changes in
vasculature. Luteal development is accompanied
by a dramatic increase in the number of blood
vessels (Augustin et al., 1995; Fraser and Lunn,
2001; Redmer et al., 2001). In the mid-stage
of CL the vascular network is so extensive that
most steroidogenic cells appear to be juxtaposed
to capillary vessels (Zheng et al., 1993). Endo-
thelial cells (EC) of the CL account for approxi-
mately half of all cell types at mid-cycle (Young
et al., 2000). Therefore, the CL provides a uni-
que model system for the study of the cellular
and molecular regulation of physiological angio-
genesis. VEGF is the main angiogenic factor
in CL. Inhibition of VEGF (or its signaling) during
the early or mid-luteal phase results in a marked
reduction in luteal angiogenesis and suppression of
luteal function (Fraser and Wulff, 2003). High
mRNA levels of VEGF and its type 2 receptor
VEGFR-2 or KDR are found during early to
mid-luteal phase and decrease at late luteal
stage (Berisha et al., 2000; Sugino et al., 2001;
Al-zi’abi et al., 2003; Fraser et al., 2005). The LH
surge augments VEGF production by granulosa cells
of the ovulatory follicle (Hazzard et al., 1999). In
addition to LH, hypoxia and IGFs further augment
VEGF expression in these cells (Martinez-Chequer
et al., 2003). Its essential role has been demon-
strated.
T. Kisliouk et al.
Blood vessel formation also involves other essen-
tial factors, including the angiopoietins (Hazzard et
al., 1999; Wulff et al., 2000; Fraser and Lunn,
2001). Angiopoietin-1 (Ang-1) is a secreted growth
factor which binds the Tie-2 receptor tyrosine
kinase. This factor enhances the stability of the
newly formed blood vessels (Wulff et al., 2000;
Fraser and Lunn, 2001). Angiopoietin-2 (Ang-2)
destabilizes capillary integrity, thus facilitating
sprouting by VEGF (Fraser and Lunn, 2001). At the
late luteal stage or during luteolysis mRNA levels of
Ang-1 sharply decrease, whereas mRNA levels of
Ang-2 increase (Fraser and Lunn, 2001; Wulff et al.,
2001; Tanaka et al., 2004).
Recently, LeCouter et al. reported the identifica-
tion of a new angiogenic mitogen that promotes
proliferation, migration, fenestration and survival
of adrenal cortical capillary endothelial cells (ACE)
(LeCouter et al., 2001). As it was found in
endocrine glands and shown to act selectively on
EC derived from these glands, it was designated
endocrine gland-derived vascular endothelial
growth factor (EG-VEGF; LeCouter et al., 2001).
This review summarizes the expression and roles of
EG-VEGF in ovarian tissue with special emphasis on
the biological functions of this peptide in steroido-
genic and EC of the CL.
Prokineticin protein family: Structure
and pattern of expression
EG-VEGF is identical to prokineticin 1 (PK-1)
which has first been identified as a stimulant of
gastrointestinal smooth muscle contraction (Li et
al., 2001). PK-1 is highly related to mamba
intestinal toxin-1 (Li et al., 2001). These proteins
together with the frog skin peptide Bv8 (Bombina
variegata) and its mammalian homolog prokineticin
2 (PK-2) belong to the same protein family (Kaser et
al., 2003). PKs share 60% amino acid identity and a
common protein structural motif (Fig. 1). They are
small secreted proteins consisting of 80–90 amino
acids, including 10 conserved cysteines. Their
amino-termini are identical; therefore the se-
quence of the first four residues, AVIT, was
suggested as a name for this family of proteins
(Kaser et al., 2003). The PKs are the cognate
ligands for two highly homologous G protein-
coupled receptors, PK receptors (PK-R1 and PK-
R2) that are about 80% identical to a previously
described mouse orphan receptor gpr73 (Lin et al.,
2002a; Masuda et al., 2002; Soga et al., 2002).
To the present date human, mouse, rat and
bovine EG-VEGF/PK-1 have been cloned (LeCouter
 ARTICLE IN PRESS
Role of prokineticin-1 and its corpus luteum 531

PK-2b MRSSRCARLLLLLLLPPLLLTPPAGDAAVITGACDRDPQCGGGMCCAVSLWVKSIRICTP 6
PK-2a MRSSRCARLLLLLLLPPLLLTPPAGDAAVITGACDRDPQCGGGMCCAVSLWVKSIRICTP 60
PK-1 ---------------------------AVITGACERDVQCRAGTCCAVSLWLRGLRVCTP 33
*******:** ** .* *******::.:*:***

PK-2b MGKVGDSCHPMTRKNHFGNGRQERRKRKRRRKKKVPFLGRRMHHTCPCLPGLACSRTSFN 120

PK-2a MGKVGDSCHPMTRK--------------------VPFLGRRMHHTCPCLPGLACSRTSFN 100
PK-1 LGRAGEECHPGSHK--------------------VPFFRKRQHHACPCLPNLLCSRGLDG 73
:*:.*:.*** ::* ***: :* **:*****.* *** .

PK-2b RYTCLAQK----- 128

PK-2a RYTCLAQK----- 108
PK-1 RYRCSTNLKNINF 86
** * ::

Figure 1. A comparison of amino-acid sequences of bovine prokineticins, PK-1 and PK-2 (a and b forms), respectively.
Signs used are as defined in CLUSTAL software W: an asterisk denotes identity in amino-acid residues; colons denote
conserved substitutions; full stops denote semi-conserved substitutions.

et al., 2001, 2003a; Masuda et al., 2002; Kisliouk et
al., 2005). The genomic structures of human and
mouse sequences have been conserved. Exon 1
contains the coding sequences for signal peptide
and the first 5 amino acids of the mature protein.
Exon 2 encodes 6 of 10 cysteins and 42 residues in
total, and exon 3 the remaining 49 amino acids
(LeCouter et al., 2003a). In human and rat EG-
VEGF/PK1 mRNA expression has been described in a
variety of tissues, in steroidogenic glands (such as
ovary, testis, and adrenal gland), but also in the
gastrointestinal tract, nervous system, bladder, and
prostate (LeCouter et al., 2001, 2003b; Li et al.,
2001; Masuda et al., 2002). Several studies have
examined the expression pattern of EG- EG-VEGF/
PK-1 in the human and primate ovaries; it was
present in ovarian stroma, follicles and corpora
lutea (LeCouter et al., 2001; Ferrara et al., 2003;
Kisliouk et al., 2003; Fraser et al., 2005). Similarly
in the bovine species, luteal steroidogenic cells
derived from mid-cycle CL as well as follicular
theca and granulosa cells expressed EG-VEGF/PK-1
mRNA. However EG-VEGF/PK-1 mRNA was not
detected in luteal EC (Kisliouk et al., 2005). It
was low at early-stage human CL (Ferrara et al.,
2003), but up-regulated during mid-luteal to late
luteal phase (Ferrara et al., 2003; Fraser et al.,
2005). Rather surprisingly, maximal expression of
EG-VEGF was found at very late luteal phase, at a
time when VEGF expression is greatly reduced or
undetectable (Fraser et al., 2005).
The expression of PK-2 mRNA is somewhat more
restricted than PK-1, PK-2 was found in suprachias-
matic nucleus of the brain where it affects
circadian rhythm (Cheng et al., 2002). High levels
of PK-2 were demonstrated in the testis where it
was confined to primary spermatocytes (Wechsel-
berger et al., 1999; LeCouter et al., 2003b). PK-2 is
encoded by four exons with the third exon
subjected to alternative splicing. Exon 3 codes for
21 amino acids, of which 11 are lysines or arginins.
Therefore, the long form of PK-2 was termed basic
PK-2 (PK-2b). The shorter variant, lacking 21 amino
acids, was consequently termed acidic PK-2 (PK-2a,
Fig. 1) (Wechselberger et al., 1999; Kaser et al.,
2003). Recently a shorter form of basic (or long) PK-
2, PK-2b, was described: PK-2b is produced by
proteolytic cleavage of PK-2b and in contrast to the
parental molecule it selectively activates PK-R1
(Chen et al., 2005).
Unlike the testis, PK-2 mRNA was almost un-
detectable in human and bovine ovaries (Ferrara et
al., 2003; Fraser et al., 2005; Kisliouk et al., 2005).
Regulation of EG-VEGF/PK-1 expression
in the ovarian steroidogenic cell types
Luteinized human granulosa cells, those har-
vested from patients undergoing IVF and SV-40
transformed granulosa cells (SVOG), as well as
bovine luteal steroidogenic cells express EG-VEGF/
PK-1 mRNA (Kisliouk et al., 2003). SVOG cells, like
non-transformed granulosa cells also produce VEGF,
interestingly however, the expression of these two
pro-angiogenic factors was inversely regulated in
these cells. cAMP elevating agent, forskolin, was
 ARTICLE IN PRESS
532 T. Kisliouk et al.

shown to be a potent stimulator of EG-VEGF/PK-1
mRNA in SVOG cells (6-fold increase after 24 h of
exposure), but it did not affect VEGF expression by
these cells (Kisliouk et al., 2003). Another study
employing luteinized human GC had in accordance
demonstrated that hCG up-regulated EG-VEGF/PK-
1 mRNA (Fraser et al., 2005). In contrast, incuba-
tion of bovine granulosa cells for 2–6 days in
luteinization promoting medium consisting of for-
skolin and insulin inhibited EG-VEGF/PK-1 while
0.6
0.5
EG-VEGF/PK-1 mRNA levels
elevating VEGF mRNA expression (Fig. 2). The
reasons for the opposite effects of these cAMP
elevating agents in human and bovine species are
yet unclear. However, this in vitro system may
correspond to luteal cells of CL at an early stage,
when VEGF levels are high and EG-VEGF/PK-1
expression still low (Fraser et al., 2005). Since
EG-VEGF/PK-1 synthesis increased in the mature
luteal tissue, it was thought that increasing levels
of progesterone might stimulate the synthesis of
EG-VEGF/PK-1. However, while progesterone ele-
vated EG-VEGF/PK-1 mRNA levels in human en-
dometrial tissue (Battersby et al., 2004) it did not
in luteinized granulosa cells (Fraser et al., 2005).
Hypoxia is generally considered to be a primary
trigger of new blood vessels formation in normal

-FI and pathologic tissues (Harris, 2002; Choi et al.,

0.4 +FI 2003). As expected VEGF, both its mRNA and
(Arbitrary units)

protein secreted into the culture media were

0.3 elevated in SVOG cells incubated with hypoxia
mimicking agents as CoCl2 and deferoxamine
mesylate (DFX), but expression of EG-VEGF/PK-1
0.2
was inhibited under the same conditions (Kisliouk
* et al., 2003). These findings contrast those re-
0.1 ported by LeCouter et al. using human adrenal
carcinoma cell lines SW13 and H295R, where both
0 VEGF and EG-VEGF/PK-1 were increased by hypoxia
2d 4d 6d (LeCouter et al., 2001). Whether or not the
different cell types may account for this discre-
100
* pancy is unknown at present.
90 * Thrombin is yet another potent activator of
angiogenesis (Tsopanoglou et al., 2004). Moreover
80
* it up-regulates VEGF receptors in ECs and induces
70 VEGF expression in several cell types (Maragoudakis
VEGF mRNA levels
(Arbitrary units)

and Tsopanoglou, 2000; Bassus et al., 2001; Lock-

60
wood et al., 2002; Wang et al., 2002). Thrombin
50 elevated VEGF expression in SVOG cells while EG-
VEGF/PK-1 expression was downregulated (Kisliouk
40
et al., 2003). In accordance increasing serum
30 concentrations up to 10% also inhibited EG-VEGF/
PK-1 expression (Kisliouk et al., 2003).
20

10

0
2d 4d
Figure 2. Inverse regulation of EG-VEGF/PK-1 (A) and
VEGF (B) mRNA expression in bovine granulosa cells. The
cells were incubating for 2–6 days (D) in basal medium
(containing 1% fetal bovine serum, FBS) or in a luteiniza-
tion promoting medium (consisting of forskolin (F) and
insulin (I) in basal medium). At each time point RNA was
extracted and mRNA levels were determined by Real-
Time RT-PCR. Results are Means 7 SEM from 4 indepen-
dent experiments. * Denotes significant difference
(Po0.05) as compared with the matching control time
point.
6d
Ovarian expression of PK-R1 and PK-R2
and their regulation
Acting via two closely related G protein-coupled
receptors, PK-R1 and PK-R2, PKs peptides regulate
diverse biological functions. However, at present it
is yet unknown whether these receptors have
selective or overlapping functions.
Expression of PK receptors in heterogeneous
systems showed that they bind to and are activated
by nanomolar concentrations of recombinant PKs
(Lin et al., 2002a; Masuda et al., 2002; Soga et al.,
 ARTICLE IN PRESS
Role of prokineticin-1 and its corpus luteum 533

2002). Activation of PK receptors overexpressed in A

cells lead to mobilization of calcium, stimulation of 200
phosphoinoside turnover and activation of p44/p42 180
MAPK cascade (Lin et al., 2002a; Masuda et al.,
2002; Soga et al., 2002). These biological effects 160

(normalized to 0.1% FBS)

PK-R1
were also observed in cells naturally expressing PK-

PK-Rs mRNA levels

140
PK-R2
Rs, namely in ACE (Lin et al., 2002b). 120
We had observed a distinct pattern of PK
100
receptors in bovine ovarian cells. Follicular and
luteal steroidogenic cells predominantly ex- 80
press PK-R1, whereas CL-derived EC express 60
high levels of both PK-R1 and PK-R2 (Kisliouk
40
et al., 2003). PK-R1 mRNA was also detected
in human luteinized granulosa cells and in SVOG 20
cell line (Kisliouk et al., 2003). Up to now, among 0
the different examined EC types PK-R2 were 0.1% FBS 2.5% FBS 10% FBS 2.5% FBS
+GF
identified only in fenestrated EC, such as those
found in the adrenal cortex, CL, kidney and liver B 120
(Masuda et al., 2002; Kisliouk et al., 2003; LeCouter
et al., 2003a), implying that PK-R2 may confer the 100

(normalized to 0.1% FBS)

selective effects of PK-1 on microvascular EC
functions.
Our recent study had demonstrated that mRNA
expression of PK receptors in luteal EC is modulated
by various stress factors such as serum starvation,
TNF-a and hypoxia induced by DFX or CoCl2
(Kisliouk et al., 2005). All treatments significantly
elevated PK-R2 mRNA expression and did not
markedly affected PK-R1 mRNA levels (Kisliouk et
al., 2005). Unlike with EC, hypoxia induced by
CoCl2 or DFX augmented PK-R1 mRNA expression in
SVOG cells (Kisliouk et al., 2003).
The relatively low mRNA level of PK-R2 in luteal
EC incubated in the medium consisting 10%
serum could suggest that different growth factors
present in serum, such as IGF-I and EGF may inhibit
PK-R2 expression in luteal EC (Fig. 3). As depic-
ted in Fig. 3, IGF-I and EGF inhibited PK-R2 mRNA
expression by 60% and 30%, respectively; FGF-b
and VEGF cultured alone did not change PK-R2
mRNA levels (data not shown), whereas the
presence of all these factors simultaneously in
the culture medium (containing 2.5% serum)
markedly decreased PK-R2 mRNA. These levels
were similar to those obtained with 10% serum
alone (inhibition almost by 90%, Fig. 3A). These
same growth factors slightly influenced PK-R1
expression in luteal EC (Fig. 3). Therefore, growth
factors present in serum exert an inhibitory
effect on PK-R2 mRNA, while stress conditions
such as serum deprivation followed by hypoxia or
exposure to TNF-a increased PK-R2 mRNA ex-
pression in luteal EC (Fig. 4) (Kisliouk et al.,
2005). Collectively, these findings tend to suggest
that PK-R2 may have a role in the survival of
microvascular EC.
PK-Rs mRNA levels
80
60
40
20
0
0.1% FBS EGF IGF-1
Figure 3. Modulation of PK-Rs expression by serum and
growth factors in luteal EC. (A) Effects of FBS and growth
factor mix on PK-Rs mRNA expression. Cells were
incubated in medium containing various concentrations
of FBS (10%, 2.5% and 0.1%) for 48 h. The mix of growth
factors (GF; Cambrex bioproducts, SingleQuots EGMTM-2)
consisted of VEGF, FGF-b, EGF and IGF-1 was added to
medium containing 2.5% FBS (2.5% FBS+GF). (B) Effects of
EGF and IGF-1 on PK-Rs mRNA expression. EGF and IGF-1
were added in to the medium containing 0.1% FBS for
48 h. PK-R1 and PK-R2 mRNA expression were determined
by Real-Time RT-PCR. Data are mean 7 range of two
separate experiments.
Effects of Prokineticins on proliferation
and survival of bovine luteal endothelial
cells
Both EG-VEGF/PK-1 and VEGF resulted in ex-
tensive angiogenesis and cyst formation when
delivered into mouse ovaries (LeCouter et al.,
2001). In adrenal cortex-derived EC, EG-VEGF/PK-1
provoked a rapid phosphorylation of p44/42 MAP
kinase and DNA synthesis in these cells (Lin et al.,
 ARTICLE IN PRESS
534
PK-R1
Steroidogenic
Cell
EG-VEGF/PK-1
VEGF
PK-R1
PK-R2 Wellbeing
Serum
GFs
Stress
Endothelial Hypoxia
Cell
Serum withdrawal
TNFα
Proliferation & survival
Figure 4. Paracrine/autocrine actions of EG-VEGF/PK-1
in luteal endothelial and steroidogenic cell types. EG-
VEGF/PK-1 is expressed by luteal steroidogenic cell
possessing PK-R1 where it augments VEGF mRNA expres-
sion. EG-VEGF/PK-1 also acts on luteal EC expressing high
levels of both PK-R1 and PK-R2. Activation of PK-Rs in
luteal EC augments their proliferation and survival. PK-R2
expression in luteal EC is inhibited by serum and growth
factors (GFs). Stress conditions such as serum with-
drawal, hypoxia and TNF-a elevate PK-R2 expression,
whereas mRNA levels of PK-R1 remain unchanged.
2002b). These studies support the concept that EG-
VEGF/PK-1 acts as selective angiogenic growth
factor for endocrine-gland derived endothelium.
Being a highly vascular endocrine gland, it was of
interest to investigate the effects of PK-1 on CL-
derived EC function. Luteal EC were isolated and
maintained in culture as described by Spanel-
Borowski and colleagues (Lehmann et al., 2000).
PKs were found to be potent mitogens for luteal EC:
they increased [3H]-thymidine incorporation by
luteal EC almost to the same level as VEGF (by
factors of 1.6, 1.7 and 2 over control, for PK-1,
PK-2 and VEGF, respectively) (Kisliouk et al., 2005).
Cell counts were in agreement with the results of
[3H]-Thymidine incorporation assay: incubation
with PKs and VEGF increased luteal EC numbers
1.5-fold and 2-fold, respectively (Kisliouk et al.,
2005). Addition of EG-VEGF/PK-1 with hypoxia
mimicking agent DFX simultaneously induced a
2.5-fold increase in [3H]-thymidine uptake as
compared with the control, whereas DFX alone
T. Kisliouk et al.
induced 2-fold increase only (Kisliouk et al.,
2005). Therefore the more pronounced effect of
EG-VEGF/PK-1 on [3H]-thymidine incorporation
during hypoxia is likely due to higher PK-R2 mRNA
expression under these conditions.
MAPK is a critical element in cell proliferation as
was shown by numerous studies (Guo et al., 1995;
Seger and Krebs, 1995; Widmann et al., 1999). It
have been demonstrated that both prokineticins
induced the phosphorylation of p44/42 MAPK in
luteal EC (Kisliouk et al., 2005) as they promoted in
ACE (Lin et al., 2002b). MAPK is one of the signaling
pathways that are known to induce expression of
the oncoproteins c-fos and c-jun, which belong to a
class of immediate early genes are rapidly acti-
vated, usually in a transient fashion, in response to
intracellular signaling cascades (Shaulian and Kar-
in, 2002; Sng et al., 2004). EG-VEGF/PK1 is a potent
inducer of c-fos and c-jun mRNA expression. It
transiently augmented c-fos mRNA expression in
luteal EC with maximum stimulation attained at 1 h
(7-fold) (Kisliouk et al., 2005). EG-VEGF/PK1 also
increased c-jun mRNA expression, however it was
less pronounced than with c-fos (2-fold increase at
1 h) (Kisliouk et al., 2005).
The immediate downstream effectors of PK-Rs
are just beginning to be resolved. Pretreatment of
luteal EC with pertussis toxin (PTX), which speci-
fically modifies the heterotrimeric G protein Gia,
blocking signaling pathways of G protein coupled
receptor involving Gia, did not inhibit the effect of
EG-VEGF/PK-1 on [3H]-thymidine incorporation
(Kisliouk et al., 2005). Therefore, Gia may not be
involved in PK-R activation in luteal EC. These
results are in agreement with those previously
reported by Lin et al. (2002a) and Soga et al.
(2002), but contradict those published by the group
led by Ferarra in adrenal gland EC (Lin et al.,
2002b). Recently, Chen et al. have reported, that
co-expression of PK-R2 with chimeric G protein
(Gqi5), which shifts receptor/Gi coupling to Ca2+
mobilization signaling, dramatically increased Ca2+
mobilization in response to ligand stimulation,
whereas co-expression of PK-R1 with Gqi5 did not
significantly affect Ca2+ mobilization (Chen et al.,
2005). These results may imply that GI can be
coupled to PK-R2 only. This study also showed PK
stimulated cAMP accumulation in PKR-expressing
cells (Chen et al., 2005) suggesting that PKRs may
be coupled to multiple G proteins as observed with
other GPCR (Hermans, 2003). These findings may
also explain why PKs are multifunctional peptides.
In addition to activating luteal EC proliferation,
prokineticins were shown to efficiently inhibit
apoptosis in these cells induced by serum starvation
(Kisliouk et al., 2005). The anti-apoptotic PKs
 ARTICLE IN PRESS
Role of prokineticin-1 and its corpus luteum
effect was demonstrated by three different
methods: nuclear morphology (DAPI staining),
detection of DNA strand breaks (TUNEL Assay),
and activation of caspase-3 (Kisliouk et al., 2005).
Serum starvation of luteal EC typically resulted in
16.572.8% apoptotic nuclei as was detected by
DAPI staining and 19.975.6% apoptotic cells as was
assessed by flow cytometry detection of TUNEL-
positive cells. EG-VEGF/PK-1 and PK-2 were able to
rescue the cells, and reduced the apoptosis rate to
7.071.5% or 8.071.2%, respectively, as detected
by DAPI staining and to 4.372.3% and 4.371.9%,
respectively, as measured by TUNEL assay (Kisliouk
et al., 2005). The amount of activated caspase-3
was determined by western blot analysis using
specific antibody to cleaved caspase-3 (17 and
19 kDa). Both PKs, like VEGF, completely inhibited
activation of caspase-3 induced by serum starvation
(Kisliouk et al., 2005). Consequently, PKs are not
only mitogens, but are also survival factors for
luteal EC.
Besides the mitogenic and anti-apoptotic effects
of prokineticins on luteal EC, EG-VEGF/PK-1 ele-
vated VEGF mRNA expression in bovine luteal
steroidogenic cells (Kisliouk et al., 2005). Augmen-
tation of VEGF expression in luteal steroidogenic
cells imply that EG-VEGF/PK-1 could also indirectly,
via VEGF, affect luteal angiogenesis.
Conclusions
EG-VEGF/PK-1 is expressed in human and bovine
ovaries where it elicits cell-specific effects in luteal
steroidogenic cells and in endothelial cells suggest-
ing that EG-VEGF/PK1 may be a novel autocrine/
paracrine regulator within the ovary (Fig. 4). PKs
promote DNA synthesis in adrenal cortex EC, as
they do in luteal EC. These cells have much in
common: irrigating endocrine glands the fene-
strated microvascular EC are particularly perme-
able to the inflow of blood-borne substances and
the outflow of specific secreted products. It is
noteworthy that adrenal cortex and luteal EC,
unlike the EC derived from the bovine aorta,
equally express the two PK receptor types -R1 and
R2 (Fig. 4). PKs also affect endocrine cells: PK-R1
were identified in ovarian steroidogenic cells where
EG-VEGF/PK-1 enhances VEGF mRNA expression.
The presence of PK-R1 and inverse regulation of
EG-VEGF/PK1 and VEGF mRNA expression by
ovarian steroidogenic cells suggest that in addition
to its angiogenic effects, EG-VEGF/PK-1 may also
have other functions in ovary, which remain to be
investigated.
535
References
Al-zi’abi, M.O., Watson, E.D., Fraser, H.M., 2003.
Angiogenesis and vascular endothelial growth factor
expression in the equine corpus luteum. Reproduction
125, 259–270.
Augustin, H.G., Braun, K., Telemenakis, I., Modlich, U.,
Kuhn, W., 1995. Ovarian angiogenesis. Phenotypic
characterization of endothelial cells in a physiological
model of blood vessel growth and regression. Am. J.
Pathol. 147, 339–351.
Bassus, S., Herkert, O., Kronemann, N., Gorlach, A.,
Bremerich, D., Kirchmaier, C.M., Busse, R., Schini-
Kerth, V.B., 2001. Thrombin causes vascular endothe-
lial growth factor expression in vascular smooth
muscle cells: role of reactive oxygen species. Arter-
ioscler. Thromb. Vasc. Biol. 21, 1550–1555.
Battersby, S., Critchley, H.O., Morgan, K., Millar, R.P.,
Jabbour, H.N., 2004. Expression and regulation of the
prokineticins (endocrine gland-derived vascular en-
dothelial growth factor and Bv8) and their receptors in
the human endometrium across the menstrual cycle.
J. Clin. Endocrinol. Metab. 89, 2463–2469.
Berisha, B., Schams, D., Kosmann, M., Amselgruber, W.,
Einspanier, R., 2000. Expression and tissue concentra-
tion of vascular endothelial growth factor, its recep-
tors, and localization in the bovine corpus luteum
during estrous cycle and pregnancy. Biol. Reprod. 63,
1106–1114.
Carambula, S.F., Pru, J.K., Lynch, M.P., Matikainen, T.,
Goncalves, P.B., Flavell, R.A., Tilly, J.L., Rueda, B.R.,
2003. Prostaglandin F2alpha- and FAS-activating anti-
body-induced regression of the corpus luteum involves
caspase-8 and is defective in caspase-3 deficient mice.
Reprod. Biol. Endocrinol. 1, 15.
Chen, J., Kuei, C., Sutton, S., Wilson, S., Yu, J., Kamme,
F., Mazur, C., Lovenberg, T., Liu, C., 2005. Identifica-
tion and Pharmacological Characterization of Prokine-
ticin 2{beta} as a Selective Ligand for Prokineticin
Receptor 1. Mol. Pharmacol. 67, 2070–2076.
Cheng, M.Y., Bullock, C.M., Li, C., Lee, A.G., Bermak,
J.C., Belluzzi, J., Weaver, D.R., Leslie, F.M., Zhou,
Q.Y., 2002. Prokineticin 2 transmits the behavioural
circadian rhythm of the suprachiasmatic nucleus.
Nature 417, 405–410.
Choi, K.S., Bae, M.K., Jeong, J.W., Moon, H.E., Kim,
K.W., 2003. Hypoxia-induced angiogenesis during
carcinogenesis. J. Biochem. Mol. Biol. 36, 120–127.
Ferrara, N., Frantz, G., LeCouter, J., Dillard-Telm, L.,
Pham, T., Draksharapu, A., Giordano, T., Peale, F.,
2003. Differential expression of the angiogenic factor
genes vascular endothelial growth factor (VEGF)
and endocrine gland-derived VEGF in normal and
polycystic human ovaries. Am. J. Pathol. 162,
1881–1893.
Fraser, H.M., Lunn, S.F., 2001. Regulation and manipula-
tion of angiogenesis in the primate corpus luteum.
Reproduction 121, 355–362.
Fraser, H.M., Wulff, C., 2003. Angiogenesis in the corpus
luteum. Reprod. Biol. Endocrinol. 1, 88.
 ARTICLE IN PRESS
536
Fraser, H.M., Bell, J., Wilson, H., Taylor, P.D., Morgan, K.,
Anderson, R.A., Duncan, W.C., 2005. Localization and
quantification of cyclic changes in the expression of
endocrine gland vascular endothelial growth factor in
the human corpus luteum. J. Clin. Endocrinol. Metab.
90, 427–434.
Guo, D., Jia, Q., Song, H.Y., Warren, R.S., Donner, D.B.,
1995. Vascular endothelial cell growth factor pro-
motes tyrosine phosphorylation of mediators of signal
transduction that contain SH2 domains. Association
with endothelial cell proliferation. J. Biol. Chem. 270,
6729–6733.
Harris, A.L., 2002. Hypoxia—a key regulatory factor in
tumour growth. Nat. Rev. Cancer 2, 38–47.
Hazzard, T.M., Molskness, T.A., Chaffin, C.L., Stouffer,
R.L., 1999. Vascular endothelial growth factor (VEGF)
and angiopoietin regulation by gonadotrophin and
steroids in macaque granulosa cells during the
peri-ovulatory interval. Mol. Hum. Reprod. 5,
1115–1121.
Hermans, E., 2003. Biochemical and pharmacological
control of the multiplicity of coupling at G-protein-
coupled receptors. Pharmacol. Ther. 99, 25–44.
Kaser, A., Winklmayr, M., Lepperdinger, G., Kreil, G.,
2003. The AVIT protein family. Secreted cysteine-rich
vertebrate proteins with diverse functions. EMBO
Reprod. 4, 469–473.
Kisliouk, T., Levy, N., Hurwitz, A., Meidan, R., 2003.
Presence and regulation of endocrine gland vascular
endothelial growth factor/prokineticin-1 and its re-
ceptors in ovarian cells. J. Clin. Endocrinol. Metab.
88, 3700–3707.
Kisliouk, T., Podlovni, H., Spanel-Borowski, K., Ovadia,
O., Zhou, Q.Y., Meidan, R., 2005. Prokineticins
(endocrine gland-VEGF and BV8) in the bovine ovary:
expression and role as mitogens and survival factors
for corpus luteum derived-endothelial cells. Endocri-
nology, June 2 (e-pub ahead of press).
LeCouter, J., Kowalski, J., Foster, J., Hass, P., Zhang, Z.,
Dillard-Telm, L., Frantz, G., Rangell, L., DeGuzman,
L., Keller, G.A., Peale, F., Gurney, A., Hillan, K.J.,
Ferrara, N., 2001. Identification of an angiogenic
mitogen selective for endocrine gland endothelium.
Nature 412, 877–884.
LeCouter, J., Lin, R., Frantz, G., Zhang, Z., Hillan, K.,
Ferrara, N., 2003a. Mouse endocrine gland-derived
vascular endothelial growth factor: a distinct expres-
sion pattern from its human ortholog suggests differ-
ent roles as a regulator of organ-specific angiogenesis.
Endocrinology 144, 2606–2616.
LeCouter, J., Lin, R., Tejada, M., Frantz, G., Peale, F.,
Hillan, K.J., Ferrara, N., 2003b. The endocrine-gland-
derived VEGF homologue Bv8 promotes angiogenesis in
the testis: localization of Bv8 receptors to endothelial
cells. Proc. Natl. Acad. Sci. USA. 100, 2685–2690.
Lehmann, I., Brylla, E., Sittig, D., Spanel-Borowski, K.,
Aust, G., 2000. Microvascular endothelial cells differ
in their basal and tumour necrosis factor-alpha-
regulated expression of adhesion molecules and
cytokines. J. Vasc. Res. 37, 408–416.
T. Kisliouk et al.
Li, M., Bullock, C.M., Knauer, D.J., Ehlert, F.J., Zhoum,
Q.Y., 2001. Identification of two prokineticin cDNAs:
recombinant proteins potently contract gastro-
intestinal smooth muscle. Mol. Pharmacol. 59,
692–698.
Lin, D.C., Bullock, C.M., Ehlert, F.J., Chen, J.L., Tian, H.,
Zhou, Q.Y., 2002a. Identification and molecular
characterization of two closely related G protein-
coupled receptors activated by prokineticins/endo-
crine gland vascular endothelial growth factor. J. Biol.
Chem. 277, 19276–19280.
Lin, R., LeCouter, J., Kowalski, J., Ferrara, N., 2002b.
Characterization of endocrine gland-derived vascular
endothelial growth factor signaling in adrenal cortex
capillary endothelial cells. J. Biol. Chem. 277,
8724–8729.
Lockwood, C.J., Krikun, G., Koo, A.B., Kadner, S.,
Schatz, F., 2002. Differential effects of thrombin and
hypoxia on endometrial stromal and glandular epithe-
lial cell vascular endothelial growth factor expression.
J. Clin. Endocrinol. Metab. 87, 4280–4286.
Maragoudakis, M.E., Tsopanoglou, N.E., 2000. On the
mechanism(s) of thrombin induced angiogenesis. Adv.
Exp. Med. Biol. 476, 47–55.
Martinez-Chequer, J.C., Stouffer, R.L., Hazzard, T.M.,
Patton, P.E., Molskness, T.A., 2003. Insulin-like growth
factors-1 and -2, but not hypoxia, synergize with
gonadotropin hormone to promote vascular endothe-
lial growth factor-A secretion by monkey granulosa
cells from preovulatory follicles. Biol. Reprod. 68,
1112–1118.
Masuda, Y., Takatsu, Y., Terao, Y., Kumano, S., Ishibashi,
Y., Suenaga, M., Abe, M., Fukusumi, S., Watanabe, T.,
Shintani, Y., Yamada, T., Hinuma, S., Inatomi, N.,
Ohtaki, T., Onda, H., Fujino, M., 2002. Isolation and
identification of EG-VEGF/prokineticins as cognate
ligands for two orphan G-protein-coupled receptors.
Biochem. Biophys. Res. Commun. 293, 396–402.
McCracken, J.A., Custer, E.E., Lamsa, J.C., 1999.
Luteolysis: a neuroendocrine-mediated event. Physiol.
Rev. 79, 263–323.
Niswender, G.D., Juengel, J.L., Silva, P.J., Rollyson,
M.K., McIntush, E.W., 2000. Mechanisms controlling
the function and life span of the corpus luteum.
Physiol. Rev. 80, 1–29.
Redmer, D.A., Doraiswamy, V., Bortnem, B.J., Fisher, K.,
Jablonka-Shariff, A., Grazul-Bilska, A.T., Reynolds,
L.P., 2001. Evidence for a role of capillary pericytes in
vascular growth of the developing ovine corpus
luteum. Biol. Reprod. 65, 879–889.
Schams, D., Berisha, B., 2002. Steroids as local regulators
of ovarian activity in domestic animals. Domest. Anim.
Endocrinol. 23, 53–65.
Schams, D., Berisha, B., 2004. Regulation of corpus
luteum function in cattle-an overview. Reprod. Do-
mest. Anim. 39, 241–251.
Schams, D., Berisha, B., Kosmann, M., Amselgruber,
W.M., 2002. Expression and localization of IGF family
members in bovine antral follicles during final growth
and in luteal tissue during different stages of estrous
 ARTICLE IN PRESS
Role of prokineticin-1 and its corpus luteum
cycle and pregnancy. Domest. Anim. Endocrinol. 22,
51–72.
Seger, R., Krebs, E.G., 1995. The MAPK signaling cascade.
Faseb J. 9, 726–735.
Shaulian, E., Karin, M., 2002. AP-1 as a regulator of cell
life and death. Nat. Cell. Biol. 4, E131–E136.
Smith, M.F., McIntush, E.W., Smith, G.W., 1994. Mechan-
isms associated with corpus luteum development. J.
Anim. Sci. 72, 1857–1872.
Sng, J.C., Taniura, H., Yoneda, Y., 2004. A tale of early
response genes. Biol. Pharm. Bull. 27, 606–612.
Soga, T., Matsumoto, S., Oda, T., Saito, T., Hiyama, H.,
Takasaki, J., Kamohara, M., Ohishi, T., Matsushime,
H., Furuichi, K., 2002. Molecular cloning and char-
acterization of prokineticin receptors. Biochim. Bio-
phys. Acta. 1579, 173–179.
Sugino, N., Kashida, S., Takiguchi, S., Karube-Harada, A.,
Kato, H., 2001. Expression of vascular endothelial
growth factor (VEGF) receptors in rat corpus luteum:
regulation by oestradiol during mid-pregnancy. Repro-
duction 122, 875–881.
Tanaka, J., Acosta, T.J., Berisha, B., Tetsuka, M., Matsui,
M., Kobayashi, S., Schams, D., Miyamoto, A., 2004.
Relative changes in mRNA expression of angiopoietins
and receptors tie in bovine corpus luteum during
estrous cycle and prostaglandin F2alpha-induced
luteolysis: a possible mechanism for the initiation of
luteal regression. J. Reprod. Dev. 50, 619–626.
Tsopanoglou, N.E., Papaconstantinou, M.E., Flordellis,
C.S., Maragoudakis, M.E., 2004. On the mode of
action of thrombin-induced angiogenesis: thrombin
peptide, TP508, mediates effects in endothelial cells
via alphavbeta3 integrin. Thromb. Haemost. 92,
846–857.
Wang, J., Morita, I., Onodera, M., Murota, S.I., 2002.
Induction of KDR expression in bovine arterial en-
dothelial cells by thrombin: involvement of nitric
oxide. J. Cell. Physiol. 190, 238–250.
537
Wang, Z., Tamura, K., Yoshie, M., Tamura, H., Imakawa,
K., Kogo, H., 2003. Prostaglandin F2alpha-induced
functional regression of the corpus luteum and
apoptosis in rodents. J. Pharmacol. Sci. 92, 19–27.
Webb, R., Woad, K.J., Armstrong, D.G., 2002. Corpus
luteum (CL) function: local control mechanisms.
Domest. Anim. Endocrinol. 23, 277–285.
Wechselberger, C., Puglisi, R., Engel, E., Lepperdinger,
G., Boitani, C., Kreil, G., 1999. The mammalian
homologues of frog Bv8 are mainly expressed in
spermatocytes. FEBS Lett. 462, 177–181.
Widmann, C., Gibson, S., Jarpe, M.B., Johnson, G.L.,
1999. Mitogen-activated protein kinase: conservation
of a three-kinase module from yeast to human.
Physiol. Rev. 79, 143–180.
Wulff, C., Wilson, H., Largue, P., Duncan, W.C.,
Armstrong, D.G., Fraser, H.M., 2000. Angiogenesis in
the human corpus luteum: localization and changes in
angiopoietins, tie-2, and vascular endothelial growth
factor messenger ribonucleic acid. J. Clin. Endocrinol.
Metab. 85, 4302–4309.
Wulff, C., Wilson, H., Rudge, J.S., Wiegand, S.J., Lunn,
S.F., Fraser, H.M., 2001. Luteal angiogenesis: preven-
tion and intervention by treatment with vascular
endothelial growth factor trap(A40). J. Clin. Endocri-
nol. Metab. 86, 3377–3386.
Young, F.M., Rodger, F.E., Illingworth, P.J., Fraser, H.M.,
2000. Cell proliferation and vascular morphology in
the marmoset corpus luteum. Hum. Reprod. 15,
557–566.
Zetser, A., Kisliouk, T., Ivakin, E., Lahav, M., 2001.
Dependence on prolactin of the luteolytic effect of
prostaglandin F2alpha in rat luteal cell cultures. Biol.
Reprod. 65, 1082–1091.
Zheng, J., Redmer, D.A., Reynolds, L.P., 1993. Vascular
development and heparin-binding growth factors in
the bovine corpus luteum at several stages of the
estrous cycle. Biol. Reprod. 49, 1177–1189.