BIOLOGY OF REPRODUCTION 70, 430–438 (2004)
Published online before print 15 October 2003.
DOI 10.1095/biolreprod.103.021139
Insulin-Like Growth Factor-I and Binding Proteins 1, 2, and 3 in Bovine Nuclear
Transfer Pregnancies1
Susan R. Ravelich,3 Bernhard H. Breier,2,3 Shiva Reddy,4 Jeffrey A. Keelan,3 David N. Wells,5
A. James Peterson,5 and Rita S.F. Lee5
Liggins Institute3 and Department of Pediatrics,4 Faculty of Medical and Health Sciences, University of Auckland,
Private Bag 92019, Auckland, New Zealand
AgResearch,5 Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand
ABSTRACT
nome are possible and, thereby, also may provide better
models for the treatment of human diseases [3–5]. Other
potential applications include the conservation and preservation of endangered breeds [6] and species [7].
An increasing variety of mammalian species, including
bovine [8], have been cloned using somatic cell NT [9–
12]. However, the efficiency of this technique is very low,
with less than 6% of reconstructed NT embryos typically
developing into live offspring [13]. Although major losses
occur during the first 14 days after transfer of cloned blastocysts, the primary cause of subsequent fetal loss appears
to be associated with functional placental deficiencies [14].
Placental abnormalities in cattle and sheep following transfer of cloned embryos have been recently documented and
include increased placental weights and low placentome
number [15, 16], enlarged umbilical vessels [8], and edematous membranes as well as increased allantoic fluid volume [8]. To our knowledge, the causes of these abnormalities remain unknown.
Placental growth and differentiation are regulated by
mechanisms that involve numerous endocrine and paracrine
signals. Cytokines, such as members of the transforming
growth factor (TGF) b family and members of the insulinlike growth factor (IGF) family, are actively synthesized by
the ruminant placenta [17]. Systemic IGF-I in the fetus is
an important determinant of the partitioning of nutrients
between the fetus and placenta to favor fetal growth [18].
In the fetus, skeletal muscle and liver are major sites of
IGF-I synthesis and potential sources of circulating IGF-I
[19], although the placenta is actively involved in the regulation of IGF-I plasma concentrations [18]. The actions of
the IGF family are regulated through their association with
high-affinity binding proteins (IGFBPs) that determine their
bioavailability. The IGFBPs also appear to mediate IGFindependent actions, such as inhibition or enhancement of
growth and induction of apoptosis. The actions of IGFs are
also regulated by IGFBP proteases, which affect the relative
affinities of IGFBPs, IGFs, and IGF-I receptor for each other [20]. To date, six different high-affinity binding proteins
(IGFBP-1 through -6) have been identified that specifically
bind IGF-I and -II. Furthermore, IGFBP-1, -3, and -4 bind
IGF-I and -II with similar affinities, whereas IGFBP-2, -5,
and -6 preferentially bind IGF-II [21]. In bovine serum, the
most abundant IGF carrier protein is IGFBP-3, which binds
more than 95% of the IGF-I and -II in the circulation [20].
In the uterus, IGFBP-1 is found solely in the luminal epithelium, where it is reported to play an important role in
regulating the transport of IGFs between the endometrium
and the uterine lumen [22, 23]. In addition, IGFBP-1 is the
predominant binding protein in amniotic fluid and a major
In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated
with low cloning efficiency. To assess differences in paracrine
and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their
binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear
transfer (NT) pregnancies at Days 50, 100, and 150 of gestation.
Fetal, maternal, and binucleate cell counts in representative
placentomes were performed on Days 50–150 of gestation in
all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of
gestation examined. Immunolocalization studies showed that
spatial and temporal patterns of expression of IGFBP-2 and -3
were markedly altered in the placentomes of NT pregnancies
compared to AI/IVP controls. Concentrations of IGF-I in fetal
plasma, as determined by RIA, were significantly higher (P 5
0.001) in NT pregnancies (mean 6 SEM, 30.3 6 2.3 ng/ml) compared with AI (19.1 6 5.5 ng/ml) or IVP (24.2 6 2.5 ng/ml)
pregnancies on Day 150 of gestation. Allantoic fluid levels of
IGFBP-1 were also increased in NT pregnancies. These findings
suggest that endocrine and paracrine perturbations of the IGF
axis may modulate placental dysfunction in NT pregnancies.
Furthermore, increased cell numbers in NT placentomes likely
have significant implications for fetomaternal communication
and may contribute to the placental overgrowth observed in the
NT placentomes.
cytokines, female reproductive tract, growth factors, placenta,
pregnancy
INTRODUCTION
The study and application of somatic cell nuclear transfer (NT) technology in mammals offers many benefits for
research, biomedicine, and agriculture [1, 2]. In addition to
providing a route for genetic modification in livestock and
production of biopharmaceuticals, NT will extend the range
of species in which desired genetic alterations of the geSupported by AgResearch and the Health Research Council of New Zealand. AgResearch also provided a Ph.D. stipend to S.R.R.
Correspondence: Bernhard Breier, Liggins Institute, University of Auckland, 2-6 Park Ave, Grafton, Auckland, New Zealand. FAX: 0064 9
3737497; e-mail: bh.breier@auckland.ac.nz
1
2
Received: 13 July 2003.
First decision: 28 July 2003.
Accepted: 1 October 2003.
Q 2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
430
PARACRINE DISTURBANCES IN CLONED CATTLE PREGNANCY
carrier protein in fetal serum, and its concentrations are
increased in the maternal circulation during pregnancy [24].
In humans, IGFBP-1 is implicated to play a role in the
abnormalities of placental development observed in preeclampsia [25]. The IGFBP-2 is primarily expressed by the
maternal caruncular tissues, and its interaction with IGF-II
may influence trophoblast function [22]. Circulating levels
of IGFBP-2 are significantly increased in large-offspring
fetuses following embryo culture. Liver expression is also
markedly increased [26]. In the sheep, IGFBP-3 mRNA is
expressed in the caruncular stroma, uterine luminal epithelium, and myometrium but is most abundant in the maternal
blood vessel walls, where it may have an important role in
angiogenesis [27, 28].
To our knowledge, no study has documented the spatial
and temporal distributions of IGFs and IGFBPs in the
cloned bovine placentome during early pregnancy. The aim
of the present study was to determine if endocrine and paracrine factors involved in the regulation of normal placental
growth, development, and function are dysregulated in cattle that are pregnant with embryos generated by NT.
MATERIALS AND METHODS
Investigations were conducted in accordance with the regulations of
the New Zealand Animal Welfare Act of 1999. The generation of embryos
for transfer to recipients or artificial insemination (AI) of the heifers was
carried out in two separate experiments, conducted approximately 2 mo
apart, with equal numbers of animals used for each experiment. All dams
and recipients were 2-yr-old heifers of predominantly dairy or dairy-cross
breeds.
Oocytes for in vitro-produced (IVP) embryos or cytoplasts for NT were
obtained from the same pool of ovaries collected from Friesian cows at
the slaughterhouse. In vitro maturation of oocytes was carried out as previously described [8].
NT Embryos
The methods used to generate the cloned (NT) embryos have been
described previously [8]. An ovarian follicular cell line (EFC) derived
from a 4-yr-old Friesian dairy cow and demonstrated to be totipotent following NT, as described by Wells et al. [8], was used in the present study.
Donor cells used for NT had been previously cryopreserved and were
obtained after at least nine cell passages in culture. Cells were cultured in
a 1:1 mixture of Dulbecco modified Eagle medium and F12 (Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal calf serum
(FCS; Invitrogen) and sodium pyruvate to a final concentration of 1 mM.
To induce the cells into a quiescent state, the FCS concentration in the
medium was reduced to 0.5%, and the cells were cultured for a further 9–
11 days before being used for NT. After injection of the donor cells into
the perivitelline space between the zona pellucida and the cytoplast, electrically mediated cell fusion was performed at 22–24 h after the start of
maturation. Two direct current electrical pulses of 2.25 kV/cm for 10 msec
each were delivered to individual, manually aligned donor cell-cytoplast
couplets in a fusion chamber comprising two parallel electrodes spaced
500 mm apart and in 0.3 M mannitol-based buffer [8]. Successfully fused
reconstructed embryos were activated 27–28 h after the start of maturation
using a combination of 5 mM ionomycin (Sigma, Sydney, Australia) for
4 min followed by incubation in 2 mM 6-dimethylaminopurine (Sigma)
for a further 4 h. After activation, in vitro culture was carried out essentially as described previously [29] in a biphasic AgResearch Synthetic
Oviduct Fluid medium (AgR SOF; AgResearch, Hamilton, New Zealand).
This medium was replaced on Day 4 of culture with fresh AgR SOF
containing 10 mM 2,4-dinitrophenol as an uncoupler of oxidative phosphorylation [30]. The fatty acid-free bovine albumin (8 mg/ml) in the AgR
SOF was ABIVP (ICP Bio, Auckland, New Zealand). On Day 7 postfusion, 49 NT embryos judged to be of suitable quality by a subjective
grading system were transferred into recipient heifers.
IVP Embryos
In vitro-matured oocytes were fertilized with frozen-thawed spermatozoa in 50-ml drops under oil for 24 h as described previously [30]. The
frozen semen used was obtained from the same Friesian bull that sired the
431
dairy cow from which the EFC cells were isolated. After fertilization, the
zygotes were cultured as described for NT embryos. On Day 7 after fertilization, embryos of suitable quality were transferred to recipients.
Artificial Insemination
Twenty-one Friesian heifers were synchronized for estrus using intravaginal CIDR progesterone-release breeding devices (Pharmacia Ltd.,
Auckland, New Zealand) inserted for 12 days. On Day 8, all heifers were
injected with 1 ml of estrumate (Schering-Plough, Union, NJ), and the
CIDR devices were withdrawn after a further 4 days. The mean onset of
estrus was approximately 48 h later. Approximately 12 h after the onset
of estrus, frozen-thawed semen obtained from the same bull described
above was used for AI.
Recipients for IVP or NT Embryos
Friesian or dairy crossbred heifers (n 5 70) were synchronized by a
single 12-day CIDR breeding device concurrent with those heifers used
for AI as described above. On the seventh day after observed estrus, 19
single-IVP and 49 single-NT embryos were transferred nonsurgically into
the uterine lumen ipsilateral to the corpus luteum of each heifer.
Pregnancy Monitoring
From Day 40 of gestation until slaughter, pregnancy establishment and
rates in all heifers were determined by monthly transrectal ultrasonographic examination using a Piemed 200 scanner with a linear 3.5- to 5-MHz
rectal probe (Philip-sweg, Maastricht, The Netherlands). Fetal heartbeats
were recorded from Day 60 onward. On Day 40, only the presence of a
gestation sac was recorded.
Tissue and Fluid Collection
Maternal blood samples were obtained from the tail vein during ultrasound scanning. A sample of pregnant animals from each group was
slaughtered on Days 50, 100, and 150 of gestation, and the reproductive
tracts were collected and transported to the laboratory within 1 h. Caruncles were removed and trimmed of their associated membranes. Fetal
fluids (amniotic and allantoic) were collected at all three gestation intervals, and fetal blood was collected at Day 150. Fetal fluids and serum
were stored at 2208C pending analysis. Both fetal and maternal cotyledon
tissues and tissue from intact placentomes were snap-frozen in liquid nitrogen or fixed in formalin-buffered PBS.
Immunohistochemistry
Two placentomes from each heifer were removed and placed in formalin-buffered PBS for sectioning, staining, and subsequent histological
and immunohistochemical analysis. Antibodies to IGF-I and -II as well as
IGFBP-1, -2, and -3 were raised in rabbits against recombinant human
(rh) IGF-I (Genentech, South San Francisco, CA), rhIGF-II (Eli Lily and
Company, Indianapolis, IN), ovine (o) IGFBP-1 (sequence amino acids
105–121; Gropep, Thebarton, Australia), and oIGFBP-2 and -3 purified
from sheep serum (Gropep) [31–33]. Antibodies were localized in placentomes by immunoperoxidase staining employing the avidin-biotin complex (ABC) for immunostaining of paraffin-embedded sections (Vector
Laboratories, Burlingame, CA). Tissues were embedded in paraffin wax,
sectioned (thickness, 5 mm), and mounted on chrome alum-coated slides.
Tissues were deparaffinized in xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was depleted by incubation with
0.3% hydrogen peroxide (H2O2) in methanol for 30 min at room temperature. Tissues were preincubated for 30 min with normal goat serum diluted 1:200 to block nonspecific binding, then incubated with the appropriate dilution of antiserum—IGF-I (1:1500), IGF-II (1:200), IGFBP-1 (1:
800), IGFBP-2 (1:800), and IGFBP-3 (1:600)—for 24 h at 48C. After
primary antibody incubations, tissues were washed in PBS and incubated
with biotinylated goat anti-rabbit immunoglobulin G for 2 h at room temperature followed by streptavidin-peroxidase conjugate for 20 min each at
room temperature. After a further PBS wash, the tissues were incubated
in diaminobenzidine until a positive reaction was observed (20–90 sec).
Normal rabbit serum and antiserum preabsorbed with excess antigen were
used as negative controls. Neutralization of immunoreactivity was defined
as a marked reduction in staining intensity in the presence of antigen.
Slides were photographed at 4003 magnification. A double-blind procedure was used to assess immunohistochemical scores based on staining
432
RAVELICH ET AL.
TABLE 1. Fetal, maternal, and binucleate cell numbers in AI, IVP, and NT placentomes on Days 50, 100, and 150 of gestation.a
Gestational
age
50
100
150
a
b
Group
n
AI
IVP
NT
AI
IVP
NT
AI
IVP
NT
31
20
62
24
24
36
30
24
48
Fetal cell number
48.8
49.6
68.2
47.1
48.1
69.6
48.9
49.5
68.3
6
6
6
6
6
6
6
6
6
0.6
1.0
0.8b
1.0
0.9
0.8b
0.9
0.7
0.7b
Maternal cell number
75.0
73.3
101.0
70.6
68.4
96.9
65.8
71.5
96.6
6
6
6
6
6
6
6
6
6
1.3
1.1
0.8b
1.3
1.7
1.1b
1.5
1.3
0.8b
Binucleate cell number
8.3
8.2
21.9
7.5
7.8
20.2
7.7
7.7
20.2
6
6
6
6
6
6
6
6
6
1.8
1.4
3.0b
0.2
0.2
0.4b
0.2
0.2
0.4b
Values are the mean 6 SEM.
P # 0.001, NT versus AI/IVP.
intensity (2, no staining; 1, minimal staining; 11, moderate staining;
111, moderate to intense staining; 1111, intense staining).
Cell Count
Paraffin-embedded sections were stained with hematoxylin and eosin
and with methyl green nuclear counterstain to assess fetal and maternal
cell numbers. Binucleate cell numbers were determined by immunostaining with bovine placental lactogen (bPL; a marker gene of binucleate cells)
[34]. Antibodies to bPL (a gift from J.C. Byatt, Monsanto Animal Agriculture Group, St Louis, MO), were raised in rabbit against purified native
bPL [35] and used at a final dilution of 1:10 000. The ABC method of
immunostaining was employed as described above. Cell counting was performed using a double-blind procedure, and random fields were assigned.
A standard grid was used over high-power-field microscopic views (1003
magnification). Six random fields were examined within a 1- 3 1-cm
ocular grid per section. All fetal trophectodermal mononucleate and binucleate and maternal uterine epithelial cells were counted at Days 50,
100, and 150 of gestation. One representative slide from each placentome
(two placentomes per cow) was assessed.
Radioimmunoassays
Levels of IGF-I and -II and IGFBP-1 in fetal and maternal serum and
fetal fluids (amniotic and allantoic) were measured using specific RIAs
[33]. The same primary antibodies used for immunohistochemistry were
used for the RIAs (see above). Cold recoveries and parallelism were performed on all bovine fluids. The limits of detection for serum IGF-I and
IGFBP-1 and for amniotic and allantoic fluid IGFBP-1 were 1.0, 1.0, 0.2,
and 0.3 ng/ml, respectively. Intra- and interassay coefficients of variation
were less than 10%. Assays for IGFBP-2 and- 3 were not available.
Statistical Analysis
Statistical analyses were carried out using a Sigma Stat (Jandel Scientific, San Rafael, CA) package. Differences between groups were determined by parametric (one-way ANOVA with post-hoc Tukey) tests. Data
are shown as the mean 6 SEM. Statistical significance was accepted at P
, 0.05.
RESULTS
Histological Examination
The cellular morphology of AI/IVP versus NT placentomes was similar at Days 50, 100, and 150 of gestation.
The bovine cotyledon consisted of numerous slender chorionic villi accommodated within the crypts of the uterine
mucosa. Each villus was composed of a core of vascular
mesoderm covered by a layer of cuboidal trophoblast cells,
many of which were binucleated. The maternal epithelium
was composed of cuboidal cells interspersed with low columnar cells having centrally located nuclei. The basic descriptions of the cytology of the maternal and fetal compartments are in general agreement with those described by
Björkman [36].
Despite similar cytological appearance, a significant in-
crease was observed in fetal, maternal, and binucleate cell
numbers in NT placentomes compared with AI/IVP controls at all gestational ages examined (Table 1). Furthermore, binucleate cell counts at Days 50, 100, and 150 of
gestation were almost threefold higher in NT placentomes
than in AI/IVP placentomes. Immunostaining with bPL allowed easy visualization of binucleated cells (which stained
dark brown in all placentomes examined at Days 50, 100,
and 150) and allowed comparison of binucleate cell numbers between AI, IVP, and NT placentomes, as illustrated
in Figure 1. Cell numbers remained relatively constant
throughout gestation, although maternal cell numbers were
consistently higher than fetal cell numbers in all groups
from Days 50 through 150 of gestation.
Immunolocalization of IGFBPs at Days 50, 100, and 150
of Gestation
The presence of IGFBP-1 was found in the deep uterine
glands of the maternal placentome. Fetal villi did not stain
for IGFBP-1. Immunoperoxidase staining was of moderate
intensity in all AI, IVP, and NT placentomes. Negative controls showed no staining. No differences were observed in
the localization of IGFBP-1 or staining intensity in AI, IVP,
and NT placentomes with advancing gestation. A representative photograph (from n 5 10 placentae) of IGFBP-1
staining at Day 50 of gestation is shown in Figure 2A.
Localization and immunohistochemical scores are shown in
Table 2.
Expression of IGFBP-2 was localized to the deep stromal tissue of the maternal placentome in all groups examined (Fig. 2, B–F). Intensity of stromal staining was
moderate in the AI placentomes at Days 50 (Fig. 2B) and
100 (Fig. 2D) of gestation, with no visible staining at Day
150 of gestation. Placentomes from IVP pregnancies
showed similar staining patterns (not shown). In contrast,
intense staining of the stroma was observed in the NT placentome (Fig. 2, C and E) at Days 50 and 100 of gestation.
The deep uterine glands and apical and medial regions (Fig.
2E) of the stromal tissue were immunonegative for IGFBP2. Maternal crypt and fetal villous tissue was also immunonegative in AI, IVP, and NT placentomes. The AI, IVP
and NT placentomes were immunonegative for IGFBP-2 at
Day 150 of gestation. No staining was detected in negative
controls (Fig. 2F).
Localization of IGFBP-3 was found in both fetal trophectodermal and maternal uterine tissue in NT placentomes, with strong, generalized staining for the three gestational periods examined (Fig. 2, H, J, and L). Little or no
staining was observed for IGFBP-3 in AI and IVP (not
shown) placentomes at all stages of gestation (Fig. 2, G, I,
PARACRINE DISTURBANCES IN CLONED CATTLE PREGNANCY
433
FIG. 1. Representative photograph comparing binucleate cell number in an AI
placentome (A) and an NT placentome (B)
at Day 100 of gestation following immunolocalization of bovine placental lactogen. Arrows illustrate binucleated cells.
CV, Chorionic villi; IC, intercrypt column.
Magnification 3400.
and K). No staining was observed in the negative control
(Fig. 2M).
calization and degree of staining intensity. Negative controls showed no staining.
Localization of IGF-I and -II on Days 50, 100, and 150
of Gestation
RIA Data
Generalized, moderate to intense staining for IGF-I was
observed in both fetal villous and maternal stromal and
epithelial tissue in all placentomes at all stages of gestation
examined, with no apparent differences between the three
groups (data not shown). The IGF-II immunoreactivity was
similar to the observations for IGF-I in terms of both lo-
Concentrations of IGF-I in fetal serum on Day 150 of
gestation were significantly higher (P 5 0.001) in NT pregnancies (30.3 6 2.3 ng/ml; n 5 8) compared with AI (19.1
6 5.5 ng/ml; n 5 5) or IVP (24.2 6 2.5 ng/ml; n 5 3)
pregnancies (Table 3). On Day 40, maternal serum concentrations of IGF-I in the three groups were similar (AI, 64.4
6 6.4; IVP, 70.5 6 9.8; NT, 62.8 6 4.8 ng/ml). Maternal
434
RAVELICH ET AL.
PARACRINE DISTURBANCES IN CLONED CATTLE PREGNANCY
concentrations were higher between Days 48 and 145 but
were not different among groups. Amniotic and allantoic
fluid concentrations of IGF-I were either at or below the
limits of detection for the assay in AI, IVP, and NT fluids
from Days 50 through 150 of gestation.
Maternal serum concentrations of IGFBP-1 were similar
in all groups from Days 40 to 145 of pregnancy. A slight
effect of stage on maternal IGFBP-1 levels was observed
in all groups examined. Concentrations of IGFBP-1 decreased slightly at Day 95, increased at Day 117, and decreased again at Day 145 of gestation. Allantoic fluid concentrations of IGFBP-1 were almost twofold higher in NT
versus AI/IVP pregnancies at Day 50 of gestation. However, no differences in amniotic fluid IGFBP-1 concentrations were found at this time point. Both amniotic and allantoic fluid IGFBP-1 concentrations at Days 100 and 150
of gestation were similar in AI, IVP, and NT pregnancies.
Amniotic fluid IGFBP-1 decreased over time in all groups,
whereas allantoic fluid IGFBP-1 levels were higher at Day
50, decreased slightly at Day 100, and increased again at
Day 150 of gestation. Both IGFBP-2 and -3 were not measured in the present study because of the unavailability of
suitable antisera. Concentrations of IGF-II in fetal serum
and in amniotic and allantoic fluid could not be determined
because of poor recovery from plasma, amniotic, and allantoic fluids.
DISCUSSION
Evidence from morphological studies of cloned pregnancies suggests that abnormal placental growth, development,
and function contribute to poor fetal viability in these pregnancies. Perhaps the most striking observation in many NT
pregnancies is the presence of large placentomes [13, 37,
38]. Histological examination of NT placentas suggests that
hyperplasia of the placentome may be a compensatory
mechanism secondary to reduced placentome number or
may arise from aberrations in trophoblastic cell differentiation and function [15, 39].
Histological examination of NT versus AI/IVP placentomes indicated that fetal and maternal cell numbers were
significantly increased in NT versus AI/IVP placentomes,
suggesting that a combination of both cotyledonary and caruncular abnormalities contributes to the aberrant development of the placenta. Furthermore, binucleate cell numbers were markedly increased in NT placentas to almost
threefold that of AI and IVP placentas. This observation
could be explained by increased binucleate cell formation
and/or decreased rate of binucleate cell migration across the
fetomaternal interface of the NT placenta. These findings
augment earlier reports by Farin et al. [40], who observed
increased volume densities of binucleate cells in bovine
placentas produced in vitro (but not in the IVP embryos of
the present study). Binucleate cells play a central role in
formation of the placental syncytium and secretions at the
fetomaternal interface that are crucial in maintaining pregb
FIG. 2. Immunolocalization of IGFBP-1 (representative Day 50 photograph [A]) and IGFBP-2 in the AI placentomes at Days 50 (B) and 100
(D) of gestation and in the NT placentome at Days 50 (C) and 100 (E) of
gestation. Immunolocalization of IGFBP-3 in AI placentomes at Days 50
(G), 100 (I), and 150 (K) of gestation and in the NT placentome at Days
50 (H), 100 (J), and 150 (L) gestation is also shown, as are representative
IGFBP-2 and -3 negative controls (F and M, respectively). Arrows indicate
uterine glands (A–E). FV, Fetal villi; S, stroma; ME, maternal epithelium;
a, apical; m, medial. Magnification 3400.
435
nancy [41]. As such, the observed increase in cell numbers
likely has significant implications for fetomaternal communication and may contribute to the placental overgrowth
observed in the NT placentome; however, the mechanisms
by which this occurs remain largely unknown.
Various components of the IGF system have been localized to the bovine uterus and placenta, where they show
spatial and temporal patterns of expression [42, 43]. In the
placenta, the IGFs promote cellular mitosis and differentiation, migration and aggregation, as well as inhibition of
apoptosis [23]. Variations in the pattern of expression of
either IGF-I or -II have a range of effects on placental
growth. Small placentas are found in IGF-II knockout mice,
whereas mice that overexpress IGF-II exhibit placental edema [44]. Furthermore, disruption of the IGF-II receptor,
with an associated reduction in IGF-II clearance, leads to
placental hypertrophy [45, 46]. Many of these features have
been reported in cloned animals [13, 15, 16]. In the present
study, we have investigated the hypothesis that differences
in the expression of IGFs and IGFBPs in NT pregnancies
may be fundamental to the placental abnormalities that are
seen in cloned cattle. To our knowledge, this is the first
study to document differences in IGFs/IGFBPs in bovine
NT pregnancies early during gestation.
The results of the present study demonstrate that serum
IGF-I levels were significantly increased in NT fetuses at
Day 150 of gestation compared to IVP and AI controls. In
contrast, Chavatte-Palmer et al. [16] found no difference in
IGF-I concentrations in cloned calves compared to controls.
However, IGF-I concentrations are dramatically reduced after birth [18]. A dominant influence on fetal IGF-I levels,
at least during the later stage of gestation, is nutrient status,
particularly glucose availability to the fetus [18]. Defects
in energy metabolism, such as hyperinsulinemia and hypoglycemia, have been reported in a number of calves following NT [47]. Increased IGF-I levels in fetal serum therefore may reflect altered energy metabolism in NT fetuses,
perhaps because of adaptations made by the conceptus to
abnormal placental function. The liver is the major source
of circulating IGF-I, and increased liver weights have been
reported in cloned sheep [38] and cattle fetuses (unpublished data). Alterations in hepatic synthesis or clearance
of IGF-I, perhaps induced by placental deficiencies, may
give rise to the increased liver weights often associated with
NT fetuses. Alternatively, elevated fetal IGF-I levels might
cause disturbances in placental growth and development.
Fetal IGF-I concentrations have been significantly and positively correlated with fetal weight and placental mass [48].
Recently, Gadd et al. [48] showed how such changes in
placental mass are correlated with alterations in the pattern
of expression of the IGF system within the uteroplacental
unit of sheep.
In the present study, maternal serum concentrations of
IGF-I and IGFBP-1 were similar between groups, and no
differences in amniotic fluid IGFBP-1 levels were observed. To the contrary, Bertolini and Anderson [37] reported increased maternal plasma concentrations of IGF-I
and -II in IVP pregnancies throughout gestation, although
no data were presented. Furthermore, those authors described alterations in the IGFBPs within amniotic and allantoic fluids of IVP pregnancies before Day 90 of gestation. Similarly, we observed significantly increased allantoic fluid IGFBP-1 concentrations at Day 50 of gestation in
NT pregnancies. These findings are coincident with an increase in allantoic fluid volumes in Day 50 NT pregnancies
(unpublished data). Allantoic fluid is normally absorbed by
436
RAVELICH ET AL.
TABLE 2. Localization and immunohistochemical scores for Day-50, -100, and -150 placentae from AI, IVP, and NT cattle.a
Treatment
AI
Ligand
IGFBP-1
IGFBP-2
IGFBP-3
IGF-I
IGF-II
a
IVP
NT
Gestation
day
F
M
F
M
F
M
Fetal
tissue
50
100
150
50
100
150
50
100
150
50
100
150
50
100
150
2/1
2/1
2
2
2
2
2/1
2/1
1
111
111
111
111
111
111
11
11
2
11
11
2
2/1
2/1
1
111
111
111
111
111
111
2/1
2/1
2
2
2
2
2/1
2/1
1
111
111
111
111
111
111
11
11
2
11
11
2
2/1
2/1
1
111
111
111
111
111
111
2/1
2/1
2
2
2
2
1/11
1/11
1/11
111
111
111
111
111
111
11
11
2
1111
1111
2
111
111
111
111
111
111
111
111
111
Villi
Villi
Villi
—
—
—
Villi
Villi
Villi
Villi
Villi
Villi
Villi
Villi
Villi
TABLE 3. IGF-I and IGFBP-1 concentrations in maternal serum and fetal
fluids (amniotic and allantoic) as determined by RIA.a
Day
AI
IVP
NT
40
48
60
69
95
117
145
40
48
60
69
95
117
145
Maternal serum (ng/ml)
64.4 6 6.4(6)
70.5 6 9.8(5)
98.4 6 5.6(8)
84.9 6 6.3(5)
75.1 6 3.0(4)
92.0 6 9.6(4)
89.1 6 7.1(7) 124.4 6 7.5(4)
84.3 6 3.5(6)
89.9 6 16.3(3)
78.2 6 8.3(5)
80.7 6 9.1(4)
84.1 6 3.6(4)
97.1 6 13.3(4)
4.0 6 0.5(6)
6.1 6 2.2(5)
4.5 6 0.5(8)
5.3 6 1.49(5)
3.8 6 1.0(4)
4.9 6 1.8(4)
3.8 6 1.1(7)
4.9 6 1.8(4)
2.1 6 0.3(6)
1.9 6 0.3(3)
4.3 6 1.1(5)
6.4 6 2.7(4)
1.2 6 0.3(4)
1.3 6 0.1(4)
IGFBP-1
50
100
150
Amniotic fluid (ng/ml)
1.20 6 0.30(5) 1.60 6 0.60(3)
1.20 6 0.20(3) 0.90 6 0.40(3)
0.40 6 0.10(4) 0.70 6 0.20(3)
0.90 6 0.10(9)
0.90 6 0.10(4)
0.70 6 0.10(7)
IGFBP-1
50
100
150
Allantoic fluid (ng/ml)
0.90 6 0.10(4) 0.80 6 0.10(3)
0.50 6 0.20(4) 0.70 6 0.20(3)
1.00 6 0.01(4) 1.00 6 0.01(3)
1.40 6 0.10(9)b
0.90 6 0.80(3)
0.90 6 0.01(9)
IGF-I
IGFBP-1
b
Maternal tissue
Uterine glands
Uterine glands
—
Uterine stroma
Uterine stroma
—
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
Throughout uterus
F, fetal; M, maternal; 2, no staining; 1, minimal staining; 11, moderate staining; 111, moderate to intense staining; 1111, intense staining.
the placenta. However, in the case of NT pregnancies, fluid
is retained, perhaps because of the inability of the lower
number of caruncles to maintain the balance [49]. An alteration in the function of the allantoic membrane, perhaps
because of altered hormonal status of the cow, also may
contribute to hydrallantois [38, 50]. No differences of amniotic or allantoic IGFBP-1 concentrations were observed
in NT versus AI/IVP at Day 100 or 150 of gestation. This
may reflect a dilution of IGFBP-1 with increased fetal fluid
volumes in NT pregnancies at later gestational stages.
In addition to the systemic actions considered above, immunoperoxidase staining of IGFBPs in NT pregnancies
showed significant differences compared to AI/IVP placentas, although staining of IGF-I and -II did not differ. The
spatial and temporal patterns of IGF-I and -II expression
a
Localization
Values are mean 6 SEM.
P # 0.001, NT versus AI/IVP.
62.8
100.2
95.8
82.8
103.2
94.4
96.3
3.4
5.2
3.2
3.2
1.8
5.2
1.6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
4.8(14)
5.9(10)
9.7(9)
11.1(3)
9.4(5)
7.3(8)
11.6(6)
1.7(14)
0.8(10)
0.4(9)
0.4(3)
0.3(5)
2.2(8)
0.3(6)
reported here are in agreement with studies described elsewhere [42, 43]. Whereas IGFBP-1 expression in AI, IVP,
and NT placentomes was similar at Days 50, 100, and 150
of gestation, expression of both IGFBP-2 and -3 was significantly increased in placental tissues from NT pregnancies. Increased expression of IGFBP-2 and -3 in NT placentas may indicate either increased production or upregulation by other growth effectors. Signals for increased production of the binding proteins may emanate from the
placenta in response to either altered availability of IGF-I
and -II or upregulation by other cytokines and cell-cycle
regulators, such as TGFb, retinoic acid, or p53 [51, 52].
However, we found no difference in immunoperoxidase
staining of IGF-I and -II between NT and AI placentas.
Increased retention of IGFs or reduced proteolysis and induction of IGF-independent action may reduce expression
of IGFs, limiting their bioavailability. Martin and Baxter
[53] showed that in T47D breast cancer cells transfected to
overexpress IGFBP-3, the cell population changed from a
state of relative cell-cycle arrest to one of insensitivity to
the inhibitory effect of IGFBP-3, in which cell growth was
uninhibited. Overexpression of IGFBP-2 and enhanced proliferation in Y-1 adrenocortical tumors via IGF-independent
actions also have been described [54]. An alternative explanation is increased induction of the regulators of IGFBP2 and -3. Fanayan et al. [55] reported that IGFBP-3 inhibitory signaling requires an active TGFb signaling pathway.
Conceivably, then, abrogation of this pathway may stimulate IGFBP-3 growth signaling effects. Consistent with this
view, Kansra et al. [56] has shown that the induction of
IGFBP-3 by TGFb is associated with enhanced growth in
colon cancer.
In summary, the results of the present study demonstrate
that cell number is significantly increased in both fetal and
maternal tissues of placentomes in NT pregnancies compared with AI/IVP controls. Increased production of
IGFBP-2 and -3 in these pregnancies may provide a causal
explanation for the increase in cell number and, potentially,
increased placental size observed in NT pregnancies.
Whether these factors are part of the cause of placental
dysfunction or merely a consequence of abnormal placental
development is yet to be established.
PARACRINE DISTURBANCES IN CLONED CATTLE PREGNANCY
ACKNOWLEDGMENTS
The authors wish to acknowledge Chris Keven and Janine Street for
their technical assistance with the RIAs and Martyn Donnison for routine
care of the animals used in the present study.
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