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Department of Farm Animal and Equine Medicine and Surgery, The Royal Veterinary College, Potters Bar, Herts, United Kingdom EN6 1NB
Address all correspondence and requests for reprints to: Prof. D. C. Wathes, Department of Farm Animal and Equine Medicine and Surgery, The Royal Veterinary College, Boltons Park, Hawkshead Road, Potters Bar, Herts, United Kingdom EN6 1NB.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IGFs mediate their effects by binding to specific (type 1 and type
2) IGF receptors (IGF-1R and IGF2R) on target cell surfaces. The type 1
receptor has an 
-subunit with binding capacity and a ß-subunit
with tyrosine kinase activity (8). The binding affinity for the type 1
receptor is greatest for IGF-I, least for insulin, and intermediate for
IGF-II. The type 2 receptor is the same molecule as the
cation-independent mannose-6-phosphate receptor (9). In contrast to the
type 1 receptor, the type 2 receptor is a single transmembrane protein
that does not have tyrosine kinase activity and does not bind IGF-I or
insulin (10). Targeted disruption of the type 2 receptor gene in mice
has effectively dismissed it as a route for signal transduction;
instead, it appears more likely to be functioning as a clearance
receptor for IGF-II (7).
The IGFs circulate bound to a family of binding proteins (IGFBPs). To date, six IGFBPs have been characterized, designated IGFBP-1 through -6. The IGFBPs specifically bind IGF-I and IGF-II, but fail to bind insulin (11, 12). IGFBP-1, -3 and -4 bind IGF-I and IGF-II with similar affinities, whereas IGFBP-2, -5, and -6 preferentially bind IGF-II. IGFBP-3 is the most abundant form of BP in serum, binding more than 95% of the IGF-I and IGF-II in the circulation. At the cellular level, IGFBPs are generally thought to inhibit the actions of the IGFs, but under some circumstances they may potentiate their metabolic and mitogenic effects (13, 14). The IGFBPs are synthesized in a wide range of tissues, including the uterus, and may, therefore, be important modulators of placental development (6, 15, 16, 17, 18).
The ewe has a cotyledonary placenta. The fetal membranes attach to the uterine endometrial epithelium on days 15–18. Interdigitation commences approximately 23 days after mating and is well established by day 30 (19). During this process fetal villi project into specialized, predetermined regions of the maternal stroma, known as caruncles, to form placentomes. These increase dramatically in size during the first third of gestation. Previous studies have revealed clear relationships among maternal nutrition, placental size, and fetal growth rate (20). The growth of individual placentomes is also influenced by litter size (20, 21). The mechanism by which either of these effects is achieved remains unknown.
Recent work from our laboratory using in situ hybridization has shown that the ovine uterus is a rich source of IGFs and IGFBPs (22, 23, 24). The present study extends these findings to examine the role of the IGF system during placental development throughout the period of its most intense proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligonucleotide probes
All probes used were single stranded oligodeoxynucleotides 45
bases in length (Table 1
). Sense probes corresponding to
each of the antisense probes [identical in sequence to the respective
messenger RNA (mRNA) targets] were always included as a negative
control, as any signal they produced could be categorized as
nonspecific.
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for exposure times).
Samples of uterus from estrous ewes, previously shown to be positive
for IGF-I, IGF-II, and the type 1 receptor mRNA (22), were included in
each experiment as a positive control.
Photographic emulsions
Slides previously exposed to x-ray film were coated with a
photographic emulsion (LM1, Amersham International, Aylesbury, UK)
according to the manufacturer’s instructions and stored at 4 C for the
required exposure time (Table 1). Slides were then developed and
counterstained with hematoxylin and eosin to confirm microscopically
the cellular localization of the radioactive signal.
Optical density (OD) quantification
An image analysis system (Seescan PLC, Cambridge, UK) was used
to convert the radioactive signals to OD units for quantification using
a linear gray scale of 0–2.1. All readings were on this part of the
scale. The antisense autoradiograph was placed under the image analyzer
lens, and a blank section of the film was measured. The region to be
quantified was outlined, and the background reading was automatically
subtracted from this. The corresponding section on the sense
autoradiograph was measured in a similar fashion, and the reading
obtained was subtracted from the antisense value to give the final OD
value for specific hybridization. A minimum of two antisense slides,
each containing at least two sections, were measured for each animal.
OD readings were taken from at least four similar regions on each
section. The average OD value for each animal was then calculated. The
detection limit was taken as an OD greater than 0.01. Coefficients of
variation for duplicate absorbance measurements between two slides were
as follows: IGF-I, 12.3%; IGF-II, 9.6%; IGF-1R, 12.6%; IGFBP-2,
15.4%; IGFBP-3, 12.7%; and IGFBP-4, 13.6%. In the majority of cases,
all slides using the same probe were analyzed in the same batch.
Statistical analysis
Values are given as the mean OD ± SEM.
Statistical analyses were performed using Unistat Statistical Package,
version 4.6 (Unistat Ltd., London, UK). Trends over time between
gestational age groups within the same region of the uterus were
analyzed by either unbalanced one-way ANOVA or ANOVA of regression.
Mann-Whitney tests were used to determine which time points differed.
If no time-related changes were detected, all data from a particular
region were pooled between time intervals. Differences between related
regions, such as the deep and superficial glands of the endometrium,
were analyzed using a paired t test. Results were considered
statistically significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
highest levels of expression were recorded around the deep glands (OD,
0.05 ± 0.009; n = 17). Low and unchanging levels of
expression were recorded in the endometrial stroma (OD, 0.02 ±
0.002; n = 17), myometrium (OD, 0.02 ± 0.002; n = 21),
and superficial glands (Fig. 1C; OD, 0.03 ± 0.004; n = 17)
throughout the period studied (days 13–55). Concentrations in the
caruncular stroma before the invasion by fetal allantochorion were also
low (OD, 0.04 ± 0.004; n = 12). After invasion of fetal
villi, IGF-I mRNA could no longer be detected in the caruncles. There
was no hybridization to fetal placental tissue.
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, D and E).
Levels of expression in this region increased up to day 35 of
gestation, then plateaued (Fig. 3; P <
0.05). Particularly intense expression was observed in mesoderm found
at the tips of the invading placentome villi. Moderately high,
unchanging levels of expression were found in the maternal caruncular
stroma. Concentrations in this region, below the area penetrated by the
fetal villi, were observed to decrease in intensity from the maternal
(OD, 0.17 ± 0.031; n = 21; P < 0.05) to the
fetal (OD, 0.11 ± 0.030; n = 21) side of the caruncle (Fig. 2, A and C). Low, stable, levels of expression were found in the
placentome capsule and endometrial stroma throughout the period studied
[Fig. 2A; OD, 0.11 ± 0.019 (n = 8) and 0.02 ± 0.003
(n = 32), respectively], whereas the concentration in the
myometrium was at the limit of detection (0.01 ± 0.003; n =
32).
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, A and D, and 5A). In
this region, concentrations were high during days 13–18 (equivalent to
values previously recorded at estrus) (22) and decreased significantly
in the fourth week of gestation (days 21–30; P <
0.01). There was a tendency to increase again from day 34 onward (Fig. 5A; P < 0.07). IGF-1R mRNA expression in the
superficial glands was significantly lower than that in the deep glands
(Fig. 5B; P < 0.05), and the concentration did not
change throughout the period studied. Photographic emulsions showed
that IGF-1R mRNA localization was mainly confined to the epithelial
cells of the glands (Fig. 4D). Low and unchanging concentrations were
also observed in the endometrial stroma, with slightly higher levels in
the myometrium (Fig. 5B).
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; OD, 0.07 ± 0.009; n = 19).
This level of receptor expression remained only in caruncles collected
from areas where significant interdigitation of the fetal
allantochorion had failed to occur, such as at the tips of the uterine
horns. In the placentomes collected after day 30, in which the
caruncles interdigitated with fetal allantochorion, IGF-1R mRNA could
no longer be detected in either fetal or maternal tissue.
Concentrations on the autoradiographs were below an OD of 0.01 (Fig. 4C), and no specific grains could be detected on the emulsion-coated
slides (Fig. 4F).
Distribution of IGFBP-2 mRNA
IGFBP-2 mRNA was absent from all uterine tissue until day 29 of
gestation, when it appeared as an intense band around the luminal space
(Fig. 6A; OD, 0.27 ± 0.031; n = 15).
Photographic emulsions confirmed the localization to the dense
caruncular-like stroma lining the luminal epithelium (Fig. 6, C and D).
The IGFBP-2 mRNA subsequently remained in a similar location and
intensity throughout the period studied (up to day 55). There was no
hybridization to any other tissue, fetal or maternal.
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and 8, C and E; OD, 0.87 ± 0.139; n = 5).
Evidence from photographic emulsions revealed that the mRNA in this
region was not uniformly distributed and instead tended to reside in
several clusters. There was insufficient information available to
enable us to establish whether these were implantation sites. After
this period of intense expression, IGFBP-3 mRNA concentrations in
the region dropped significantly (P < 0.001) and
remained below this original level (Fig. 9A).
Concentrations in the myometrium were initially modest and showed a
gradual decrease with time (Fig. 9B). Low, unchanging levels of IGFBP-3
mRNA were found throughout the endometrium (Fig. 9C; OD, 0.07 ±
0.010; n = 27).
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; OD, 0.62 ± 0.057; n = 6). Careful analysis of this
region using photographic emulsions revealed localization of the
IGFBP-3 mRNA to maternal blood vessel walls (Fig. 8A). Moderate levels
of expression were detected in the placentome capsule (OD, 0.14 ±
0.040; n = 6). No IGFBP-3 mRNA was found in any fetal tissue in
this study.
Distribution of IGFBP-4 mRNA
Uniformly high levels of IGFBP-4 mRNA expression (OD, 0.25 ±
0.024; n = 27) were colocalized with IGFBP-2 mRNA in the dense
caruncular-like stroma lining the luminal space. Very low levels of
IGFBP-4 gene expression were detected throughout the endometrium and
myometrium [Fig. 10A; OD, 0.05 ± 0.006 (n =
29) and 0.04 ± 0.005 (n = 27), respectively]. No evidence
of hybridization to any fetal tissue was found. Levels of IGFBP-4 mRNA
expression in caruncular stroma were moderate (OD, 0.20 ± 0.015;
n = 27). In this same region after fetal allantochorion
interdigitation/placentome formation, levels tended to drop (OD,
0.06 ± 0.017; n = 8). Higher levels of expression were
detected in the placentome capsule (Fig. 10, C, E, and F; OD, 0.28
± 0.068; n = 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The technique of in situ hybridization allows both quantification and accurate localization of potential sites of protein production within the tissues examined. Previous studies have generally found good agreement between mRNA analyses for the genes studied and protein expression. Although immunocytochemistry can be used to localize protein, this technique is unable to distinguish between local synthesis and proteins reaching the uterus via the circulation.
Uterine concentrations of IGF-I mRNA during gestation were lower than those previously reported at estrus (22). Peak levels of expression were found around the deep endometrial glands contrasting with studies performed in the pregnant rat uterus (6) in which IGF-I mRNA expression was restricted to the myometrium. In the mouse, peak IGF-I mRNA expression occurred in the endometrial stroma on day 4 of gestation, but then decreased after implantation (39). The pattern of expression in the pig is again different, peaking in the endometrium on day 12, coincident with the production of estrogen by the conceptus (40). A decline in porcine endometrial expression does, however, occur later in pregnancy (16), mimicking the reported decline in expression for sheep and mice. The large discrepancies in the spatiotemporal expression across species in early gestation presumably reflects the different roles for this peptide and may also be due to differences in the type of placentation. Although locally derived IGF-I may stimulate the growth and differentiation of the conceptus in the pig and perhaps the mouse, a similar role in the postimplantation sheep and rat seems unlikely at present.
In contrast to IGF-I mRNA, IGF-II mRNA concentrations during pregnancy were higher than those found at estrus. Peak IGF-II gene expression was recorded in the fetal mesoderm and maternal caruncular stroma, supporting a role in stimulating proliferation within the developing placentomes. This finding is in agreement with previous studies performed in rats and humans (6, 18, 41), in which IGF-II was detected in the embryonic and extra embryonic mesoderm, and is further supported by experiments that used the targeted mutagenesis of the genes encoding IGF-II in mice to show placental growth deficiency (42).
Targeted disruption experiments have effectively eliminated the IGF-2R as a candidate for signal transduction, leading to suggestions that the growth-promoting effects of IGF-II might be mediated through the IGF-1R (43). Our data are hard to reconcile with this view, as we have shown loss of IGF-1R mRNA from the maternal caruncular stroma as the fetal allantochorion starts to invade. This invasion of fetal tissue would contribute to the reduction in overall concentration within the placentomes. Nevertheless, maternally derived caruncular tissue continues to occupy a significant proportion of the space, and no IGF-1R mRNA was detected in this on either autoradiographs or emulsions, even if the exposure time was increased to 28 days. It is possible that the in situ technique was too insensitive to detect low levels of IGF-1R mRNA expression. We think that this is improbable, however, as IGF-1R mRNA was clearly present in the superficial and deep glands throughout the period studied, although these are situated too far away to make them a likely target for fetal or maternal caruncular IGF-II. Alternatively, protein for the IGF-1R could be retained from synthesis earlier in gestation. Another explanation could be a separate, as yet uncharacterized, receptor capable of binding IGF-II and mediating uterine growth. Several previous reports have postulated the existence of such a receptor (7, 44, 45, 46, 47), but it has yet to be identified. This might explain the results of Lacroix et al. (36), who used affinity cross-linking studies and immunohistochemistry to demonstrate IGF-1R (1 pmol/mg) in the trophoblast cells of cotyledons taken from ewes on days 40, 50, and 75 of gestation.
Studies performed in baboons during early pregnancy have also found IGF-1R mRNA localized to the glandular and luminal epithelia (48). In these species IGF-1R is thought to play a role in the decidualization process as well as possibly mediating the growth-promoting effects of the IGFs at the fetal maternal interface. In the sheep we believe that the IGF-1R mRNA localized to both the deep and superficial glands in conjunction with either locally produced or systemic IGF-I may facilitate glandular secretions contributing to the uterine milieu. The ovine conceptus is dependent on uterine secretions for nutritional support during preimplantation development, and histological evidence has shown that they are capable of pinocytotic capture of such proteins (49). The increase in the IGF-1R mRNA concentration in the deep glands from day 34 onward coincides with the onset of ovine uterine milk protein secretion, which begins around day 30, and thus supports a possible role for this receptor and IGF-I in this process (50).
IGFBP-2 mRNA appeared exclusively in the dense caruncular-like stroma lining the luminal epithelium from day 29 of gestation onward. Synthesis at this time could be initiated by the rising levels of IGF-II in the adjacent placentome region and might serve as a barrier blocking the transport of IGF-II into the endometrium. These findings reflect those of previous reports in which IGFBP-2 mRNA was found in close proximity to IGF-II in tissues that were rapidly proliferating (28). Previous studies in the rat using Northern blot analysis also failed to detect any IGFBP-2 during the estrus cycle (15), and in pregnancy, IGFBP-2 gene expression was barely detectable during early placental development, but increased significantly toward term (6). In contrast, IGFBP-2 mRNA was found in the endometrium of cyclic gilts, although levels were significantly higher during pregnancy (16). In the bovine endometrium, IGFBP-2 mRNA levels were high between days 10–18 of both the estrous cycle and early pregnancy, with expression seemingly up-regulated by progesterone (51).
IGFBP-3 mRNA was intensely expressed in specific regions of the luminal epithelium during early pregnancy (days 13–15), suggesting that IGFBP-3 may play a role in ovine implantation. Unfortunately, this remains impossible to verify, as the exact location of the implantation sites was unknown. Alternatively, IGFBP-3 in this region could act to create a local reservoir of IGFs in the luminal fluid. IGFBP-3 has also been detected in the uteri of rats, humans, and baboons during early pregnancy, but its function is still unknown (17, 18, 52). IGFBP-3 mRNA expression was also intense in the maternal caruncular stroma, particularly after invasion by the fetal allantochorion. Photographic emulsions showed that the majority of the signal emanated from the blood vessel walls. This observation corroborates a previous study in which cultured bovine endothelial cells secreted IGFBP-2, -3, and -4 (53). Endothelial IGFBP-3 could assist transfer of the IGFs from the circulation to the target tissue.
IGFBP-4 mRNA was expressed intensely in the caruncular capsule and the dense caruncular-like stroma lining the luminal epithelium. A previous study in the mouse also located IGFBP-4 mRNA in the stroma underlying the luminal epithelium, thus corroborating our findings (54). In mice, there was also intense IGFBP-4 expression in the areas adjacent to implantation sites, so IGFBP-4 has been suggested to play a physiological role in the implantation process. In the ovine uterus, because IGFBP-4 mRNA expression was uniformly distributed throughout the endometrium, a similar role seems unlikely. IGFBP-4 will bind IGF-II, so its location in the caruncular capsule may instead have an inhibitory action to prevent the fetal IGF-II, which concentrates at the tips of the invading villi, leaching out into the endometrium via the placentome region. IGFBP-4 has been shown to inhibit the actions of the IGFs in other systems; for example, it will block IGF-stimulated cell proliferation in chicken bone cells (55) and rat neuronal cells (56).
We have to date been unable to study the remaining binding proteins (IGFBP-1, -5, and -6) because of a lack of suitable reagents for the ewe. However, work in other species, particularly in primates, has shown IGFBP-1 to be the most abundant binding protein present during early pregnancy (17, 18). In baboons, IGFBP-1 is synthesized by both the glandular epithelium and stromal cells and is thought to facilitate trophoblast penetration at the maternal interface (48).
In summary, our data have shown intense IGF-II expression by the fetal mesoderm during a period of rapid and prolific growth of the ovine placentomes. Furthermore, it has demonstrated for the first time the loss of IGF-1R mRNA from the placentome region at this time. Uterine IGF-I mRNA expression is down-regulated in the first half of pregnancy. Increased expression of IGFBP-3 mRNA in the luminal epithelium on days 13–15 suggests a role in the implantation process. Local production of IGFBP-2, -3, and -4 mRNA by the maternal caruncular stroma suggests that the binding proteins are involved in organizing and controlling the growth of the invading fetal villi into the placentomes.
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Acknowledgments |
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Footnotes |
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Received August 1, 1996.
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