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Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903
Address all correspondence and requests for reprints to: Dr. Carol A. Bagnell, Department of Animal Sciences, P.O. Box 231, Rutgers University, New Brunswick, New Jersey 08903. E-mail: bagnell{at}aesop.rutgers.edu
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Introduction |
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In the pig, the uterine IGF system is important for implantation and uterine and conceptus growth during pregnancy (8). IGF-I is present in the uterine flushes (UF) of pregnant, cyclic, and prepubertal animals (9, 10). In addition, IGF and IGF-binding protein (IGFBP) messenger RNAs (mRNAs) are differentially expressed in the pig uterus during pregnancy and the estrous cycle (10, 11). Elevated levels of uterine IGF-I mRNA and protein indicate the importance of IGF-I in promoting uterine growth during early pregnancy (8, 10). Additionally, the significance of uterine IGF-II and IGFBP-2 and -3 in later pregnancy is supported by the prevalence of their mRNAs after implantation (10, 11).
Regulation of the uterine IGF system by steroids is well established. In sexually mature, ovariectomized pigs, estradiol and progesterone increase uterine IGF-I protein and mRNA and decrease IGF-II mRNA. Expression of uterine IGFBP-2 mRNA is dependent on the ratio of systemic estrogen and progesterone (10, 11). In the human endometrium, estradiol and progesterone increase IGFBP-2 and -3 secretion in vitro and gene expression in vivo (12). In addition to steroids, relaxin stimulates uterine IGFBP production. Basal and progesterone-induced IGFBP-1 secretion, gene expression and promoter activity are enhanced by relaxin in cultured human endometrial stromal cells (13, 14, 15). Relaxin also increases the secretion of IGFBP-1 and a 24-kDa IGFBP from human decidual cells in vitro (16).
The hypothesis that the pig uterine IGF system mediates relaxin-induced uterine growth was tested in the present study by monitoring the effects of relaxin on uterine IGFs and IGFBPs, independent of estradiol, in a prepubertal gilt model (1).
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Materials and Methods |
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-globulin and normal
rabbit serum were obtained from Antibodies, Inc. (Davis, CA) and Vector
Laboratories (Burlingame, CA), respectively. The vector used to
transcribe a riboprobe that protects 210 bp in exons 1 and 3 of the
IGF-I gene, the class 1 probe as described by Weller et al.
(20), was a gift from Dr. R. S. Gilmour (AFRC Babraham Institute,
Cambridge, MA). A rat IGF-II complementary DNA (cDNA) clone (21) was
provided by Dr. M. M. Rechler (NIH, Bethesda, MD). The RPA II kit, 18S
ribosomal RNA template, Century RNA Markers, and ribonuclease-free
deoxyribonuclease were purchased from Ambion (Austin, TX). Polyclonal
antihuman IGFBP-1 and anti-bovine IGFBP-2 antisera were obtained from
Upstate Biotechnology (Lake Placid, NY), and protein A-Sepharose was
purchased from Pharmacia Biotech (Piscataway, NJ). Human amniotic fluid
was donated by Dr. L. Sciorra (Robert Wood Johnson Medical School, New
Brunswick, NJ). Monotyrosylated relaxin was a gift from Dr. R. V.
Anthony (Colorado State University, Fort Collins, CO), and the rabbit
anti-porcine relaxin antibody (P5) was a gift from Dr. D. G. Porter
(University of Guelph). 17ß-Estradiol antibody and
[2,4,6,7-3H]estradiol were obtained from ICN Biomedicals
(Costa Mesa, CA) and Amersham Corp. (Arlington Heights, IL),
respectively. All other chemicals were purchased from Sigma Chemical
Co. (St. Louis, MO) unless otherwise specified.
Animal model and sample collection
Prepubertal (
115-day-old) Yorkshire-Landrace gilts (Swine
Unit of the New Jersey Agricultural Experiment Station, Rutgers
University, New Brunswick, NJ) were injected im with 0.5 mg porcine
relaxin or saline every 6 h for 54 h (1). Plasma was
collected 1) from all animals before and after the entire 54-h
treatment regimen and 2) hourly during one 6-h treatment period from an
indwelling jugular catheter of one control and one relaxin-treated
animal. Three hours after the last injection, animals were killed by
exsanguination after stunning. This animal experimental protocol was
reviewed and approved by the Rutgers University animal care advisory
committee. Uteri were removed, separated from the cervices, trimmed of
fat, and weighed. Each uterine horn was flushed with 20 ml PBS to
obtain UF, which were collected on ice and stored at -20 C. Uterine
horns were then cut into 1-cm pieces, frozen in liquid nitrogen, and
stored at -80 C. Ovaries were weighed, and follicular fluid (FF) was
aspirated from morphologically healthy follicles. Protein in uterine
and ovarian tissue homogenates (22) and UF was determined using the
Bio-Rad protein assay (Bio-Rad, Hercules, CA). To determine whether
relaxin alters IGF-I receptor binding, uterine membrane fractions were
isolated and processed as described by Hofig et al. (23),
and the percentage of maximum binding of IGF-I to its receptor was
assessed in receptor-ligand binding experiments.
RIAs
Before measuring IGFs by RIA, IGFBPs were removed by Sep-Pak
(Millipore Corp., Milford, MA) extraction (24) with modifications (7).
To validate this extraction procedure, preliminary experiments were
performed in which IGFBPs were removed from 2- and 4-ml aliquots of the
same sample. When the IGF values were compared between these samples,
there was twice as much IGF-I from the 4-ml aliquot as in the 2-ml
aliquot, as would be expected if the extraction method removed the
majority of the IGFBPs from the samples. Lee et al. (24)
report that this method removes approximately 80% of the binding
proteins from serum samples. In addition, we determined an IGF recovery
of 75% using this method to extract IGF-I from culture medium (7).
Although IGF-I was measured as previously described (7), IGF-II was detected with an IGF-II antibody (GroPep; final concentration, 1:7,500) incubated with [125I]IGF-II (15,000 cpm/tube) overnight at 4 C. Antibody-antigen complexes were precipitated by conventional double-antibody methods. All samples for IGF-II were measured in a single RIA. Interassay variation was 7.2 ± 2.8% for IGF-I, and intraassay variation was 4.2 ± 0.3% and 5.8 ± 0.3% for IGF-I and IGF-II, respectively. The minimum detection limits for the IGF-I and IGF-II assays were 5 and 80 pg/tube, respectively. Monotyrosylated relaxin was iodinated with Iodogen according to the method of Hall et al. (4) and was used in one assay to measure relaxin in plasma, FF, and UF according to the method of Afele et al. (25) with modifications. Samples were serially diluted in 0.05 M barbitone buffer containing 0.5% BSA and incubated with a polyclonal relaxin antibody (P5; final concentration, 1:30,000) overnight at 4 C. [125I]Relaxin (12,000 cpm) was added for an additional 48 h at 4 C. Bound and free hormone were separated by conventional double-antibody procedures. Intraassay variation was 7.2 ± 1.1%, and assay sensitivity was 22 pg/ml. 17ß-Estradiol was measured after extraction from plasma and UF with an ethyl acetate-hexane mixture and was determined in FF without extraction (26). Intra- and interassay variations were 4.5 ± 1.0% and 14.9 ± 0.1%, respectively, and assay sensitivity was 1.73 pg/ml.
Ligand blotting and immunoprecipitation
Ligand blotting was performed to monitor IGFBPs in UF, FF, and
plasma as previously described (7). Equal volumes of fluids were
examined to obtain a measure of IGFBP concentrations in the UF, FF, and
plasma. Before ligand blotting, UF were concentrated 4-fold with
Centriprep-10 devices (Amicon, Beverly, MA). Densitometry was used to
quantify IGFBP levels for statistical analysis. The identity of an
IGFBP doublet (33 and 34.5 kDa) in the UF was determined by
immunoprecipitation. Protein A-Sepharose (50 µl) was preincubated
with 50 µl concentrated UF in 450 µl immunoprecipitation buffer (20
mM HEPES, 150 mM NaCl, 1.5 MgCl2
mM, 1 mM EGTA, 10% glycerol, 1% Triton-X, 100
µg/ml phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin) for 30
min at 4 C. The supernatant was subsequently incubated with an IGFBP-1
or -2 antibody for 2 h at 4 C, after which protein A-Sepharose (40
µl) was added and incubated overnight at 4 C. Sepharose beads were
washed twice with immunoprecipitation buffer, and loading buffer was
added to the beads. After boiling, the beads were pelleted, and the
supernatant was subjected to ligand blotting. Human amniotic fluid and
porcine FF, which contain IGFBP-1 and -2, respectively (27), were used
as positive controls to check IGFBP antibody specificity.
Dephosphorylation experiments were performed to determine whether the
presence of the doublet was the result of IGFBP phosphorylation. Before
ligand blotting, immunoprecipitated UF were incubated with alkaline
phosphatase (15 U; Boehringer Mannheim, Indianapolis, IN) for 3 h
at 37 C in the presence or absence of alkaline phosphatase inhibitors
(28).
Ribonuclease protection assay and Northern analysis
Total RNA was isolated from uterine tissue as previously
described (22). The quantity and purity of the RNA were assessed
spectrophotometrically. Expression of IGF-I mRNA was monitored using a
ribonuclease protection assay (RPA) with a riboprobe complementary to
210 bp in exons 1 and 3 of the IGF-I gene (class 1) (20). An 18S
ribosomal RNA (rRNA) probe was used to correct for equal loading. The
RNA probes were transcribed using the Gemini System from Promega
(Madison, WI) according to the manufacturer’s directions. The reaction
consisted of 1 x transcription buffer (40 mM Tris
HCl, pH 7.5; 30 mM MgCl2; 10 mM
spermidine; 10 mM NaCl); 50 mM dithiothreitol;
40 U RNAsin; 500 µM each of ATP, CTP, and GTP; 6
µM UTP; 1 µg linearized plasmid template; 50 µCi
[32P]UTP (800 µCi/nmol); and 20 U T7 polymerase and was
incubated for 1 h at 37 C. As 18S rRNA is expressed much higher
relative to IGF-I RNA, an 18S rRNA probe of lower specific activity was
synthesized with 0.5 µCi [32P]UTP and 500
µM cold UTP in the above reaction. Subsequently,
riboprobes were treated with ribonuclease-free deoxyribonuclease (1 U)
for 15 min at 37 C and purified on an 8 M urea-5%
polyacrylamide gel.
The RPA was performed with the RPA II kit according to the manufacturer’s directions. Specifically, total RNA (10 µg) was coprecipitated with 32P-labeled IGF-I (50,000 cpm; 5 x 108 cpm/µg) and 18S rRNA (150,000 cpm; 5 x 105 cpm/µg) probes and dissolved in hybridization solution. The RNA was heat denatured and hybridized at 45 C for 18 h. Ribonuclease (1:50) was added to digest unprotected RNA for 30 min at 20 C. After heat denaturing, hybridized fragments were subjected to electrophoresis on an 8 M urea-5% polyacrylamide gel at 275 V for 2.5–3 h, and gels were exposed to Hyperfilm-MP (Amersham Corp., Arlington Heights, IL). RNA markers (100–500 bp) were used to determine the size (base pairs) of the protected fragments. To validate the assay, the IGF-I and 18S rRNA probes were hybridized with increasing amounts (2.5, 5, 10, 15, and 20 µg) of pig liver RNA. For Northern analysis, uterine RNAs were fractionated on a 1% agarose-formaldehyde gel and transferred to nylon membranes. IGF-II mRNA was detected with a 32P-labeled IGF-II rat cDNA probe (21) using prehybridization, hybridization, and low stringency wash techniques as described by Straus and Takemoto (29). Pig liver from 15-day-old animals and placenta were used as positive controls for IGF-II mRNA.
Statistics
Data are expressed as the mean ± SEM of
samples from control and relaxin-treated gilts using at least three
animals per group. Student’s t test was used to analyze the
data with the StatView 4.5 program (Abacus Concepts, Berkeley, CA).
P 
0.05 was accepted as significant.
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Results |
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To verify the uterotropic effects of relaxin in this model,
uterine wet weight, length, and protein were measured and were
significantly higher in relaxin-treated gilts than those in control
animals (P < 0.05; Table 1
). UF of
relaxin-treated animals also had significantly higher protein
concentration and content than those of control animals (Table 1).
Despite uterine growth, changes in ovarian wet weight or protein
content with relaxin administration were not evident (Table 1).
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). These increases in uterine lumen concentrations of
IGF-I and IGF-II were 2.5- and 8-fold above control, respectively.
Relaxin did not alter IGF-I gene expression in the uterus, as shown by
RPA (Fig. 1B). Validation of the RPA was demonstrated by a linear
increase in IGF-I and 18S ribosomal mRNAs when increasing amounts of
pig liver RNA were hybridized with the IGF-I and 18S rRNA probes (Fig. 1C). Unprotected complementary RNA probes for IGF-I and 18S rRNA were
270 and 116 bp, respectively, whereas the protected fragments were 210
and 80 bp, respectively. Northern analysis detected two IGF-II
transcripts (3.6 and 1.8 kb), which were not influenced by relaxin
treatment (data not shown). Plasma IGF-I and -II concentrations did not
differ when relaxin-treated animals were compared with control animals
or when IGF levels were compared before and after relaxin
administration within a treatment group (Fig. 2). In
addition, FF concentrations of IGF-I (control, 47.2 ± 5.9;
relaxin-treated, 43.3 ± 4.2 ng/ml) or IGF-II (control, 139.2
± 32.9; relaxin-treated, 123.2 ± 33.7 ng/ml) were not affected
by relaxin administration. Furthermore, relaxin did not influence the
percent maximum binding of IGF-I to the uterine IGF receptor (control,
19.73 ± 0.97%; relaxin-treated, 18.10 ± 1.18%).
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), as demonstrated by ligand blotting and
densitometry. Plasma and FF IGFBPs were unaffected by relaxin treatment
(data not shown). Figure 3B illustrates the IGFBP profiles of
representative FF, UF, and plasma samples. The 33-kDa band in the FF
and plasma lanes represents IGFBP-2, as previously described in FF and
plasma (24, 29). The bottom band of the relaxin-stimulated doublet (33
kDa) in the UF migrated similarly to IGFBP-2 in FF and plasma (Fig. 3B). Immunoprecipitation experiments, using a specific IGFBP-2
antibody, identified the doublet in the UF from control and
relaxin-treated gilts as IGFBP-2 (Fig. 4). Although
IGFBP-2 was immunoprecipitated as a doublet in the UF (Fig. 4, lanes 3
and 5; 33 and 34.5 kDa), the antibody precipitated a single band of
IGFBP-2 from FF (Fig. 4, lane 1; 33 kDa). No IGFBPs were precipitated
when normal rabbit serum was substituted for the specific IGFBP
antibodies (Fig. 4, lanes 2, 4, and 6). Although the IGFBP-1 antibody
immunoprecipitated IGFBP-1 from amniotic fluid, there was no evidence
for immunoreactive IGFBP-1 in either FF or UF (data not shown).
Dephosphorylation of the immunoprecipitated UF with alkaline
phosphatase did not reduce the IGFBP-2 doublet to a single band (data
not shown), indicating that uterine IGFBP-2 was not a doublet due to
phosphorylation.
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Discussion |
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Paracrine changes in the IGF system are pivotal in stimulating uterine growth during pregnancy in the pig (10, 11, 12). The concept that uterine growth is dependent upon stimulation of local growth factors is supported by several reports. For example, it has been suggested that growth factors mediate the trophic actions of estrogens on the uterus, as estrogen stimulates uterine production of IGFs (9, 30, 31) and their binding proteins (32). Likewise, we propose that the growth-promoting effects of relaxin on the uterus are mediated by local IGFs and IGFBPs. In addition to enhancing uterine growth in early pregnancy, uterine IGF-I is critical for implantation (8, 9, 10, 11). Therefore, factors that regulate uterine IGF-I will also impact on implantation and conceptus growth. For instance, conceptus-derived estrogens are reported to stimulate uterine IGF-I production and thus indirectly facilitate implantation (8, 9). Similarly, local production of relaxin by the pig endometrium around the time of implantation (33) together with the relaxin-induced increase in IGF-I secretion presented here support a role for relaxin in conceptus growth and in creating a suitable uterine environment for the establishment of pregnancy. In humans, relaxin may also be important around the time of implantation because it is detected in the plasma of women during the periimplantation period (34). In addition, relaxin alters the ultrastructure of human endometrial stromal cells during the progestin-induced differentiation that occurs with decidualization (35).
Given that estradiol has potent uterotropic effects in the pig and stimulates the uterine IGF system (9), it was important to rule out the possibility that endogenous estradiol was contributing to the observed effects of relaxin. Estradiol was not detected systemically, and FF estradiol levels in both control and relaxin-treated prepubertal gilts were at least an order of magnitude lower than those in cyclic animals (10–500 ng/ml) (36). These data suggest that the effects of relaxin on uterine growth and the IGF system reported here are independent of endogenous estradiol. This is consistent with the work of Hall et al. (4), who found that although estradiol enhanced the uterotropic effects of relaxin in the pig, it is not required for relaxin to promote growth of the uterus.
Whereas the effects of relaxin on IGFBP production in human endometrial and decidual cells are well documented (13, 14, 15, 16), the present report is the first to demonstrate an effect of relaxin on porcine uterine IGFBPs. The idea that IGFBPs modulate IGF activity in the uterus is supported by the fact that endometrial IGFBP-2 and -3 mRNAs are abundant when IGF-II mRNA levels are high at mid- to late pregnancy (10, 11). A similar temporal relationship between increased levels of IGF-II and IGFBP-2 are also found during pregnancy in human amniotic fluid (37). The evidence for regulation of uterine IGF-II and IGFBPs by relaxin reported here together with the high uterine IGF-II and IGFBP mRNA levels after implantation (10, 11) suggest coordinate actions of relaxin and the IGF system on uterine growth as pregnancy progresses. In addition, IGFBP-2 can act directly on uterine cells by binding to the cell membrane via its Arg-Gly-Asp (RGD) site (8, 11). In this fashion, IGFBP-2 can also potentiate IGF activity (reviewed in 38 .
In addition to the relaxin-induced increase in uterine IGFBP-2, we identified this IGFBP as a doublet in the UF by immunoprecipitation. This is the first report of IGFBP-2 as a doublet in the pig, as it is reported as a single band in porcine FF and plasma (24, 27). One possibility for the existence of an IGFBP-2 doublet is that the upper IGFBP-2 band (34.5 kDa) is a phosphorylated form of IGFBP-2. Phosphorylation of some of the IGFBPs, including IGFBP-2, has been reported (reviewed in 39 . However, our dephosphorylation experiments did not reduce the doublet to a single IGFBP-2 band, as would be expected if the binding protein was phosphorylated. In addition, glycosylation of IGFBP-2 is unlikely, as the IGFBP-2 cDNA sequence does not indicate any glycosylation sites on this binding protein (40). Furthermore, the appearance of the IGFBP-2 doublet in relaxin-treated animals cannot be explained by relaxin treatment because IGFBP-2 appears as a doublet in control animals. As the IGFBP-2 doublet is not a result of phosphorylation, as shown here, or glycosylation the reason for its presence is unknown. The absence of IGFBP-1 protein in the UF, as shown by immunoprecipitation experiments, is supported by the lack of IGFBP-1 transcript in the porcine endometrium (11).
Although we were able to demonstrate an increase in IGF-I and -II protein in the UF of relaxin-treated, prepubertal gilts, a simultaneous increase in uterine IGF-I and -II mRNA was not observed. A similar observation with IGF-I was made by Simmen et al. (9) in the unilaterally pregnant pig, when gravid and nongravid uterine horns were compared. Although gravid horns had higher IGF-I in UF than nongravid horns, there was no difference in endometrial IGF-I mRNA between the two horns. In contrast, steroidal regulation of IGF-I gene expression in the porcine uterus was highly correlated with IGF-I protein in UF of mature, ovariectomized gilts (9). The explanation for this difference in hormonal regulation of IGF-I secretion and mRNA in these animal models is not clear. However, the data presented here and for the unilaterally pregnant pig show that IGF-I secretion can be stimulated independent of IGF-I gene expression. The relaxin-induced enhancement of IGF protein in the absence of an increase in IGF-I and -II mRNA is probably due to translational regulation of IGF synthesis and/or an increase in IGF secretion by relaxin. Although storage of IGFs in secretory vesicles of the endometrial glandular epithelium has yet to be demonstrated, Geisert et al. (41) shows an exocytosis of these vesicles during early pregnancy. Secretory activity of the pig endometrium is stimulated by progesterone and conceptus-derived estrogens (41, 42, 43). Similarly, we found relaxin to significantly increase uterine lumen protein, indicating that relaxin also enhances the secretory activity of the porcine uterus. It is also possible that high levels of IGFBP-2 in the uterine lumen of relaxin-treated animals acted as a reservoir for the IGFs, extending the half-life of the protein during relaxin administration, resulting in higher IGF-I and -II concentrations in the UF of relaxin-treated gilts.
Given the influence of relaxin on uterine IGF-I and IGFBPs reported here, we were interested in determining whether relaxin had any influence on the uterine IGF-I receptor in this animal model. In the periimplantation pig uterus, Hofig et al. (23) found that relaxin did not interact with the uterine IGF-I receptor, however, an effect of relaxin on IGF-I binding to its receptor in the uterus has not been reported. The data presented here indicate that relaxin administration in vivo does not alter the percent maximum binding of IGF-I to its uterine receptor. Therefore, the interaction of relaxin and the IGF system in the porcine uterus involves IGFs and IGFBPs, and not the IGF-I receptor.
The data presented indicate that the effects of relaxin on the uterine
IGF system were local and independent of the endocrine IGF system, as
relaxin did not affect systemic IGFs and IGFBPs. Local uterine
production of IGFs and their binding proteins has been well documented
in the pig (8, 9, 10, 11). IGF transcripts are hormonally regulated and
differentially expressed in the porcine endometrium and myometrium
during the cycle and pregnancy (10, 11). Although IGF proteins have not
been localized in the pig endometrium to a particular cell type, it is
likely that IGFs are synthesized by the luminal epithelial cells and
then secreted into the uterine lumen. This is supported by the presence
of IGF-II mRNA in the endometrial glandular and epithelial cells in the
pig uterus at midpregnancy (11). In addition, IGFBP-2 protein has been
localized to both glandular and luminal epithelial cells of the porcine
endometrium (11). The possibility that the increase in uterine IGFs and
IGFBPs is due to diffusion from the plasma through the uterine
epithelial lining to the uterine lumen is unlikely for several reasons.
First, the percentage of plasma proteins that transfer to the uterine
lumen is low (7%) (44). Secondly, when electrophoresis or
chromatography patterns of uterine fluid proteins are compared with
those of serum or plasma of rats, humans, and pigs, strikingly
different protein profiles are observed (44, 45). Thirdly, the idea of
a blood-uterine lumen barrier has been proposed (46). In our study,
this barrier is clearly illustrated by the fact that relaxin
concentrations in the plasma ranged from 3.4–19.9 ng/ml, whereas those
in the UF were only 65 pg/ml. In contrast, in FF, which actively
exchanges substances with the blood, the relaxin concentration was 8.8
ng/ml, comparable to that in plasma. If a large amount of plasma
proteins diffused from the plasma to the uterine lumen, a much higher
concentration of relaxin would be expected in the uterine lumen.
Previously, we reported that relaxin stimulates granulosa cell IGF-I secretion in vitro (7). However, an effect of relaxin on the ovarian IGF system in vivo was not evident here. This was reflected in the absence of ovarian growth after relaxin administration in vivo in the face of a dramatic growth-promoting effect of relaxin on the uterus. We suspect that the lack of an in vivo effect of relaxin on the ovary in this prepubertal gilt model may be due to the high levels of relaxin achieved as a result of relaxin treatment. Although the circulating relaxin level was similar to that reported by Hall et al. (4), the systemic and local concentrations of relaxin measured here were 10-fold higher than those reported during the cycle (47, 48), when follicular growth occurs. In fact, plasma and follicular relaxin levels in the treated gilts were closer to those reported in pregnancy (47, 49), when the ovary is relatively quiescent. Furthermore, at higher concentrations in vitro, relaxin is less effective in promoting growth of porcine thecal cells (6) and inhibits growth of MCF-7 mammary cancer cells (50). Thus, the high local and systemic relaxin levels attained in this in vivo model, as in pregnancy, would not be expected to promote follicular growth or stimulate the ovarian IGF system. On the other hand, the uterus, during pregnancy and in the present study, is highly sensitive to the growth-promoting effects of relaxin. Collectively, these data suggest that the trophic effects of relaxin are tissue specific and concentration dependent and may be contingent upon the reproductive status of the animal.
In summary, the present study is the first to demonstrate an effect of relaxin on the porcine uterine IGF system in vivo. In granulosa cells in vitro, we established that the trophic effects of relaxin were dependent at least in part on IGF-I, as IGF-I immunoneutralization blocked relaxin-induced DNA synthesis (7). Although the relaxin-induced increases in uterine IGFs and IGFBPs suggest that the uterotropic effects of relaxin are mediated indirectly by the uterine IGF system, relaxin may also promote uterine growth independent of the IGF system. Recently, it has been suggested that relaxin elicits its uterine growth effects through specific relaxin-binding sites localized in the uterus by Min et al. (2). Therefore, future studies are needed to determine whether relaxin stimulates uterine growth directly through the relaxin receptor or by enhancing the production of other uterine growth factors.
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Acknowledgments |
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Footnotes |
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Received January 27, 1997.
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