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Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia
Address all correspondence and requests for reprints to: Dr. E. Marelyn Wintour, Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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The interpubic ligament does not relax during pregnancy in rlx-/- mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx-/- mice than in wild-type controls.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For many years, there were no pure preparations to establish tests for bioactivity and no sensitive homologous assays for measuring changes in plasma relaxin concentrations during pregnancy. When heterologous or homologous assays did become available for some species, it became clear that the patterns of relaxin secretion differed markedly between species. In the rat and mouse, the relaxin concentration is elevated only during the last half of pregnancy (3), whereas the human circulating concentrations of relaxin rise to a peak toward the end of the first trimester, then decrease to a plateau that is maintained for the rest of pregnancy (4).
The molecular structure of relaxin is known for many species, and several important points have emerged (5). The relaxins of all species in which it exists are peptides of between 43–64 amino acids, containing two chains, A and B, covalently linked by two disulfide bonds (the C peptide is trimmed off in the processing of prorelaxin to produce relaxin). There is remarkable heterogeneity in the relaxin family, with, in general, no more than 50% structural homology between relaxins of various species (5). Some ruminants (sheep and cow) do not express a functional relaxin gene (6, 7); however they do express a relaxin-like factor (Leydig cell insulin-like peptide) in ovarian cells.
In mice and rats, the corpus luteum is the major source of circulating relaxin during pregnancy (8, 9); however, relaxin expression has also been detected in several other tissues. In the rat, moderate levels of relaxin messenger RNA (mRNA) and protein have been detected in the uterus (10, 11), and relaxin gene expression has also been observed in the brain and prostate gland (10, 12). Binding sites for relaxin have been identified in tissues of the reproductive tract, such as the uterus (13), cervix (14, 15), mammary gland, and nipple (15, 16) as well as in the pubic symphysis (17), cardiac atrium, and brain (13, 18, 19). In the mammary gland the binding sites are located in the epithelial cells of the lobulo-alveolar elements as well as in smooth muscle and in epithelial cells of the skin of the nipple (15, 20).
The biological actions of relaxin in the pregnant rat have been characterized extensively. They include inhibition of myometrial contraction and connective tissue remodeling at target sites, including the pelvic ligaments and the cervix. Studies in which the source of circulating relaxin was removed by bilateral ovariectomy (2, 21) have indicated that relaxin in combination with estrogen is required for normal parturition in rats. In the absence of circulating relaxin, gestation and labor were prolonged or prevented, and fetal survival was reduced. Similar results have been obtained in more recent studies in which endogenous relaxin activity was neutralized by passive immunization (22). Additionally, in passively immunized rats, underdeveloped mammary glands and nipples resulted in poor lactation performance (23, 24), whereas direct administration of relaxin promoted nipple growth (16).
Relaxin treatment caused changes in fluid balance in nonpregnant rats (25). Synthetic relaxin has potent effects on isolated heart atria, increasing both the force and the rate of contraction (26). There is considerably less information on potential physiological roles of relaxin in other species.
To study the important roles of relaxin in the mouse we have investigated the effect of inactivation of the gene by homologous recombination in embryonic stem (ES) cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Production of animals homozygous for a mutant relaxin gene
We used homologous recombination in ES cells to disrupt the
murine relaxin gene. The mouse relaxin gene is a single copy gene
consisting of two exons encoding a B chain, a C peptide, and an A chain
(9). A targeting construct was made (in the cloning vector pBluescript
KS, (Stratagene, La Jolla, CA)) using a relaxin genomic
DNA fragment containing exons I and II. A 750-bp
EcoRI/BglII fragment was replaced with a neomycin
transferase gene (PGKNeo; Fig. 1
). This
deleted fragment encodes 90 C-terminal amino acids of the 103 amino
acids that make up the C peptide and 17 N-terminal amino acids of the
25 amino acids that comprise the A chain. Previous data indicate that
these regions are essential for the biological activity of relaxin
(5).
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and 2, a and b). DNA was also digested with
EcoRI and probed with the internal neomycin gene cDNA probe
to ensure that a single integration event had occurred (Figs. 1 and 2c).
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) was used for microinjection into 3.5-day-old
(C57BL/6J x C57BL/10ScSn)F1 blastocysts to generate
chimeras (29). Strong male chimeras in which a large number of ES cells
had become incorporated were mated to C57BL/6J females to produce
offspring that were heterozygous for the mutated allele. Heterozygous
animals were set up as breeding pairs to generate animals homozygous
for the altered allele. Heterozygous and homozygous mutant mice were
identified by Southern blot analysis and application of PCR (Fig. 3).
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DNA was isolated for PCR by lysing toe tissue in 200 µl PCR lysis buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 0.45% Nonidet P-40, and 0.45% Tween-20] with proteinase K (200 µg/ml) at 56 C overnight (28). Digested DNA (1.5 µl) was used as a template in a 25-µl reaction mix containing 2.5 nM of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 µM deoxy-NTPs, and 2.5 U Taq DNA polymerase (Perkin Elmer, Branchburg, NJ). Amplification of the samples was carried out using the following protocol: denaturation for 3 min at 94 C, followed by 40 cycles consisting of denaturing at 94 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 30 sec, and concluding with an additional 10-min extension at 72 C. Ten microliters of PCR reaction product were analyzed by electrophoresis on an agarose gel consisting of 2% SEAKEM HGT (high gelling temperature) and 2% LMT (low melting temperature) agarose (FMC BioProducts, Rockland, ME).
A 235-bp amplification product was generated from the wild-type
allele between relaxin primers P1 and P2, and a product of 170-bp was
generated from the mutant allele between primers P3 and P4 (Figs. 1 and 3).
Northern blot analysis
To confirm the presence or absence of relaxin mRNA, ovaries were
removed from 17.5 to 18.5 day-pregnant homozygous
(rlx-/-), heterozygous (rlx+/-),
and wild-type (rlx+/+) mice; rapidly frozen in
liquid nitrogen; and stored at -80 C for RNA extraction.
Total RNA was extracted using the acid-phenol single step method (30).
The RNA was transferred to a Hybond-N+ membrane (Amersham
International plc, Aylesbury, UK) and probed for relaxin mRNA with a
synthetic relaxin probe produced by annealing two 30-mer
oligonucleotides that overlap in the underlined region
(5'-TCACGGAAAAAGAGGGAGTCTGGTGGATTG-3' and
5'-TGGCAACATTGCTGGCTCATCAATCCACCA-3'). The annealed
oligonucleotides were end-filled using Klenow DNA polymerase (31) in
the presence of [
-32P]deoxy-CTP to generate a 50-bp
double-stranded probe [that corresponds to amino acids 134–150 at the
C peptide/A chain junction of mouse relaxin (9)] that was hybridized
to the filter in 5 x Denhardt’s, 5 x SSC (standard saline
citrate), 50% formamide (Merck, Darmstadt, Germany), 100 µg/ml
herring sperm DNA, and 1% SDS at 42 C overnight and washed in 2
x SSC, 0.1% SDS at 42 C three times for 15 min each time, then in
1 x SSC, 0.1% SDS at room temperature three times for 15 min
each time. The blot was exposed to BioMAX film (Eastman Kodak Co., Rochester, NY) for 24 h at -80 C with an intensifying
screen. The same filter was stripped (0.1 x SSC, 1% SDS, and 40
mM Tris-HCl, pH 7.8, at 100 C, 10 min) and reprobed with a
rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe (500 bp) as a
loading control, and the filter was exposed to BioMAX film
(Eastman Kodak Co.) for 18 h at -80 C without an
intensifying screen.
Specimen collection and analysis
Blood was taken (0.5–1.5 ml) by cardiac puncture into a
heparinized syringe under anesthesia with penthrane (methoxyflurane,
Abbott Laboratories, North Chicago, IL). Plasma
osmolality (Posmol) was measured using an Advanced Cryomatic Osmometer
(model 3C2, Advanced Instruments, Inc., Norwood, MA). Samples
were collected between 14.5–18.5 days of pregnancy from
rlx-/- mice (n = 22; 11 at 18.5 days; 6 at
15.5–17.5 days; 5 at 14.5 days), rlx+/+ mice
(n = 11; 5 at 18.5 days; 2 at 16.5–17.5 days; 4 at 14.5 days),
and rlx+/- mice (n = 10; 5 at 18.5 days; 5 at
14.5–15.5 days).
After blood collection, mice were killed with an overdose of penthrane; pubic symphyses were dissected clean, and the length of the pubic ligament was measured with a vernier caliper. Pubic symphyses were measured in virgin mice (n = 10); in full-term (day 18.5) wild-type (n = 5), heterozygotes (n = 6) and null-mutant mice (n = 11); as well as in day 14.5–16.5 wild-type (n = 6). Mammary tissues and nipples were removed and fixed in 4% paraformaldehyde (Pharmacia Biotech, Uppsala, Sweden) overnight. The right abdominal nipple and two associated mammary glands were collected from wild-type (+/+) and mutant (-/-) on gestation days 14.5 (n = 5) and 18.5 (n = 4) and postpartum day 2 (n = 4). They were then dehydrated, embedded in paraffin, and sectioned at 5 µm. Staining was with hematoxylin and eosin or by the Masson trichrome procedure (32).
Myoepithelial cells were demonstrated immunocytochemically in sectioned
mammary tissue using mouse monoclonal anti--smooth muscle actin
antibody (clone 1A4, Sigma Chemical Co., St. Louis, MO) at
a concentration of 1:400. Goat antimouse IgG (1:200; Dako Corp., Copenhagen, Denmark) was used as a secondary antibody.
Localization was performed with avidin and biotinylated horseradish
peroxidase/diaminobenzidine tetrahydrochloride (Dako Corp.). Sections were counterstained with hematoxylin.
One hundred sections were cut from each mammary gland. Every 10th section from each mammary gland was selected for morphometry using a Projectoscope (Olympus Corp., New Hyde Park, NY; Bh-2) and an autostage (Fauling Imaging, Mulgrave North, Australia). Fields were selected by setting the stage to make the slide move 1.5 mm/step on the y-axis and 2.5 mm/step on the x-axis. The tissue types were scored at all intersections of a 9-point grid. A minimum of 1000 points were scored per section. Alveoli, mammary duct, and adipose tissues were classified separately, whereas connective tissue and blood vessels were counted as "others."
Statistical analysis
Statistical data were analyzed using Microsoft Corp. (Redmond, WA). Exel software to calculate two-tailed
Student’s t test for unpaired data. All data in this paper
are presented as the mean ± SEM. P <
0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No full length relaxin mRNA expression was demonstrable on Northern
blotting from ovaries (embryonic days 17.5–18.5) of
rlx-/- mice (n = 2), whereas the level of
relaxin mRNA in rlx+/- mice (n = 2) was much less
than that in the wild-type (n = 2) animals (Fig. 4). The 1-kb band corresponds to the
mature mouse relaxin mRNA (9). A relaxin-hybridizing band at 4.5 kb is
the expected size for the relaxin precursor RNA containing the intron
sequence (9).
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Both male and female rlx-/- mice were fertile. Sixteen rlx-/- females were crossed with 3 rlx+/+, 3 rlx+/-, and 10 rlx-/- males, and all became pregnant, indicating that rlx-/- mice were fertile. The distribution of genotypes of 298 offspring of rlx+/- x rlx+/- matings followed the Mendelian ratio. Average litter sizes were 8.0 ± 0.7 (n = 22) from rlx+/+ mothers, 8.7 ± 0.4 (n = 33) from rlx-/- mothers, and 9.3 ± 0.4 (n = 40) from rlx+/- mothers; mean gestation length was 19.7 ± 0.26 days for rlx-/- mothers (n = 17) compared with 19.5 ± 0.20 days for rlx+/+ animals (n = 9). In a further experimental series, the delivery time (from the first pup to the last) was observed for 8 rlx-/- mice and 7 age-matched controls. For rlx+/+ animals, the average time was 1.7 ± 0.3 h. Six of the mutant animals delivered their young in 2.0 ± 0.3 h, but 2 were unable to deliver normally. One had a protracted labor lasting for 15 h, and the other had to be killed because she was in obvious distress having given birth to 2 dead pups some 12 h earlier. Ten further pups (fresh dead) were found in the uterus postmortem.
The rlx-/- mothers exhibited normal nursing behavior, but, with the exception of only one litter, no pups survived for more than 24 h. These pups were examined and found to have no milk in their stomachs. In the one rlx-/- mouse whose pups survived, the nipples were smaller than normal, but capable of delivering milk to the young. After 3 days of suckling the nipples had increased in size. The litter survived to weaning. The fact that the dam was indeed -/- was confirmed in three separate PCR reactions. This female mouse was mated a second time and was also able to feed the second litter.
Suckling of pups
Twelve newborn wild-type pups and 6 2-day-old wild-type pups were
fostered to 3 rlx-/- mothers; all of these
animals failed to suckle, and the pups were moribund within 24 h,
without milk in their stomachs. On the other hand, when 40
rlx-/- pups were fostered to 6
rlx+/+ mothers, 26 of them survived and grew to
maturity.
Histology of mammary glands
There was no difference in the histology of the mammary gland
between nonpregnant rlx-/- mice and their
wild-type littermates. Breast development is minimal, consisting of
sparse duct tissue embedded in connective tissue together with adipose
tissue. Alveoli are minimally developed and hardly recognizable by
light microscopy (results not shown).
At 14.5 days gestation, there was slightly less glandular tissue in
rlx-/- animals compared with that in
rlx+/+ mice, whereas the ducts were somewhat
dilated, but this did not reach statistical significance at the 0.05
level (Table 1).
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and
Fig. 5, A and B). There was much more
glandular tissue and less fat in the rlx+/+ than
in the rlx-/- animals, and the ducts of the
rlx-/- mice were grossly dilated compared with
those of rlx+/+ mothers. Secretion was observed
in the alveoli and ducts of both rlx+/+ and
rlx-/- mice. Mammary glands from heterozygotes
appeared similar to those from the wild-type mice, although
morphometric studies were not carried out in these animals.
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) animals remained very dilated.
The alveoli were in the early stage of involution (Fig. 5E) compared
with the alveoli of rlx+/+ mice (Fig. 5F). They
were often smaller and lined by flattened cells with heterochromatic
nuclei compared with the cubical cells and euchromatic nuclei seen in
normally lactating glands from wild-type mothers (Fig. 5F). The ducts
in rlx-/- animals (Fig. 5C) were even more
dilated than those at 18.5 days (Fig. 5A) and were lined by squamous
epithelial cells compared with the more cubical cells lining the ducts
of wild-type control animals.
Morphology of nipple
The nipples from rlx-/- mice 2 days after
delivery were much smaller than those from postpartum day 2
rlx+/+ animals (Fig. 6), but were similar to those of virgin
mice in both size and histology (data not shown). The pups were unable
to suckle from these or to stimulate milk let down, as shown by the
absence of milk in their stomachs. Nevertheless, the
rlx-/- mothers showed nursing behavior, and the
pups actively searched for the nipples. Even though the mammary glands
of the rlx-/- mice did not develop as well as
those of the rlx+/+ animals, secretion was
observed in the dilated ducts in sectioned material (Fig. 5, A and
C).
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More detailed histological observations were made on nipples stained
with Masson’s trichrome stain (Fig. 7).
In rlx+/+ animals, the epithelium of the skin was
greatly thickened and thrown into folds compared with that in virgin
animals, whereas the underlying connective tissue was much looser.
Neither of these changes had occurred in the
rlx-/- animals.
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We observed that in the rlx-/- animals (n
= 11) at full term, the symphysis (1.65 ± 0.22 mm) was wider
(P = 0.056) than that in virgin mice (n = 10;
1.17 ± 0.65 mm), but was much narrower (P <
0.01) than that in rlx+/+ animals (n = 5;
5.37 ± 0.64 mm) at full term. Symphyses from rlx+/-
mice (n = 6) at full term gave intermediate values (3.67 ±
0.52 mm) significantly different (P < 0.01) from those
of the rlx-/- animals. Figure 8 shows the very obvious difference
between the pubic ligament of a rlx-/- compared
with that of a rlx+/+ 18.5-day-pregnant
mouse.
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10 mosmol/kg water) lower than early
pregnancy values by day 14 (25). We therefore combined values for all
samples from 14.5–18.5 days gestation for statistical analysis. Posmol
in 22 samples from rlx-/- mice was 316 ±
1 mosmol/kg water, which was significantly greater (P
< 0.001) than the value from 11 wild-type mice (306 ± 2
mosmol/kg water). In 10 samples from heterozygote mice, the osmolality
was 311 ± 3 mosmol/kg water, an intermediate value, which was not
significantly different at the 0.05 level from that in either
null-mutant or wild-type mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bearing in mind published observations attesting to a role for relaxin in rodent parturition, one would have expected that delivery would be compromised in the null-mutant mice. In previous studies in which circulating relaxin was neutralized with a purified monoclonal antibody in pregnant rats, the delivery of normal litters was compromised (22, 34). The time from insemination to the onset of abdominal straining was not altered by the antibody treatment, but the duration of straining to the onset of delivery of the first pup was increased, as was the duration of litter delivery, wheras the number of live pups delivered was reduced. The dystocia was associated with a slower than normal decrease in plasma progesterone and small, inextensible cervices.
The small size of the nipple and decreased mammary gland epithelial tissue in rlx-/- mice can be explained in terms of the absence of connective tissue changes and the lack of smooth muscle relaxation, which are consequent upon changes in the level of circulating relaxin during pregnancy in wild-type mice (3). Relaxin has been shown to alter collagen metabolism in the cervix and interpubic ligament in mice and rats, decreasing total collagen content without altering the proportions of collagen types (35, 36). In cultured human dermal fibroblasts, relaxin treatment was able to decrease collagen synthesis and increase collagen degradation (37). Procollagenase mRNA and protein levels were increased by relaxin, and the level of tissue inhibitor of metalloproteinase was decreased somewhat. In vivo, in mice and rats, relaxin treatment decreased collagen accumulation and altered the organization of mesenchymal cells in two models of fibrosis (38). It has been shown that apoptosis of the alveolar epithelial cells of the mammary gland in mice depends on a balance between the activities of matrix metalloproteinases and their inhibitors (39) as well as on the interaction between components of the extracellular matrix and the ß1 integrin transmembrane receptor complex (40). In addition to well described changes in the pubic ligament (33), loosening of connective tissue also accompanies enlargement of the nipple. Similar connective tissue changes are apparent around the lactiferous ducts, and conceivably their absence together with continued high tone in associated smooth muscle result in impaired drainage and consequent dilatation of these ducts. All of these features together with the thickening and increased rugosity of the overlying skin observed in wild-type mice but absent in homozygous mutants correlate with the demonstration of relaxin-binding sites in these areas (15, 20). Normal expansion of the mammary parenchyma is also dependent to some extent on surrounding connective tissue and might thus be inhibited in the rlx-/- animals. Of course, relaxin might also act directly (as a growth factor) or indirectly (through the mediation of other endocrines) upon the mammary system, and this is the subject of further investigation in our laboratory.
In the pregnant rat during the second half of pregnancy, immunization with monoclonal antibodies to rat relaxin prevented the development of normal nipple morphology and the ability of the young to suckle efficiently (24). Relaxin-binding sites had been demonstrated in the epithelial cells of lobulo-alveolar elements of the mammary gland and in the smooth muscle epithelial cell and skin of the nipple of rats (15). Moreover, when porcine relaxin was administered sc to the left abdominal nipple of ovariectomized rats, it caused a significant increase in nipple length and wet weight (16).
The finding that Posmol was higher in null mutant mice than in wild-type animals over the last few days of pregnancy suggests that relaxin may exert the same effects in mice as those previously shown in rats (25). In the rat, Posmol decreases by about 10 mosmol/kg water between days 10–14 of pregnancy and remains lower until term due to a decrease in the threshold for water drinking and for plasma arginine vasopressin (PAVP) release, such that there is a resetting of the relationship between Posmol/PAVP, resulting in a decrease in Posmol without the normal suppression of AVP. These phenomena can be mimicked in ovariectomized rats by infusion of relaxin alone to produce normal late pregnancy plasma relaxin levels (25). The effect of pregnancy on the Posmol/PAVP relationship has not yet been determined in mice, but it is known that the ovarian relaxin content is increased from approximately day 11 of pregnancy (41), and plasma relaxin levels, measured with a heterologous assay, are also increased about this time (3). The lack of decrease in Posmol in the null mutant mice may reflect the absence of Posmol/PAVP resetting in the absence of circulating relaxin.
Many studies have been performed on mammary gland development (42, 43, 44). A number of genes, when deleted by gene targeting, give rise to abnormalities of mammary development and/or lactation. These include colony-stimulating factors I (CSF-I) (45), cyclin D1 (46), oxytocin (47), activin AB and inhibin ßB (48), the transcription factor STAT5a (49) (in the second messenger pathway for PRL, GH, and CSF-I), and a cell-surface receptor-like protein tyrosine phosphatase, leukocyte common antigen-related factor (LAR) (50). Although all mutant mothers have in common a failure to be able to nurse their young, the reasons why this occurs differ. In some cases, the branching morphogenesis that typically occurs in midpregnancy fails, and precocious lobulo-alveolar differentiation occurs (CSF-I-/-, ß-subunit of activin/inhibin-/-, and overexpression of WAP, whey acid protein). In others (cyclin D1-/- and PRL receptor+/-) (51), there is no lobulo-alveolar development and no lactogenesis. These are clearly different from the phenotype of the relaxin null-mutant mice. A decrease in the number and size of alveoli occurred in the LAR-/- mice, although the ducts were not dilated, as secretory vesicles accumulated in epithelial cells. There was also interstitial fibrosis in these mice; however, oxytocin injection allowed milk let-down and withdrawal. This has also been observed in rlx-/- mice in a preliminary trial. Thus, the LAR-/- mice, although not identical in phenotype to the relaxin-/- mice, most closely resembled them. It would be interesting to speculate that this tyrosine phosphatase receptor-like protein (LAR) might form one component of the mammary gland relaxin receptor. It has been reported recently that when fibroblasts from human lower uterine segments were cultured with various concentrations of human H-2 relaxin, there was an increasing tyrosine phosphorylation of a protein with a Mr of approximately 220 kDa in the fibroblast cells (52).
In conclusion, the results from the rlx-/- mice show some, but not all, of the features that could have been anticipated from the use of relaxin antibodies in the rat.
Apart from possible species differences, two plausible explanations for the diversity of results exist. First, it must be pointed out that the relaxin receptor has not yet been isolated and characterized, and consequently, its physiological activity in the presence of relaxin antibody cannot be assessed. Secondly, the creation of rlx-/- mutant mice could allow compensatory mechanisms to develop ab initio, thus producing a different physiological picture from that which might occur after acute abrogation of relaxin activity.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Medical Laboratory Science, RMIT
University, Melbourne, Victoria 3001, Australia.
3 Present address: Department of Biochemistry, University of
Leicester, University Road, Leicester, LEI7RH United Kingdom.
Received June 2, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Feng, N. V. Bogatcheva, A. A. Kamat, A. Truong, and A. I. Agoulnik Endocrine Effects of Relaxin Overexpression in Mice Endocrinology, January 1, 2006; 147(1): 407 - 414. [Abstract] [Full Text] [PDF]
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 C. S. Samuel Relaxin: Antifibrotic Properties and Effects in Models of Disease Clin. Med. Res., November 1, 2005; 3(4): 241 - 249. [Abstract] [Full Text] [PDF]
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 E. Dimitriadis, C. Stoikos, M. Baca, W. D. Fairlie, J. E. McCoubrie, and L. A. Salamonsen Relaxin and Prostaglandin E2 Regulate Interleukin 11 during Human Endometrial Stromal Cell Decidualization J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3458 - 3465. [Abstract] [Full Text] [PDF]
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 B. T. Nguyen and C. W. Dessauer Relaxin Stimulates Protein Kinase C {zeta} Translocation: Requirement for Cyclic Adenosine 3',5'-Monophosphate Production Mol. Endocrinol., April 1, 2005; 19(4): 1012 - 1023. [Abstract] [Full Text] [PDF]
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 A. H. Bogzil, R. Eardley, and N. Ashton Relaxin-induced changes in renal sodium excretion in the anesthetized male rat Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R322 - R328. [Abstract] [Full Text] [PDF]
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 E.S. Hayes, E.C. Curnow, A.O. Trounson, L.A. Danielson, and E.N. Unemori Implantation and Pregnancy Following In Vitro Fertilization and the Effect of Recombinant Human Relaxin Administration in Macaca fascicularis Biol Reprod, November 1, 2004; 71(5): 1591 - 1597. [Abstract] [Full Text] [PDF]
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A. A. Kamat, S. Feng, N. V. Bogatcheva, A. Truong, C. E. Bishop, and A. I. Agoulnik Genetic Targeting of Relaxin and Insulin-Like Factor 3 Receptors in Mice Endocrinology, October 1, 2004; 145(10): 4712 - 4720. [Abstract] [Full Text] [PDF]
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K. P. Conrad and J. Novak Emerging role of relaxin in renal and cardiovascular function Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R250 - R261. [Abstract] [Full Text] [PDF]
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 O. D. Sherwood Relaxin's Physiological Roles and Other Diverse Actions Endocr. Rev., April 1, 2004; 25(2): 205 - 234. [Abstract] [Full Text] [PDF]
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 M. A. M. Krajnc-Franken, A. J. M. van Disseldorp, J. E. Koenders, S. Mosselman, M. van Duin, and J. A. Gossen Impaired Nipple Development and Parturition in LGR7 Knockout Mice Mol. Cell. Biol., January 15, 2004; 24(2): 687 - 696. [Abstract] [Full Text] [PDF]
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A. L. Siebel, H. M. Gehring, I. G. T. Reytomas, and L. J. Parry Inhibition of Oxytocin Receptor and Estrogen Receptor-{alpha} Expression, But Not Relaxin Receptors (LGR7), in the Myometrium of Late Pregnant Relaxin Gene Knockout Mice Endocrinology, October 1, 2003; 144(10): 4272 - 4275. [Abstract] [Full Text] [PDF]
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 X.-J. Du, C. S Samuel, X.-M. Gao, L. Zhao, L. J Parry, and G. W Tregear Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype Cardiovasc Res, February 1, 2003; 57(2): 395 - 404. [Abstract] [Full Text] [PDF]
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 Y. Kubota, C. Temelcos, R.A.D. Bathgate, K.J. Smith, D. Scott, C. Zhao, and J.M. Hutson The role of insulin 3, testosterone, Mullerian inhibiting substance and relaxin in rat gubernacular growth Mol. Hum. Reprod., October 1, 2002; 8(10): 900 - 905. [Abstract] [Full Text] [PDF]
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 I. P. Gorlov, A. Kamat, N. V. Bogatcheva, E. Jones, D. J. Lamb, A. Truong, C. E. Bishop, K. McElreavey, and A. I. Agoulnik Mutations of the GREAT gene cause cryptorchidism Hum. Mol. Genet., September 15, 2002; 11(19): 2309 - 2318. [Abstract] [Full Text] [PDF]
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C. Binder, Th. Hagemann, B. Husen, M. Schulz, and A. Einspanier Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases Mol. Hum. Reprod., September 1, 2002; 8(9): 789 - 796. [Abstract] [Full Text] [PDF]
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 S. Y. Hsu, K. Nakabayashi, S. Nishi, J. Kumagai, M. Kudo, O. D. Sherwood, and A. J. W. Hsueh Activation of Orphan Receptors by the Hormone Relaxin Science, January 25, 2002; 295(5555): 671 - 674. [Abstract] [Full Text] [PDF]
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S. Hombach-Klonisch, S. Seeger, G. Tscheudschilsuren, J. Buchmann, B. Huppertz, G. Seliger, B. Fischer, and T. Klonisch Cellular localization of human relaxin-like factor in the cyclic endometrium and placenta Mol. Hum. Reprod., April 1, 2001; 7(4): 349 - 356. [Abstract] [Full Text] [PDF]
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L. A. Danielson, L. J. Kercher, and K. P. Conrad Impact of gender and endothelin on renal vasodilation and hyperfiltration induced by relaxin in conscious rats Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1298 - R1304. [Abstract] [Full Text] [PDF]
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 G. Gross, T. Imamura, and L. J. Muglia Gene Knockout Mice in the Study of Parturitions Reproductive Sciences, March 1, 2000; 7(2): 88 - 95. [Abstract] [PDF]
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L. Zhao, C. S. Samuel, G. W. Tregear, F. Beck, and E. M. Wintour Collagen Studies in Late Pregnant Relaxin Null Mice Biol Reprod, March 1, 2000; 63(3): 697 - 703. [Abstract] [Full Text]
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S. Y. Hsu Cloning of Two Novel Mammalian Paralogs of Relaxin/Insulin Family Proteins and Their Expression in Testis and Kidney Mol. Endocrinol., December 1, 1999; 13(12): 2163 - 2174. [Abstract] [Full Text]
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R. A. D. Bathgate, C. S. Samuel, T. C. D. Burazin, S. Layfield, A. A. Claasz, I. G. T. Reytomas, N. F. Dawson, C. Zhao, C. Bond, R. J. Summers, et al. Human Relaxin Gene 3 (H3) and the Equivalent Mouse Relaxin (M3) Gene. NOVEL MEMBERS OF THE RELAXIN PEPTIDE FAMILY J. Biol. Chem., January 4, 2002; 277(2): 1148 - 1157. [Abstract] [Full Text] [PDF]
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indicates the
BglII sites that were deleted when the construct was
made. The SacI restriction enzyme sites (S*) were used
for genomic Southern detection of the mutant ES cells and animals. The
loss of one of these sites in exon II resulted in an 8-kb
SacI fragment from the mutant allele, whereas a 4-kb
band was generated from the endogenous allele when the 1.3-kb relaxin
probe (a) external to the 3'-end of the targeting construct was used. A
352-bp neomycin cDNA probe (b) was used to check the number of
integration sites. Using this probe, a single 8-kb band (after
SacI digestion) or a 5-kb band (after
EcoRI digestion) will be detected if homologous
recombination has occurred. Bar = 1 kb.
