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Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Dr. Daniel I. H. Linzer, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois 60208. E-mail: dlinzer{at}nwu.edu
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Introduction |
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The biological and physiological functions of most of these orphan hormones have not been identified. We have shown that the midpregnant mouse placenta secretes significant angiogenic activity that can be attributed primarily to PLF, and that later in pregnancy an antiangiogenic activity is released that corresponds to PRP (15). PLF appears to stimulate decidual neovascularization in the region of implantation (16), whereas PRP is predicted to be essential for preventing maternal and fetal vessels from growing across the junctional (basal) zone of the placenta. Although a PLF homolog has not yet been reported in other species, a hamster placental complementary DNA (cDNA) clone encoding a protein very similar to PRP has recently been characterized (17).
Given the wide range of actions of PRL and GH, and the already demonstrated activities for some of the orphan members of this family, it seemed important to us to establish which, if any, of these hormones discovered in the rat had homologs in the mouse. We, therefore, conducted a search of a mouse expressed sequence tag (EST) database for novel cDNAs in the PRL/GH family. We report here the characterization of three of these clones that are very closely related in amino acid sequence to members of this hormone family in the rat. In a second paper, we present our results on two other clones that encode novel members of the PRL/GH hormone family (18).
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Materials and Methods |
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Database screening and DNA sequence analysis
An Expressed Sequence Tag (EST) database available through the
National Center for Biotechnology Information (accessed at
http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-blast?Jform = 0) was
searched for sequences similar to mouse PRL-like cDNAs using BLAST.
GeneWorks (Intelligenetics, Mountain View, CA) was then used to compare
the partial cDNA sequences retrieved from the database to the cDNA
sequences of known members of the mouse PRL family. The cDNAs that
represented novel messenger RNAs (mRNAs) were obtained from Genome
Systems (St. Louis, MO); these are EST mo09e06.r1 (GenBank no. AA096890
and clone identification no. 553090), EST mo52c12.r1 (GenBank no.
AA110117 and clone identification no. 557206), and EST mp09c02.r1
(GenBank no. AA107907 and clone identification no. 568706). Plasmid
DNAs were purified from bacterial lysates by CsCl ultracentrifugation.
The sequences at the ends of the inserts were determined initially
using vector-specific oligonucleotides as primers. Internal primers
were then synthesized to obtain the remaining sequence. The major open
reading frame and predicted translation product were identified using
GeneWorks.
RNA filter and in situ hybridization
Tissues used for RNA extraction were collected from pregnant
female mice approximately 2 months of age; the tissues were rapidly
frozen on dry ice and stored at -80 C until use. RNA was isolated from
these mouse tissues by homogenization in Tri-Reagent (Sigma Chemical
Co., St. Louis, MO), chloroform extraction, and isopropanol
precipitation. For filter hybridizations, 20 µg total RNA/lane were
separated on 1% formaldehyde-agarose gels, transferred to nylon
membranes (Schleicher and Schuell, Keene, NH), and exposed to UV light
to cross-link the RNA to the membranes. Radiolabeled probes were
synthesized by random primer extension of cDNA inserts that had been
separated from the plasmid vector by restriction endonuclease digestion
and agarose gel electrophoresis. Radiolabeled products were purified
away from unincorporated nucleotides using MicroSpin S-300 HR columns
(Pharmacia, Piscataway, NJ). Hybridization was carried out for 12–16 h
at 42 C in 50% formamide, 5 x SSC (standard saline citrate),
0.5% SDS, and 5 x Denhardt’s solution (1x is 0.02% each BSA,
Ficoll, and polyvinylpyrrolidone). After hybridization, membranes were
rinsed in 2 x SSC once for 10 min and washed twice in 0.1 x
SSC-0.1% SDS at 50 C.
Tissues for in situ hybridization analysis were rapidly frozen before preparing 25-µm sections on a cryostat. Tissue sections were air-dried for 45 min at room temperature and fixed in 4% paraformaldehyde in PBS for 5 min. After incubating in 2 x SSC for 5 min, the sections were dehydrated through a series of 50%, 70%, 90%, and 100% ethanol. Digoxigenin-labeled riboprobes were synthesized using T7 or SP6 RNA polymerase in a reaction of 20 µl containing 2 µg linearized template DNA, 1 x transcription buffer (Promega, Madison, WI), 10 mM dithiothreitol, 1 µl ribonuclease inhibitor (40 U/µl), 500 µM NTPs (ATP, CTP, and GTP mix), 100 µM UTP, and 400 µM digoxigenin-11-UTP (Boehringer Mannheim, Indianapolis, IN) at 37 C (for T7 RNA polymerase) or 40 C (for SP6 RNA polymerase) for 90 min. Template DNA was removed by adding 1 µl RQ1 deoxyribonuclease (Promega) and incubating at 37 C for 10 min. Probes were isolated by ethanol precipitation, and the labeling efficiency was determined by dot blot, followed by detection with alkaline phosphatase-conjugated digoxigenin antibody (Boehringer Mannheim).
In situ hybridization was performed in a humidified chamber at 47 C for 12–16 h in a solution containing 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 x Denhardt’s solution, 10% dextran sulfate, 10 mM dithiothreitol, 0.5 mg/ml yeast transfer RNA, 0.5 mg/ml polyA, and 10 ng/ml denatured riboprobe. Slides were rinsed twice in 2 x SSC at room temperature followed by 30 min washes in 1, 0.5, and 0.1 x SSC at 65 C; slides were then incubated in blocking buffer (2 x SSC, 0.05% Triton X-100, and 0.1% BSA) at 30 C for 1 h. Alkaline phosphatase-conjugated antibody against digoxigenin was diluted 1:500 in blocking buffer and added to the tissue sections for 1 h at 37 C. Slides were washed for 10 min each in 100 mM Tris-HCl (pH 7.5) and 150 mM NaCl, and then in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2, and the chromogenic reaction was performed in the dark at room temperature in the latter solution containing 250 µg/ml nitro blue tetrazolium and 225 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate (Sigma). Slides were either counterstained with hematoxylin or directly mounted for microscopy.
Yeast artificial chromosome isolation and analysis
A yeast artificial chromosome (YAC) library (19) of mouse
genomic DNA fragments was obtained from the Imperial Cancer Research
Fund (London, UK) and screened with a mixture of radiolabeled mouse
PRL, PL-I, PL-II, PLF, and PRP cDNAs. The sizes of the genomic inserts
from positive clones were determined by pulsed field gel
electrophoresis and Southern blot hybridization. YAC-899 contained a
700-kilobase (kb) insert that hybridizes to the PRL, PL-I, PL-II, and
PRP cDNAs, but not to the PLF cDNA; YAC-121 had a 1000-kb insert that
hybridizes to the PLF cDNA, but not to the other cDNAs in this family.
Subsequent hybridizations were to DNA dot blots prepared using a
96-well chamber (Schleicher and Schuell). Briefly, total yeast DNA was
purified by banding in a 10–50% sucrose gradient followed by
overnight dialysis against 10 mM Tris-HCl (pH 8.0) and 1
mM EDTA. Samples were prepared by heating 1.5 µg total
yeast DNA or 0.5 ng plasmid DNA in 0.2 M NaOH and 1
mM EDTA at 85 C for 15 min. The samples were subsequently
applied to a nylon membrane assembled in the vacuum chamber followed by
a wash with 0.2 M NaOH and exposure to UV light to
cross-link DNA to the membrane. Hybridization and wash conditions were
as described above for the RNA filter hybridization.
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Results |
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EST clone mo09e06.r1 encodes a translation product of 227 amino acids
(Fig. 1
). The first 31 amino acids are
predicted to comprise the secretion signal sequence, based on the known
site of signal sequence cleavage for rat PLP-A (20). The mature protein
of 196 residues, which contains a single site for N-linked
glycosylation at Asn144, displays 78% amino acid sequence
identity with rat PLP-A (Fig. 2) and much
less identity to any other mouse or rat family member (see Fig. 3A in
Ref.18). The mouse and rat PLP-A proteins, which are the same length,
also have the same locations of the single N-linked
glycosylation site and four cysteine residues (positions 101, 172, 189,
and 196).
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). A comparison of the predicted protein
to previously identified PRL-related hormones revealed 66% amino acid
sequence identity with rat PLP-B (Fig. 4). In contrast, this protein is 32%
identical to mouse PRL. One consensus sequence for N-linked
glycosylation is present at Asn28 in both the mouse protein
and rat PLP-B, and four cysteines are present in each protein at the
same positions (60, 176, 193, and 201).
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); the predicted cleavage site is
consistent with that found for rat d/tPRP (22). The secreted product of
211 amino acids is 64% identical to rat d/tPRP (Fig. 6) compared with only 22% identity to
mouse PRL. Both the rat and the mouse proteins, which are the same
length, have two sites for N-linked glycosylation at
Asn74 and Asn183, and both have cysteine
residues at positions 4, 11, 71, 187, 204, and 211. Based on the
expression pattern of this mRNA (see below), we currently refer to this
mouse clone as decidual PRP (dPRP) rather than d/tPRP.
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), including pituitary (data not
shown). In this respect, the three mouse mRNAs and the corresponding
rat mRNAs are again similar. However, some striking differences are
detected between the mouse and the rat in the temporal pattern of
expression of these mRNAs. Mouse PLP-A mRNA is at a low level on day 8,
is abundant on days 10–14, and decreases by day 16 (Fig. 8). This pattern is very similar to that
for mouse PLF mRNA (7, 23), but is different from that for rat PLP-A
mRNA, which is first detected on day 14 (equivalent to about day 12 in
mouse gestation) and continues to increase in amount through day 18
(9).
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). Again, this temporal pattern contrasts
with that of the rat PLP-B mRNA, which is first detected on day 15
(equivalent to about day 13 in the mouse) and increases in amount
through day 18 (10). Another distinction between mouse and rat PLP-B is
that two PLP-B mRNAs that differ in length by approximately 300
nucleotides are present in the rat placenta in approximately equal
abundance (10); a small amount of a higher mol wt RNA that hybridizes
to mouse PLP-B is detected in some samples (Fig. 7), but typically only
a single mRNA species is seen (Fig. 8).
The amount of dPRP mRNA is high on days 8–12 and then decreases in the
latter part of gestation (Fig. 8). A similar pattern of d/tPRP
expression is seen in the pseudopregnant rat; d/tPRP mRNA accumulates
to high levels from days 9–12, then decreases (24). However, the
pattern of rat d/tPRP synthesis is complicated by the production of
this mRNA in placental trophoblast cells, with high levels of placental
d/tPRP transcripts detected from day 15 to term (25); a similar
high level of dPRP synthesis late in mouse pregnancy was not seen.
Cell type-specific expression of PLP-A, PLP-B, and dPRP
An in situ hybridization analysis of the PLP-A, PLP-B,
and dPRP mRNAs was carried out for days 10 and 12 of gestation, as peak
levels of each of these mRNAs were detected on these days. Mouse PLP-A
is expressed exclusively in placental trophoblast cells (Fig. 9, A and B), as has been found in the rat
(26). On day 10, this mRNA was only detected in giant cells, with some
of these cells strongly positive (Fig. 9A, arrow) and others
weakly positive. On day 12, two unique aspects of PLP-A expression
(compared with other related hormones in the mouse) were evident: the
junctional zone contained adjacent giant cells that were, respectively,
positive (downward arrow) and negative (upward
arrow) for PLP-A mRNA, and the positive cells were primarily
clustered around maternal blood sinuses (horizontal arrow).
Some smaller cells in this region also synthesized PLP-A; these cells
may be spongiotrophoblasts that have begun to differentiate into giant
cells, as very little PLP-A mRNA was seen in the spongiotrophoblast
layer beneath the giant cells.
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), with higher expression in the lateral regions compared
with the antimesometrial pole (data not shown); a few PLP-B-expressing
trophoblast cells were also seen scattered in the junctional zone (data
not shown). On day 12, though, cells synthesizing PLP-B were mostly in
the placenta (Fig. 9D) and not in the antimesometrial decidua (data not
shown). Placental expression at this time was restricted to the
spongiotrophoblast layer of the junctional zone and was not detected
within the trophoblast giant cells that form the boundary layer between
the maternal and extraembryonic compartments. Positive
spongiotrophoblasts formed columns that extended into the labyrinth
region and were adjacent to morphologically similar cells that did not
express PLP-B (Fig. 9D). The localization of PLP-B mRNA exclusively in
the junctional zone spongiotrophoblasts on day 12 in the mouse was also
seen in the rat on day 16 (26), and rat PLP-B has been reported to be
expressed in decidual tissue earlier in gestation (27).
In addition to synthesizing PLP-B, antimesometrial decidual cells on
day 10 of gestation synthesized dPRP (Fig. 9E), and expression remained
confined to these decidual cells on day 12 (Fig. 9F). In contrast, no
dPRP mRNA was detected in the mesometrial decidual cells (data not
shown). In the rat, a switch in synthesis of this hormone from maternal
decidual cells to embryo-derived trophoblast cells occurred during
pregnancy, hence the designation as d/tPRP (25). It is possible that
dPRP was also synthesized in trophoblast cells later in mouse
pregnancy, but if so, expression was below the level of detection in
these in situ hybridizations (data not shown). We,
therefore, propose to call this protein mouse dPRP rather than
d/tPRP.
Chromosomal mapping of PLP-B and dPRP
All of the genes in the PRL/GH family in the mouse, with the
exception of the GH gene, have been mapped to chromosome 13 at 14.0
centimorgans (28, 29). As a rapid means of mapping additional members
of this family, YACs containing this region of the mouse genome were
identified by screening a library for hybridization to the previously
identified family members. YAC-899 had an insert of 700 kb that
hybridizes to the PRL, PL-I, PL-II, and PRP cDNA clones (shown for PRP
in Fig. 10), whereas YAC-121, with an
insert of 1000 kb of mouse DNA, hybridized to the PLF cDNA [data not
shown, but note that the localization of the PLF genes to one end of
the gene family cluster is consistent with previous data (28)].
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). The PLP-A cDNA did not hybridize to either YAC, but both PLP-B and
dPRP cDNAs hybridized specifically to YAC-899 (Fig. 10). Thus, the
PLP-B and dPRP genes colocalize with most of the other family members
on chromosome 13. |
Discussion |
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Although all three of these mRNAs are restricted in their expression to decidual/placental tissue in both mouse and rat, some aspects of the expression of these three mRNAs are distinct between species; mouse PLP-A and PLP-B expression declines at a stage when rat PLP-A and PLP-B synthesis increases (9, 10), and synthesis of dPRP in the mouse apparently corresponds only to the decidual phase, not the later placental phase, of d/tPRP expression in the rat (25). Differences in the regulation of expression of PLP-A, PLP-B, and dPRP, and therefore, the times and concentrations at which these hormones appear during pregnancy, may contribute to the species-specific physiological effects of these hormones.
PLP-A expression is restricted to the extraembryonic trophoblast cells. An important feature of PLP-A synthesis is that the trophoblasts harboring the most PLP-A mRNA surround maternal blood sinuses. Thus, PLP-A may be a marker of a subclass of trophoblasts that have differentiated from morphologically indistinguishable neighboring trophoblasts. The uneven distribution of PLP-A expression in the junctional zone may result from the induction of PLP-A gene expression by a maternal factor, such that trophoblast cells in direct contact with maternal blood would be exposed to the highest levels of the putative inducer. The location of the PLP-A-expressing cells may also result in the more efficient delivery of PLP-A into the maternal circulation.
The most remarkable aspect of PLP-B synthesis is the switch from decidual cells early in gestation to trophoblasts later in pregnancy; this switch has also been reported to occur in the rat (27). As the d/tPRP gene undergoes a similar switch in expression in the rat (25), the change in hormone gene activity from a maternal to an extraembryonic site, therefore, appears to be a conserved theme in pregnancy. It is possible that mouse dPRP synthesis also moves to the placenta later in pregnancy, as it does in the rat, but we have not yet been able to detect such a transition. The PLP-B and dPRP genes both map to a 700-kb region on mouse chromosome 13 at 14.0 centimorgans, so it may be that they are sufficiently close to share regulatory elements involved in maternal/extraembryonic switching.
The timing of the decidual (day 10) to trophoblast (day 12) switch for PLP-B is also noteworthy. These stages of gestation also correspond to the placental switch from the synthesis of PL-I to PL-II (1), and from maximal secretion of the angiogenic hormone PLF to maximal secretion of the antiangiogenic hormone PRP (15). Thus, a number of genes in this family may be coordinately activated or inactivated by signals that reach the placenta during midgestation.
Even with the results reported here and in the accompanying paper (18), the roster of the mouse PRL/GH family has almost certainly not yet been completed. It is now reasonable to expect that mouse homologs to rat PLP-C, PLP-C-variant, and PLP-D will soon be found as well as additional hormones that have not yet been detected in either species. These genes should provide valuable markers for stages of placental and uterine development, and the identification of factors that control their expression may, therefore, reveal important regulators of the broader programs of trophoblast and decidual cell differentiation. The evolution of a large array of distinct PRL-like hormones that are expressed specifically in the decidua or placenta argues that these hormones provide significant advantages for reproduction and fetal development. A major challenge now will be to explain how the abundance and diversity of these hormones provide unique and overlapping functions in the regulation of pregnancy and fetal development.
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Acknowledgments |
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Footnotes |
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Received May 19, 1997.
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