Inhibin Binding Protein in Rats: Alternative Transcripts and Regulation in the Pituitary across the Estrous Cycle
Daniel J. Bernard and
Teresa K. Woodruff
Department of Neurobiology and Physiology (D.J.B., T.K.W.)
Northwestern University and Department of Medicine
(T.K.W.) Northwestern University Medical School Evanston,
Illinois 60208-2850
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ABSTRACT
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Inhibin binding protein (InhBP) and the
transforming growth factor-ß (TGFß) type III receptor, betaglycan,
have been identified as putative inhibin coreceptors. Here we cloned
the InhBP cDNA in rats and predict that it encodes a large
membrane-spanning protein that is part of the Ig superfamily, as has
been described for humans. Two abundant InhBP transcripts (4.4 and 1.8
kb) were detected in the adult rat pituitary. The larger transcript
encodes the full-length protein while the 1.8-kb transcript
(InhBP-short or InhBP-S) corresponds to a splice variant of the
receptor. This truncated isoform contains only the N-terminal signal
peptide and first two (of 12) Ig-like domains observed in the
full-length InhBP (InhBP-long or InhBP-L). InhBP-S does not contain a
transmembrane domain and is predicted to be a soluble protein.
Betaglycan was also detected in the pituitary; however, it was most
abundant within the intermediate lobe. Although we also observed
betaglycan immunopositive cells in the anterior pituitary, they rarely
colocalized with FSHß-producing cells. We next examined physiological
regulation of the coreceptors across the rat estrous cycle. Like
circulating inhibin A and inhibin B levels, pituitary InhBP-L and
InhBP-S mRNA levels were dynamically regulated across the cycle and
were negatively correlated with serum FSH levels. Expression of both
forms of InhBP was also positively correlated with serum inhibin B, but
not inhibin A, levels. These data are particularly interesting in light
of our in vitro observations that InhBP may function as an
inhibin B-specific coreceptor. Pituitary betaglycan mRNA levels did not
fluctuate across the cycle nor did they correlate with serum FSH. These
observations, coupled with its pattern of expression within the
pituitary, indicate that betaglycan likely functions as more than
merely an inhibin coreceptor within the pituitary. A direct role for
InhBP or betaglycan in regulation of pituitary FSH by inhibin in
vivo has yet to be determined, but the demonstration of dynamic
regulation of pituitary InhBP and its negative relation to serum FSH
across the estrous cycle is an important step in this direction.
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INTRODUCTION
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The inhibins are produced in the gonads and act in an endocrine
manner to down-regulate pituitary FSH synthesis and secretion, but the
mechanisms underlying this regulation are not well understood. The
activins are functional antagonists of the inhibins and potently
stimulate pituitary FSH in a paracrine or autocrine fashion. Activins
act on target cells by binding two membrane-bound receptor
serine/threonine kinases, the activin type I and type II receptors.
Activins, which are homo- or heterodimers of two closely related
ß-subunits, bind the type II receptor (ActRII or ActRIIB). After
binding, the type II receptor recruits and transphosphorylates the type
I receptor (Alk4), which in turn stimulates Smad-dependent signal
transduction (1). Inhibins are produced through the heterodimeric
assembly of an
-subunit and one of the two ß-subunits they share
with activin. Inhibins can bind the activin type II receptors via the
ß-subunit, but this ligand-receptor interaction does not lead to
recruitment or phosphorylation of the type I receptor (2, 3, 4, 5, 6, 7). Based on
this observation, it has been argued that inhibins may act by competing
with activin for binding to the type II receptor. However, given that
activin has a higher affinity for the type II receptor, this mode of
antagonism would occur only in those contexts where inhibin levels
exceed those of activin.
Recently, two inhibin receptors or coreceptors were identified, raising
the possibility that different mechanisms of inhibin action may also
exist (7, 8, 9). Inhibin binds the transforming growth factor ß (TGFß)
type III receptor, betaglycan, with low affinity. However, in the
presence of the activin type II receptor, inhibin, betaglycan and the
type II receptor form a highly stable complex that is not disrupted by
activin (7). Thus, in those situations where betaglycan and the activin
type II receptor are coexpressed, inhibin can antagonize activin action
even at low concentrations. Although this mode of antagonism requires
the presence of an additional cell surface-binding protein, the
mechanism of antagonism is similar to that described above in which
activin signaling is abrogated through competition for the type II
receptor. A second, recently identified, receptor, known as
inhibin-binding protein (InhBP; formerly called p120; Ref. 10),
provides a novel mechanism for the antagonism of activin by inhibin (8, 11). Unlike the case for betaglycan, InhBP forms a complex with the
activin type I receptor, Alk4, but does so in a ligand-independent
manner. In the absence of inhibin, InhBP does not disrupt
activin-dependent signal transduction; however, in the presence of
InhBP, inhibin B, but not inhibin A, blocks activin-stimulated gene
transcription (11). These data suggest that InhBP may function as an
inhibin B-specific receptor.
Thus far, the majority of data indicating a role for both InhBP and
betaglycan in inhibin action has been gathered using artificial
in vitro model systems. To establish either or both proteins
as bona fide inhibin receptors, their function must be characterized
in vivo and in particular with respect to the effects of
inhibin on pituitary FSH release. Toward this end, we first
characterized the InhBP cDNA in rats. We next examined InhBP and
betaglycan expression in female rat pituitaries over the rat estrous
cycle, a period during which both inhibin A and B levels fluctuate
dramatically (12). We observed that InhBP, but not betaglycan, mRNA
levels varied over the estrous cycle and that they were negatively
correlated with circulating FSH levels. These data are consistent with
a role for pituitary InhBP in the regulation of FSH by inhibin
in vivo.
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RESULTS
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Cloning of the Rat InhBP cDNA
The full-length rat InhBP cDNA was cloned by a combination of cDNA
library screening, RT-PCR, and both 5'- and 3'-rapid amplification of
cDNA ends (RACE). The cDNA was determined to be 4,407 bp, containing
an open-reading frame (ORF) of 3,960 bp, flanked by 171 bp and
276 bp of 5'- and 3'-untranslated regions (UTRs), respectively (GenBank
accession no. AF322216). The ORF begins with tandem ATG codons, both of
which lie in an appropriate context for translation initiation (Fig. 1
; Ref. 13). There is an in-frame stop
codon 84 bp upstream of the first ATG in the 5'-UTR and a consensus
polyadenylation signal (AATAAA) 29 bp upstream of the poly A tail in
the 3'-UTR. The 5'-RACE procedure used [RNA ligase-mediated
(RLM)-RACE] to clone the 5'-end of the cDNA only amplifies capped RNA
species and therefore can be used to map transcription initiation
sites. Five putative transcription start sites were mapped -152,
-161, -162, -169, and -171 bp relative to the first start
codon.

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Figure 1. Alignment of the Human (GenBank Accession No.
Y10523) and Rat inhBP (GenBank Accession No. AF322216) cDNA Sequences
around the Predicted Start of Translation
The previously reported start codon for humans is
underlined (14 15 19 ). Note that this sequence does
not conform to the Kozak consensus sequence: GCC(A/G)CCATGG (13 ).
Notably absent is a purine in position -3. The next in-frame start
codon (boxed) conforms almost exactly to the consensus
sequence for translation initiation. This codon aligns with the second
of tandem ATG codons at the beginning of the rat InhBP ORF. We predict
this codon is used for translation initiation in both species.
Conserved nucleotides are shaded and predicted amino
acids are shown above (human) and below
(rat) the nucleotide sequences.
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The ORF is predicted to encode a protein of 1,320 amino acids with a
calculated molecular mass of 147.2 kDa. After cleavage of the
N-terminal signal peptide (encoded by the first 20 amino acids) the
molecular mass would be 144.9 kDa. The human InhBP cDNA (also known as
IGSF1 or IGDC1; Refs. 14, 15) was reported to encode a protein of
1,336 amino acids (14). The major difference between the two species
exists at the N termini of the molecules (Fig. 1
). The human protein
was predicted to have an additional 10 amino acids at its N terminus.
However, our analysis of the human sequence indicates that the putative
start of translation does not conform well to the Kozak consensus
sequence, unlike the ATG codon at the 12th amino acid position (Fig. 1
). This latter start codon, in fact, aligns to the second ATG of the
rat sequence and suggests that it may be used for translation
initiation in both species, which would yield proteins of 1,325 and
1,319 amino acids in humans and rats.
The other differences appear to arise from the use of different splice
sites in the two species. In humans, the InhBP cDNA is encoded by 19
exons (14) and the human nomenclature is used here. In about half of
all rat clones analyzed, we observed an additional 3 bp (CAG) at the
exon 3-exon 4 junction, which produces an in-frame alanine
(GCA; see Fig. 5
). The sequence deposited in GenBank
includes these additional 3 bp. The rat protein lacks the first nine
amino acids encoded by exon 8 in humans. This appears to result from
the use of an alternative 5'-splice acceptor site, which leads to an
in-frame deletion of 27 bp. The same deletion occurs in mouse and we
have confirmed the use of an alternative AG splice acceptor site. It is
worth noting that the AG used in humans is conserved in the mouse
sequence (D. J. Bernard and T. K. Woodruff, unpublished).
Finally, the rat cDNA sequence has an additional 6 bp at the beginning
of exon 9, which results in the in-frame addition of a valine and a
threonine residue.

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Figure 5. Nucleotide and Predicted Amino Acid Sequence of Rat
InhBP-S
The boxed amino acids correspond to the predicted
N-terminal signal peptide. The 5'-ends of the three clones used to
determine this sequence are indicated in bold within the
5'-untranslated sequence. The point at which the InhBP-S sequence
diverges from that of InhBP-L is indicated by an
arrowhead. Note that a GT, characteristic of a splice
donor site, appears at this point. An in-frame stop codon upstream of
the first ATG of the ORF is solid underlined. The
additional CAG observed in about half of all clones is denoted by a
dotted underline. The consensus polyadenylation signal
sequence is double underlined in the 3'-UTR. Nucleotides
are numbered at the left and amino acids at the
right.
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Overall, the rat and human amino acid sequences are 85% identical, and
the structure of the two molecules appears to be conserved. That is,
rat InhBP protein is predicted to consist of 12 C-type Ig-like domains,
organized in groups of five and seven domains separated by a
hydrophobic linker region. After the 12th Ig domain there is a
transmembrane domain followed by a short serine/threonine-rich
(
21%) cytoplasmic tail. Like the human protein, the rat
intracellular domain does not contain any known signaling motifs.
Interestingly, several Web-based analysis programs predict two
transmembrane domains within the hydrophobic linker region, but we have
not yet confirmed nor refuted this experimentally.
Expression of InhBP and Betaglycan in Rat Tissues
RNA blot analyses were used to examine both InhBP and
betaglycan mRNA expression in various rat tissues. A high level of
InhBP expression was observed in pituitary and testis total RNA (Fig. 2A
, left panel), while no or
little expression was detected in ovary, adrenal, or liver. Two major
InhBP mRNA species (
4.4 and 1.8 kb) were observed in rat pituitary
using a probe directed against the 5'-end of the rat cDNA. The 4.4-kb
transcript likely corresponds to the full-length InhBP described above,
while the 1.8-kb transcript may encode a truncated form of the receptor
(see below). In testis, two major transcripts were also detected. The
1.8-kb transcript appeared identical to that observed in pituitary. The
larger form, however, appeared smaller than the 4.4-kb transcript
detected in pituitary. We repeated this analysis with different
pituitary and testis RNA samples to ensure that the discrepancy did not
arise from aberrant migration of the RNAs in the original gel. The same
migration pattern was observed, indicating that there is a real
difference between the larger transcripts in testis (
3.7 kb) and
pituitary (
4.4 kb) (Fig. 2B
, left panel; data in
right panel described below). Interestingly, when these
blots were hybridized with probes from the middle and 3'-portions of
the full-length cDNA, the 4.4-kb band was still observed in the
pituitary but the 3.7-kb band was not detected in the testis lane (data
not shown). Thus, the 3.7-kb transcript observed in testis appears to
represent a form of InhBP containing some, but not all, of the 4.4-kb
sequence (see below). Larger transcripts (>6 kb) were also observed in
pituitary and testis, but they have not yet been characterized.

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Figure 2. InhBP and Betaglycan Expression in Rat
Tissues
A, RNA blots show InhBP (left panel) and betaglycan
(right panel) mRNA expression in various rat tissues.
Fifteen micrograms of total RNA derived from the indicated tissues were
hybridized with 32P-labeled cDNA probes from the 5'-end of
InhBP (corresponding to nucleotides 172839 of the full-length rat
cDNA; see Fig. 7 ) or the ApaISacI
fragment of the rat betaglycan cDNA (GenBank accession no. M77809 and
Ref. 31). B, Rat pituitary and testis RNA were hybridized with the same
probe used in A (left panel) or a PCR-generated probe
from intron 5 (which corresponds to part of the 3'-UTR of InhBP-S, see
text). Note that the RNA samples in A (left panel) and B
were derived from different animals and the gel in B was run longer to
better distinguish the difference in size of the larger molecular
weight transcripts. Molecular size markers (in kilobases) are
indicated.
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We next examined the distribution of InhBP mRNA within the pituitary
using in situ hybridization histochemistry (Fig. 3
). Like FSHß mRNA (Fig. 3A
), InhBP
mRNA was detected exclusively within the anterior pituitary (Fig. 3
, B
and C). However, the pattern of labeling differed between the two
probes. For FSHß, a high density of silver grains was clustered over
many, but not all, cells within the anterior pituitary. This pattern is
consistent with previous estimates of the proportion of anterior
pituitary cells producing FSH. With the InhBP antisense probe, silver
grains were observed in a consistent, diffuse pattern across the
majority of cells within the anterior pituitary. No labeling was
observed in adjacent sections hybridized with an InhBP sense riboprobe
(data not shown). These data suggest that InhBP is produced in many
cell types within the rat anterior pituitary, including
gonadotropes.

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Figure 3. In Situ Hybridization Histochemistry
Was Used to Localize FSHß and InhBP mRNA in the Rat Pituitary Gland
The dark-field images presented in panels A and B show adjacent
sections hybridized with antisense 33P-UTP-labeled
riboprobes directed against FSHß or InhBP, respectively. For both
probes, the highest density of silver grains is observed within the
anterior pituitary with no or little labeling in the intermediate or
posterior lobes. Scale bar in panel B = 500 µm
and also applies to A. C, Higher magnification of the image shown in
panel B, centered at the border of the anterior and intermediate lobes.
Note the greater silver grain density in the anterior lobe. D,
Bright-field view of the image pictured in panel C. Scale
bar in panel D = 50 µm and also applies to
C. P, Posterior, i, intermediate, and a, anterior lobes
of the pituitary.
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Betaglycan was detected in the majority of tissues examined (Fig. 2A
, right panel). The highest level of expression was observed
in the adrenal gland and ovary, but importantly betaglycan was
expressed at detectable levels in the pituitary. We also observed a
second, smaller transcript in testis (
2.7 kb) and a larger
transcript in adrenal (>7.5 kb). These transcripts have not yet been
characterized, nor have they been described in the literature. We next
localized betaglycan protein within the pituitary using
immunofluorescence techniques and examined its distribution relative to
that of FSHß (Fig. 4
).
FSHß-immunoreactive cells were detected exclusively within the
anterior pituitary (Fig. 4
, A and C). The highest level of betaglycan
immunoreactivity was observed in the intermediate lobe (Fig. 4B
), but
betaglycan-positive cells were also detected in the anterior lobe (Fig. 4D
). Double-immunofluorescence analyses indicated that betaglycan was
expressed near, but rarely in, FSHß-positive cells within the
anterior lobe (Fig. 4E
). No double-labeled cells are pictured in Fig. 4E
, and this was typical of the majority of fields examined.

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Figure 4. FSHß and Betaglycan Were Localized in the Rat
Pituitary Using Immunofluorescence
A, FSHß (green) is expressed exclusively within the
anterior lobe. B, Betaglycan (red) is expressed most
highly in the intermediate lobe, while some labeled cells are also
detected in the anterior lobe. Nuclei are stained blue
with DAPI. FSHß (C) and betaglycan-positive (D) cells alone and
together (E) in the anterior pituitary are shown at higher
magnification. Note that FSHß (arrowhead) and
betaglycan (asterisk) containing cells are often in
close proximity, but double-labeled cells (not visible in this figure)
are rarely, if ever, observed. The scale bar in B = 200 µm
and also applies to A. The scale bar in E = 20 µm and also
applies to C and D. P, posterior, i, intermediate, and a, anterior
lobes of the pituitary.
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Characterization of the InhBP Splice Variants in Pituitary and
Testis
As described above, an abundant 1.8-kb mRNA InhBP transcript was
detected in both rat pituitary and testis. To characterize this
isoform, we first performed RNA blot analyses on testis RNA using
contiguous, nonoverlapping probes spanning the entirety of the ORF of
the full-length InhBP. Only those probes containing sequences encoding
the first five exons hybridized to the 1.8-kb transcript (data not
shown), suggesting that this isoform may represent a truncated form of
InhBP. We next used 5'-probes to screen a rat testis
ZAP cDNA
library. Three independent clones containing inserts of approximately
1.4 to 1.8 kb were isolated. The largest of the three clones contained
an insert of 1,745 bp (not including the poly A tail), with an ORF of
699 bp flanked by 169 and 877 bp of 5'- and 3'-untranslated sequences
(Fig. 5
). The three clones begin -59,
-161, and -169 bp relative to the first ATG. It is notable that the
-161 and -169 bp ends correspond exactly to two start sites mapped by
RLM-RACE (see above). A consensus polyadenylation signal is observed 17
bp upstream of the poly A tail in the 3'-UTR. The sequence of this
clone was identical to that of the full-length transcript from the
5'-end through the end of exon 5. Thereafter, the sequences
diverged.
Unlike the full-length cDNA, the 1.8-kb transcript appears to retain
part of the intron 5 sequence (Figs. 6
and 7
). We have not yet sequenced intron
5 in rats, but several pieces of data support this hypothesis. First,
at the point where the two sequences diverge, the first two nucleotides
of the 1.8-kb form are GT, which correspond to the consensus 3'-splice
donor site. Second, the point of divergence occurs precisely at the
exon 5/intron 5 boundary defined in humans (14). Third, 41 bp at the
5'-end of the human intron 5 have been reported. Thirty-seven of the
first 41 bp (90%) after the divergence point in the 1.8-kb isoform
match the human intron 5 sequence identically. After the exon 5-intron
5 boundary the sequence continues 59 bp before reaching an in-frame
stop codon. As a result, this transcript is predicted to encode a
233-amino acid protein with a molecular mass of 26.4 kDa. The predicted
protein shares the N-terminal signal sequence and first two Ig-like
domains with the full-length protein, but has 20 novel amino acids at
its C terminus. Interestingly, these novel amino acids are not
predicted to encode a transmembrane domain, nor do we believe the
protein is anchored to the membrane by a GPI moiety (16, 17, 18).
Therefore, we predict that the 1.8-kb transcript may encode a soluble
form of the protein, containing the first two Ig-like domains (Fig. 7
).
We refer to this form as InhBP-short (or InhBP-S; GenBank accession
no. AF322217) and now call the full-length form InhBP-long (or
InhBP-L).

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Figure 6. InhBP-L and InhBP-S Are Generated through
Alternative Splicing of Intron 5
In this schematic, InhBP-L and InhBP-S sequences are compared with the
human genomic sequence around intron 5 (14 ). The corresponding genomic
sequence is not yet available in rat. Exon sequences are
boxed and in uppercase, while intronic
sequences are pictured as white on a black background.
Nucleotides in the rat sequence that differ from those in the human
sequence are shaded in gray. The arrow
indicates where the available sequence at the 5'-end of human intron 5
ends. Predicted amino acids are displayed above
(InhBP-L) and below (InhBP-S) the nucleotide sequences.
In InhBP-L, intron 5 is spliced from the pre-mRNA in a manner
consistent with exon-intron organization described in humans. InhBP-S
appears to retain the intron 5 sequence. Note the high sequence
conservation between the human intron 5 sequence and the sequence in
InhBP-S after it diverges from that of InhBP-L. Retention of intron 5
produces 19 novel amino acids at the C terminus of InhBP-S before
terminating at an in-frame stop codon (*).
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Figure 7. Schematic Representation of the InhBP-L and InhBP-S
Proteins
As described in the text, InhBP-L contains an N-terminal signal peptide
(box with diagonal lines) followed by 12 Ig-like
domains, a transmembrane domain, and a short intracellular domain.
InhBP-S shares the signal peptide and first two Ig-like domains with
InhBP-L. The novel 19 amino acids at the C terminus of InhBP-S are
shown as a white box. Our model of how InhBP-S is
produced is shown below the schematic illustration of
the protein. The exon/intron structure and exon numbering for human
InhBP are shown at the bottom (adapted from Ref. 14).
Exons are depicted as open boxes and introns are drawn
as horizontal lines (a scale bar is shown
below). The shaded region after exon 5 represents the
intron 5 sequence that is retained in InhBP-S, but not in InhBP-L. In
the mature mRNA, exon 1 and part of exon 2 encode the 5'-UTR
(stippled box at left). The remainder of exon 2 and exon
3 encodes the N-terminal signal peptide (box with diagonal
lines). Exons 4 and 5 encode Ig-like domains 1 and 2,
respectively (black boxes). The C-terminal 19 amino
acids (white box) and 3'-UTR (right stippled
box) are encoded by the intron 5 sequence retained within this
transcript. The dotted line indicates where exon 5
sequence terminates in InhBP-L. The location of the 5'- and intron 5
probes used in the RNA blot analyses (Fig. 2 ) are shown.
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We used a probe derived from intron 5 (which corresponds to part of the
3'- UTR of InhBP-S) in a RNA blot analysis of rat pituitary and testis
RNA (Fig. 2B
, right panel; see also Fig. 7
). As predicted,
the 1.8-kb transcript was detected in both tissues. Interestingly,
while the 4.4-kb transcript was not detected in the pituitary, the
3.7-kb transcript was observed in testis (and in pituitary, although at
lower levels). We have not yet characterized this transcript in its
entirety; however, RNA blot analyses suggest that it encodes a form of
InhBP similar to InhBP-S at its 3'-end, but contains additional
5'-sequence. That is, probes downstream of exon 5 (other than the
intron 5 probe) fail to detect the 3.7- or 1.8-kb transcripts (data not
shown). We predict that the 3.7-kb isoform has about 2 kb of
5'-sequence that it shares neither with InhBP-L nor InhBP-S. In humans,
three InhBP sequences have been deposited in GenBank. One of these
sequences (GenBank accession no. AB002362; Ref. 19) contains an
additional 1 kb of 5'-UTR relative to the other two sequences (Genbank
accession no. Y10523 and AF034198). Thus, an alternative promoter
may drive transcription initiation from an upstream site, and this
promoter appears to be more active in testis than in any of the other
rat tissues examined to this point. Nonetheless, given the in-frame
stop codon upstream of the ATG in InhBP-S (Fig. 5
), the 3.7-kb
transcript is predicted to contain an ORF identical to that of InhBP-S.
We are currently rescreening the testis cDNA library to determine the
complete sequence of this transcript.
Pituitary InhBP Regulation across the Rat Estrous Cycle
We used RNA blot analyses to examine pituitary mRNA levels of
InhBP-S, InhBP-L, and betaglycan across the rat estrous cycle.
Pituitaries were collected at seven stages of the cycle (n = 3 per
group): at 1000 h on metestrus, diestrus, proestrus, and estrus;
at 1800 and 2400 h on proestrus; and at 0400 h on estrus. The
rats showed the typical changes in serum hormone levels across the
cycle (Fig. 8
). The only notable
exception was the lack of a large primary FSH surge on the afternoon of
proestrus. While the levels at this time were slightly higher than
observed on proestrous morning, they were below the levels attained
during the secondary FSH surge on early estrous morning (Fig. 8A
).
Previously, we observed a decline in serum inhibin B levels from the
morning to the afternoon of proestrus (12). Here, however, inhibin B
levels did not decline until after the LH surge (Fig. 8C
). This may,
therefore, account for the blunted primary FSH surge in this cohort of
animals.

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Figure 8. Serum Hormone Levels across the Estrous Cycle
A, Both serum FSH and LH varied significantly across the cycle
(P < 0.001). Points with an
asterisk (*) differed significantly from points without
an asterisk. B, Serum estradiol (E2, P
< 0.001) and progesterone (P4, P <
0.02) varied significantly across the cycle. For the estradiol data,
the point within an (a) differed from all other groups and the point
with a (b) differed from all groups except for 1000 h diestrus.
For the progesterone data, the point with a (c) differed from all other
points except 2400 h proestrus. The point with a (d) differed
significantly from the 1000 h time points on proestrus and estrus.
C, Both serum inhibin A (P < 0.001) and inhibin B
(P < 0.04) fluctuated significantly across the
cycle. For the inhibin A data, the point with an (a) differed from all
other points except 1000 h proestrus; the point with a (b)
differed from all others except 1800 h proestrus and 1000 h
diestrus; the point with a (c) differed from 2400 h proestrus and
0400 h estrus. For the inhibin B data, the point with a (d)
differed from all others except 0400 h and 1000 h on estrus;
the point with an (e) differed from 1000 h metestrus and 1800
h proestrus; the point with an (f) differed from 1000 h metestrus.
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InhBP-L and InhBP-S mRNA levels were highly correlated
(r = 0.858, P = 0.0001; Table 1
) and both varied significantly across
the cycle (P < 0.03 and 0.001, respectively; Fig. 9A
). Levels declined from their peak on
the morning of metestrus to their nadir on the late evening of
proestrus into the early morning of estrus. Thus, like serum inhibin A
and B, InhBP expression was lowest at the time of the secondary FSH
surge. In fact, expression of both forms of InhBP showed a significant
negative correlation across the cycle with serum levels of FSH
(rs > -0.69; Table 1
). InhBP mRNA levels (for both
isoforms) also showed a significant positive correlation with serum
inhibin B, but not inhibin A, levels across the cycle (Table 1
).
Pituitary betaglycan levels did not vary significantly across the cycle
(P > 0.8; Fig. 9B
); however, there was a trend toward
lower levels from 1000 h proestrus through 0400 h estrus, and
the correlation between betaglycan mRNA and serum FSH levels just
failed to reach statistical significance (r = -0.38,
P = 0.089).
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Table 1. Correlations between Serum Hormone Levels and
Pituitary Inhibin Coreceptor mRNA Levels across the Rat Estrous
Cycle
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Figure 9. InhBP and Betaglycan mRNA Levels across the Rat
Estrous Cycle
A, Pituitary InhBP-L (P < 0.03) and InhBP-S
(P < 0.001) mRNA levels fluctuated significantly
across the rat estrous cycle. Symbols above and below the
lines refer to InhBP-L and InhBP-S data, respectively. An (a)
denotes a statistically significant difference from the 1000 h
time points on metestrus and diestrus. The point with a (b) differed
from 1000 h metestrus. Points with a (c) are equivalent to each
other and differed from all other time points. Points with a (d) were
equivalent to each other, but differed from all other time points. B,
Pituitary betaglycan mRNA levels did not change significantly across
the estrous cycle (P = 0.8). The data are derived
from RNA blot analyses as described in the Materials and
Methods. The blots were hybridized with the same probes used in
Fig. 2A as well as with a probe directed against rat ribosomal protein
L19 to control for equal loading. Data in both panels reflect
normalized values expressed relative to the 1000 h metestrus time
point.
|
|
 |
DISCUSSION
|
---|
Two putative inhibin receptors or coreceptors have been
identified, InhBP and betaglycan (7, 8, 9). Here we characterized two
forms of InhBP in rats. The long form, InhBP-L, encodes a predicted
protein of 1,3191,320 amino acids that shares a high degree of
sequence conservation with the orthologous protein described previously
in humans (14, 15, 19). This protein is a member of the Ig superfamily
and contains 12 Ig-like domains, a single transmembrane domain, and
short intracellular domain with no identified signaling motifs. The
second form of InhBP, InhBP-S, which is highly expressed in pituitary
and testis, is predicted to be a soluble protein containing the first
two Ig-like domains of InhBP-L. We have not yet characterized the
inhibin-binding domain within InhBP-L, but many receptors within this
family bind ligand within the first two to three Ig-like domains (20).
If this is also the case for InhBP, then InhBP-L and InhBP-S may encode
membrane-bound and soluble inhibin binding proteins, respectively.
The generation of multiple isoforms by alternative splicing of
pre-mRNAs is frequently observed in Ig superfamily proteins. For
example, alternative splicing produces both membrane and soluble forms
of a natural killer (NK) cell receptor, 2B4R (21). Similar to what we
describe here for rats, different InhBP transcripts are also observed
in humans. Using probes directed against 5'-sequences, transcripts of
approximately 4.4 and 1.8 kb are detected in several human tissues
(14). The molecular nature of the smaller human transcript is not yet
known, but the similarity in size to rat InhBP-S suggests that it may
similarly encode a truncated form of InhBP. Using probes directed
against 3'-sequences, the 4.4-kb species is again detected and an
additional 2.7-kb transcript is observed in some human tissues,
including heart (15). We have similarly detected a 2.6-kb transcript in
rat pituitary, testis, and adrenal (data not shown). Clearly, there are
several InhBP isoforms expressed in different tissues and in different
species. A thorough understanding of the role of each of these isoforms
in inhibin action must await their molecular characterization.
At this point, one can speculate regarding the possible function of
InhBP-S. If this protein, like InhBP-L, binds inhibin, then it may
either potentiate or inhibit inhibin action in target cells. The
interleukin 6 receptor (IL-6R) is produced in both transmembrane and
soluble forms (22, 23). Cells that lack the transmembrane form of
IL-6R, but express the coreceptor, gp130, can respond to IL-6 in the
presence of the soluble form of IL-6R (sIL-6R) (e.g. Ref.
24) through a process called trans-signaling (25). The nature of the
cellular response is similar to that seen with the transmembrane form
of IL-6R. In contrast, for other growth factors, soluble forms of the
receptor act to antagonize or block action by competitively binding
ligand and thereby preventing binding to the membrane form of the
receptor (e.g. Refs. 26, 27, 28). Interestingly, if we observe
that InhBP-S potentiates inhibin action on target cells, then this may
indicate the presence of an additional membrane- bound signaling
coreceptor for InhBP. We have shown that InhBP-L interacts with Alk4
(and other TGFß superfamily type I receptors), and this interaction
occurs independently of the intracellular domains of the proteins (11).
It remains to be determined whether this interaction alone provides the
mechanism for inhibins antagonism of activin signaling or whether an
additional coreceptor, similar to gp130 for the IL-6 family, is
involved.
RNA blot analyses indicate that both InhBP and betaglycan are expressed
in the pituitary gland, as one would predict for bona fide inhibin
receptors (29). Immunofluorescence analyses further show that
betaglycan is most abundant in the intermediate lobe, but expression is
also observed in the anterior pituitary. Double-labeling experiments
indicate, however, that few, if any, of the betaglycan-producing cells
within the anterior pituitary are FSH-producing gonadotropes. Thus,
betaglycan-dependent effects of inhibin on FSH may not be direct.
Betaglycan exists in both transmembrane and soluble forms (30, 31).
Therefore, if betaglycan is involved in inhibin regulation of FSH, then
it is likely through its soluble form. It is worth noting, however,
that soluble betaglycan has been shown to inhibit rather than
potentiate ligand binding to membrane receptors (27). Currently
available antisera do not permit a similar analysis of InhBP protein
distribution within the rat pituitary (at least not with the method of
tissue fixation used here), but in situ hybridization
analyses indicate that InhBP mRNA is expressed exclusively in the
anterior pituitary. Future experiments will identify whether this
expression is observed within FSH-producing gonadotropes.
Inhibin is best known for its endocrine and paracrine actions in the
pituitary and gonads, respectively. Given the rather restricted number
of tissues in which the inhibins act, one might predict tissue
specificity in expression of the inhibin receptor. Consistent with this
argument, we observe highest levels of InhBP expression in the rat
pituitary and testis. With longer exposure times, we also detect low
levels of expression in rat ovaries in RNA blot analyses (data not
shown). No expression is detected in rat liver (at least not with the
probes used here). A similar pattern of results is observed in adult
human testis, ovary, and liver (15). Betaglycan, on the other hand, is
expressed more broadly, with highest levels observed in rat adrenal and
ovary. Previous analyses have also revealed high levels of expression
in rat lung, kidney, heart, and muscle (32). At first glance, this
broad pattern of expression may appear inconsistent with inhibins
limited number of target tissues. However, betaglycan plays an
important role in TGFß signaling, particularly for
TGFß2 (33), and its expression is therefore
predicted in TGFß-responsive as well as inhibin-responsive tissues.
Perhaps more importantly, inhibin A binds betaglycan with high affinity
only in the presence of the activin type II receptor (7). Therefore,
inhibins may only act via betaglycan in those cells coexpressing the
type II receptor. In this context, it will be important to examine
coexpression of betaglycan and actRII in relation to FSH-producing
gonadotropes in the anterior pituitary. This will provide a more
compelling argument for betaglycans role as an inhibin
coreceptor in vivo.
Both serum inhibin A and B levels are regulated across the rat estrous
cycle, and levels of both hormones are negatively correlated with serum
FSH (current study and Ref. 12). Here we demonstrate that pituitary
InhBP-L and InhBP-S mRNA levels are also dynamically regulated across
the 4-day cycle and are negatively correlated with serum FSH. In fact,
pituitary InhBP levels are more strongly correlated with serum FSH
levels than are inhibin A or inhibin B levels. Because we are currently
unable to measure InhBP protein levels in rats, we do not know whether
these changes in mRNA levels produce similar changes in protein
expression across the cycle. However, if we assume that the large
changes in mRNA levels lead to similar changes in the receptor protein
levels, then both circulating inhibin levels and gonadotrope
responsiveness (via InhBP) to inhibin are minimized at precisely the
time of the secondary FSH surge on the early morning of estrus.
It is not yet clear what regulates InhBP expression across the cycle.
One might propose a role for progesterone and estradiol, but
circulating levels of these hormones do not correlate with pituitary
InhBP mRNA levels. Interestingly, inhibin B, but not inhibin A, levels
are positively correlated with InhBP expression across the cycle,
suggesting that the ligand may play some role in the regulation of its
own coreceptor. Unlike InhBP, pituitary betaglycan mRNA levels do not
vary significantly across the cycle, nor do they correlate
significantly with serum FSH (although there is a trend in this
direction). As described above, betaglycan is most highly expressed in
the intermediate lobe of the pituitary. It is, therefore, possible that
betaglycan may be regulated in the anterior lobe across the cycle, but
this effect may be masked by high and stable levels of expression in
other parts of the pituitary (in particular within the intermediate
lobe).
A growing body of literature indicates that the two forms of inhibin
subserve different biological functions and that inhibin B may be the
main regulator of FSH in vivo. First, inhibin B alone is
produced in male mammals and, in nonhuman primates, appears to be the
primary endocrine regulator of pituitary FSH (12, 34, 35, 36). Second,
during the early part of the rat estrous cycle on metestrus and
diestrus when FSH levels are low, inhibin B, but not inhibin A, levels
are elevated (present study and Ref. 12). Third, during the follicular
phase of the menstrual cycle, inhibin B levels alone are increased in
response to elevated FSH (e.g. Refs. 37, 38, 39). Fourth,
treatment of metestrous rats with exogenous inhibin A fails to decrease
FSH levels (40), while treatment with porcine follicular fluid (a
source of both inhibin A and B) decreases FSH in metestrous rats (41).
Based on in vitro data from our laboratory, we proposed that
InhBP may function as an inhibin B receptor and may therefore provide
one mechanism through which inhibin B-specific signals are conveyed to
target cells (11). Our observations that pituitary InhBP mRNA levels
are negatively correlated with serum FSH and positively correlated with
serum inhibin B across the estrous cycle are consistent with this
idea.
In summary, two inhibin coreceptors have recently been identified,
InhBP and betaglycan. Both are expressed in the adult rat pituitary
where they may mediate inhibins antagonism of FSH synthesis and
secretion. InhBP is expressed in two forms within the anterior
pituitary gland. InhBP-S is produced through alternative splicing of
the InhBP pre-mRNA, and both isoforms are dynamically regulated across
the rat estrous cycle. Pituitary levels of InhBP-L and InhBP-S mRNA are
high when serum FSH is low on the morning of metestrus and then decline
significantly by the early morning of estrus at the time of the
secondary FSH surge. Pituitary betaglycan mRNA levels do not change
significantly over the estrous cycle, and the protein is most abundant
in the intermediate lobe. Although also expressed in the anterior
pituitary, betaglycan does not appear to be expressed in FSH-producing
gonadotropes. The function of both InhBP and betaglycan in mediating
inhibins suppression of FSH has yet to be determined. However, we
propose that InhBP plays an important role in inhibin Bs regulation
of pituitary FSH in vivo.
 |
MATERIALS AND METHODS
|
---|
Animals and Experimental Procedures
Adult male and female Sprague Dawley rats were purchased from
Charles River Laboratories, Inc. (Wilmington, MA) and
housed on a 14-h light,10-h dark photoperiod in the animal facility at
Northwestern University (Evanston, IL). Animals were housed up to five
per cage and were provided with food and water ad libitum.
For cloning and gene expression studies, various tissues (including
pituitaries, testes, ovaries, adrenals, and livers) were collected from
animals after asphyxiation in a CO2 chamber.
Animals were decapitated and trunk blood was collected. Blood was
allowed to clot overnight at 4 C and serum was collected after
centrifugation. Dissected organs were immediately frozen on powdered
dry ice. For the estrous cycle study, vaginal smears were performed
daily for at least 10 days, and cell morphology was used to determine
cycle stage. Pituitaries collected from these animals were used in RNA
blot analyses of InhBP and betaglycan mRNA levels (see below). All
animals were treated in accordance with the NIH Guide for the Care and
Use of Laboratory Animals.
InhBP Cloning: cDNA Library Screening, RT-PCR, and RACE
Initially, the human InhBP cDNA sequence was used to identify
mouse expressed sequence tag (EST) clones within GenBank. Several
clones with high sequence homology were identified and obtained from
the Image Consortium. The insert from one clone (GenBank accession no.
AI035698) was isolated, random prime labeled, and used to screen a rat
testis cDNA library in
gt11 (CLONTECH Laboratories, Inc., Palo Alto, CA) using standard techniques. DNA from one of
several clones isolated (N-1) was purified, and its insert was excised
from the
DNA by EcoRI digestion. This yielded three
fragments in addition to the phage arms (indicating the presence of two
EcoRI sites within the insert), which were subcloned into
pcDNA3.0 (Invitrogen, Carlsbad, CA). The resulting
subclones were sequenced with T7 and SP6 primers using BigDye
Terminator Cycle Sequencing (ABI Prism, Foster City, CA). Additional
gene-specific primers were designed to complete sequencing of both
strands of the subcloned fragments. All sequences were aligned using
Sequencher (version 3.1.1, Gene Codes Corp., Ann Arbor, MI) on a
Macintosh G3 computer (Apple, Cupertino, CA).
The 5'-end of the cloned rat InhBP cDNA corresponded to bp 1,619 of the
human sequence (GenBank accession Y10523), which lies approximately
1,539 bp downstream of the putative start of translation. Therefore, to
obtain additional 5'-sequence, we screened rat testis and pituitary
cDNA libraries (both in
gt11; CLONTECH Laboratories, Inc.) using probes derived from the 5'-end of the available rat
sequence or from PCR-generated fragments from the 5'-end of the human
cDNA. No additional sequence was obtained using these approaches. We
next used RT-PCR to extend the sequence in the 5'-direction. Various
sense primers were designed from the human sequence and were used in
combination with antisense primers designed from the 5'-most sequence
of the rat cDNA. Rat pituitary total RNA was reverse transcribed into
cDNA using Maloney murine leukemia virus reverse transcriptase in the
presence of random hexamer oligonucleotides and deoxynucleotide
triphosphates (dNTPs) (Promega Corp.; Madison, WI). PCR
was performed on one-fifth of the RT reaction using standard
techniques. PCR products were cloned using a T/A Cloning Kit
(Invitrogen, San Diego, CA) and sequenced using T7 or
PCR3.1 reverse primers as described above. This approach yielded
additional sequence; however, PCR primers directed against the putative
start of translation in the human sequence failed to produce detectable
products. We therefore used RLM-RACE following the manufacturers
instructions (Ambion, Inc. Austin, TX) to obtain the
remaining 5'-sequence.
The 3'-end of the original subcloned rat InhBP cDNA did not contain a
poly A tail and appeared truncated relative to the human (GenBank
accession no. Y10523) and mouse 3'-ends (Mouse EST, GenBank accession
no. AI035698). We, therefore, used 3'-RACE according to the
manufacturers instructions (Life Technologies, Inc.,
Gaithersburg, MD) to obtain the remaining 3'-sequence. RACE products
were cloned and sequenced as described above.
RNA Extractions and RNA Blot Analyses
Total RNA was extracted from various tissues using Trizol
following the manufacturers instructions (Life Technologies, Inc.) and was resuspended in TE buffer (10 mM Tris,
pH 8.0, 1 mM EDTA). RNA used in RT-PCR or RACE analyses
(above) was treated with RQ1 RNase-free DNase (Promega Corp.). RNA concentration was estimated by measuring absorbance
at 260 nm.
For RNA blot analyses, 15 µg of total RNA per lane were
electrophoresed through formaldehyde-MOPS
[3-N-morpholino)propanesulfonic acid] gels using standard
procedures. RNA was then transferred overnight with 20xSSC to
nylon membranes (Nytran; Schleicher & Schuell, Inc.,
Keene, NH) by capillary action. The membranes were hybridized overnight
at 42 C with the indicated 32P-dCTP (3,000
Ci/mmol, 10 mCi/ml; NEN Life Science Products, Boston, MA)
labeled cDNA probes in 50% formamide, 5xSSC, 1xDenhardts, 20
mM NaPO4 (pH 6.8), 1% SDS, 5%
dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. Membranes
were washed for 30 min each in 2xSSPE/0.5% SDS at room
temperature and 65 C, and 0.2x SSPE/0.1% SDS at 65 C, and then
exposed to X-OMAT film (Eastman Kodak Co., Rochester, NY)
at -80 C with intensifying screens.
For the estrous cycle study, a total of three gels were run such that
pituitary RNA from one rat in each cycle stage appeared on each
gel. All three of these membranes were hybridized, washed, and analyzed
at the same time. For analyses, the membranes were exposed to the same
phosphoimaging screen (Molecular Dynamics, Inc.;
Sunnyvale, CA) to help minimize variability in the densitometry
measures between blots. To quantify relative mRNA levels, densitometry
values obtained with the InhBP and betaglycan probes were divided by
the value obtained with a loading control, ribosomal protein L19
(RPL19). So that data from the different blots could be compared in the
same statistical analysis, the data were normalized by expressing RNA
level for a given time point relative to the 1000 h metestrous
time point on the same gel.
In Situ Hybridization
In situ hybridization was performed as previously
described (42). Briefly, 10-µm pituitary sections were cut on a
cryostat and thaw mounted onto Vectabond-coated (Vector Laboratories, Inc., Burlingame, CA) microscope slides.
Sections were fixed in 4% paraformaldehyde (pH 7.4), acetylated with
0.25% acetic anhydride in 1x triethanolamine (pH 8.0), and
dehydrated in a graded series of ethanol. Adjacent sections were
hybridized with antisense or sense riboprobes directed against Siberian
hamster FSHß (GenBank accession no. AF106914 and Ref. 43) or mouse
InhBP. The hamster FSHß probe was 380 nucleotides (nt) and 90%
conserved with the corresponding rat sequence. The mouse InhBP probe
was 1) 677 nt, 2) directed against the last 94 bp of the ORF and the
entirety of the 3'-UTR, and 3) 92% conserved with the corresponding
rat sequence. Probes were transcribed in vitro from
linearized plasmids with T7 or T3 RNA polymerase (MAXIscript,
Ambion, Inc. Austin, TX) in the presence of
33P-UTP (2,000 Ci/mmol, 10 mCi/ml; NEN Life Science Products). Probes were applied to sections in 50%
formamide, 300 mM NaCl, 10
mM Tris (pH 8.0), 1 mM EDTA
(pH 8.0), 1x Denhardts, 10% dextran sulfate, 10
mM dithiothreitol, 500 µg/ml yeast tRNA, and
500 µg/ml poly(A)+ RNA. Coverslips were applied and hybridization
proceeded overnight at 51 C.
Coverslips were removed in two changes of 4xSSC. Sections were then
treated with 20 µg/ml RNase A in 2xSSC at 37 C for 30 min, rinsed in
2xSSC for an additional 30 min at 37 C, washed in 0.1xSSC at 65 C for
30 min, rinsed in 1xSSC at room temperature, and dehydrated in a
graded series of ethanol. After exposure to Biomax film, slides were
dipped in NTB-2 emulsion (Kodak), air dried, and stored
with desiccant at 4 C until developed using standard techniques.
Sections were counterstained with hematoxylin and viewed using both
dark-field and bright-field microscopy. Digital images were collected
on a PC computer using the Metamorph image analysis system (v. 4.5;
Universal Imaging Corp., West Chester, PA).
Immunofluorescence
Whole pituitaries were extracted and immersed in 4% buffered
paraformaldehyde (pH 7.4) overnight at 4 C. Tissues were then
cryoprotected in 30% sucrose before freezing on dry ice.
Ten-micrometer sections were cut on a cryostat and thaw mounted onto
Vectabond-coated slides. Sections were washed in 1x PBS and then
incubated in 10% normal donkey serum (Sigma, St. Louis,
MO) in 1x PBS, 1% BSA, and 0.05% Tween (PBT) for 1 h at room
temperature. After a brief rinse in PBS, endogenous biotin was blocked
using an Avidin/Biotin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA). Sections were then incubated overnight at
4 C in either rabbit antirat FSHß (provided by Dr. A. Parlow, NHPP)
(1:50 in PBT) or affinity purified goat antihuman TGFß type III
receptor IgG (40 µg/ml in PBT; R&D Systems; Minneapolis, MN). After
two washes in PBS, sections were incubated for 1 h at room
temperature with biotinylated donkey antirabbit IgG and Texas Red
donkey antigoat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:250 in PBT. Sections were then
washed in PBS and incubated with fluorescein-conjugated Avidin DCS
(Vector Laboratories, Inc.). Sections were washed in PBS
and coverslipped with Vectashield containing DAPI (Vector Laboratories, Inc.). Slides were viewed using fluorescence
microscopy and digital images were collected as described above.
Colocalization was determined using the overlay feature of the
Metamorph software package.
Hormone Assays
Serum LH, FSH, estradiol, and progesterone were measured by RIA.
Serum inhibin A and inhibin B were measured by enzyme-linked
immunosorbent assays using kits from Serotec (Oxford, UK).
These assays have previously been validated for use in rats
(12). Intraassay coefficients of variation were less than 5%
for the steroids, less than 18% for the gonadotropins, and less than
10% for the inhibins.
Statistical Analyses
Hormone and mRNA levels across the cycle were compared
using one-way ANOVAs. Post-hoc comparisons were made using Fishers
least significant difference procedure. Correlations were made using
simple linear regression analyses. In all cases, statistical
significance was determined relative to P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Fernando Lopez-Casillas, who
provided the rat betaglycan cDNA, and Brigitte Mann, who conducted the
gonadotropin and steroid RIAs. Wei Chen and Hilary Rainey provided
valuable assistance with the initial cDNA cloning.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Teresa K. Woodruff, Associate Professor, Northwestern University, Department of Neurobiology and Physiology, O.T. Hogan 4150, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: tkw{at}nwu.edu
This research was support by a Lalor Foundation Postdoctoral Fellowship
(D.J.B.), and NIH Grants HD-37096, HD-28048, and HD-21921 (T.K.W.).
Received for publication December 1, 2000.
Accepted for publication January 16, 2001.
 |
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