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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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; 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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). 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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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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). 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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, 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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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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, 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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) 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 inhibin’s 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 inhibin’s 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 betaglycan’s 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 inhibin’s 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 inhibin’s suppression of FSH has yet to be determined. However, we propose that InhBP plays an important role in inhibin B’s regulation of pituitary FSH in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 manufacturer’s
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 manufacturer’s 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 manufacturer’s 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, 1xDenhardt’s, 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 Denhardt’s, 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 Fisher’s
least significant difference procedure. Correlations were made using
simple linear regression analyses. In all cases, statistical
significance was determined relative to P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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