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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go; 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.

 
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. 1Go). 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. 1Go). 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. 5Go). 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.

 
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. 2AGo, 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. 2BGo, 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 172–839 of the full-length rat cDNA; see Fig. 7Go) or the ApaI–SacI 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.

 
We next examined the distribution of InhBP mRNA within the pituitary using in situ hybridization histochemistry (Fig. 3Go). Like FSHß mRNA (Fig. 3AGo), InhBP mRNA was detected exclusively within the anterior pituitary (Fig. 3Go, 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.

 
Betaglycan was detected in the majority of tissues examined (Fig. 2AGo, 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. 4Go). FSHß-immunoreactive cells were detected exclusively within the anterior pituitary (Fig. 4Go, A and C). The highest level of betaglycan immunoreactivity was observed in the intermediate lobe (Fig. 4BGo), but betaglycan-positive cells were also detected in the anterior lobe (Fig. 4DGo). Double-immunofluorescence analyses indicated that betaglycan was expressed near, but rarely in, FSHß-positive cells within the anterior lobe (Fig. 4EGo). No double-labeled cells are pictured in Fig. 4EGo, 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.

 
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 {lambda}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. 5Go). 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. 6Go and 7Go). 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. 7Go). 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. 2Go) are shown.

 
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. 2BGo, right panel; see also Fig. 7Go). 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. 5Go), 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. 8Go). 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. 8AGo). 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. 8CGo). 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.

 
InhBP-L and InhBP-S mRNA levels were highly correlated (r = 0.858, P = 0.0001; Table 1Go) and both varied significantly across the cycle (P < 0.03 and 0.001, respectively; Fig. 9AGo). 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 1Go). 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 1Go). Pituitary betaglycan levels did not vary significantly across the cycle (P > 0.8; Fig. 9BGo); 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. 2AGo 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1,319–1,320 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 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {lambda}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 {lambda} 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 {lambda}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.


    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 4–150, 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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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