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U.S. Department of Agriculture, Agricultural Research Service, RLH U.S. Meat Animal Research Center (M.D.L., J.J.F.), Clay Center, Nebraska 68933; Department of Neuroscience and Cell Biology, University of Medicine and Dentistry, Robert Wood Johnson Medical School (G.J.M.), Piscataway,New Jersey 08854
Address all correspondence and requests for reprints to: Dr. J. J. Ford, P.O. Box 166, Clay Center, Nebraska 68933. E-mail: ford{at}marcvm.marc.usda.gov
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Abstract |
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-, ßA-, and ßB-subunits in porcine
anterior pituitary glands using quantitative reverse transcription-PCR
and ribonuclease protection techniques. We found that 1) the
inhibin/activin ßA- and ßB-subunits and
follistatin were expressed in porcine pituitary; 2) the -subunit was
not detected in the porcine pituitary, but was highly expressed in
porcine follicles; and 3) the ßB-subunit gene is more
abundantly expressed (2-fold greater) in MS boar pituitaries than in
pituitaries of White Composite boars. We conclude that this is not due
to a breed difference, because the expression levels of this gene were
similar in pituitaries of females of these breeds. No breed differences
were detected for other genes screened in this study. From these
observations, we propose that activin B, a dimer of
ßB-subunits and a stimulator of FSH secretion, may be
partially responsible for the elevated plasma FSH concentrations in MS
boars, and intrapituitary inhibin plays no or a very minimal role. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Markedly greater plasma FSH concentrations and pituitary FSHß messenger RNA (mRNA) have been detected in MS boars than in contemporary boars (4, 5, 6). Although a direct relationship between plasma FSH concentrations and reproductive efficiency has not been established experimentally, results from a long term selection experiment in pigs indicated a positive association between ovulation rate in females and FSH secretion in boars (7). Similar to man, a negative relationship between elevated plasma FSH concentrations and testicular size has been documented recently in boars (8, 9, 10). However, the cause(s) of such a relationship remains to be characterized.
Activins and inhibins were initially isolated from porcine and bovine
follicular fluids as gonadal proteins with the capacity to stimulate
and inhibit the release of FSH secretion and synthesis in anterior
pituitary glands, respectively (11, 12). Inhibins are dimeric proteins
consisting of a common -subunit and one of two ß-subunits and are
present in two forms: inhibin A (/ßA) and inhibin B
(/ßB) (13, 14, 15, 16). Activins are also dimeric proteins,
but they are composed of only ß-subunits. Three forms of activin have
been isolated from follicular fluids and determined to stimulate FSH
secretion in pituitaries; they are activin A
(ßA/ßA), activin AB
(ßA/ßB), and activin B
(ßB/ßB), respectively (17, 18, 19, 20). The
complementary DNAs (cDNAs) encoding the -, ßA-, and
ßB-subunits have been cloned from several species, and
each subunit is encoded by a distinct gene. The genomic organization of
rat, mouse, and human subunits has been reported (21, 22, 23, 24, 25).
The transcripts for these subunits have been detected in the gonads and
several extragonadal tissues, such as placenta, pituitary, adrenal, and
spleen. In the rat pituitary, only mRNAs for the - and
ßB-subunits were detected by S1 nuclease analysis (26).
Although immunohistochemical techniques show nuclear staining for the
protein in some cells (27), the mRNA for the ßA-subunit
has never been detected in the rat pituitary. In humans, mRNAs for all
three subunits were detected in a normal human pituitary cDNA library
(28), suggesting that they are all expressed in human pituitaries.
Follistatin (FS), another protein isolated from porcine and bovine follicular fluid, possesses an inhibin-like activity, i.e. to suppress pituitary FSH secretion (29, 30). However, its structure differs greatly from inhibins and activins, as it consists of a single chain polypeptide. Its ability to suppress pituitary FSH secretion was 10–30% less than that of inhibin and is due to its binding of activin (30). Additionally, both activins and FS play a role in multiple systems, such as erythropoiesis and the immune system, acting as paracrine and autocrine regulators (25, 29).
To determine the cause(s) of the elevated FSH secretion in MS boars, we
examined expression differences for the inhibin/activin (I/A) -,
ßA-, and ßB-subunit and FS genes in MS
anterior pituitary glands in comparison with European White Composite
(WC) boars. Our results indicated that the ßB-subunit is
more highly expressed in MS boars than in WC boars, but not in the
females of these two breeds. The transcript for the -subunit could
not be detected in the anterior pituitary glands of either breed. In
contrast, the mRNA for the -subunit was detected easily in porcine
follicles, particularly in the midstages of the follicular phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation and preparation
Total RNA was isolated from individual frozen anterior
pituitaries or follicles by guanidine isothiocyanate extraction and
CsCl centrifugation (31). Before use, RNA was treated with ribonuclease
(RNase)-free deoxyribonuclease I at 37 C for 30 min. After extraction
with phenol/chloroform and ethanol precipitation, the RNA was
redissolved into diethyl pyrocarbonate-treated water.
Primers used in quantitative reverse transcription-PCR (RT-PCR) and
subcloning
Based upon the reported nucleotide sequences for the porcine
genes of I/A -, ßA-, and ßB-subunits,
FS, and FSHß genes (32, 33, 34), five sets of oligonucleotide primers
corresponding to each gene were designed and synthesized with an Oligo
1000 DNA Synthesizer (Beckman, Palo Alto, CA). The primer sequences and
the expected PCR product sizes amplified by them are given in Table 1
. A set of primers for porcine ß-actin gene (GenBank
Accession U07786) is also given.
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-9, pßA-21, pßB-5,
pFSHß, and pß-actin containing partial FS; -, ßA-,
and ßB-subunits; FSHß; and ß-actin gene sequences
were verified by sequence analysis from both directions by the dideoxy
method of Sanger et al. (35) adapted for double stranded DNA
templates (U.S. Biochemical Corp., Cleveland, OH) compared with the
corresponding sequence region of each gene (32, 33, 34).
Preparation of oligonucleotide primer, sense, and antisense
riboprobes
Oligonucleotide primer was end labeled with T4 polynucleotide
kinase (Promega Corp., Madison, WI) in the presence of
[
-32P]deoxy-ATP and 1 x T4 polynucleotide kinase
buffer (0.5 M Tris-HCl, pH 7.5; 0.1 M
MgCl2; and 50 mM dithiothreitol). The reaction
mixtures were incubated at 37 C for 30 min and stopped by adding 0.5
M EDTA, pH 8.0, to a final concentration of 40
mM. Free nucleotides were removed by Sephadex G-25 spin
column (5 Prime-3 Prime, Boulder, CO).
Sense and antisense porcine ßB and ß-actin riboprobes
were transcribed from the linearized plasmids pßB-5 and
pß-actin DNA by EcoRV or HindIII in the
presence of [-32P]UTP with DNA-dependent SP6 or T7 RNA
polymerases under the conditions reported previously (36). The plasmid
DNA templates were removed by incubation with RNase-free
deoxyribonuclease I and extraction with phenol-chloroform.
Quantitative RT-PCR
RT-PCR were performed as previously described (36). Briefly, the
RT was performed in a final concentration of 20 µl with 1 µg total
RNA, 4 µl 5 x reverse transcriptase buffer (0.1 M
Tris-HCl, pH 8.8; 0.5 M KCl; and 1% Triton X-100), 10
mM dithiothreitol, 1 mM of each deoxy-NTP, 20 U
RNasin, 0.1 µg oligo(deoxythymidine)15, and 200 U Moloney
murine leukemia virus reverse transcriptase (Pharmacia Biotech,
Piscataway, NJ). Reaction mixtures were incubated at 37 C for 1 h
and heated at 95 C for 5 min to inactivate the reverse transcriptase.
Amplification of 4 µl RT mixture (equal to 0.2 µg total RNA) or the
amounts specified in the text was carried out with 5 µl 10 x
PCR buffer [.5 M KCl; 0.1 M Tris-HCl, pH 8.3;
and 0.01% (wt/vol) gelatin], 2.0 mM MgCl2,
0.1 µg [-32P]ATP end-labeled sense and unlabeled
antisense primers, and 2.5 U Ampli-Taq DNA polymerase (Promega,
Madison, WI) in a 50-µl total volume. The PCR reaction mixtures were
overlaid with mineral oil and initially denatured at 94 C for 3 min,
then subjected to 15–40 cycles of denaturation (94 C, 1 min),
annealing (50–60 C, depending on the primer pairs used, 45 sec), and
extension (72 C, 45 sec). After the last cycle, the extension phase was
continued for 7 more min at 72 C, and a 15-µl sample was resolved on
composite gels of 1.5% NuSieve GTG (FMC Bioproducts, Rockland, ME)
-1% agarose containing 25 µl ethidium bromide at 500 µg/ml.
Appropriate bands were cut from the agarose gel, and the radioactivity
of each band was determined. For every gene studied, the mRNA level of
each sample was normalized to ß-actin RNA. The ß-actin
determinations were conducted simultaneously under identical condition,
except different primers were used.
RNase protection assay
Hybridizations and RNase digestions were performed with a RPA-II
kit from Ambion (Austin, TX) under conditions previously described (36, 37). For every riboprobe used, two yeast RNA control tubes with one
containing RNase A/T1 (negative control) and the other containing no
RNase A/T1(positive control) were always included in the assay.
Statistical analysis
The mRNA level of every gene of interest was adjusted first to a
constant amount of ß-actin mRNA. The t test was used to
test significance between breeds or sexes. Females from which follicles
were collected were grouped with the complete linkage method (38) based
on plasma estradiol and progesterone concentrations and the mean and
SE of follicle number for each animal. The data are
presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). After logarithmic transformation, a linear
relationship between the radioactivity incorporated into PCR products
and amplification cycles was obtained for the first 35 cycles (see Fig. 1B). After 35 cycles, the rate decreased and approached a plateau (data
not shown). Also, aliquots of the cDNA mixture equivalent to 0.0–0.8
µg total RNA were amplified for 25 cycles for the
ßB-subunit-specific primers, and the rates of
amplification were exponential between 0.2–0.6 µg total RNA (see
Fig. 1, C and D). Based upon these observations, it was determined that
with 0.2 µg pituitary total RNA, 25 amplification cycles were optimal
for quantification of the I/A ßB-subunit mRNA.
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-subunit, no products were amplified from porcine
pituitary RNA even after 35 cycles of amplification, but 25 cycles
produced quantifiable product from follicular RNA.
FSHß gene expression and pituitary FSH concentration
A significantly higher expression of FSHß gene (
3.2-fold) was
detected in MS boar pituitary RNA than in pituitary RNA of WC boars
(P < 0.01; Fig. 2A). No difference was
detected between females of these breeds (P > 0.10;
Fig. 2B). Pituitary FSH concentrations were greater in MS than in WC
boars (P < 0.01; MS, 36.5 ± 4.0 µg/mg; WC,
7.4 ± 1.6 µg/mg), but were similar in these two groups of
females (P > 0.10; MS, 26.2 ± 3.9 µg/ml; WC,
18.3 ± 5.6 µg/mg).
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I/A ßB-subunit expression (by RT-PCR)
When primers specific for the ßB-subunit were
used, we found that the I/A ßB-subunit was expressed in
the pituitaries of both breeds, but the expression in MS boar pituitary
was significantly greater than that in WC boars (P <
0.01; Fig. 3A). However, no differences were detected in
the females of these two breeds (P > 0.10; Fig. 3B). A
significant difference existed between MS boars and females
(P < 0.01), but not between sexes in WC pigs
(P > 0.10).
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-subunit expression-subunit RNA in either sex of both breeds
even after 35 cycles of amplification. However, when porcine follicular
RNA was used, the expected PCR products were amplified with 25 cycles
of amplification. Expression of -subunit was about 3- to 5-fold
greater in midstage follicles than in follicles during the early and
late follicular phases (early, 0.82 ± 0.23; mid, 4.25 ±
0.10; late, 1.34 ± 0.16; P < 0.01).
FS gene expression
Primers of 5'-FS and 3'-FS-2 or 3'-FS-1 were used to quantify the
expression in anterior pituitaries of mRNAs for FS-315 and FS-315 plus
FS-288, which are generated by alternative splicing (39). No
significant differences were detected between MS and WC pig breeds for
either FS-315 mRNA (MS, 0.52 ± 0.08; WC, 0.65 ± 0.12;
P > 0.10) or FS-315 plus FS-288 mRNAs (MS, 0.85
± 0.22; WC, 1.22 ± 0.25; P > 0.10).
I/A ßB-subunit expression (by RNase
protection assay)
The RNase protection assay was employed to confirm results
obtained with quantitative RT-PCR. Schematic diagrams of the assay and
the expected protected fragments for ßB RNAs are
given in Fig. 4A. When the antisense pßB-5
riboprobe was hybridized with 10 µg boar pituitary total RNA,
followed by digestion with RNase A/T1, an expected band of 243
nucleotides for the ßB-subunit mRNA was protected in all
samples (Fig. 4C). In contrast, when the sense pßB-5
riboprobe was used, no fragments were protected (Fig. 4B), indicating
that the protected fragment for antisense ßB riboprobe
was specific for the ßB-subunit mRNA. Relative
intensities of the protected fragment for ßB-subunit were
clearly greater (2-fold; P < 0.05) in MS boars than
in WC boars (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of the inhibin -subunit gene could not be detected in
porcine pituitaries of either breed or sex. However, with the same
conditions and primers, the transcript of -subunit could be detected
in porcine follicles, particularly in the midstage of the follicular
phase of the estrous cycle, similar to that reported previously (40).
This indicated that nondetection of the inhibin -subunit was not due
to improper PCR conditions or other technical problems.
By RT-PCR, FSH-ß mRNA was 3.4 times more abundant in pituitaries of
MS than WC boars and confirmed our previous observations (6). This
difference in FSHß gene expression corresponded with greater FSH
levels in plasma and pituitaries of MS boars (4, 5, 6). Such differences
have never been detected in females of these two breeds at both the
protein (41) and RNA levels (see Fig. 2
). Currently, one of the major
research objectives in our laboratory is to identify genes that are
responsible for such elevated FSH concentrations in MS boars. One of
the most straightforward approaches was to determine whether expression
differences exist for the I/A -, ßA-, and
ßB-subunit and FS genes, because the proteins coded by
these mRNAs are involved in the regulation of FSH synthesis and
secretion in anterior pituitary glands (29, 42). Expression of the
ßA- and ßB-subunit genes in porcine
pituitary glands coupled with low or no expression of -subunit
indicate that activins may be present in this tissue. It is expected
that greater ßB expression would lead to greater FSH
synthesis and secretion in MS than in WC boars.
In addition to activin A (ßA/ßA) (17) and activin AB (ßA/ßB) (18, 19), activin B (ßB/ßB) has been identified in porcine follicular fluid (20) and rat pituitary cells (12, 43), supporting the hypothesis given above. However, whether ßB gene expression translates into protein that has access to FSH-producing gonadotrophs in porcine pituitaries remains to be determined. With the limitations of abundance of protein and sensitivity of antibodies against activin B, we were unable in preliminary studies to determine tissue levels of activin B. A more reliable and sensitive assay to measure the levels of activin B is required. Additionally, activin B must avoid the neutralizing action of FS either through an excess concentration or by regional localization. Further experiments are required to address these possibilities.
Expression of the activin/inhibin subunits and FS genes is widespread in both the embryo and the adult (26, 44, 45), and activin’s effects have been noted in a wide range of tissue and cell types (11, 25, 42). Most of these effects have been characterized with respect to activin A, because it is the most commonly purified molecule and the only activin produced in significant quantity by recombinant means. Native activin B has been purified from follicular fluids and was significantly less potent than activins A and AB in all bioassays tested, with the exception of the induction of mesoderm in Xenopus embryos (20). However, these results contrast with findings for proteins expressed recombinantly, in which activins A and B were equipotent in several mammalian cell assays (23). Such differences could be due to differences in pituitary tissue or different receptors mediating the actions of different activins. Two types of activin receptors (ActRI and ActRII) are involved in postreceptor signaling, and both have been identified in several species. These receptor subunits are thought to form heteromeric complexes to create functional receptors (46); moreover, mice deficient in ActRII have reduced plasma concentrations of FSH (47). It remains to be determined how many types of activin receptor are present in the porcine pituitary and if expression differences in these receptor subunits exist between these two breeds.
The expression patterns of activin/inhibin subunits in porcine
pituitary differ greatly from those in rat or human pituitary. In rats,
only the - and ßB-subunit mRNAs were detected (26),
whereas in humans, all three subunits were reported to be expressed in
normal pituitary cDNA library (28). Here we report that only mRNAs for
the ßA- and ßB-subunits, but not
-subunit, can be detected in pig pituitary. Therefore, it is
predicted that no inhibin or very small amounts of inhibin could be
synthesized in pig pituitary. This suggests that the regulation of FSH
synthesis and secretion in pig might differ from that in rats or
humans, and locally produced inhibin may not be a major regulator of
FSH secretion in the pig pituitary, as reported in other species.
Similar to the ßA-subunit RNAs, no differences were
detected for the FS transcript between the pituitaries of these two
breeds. In addition, it has been reported that FS RNA can be detected
in all cell types of the rat pituitary and is not restricted to only
FSH-producing cells (48). This coupled with our inability to detect
major differences in negative regulation of FSH secretion and synthesis
between MS and WC boars (5, 6) prompted the current studies. We
hypothesized that the elevated FSH concentrations in MS boars result
from increased local expression of activin B in the pituitary. This
hypothesis has been supported by a gene knockout experiment in mice in
which inhibin--deficient mice showed elevated (2- to 3-fold) serum
FSH concentrations (49). Because the -subunit was absent, no
inhibins would be formed; consequently, the ßB-subunit
that would normally be found in inhibin dimers would give rise to
increased activin concentrations. Thus, these transgenic mice developed
similar to the situation observed naturally in male pigs, except that
testicular inhibin production is present in male pigs.
Collectively, with the genes screened in this study, we found that the
I/A ßA- and ßB-subunit, but not
-subunit, genes were expressed in the porcine anterior pituitary.
Additionally, we found that the ßB-subunit was more
highly expressed in anterior pituitaries of MS boars than in
pituitaries of WC. No difference was detected in pituitaries of females
of these breeds; therefore, the finding in boars was not due to
inherent breed differences. It is hypothesized that elevated plasma FSH
concentrations in MS boars result partially from the increased
expression of ßB-subunit in the anterior pituitary, but
no inhibin or only a very small amount could be synthesized in porcine
anterior pituitary glands.
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Acknowledgments |
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Footnotes |
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Received August 26, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-B-subunit genes
suggests two different regulatory mechanisms. Mol Endocrinol 3:1914–1925[Abstract]
gene and its promoter. Biochem Biophys Res Commun 186:293–300[CrossRef][Medline]
, ßA, and
ßB subunits in various tissues predicts diverse
functions. Proc Natl Acad Sci USA 85:247–251- and
ßA-subunit messenger ribonucleic acids and inhibin
immunoactivity during preovulatory maturation in the pig. Biol Reprod 47:1018–1025[Abstract]
-inhibin is a tumour-suppressor gene with gonadal
specificity in mice. Nature 360:313–319[CrossRef][Medline]
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