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Original Studies |
Department of Obstetrics and Gynecology, University of Newcastle upon Tyne, Royal Victoria Infirmary (A.J.P., C.S., S.C.R., G.N.E.-F.), Newcastle upon Tyne NE1 4LP, United Kingdom; and Cold Spring Harbor Laboratory (A.R.K.), Cold Spring Harbor, New York 11724
Address all correspondence and requests for reprints to: Drs. Alison J. Pollard and G. Nicholas Europe-Finner, Department of Obstetrics and Gynecology, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom. E-mail: a.j.pollard{at}ncl.ac.uk and g.n.europe-finner@ncl.ac.uk.
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Introduction |
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) (9), PGE2
receptors (EP1 and EP3),
PGF2 receptors, thromboxane receptors
(10, 11, 12, 13), progesterone receptors (14), estrogen receptors (15, 16),
caldesmon (17), and CRH receptors (18, 19). Most of these proteins
exist as alternatively spliced isoforms whose tissue-specific
expression may be regulated by posttranscriptional premessenger
ribonucleic acid (mRNA) processing. The adenylyl stimulatory G protein
Gs provides one such example, where two
specific isoforms, Gs-Large (with an
additional serine after amino acid 86) and
Gs-Small (with an additional serine after
amino acid 71) are up-regulated during pregnancy and subsequently
down-regulated at the onset of labor (20).
Many of the proteins associated with smooth muscle myogenesis and
contractility are also regulated by alternative splicing (reviewed in
Ref. 21). These include ß-tropomyosin, -tropomyosin, and the
troponin genes (21, 22). The coding capacity of these genes is
increased manyfold as a result of alternate pre-mRNA processing. Both
ß-tropomyosin and troponin have been used as model proteins to study
alternative splicing mechanisms and their regulation in different
tissues (23, 24, 25, 26). In this respect there have also been comparable
studies on -tropomyosin (21).
There is now extensive evidence indicating that alternative precursor mRNA splicing is regulated by trans-acting factors, which include small nuclear ribonuclear proteins (snRNPs) that are ubiquitously expressed, the serine-arginine protein family, and heterogeneous ribonuclear proteins (hnRNPs) (27, 28, 29, 30). Two of the major trans-acting factors that govern splice site selection, exon inclusion, and skipping within pre-mRNA transcripts have been shown to be the protein SF2/ASF, a member of the serine-arginine protein family, and the alternatively spliced hnRNP proteins hnRNP A1/A1B (31, 32, 33). Several studies have indicated that altering the concentrations of SF2/ASF and hnRNP A1/A1B results in the formation of different spliced isoforms of a number of precursor mRNAs (34, 35, 36, 37). The concentration ratios of hnRNP A1/A1B to SF2/ASF in vivo may therefore be critical in defining the expression of specific spliced protein isoforms in different tissues (38, 39).
As tissue-specific regulation of alternative splicing can be attributed to ratios of hnRNP A1/A1B to SF2/ASF, the question arises as to whether these trans-acting splicing factors are also involved in modulating the expression of specific isoforms of myometrial proteins involved in regulating uterine activity during pregnancy and labor. Consequently, the purpose of this present study was to address this question by investigating the expression of SF2/ASF, hnRNP A1/A1B, and several snRNP proteins (as controls) within the human myometrium during gestation and parturition using Western immunodetection/immunohistochemical staining with monoclonal antibodies of defined specificity. There is also increasing evidence to indicate that proteins involved in controlling uterine activity are not only temporally, but also spatially, regulated within different regions of the uterus (6, 18, 40). Fuchs et al. (40) have observed a fundus to cervix gradient of myometrial OTRs at term in the uterus. Similarly, Sparey et al. (6) have shown that levels of connexin-43 are more abundant in the upper region of the uterus than in the lower uterine segment, whereas the converse was observed for the expression of COX-1 and -2. A further aim of the study was to determine whether SF2/ASF and hnRNP A1/A1B were also spatially expressed within different regions of the uterus during gestation and parturition. To accomplish this, paired myometrial tissue samples from both the lower and upper (corpus) regions of the uterus from individual patients in and not in spontaneous labor at term were analyzed for the expression of both of these trans-acting splicing components in conjunction with the control snRNP proteins U1–70K, U1-A, and U2-B'', and a comparison was made with nonpregnant control tissue samples.
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Materials and Methods |
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The monoclonal antibodies used in this study were anti-SF2/ASF (mAb96) and anti-hnRNP A1/A1B (4B10). The specificity of these antibodies has been previously reported (39, 41). Expression of three snRNP proteins, U1–70K, U1-A, and U2-B'', were also included as positive controls, because they have been reported to have ubiquitous roles in both constitutive and alternative pre-mRNA splicing (29). Anti-U1–70K (H111), anti-U1-A (H304), and anti-U2-B'' (4G3) were all obtained from Euro-Diagnostica (Arnhem, The Netherlands). Mouse IgG1 (MOPC-21) was obtained from Sigma (Poole, UK) to use as a negative control monoclonal antibody.
Myometrial samples
Myometrium was obtained from nonpregnant, pregnant nonlaboring, and spontaneous laboring women undergoing surgery. Written consent was obtained from all women, and ethical approval was granted by the Newcastle and North Tyneside Health Authority ethics committee. Samples of myometrium from nonpregnant premenopausal women (age range, 32–46 yr) were obtained from hysterectomies performed for benign gynecological disorders. The uteri were excised longitudinally, and samples of myometrium were taken from the middle of the corpus (termed upper segment) and from close to the cervix (termed lower segment).
Myometrium was also obtained from 12 pregnant nonlaboring women undergoing elective lower segment cesarean section at term and from 12 women in spontaneous labor at term who required emergency cesarean section for fetal distress or failure to progress. Spontaneous labor was defined as regular uterine contractions and cervical dilatation of more than 3 cm on admission. Augmentation of labor with oxytocin was not a reason for exclusion, providing the onset of labor had been spontaneous. Cesarean sections were carried out under subarachnoid block or general anaesthetic. After delivery of the infant the placental site was confirmed manually, and the placenta was delivered.
Five nonfundal, upper segment (corpus) biopsies were taken from the side opposite the placental bed using laparoscopic biopsy forceps (Wolf) introduced through the lower segment incision. In addition, a 1- to 2-cm3 sample of myometrium was taken from the upper margin of the lower uterine segment using tissue forceps and scissors. Paired upper and lower segment samples were obtained from all women in each group. Myometrial samples were snap-frozen in liquid nitrogen and stored at -70 C until required. Blocks of tissue (0.5–1.0 cm3) to be used for immunohistochemical analysis were placed in Cryo-M.Bed compound (Brights Instrument Co., Huntington, UK), frozen in liquid nitrogen with cooled iso-pentane (BDH, Poole, UK), and stored at -70 C.
Preparation of myometrial homogenates
Samples were homogenized at a ratio of 1:10 in 25 mmol/L Tris buffer (pH 7.6) containing 0.25 mol/L sucrose and 1 mmol/L ethylenediamine tetraacetate in the presence of pepstatin, leupeptin, aprotinin, and phenylmethylsulfonylfluoride (Sigma). Homogenates were subsequently centrifuged at 1000 x g to remove tissue debris, and the resultant supernatants were stored at -70 C. The protein concentration was assayed using the DC protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA) with BSA as a protein standard.
Western blot immunodetection
SDS-PAGE was performed using 500 µg total protein from each myometrial homogenate solubilized in sample loading buffer (0.5 mol/L Tris, 5 mol/L urea, 2.5% SDS, and 3.5% ß-mercaptoethanol) and resolved on 12.5% or 7.5% polyacrylamide gels. Transfer of proteins was performed using a Bio-Rad Laboratories, Inc. Trans-Blot electrophoretic cell system. Samples were transferred onto either polyvinylidene difluoride (Hybond-P, Amersham Pharmacia Biotech, Aylesbury, UK) or nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) at 90 V for 2.5 h. To check that similar levels of smooth muscle protein were loaded in each well, all nitrocellulose membranes were stained with Ponceau-S solution (Sigma) and densitometrically scanned before immunodetection. Membranes were preblocked with 10% Marvel milk powder/phosphate-buffered protein (PBS) for 1 h and then probed with primary mAbs (diluted 1:10–100 in PBS containing 1% Marvel) for 1–2 h at room temperature (mAb96, 4G3, H304) or overnight at 4 C (4B10, H111). After three 10-min washes, membranes were incubated for 1 h with horseradish peroxidase-conjugated antimouse IgG (DAKO Corp., High Wycombe, UK), diluted 1:2000, and detection was carried out using an enhanced chemiluminescence assay system (ECL, Amersham Pharmacia Biotech). In control experiments all antibodies were substituted with nonimmune mouse serum (data not shown). In all cases, immunodetected bands were specific. Data were obtained under conditions where a linear relationship existed between the amount of protein loaded and the intensity of the ECL signal from the immunoblots.
Quantification
ECL signals were quantified by scanning densitometry using a UMAX PS 2400 scanner at 700 dpi coupled to the Intelligent Quantifier software package from BioImage (Ann Arbor, MI). The data presented are the mean ± SEM (n = 12 for all nonpregnant, pregnant, and laboring samples). Quantification was performed using Prism 2.01 software (GraphPad Software, Inc., San Diego, CA), and data were subsequently analyzed using a one-way ANOVA with Bonferroni’s multiple comparison test.
Immunohistochemical analysis
Six-micron serial sections were cut from the frozen blocks in an Anglia Scientific Cryotome-620 cryostat at -20 C, mounted on 3-amino-propytriethoxy-silane (Sigma)-pretreated slides, and set aside to dry at room temperature for over 1 h. Tissue sections were fixed in cold acetone (BDH) for 10 min and either stained immediately or stored at -20 C. Primary antibodies diluted in PBS (1:100) were applied to each tissue section and incubated at room temperature for 1–2 h. To quench any endogenous peroxidase activity, tissue sections were incubated with 0.6% hydrogen peroxide in methanol for 3 min and then rinsed three times in PBS. Immunodetection was performed with a Histostain-Plus antimouse SP (peroxidase) staining kit (Zymed Laboratories, Inc., San Francisco, CA) using the recommended labeled biotinylated streptavidin (LAB-SA) method with a horseradish peroxidase-aminoethyl-carbazole substrate/chromogen kit.
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Results |
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). Quantification by densitometric
analysis indicated that levels of SF2/ASF were similar in upper and
lower uterine myometrium in nonpregnant women. However, during
pregnancy myometrial SF2/ASF expression increased significantly in the
lower uterine segment (P < 0.001), whereas there was a
small, although not significant, increase in the upper uterine region
compared with similar nonpregnant tissues (see Fig. 1B). During
parturition, levels of SF2/ASF in the lower uterine segment decreased
approximately 2-fold (P < 0.05); the data also suggest
that there was a small concurrent reduction in levels of SF2/ASF in the
upper uterine segment (see Fig. 1B).
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). Quantification demonstrated that
levels of hnRNP A1/A1B were similar in both lower
and upper nonpregnant myometrium. However, hnRNP
A1/A1B expression increased during gestation in
the upper uterine region compared with that in nonpregnant tissues
(P < 0.01). Levels of expression of hnRNP
A1/A1B proteins appeared to increase further
within the upper uterine region when comparing laboring with pregnant
tissues (see Fig. 2B), although the difference was not significant
(P > 0.05). A substantial decrease in the levels of
expression of hnRNP A1/A1B also occurred in lower
segment myometrium during both pregnancy (P < 0.001)
and spontaneous labor (P < 0.001) compared to levels
in nonpregnant myometrium.
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). Similar levels of expression were
observed in lower and upper uterine samples from nonpregnant, pregnant,
and laboring samples (Fig. 3B). Immunodetection using the H111
monoclonal antibody to a core U1 snRNP protein, U1–70K, detected bands
of 70 kDa in all tissues (Fig. 4A).
Quantification of U1–70K confirmed that similar levels of U1–70K were
expressed in both upper and lower samples of myometrial tissues from
all three patient groups (Fig. 4B).
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). Quantification of U1-A protein
levels indicated that U1-A expression decreased significantly during
pregnancy and parturition in both the lower (P < 0.01)
and upper (P < 0.01) segments of the myometrium (Fig. 5B) compared to that in nonpregnant samples.
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. The
data clearly indicate that a switch in the ratios of hnRNP
A1/A1B to SF2/ASF occurs in both lower and upper
uterine segments during gestation and parturition. The increase in the
ratio of hnRNP A1/A1B to SF2/ASF during labor in
the upper region reflects the further increase in hnRNP
A1/A1B during labor and the corresponding
decrease in SF2/ASF.
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, A and B, ii) and weak in
sections taken from the upper uterine regions during pregnancy (Fig. 7D). However, strong nuclear staining for SF2/ASF was observed in the
lower uterine regions prepared from pregnant samples (Fig. 7C),
providing further verification that SF2/ASF is spatially regulated
within the myometrium during pregnancy (Fig. 7C, ii). hnRNP
A1/A1B staining, using 4B10 mAb, was moderate in
lower and upper nonpregnant samples (Fig. 7, A and B, i). Very strong
nuclear staining for hnRNP A1/A1B was observed in
upper uterine sections from pregnant samples (Fig. 7D), consistent with
the Western blot data; negligible staining for hnRNP
A1/A1B was observed in sections taken from the
lower uterine region during pregnancy. Immunostaining for U2-B'', using
4G3 mAb, confirmed that U2-B'' protein levels were similar in upper and
lower uterine regions in nonpregnant and pregnant-nonlaboring states
and that U2-B'' expression was confined to the nucleus of myometrial
cells. Control reactions for each tissue section using a negative
control IgG1 mAb (MOPC 21) were all negative
(Fig. 7, A–D, iv).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is further confirmed by the change in
ratios of hnRNP A1/A1B to SF2/ASF.
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There have been numerous reports demonstrating that altering the ratios of hnRNP A1/A1B to SF2/ASF in vitro promotes the formation of different spliced mRNA isoforms from the same pre-mRNA transcript (33, 34, 36). Subtle fluctuations in the concentrations or ratios of SF2/ASF and hnRNP A1/A1B can alter the splicing patterns; notably, these two regulators have an antagonistic relationship and can counteract each other in a concentration-dependent manner in selecting 5'- and 3'-splice sites (34, 36, 37, 44). These studies included the use of several reporter gene constructs and have demonstrated by overexpression of SF2/ASF and/or hnRNP A1/A1B that different alternatively spliced mRNA isoforms could be generated. Furthermore, SF2/ASF and hnRNP A1/A1B levels have been shown to fluctuate in different cell types (39, 45), and two studies have looked at the variability of hnRNP A1/A1B and SF2/ASF expression in different tissues in the mouse (38) and rat (39). However, no previous studies have as yet looked at SF2/ASF and hnRNP A1/A1B levels within different human organs and tissues.
The present study supports the previously published findings that
fluctuations in the concentration and/or ratios of hnRNP
A1/A1B to SF2/ASF govern splice site selection
in vitro. The data presented in this study clearly
complement results of the in vitro studies and also provide
definitive evidence to demonstrate that this posttranscriptional
regulatory process occurs in vivo. The polarization of
SF2/ASF and hnRNP A1/A1B within the lower and
upper segments of the myometrium, respectively, may be important in
defining the functions of these different regions of the uterus at the
onset of parturition. Different regions of the uterus have distinct
functions; i.e. the upper region governs contractility, and
the lower region governs dilatation. Sparey et al. (6) have
recently shown that the myometrial gap junction protein connexin-43,
which is associated with cell-cell communication, is up-regulated in
the upper region at term, and in conjunction with the fundus to cervix
gradient of OTRs (40) is probably involved in the propagation of
contractions during parturition. Conversely, these investigators
observed the increased expression of COX-1 and COX-2. COX-2 exists as
two spliced isoforms in the lower uterine segment. The high levels of
COX-1 and -2 in the lower uterine region may be responsible for the
increased synthesis of prostacyclin/PGE2,
resulting in cervical ripening and dilatation before and during
parturition, respectively (46). Stevens et al. (18) also
reported that CRH receptor R1 levels increased significantly in the
lower region compared to the upper uterine region at preterm and term
labor. Another study comparing the regional variations of PG receptors
in the baboon myometrium (12) demonstrated that
EP3 receptor mRNA was significantly decreased in
the lower region compared to the upper uterine region, whereas
EP2 receptor mRNA was more abundant in the lower
region than the upper region during pregnancy. It is worthy of note
that EP1, EP3,
PGF2, and thromboxane receptors can all exist
as alternatively spliced isoforms (11). In this context it is also
probable that SF2/ASF and hnRNP A1/A1B are
involved in regulating the spatial expression of specific protein
isoforms of many more genes that are involved in controlling uterine
activity during gestation and labor. Future studies will be undertaken
to further elucidate the involvement of SF2/ASF and hnRNP
A1/A1B in regulating the expression of specific myometrial
proteins within the uterus.
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Acknowledgments |
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Footnotes |
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Received September 29, 1999.
Revised December 15, 1999.
Accepted December 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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proteins in the upper
and lower segments of the human uterus during pregnancy and labour. J Clin Endocrinol Metab. 84:1705–1710.s-small (with serine) and Gs-large (with
serine) messenger ribonucleic acid splice variants promote the
increased expression of 46- and 54-kilodalton Gs protein
isoforms in pregnancy and their down-regulation during labour. J
Clin Endocrinol Metab. 81:1069–1075.[Abstract]
gene are generated by alternative splicing
and promoter usage. Mol Endocrinol. 12:1939–1954.s messenger ribonucleic acid transcript during pregnancy and
labour: involvement of alternative splicing pathways. J Mol Endocrinol. 18:15–25.[Abstract]
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