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Mechanisms of Hormone Action |
a Department of Tissue Physiology
b Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
c Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan
d Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, follistatin, growth factors, placenta
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 amino acids, have 10 conserved cysteine residues [15, 16], and are thought to have growth factor-binding motifs. Mouse FLRG fusion proteins bind to activin A and BMP-2 [8], and blocked the activities of acitvin A, activin B, activin AB, and BMP-2 [8, 17]. Human FLRG protein also binds to activin [9, 10]. Therefore, FLRG protein has been postulated as a new TGFß family binding protein, which modulates actions of the growth factors. However, little is known about the physiology of FLRG and its roles in reproductive events. In humans, FLRG mRNA is abundantly expressed in the placenta compared with other tissues [7, 8]. In contrast, expression of follistatin in the rat placenta is very low, although the decidua abundantly expresses follistatin mRNA [18, 19]. These data suggest that FLRG rather than follistatin has an important role in the regulation of TGFß family members in the placenta. FLRG is also expressed in the mouse ovary [8, 11], suggesting that FLRG plays physiological roles in the regulation of ovarian function as follistatin does. To elucidate the physiological aspects of FLRG in reproductive tissues, we characterized rat FLRG cDNA and examined expression of FLRG mRNA in the rat placenta and decidua during the second half of pregnancy. We also examined changes in FLRG expression in the ovary during the estrous cycle. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult cycling female rats of the Wistar strain weighing 250–300 g (3–4 mo of age) were used in this study. Rats were kept under controlled temperature (25°C ± 2°C) and lighting (14L:10D, lights-on at 0500 h) conditions, and food and water were available ad libitum. Vaginal smears were checked daily, and only rats that showed at least two consecutive 4-day estrous cycles were used. To obtain pregnant rats, each female rat was transferred to the cage of a single male on the day of proestrus and was left there overnight. Mating was indicated the next morning by the presence of spermatozoa in the vaginal smear or a vaginal plug; this day was designated as Day 0 of pregnancy. On Days 12, 14, 16, 18, and 20 of pregnancy, rats were decapitated under ether anesthesia, and placentas and decidual tissues were collected. On each sampling day, five placentas and five decidual tissue samples were collected from three pregnant rats. The tissues were immediately frozen in liquid nitrogen and were stored at -80°C until isolation of total RNA. To examine tissue distribution of FLRG mRNA in the rat, the adrenal gland, brain, heart, small intestine, kidney, liver, lung, skeletal muscle, ovary, and pituitary gland were collected from cycling female rats at random stages of the estrous cycle. Testis was collected from a 4-mo-old male rat. The tissues were immediately frozen in liquid nitrogen and stored at -80°C until isolation of total RNA. Total RNA was isolated with TRIzol reagent (Life Technologies, Tokyo, Japan) and quantified by spectrophotometric measurement at 260 nm. The ovarian samples used to qunatify FLRG mRNA during the rat estrous cycle had been obtained and examined previously [20].
The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared at Tokyo University of Agriculture and Technology.
Cloning of Rat FLRG cDNA
Rat FLRG cDNA was obtained using polymerase chain reaction (PCR)-based methods, i.e., 5' and 3' rapid amplification of cDNA ends (RACE). Placental total RNA was isolated with TRIzol reagent from placentas on Day 20 of pregnancy, then poly(A)+ RNA was prepared with Oligotex-dT30 Super (Roche Diagnostics, Tokyo, Japan). For 3'-RACE, 1 µg placental poly(A)+ RNA sample was primed with a 3' adapter primer (5'-GGCCACGCGTCGACTAGTAC(T)17-3') and reverse transcribed using superscript II reverse transcriptase (Stratagene, La Jolla, CA). The resultant single-stranded cDNA was used as the template for 3'-RACE. For 5'-RACE, placental poly(A)+ RNA was reverse transcribed with superscript II transcriptase in the presence of oligo (dT)15 primer, and double-stranded cDNA was prepared using the Universal Riboclone cDNA Synthesis System (Promega, Tokyo, Japan). Both ends of the double-stranded cDNA were blunted with T4 DNA polymerase (Promega) and phosphorylated with T4 polynucleotide kinase (Promega). Thereafter, an adapter was ligated to both ends of the cDNA with T4 DNA ligase (Promega). The structure of the adapter was as follows: 5'-GGCCACGCGTCGACTAGTACGGGCAGGT-3', 3'-NH2-CCCGTCCA-5'. For both 3'- and 5'-RACE, the identical adaptor primer (5'-GGCCACGCGTCGACTAGTAC-3') was used to amplify FLRG cDNAs. The gene-specific primers for rat FLRG were designed with reference to two rat EST clones (GenBank/EMBL/DDBJ accessions AA965262, AA997007), which were very similar to human FLRG cDNA (Fig. 1A). The sequences of the rat FLRG-specific primers are as follows: sense, 5'-TTCCGGCAACATCAACACAGC-3'; antisense, 5'-TCTGATCCACAAGGCACGACTG-3'. Resultant RACE products were subcloned into a pGEM-T easy Vector (Promega) and subjected to sequence analysis.
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Northern Blot Hybridization
Northern blot hybridization was done using a digoxigenin (DIG)-labeled cDNA probe. The 371-base pair (bp) rat FLRG cDNA probe was labeled with DIG PCR labeling mix (Roche). The rat FLRG sense and antisense primers were used for labeling. The plasmid that contains the 5'-RACE product was used as a template.
Five micrograms of rat placental poly(A)+ RNA was fractionated by electrophoresis through a 1.2% agarose gel containing 0.74 M formaldehyde under denaturating conditions. Fractionated RNA was transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Tokyo, Japan) by a capillary technique and fixed on the membrane by ultraviolet light (1200 mJ/cm2). The membrane was incubated in a prehybridization buffer (50% formamide, 1 M NaCl, 1% SDS, 10 mM Tris-HCl, pH 7.6, 200 µg/ml sheared salmon sperm DNA, 10% dextran sulfate) for 4 h at 42°C and then hybridized overnight to a DIG-labeled double-stranded rat FLRG cDNA probe at a conentration of 50 ng/ml at 42°C in prehybridization buffer. After washing three times with 2x saline-sodium citrate (SSC; 1x SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) at 70°C, the membrane was reacted with ovine anti-DIG antibody conjugated with alkaline phosphatase (Roche) according to the procedures recommended by the manufacturer. Disodium 3-(4-methoxyspiro {1, 2 dioxetane-3,2'-(5'-chlolo)triclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD; Roche) was used as a substrate. Membranes were exposed to x-ray film RXU (Fujifilm, Tokyo, Japan) for 12 h.
RNA Probes
The rat FLRG cDNA obtained by the 3'-RACE was digested with PvuII, and the 289-bp fragment was subcloned into a pBluescript II SK(+) vector (Stratagene). The pBluescript KS(-) vector containing the 393-bp rat follistatin cDNA was prepared as described previously [20]. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clone was obtained by reverse transcription PCR using the following rat GAPDH primers: sense, 5'-ACCACAGTCCATGCCATCAC-3'; antisense, 5'-TCCACCACCCTGTTGCTGTA-3'; and the PCR product was subcloned into a pGEM-T easy vector. The GAPDH cDNA was digested with ApaI, and the 171-bp fragment of rat GAPDH cDNA was subcloned into a pBluescript KS(-) vector as a template for the RNA probe. For in vitro transcription, the vectors containing the 289-bp rat FLRG cDNA and 393-bp rat follistatin cDNA were linearized with BamHI. DIG-labeled antisense probes were synthesized with T3 or T7 RNA polymerase in the presence of DIG RNA Labeling Mix (Roche; final concentration: 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.65 mM UTP, 0.35 mM DIG-UTP) according to the procedure recommended by the manufacturer. For the synthesis of DIG-labeled GAPDH antisense RNA probe, the vector was linearized with Asp718 (Roche), and T7 RNA polymerase was used. Incorporation of DIG into the GAPDH antisense probe was decreased by using an NTP solution containing a low concentration of DIG-UTP (final concentration: 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.93 mM UTP, 0.07 mM DIG-UTP). The reaction was terminated by adding RNase-free DNase I (Roche). The probe was purified by passing it through a 5% acrylamide denaturating gel. Probe RNA was eluted out of the gel with probe elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.2% SDS) for 2 h at 37°C.
RNase Protection Assay
RNase protection assays using DIG-labeled RNA probes were conducted according to the procedure of Wundrack and Dooley [21], with modifications as previously described [20, 22]. Aliquots of the DIG-labeled RNA probes (2 ng for FLRG and follistatin, 5 ng for GAPDH) were added to a known amount of total RNA (40 µg) and precipitated with ethanol. GAPDH probe was added to all tubes as an internal standard control. The resulting pellet was air dried and then dissolved in 10 µl hybridization solution containing four parts deionized formamide and one part 5x stock solution (200 mM piperazine-N,N'-bis[2-ethane-sulfonic acid], pH 6.4, 2 M NaCl, 5 mM EDTA). Samples were denatured for 5 min at 90°C and then hybridized at 45°C overnight. After hybridization, 100 µl RNase digestion buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM sodium acetate, 0.33 kunitz units/ml RNase A, 100 units/ml RNase T1) was added. Single-stranded RNA was then digested for 30 min at 37°C. RNases were inactivated by adding 5 µl 10% SDS and 1.25 µl proteinase K solution (10 mg/ml in 10 mM Tris-HCl, pH 7.5). After incubation at 37°C for 30 min, protected RNA probes were precipitated with ethanol in the presence of 10 µg yeast RNA. The resulting precipitate was redissolved in 8 µl gel loading buffer (95% deionized formamide, 0.5% SDS, 0.5 mM EDTA, 0.025% xylene cyanol, 0.025% bromophenol blue) and then denatured at 90°C for 5 min. The sample was then separated on a 6% denaturating polyacrylamide gel, electrotransferred to a nylon membrane (Highbond N+), and cross-linked by ultraviolet illumination onto the membrane. The membrane was then reacted with ovine anti-DIG antibody conjugated with alkaline phosphatase (Roche) according to the procedures recommended by the manufacturer. Signals were detected according to the recommended procedures using CSPD as a substrate. Membranes were exposed to x-ray film RXU for 6 h. The intensity of the bands was measured using a laser densitometer.
Statistical Analysis
Values are presented as means ± SEM. To compare the mean values, results were subjected to an ANOVA, followed by a Student-Neuman-Keuls test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Schemes of 5'- and 3'-RACE products and rat FLRG mRNA are illustrated in Figure 1B. The nucleic acid sequence of rat FLRG mRNA has been registered in the DNA database of Japan (DDBJ, accession no. AB071603). The RACE products revealed that rat FLRG mRNA contains an open reading frame consisting of 771 bases. A polyadenylation signal was found 1073 bases downstream from the stop codon. The nucleic acid sequence of the coding region is identical to the previously registered rat FLRG mRNA sequence (GenBank/EMBL/DDBJ accession no. AB021295). The 3' untranslated region (UTR) of rat FLRG mRNA contains three copies of the AUUUA pentamer, which is thought to destabilize mRNAs [23, 24], within 52 bases (Fig. 1B, arrowheads). Northern blot hybridization revealed that FLRG mRNA in the rat placenta is approximately 2.0 kilobases (kb) in length, and the size is consistent with that of the RACE products (Fig. 1C). Rat and mouse FLRG mRNA encodes 256 amino acid residues, whereas human FLRG encodes 263 amino acid residues (Fig. 2A). Although rat, mouse, and human FLRGs show noticeable similarities, they are rather diverse compared with the follistatins. The deduced amino acid sequence of rat FLRG shares 96.9% and 84.7% homology with those of mouse and human FLRGs, respectively, whereas the amino acid sequence of rat follistatin shares 99.1% and 97.7% homology with those of mouse and human follistatins, respectively. The amino acid sequences of rat FLRG and rat follistatin are aligned in Figure 2B. Positions of the cysteine residues are highly conserved between these two proteins. As in mouse and human FLRGs [8], rat FLRG protein contains two FS domains characterized by 10 conserved cysteine residues and lacks the heparin-binding site [25], which is found in follistatins. FS domains of rat FLRG protein and follistatin are aligned in Figure 2C. As observed in mouse FLRG [8], the first and second FS domains of rat FLRG most resemble the first and second FS domains of follistatins, respectively.
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Distribution of Rat FLRG mRNA
The RNase protection assay revealed that rat FLRG mRNA was abundantly expressed in the placenta (Fig. 3). Relatively strong expression of FLRG mRNA was also observed in the adrenal gland, lung, ovary, testis, and uterus. FLRG mRNA was weakly expressed in the heart, kidney, pituitary gland, and small intestine and was not detected in the brain, liver, and skeletal muscle.
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Changes in FLRG and Follistatin mRNAs in the Placenta and Decidua During the Second Half of Rat Pregnancy
During the second half of pregnancy, expression of FLRG mRNA in the rat placenta continuously increased (Fig. 4), whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter (Fig. 5). FLRG was also expressed in the decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20 (Fig. 4). In contrast, follistatin mRNA was strongly expressed in the decidua on Day 12 of pregnancy, but expression continuously decreased until Day 16 and remained at relatively low levels thereafter (Fig. 5). As observed in previous studies [18, 19], follistatin mRNA was strongly expressed in the decidual tissue but weakly expressed in the placenta. However, FLRG mRNA was abundantly expressed in both the placenta and decidua during late pregnancy.
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Changes in FLRG mRNA in the Ovary During the Rat Estrous Cycle
In rats used in the present study, the primary FSH surge was observed at 1700 h on the day of proestrus, and the secondary surge of FSH occurred during the early morning of estrus (Fig. 6C). Levels of ovarian FLRG mRNA were higher during the day than at night, and the changes likely depended on the time of day rather than the stage of the estrous cycle (Fig. 6, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With respect to the amino acid sequences, rat, mouse, and human FLRGs are more diverse than the follistatins, suggesting that functions of FLRG proteins are less susceptible to replacement of amino acid residues than are follistatins. As in the human and mouse, the positions of cysteine residues are highly conserved between FLRG and follistatin in the rat. The conserved positions of cysteine residues suggest similarities in their tertiary structures and, therefore, functions. As in human and mouse FLRGs, rat FLRG protein lacks the third FS domain, which is found in follistatins.
As observed in humans [7], rat FLRG mRNA was highly expressed in the placenta. In addition, the distribution pattern of FLRG is generally consistent with that of human and mouse FLRG [7, 8, 11]. Although the follistatin-expressing tissues [26, 27] and FLRG-expressing tissues overlap, the expression pattern of FLRG is apparently different from that of follistatin. Furthermore, changes in FLRG mRNA in the placenta and decidua were quite different from that of follistatin mRNA. These data suggest that FLRG and follistatin differently regulate physiological events. This hypothesis is supported by results of a previous study, in which changes in FLRG and follistatin mRNAs were observed in mouse skin during wound healing [11].
Previous studies revealed that activin subunits are expressed in both the placenta [28–31] and decidua [31]. Furthermore, activin affects functions of placental cells [32–34]. In addition, mRNAs for BMPs have been found in mouse decidua [35–37]. These previous findings suggest that activins and BMPs play important roles in the regulation of placental and decidual function and that their binding proteins should play roles in inhibiting their effects.
In the present study, follistatin mRNA was abundantly expressed in the decidua, but its expression in the placenta was relatively weak. These results are consistent with those of previous reports [18, 19]. The changing patterns of folllistatin mRNA expression in these tissues are also consistent with previous data [19]. In contrast, FLRG mRNA in the placenta and decidua increased during the second half of pregnancy. Because follistatin and FLRG share common ligands, the results suggest that FLRG protein may compensate for the decrease in follistatin in the placenta and decidua during the second half of pregnancy. However, our previous results suggest that affinities of these proteins for TGFß family proteins are probably different [8], and our preliminary data suggest that human and rat FLRG proteins do not suppress pituitary FSH secretion. In addition, FLRG protein lacks a heparin-binding site, which is found in the first FS domain of follistatin [25]. Follistatin binds to cell-surface heparan sulfate proteoglycans via the heparin-binding site [38], and the biological activities of follistatins are considerably affected by this heparin-binding ability [5]. Sidis et al. [13] also reported differences in biological activities between follistatin and human FLRG fusion proteins. Furthermore, it is not yet known whether the growth factors to which follsitatin and FLRG proteins can bind overlap. Therefore, the shift from follistatin to FLRG may alter the growth factor milieu and may produce a more preferable environment for placental and decidual function during the second half of pregnancy.
The changing pattern of FLRG mRNA in the ovary during the estrous cycle was also different from that of follistatin mRNA [20]. Ovarian FLRG mRNA did not show clear cyclic changes, while ovarian follistatin mRNA showed estrous stage-dependent changes [20]. Changes in ovarian FLRG mRNA likely depend on time of day rather than stages of the estrous cycle. Although the factors regulating ovarian FLRG mRNA expression are unknown, humoral factors, which show circadian changes, may be involved this regulation. Because both melatonin receptor [39, 40] and glucocorticoid receptor [41] are expressed in the ovary, ligands of these receptors are possible factors regulating ovarian FLRG mRNA expression. Identification of cell types that express FLRG mRNA will be help elucidate factors regulating ovarian FLRG mRNA expression, and such investigations are currently being conducted. In addition to identifying FLRG-expressing cells in the ovary, measurement of ovarian FLRG protein levels will be of interest because ovarian follistatin-like immunoreactivity levels are not always correlated with ovarian follistatin mRNA levels [19].
In the present study, we determined the entire structure of rat FLRG mRNA. The results suggest that rat FLRG plays important roles in the regulation of reproductive events, especially in the placenta and decidua during the latter half of pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Koji Y. Arai, Center for Reproductive Sciences, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 7180; karai{at}kumc.edu, kojiarai{at}cc.tuat.ac.jp
Received: 17 June 2002.
First decision: 15 July 2002.
Accepted: 5 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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