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a Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
b Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the ß subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHß, LHß, and the subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHß and LHß in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHß mRNA levels, but there was no significant change in LHß or subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHß is negatively regulated by the ovaries.
follicle-stimulating hormone, gene regulation, luteinizing hormone, ovary, pituitary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß. The subunit is common among these hormones within a species and is highly conserved between species, whereas the ß subunit is specific to each hormone and confers biological specificity [1]. The presence of the two chemically distinct kinds of GTHs, FSH and LH (GTH-I and GTH-II in teleost) in nonmammalian pituitary glands has been demonstrated by purification and/or molecular cloning in birds [2–5], chelonian and crocodilian reptiles [6, 7], anuran amphibians [8–16], and teleosts [17–21]. Regarding urodele amphibians, Licht et al. [9, 22] documented the presence of FSH and LH in the salamander (Ambystoma tigrinum) but did not fully confirm the biochemical or biological properties because of the difficulty of collecting pituitaries in sufficient numbers. In our previous studies, we showed that the isoelectric focusing (IEF) profiles of pituitary GTH activity of the red-bellied newt (Cynops pyrrhogaster) determined by Xenopus and Anolis radioreceptor assays (RRAs) are markedly different from those of the bullfrog [23, 24]. Furthermore, the IEF profiles of newt GTH vary according to seasonal reproductive states; for example, the Anolis RRA gave one or two exceptional components in an acidic region only during the month of July, when spermatogenesis is active [23]. The isoelectric points of these additional components agree with those of Ambystoma FSH prepared by Licht et al. [22] when estimated by chromatographic behavior, but the acidic components are potent in stimulating testosterone production from newt testes, similar to the function of the alkaline to neutral components detected by both Xenopus and Anolis RRAs [25]. Accordingly, it is of interest to isolate cDNAs of the newt GTH subunits to ascertain the duality of the GTH molecules and to understand the physiologic role of GTH in urodele reproductive processes. The red-bellied newt is an especially good model for studying the regulatory mechanism of the amphibian hypothalamic-pituitary-gonadal axis because its gonadal function reflects the marked seasonal changes seen in many amphibian species living in the temperate zone [26–28]. In this study, we identified cDNAs encoding the subunits of FSH and LH from pituitary glands of the red-bellied newt, localized FSH and LH mRNAs in the pituitary, and examined the effects of ovariectomy on the mRNA levels in the pituitary glands.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually mature newts (Cynops pyrrhogaster) of both sexes, purchased from a commercial dealer (Wonder-up, Tokyo, Japan) and collected in Murakami, Niigata Prefecture, were kept in laboratory conditions and fed earthworms. The pituitary glands were removed under anesthesia with MS 222 (Nacalai Tesque, Kyoto, Japan) for cDNA cloning, Northern blot analysis, and in situ hybridization histochemistry. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Shizuoka University.
Construction of Pituitary cDNA Library
Total RNA was extracted from 280 pituitary glands of newts using TRIZOL RNA extraction reagent (Gibco BRL, Rockville, MD), and then 5.4 µg of poly(A)+ RNA was separated from about 160 µg of the total RNA using Oligotex-dT30 super (Takara, Kyoto, Japan). We constructed a 
ZAP cDNA library (1.4 x 106 pfu/µg of arms) from the poly(A)+ RNA using a ZAP Express cDNA synthesis kit and a Gigapack III Gold cloning kit (Stratagene, La Jolla, CA) in accordance with the manufacturer's instructions.
Oligonucleotide Primers for Polymerase Chain Reaction
Degenerate primers for the original amplification of newt FSHß and LHß fragments were designed based on the conserved regions of FSHß and LHß from other species. We also designed primers for newt subunit cDNA, which have been previously reported [29]. The following primers were commercially synthesized (Gibco BRL): FSHß primer 1 (F1), 5'-TGGTG(C/T)(A/T)(G/C)AGG(A/C/G/T)TA(C/T)TG(C/T)TA(C/T)AC-3'; FSHß primer 2 (F2), 5'-AC(A/C/G/T)GT(A/C/G/T)AA(A/G)(A/G)T(A/C/G/T)CC(A/C/G/T)GG(A/G/T)TG-3'; FSHß primer 3 (F3), 5'-CC(A/C/G/T)A(A/G)(A/C/G/T)(C/G)C(A/C/G/T)C(G/T)(A/C/G/T)AC(A/C/G/T)GT(A/G)CA(A/G)TC-3'; LHß primer 1 (L1), 5'-TG(C/T)CC(A/G/C/T)GT(A/C/G/T)TG(C/T)AT(A/C/T)AC(A/C/G/T)TT(C/T)AC-3'; LHß primer 2 (L2), 5'-CA(A/C/G/T)(C/G)(A/T)(A/C/G/T)A(A/T)(A/C/G/T)GC(A/C/G/T)AC(A/C/G/T)GG(A/G)(A/T)A-3'; subunit primer 1, 5'-CACTCTGTTCTTGCTGCTCA-3'; and subunit primer 2, 5'-GAGTCTTCTGAACTGGTGTC-3'.
Polymerase Chain Reaction Amplification and Cloning
We performed polymerase chain reaction (PCR) using the DNA prepared from the newt pituitary cDNA library in 25 µl of Ex-taq buffer containing 0.2 mM of each dNTP and 50 pmol of each of primers 1 and 2 with 0.5 units of Ex-taq polymerase (Takara), basically as described previously [30]. Nested PCR amplification was further performed using the F2 and F3 primers. The procedure of PCR amplification was an initial denaturation step of 95°C for 5 min followed by denaturation (94°C, 90 sec), annealing (50°C, 90 sec), and extension (72°C, 150 sec) for 30 cycles in a thermal cycler (ASTEC, Fukuoka, Japan). Amplified fragments were cloned into the pGEM-3z vector (Promega, Madison, WI).
Screening of the Newt cDNA Library
We synthesized DNA probes, obtained from the PCR products as described previously, using a digoxigenin (DIG)-High Prime kit (Roche Molecular Biochemicals, Meylan, France) and used them to screen the cDNA library of the newt pituitary gland, in accordance with the manufacturer's instructions. Hybridization signals were detected with 25 mM CSPD [disodium 3-(4-methoxyspiro{1, 2-dioxetane-3, 2'-(5'-chloro)tricyclo[3.3.13.17]decan}-4-yl)phenyl phosphate) chemiluminescent substrate (Tropix Inc., Bedford, MA) on Hyperfilm-ECL (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) after incubation with alkaline phosphatase-labeled anti-DIG antibody (Roche).
DNA Sequence Analysis
The cDNAs were sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA). The sequencing reactions were analyzed by an Applied Biosystems DNA sequencer model 377 (PE Biosystems). A phylogenetic tree of amino acids sequences of ß subunits of the vertebrate GTH family was constructed by the unweighted pair-group method with arithmetic mean using the Genetyx-Mac software (Software Development Co., Ltd., Tokyo, Japan).
Northern Blot Analysis
Total RNA was isolated from 10 pituitary glands and 100 mg of newt organs (brain, heart, liver, lung, stomach, testis, and ovary) using TRIZOL reagent. Seven micrograms of the total RNA from each organ was electrophoresed on a denatured gel containing 1% agarose and 2 M formaldehyde and blotted onto a nylon membrane (Roche). The RNAs were fixed on the membrane by UV cross-linking.
The membrane was prehybridized for 1 h at 50°C in prehybridization solution. Hybridization with DIG-labeled cDNA probe was performed for 15 h at 50°C, with the probe added to the prehybridization solution. The DIG-labeled cDNA probe was prepared from the full coding region of the cDNAs using of a DIG-High Primer kit, in accordance with the manufacturer's instructions (Roche). The membrane was washed once in 2x saline-sodium citrate (SSC) containing 0.1% SDS at room temperature and twice in 0.1x SSC containing 0.1% SDS for 30 min at 65°C. After a blocking step, the membrane was incubated with alkaline phosphatase-labeled anti-DIG antibody (Roche) for 30 min at room temperature, reacted with CSPD for 5 min, and then exposed on Hyperfilm-ECL (Amersham). For sequential expression analysis on the same membrane, after the first hybridization with the FSHß probe, the membrane was rehybridized with the LHß probe, the subunit probe, and then with the newt ß-actin probe. Before the membrane was hybridized with another cDNA probe, it was incubated in 50 mM Tris-HCl (pH 8.0) containing 50% formamide and 0.1% SDS for 30 min at 68°C to strip off the previously hybridized cDNA probe.
The dried films were image-scanned, and the signal intensity was quantified with a computer image analyzer (NIH-Image, version 1.62, NIH, Bethesda, MD). The relative density was expressed as the ratio of the signal intensity of each band of the GTH subunits to the ß-actin band to normalize any variation in RNA loading and transfer.
In Situ Hybridization Histochemistry
DIG-labeled antisense and sense complementary RNA (cRNA) probes were prepared from the full coding region of LHß and FSHß cDNAs by in vitro transcription, as described previously.
Newt pituitary glands were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4, overnight at 4°C. After fixation, the tissues were dehydrated through a graded alcohol series, cleared in methyl benzoate-celloidin, and embedded in Paraplast (Oxford Labware, St. Louis, MO). Sections were cut at 4 µm thickness and mounted on silane-coated slides. In situ hybridization was carried out according to a method described previously [31]. Briefly, the deparaffinized sections were digested with 5 µg/ml proteinase K for 20 min, fixed in 4% PFA for 20 min, and then incubated with the DIG-labeled cRNA at 55°C for 15 h. After hybridization, the sections were treated with 1 µg/ml RNase solution for 30 min and then incubated with alkaline phosphatase-conjugated sheep anti-DIG Fab antibody (Roche) for 15 h. The label was detected with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate (Roche).
Dual mRNA and Protein Staining
After mRNA was stained as described previously, the sections were washed with PBS and incubated with primary antibody against anti-bullfrog LHß (BL4B11) [32] or human TSHß (supplied by Dr. A.F. Parlow, Science Director, National Hormone & Pituitary Program, Torrance, CA) overnight, followed by secondary antibody conjugated with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF; Jackson Immunoresearch, West Grove, PA) or with lissamine rhodamine sulfonyl chloride (LRSC) (Jackson Immunoresearch) for 2 h. The sections were washed with PBS and then mounted in PermaFluor (Immunon, Pittsburgh, PA) and examined with an Olympus BX50 microscope equipped with a BX-epifluorescence attachment (Olympus Optical Co., Tokyo, Japan).
Experimental Protocol for Ovariectomy
To determine the effect of ovariectomy on the expression of FSH and LH by the pituitary glands, the ovaries of adult female newts were removed in the month of August. Experimental and control newts were sampled 3 and 7 days after ovariectomy, and their pituitaries were extracted.
Northern blot analysis was performed for sequential expression analysis. To examine the degree of changes in the number of mRNA-expressing cells, we performed semiquantitative analysis 7 days after ovariectomy by double-staining with in situ hybridization for FSHß or LHß and immunofluorescence staining for LHß. We counted the number of FSHß mRNA- or LHß mRNA- and LHß protein-positive cells in each section and calculated the ratio of the number of FSHß mRNA- or LHß mRNA-positive cells per total number of FSHß mRNA- or LHß mRNA- and LHß protein-positive cells in each section. The results are expressed as the means ± SEMs of five sections from five animals.
Statistical Analysis
All data are presented as means ± SEMs. We used the Student t-test for statistical analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We amplified one major fragment from the newt pituitary cDNA library by the first PCR using the F1 and F2 primers, followed by nested PCR using the F2 and F3 primers. We obtained a 122-base pair (bp) fragment as a candidate for the putative newt FSHß. The amino acid sequence deduced from this fragment showed a higher homology to mammalian and bullfrog FSHß than to the ß subunits of mammalian LH and TSH, suggesting that this fragment encodes a portion of the newt FSHß subunit. Therefore, we used this cDNA fragment as an FSHß probe for cDNA library screening. From the approximately 2 x 104 plaques screened, four positive clones for FSHß were identified, isolated, and sequenced. Since these four clones have identical open reading frames with different lengths of 3'-nontranslational regions, we show the sequence of the longest clone (Fig. 1). This clone contained a cDNA insert 2231 bp long that had an open reading frame of 387 bp. The cleavage site for the signal peptide was inferred by comparison with the data (N-terminal cysteine residue) obtained from N-terminal sequencing of bullfrog FSHß [15]. When a signal sequence of 20 amino acids was located, the proposed mature ß subunit of newt FSH started with cysteine and consisted of 109 amino acids. A putative N-linked glycosylation site was located at Asn-5 and Asn-22 from the N-terminus of the predicted mature peptide. Figure 2 shows the alignment of amino acid sequences of newt FSHß and other vertebrate FSHß. All of the 12 cysteines and the putative two N-linked glycosylation sites of newt FSHß were completely conserved among tetrapods and the Japanese eel. Similarly, 11 of 12 cysteines and the putative N-linked glycosylation site at Asn-5 were conserved among other teleost GTH-Iß, although the common carp had the additional glycosylation site at Asn-22.
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Figure 3 shows the phylogenic analysis of the ß subunits of GTHs in vertebrates. Newt FSHß belongs to the cluster of tetrapod FSHß. The homology between the newt FSHß and the bullfrog [15], Japanese quail [5], rat [33], pig [34], bovine [35], sheep [36], or human [37, 38] FSHß subunits was 56.0%, 63.4%, 59.1%, 52.3%, 59.1%, 60.0%, and 58.1%, respectively. The newt FSHß displayed respective 33.3%, 36.2%, 31.0%, and 43.1% homology with chum salmon [18], common carp (AB003583), the Japanese flounder [21], and the Japanese eel [39] GTH-Iß, corresponding to tetrapod FSHß (Fig. 3).
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Complementary DNA Cloning of the Newt LHß Subunit
We obtained a 197-bp fragment as a candidate for the putative newt LHß cDNA, which we amplified from the newt pituitary cDNA library using L1 and L2 primers. The deduced amino acid sequence from this fragment showed a higher homology to mammalian and bullfrog LHß than to the pituitary glycoprotein hormone ß subunit of other vertebrates, suggesting that this fragment is an encoded newt LHß subunit. Therefore, we used this cDNA fragment as the LHß probe for cDNA library screening. From the approximately 2 x 104 plaques screened, we identified, isolated, and sequenced three positive clones for LHß. Since these three clones have identical open reading frames with the same 3'-nontranslational regions, we show the sequence of one clone. This clone contained a cDNA insert 1780 bp long (Fig. 4) with an open reading frame of 393 bp. By comparing the sequence with that of other vertebrate LHß subunits, a signal peptide of 19 amino acids and a mature peptide of 112 amino acids were predicted for the newt LHß subunit. A putative N-linked glycosylation site was found at Asn-8 from the N-terminus of the putative mature peptide. All of the 12 cysteines and the putative N-linked glycosylated site of newt LHß were completely conserved among tetrapod LHß and teleost GTH-IIß (Fig. 5). A phylogenic analysis showed that newt LHß belongs to the cluster of teleost GTH-IIß (Fig. 3).
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The amino acid sequence homology between the newt LHß and the bullfrog [16], Japanese quail [4], rat [40], pig [41], bovine [42], sheep [43], and human [44–46] LHß subunits was 67.9%, 45.6%, 46.6%, 46.6%, 45.7%, 45.7%, and 42.2%, respectively. The newt LHß displayed respective 49.6%, 56.4%, 49.6%, and 57.8% homology with chum salmon [18], common carp [47], the Japanese flounder [21], and the Japanese eel [39, 48] GTH-IIß, corresponding to tetrapod LHß (Fig. 3).
The newt FSHß and LHß showed a similarity of 56.5% in the nucleotide sequence and 41.6% in the amino acid sequence.
Northern Analyses of FSHß and LHß Expression in Various Tissues
To determine whether the newt FSHß and LHß cDNAs were specific to the pituitary, we performed Northern blot analysis using various organs from the newt. FSHß mRNA was revealed to be 2.7 and 0.8 kilobase (kb) long, whereas LHß mRNA was 10.3 and 2.2 kb long (Fig. 6A). In addition, the subunit mRNA was 0.8 kb long. The mRNAs of these subunits were expressed exclusively in the pituitary gland; no signals were detected for mRNAs in other organs such as the brain, heart, liver, lung, stomach, testis, and ovary (Fig. 6B).
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Distribution of GTH Subunit mRNAs in the Pituitary Gland
We determined the sites of LHß mRNA expression by in situ hybridization histochemistry with a DIG-labeled antisense RNA probe (Fig. 7, a–d). The hybridization signal for LHß mRNA was distributed throughout the pars distalis, and the more intense staining was seen in the centroventral region (Fig. 7a). No distinct signal was detected in the pars intermedia and nervosa. The hybridization signal was confined to the cytoplasm, while the nucleus remained unstained (Fig. 7b). The positive cells were often round or ovoid. The number and intensity of reactions varied among the hybridization-positive cells, probably reflecting differences in mRNA expression. A similar distribution of hybridization signals was observed using the FSHß cRNA probe (Fig. 7, e–h). However, the signal was more intense in the dorsal region of the pars distalis than in the centroventral region. When the tissue section was incubated with sense LHß or FSHß probes, no hybridization signal was detected (data not shown).
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To identify cells that express LHß mRNA or FSHß mRNA, we applied fluorescence staining with anti-LHß and anti-TSHß to the same sections because no antibody against FSHß is available for staining of newt pituitary gland [49]. We observed cells that contained LHß or FSHß mRNAs in LHß-immunopositive cells, but not in TSHß-immunopositive cells, indicating that one cell could express both LHß and FSHß genes (Fig. 7, b–d, f–h).
Effect of Ovariectomy on the Expression of Subunit mRNAs of GTH in the Pituitary Gland
To elucidate a functional relationship between pituitary GTH and the ovary, we examined the effect of ovariectomy on levels of FSHß, LHß, and subunit mRNAs in the pituitary by Northern blot analysis. A representative pattern of the bands on the membranes is shown in Figure 8A. The 2.7- and 0.8-kb mRNA levels, corresponding to FSHß mRNA, increased 4- to 7-fold (P < 0.001) 3 days after ovariectomy and 3- to 4-fold (P < 0.001) 7 days after ovariectomy compared with those of the control group (Fig. 8B). For LHß mRNA levels, since the 2.2-kb LHß mRNA band was recognized as a doublet after ovariectomy, we used the sum of the two bands as the value of the 2.2-kb mRNA level. Both the 10.3- and 2.2-kb mRNA levels appeared to increase slightly 3 and 7 days after ovariectomy, except for the 10.3-kb mRNA level 3 days after ovariectomy, but there was no significant difference in mRNA levels between the ovariectomized and the control groups. Alpha subunit mRNA levels were similar in the pituitary glands in the ovariectomized and the control groups 7 days after ovariectomy.
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To confirm the Northern blot data, we examined the pituitary glands by in situ hybridization histochemistry. As shown in Figure 9, signals for FSHß mRNA increased in GTH-producing cells in the dorsal region of the pituitary after ovariectomy, whereas the signal for LHß mRNA showed a slight increase in the number and staining intensity of GTH-producing cells between the control and ovariectomized newts. A semiquantitative analysis of the data from the double-staining showed that the ratio of the number of FSHß mRNA-positive cells to the total number of FSHß mRNA- and LHß protein-positive cells increased from 12.3% ± 2.2% in control newts to 69.7% ± 1.7% in ovariectomized newts. On the other hand, the ratio of the LHß mRNA-positive cells to the total number of LHß mRNA- and LHß protein-positive cells did not change, with values of 90.0% ± 5.7% in the control newts and 93.1% ± 4.7% in the ovariectomized newts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GTH ß subunit is structurally characterized by 12 cysteine residues and N-glycosylation sites, and these structures are considered to play important roles in maintaining the molecular conformation. The crystalline structure of human chorionic gonadotropin reveals that the disulfide bond between the 3rd and 12th cysteines is essential for the assembly of the heterodimers of and ß subunits and for receptor binding [50]. On the other hand, glycosylation imparts a longer half-life to the GTH dimers to decrease their rate of clearance from circulation [51]. In newt FSHß and LHß, the number and position of cysteine residues and N-glycosylations is well conserved. In some teleost GTH-Iß, however, the third cysteine is replaced by Ser or Thr, and the glycosylation site at Asn-22 is deleted or replaced by Gln or Ser (Fig. 2). The CAGYC sequence, conserved in the ß subunits of almost all mammalian FSH and LH, has been suggested to be involved in the subunit binding site [1]. However, the sequence is replaced by CSGYC in both newt FSHß and LHß as well as in bullfrog FSHß. Bullfrog LHß has the CTGYC sequence. Similarly, some variations are seen in other animals: CGGYC in quail LHß, CAGLC or CAGQC in teleost GTH-IIß, and CSGHC in teleost GTH-Iß. Thus this sequence is slightly modified among species, but it is thought to be important for the binding site of and ß subunits. In future studies, the exact role of this sequence should be clarified. Certainly, the highly conserved structure between the newt LHß and FSHß implies that they are derived from a common ancestral molecule. From phylogenetic and homology analysis, the deduced amino acid sequences of newt FSHß showed higher homology to tetrapod FSHß than to teleost GTH-Iß, and the newt LHß showed higher homology to bullfrog LHß and teleost GTH-IIß than to other tetrapod LHß. This suggests that the newt FSH and LH each locates phylogenetically at a transitional position for linking between teleost GTH and higher tetrapod GTH. Newt FSHß has a 41.6% homology with newt LHß, slightly greater than the homology of FSHß and LHß in other tetrapod animals (32.3%–39.5%). Since the ß subunit determines the biological specificity of FSH and LH, the homology among ß subunits within a species is expected to be low. Therefore, this finding supports previous physiologic data showing less specificity of amphibian FSH and LH in gonadal function [9, 25, 52].
In this study, we investigated the expression of newt GTHß mRNAs using the present cloned cDNA probes. Northern blot analysis revealed that the ß subunits of FSH and LH are expressed specifically in pituitary glands among the various newt tissues and showed the presence of two mRNAs of different sizes for LHß and FSHß. The mechanism of production of mRNAs of different sizes is currently unknown. In the present study, however, we obtained two clones with 0.8- and 2.2-kb sizes (data not shown) during the cloning of newt FSHß that were consistent with the mRNAs (0.8 and 2.7 kb) detected by Northern blot analysis. These two clones have identical coding regions with poly(A)-tailed sequences added to different sites of nontranslational regions. In addition, two sites of poly(A) signal sequence are found in the 3'-nontranslational region of FSHß cDNA (Fig. 1, boxed). Therefore, the two different sizes of mRNAs may be derived from differential uses of the poly(A) sites or differences in the length of 5'-nontranslational region and polyadenylation. On the other hand, LHß mRNAs were detected at the 2.2- and 10.3-kb bands on the Northern blot. Since in situ hybridization histochemistry for LHß mRNA did not stain the nucleus, it is not possible that the 10.3-kb band is a presplicing type. The band may be derived from differences in the 5'-nontranslational region, in the 3'-nontranslational region and/or in polyadenylation. The Northern blot data showing that two sizes of mRNA for FSHß and LHß behave similarly after ovariectomy suggest that these two mRNAs are specific expressions.
Immunocytochemical studies have shown that both ß subunits of FSH and LH coexist in the same GTH-producing cells of anuran amphibian pituitary glands as well as in those of mammalian species [53–56]. However, FSHß-immunopositive cells have not been identified in urodele pituitary glands because no anti-FSHß sera reacts with any urodele pituitary cells [49]. In the present study, we clearly demonstrated that LHß and FSHß were coexpressed in the same GTH-producing cells of newt pituitary glands when in situ hybridization was followed by immunohistochemistry for LHß. The present in situ hybridization also indicated that the number of FSHß mRNA-positive cells is less than the number of LHß mRNA-positive cells and that expression of each ß subunit varies from cell to cell, suggesting that FSHß and LHß are synthesized by different regulatory factors in individual cells. Moreover, this finding may reflect seasonal variation, as we found seasonal differences in the IEF profiles of newt GTH [23]. Further studies are necessary to clarify seasonal changes on the expression of FSHß and LHß mRNAs.
In mammals, the synthesis and secretion of pituitary GTHs are regulated by gonadal steroids and some peptide factors including inhibin [57]. In amphibians, McCreery and Licht [58] and Pavgi and Licht [59] found direct evidence for gonadal negative feedback control of plasma GTH in anuran species using a radioimmunoassay, but little is known of the regulation of biosynthesis of the GTH subunits at the pretranslational levels [60]. In this study, we demonstrate different regulation of FSHß and LHß at the mRNA levels by the patterns of changes in FSHß and LHß mRNA levels. In ovariectomized newts, FSHß mRNA levels increased dramatically, whereas LHß and subunit mRNA levels rose slightly by 7 days postovariectomy, although there were no significant differences compared with the control group. In addition, we detected the 2.2-bp band of LHß mRNA as a doublet after ovariectomy. The appearance of this band may be due to differential promoter use and/or alternative splicing for LHß mRNA expression. The Northern blot data is confirmed by the semiquantitative data from the in situ hybridization indicating that the number of FSHß mRNA-positive cells increased after ovariectomy, whereas that of LHß mRNA-positive cells did not differ between the control and ovariectomized groups. In mammals, it is accepted that gonadal steroid hormones including testosterone, dihydrotestosterone, and estradiol-17ß negatively regulate FSHß and LHß mRNA levels, whereas gonadal inhibin negatively regulates only FSHß mRNA levels. In the newt ovary, therefore, the FSHß mRNA expression may be regulated negatively not only by steroid hormones, but also by inhibin. Recently, Uchiyama et al. [61] reported that inhibin B suppresses activin-induced release of FSH from dispersed anterior pituitary cells of the bullfrog, although inhibin B also suppresses the release of LH and has not yet been identified in amphibian ovary in situ. In our study in newts, on the other hand, LHß mRNA levels did not fully increase after ovariectomy. This may occur because the reduction in plasma steroid hormones induced by ovariectomy is insufficient to increase LHß mRNA levels, but there is evidence that in amphibians, plasma steroid hormone levels are very low or undetectable 7 days after gonadectomy [62, 63]. On the other hand, the change in mRNA levels observed in this study correlates well with the secretory changes of FSH and LH from bullfrog pituitary: in ovariectomized bullfrogs, plasma FSH increases after 4 days, whereas plasma LH only barely increases after a month [58]. Taken together, these data indicate that the negative feedback system by steroid hormones may be weakly regulated in amphibians, including newts. Indeed, the steroidogenic activity in the newt persists at high levels over several months [29, 64, 65]. To clarify this issue, it is necessary to perform the same experiments with different periods at different stages of the ovarian cycle.
It has been shown that the pituitary glands of the urodeles, including the newt, possess activities in a Xenopus ovulation assay and in testosterone production by the testes that are similar to that of bullfrog LH [52, 66]. Newt pituitary LH, therefore, should be involved in the regulation of gonadal function, even though LHß mRNA levels did not change after ovariectomy in this study.
Taken together, these data suggest that the reproductive process of the urodele is regulated by two kinds of GTH, namely FSH and LH, both of which are secreted by the same GTH-producing cells in the pars distalis.
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FOOTNOTES |
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1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan to S.T. 
2 Correspondence: Shigeyasu Tanaka, Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan. FAX: 81 54 238 0986; sbstana{at}ipc.shizuoka.ac.jp
Accepted: November 11, 2001.
Received: September 13, 2001.
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 B. Querat, Y. Arai, A. Henry, Y. Akama, T. J. Longhurst, and J. M.P. Joss Pituitary Glycoprotein Hormone {beta} Subunits in the Australian Lungfish and Estimation of the Relative Evolution Rate of These Subunits Within Vertebrates Biol Reprod, February 1, 2004; 70(2): 356 - 363. [Abstract] [Full Text] [PDF]
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Y. Yaoi, M. Suzuki, H. Tomura, S. Kurabuchi, Y. Sasayama, and S. Tanaka Expression and Localization of Prohormone Convertase PC1 in the Calcitonin-producing Cells of the Bullfrog Ultimobranchial Gland J. Histochem. Cytochem., November 1, 2003; 51(11): 1459 - 1466. [Abstract] [Full Text] [PDF]
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 Y. Yaoi, M. Suzuki, H. Tomura, Y. Sasayama, S. Kikuyama, and S. Tanaka Molecular Cloning of Otoconin-22 Complementary Deoxyribonucleic Acid in the Bullfrog Endolymphatic Sac: Effect of Calcitonin on Otoconin-22 Messenger Ribonucleic Acid Levels Endocrinology, August 1, 2003; 144(8): 3287 - 3296. [Abstract] [Full Text] [PDF]
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H.F. Vischer, A.C.C. Teves, J.C.M. Ackermans, W. van Dijk, R.W. Schulz, and J. Bogerd Cloning and Spatiotemporal Expression of the Follicle-Stimulating Hormone {beta} Subunit Complementary DNA in the African Catfish (Clarias gariepinus) Biol Reprod, April 1, 2003; 68(4): 1324 - 1332. [Abstract] [Full Text] [PDF]
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