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a Center for Reproductive Science, Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and the unique FSHß and LHß subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHß, LHß, and subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHß mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHß. Neither serum LH nor pituitary LHß mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHß and subunit mRNA accumulation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FSH and LH are glycoprotein hormones comprised of a common subunit and distinct ß subunits that confer biological specificity (FSHß and LHß) (see [9] for review). In most, but not all, situations where serum levels of FSH or LH are elevated, there are increases in mRNA levels of their corresponding ß subunits. For example, in rats, postcastration increases in serum FSH and LH are associated with elevated pituitary FSHß and LHß mRNA levels [10,11]. In addition, gonadotropin ß subunit mRNA levels are increased in association with the gonadotropin surges on the afternoon of proestrus in female rats [12,13].
The subunit cDNA has recently been cloned from the pars tuberalis of Siberian hamsters [14]; however, neither gonadotropin ß subunit has been characterized in this species. As a result, the extent to which changes in pituitary subunit mRNA accumulation contribute to the observed differential regulation of LH and FSH secretion is unknown. The goals of the present study were 2-fold. First, we characterized partial cDNAs for all three gonadotropin subunits in Siberian hamsters. Second, we measured pituitary mRNA levels for all three subunits during the transition from short to long days when FSH, but not LH, levels are elevated. The results show that and FSHß subunits, but not LHß subunit, mRNA levels increase in the pituitary of Siberian hamsters transferred from short to long days.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Siberian hamsters derived from our breeding colony at Northwestern University were used in all experiments. Specific treatments of the animals are presented below where appropriate. In general, animals were born into long days (16L:8D; lights-on 0500 h CST) and weaned at postnatal day 18. Thereafter, animals were group-housed with same-sex siblings. Food (laboratory chow; Harlan Teklad, Madison, WI) and water were provided ad libitum.
To examine photoperiodic changes in gonadotropin subunit mRNA levels, long-day male hamsters were weaned 18 days after birth and immediately transferred to short days (6L:18D; lights-on 0900 h CST). After 4 wk animals were transferred back to long days by advancing light onset by 4 h and delaying light offset by 6 h. Hamsters were killed 2 h after lights were illuminated on the morning of the 2nd, 5th, 7th, 10th, 15th, or 20th long day. From the time of weaning, long-day (LD) controls remained on long days while short-day (SD) controls were maintained in short days. Both LD and SD controls were age-matched to animals killed after stimulation with 20 long days. Animals (N = 3–5 per group) were killed with CO2 and exsanguinated via cardiac puncture. Blood was allowed to clot overnight at 4°C, and serum was collected following centrifugation. Serum FSH and LH levels were measured in duplicate by radioimmunoassay using previously described methods (e.g., [8]). Intraassay coefficients of variation were between 8% and 16% for the FSH and LH assays. Pituitaries were extracted, frozen immediately on dry ice, and stored at -80°C until gonadotropin subunit mRNA levels were measured by lysate ribonuclease protection assay (RPA) (see below). Testes were removed and weighed.
All hamsters were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
RNA Extraction
Total RNA was extracted from a variety of adult Siberian hamster tissues, including whole pituitary glands, hypothalami, testes, kidneys, adrenals, and livers. Pituitaries and adrenals were pooled from several adult, long-day males and females. RNA was extracted with Trizol (Gibco BRL, Life Technologies, Gaithersburg, MD) following the manufacturer's instructions and resuspended in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). RNA used in RT-PCR analyses (see below) was further treated with RQ1 RNase-free DNase (Promega, Madison, WI). RNA concentration was determined spectrophotometrically at 260 nm. Samples with A260/A280 < 1.6 were not used.
Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR)
Partial cDNAs for the three gonadotropin subunits and ribosomal protein L19 (RPL19; see [15]) in Siberian hamsters were generated using an RT-PCR-based cloning strategy. Pituitary or hypothalamic (RPL19) RNA (3–5 µg) was reverse transcribed into single-stranded DNA using 50 ng random hexamer oligonucleotide primers and 200 U Maloney murine leukemia virus-RT (Promega) in 50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM deoxynucleotide triphosphates (dNTPs), and 20 U RNasin (Promega) at 23°C for 10 min, 37°C for 60 min, and 70°C for 10 min.
Four microliters of the 20-µl RT reaction was used in a 50-µl PCR reaction containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 600 nM forward and reverse gene specific deoxyoligonucleotide primers (Oligo-To-Go; Cruachem, Dulles, VA), and 2.5 U Amplitaq polymerase (Perkin-Elmer, Roche, Branchburg, NJ). Primers were designed from highly conserved regions of the rat, mouse, and human cDNA sequences. The primer sequences and predicted product sizes are shown in Table 1. For PCR, tubes were incubated at 94°C for 2 min and then subjected to 35 cycles of 94°C for 30 sec to 1 min, 55 or 60°C for 30 sec to 1 min, and 72°C for 1 min. After a final 10-min extension step at 72°C, 10–40 µl were run on low-melt agarose gels. Bands of the predicted size were cut out and purified. Corresponding bands were not observed in tubes from which RT enzyme or RNA was omitted.
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Cloning and Sequencing
Purified PCR products were blunt-end cloned into the Srf I site of pCR-Script Amp SK+ following the manufacturer's instructions (Stratagene, La Jolla, CA). Briefly, PCR products were blunt-ended with Pvu polymerase and then ligated into the vector in the presence of T4 DNA ligase and Srf I for 1 h at room temperature. DNA was then introduced into XL-1 Blue MRF' Ultracompetent cells by heat-shock transformation. Recombinant bacterial colonies were screened by blue-white color selection and subsequent restriction digest of purified plasmid DNA. DNA from positive colonies was sequenced from both strands by Big Dye Terminator cycle sequencing (ABI; Perkin-Elmer).
Northern Blot Analysis
Expression of the gonadotropin subunits in various tissues was determined by Northern blot analysis. Hypothalamic, pituitary, adrenal, kidney, liver, and testis RNA (20 µg each) were electrophoresed through a 1.5% agarose, 3.3% formaldehyde gel. RNA was transferred overnight to a nylon membrane (Nytran; Schleicher & Schuell, Keene, NH) by capillary action. The filter was UV-cross-linked (Stratalinker; Stratagene) and baked at 80°C for 2 h. The blot was sequentially hybridized with probes made from the species-specific clones random prime-labeled with [32P]dCTP (3000 Ci/mmol, 10 mCi/ml; Amersham, Arlington Heights, IL) using Ready-To-Go Beads (Pharmacia Biotech, Piscataway, NJ). Hybridization was performed overnight at 42°C in 50% formamide, 5-strength saline-sodium citrate (SSC; single strength is 0.15 M NaCl and 0.015 M sodium citrate), 1-strength Denhardt's, 20 mM NaPO4 (pH 6.8), 1% SDS, 5% dextran sulfate, denatured 100 µg/ml salmon sperm DNA, and 1–2 x 106 cpm/ml probe. Filters were washed consecutively for 30 min each in double-strength SSPE (0.36 M NaCl, 20 mM NaH2PO4, 20 mM EDTA)-0.5% SDS at room temperature, double-strength SSPE-0.5% SDS at 65°C, and 0.2-strength SSPE-0.1% SDS at 65°C. Filters were exposed to X-OMAT film (Eastman Kodak, Rochester, NY) for 7–20 h at -80°C with two intensify screens. The filter was probed consecutively with the LHß, FSHß, RPL19, and probes. Between hybridizations, probe was removed by washing the blot in 55% formamide, double-strength SSPE, and 1% SDS at 65°C for 1 h. The filter was then rinsed briefly in single-strength SSC-0.1% SDS.
Lysate Ribonuclease Protection Assay
The three gonadotropin subunit mRNA levels were measured from individual pituitaries of photostimulated hamsters (see Experimental Design above) in a lysate RPA using the Direct Protect reagents from Ambion (Austin, TX). Briefly, each pituitary was homogenized in 50 µl of lysis/denaturation buffer. Thirty microliters of lysate was transferred to a fresh 1.5-ml tube and 20 µl of lysis/denaturation buffer containing FSHß (105 cpm), LHß (105 cpm), (5 x 104 cpm), and RPL19 (105 cpm) antisense riboprobes (see below) was added to each tube. Following an overnight hybridization at 37°C, samples were digested with RNase A/T1 cocktail at 37°C for 30 min, treated with sodium sarcosyl and proteinase K for 30 min at 37°C, and precipitated with isopropanol for 30 min at -20°C. Following centrifugation, pellets were resuspended in 20 µl RNA gel loading dye and heated for 3 min at 85°C. Fifteen microliters of each sample was run on 5% acrylamide, 8 M urea gels. Gels were dried and exposed to BIOMAX film (Kodak) for 2 days. Films were then aligned to the dried gels, the bands cut out, and cpm determined by scintillation counting. Values for FSHß, LHß, and were normalized by dividing by the RPL19 value in the same lane. Pituitary RPL19 mRNA levels were shown in previous analyses to be unaffected by photoperiod manipulation (data not shown). Data are presented as fold-change from short-day control values.
Riboprobe Preparation
Plasmids containing the gonadotropin subunits and RPL19 cDNAs were linearized with either NotI or BamHI, and probes were transcribed in vitro in the presence of [32P]UTP (800 Ci/mmol, 10 mCi/ml; NEN, Boston, MA) with T3 or T7 RNA polymerase (T3/T7 MAXIscript kit; Ambion). The FSHß, LHß, and RPL19 probes were synthesized to a specific activity of 4 x 108 cpm/µg and to 1.9 x 108 cpm/µg. Because the FSHß and LHß clones contain inserts of roughly equivalent size and would protect fragments of the similar size, an LHß clone containing a 341-base pair (bp) insert was used to generate riboprobes for the assay. This clone was generated in the course of the original cloning but was found to be truncated 35 bp at the 5' end and 8 bp at the 3' end relative to the full-length 384-bp LHß PCR product. The sequences were otherwise identical.
Statistical Analyses
Serum FSH and LH, paired testis mass, body mass, and relative gonadotropin subunit mRNA levels were all analyzed by one-way ANOVA. Post-hoc comparisons were made with Fisher's Least Significant Difference procedure. Hormone and mRNA data were log-transformed prior to statistical analysis. A P value of 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Siberian hamster FSHß, LHß, and cDNA sequences (GenBank accession numbers: AF106914 [FSHß], AF106915 [LHß], and AF106916 []) are highly conserved with orthologous sequences in other mammalian species and correspond to sequences encoding amino acids 2–127 of FSHß, 1–128 of LHß, and 8–113 of in rats [16–18]. The hamster FSHß, LHß, and gonadotropin cDNAs all share 90% identity with published rat nucleotide sequences [16–18]. The predicted amino acid sequences are also highly conserved (91%, 95%, and 96% identity, respectively). It should be noted that partial sequence for the subunit cDNA in Siberian hamsters has previously been characterized ([14]; GenBank accession X90776). Our clone extends this sequence 174 bp in the 5' direction. The sequences are identical where they overlap, with two exceptions. Nucleotide number 2 in X90776 is C, but it is A in our clone (nucleotide 176). This difference results in a predicted histidine rather than proline. Nucleotide number 6 in X90776 is T but is A in our clone (nucleotide 180). In both cases, threonine is predicted. Both of these discrepancies fall within the 5' PCR primer used previously [14], but they are in the middle of our clone. Because the primer in X90776 defines the sequence, our clone more likely represents the true Siberian hamster sequence at these two positions. The hamster RPL19 cDNA sequence (GenBank accession: AF106917) is also highly conserved.
Northern Blot Analysis
As shown in Figure 1, all three gonadotropin subunits were abundantly expressed in the hamster pituitary gland. There was a lower but detectable level of subunit expression in the hypothalamus. This is likely attributable to expression of this gene within the pars tuberalis, as has been described previously [14]. Our hypothalamic dissections most often include the pars tuberalis. Predominant transcripts of approximately 1.7, 0.6, and 0.75 kilobases were detected for the FSHß, LHß, and subunits, respectively. A smaller, less-abundant transcript was also noted for FSHß. Overall, these transcript sizes correspond to those reported in other species (see [9] for review).
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Photostimulated Changes in Gonadotropin Subunit mRNA Levels
Transferring short-day hamsters to long days caused the characteristic increases in paired testis mass and body mass (F[7,29] = 37.8, P < 0.0001 and F[7,29] = 3.7, P < 0.01; Fig. 2). Testes were significantly larger in LD controls than in all other groups. Testes were significantly enlarged from SD values by the 20th long day (Fig. 2A). Body mass was significantly greater in LD controls than in all of the other groups, which did not differ from one another (Fig. 2B). Serum FSH varied significantly (F[7,29] = 3.1, P < 0.02; Fig. 3A). FSH levels peaked on the fifth long day and decreased to stable long-day levels over the following 2 wk. In the context of the overall ANOVA, FSH levels did not differ between SD and LD controls. However, when the means were compared separately with a two-tailed t-test, LD values were significantly greater than SD values (t[8] = 2.95, P < 0.02). Serum LH levels did not vary significantly over the course of the experiment, nor did they differ between LD and SD controls (Fig. 3A).
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We developed a lysate RPA in order to measure simultaneously all three gonadotropin subunit mRNAs as well as a loading control (RPL19) from individual hamster pituitaries. Figure 4 shows an example of protected fragments for the four mRNA species from a SD and a LD control as well as an animal stimulated with five long days. The analysis of the gonadotropin subunit mRNA levels indicated that FSHß mRNA levels increased following long-day transfer (F[7,29] = 3.3, P < 0.02; Fig. 3B). In general, serum FSH and pituitary FSHß mRNA levels were highly correlated (r = 0.65, P < 0.001). Similar to serum FSH levels, the mRNA levels peaked (3.6-fold relative to SD values) after five days and remained above SD control levels through 20 long days. FSHß mRNA levels were 1.6-fold greater in LD control than SD control animals, but this difference was not significant in the context of the ANOVA. However, FSHß mRNA levels were significantly greater in LD controls than SD controls when compared separately (t[8] = 2.65, P < 0.03, two-tailed). Subunit mRNA levels also increased following long-day transfer, although to a lesser extent than FSHß (F[7,29] = 2.9, P < 0.03; Fig. 3B). Levels were significantly increased from SD control values on the morning of the second long day and peaked after seven long days (2-fold relative to SD controls). LHß mRNA levels did not vary significantly over the course of the study nor did they differ between SD and LD controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(2-fold) mRNA levels were significantly increased shortly after transfer from short to long photoperiods. In addition, the patterns of change in serum FSH and pituitary FSHß mRNA levels were highly correlated. In contrast, serum LH and LHß mRNA levels did not change significantly following photostimulation. These data indicate that the singular increase in serum FSH following photostimulation reflects increases in FSHß and subunit mRNA accumulation, and that serum LH remains unchanged, at least in part, because of stable LHß mRNA levels.
While both FSHß and mRNA levels increase significantly following photostimulation, it seems likely that the increase in FSHß mRNA (and subsequently protein) accumulation is the major determinant of increased FSH levels. First, as indicated by both the Northern blot and RPA analyses, pituitary subunit mRNA levels were significantly higher than were FSHß mRNA levels. This is not surprising given that sufficient subunit must be produced to dimerize with FSHß, LHß, and thyrotropin stimulating hormone ß (TSHß). Second, the increase in mRNA levels following photostimulation was modest compared to the increase in FSHß mRNA. Because basal pituitary subunit mRNA (and protein) levels are relatively high, additional synthesis may not be necessary to accommodate the increased FSHß levels and subsequent FSH production. Third, the observed increases in subunit mRNA levels may be unrelated to increased FSH levels. For example, these increases may relate to seasonal changes in TSH production. Within the pars tuberalis, and TSHß mRNA levels are significantly higher in long-day than short-day hamsters [14].
Overall, serum FSH and pituitary FSHß showed similar patterns of change following long-day transfer. Levels of FSHß mRNA were lowest in SD controls and began to rise between 2–5 long days. Both serum FSH and FSHß peaked after 5 long days. Thereafter, levels of both declined over the next 2 days, although at a faster rate for serum FSH. The rapid drop in serum FSH was unexpected given that previous reports have shown that FSH continues to increase between 5–7 long days [6,8,19]. The FSH response varies from individual to individual, however, and the Day 7 group in the present study had only three animals. Therefore, these data should be interpreted in that light. Regardless of the cause of the early decline in FSH levels, both FSH and FSHß mRNA levels showed a consistent decline. From Day 10 through Day 20 of photostimulation both serum FSH and pituitary FSHß mRNA levels remained relatively unchanged and above SD values.
At this point, we do not know what mechanisms drive the increases in FSHß mRNA levels; however, GnRH may be involved. We have shown previously that photostimulated increases in serum FSH levels are GnRH-dependent [19]. In addition, preliminary data indicate that a GnRH antagonist also attenuates photostimulated increases in pituitary FSHß mRNA levels (unpublished observations). Several other laboratories have shown that GnRH regulates gonadotropin secretion and subunit synthesis in a frequency-dependent fashion (e.g., [20–23]). In general, fast GnRH pulses increase LHß and mRNA levels, while slower pulses stimulate FSHß transcription. If this is true for Siberian hamsters, then during the transition from short to long days, GnRH pulse frequency may increase enough to stimulate FSH, but not LH, synthesis and secretion. Indeed, exogenous GnRH infusions of different pulse frequencies differentially stimulate FSH and LH secretion in short-day hamsters [8]. After 10 days, s.c. delivery of GnRH maximally stimulates serum FSH and LH levels at pulse frequencies of 1 per 90 and 1 per 45 min, respectively. However, even pulses as slow as 1 per 180 min are sufficient to stimulate both LH and FSH release. Therefore, this mode of GnRH delivery does not recapitulate the selective release of FSH following photostimulation.
These data suggest that GnRH pulse frequency does not account completely for the selective increase in FSH. However, it is possible that pulse frequencies slower than those used (< 1 per 180 min) may be sufficient to stimulate FSH selectively. In fact, in some rat models, very slow GnRH pulses (1 per 240–480 min) stimulate FSHß but not or LHß mRNA levels, although serum LH levels are still elevated (e.g., [20]). Future experiments should examine gonadotropin secretion in short-day hamsters administered GnRH at these slower pulse frequencies. Still, it is worth noting that we observed 1.5- to 2-fold increases in mRNA subunit levels following photostimulation. Therefore, it is not clear that even very slow GnRH pulses will mimic long-day transfer given that previous studies show that fast or continuous, but not slow, GnRH pulses increase mRNA levels. It seems likely that gonadotropin subunit regulation in Siberian hamsters will involve both GnRH-dependent and -independent mechanisms. As a result, we are currently exploring the roles of pituitary and gonadal activin, inhibin, and follistatin in the selective regulation of FSH.
In general, we observed the same pattern of change in FSH and LH following photostimulation as has been reported previously. That is, FSH levels increased, while LH levels remained unchanged after long-day transfer and did not differ between SD and LD controls [6,8,19]. While FSH levels increased in photostimulated animals, we observed greater variability than described previously. Depending on the experiment, serum FSH levels increase somewhere between 3 to 10 days after long-day transfer, remain elevated for 1–2 wk, and then decline to stable LD levels [4,6-8,19]. In the present study, FSH levels were significantly elevated (and peaked) after 5 long days. Other than this elevation, however, serum FSH levels did not differ significantly between SD controls and any of the other groups. This stems from the fact that the SD controls in this study had higher levels of FSH (~3.7 ng/ml) relative to what we have reported previously (2.0–3.0 ng/ml) [6-8,19]. Hamsters sampled on the morning of the second long day had FSH levels within this range (~2.7 ng/ml), and these levels differed significantly from all of the other long-day transfer groups.
While we do not know why serum FSH levels were higher than usual in the SD controls in this study, the levels were still below those of age-matched LD controls (~5.1 ng/ml) and were insufficient to promote gonadal growth. In this species, long-day-induced gonadal recrudescence is FSH-dependent [5]. It seems likely, therefore, that the FSH levels in the SD controls in this study did not reach some threshold necessary to drive testicular growth. What this threshold is remains to be determined; however, it is worth noting that the mean FSH levels in all of the photostimulated groups were greater than 5 ng/ml.
Interestingly, the testes were significantly increased in mass relative to SD controls only after 20 long days. This is somewhat slower than described previously [6,8,19]. Using the same paradigm, we have observed increases in testes mass after as few as 10 long days. In that study, however, the elevation of FSH following long-day transfer was of greater amplitude (maximum of 8.5 ng/ml in the present study relative to ~14 ng/ml in ref. [8]). These data suggest that a rapid and sustained elevation of FSH to a high amplitude will result in rapid gonadal recrudescence. Gonadal growth will occur more slowly, however, as long as FSH levels are elevated above some threshold and remain there for several weeks (present data).
While this may account for the observed pattern of gonadal recrudescence in the present study, we do not know why the testes of LD controls were larger than those of SD controls when there were small or no significant differences in serum FSH and LH or in pituitary FSHß, LHß, and mRNA levels between these two groups. Given the number of experimental groups compared in the ANOVA, true differences in serum FSH and pituitary FSHß levels between LD and SD controls may have been obscured. A re-examination of these data using two-tailed t-tests indicated that both FSH and FSHß levels were significantly higher in LD than in SD controls. Differences in serum FSH may therefore account for the differences in testes mass. Alternatively, LD and SD hamsters may show different sensitivities to comparable levels of FSH. Indeed, SD hamsters have fewer testicular FSH receptors than do LD hamsters [24].
In summary, we report that photostimulated increases in FSH secretion in Siberian hamsters reflect selective increases in pituitary FSHß and subunit mRNA levels. These messages are significantly elevated after 2–5 long days, coincident with increases in serum FSH levels. Thereafter, both FSHß mRNA and serum FSH levels decrease gradually and in synchrony over the following weeks. Because both the ascending and descending phases of FSH synthesis and secretion occur over a period of days to weeks, Siberian hamsters provide a powerful system in which to investigate the mechanisms controlling the differential regulation of FSH and LH.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The research was supported by NIH grants F32-MH11493 (D.J.B.), R01-HD09885, P01-HD2192, and P30-HD28048 (F.W.T.). 
2 Correspondence: Daniel J. Bernard, Department of Neurobiology and Physiology, Northwestern University, 2153 N. Campus Drive, Evanston, IL 60208. FAX: 847 467 4065; dbernard{at}nwu.edu
Accepted: August 27, 1999.
Received: March 19, 1999.
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