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Endocrinology Vol. 138, No. 10 4101-4108
Copyright © 1997 by The Endocrine Society

ARTICLES

Initial Expression of the Common {alpha}-Chain in Hypophyseal Pars Tuberalis-Specific Cells in Spontaneous Recrudescent Hamsters1

T. M. Böckers, J. Bockmann, A. Salem, P. Niklowitz, A. Lerchl, M. Huppertz, W. Wittkowski and M. R. Kreutz

AG Molecular Neuroendocrinology (T.M.B., J.B., A.S., M.H., W.W.), Institute of Anatomy, and Institute of Reproductive Medicine (P.N., A.L.), University of Münster, D-48149 Münster, Germany; and AG Molecular and Cellular Neurobiology (M.R.K.), Institute of Medical Psychology, University of Magdeburg, 39120 Magdeburg, Germany

Address all correspondence and requests for reprints to: Professor Dr. W. Wittkowski, AG Molecular Neuroendocrinology, Institute of Anatomy, Vesaliusweg 2–4, D-48149 Münster, Germany. E-mail: bockers{at}uni-muenster.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When exposed to short-day conditions, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure to short days, however, the animals become photorefractory and gonadal recrudescence occurs. The underlying mechanism for this insensitivity is still unknown. There is growing evidence, however, that specific cells of the pituitary pars tuberalis (PT) mediate these photoperiod/nonphotoperiod-dependent changes as a direct or indirect "Zeitgeber" for the endocrine system.

We investigated messenger RNA (mRNA)/protein formation for several hypophyseal hormones (ß-TSH, ß-LH, PRL, common {alpha}-chain) in the pars distalis (PD) and PT of female Djungarian hamsters in long photoperiod (LP) and after 18, 28, and 38 weeks of short photoperiod (SP). As indicated by gonadal and body weight, the hamsters displayed gonadal regression after 18 and 28 weeks of SP; after 38 weeks of SP, all animals showed recrudescence. At 18 and 28 weeks of SP, only PRL mRNA and protein levels were significantly reduced in the PD and returned to LP values after 38 weeks of SP. The expression of hypothalamic tyrosine hydroxylase in the arcuate nucleus that was determined by immunocytochemistry and by in situ hybridization was also down-regulated in SP18 and SP28 with increasing levels at SP38. Urinary 6-sulfatoxymelatonin levels were elevated in SP with highest levels in the SP18 group.

In the PT, ß-TSH mRNA and protein were not detectable in all SP groups compared with the moderate signal intensity in LP. The common {alpha}-chain mRNA and protein, however, which were also reduced in the animals of the SP18 group, were already elevated after 28 weeks of SP and nearly reached LP-levels after 38 weeks of SP.

These results show that, in contrast to LH and TSH, PRL expression in the PD is a sensitive indicator for photoperiod dependent changes of the endocrine system and seems to be tyrosine hydroxylase independent. The increase of common {alpha}-chain expression in PT-specific cells depending upon duration of SP that precedes the hormonal changes in the PD leads us to speculate that PT-specific cells initiate spontaneous recrudescence via a PT-PD pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN HAMSTERS and other long-day breeders, exposure to short-day type photoperiods induces gonadal regression with a latency of several weeks. These dramatic changes are caused by the photoperiod-dependent secretion pattern of the pineal hormone melatonin (see Refs. 1–3 for reviews). The repression of sexual functions, however, can only be maintained for about 4–6 months. After a chronic exposure to short photoperiod (SP) for several months, testicular regrowth or a regular estrous cycle occurs (4, 5, 6). This stage of full fertility is maintained under SP unless the animals are exposed to long-day photoperiods (LP) for several weeks before their reexposure to SP (7, 8, 9). It is still a matter of speculation by which mechanism these changes of the endocrine system are mediated (10). Several experiments have shown that spontaneous gonadal recrudescence is not due to a failure to produce an adequate melatonin rhythm (7, 9, 11). Thus, it could be shown that the insensitivity to melatonin also occurs in pinealectomized hamsters injected with an SP melatonin pattern. Therefore, an insensitivity of the target tissue for melatonin has been proposed (7). Studies by Weaver et al. (12), who thoroughly investigated the physiology of the melatonin receptor expressed in the pars tuberalis (PT) of spontaneous recrudescent hamsters, showed that the receptor density and function of the melatonin receptor itself is most likely not the cause of target tissue insensitivity. Hence, the authors suggest that the sensitivity to melatonin is regulated at levels downstream of the response to acute receptor occupation.

The adenohypophyseal cell type that most densely expresses melatonin receptors are PT-specific cells, a cell type that covers the hypophyseal stalk and the median eminence (10). These cells are also characterized by the expression of both TSH subunits (13, 14). Morphological and immunocytochemical investigations, however, clearly demonstrate that PT-specific cells do not resemble PD-thyrotropes (15, 16). Recent studies strongly suggest that this cell type plays a key role in mediating photoperiodic responses to the endocrine system. PT-specific cells display marked alteration of ultrastructure and expression pattern of TSH-subunits, depending upon photoperiod and circulating melatonin levels (14, 17).

Interestingly, the expression pattern and the ultrastructural appearance of PT-specific cells was always reflected by the morphological and functional adaptations to photoperiod in all experiments carried out so far, i.e. gonadal regression was always correlated with a low hybridization signal for TSH subunits in PT-specific cells and vice versa (17, 18, 19).

To elucidate the possible role of PT-specific cells in spontaneous recrudescence, we closely investigated the morphology and expression pattern of pituitary PT-specific cells in hamsters exposed to LP as well as to SP for 18, 28, and 38 weeks. Because the insensitivity to SP is thought not to be a sudden event (7), this experimental design allows to monitor subtle changes of hormonal expression in the pituitary before the actual gonadal recrudescence occurs. To determine the time course of hormonal regulation in the PD and the PT, we examined several PD hormones/subunits (PRL, LH, TSH) at different time points by in situ hybridization and immunocytochemistry. In this respect, the regulation of PRL expression is of special interest because the photoperiod-induced changes of hypophyseal and serum PRL-concentrations are only poorly understood (20, 21, 22, 23). The fact that dopamine is known to be a potent PRL inhibiting factor (PIF) that might play a key role in the hormonal changes during the annual cycle prompted us to determine steady state mRNA and protein levels of hamster tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis, in the hamster arcuate nucleus. The partial cloning of hamster TH was a prerequisite for these studies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Female Djungarian hamsters Phodopus sungorus were raised in the colony of the Institute of Reproductive Medicine, Muenster, under LP with a controlled light regimen (16-h light, 8-h dark, lights-on from 0400 h to 2000 h). Standardized food and tap water were available ad libitum. The temperature in the animal rooms was 22 C ± 2 C, relative humidity was 60–80%. At the beginning of the experiments, body weight was measured and sexual maturation of the hamsters ranging in age from 4–5 months was assessed by the opening of the vagina indicating the onset of regular estrous cycles (24).

Sixty females were randomly divided into four subgroups of 15 animals each. While the LP controls remained at LP (16:8) for 38 weeks, group 2 (SP18) was exposed to SP with 8 h of light per day (SP 8:16, lights-on from 0800 h to 1600 h) for 18 weeks, groups 3 and 4 were exposed to SP for 28 (SP28) or 38 (SP18) experimental weeks. To avoid age-associated changes, the groups were set up in a delayed fashion. Body weight, the appearance of the outer genitals, and coat color were controlled in intervals of 2 weeks. In the last experimental week, 8 hamsters of each group were transferred for 24 h into metabolic cages, and urine was sampled in every 3 h and stored at -20 C for the measurement of 6-sulfatoxymelatonin. At the end of the experiment, the animals were killed by decapitation between 0900 h and 1200 h, skulls were opened and the brain removed. The ovaries and uteri were dissected out and weighed.

The study was performed in accordance with the regulations of the German Federal Law on the Care and Use of Laboratory Animals (License: Münster, 72/92-Teilprojekt 5).

Determination of urinary 6-sulfatoxymelatonin levels
6-Sulfatoxymelatonin concentrations in urine were determined using a specific commercially available RIA (Stockgrand Ltd., Surrey, UK). The samples were diluted 1:250 with destilled water and analyzed in duplicate in three assays. Standards ranged from 1–100 pg/tube. Mean intraassay variations were determined by coefficients of variations (CVs) of the duplicate measurements. At 20% binding corresponding to 4.2 pg/tube, the CV was 11.5% at 50% binding (14.3pg/tube), the CV was 10%, and at 80% binding (42.6 pg/tube) the CV was 12%. Sensitivity was always better than 1 pg/tube. Total daily excretion was determined by summing the amount of 6-sulfatoxymelatonin over the 24-h collection period.

Partial cloning and sequence analysis of hamster tyrosine hydroxylase
Total RNA was extracted from hamster hypothalamus using the guanidine isothiocyanate method (25, 26) with minor modifications. Briefly, 330 mg of tissue were homogenized with an Ultra Turrax in 4 ml 5 M guanidine isothiocyanate, 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0, and 8% ß-mercaptoethanol. After adding 7 vol of 4 M LiCl followed by an overnight incubation at 4 C, the RNA was precipitated at 10,000 x g for 90 min. The pellet was dissolved in 7 ml 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, and 0.1% SDS for 45 min using a magnetic stirrer. The RNA was recovered by sequential extractions with equal volumes of TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0) saturated phenol and chloroform/isoamyl alcohol (24:1), followed by an ethanol precipitation. Poly(A)+ RNA was separated using oligo(dT)+-cellulose columns (Pharmacia Biotech, Uppsala, Sweden).

The PCR method was used to generate complementary DNA (cDNA) fragments from hamster tyrosine hydroxylase. Briefly, poly(A)+ RNA from hamster hypothalamus was reversely transcribed with random primers (GIBCO-BRL, Life Technologies, Eggenstein, Germany). Subsequently, PCR amplification was carried out using specific primers that were chosen from rat tyrosine hydroxylase mRNA sequence (27): Sense (1027–1048):-5'-TGT TGG CTG ACC GCA CAT TTG C-3'-Antisense (1378–1357):-5'- AAT GGG CGC TGG ATA CGA GAG G-3'-

Subsequently, the cDNA fragments (size: 351bp) were analyzed on an agarose gel, eluted and subcloned into a pGEM-T vector (Promega, Madison, WI). The nucleotide sequences of these clones were determined from double-strand plasmids according to the dideoxy termination method using the T7 Sequencing Kit (Pharmacia Biotech, Uppsala, Sweden). Several clones generated by three independent RT/PCR amplifications were sequenced.

GenBank accession number
The GenBank accession number for the sequence reported in this paper is Y09294 (Phodopus sungorus, tyrosine hydroxylase).

In situ hybridization
For in situ hybridization, brains (n = 4/experimental group) were frozen on dry ice in isopentan at -40 C. The brains were cut on a cryostat in frontal or sagittal sections (18 µm), mounted on Superfrost Plus slides (Menzel, Braunschweig, Germany), and stored at -70 C until used. The mRNAs encoding the hormonal subunits were detected with cDNA antisense oligonucleotides purchased from MWG-Biotech (Ebersberg, Germany): 1) {alpha}-subunit sequence (Djungarian hamster) complementary to the 82–47 bp region (14) -5'-ATG-CTT-TGG-CCA-CAC-AGC-ATG-TGG-CCT-CTG-AGG-TGA-3'-2) ß-TSH subunit sequence (Djungarian hamster) complementary to the 67–32 bp region (14) -5'-CTT-GCC-ATT-GAT-GTC-CCG-TGT-CAT-ACA-ATA-CCC-GGC-3'-3) PRL sequence (Golden hamster) complementary to the 359–326 bp region (28) -5'-GAC-TTC-CGG-AGG-GAC-CTG-CTG-GGC-TTC-TTC-CTT-3'-4) ß-LH subunit (rat) complementary to the 167–134 bp region (29) -5'-GAC-AGT-AGC-CGG-CAC-AGA-TGC-TGG-TGG-TGA-AGG-3'-5) Tyrosine hydroxylase (Djungarian hamster) complementary to the 1088–1055 (rat) bp region -5'-GCT-CCC-AGA-GAT-GCA-AGT-CCA-ATG-TCC-TGG-GAG-3'-The oligonucleotides were 3'end-labeled with terminal deoxynucleotidyl-transferase using [{alpha}]-35S dATP (Amersham Buchler, Braunschweig, Germany).

Frozen sections [6 sections/animal (hormonal subunits) 12 sections/animal (TH)] were airdried at room temperature (RT). The oligonucleotides were preheated at 90 C (3 min) and placed on ice before being diluted in the hybridization buffer [50% formamide; 20% 20 x SSC; 10% 0.2 M phospate buffer (pH 7.6); 10% dextran sulphate; 5% sarcosyl (20%); 500 µg/ml sheared salmon sperm DNA; 250 µg/ml yeast tRNA, 100 mM DTT] to a final concentration of about 5 x 105 cpm/slide, corresponding roughly to 0.2 ng/slide. Sections were incubated in a humidified box at 42 C for 16 h. Posthybridization steps were as follows: 2 times 2 x SSC for 10 min at RT, followed by 6 times 1 x SSC/10 mM ß-mercaptoethanol for 15 min at 55 C and 1 x SSC for 15 min at RT. Subsequently, sections were dehydrated, airdried, and dipped in NTB3 nuclear track emulsion (Kodak, Fernwald, Germany, diluted 1:1 with water), stored for 2–3 weeks in the dark at 4 C, developed, and counterstained with hematoxylin.

Controls were performed as follows: 1) omission of the antisense oligonucleotide; 2) posthybridizational washing steps above the calculated melting point of the hybrid; and 3) hybridization in the presence of 100-fold excess of unlabeled oligonucleotide.

Immunocytochemistry
For immunocytochemistry, brains (n = 4/experimental group) were fixed by immersion in Bouin‘s fluid for 48 h, dehydrated, and embedded in Paraplast. Seven-micrometer sections were cut on a microtome in frontal orientation.

The common {alpha}-chain subunit for the glycoprotein hormones was detected using an anti (r) {alpha}-LH polyclonal antibody (diluted 1:2.000) that was generously provided by the National Hormone And Pituitary Program (NIH, University of Maryland, School of Medicine, Baltimore, MD). The anti (r) ß-TSH (diluted 1:1.000), anti (r) PRL (diluted 1:3.000) and anti (r) ß-LH (diluted 1:5.000) polyclonal antibodies were purchased from UCB bioproducts (Braine-L‘Alleud, Belgium). Frontal sections (8 sections/animal) of the rostral PT and PD, respectively, of each animal were processed in parallel experiments. According to the PAP-method (30), sections were deparaffinized in xylene, hydrated through a graded ethanol series, and equilibrated in 0.1 M Tris-HCl buffer (pH 7.6) for 10 min. After preincubation with 5% normal swine serum in 0.1 M Tris-HCl (pH 7.6), 0.2% Triton x for 30 min, the primary antibody was applied in preincubation buffer for 22 h at room temperature (RT). Antibody binding was visualized by incubating sections with 1) swine antirabbit IgG (DAKO, Hamburg, Germany) diluted 1:50 for 30 min, and subsequently with 2) rabbit PAP complex (DAKO) diluted 1:100 for 30 min. All secondary antibodies were diluted in the preincubation buffer. Thereafter, 3) the color solution, 3,3-diaminobenzidine (0.05%)/H2O2 (0.001%) (Sigma, Munich, Germany), was applied to the sections for 6 min. After completion of the staining procedure, sections were dehydrated and mounted in DePeX (Serva, Heidelberg, Germany). Some sections were counterstained with hematoxylin for morphological orientation.

For the detection of tyrosine hydroxylase (12 sections/animal), we used a monoclonal antibody (diluted 1:3.000; Boehringer Mannheim, Germany). Antibody binding was visualized employing the ABC Vectastain Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions.

Controls were performed as follows: 1) omission of the first or the secondary antibody and (ii) preabsorption of the first antibody with the antigen overnight (ß-TSH, ß-LH).

Quantitative evaluation
Staining intensities from in situ hybridization and immunocytochemical experiments were analyzed with a computer assisted image analysis system (Optimas, Bothell, WA) under standardized conditions as described previously (13).

Briefly: After in situ hybridization, sections were evaluated with the following protocol: 1) The PD or the glandular cell layer of the PT was outlined with a cursor on a digitizer tablet. 2) After subtraction of background, the optical density of the silver grains on the PD/PT was determined and expressed as inverted median gray levels ranging from 40 (white) to 200 (black). 3) Median gray levels of the PT in the different groups were compared and tested for statistical significance as described below.

For the evaluation of immunocytochemical stainings the following protocol was used: 1) The PD or the glandular cell layer of the PT were outlined with a cursor on a digitizer tablet. 2) After subtraction of background, the immunostained area with a gray-level lower than 150 was measured. 3) The stained PD/PT-cell areas in each group were compared and tested for statistical significance as described below.

Tyrosine hydroxylase expression in the arcuate nucleus was determined by counting labeled cells after in situ hybridization and measurement of the immunopositive cell area after immunocytochemical staining.

Statistical analysis
The differences among group means (body parameters, 6-sulfatoxymelatonin concentrations) were determined using one-way ANOVA followed by a multiple range test (LSD, least significant difference) with 95% confidence intervals.

Differences between median gray levels or immunopositive PD/PT-area, were tested applying the t test. Significance level was set as P < 0.05 unless stated otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After 38 experimental weeks, the following inclusion criteria were chosen: LP: hamster with open vaginas, n = 9 SP18: photoinhibited hamsters whose vaginas had closed at the experimental week 34 or earlier, n = 9. Four nonresponsive hamsters and two hamsters with a reduced body weight but still open vaginas were excluded. SP28: photoinhibited hamsters whose vaginas had closed at experimental week 24 or earlier, n = 10. Three nonresponsive hamsters and two hamsters (closing of the vagina after experimental week 24) were excluded. SP38: spontaneous recrudescent hamsters whose vaginas had opened again at the experimental week 34 or earlier, n = 8. Two hamsters nonresponsive to the inhibitory influence of SP and five hamsters with still closed vaginas were excluded.

The LP group, as well as the three SP groups (SP18, SP28, SP38) of female Djungarian hamsters are well characterized by their body parameters. After 18 weeks of SP, the animals displayed all features of SP exposed hamsters. The coat color had changed from brown to white (data not shown), and the body weight as well as the weight of the ovaries and uteri of the hamsters were markedly diminished (Figs. 1Go and 2Go). In addition, the closure of the vaginas in all animals indicates that the animals had no longer a regular estrous cycle (24) (data not shown). After 28 weeks of SP, the hamsters were at the turning point toward gonadal recrudescence. The body weight, weight of the uteri, and coat color were slightly changing toward LP parameters. The weight of the uteri and the closure of the vaginas, however, showed that the animals were still photoinhibited (Figs. 1Go and 2Go). After 38 weeks of SP, all body parameters were not significantly different from hamsters kept in LP. The opening of the vaginas in these animals indicates that a regular estrous cycle had reoccurred in these hamsters (data not shown).



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  		Figure 1. Changes of body weight of female hamsters kept under LP and under different durations of SP. The body weight in the SP18 (circle) as well as in the SP28 (square) group is significantly reduced compared with LP conditions (triangle, top down). The weight of the photorefractory hamsters of the SP38 group (triangle, top up) does not differ from controls in LP. Note that open symbols indicate LP, closed symbols SP. Data were examined by ANOVA followed by a multiple range test (LSD) with a 95% confidence interval. Significance (*) was assumed if P < 0.01.

 


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  		Figure 2. Weight of ovaries and uteri at the end of the experiment. The weight of the ovaries that is significantly reduced after 18 weeks of SP compared with LP conditions increases steadily in hamsters kept in SP for 28 or 38 weeks. The weight of the uteri (significantly diminished in SP18), however, shows a further reduction after 28 weeks of SP. After 38 weeks of SP, the weight nearly reaches values of hamsters kept in LP. Data were examined by ANOVA followed by a multiple range test (LSD) with a 95% confidence interval. Data with different superscripts are significantly different (P < 0.05).

 
6-Sulfatoxymelatonin levels
6-sulfatoxymelatonin levels per 24 h measured in urine showed that the overall melatonin production (24 h) after 18 weeks of SP exposure was significantly higher compared with animals kept in LP. After 28 weeks and 38 weeks of SP, the amount of excreted 6-sulfatoxymelatonin per day was still elevated but not significantly different from levels obtained under LP (Fig. 3Go).



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  		Figure 3. 6-Sulfatoxymelatonin levels (ng/24 h) in female hamsters kept in LP or in SP for 18, 28, and 38 weeks. While 6-sulfatoxymelatonin levels are elevated in all SP groups, the amount of secreted 6-sulfatoxymelatonin is significantly higher in the SP18 group compared with SP28 and SP38. Data were examined by ANOVA followed by a multiple range test (LSD) with a 95% confidence interval. Data with different superscripts are significantly different (P < 0.05).

 
Cloning and sequencing of hamster tyrosine hydroxylase
For the hamster tyrosine hydroxylase, several clones resulting from three independent RT-PCR amplifications were isolated. They contained a 351-bp insert (including primers) that was identified to encode for the hamster tyrosine hydroxylase molecule. The sequence homology between the partial sequence of rat and hamster tyrosine hydroxylase is 91% on cDNA level and 95% on the protein level (27).

Expression pattern of hypophyseal hormones in PT and PD
In situ hybridization with antisense oligonucleotides and immunocytochemistry detecting common LH-ß mRNA and protein revealed a strong labeling of gonadotropes in the PD (Fig. 4Go). The comparison of staining intensities in the experimental groups (LP vs. SP18–38) showed a decrease of mRNA levels in SP18 and 28, but these differences were not statistically significant (Figs. 4Go and 6Go).



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  		Figure 4. Expression of PRL (A, B, E, F, I, J, M, N) and LH-ß (C, D, G, H, K, L, O, P) in the hypophyseal pars distalis (PD) from female hamsters kept in LP (A–D) and in 18 (E–H), 28 (I–L) and 38 (M–P) weeks of SP as revealed by in situ hybridization (A, C, E, G, I, K, M, O) and immunocytochemisty (B, D, F, H, J, L, N, P). Note the significant reduction of PRL mRNA and protein in the SP18 and SP28 group with increased levels in spontaneous recrudescent hamsters in the SP38 group. In contrast, LH-ß mRNA and protein is not obviously altered under these experimental conditions. Magnification, x150.

 


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  		Figure 6. Median gray levels after in situ hybridization in the PT and PD. Note that the median gray levels range from 40 (white) to 200 (black). The hybridization signal intensities for TSH-ß and LH-ß do not significantly differ in the PD under the experimental conditions. In the PT, TSH-ß expression is not detectable in SP (18–38) compared with LP. The common {alpha}-chain expression is also reduced in SP compared with LP conditions, but the expression gradually increases depending upon duration of SP. Differences in gray scale value (SP18, 28 and 38 compared with LP) were tested applying the t test. Significance was assumed if (**) P < 0.005 (*) P < 0.05.

 
The in situ hybridization signal of the common {alpha}-chain was increased after 38 weeks of SP; protein steady-state levels did not differ significantly in the experimental groups (Fig. 7Go). Immunocytochemical staining for TSH-ß in the PD resulted in a slight increase of detectable protein in the SP28 and 38 groups; steady-state mRNA levels, however, were unaffected (Figs. 6Go and 7Go). In situ hybridization experiments and immunocytochemical staining for PRL displayed significant changes of detectable mRNA and protein under SP in the PD. After 18 and 28 weeks of SP, the in situ hybridization signal as well as the detectable PRL antigen in the PD were significantly reduced. In spontaneous recrudescent hamsters after 38 weeks of SP, steady-state PRL mRNA and protein were increased, nearly reaching LP levels (Figs. 4Go, 6Go, and 7Go).



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  		Figure 7. Stained PT and PD area after immunocytochemical labeling of different hypophyseal hormones. TSH-ß as well as PRL show a significant reduction in SP18 and 28 reaching LP levels after 38 weeks of SP. In the PT, TSH-ß was not detectable in all SP groups; the common {alpha}-chain, however, that is reduced in SP18 and 28 displayed a steady increase of expression and reached LP values after 38 weeks of SP. Differences in stained PT and PD area (SP18, 28 and 38 compared with LP) were tested applying the t test. Significance was assumed if (**) P < 0.005 (*) P < 0.05.

 
In the PT, the expression patterns of both TSH subunits were different under these experimental conditions. The TSH-ß-chain (mRNA/protein) in PT-specific cells was not detectable in any of the SP groups ( Figs. 5–7GoGoGo). The common {alpha}-chain, however, that was initially low in the SP18 group exhibited increased signal intensity as revealed by immunocytochemistry and in situ hybridization, depending upon the duration of SP. Low steady-state mRNA and protein levels after 18 weeks of SP were elevated already at week SP28 and nearly reached LP-levels after 38 weeks of SP ( Figs. 5–7GoGoGo).



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  		Figure 5. Expression of TSH-ß (A–D) and common {alpha}-chain (E–H) in the hypophyseal PT as revealed by in situ hybridization (upper subsets, A1–H1) and immunocytochemistry (lower subsets, A2–H2). Note that, in contrast to LP (A), TSH-ß expression is not detectable in the SP18 (B), SP28 (C), and SP38 group (D). The expression of the common {alpha}-chain increases depending upon duration of SP. In comparison to the expression pattern in LP (E), the in situ hybridization signal and the amount of detectable protein that is still weak in the SP18 group (F) is enhanced in hamsters kept in SP for 28 (G) or 38 (H) weeks. me, Median eminence, Magnification, x270.

 
Expression of TH
The detection of hamster TH mRNA (average cell number of labeled cells) and protein (% area of arcuate nucleus) in the arcuate nucleus revealed a significant reduction of labeling of cell bodies and axonal processes in the SP 18 (% area, 1.7 ± 0.2; cell number, 3 ± 1) and SP28 group (% area, 1.5 ± 0.3; cell number, 1 ± 1) compared with animals kept in LP (% area, 3.7 ± 0.5, cell number, 17 ± 4) (Fig. 8Go). After 38 weeks of SP, both parameters of TH expression in cells of the arcuate nucleus (% area, 2.9 ± 0.7; cell number, 6 ± 1) were increased but did not reach LP levels.



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  		Figure 8. TH expression in the arcuate nucleus in LP (A, B) and in hamsters kept in SP for 18 (C, D), 28 (E, F) and 38 weeks (G, H) as revealed by immunocytochemistry (B, D, F, H) and in situ hybridization (A, C, E, G). Note that the mRNA as well as the protein levels are clearly reduced in the SP18 and 28 group compared with LP conditions, but the expression of TH clearly increases in the SP38 group. Magnification, x280.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of different hormonal subunits was monitored in the pituitary PD and PT from female hamsters kept in LP, in gonadal regression at 18 and 28 weeks of SP, and in spontaneous recrudescence (38 weeks of SP). Despite the marked changes of several body parameters and gonadal function in SP (18/28), only PRL mRNA levels and immunoreactivity were significantly altered in the pituitary PD. In the PT, the TSH-ß-chain stayed suppressed in SP. The common {alpha}-chain expression was down-regulated at 18 weeks of SP but increased steadily after 28 and 38 weeks of SP, thereby preceding the hormonal changes in the PD.

The phenomenon that PRL is the only hormone that displays a significant reduction of both the mRNA and the protein in SP has already been described by Hegarty et al. (31), who measured mRNA and protein levels of several hormones in golden hamsters kept in SP or LP. The authors found no significant photoperiod dependent alteration of mRNA levels for LH-ß, and POMC; only the steady-state mRNA for PRL was down-regulated. In the serum, however, PRL, LH-ß, and testosterone concentrations were significantly reduced. Therefore, it is most unlikely that only a hypothalamo-hypophyseal reduction of LH expression causes low serum LH and testosterone levels with subsequent involution of the gonads in SP. It needs to be discussed whether an altered hypophyseal secretion pattern, a different half-life of the hormones, or an altered receptor expression of the target organs could account for the physiological changes of the reproductive system that occurs in SP (32, 33).

With respect to these data, the question arises how PRL expression is controlled according to photoperiod. The cloning of the hamster tyrosine hydroxylase enabled us to investigate the expression of TH in the hamster arcuate nucleus, giving indirect evidence for the production of dopamine as a PRL inhibiting factor (PIF) by the tuberoinfundibular dopaminergic (TIDA) neurons (34). In lactating rats with high levels of PRL expression, TH levels in TIDA neurons are low (34). Interestingly, in our experiment the immunoreactivity as well as the mRNA levels of TH were significantly down-regulated during SP (SP18 and 28) when hypophyseal PRL expression is clearly suppressed. Several studies have shown that TH, as well as the dopamine content of the median eminence, is significantly diminished already after 1 day of exposure to SP (20, 23, 35, 36). Therefore, low PRL levels induced by SP are accompanied by low PRL inhibiting factor (dopamine) levels. The authors explain this phenomenon by the proposal of a novel dopaminergic system that could be stimulatory for PRL (20) or by an increased dopamine turnover of cells of the neurointermediate lobe in SP (23) controlling PRL expression.

Our data also show the SP-dependent reduction of PRL and TH over a long period of time (at least 28 weeks) followed by an increase of TH expression in the TIDA neurons after the occurrence of spontaneous recrudescence with significantly higher PRL levels. Furthermore, we found that the increased {alpha}-chain expression of PT-specific cells that occurs after a prolonged SP is followed by raised PRL mRNA and protein levels in the PD. Recent studies on the physiological role of the common {alpha}-chain on PRL cell differentiation and secretion (37, 38) strongly support initial evidence by Begeot et al. (39) that the common {alpha}-chain of the glycoprotein hormones alone exerts a physiological effect on lactotrophic cells. In addition, Morgan et al. (40) recently reported the secretion of one or more PRL-releasing factors from ovine PT-specific cells that enhance the PRL-levels released from PD primary culture (41). Further evidence that photoperiod dependent PRL secretion from the ovine PD occurs independently of the brain but most likely under the control of the hypophyseal PT came from a study of Lincoln and Clarke (42) with hypothalamo-pituitary disconnected rams. Because PT-specific cells that cover the median eminence and hypophyseal stalk are in close spatial relationship to the primary plexus of the portal system, even small amounts of protein released reach the PD at relatively high concentrations (10) via a PT-PD pathway. In addition, the expression of the common {alpha}-chain in PT-specific cells could be shown in all animals investigated so far and occurs very early in fetal development before other hypophyseal hormones are detectable (10, 43, 44, 45).

Taken together, these data would favor the idea of the absence of a factor that promotes PRL expression (i.e. common {alpha}-chain) or a PRL releasing factor during SP that is released by PT-specific cells. A comparable physiological situation for SP-induced PRL repression is the chronic treatment of rats with bromocriptine that causes a reduction of PRL due to a TH independent mechanism (46). In those animals, the dopamine content and TH expression in TIDA neurons is also significantly reduced via a negative feedback mechanism. Therefore, the concept of a central, PT-dependent but TH-independent mechanism of SP-induced PRL reduction would readily explain our observations. However, not all alterations of body parameters can solely be explained by the changes of PRL levels (22). Therefore, it needs to be elucidated which role PRL has directly on the target tissue, as a cofactor in hormonal responses of target tissue (i.e. testis, 47 or as a paracrine modulator of hormone expression in the pituitary.

It is a matter of speculation why the gene expression of the common {alpha}-chain that is mainly regulated via a cAMP dependent pathway (48) slowly increases during prolonged SP exposure. Several studies have shown that both TSH subunits are hardly detectable in the hamster PT treated with a daily afternoon melatonin injection (19) or after 13 weeks of SP (14, 19). Interestingly, the complete inhibition of expression could still be observed for ß-TSH in this study after 18, 28, and even after 38 weeks of SP when spontaneous recrudescence has already occurred. The common {alpha}-chain expression that was already slightly enhanced after 18 weeks of SP compared with the expression pattern after 13 weeks (14, 19) shows a further steady increase of steady-state mRNA and protein after 28 and 38 weeks of SP. This phenomenon suggests that the inhibitory effect of SP and circulating melatonin levels on common {alpha}-chain expression becomes ineffective depending upon duration of SP in this cell type. Because the density and physiological function of melatonin receptors are not altered in the PT under prolonged SP (12) and TSH-ß expression is still repressed after 38 weeks of SP, it is tempting to speculate that one or more specific intracellular factors that inhibit {alpha}-chain expression become less effective during chronic SP treatment or that the concentration of a transcriptional promotor for the {alpha}-chain gene slowly rises.

The overall amount of 6-sulfatoxymelatonin levels (24 h) showed that the total amount of secreted melatonin is significantly enhanced after 18 weeks of SP and slowly decreases after 28 or 38 weeks of SP. Therefore, the decrease of total melatonin or a different secretion pattern of melatonin after prolonged SP (49) could be responsible for the partial lack of repression in PT-specific cell transcription and translation. On the other hand, gonadal recrudescence occurs even in pinealectomized hamsters injected with exogenous melatonin (7, 8, 9), pointing toward a target tissue insensitivity toward SP melatonin pattern.

Furthermore, our data show that despite an LP-like appearance of hamsters in spontaneous recrudescence, a longer photoperiod is needed to achieve an expression pattern of PT-specific cells that is identical to animals kept in LP. The concept that the lack of {alpha}-chain repression in the PT could induce the hormonal changes toward spontaneous recrudescence would explain why recrudescent hamsters have to be exposed to LP for several weeks before gonadal regression can occur after reexposure to SP (8). Only PT-specific cells that are sensitive to the SP melatonin signal, i.e. display an LP-like ultrastructural morphology and expression pattern, can be forced to down-regulate TSH subunit expression with subsequent hormonal changes in the PD via a PT-PD pathway.

In summary, our data show that only the TSH subunits in the PT and PRL in the PD show a significant reduction of steady state mRNA and protein during SP. In contrast to the TSH-ß-chain, the expression of the common {alpha}-chain uncouples from its inhibited state during chronic SP and displays a steady increase of expression depending upon duration of SP. Because these changes are closely followed by the enhanced expression of PRL mRNA and protein in the PD, it is tempting to speculate that the time-dependent disinhibition of the common {alpha}-chain expression or other secretory products from PT-specific cells cause the stimulation of PRL expression in the PD. Because it is still unknown how PRL levels could cause and/or mediate the endocrine responses according to photoperiod, further studies should primarily focus on the endocrine/paracrine effects of PRL on the hypophysis and on target tissue. In this respect, the proposed synergistic action of PRL and gonadotropins on reproductive organs has to be proved (50).


   Acknowledgments
 
The authors would like to thank Mrs. I. Sinha, Mrs. A. Ahle, and Mrs. E. Langener for their technical assistance, and Mrs. S. Loheide for her photographical work.


   Footnotes
 
1 This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Wi 558/5–1). Back

Received February 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reiter R 1991 Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12;2:151–180
  2. Karsch FJ, Bittmann EL, Foster DL, Goodman RL, Legan SJ, Robinson JE 1984 Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 40:185–232
  3. Bartness TJ, Goldman BD 1989 Mammalian pineal melatonin: a clock for all seasons. Experientia 45:939–945[CrossRef][Medline]
  4. Reiter RJ 1972 Evidence for refractoriness of the pituitary-gonadal axis to the pineal gland in golden hamsters and its possible implications in annual reproductive rhythms. Anat Rec 173:365–372[CrossRef][Medline]
  5. Hoffmann K 1979 Photoperiod, pineal, melatonin and reproduction in hamsters. Prog Brain Res 52:397–415[Medline]
  6. Lerchl A, Schlatt S 1993 Influence of photoperiod on pineal melatonin synthesis, fur color, body weight, and reproductive function in the female Djungarian hamster, Phodopus sungorus. Neuroendocrinology 57:359–364[Medline]
  7. Bittman EL 1978 Hamster photorefractoriness: the role of insensitivity of pineal target tissue. Science 202:648–650[Abstract/Free Full Text]
  8. Reiter RJ, Petterborg LJ, Philo RC 1979 Refractoriness to the antigonadotropic effects of melatonin in male hamsters and its interruption by exposure of the animals to long daily photoperiods. Life Sci 25:1571–1576[CrossRef][Medline]
  9. Watson-Whitmyre M, Stetson MH 1985 Induction of refractoriness to melatonin in pinealectomized hamsters. In: Brown GM, Wainwright SD (eds) The Pineal Gland: Endocrine Aspects. Pergamon, Oxford, pp 121–126
  10. Wittkowski W, Schulze-Bonhage AH, Böckers TM 1992 The pars tuberalis of the hypophysis: a modulator of the pars distalis? Acta Endocrinol (Copenh) 126:285–290[Medline]
  11. Rollag MD, Panke ES, Reiter RJ 1980 Pineal melatonin content in male hamsters throughout the seasonal cycle. Proc Soc Exp Biol Med 165:330–334[Medline]
  12. Weaver DR, Provencio I, Carlson LL, Reppert SM 1991 Melatonin receptors and signal transduction in photorefractory Sibirian hamsters (Phodopus sungorus). Endocrinology 128:1086–1092[Abstract]
  13. Böckers TM, Sourgens H, Wittkowski W, Jekat A, Pera F 1990 Changes in TSH-immunoreactivity in the pars tuberalis and pars distalis of the fetal rat hypophysis following maternal administration of propylthiouracil and thyroxine. Cell Tissue Res 260:403–408[CrossRef][Medline]
  14. Bockmann J, Böckers TM, Vennemann B, Niklowitz P, Müller J, Wittkowski W, Sabel BA, Kreutz MR 1996 Short photoperiod-dependent down-regulation of thyrotropin-{alpha} and -ß in hamster pars tuberalis-specific cells is prevented by pinealectomy. Endocrinology 137:1804–1813[Abstract]
  15. Bockmann J, Böckers TM, Winter C, Wittkowski W, Winterhoff H, Deufel T, Kreutz MR 1997 Thyrotropin expression in hypophyseal pars tuberalis-specific cells is 3,5,3'-triiodthyronine, thyrotropin releasing hormone and Pit-1 independent. Endocrinology 138:1019–1028[Abstract/Free Full Text]
  16. Stoeckel ME, Porte A 1984 Fine structure and development of the pars tuberalis in mammals. In: Motta PM (ed) Ultrastructure of Endocrine Cells and Tissues. Martin Nijhoff, Boston, pp 29–38
  17. Wittkowski W, Hewing M, Hoffmann K, Bergmann M, Fechner J 1984 Influence of photoperiod on the ultrastructure of the hypophyseal pars tuberalis of the Djungarian hamster, Phodopus sungorus. Cell Tissue Res 238:213–216[Medline]
  18. Wittkowski W, Bergmann M, Hoffmann K, Pera F 1988 Photoperiod-dependent changes in TSH-like immunoreactivity of cells in the hypophyseal pars tuberalis of the Djungarian hamster, Phodopus sungorus. Cell Tissue Res 251:183–187[CrossRef][Medline]
  19. Böckers TM, Niklowitz P, Bockmann J, Fauteck J-D, Wittkowski W, Kreutz M 1995 Daily melatonin injections induce morphological changes in hamster pars tuberalis-specific cells similar to short photoperiod. J Neuroendocrinol 7:607–613[CrossRef][Medline]
  20. Viguie C, Thibault J, Thiery J-C, Tillet Y, Malpaux B 1996 Photoperiodic modulation of monoamines and amino-acids involved in the control of prolactin and LH secretion in the ewe: evidence for a regulation of tyrosine hydroxylase activity. J Neuroendocrinol 8:465–474[CrossRef][Medline]
  21. Blask DE 1989 Sexually dimorphic effects of melatonin on prolactin cell function in male and female Syrian hamsters. J Pineal Res 7:221–230[Medline]
  22. Bartness TJ, Wade GN, Goldman BD 1987 Are the short-photoperiod-induced decreases in serum prolactin responsible for the seasonal changes in energy balance in Syrian and Sibirian hamsters? J Exp Zool 244:437–454[CrossRef][Medline]
  23. Steger RW, Juszczak M, Fadden C, Bartke A 1995 Photoperiod effects on neurohypophyseal and tuberoinfundibular dopamine metabolism in the male hamster. Endocrinology 136:3000–3006[Abstract]
  24. Schlatt S, Niklowitz P, Hoffmann K, Nieschlag E 1993 Influence of short photoperiods on reproductive organs and estrous cycles of normal and pinealectomized female Djungarian hamsters, Phodopus sungorus. Biol Reprod 49:234–250
  25. Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD 1983 Laboratory methods: a method for isolation of intact, translationally active ribonucleic acid. DNA 2:329–335[Medline]
  26. Mac Donald RJ, Swift CA, Przybyla AE, Chirgwin JM 1987 Isolation of RNA using guanidinium salts. Methods Enzymol 152:219[Medline]
  27. Grima B, Lamouroux A, Blanot F, Biguet NF, Mallet J 1985 Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc Natl Acad Sci USA 82:617–621[Abstract/Free Full Text]
  28. Southard JN, Sanchez-Jimenez F, Campbell GT, Talamantes F 1991 Sequence and expression of hamster prolactin and growth hormone messenger RNAs. Endocrinology 129:2965–2971[Abstract]
  29. Kato Y, Ezashi T, Hirai T, Kato T 1990 Strain differences in nucleotide sequences of rat glycoprotein hormone subunit cDNAs and gene fragments. Zool Sci 7:877–885
  30. Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG 1970 The unlabelled antibody-enzyme method of immunohistochemistry: preparation and properties of a soluble antigen-antibody complex (horseradish-peroxidase-antiperoxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315–345[Abstract]
  31. Hegarty CM, Jonassen JA, Bittman EL 1990 Pituitary hormone gene expression in male golden hamsters: interactions between photoperiod and testosterone. J Neuroendocrinol 5:567–573[CrossRef]
  32. Goldman B, Nelson RJ 1993 Melatonin and seasonality in mammals. In: Yu HS, Reiter RJ (eds) Melatonin: Biosynthesis, Physiological Effects, and Clinical Applications. CRC Press, Boca Raton, pp 225–252
  33. Hoffman RA, Reiter RJ 1965 Pineal gland: influence on gonads of male hamsters. Science 148:1609–1611[Abstract/Free Full Text]
  34. Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564–589[CrossRef][Medline]
  35. Steger RW, Bartke A, Goldman BD 1982 Alteration in neuroendocrine function during photoperiod induced testicular atrophy and recrudescence in the golden hamster. Biol Reprod 26:437–444[Abstract]
  36. Benson B 1987 Temporal changes in medial basal hypothalamic catecholamines in male Syrian hamsters exposed to short photoperiod. Exp Brain Res 65:371–376[Medline]
  37. Van Bael A, Denef C 1996 Evidence for a trophic action of the glycoprotein hormone {alpha}-subunit in rat pituitary. J Neuroendocrinol 8:99–102[CrossRef][Medline]
  38. Blithe D, Richards R, Skarulis M 1991 Free alpha molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy. Endocrinology 129:2257–2259[Abstract]
  39. Begeot M, Hemming FJ, Dubois PM, Combarnous Y, Dubois MP, Aubert ML 1984 Induction of pituitary lactotrope differentiation by luteinizing hormone {alpha}-subunit. Science 226:566–568[Abstract/Free Full Text]
  40. Morgan PJ, Webster CA, Mercer JG, Ross AW, Hazlerigg DG, MacLean A, Barrett P 1996 The ovine pars tuberalis secretes a factor(s) that regulate(s) gene expression in both lactotrophic and nonlactotrophic pituitary cells. Endocrinology 137:4018–4026[Abstract]
  41. Hazlerigg DG, Hastings MH, Morgan PJ 1996 Production of a prolactin releasing factor by the ovine pars tuberalis. J Neuroendocrinol 8:489–492[CrossRef][Medline]
  42. Lincoln GA, Clarke IJ 1994 Photoperiodically-induced cycles in the secretion of prolactin in hypothalamo-pituitary disconnected rams: evidence for translation of the melatonin signal in the pituitary gland. J Neuroendocrinol 6:251–260[Medline]
  43. Stoeckel ME, Hindelang C, Klein M-J, Poissonnier M, Felix J-M 1993 Early expression of the glycoprotein hormone alpha-subunit in the pars tuberalis of the rat pituitary gland during ontogenesis. Neuroendocrinology 58:616–624[Medline]
  44. Böckers TM, Bockmann J, Fauteck J-D, Kreutz MR, Bock R, Wittkowski W 1994 Pars tuberalis-specific cells in the ovine pituitary do express the common {alpha}-chain of glycoprotein hormones: an in situ hybridization and immunocytochemical study. Eur J Endocrinol 131:540–546[Abstract/Free Full Text]
  45. Böckers TM, Bockmann J, Fauteck JD, Wittkowski W, Sabel B, Kreutz MR 1996 Evidence for gene transcription of adenohypophyseal hormones in the ovine pars tuberalis. Neuroendocrinology 63:16–27[Medline]
  46. Arbogast LA, Voogt JL 1995 Hyperprolactinemia decreases tyrosine hydroxylase activity in the tuberoinfundibular dopaminergic neurons acutely by protein dephosphorylation and chronically by changes of gene expression. Endocrine 3:801–806
  47. Bex FJ, Bartke A 1977 Testicular LH binding in the hamster: modification by photoperiod and prolactin. Endocrinology 100:1223–1226[Abstract]
  48. Delegean AM, Ferland LH, Mellon PL 1987 Tissue specific enhancer of the human glycoprotein hormone {alpha}-subunit gene: dependence on cyclic AMP-inducible element. Mol Cell Biol 7:3994–4002[Abstract/Free Full Text]
  49. Lerchl A, Nieschlag E 1992 Interruption of nocturnal pineal melatonin synthesis in spontaneous recrudescent Djungarian hamsters (Phodopus sungorus). J Pineal Res 13:36–41[Medline]
  50. Lincoln GA, Clarke IJ, Sweeney T 1996 "Hamster-like" cycles in testicular size in the absence of gonadotrophin secretion in HPD rams exposed to long-term changes in photoperiod and treatment with melatonin. J Neuroendocrinol 8:855–866[CrossRef][Medline]



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