Temporal Sequence of Neuroendocrine Events Associated with the Transfer of Male Golden Hamsters from a Stimulatory to a Nonstimulatory Photoperiod1

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1 BIOLOGY OF RPRODUTION 44, (1991) Temporal Sequence of Neuroendocrine vents Associated with the Transfer of Male Golden Hamsters from a Stimulatory to a Nonstimulatory Photoperiod1 RIHARD W. STGR2 and ANDRZJ BARTK Department of Physiology, Southern Illinois University School of Medicine arbondale, Illinois, ABSTRAT The transfer of male golden hamsters from long day (LD) to short day (SD) conditions results in gonadal atrophy within 8 weeks and significant reductions in LH, FSH, and prolactin (Prl) secretion as early as 4 weeks, hanges in hypothalamic neurotransmitter metabolism precede these changes in pituitary hormone secretion. Thus median eminence norepinephrine (N) turnover declines steadily after SD exposure, although the differences as compared to turnover in LD hamsters are not significant until Week 4. Median eminence dopamine (DA) turnover is reduced significantly within 1 week. Turnover of N and DA in the medial basal hypothalamus also changes significantly within 1 or 2 weeks of SD exposure, but the changes are not maintained through Week 8, despite continued reductions in levels of circulating LH, FSH, and PrI. Reductions in median eminence N metabolism appear to be responsible for the decrease in LH and FSH release. Initial decreases in Prl release appear to be hypothalamic in origin, but the hypothalamic factor(s) responsible for this change is not evident. An increase in inhibitory input from tuberoinfundibular dopaminergic neurons is clearly not involved. INTRODUTiON The golden (Syrian) hamster is a seasonal breeder that shows well-characterized endocrine responses to changes in photoperiod. When adult male or female hamsters are transferred from a long to a short photoperiod (less than 12.5 h light per day), gonadal function is suppressed and the reproductive tract involutes [1-6]. Reproductive function undergoes spontaneous recovery after approximately 25 weeks in short photoperiod and it can be restored earlier by exposure to long photoperiods (8]. In the male golden hamster, short photoperiod-induced testicular regression is preceded by decreases in serum LH, FSH, and prolactin (Prl) levels (5, 6, 8-1]. The reduction in the concentration of gonadotropins appears to be due to a suppression of LHRH release since pituitary LHRH responsiveness does not decrease in short-day-exposed male hamsters (ii, 12]. We have previously demonstrated that endocrine changes associated with photoperiod-induced testicular regression and recrudescence are associated with changes in hypothalamic catecholamine metabolism [1, 13, 14]. Thus exposure to a short photoperiod causes a decrease in hypothalamic norepinephrine (N) and dopamine (DA) turnover, and the exposure of short-day-exposed, testicularly regressed hamsters to a stimulatory photoperiod (1 4L: 1OD) increases hypothalamic N and DA turnover. More recently, Benson [15] has demonstrated temporal increases followed by decreases in hypothalamic N and DA metabolism pre- Accepted August 29,199. Received June 29, 199. Supported by NSF grant DB and NIH grant HD orrespondence: Dr. R W. Steger. Department of Po siology, SIU School of Medicine, arbondale, IL ceding endocrine and gonathl changes associated with the transfer of male hamsters from a stimulatoty to a nonstimulatory photoperiod. In the present study, we report temporal changes in monoamine metabolism and pituitary function in three distinct hypothalamic regions of male hamsters undergoing photoperiod-induced testicular regression. MATRIALS AND MTHODS Adult golden (Syrian) hamsters [Lak: LVG(SYR) strain] were purchased from harles River Breeding Laboratories, (Wilmington, MA) and housed in a long photoperiod (LD, 16L:8D) or in a short photoperiod (SD, 8L: 16D). The temperature was maintained at 24 ± 1 #{176} and food and water were available ad hibitum. At 1, 2, 4, or 8 wk after transfer from LD to SD conditions, LD and SD animals (8-1 hamsters/group) were injected with saline or the tyrosine hydroxylase inhibitor, alpha methylp-tyrosine (ampt, 25 mg/kg i.p.) and were killed 6 mm later by decapitation. The transfer to SD was timed so that all animals were killed on the same morning. Blood was collected from the animals for measurement of serum LH, FSH, and Prl concentrations. After decapitation, the brain of each hamster was rapidly removed and the median eminence (M) was rapidly dissected free and frozen on dry ice. The remaining neural tissue was also rapidly frozen and stored at -7#{176}.Within several weeks, the brains were allowed to thaw partially and the medial basal hypothalamus (MBH) and anterior hypothalamus (AH) were dissected from the brain and weighed as previously described [14, 16]. Tissue samples were homogenized in.2 N perchloric acid containing i M sodium bisulfite. N-methylserotonin and dihydroxybenzylamine were added to each sample as 76 on 11 April 218

2 PHOTOPRIOD AND NURONDORIN FUNTION 77 internal standards with which procedure-related amine losses could be estimated. Internal standard recovery in the hydroxytryptamine (HT) assay averaged 98 ± 4% while recovery in the catecholamine assay averaged 83 ± 8%. After centrifugation for 1 mm at 12 x g, levels of HT and hydroxymndoleacetic acid ( HIM) in the supernatant fluids were determined by electrochemical detection following separation by high-performance liquid chromatography (HPL-) [14, 16]. Additional aliquots of the supernatant fluid were subjected to alumina extraction and measurement of N and DA levels using HPL- according to previously described procedures [14, 16]. atecholammne turnover rates were estimated using the formula K = k(a], where [A] equals the mean catecholamine concentration at zero time (uninjected controls), and the rate constant, k, represents the -log of the slope of the line describing the decline of N or DA concentration during the 1 h following the blockade of tyrosine hydroxylase with ampt (16]. It should be mentioned that the turnover rates are only estimates, because the linearity of the response to ampt over time was not determined. Pituitaries were removed at autopsy and the posterior lobe of each was separated and discarded. The anterior pituitaries of saline-treated hamsters were hemisected, weighed, and placed in individual 12 X 7mm glass culture tubes [12]. One milliliter of Medium 199 (M199 + bicarbonate, ph 7.3; Gibco Labs, Grand Island, NY) was added and the glands were preincubated for 1 h at 37#{176} in a Dubnoff Metabolic Shaker (Precision Scientific, hicago, IL). The tubes were maintained in an atmosphere of 5% O2 : 95%2. After 6 mm, the medium was removed and discarded and fresh M-199 was added. Sixty minutes later, medium was removed and frozen for subsequent measurement of basal hormone release and medium containing 18 M DA was added. The pituitaries were incubated for an additional hour, after which the medium was removed and frozen and the pituitaries were sonicated in I ml of saline. The concentration of Pr! in the medium, pituitary homogenates, and plasma was measured in a homologous hamster Prl assay (reagents kindly provided by Dr. Frank Talamantes), according to previously published procedures [12]. LH and FSH concentrations were measured using the rat LH and FSH assay kits provided by the NIAMMD (Baltimore, MD) as validated for use in the hamster [17]. All hormone measurements were run in a single assay. The data were analyzed by analysis of variance and statistical significance among means was determined using the Student-Newman-Keuls test. Differences were considered significant if p <.5. U, 2.O tes * naivesi AG. 1. The effects of time in a short (8L:16D) photoperiod on testicular and seminal vesicle weights. Values expressed as mean ± SM (n = 16-2 hamsters/group). The asterisk denotes statistical significance (p <.5) vs. the long photoperiod controls ( weeks in BL:16D). not to the degree seen in previous studies [1, 13, 14]. No changes in these organ weights were seen between and 4 weeks of SD exposure. Serum levels of LH, FSH, and Prl were unchanged after 2 weeks of SD exposure, but by 4 weeks serum concentrations of all 3 hormones were significantly less than seen in the LD controls (Fig. 2). Injection of ampt resulted in a significant rise in Prl levels in all groups. The absolute increase in PrI levels was significantly smaller in the animals exposed to SD for 8 weeks than in any of the other groups, but the relative increase in Pr! was twice as high at 8 weeks as at weeks (interaction between length of SD exposure and ampt had a p <.1 by 2-way ANOVA). In Vitro Pri Secretion In vitro Prl secretion was not altered by exposure of the animals to SD for 1, 2, or 4 weeks but declined significantly by 8 weeks of SD exposure (Fig. 3). xposure to 1-8 M DA appeared to reduce in vitro PrI secretion, but this effect was significant only after 8 weeks of SD. The overall effect of DA was significant (p <.5) by ANOVA, but the interaction of DA and length of SD exposure was not. Pituitary PrI content measured after 3 h of incubation was significantly reduced after 8 weeks of SD exposure as compared to, 1, 2, and 4 weeks of SD exposure. Values at 4 weeks tended to be lower than those seen at earlier times, but the differences were not statistically significant. RSULTS Organ Weights and Plasma Hormone Levels xposure to 8L: 16D for 8 weeks led to a significant decline in testes and seminal vesicle weights (Fig. 1) although H)pothalamic Amine Metabolism M-N turnover declined steadily during SD exposure but the reduction did not reach significance until 4 weeks (Fig. 4). DA turnover in the M was reduced within 1 week of SD exposure and remained low through Week 8. N on 11 April 218

3 78 STGR AND BARTK L25 S.- -S 1..z _J.5 /) U) I U) U. U) a FIG. 3. The effects of time in a short (8L:16D) photoperiod on pituitary PrI content (bottom panel) and in vitro PrI secretion (top panel). Values expressed as mean ± SM (n = 8-1 hemipituitaries/group). Only hemipituitaries from saline-treated hamsters were used. The asterisk denotes statistical significance (p <.5 by ANOVA) vs. the long photoperiod controls ( weeks in 8L:16D). a. U) FIG. 2. The effects of time in a short (8L: 16D) photoperiod on serum hormone levels. Values expressed as mean ± SM (n = 8-1 hamsters/ group). LH and FSH levels are for saline-injected hamsters. The asterisk denotes statistical significance (p <.5) vs. the respective long photoperiod control ( weeks in 8L:16D). turnover in the MBH was reduced after 1 week of SD exposure and remained low at 2 and 4 weeks. However, after 8 weeks in SD, MBH-N turnover was similar to values measured in the LD controls (Fig. 5). DA turnover showed almost an opposite pattern of change with values elevated after 2 and 4 weeks of SD exposure and a return to LD levels by 8 weeks. Serotonin levels were unchanged at the times measured, but HIM levels showed a transient but significant increase at Weeks 1 and 2 (Fig. 6). A marked increase in N turnover was seen in the AH within 1 week of SD exposure (Fig. 7). Although AH-N turnover decreased between 1 and 2 weeks, values seen at 2, 4, and 8 weeks were still significantly higher than those in the LD controls. DA turnover in the AH decreased significantly between 1 and 2 weeks of SD exposure and remained low through the course of the experiment. Serotonin and HIM levels in the AH were unchanged during exposure to SD (data not shown). on 11 April 218

4 PHOTOPRIOD AND NURONDORIN FUNTION 79 w ) Medial Basal Hypothalamus ) > w z Ui z -c ) FIG. 5. The effects of time in a short (8L:16D) photoperiod on medial basal hypothalamic N and DA turnover. (See legend of Fig. 4 for details.) FIG. 4. The effects of time in a short (8L: 16D) photoperiod on median eminence N and DA turnover. Turnover was calculated from the rate of catecholamine depletion following the inhibition of tyrosine hydroxylase with ampt. Values expressed as mean ± SM (n = 8-1 hamsters/group). The asterisk denotes statistical significance (p <.5) vs. the long photoperiod controls ( weeks in 8L:16D). 8 previous observations of reduced M and whole hypothalamic N turnover in male hamsters exposed to an SD photoperiod for weeks [1, 18]. Based on these observations and the demonstration that increases in LH levels after transfer of gonadally regressed hamsters to a stimu- ) Medial Basal Hypothalamus DISUSSION The present study documents significant changes in hypothalamic monoamine metabolism that are associated with SD-induced decreases in gonadotropmn and Prl secretion and in the weights of testes and the accessory reproductive glands in male golden hamsters. The temporal changes described in the present study partially confirm and extend the results of a similar study by Benson [15]. Significant changes in serum W, FSH, and PrI levels were first seen at 4 weeks, which coincided with the first significant decrease in N turnover in the M. These results are in good agreement with Benson [15], who observed no change in LH or N turnover in hypothalamic fragments containing both the M and the MBH after 3 weeks of SD exposure but found that both of these parameters were significantly decreased at Week 6. They are also consistent with ) ).) I U, U, FIG. 6. The effects of time in a short (8L:16D) photoperiod on medial basal hypothalamic content of HT and HIAA. Values expressed as mean ± SM (n = 8-1 hamsters/group). The asterisk denotes statistical significance (p <.5) vs. the long photoperiod controls ( weeks in 8L:16D). 8 on 11 April 218

5 8 STGR AND BARTK -S I.. a > w z > Anterior Hypothalamus FIG. 7. The effects of time in a short (8L:16D) photoperiod on anterior hypothalamic N and DA turnover. (See legend of Fig. 4 for details.) latory photoperiod are preceded by increases in N turnover [13], there is good reason to believe that reductions in N turnover are responsible for decreased gonadotropin secretion in the gonadally regressed hamster. This hypothesis is further strengthened by the demonstration that pinealectomy blocks the effect of SD on LH secretion and M- N turnover and that administration of the N precursors, 1-DOPA or 1-DOPS, stimulates LH release when given to SD hamsters [13, 14, 19; Steger unpublished observations]. The stimulatory effect of N on LHRH and consequently LH secretion in the rat is well documented [2, 211, and we have previously reported that similar effects occur in other species, including the hamster [6, 22]. N changes in the MBH did not parallel changes in the M. Whereas, M-N turnover did not decrease until Week 4, N turnover in the MBH decreased signfficantly after only 1 week of SD exposure. In addition, at 8 weeks, MBH-N turnover had returned to control levels despite the continued reduction of M-N turnover. These data are difficult to compare with those of Benson [15], since he did not separate the MBH and the M and his dissection was slightly different. Benson s data for the whole hypothalamus do indicate that N turnover reaches a nadir at 6 weeks and then doubles by Week 12, which is in agreement with the results we would obtain if we combined our M and MBH data. The MBH-N data does not correlate with serum gonadotropin levels and it is unclear what these changes represent. However, it was previously demonstrated that pinealectomy does not block photoperiod-induced changes in MBH-N turnover despite the fact that it blocks changes in gonads! weight, LH levels, and M-N turnover [14]. Furthermore, it has also been shown that pinealectomy does not completely block photoperiod-induced changes in Prl synthesis and release [231, suggesting that pineal-independent changes in MBH-N metabolism may be involved in the regulation of Pr! release. In the rat, N has been shown to have both inhibitory and stimulatory effects on Pr! secretion [2, 24]. Serum Prl levels in the present experiment were significantly depressed by Week 4, but in vitro Pr! secretion and pituitary Prl content were not significantly depressed until Week 8. These data suggest-but certainly do not provethat hypothalamic rather than pituitary factors were responsible for the initial changes in PrI secretion. On the basis of an initial increase in hypothalamic DA turnover seen at Weeks 3 and 6, Benson [151 concluded that DA was involved in the initial decline of Prl but that other mechanisms must be responsible for the suppressed Prl levels at Weeks 9 and 12 when hypothalamic DA turnover was decreased to rates lower than those in LD animals. In agreement with Benson s data [15] for the whole hypothalamus, our data for the MBH also showed an initial increase in DA turnover followed by a return to LD levels by Week 8. However, it does not appear that increases in tuberoinfundibular DA (TIDA) activity are responsible for the decline in Pr!, since DA turnover in the M, the terminal field for TIDA neurons, was significantly decreased after 1 week and remained depressed for at least 8 weeks after transfer to SD. Furthermore, the proportional Pr! response to inhibition of DA synthesis with ampt did not differ between LD hamsters and hamsters exposed to SD for 1, 2, or 4 weeks. Previously, we suggested that an increase in the pituitary s response to DA might explain the decrease in Prl levels in SD-exposed male hamsters despite observed decreases in TIDA activity [12, 14], but an increase in response to this particular dose of DA was not seen until Week 8 and therefore does not explain the decrease in serum Prl at Week 4. The data from the present experiment also question the previous hypothesis that reductions in TIDA activity were secondary to reduced PrI feedback [18] since the decline in M-DA turnover preceded the decrease in Pr! by several weeks. Numerous other hypothalamic neurotransmitters and peptides-such as gamma-aminobutyric acid, vasoactive intestinal polypeptide, and thyrotropin-releasing hormone (TRH)-and opioid peptides are known to effect Prl release [24] and could also be involved in photoperiod-in- on 11 April 218

6 PHOTOPRIOD AND NURONDORIN FUNTION 81 duced changes in Pr! release. The observation that SD exposure also reduces thyroid function [25] suggests that reduction in TRH release might be involved. It is unclear what the role of HT might be in the transition from a gonadally active to a gonadally regressed state. Previously we have shown that fully regressed SD hamsters have an increased rate of MBH-HT synthesis that is decreased within 2 days of LD exposure (26], but we have not established whether these events have any consequences with regard to pituitary hormone secretion or other events associated with changes in photoperiod. In the present study, we demonstrated a transient increase in HIM levels at Weeks 1 and 2 of SD exposure that may represent increased HT release. Serotonin was shown to be generally stimulatory to Prl release while inhibitory to LH release, but there was no temporal relationship between HT and hormone changes demonstrated in this experiment. Additional studies involving direct measurements of HT synthesis or release could shed further light on the role of HT in mediating hormonal changes associated with response to photoperiod. Data from the present and previous studies indicate that there are complex changes in neuroendocrine control systems as animals are exposed to changes in day length. It has previously been demonstrated that SD male hamsters are exquisitely sensitive to testosterone negative feedback effects on LH release and hypothalamic N metabolism [27, 28]. These changes may in turn be partially due to alterations in Prl secretion since Pd is able to decrease the rate of testicular regression, restore testicular function in regressed SD hamsters, and decrease feedback sensitivitypossibly through alterations in androgen receptors [ A number of SD-associated changes in pituitary PrI synthesis, storage, and release have been described [12, 23,34,351, but it is still unclear why these changes occur. However, it appears that these changes may be due to a series of complex changes in inhibitory input from the TIDA system as well as changes in other putative Pr! releasing and/or inhibiting factors. Furthermore, it appears that the relative importance of these factors may change considerably during the course of gonada! regression and recrudescence, emphasizing the need for studying PrI regulation during transitions between peaks and nadirs of reproductive function. In conclusion, it is evident that during the response of adult male golden hamsters to SD, changes in the metabolism of hypothalamic neurotransmitters occur well in advance of the changes in adrenohypophyseal hormone release. However, additional studies are necessary to identify the cause:effect relationships involved. AKNOWLDGMNTS We gratefully acknowledge the expert technical assistance of Jeanie Rathert and Sherrie Hodges. Materials for the LH and FSH assay were kindly provided by the NIDDKD, Baltimore, MD. RFRNS 1. 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