- Email: [email protected]
Photic threshold for stimulation of testicular growth and pituitary FSH release in male Djungarian hamsters
304
Brain Research. ,, 12 (1990) 304- ~ll8 Elsevier
BRES 15319
Photic threshold for stimulation of testicular growth and pituitary FSH release in male Djungarian hamsters Jill J. Milette, Joseph S. Takahashi and Fred W. Turek Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208 (U.S.A.)
(Accepted 22 August 1989) Key words. Djungarian hamster; Siberian hamster; Photoperiodic stimulation; Light; trradiance; Photoreceptor
While the effects of photoperiod on neuroendocrine-gonadal activity have been extensively studied in a number of species, surprisingly little information concerning the quantitative aspects of light regulating reproductive activity is available. In the present experiment, Djungarian hamsters were exposed to two 10 min pulses of light per day and the light irradiance was systematically varied to determine the threshold for photostimulation by white light. After 10 days testes weight and serum follicle-stimulating hormone (FSH) levels were determined. The data indicate that the irradiance threshold necessary for induction of significant increases of both serum FSH levels and testes weight lies between 0.1 and 0.34/tW/cm 2 for 10 min pulses of light. These results demonstrate a strong correlation between the effects of light on serum FSH levels and testes weight and provide the first quantitative assessment of the irradiance threshold for light involved in photoperiodic stimulation of the hypothalamic-pituitary gonadal axis of a mammalian species. INTRODUCTION Many seasonally breeding animals synchronize their reproductive cycles to the appropriate time of the year by evaluating the length of the day, or photoperiod. In many small rodent species, the long days of spring and summer are stimulatory to reproductive function while the short days of fall are inhibitory 8'22. The importance of light for stimulating reproductive function is particularly clear in young male Djungarian hamsters, a species that depends primarily upon photoperiodic information for the timing of its seasonal breeding cycle 9'2°. An increase in serum follicle-stimulating hormone (FSH) levels and initiation of testicular growth can be induced by exposing the hamsters to long day lengths. Furthermore, these responses occur very rapidly in the male Djungarian hamster. For example, within one week of photostimulation, serum FSH levels and testicular weight have increased significantly over initial control values 19. The Djungarian hamster, like other small photoperiodic rodents, relies upon a circadian time-keeping system for induction of these neuroendocrine events; even brief pulses of light presented at critical times relative to the phase of the hamster's circadian clock can induce a significant neuroendocrine response 5121415. Light reception is the first step in the cascade of physiological events involved in the initiation of neuroendocrine-gonadal function in photoperiodic animals. How-
ever, very little is known about the quantitative aspects of light responsible for initiating the photoperiodic response. In order to study the properties of the retinal photoreceptors mediating photoperiodic reproductive responses, the response of the system must first be assessed using brief pulses of light on a background of darkness in order to avoid complications that can result from light adaptation of the photoreceptors due to prolonged light exposure. We have previously shown that significant increases in both paired testes weight and serum FSH levels occur within 10 days after exposure of male Djungarian hamsters to a skeleton photoperiod consisting of two 10-min pulses of white light per day given 8 and 16 h apart 15. Because such short periods of light can stimulate neuroendocrine-gonadal activity so rapidly in the Djungarian hamster, this species is a useful model to quantify the irradiance of light necessary to stimulate reproductive function. In the present experiment, the irradiance of white light presented to groups of hamsters during two 10-min pulses of light per day was varied systematically to define the threshold irradiance necessary to induce a neuroendocrine-gonadal response. MATERIALS AND METHODS Male Djungarian hamsters (Phodopus sungorus) were born in our breeding colony under a 16 h light (L)-8 h dark (D) photoperiod
Correspondence: KW. Turek, Department of Neurobiology and Physiology, Hogan Hall, Northwestern University, Evanston, IL 60208, U.S.A.
01~6-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
305 (lights on 10.00-02.00 h). At 18 + 2 days of age the males were weaned, group housed 3-5 per cage, and transferred to a light-tight box with an 8L:16D photoperiod (lights on 22.00-06.00 h). After 21 + 3 days in the short photoperiod, testis regression was assessed by gentle palpation of the scrotal area of unanesthetized animals, Animals with palpable testes were removed from the study. The estimated paired testes weight was less than 50 mg for the remaining animals with nonpalpable testes. During the 10 day experimental period, the rhythm of locomotor activity was recorded from all animals. Each cage was equipped with a running wheel connected to an Esterline-Angus event recorder via a microswitch. All activity data from the animals was recorded as previously described 15. The animals were only exposed to light when they were placed into the photic stimulation apparatus consisting of a light-tight box with three compartments to allow separate access to the light source, the filter compartment and the animal chamber. The light was projected downward such that the animals received the light from overhead. The light source compartment was separated from the filter and animal compartments by a hand-operated metal shutter. Light from a 500-W tungsten-halogen lamp (General Electric, BCK) was condensed and projected with glass lenses through a series of infrared absorbing filters (Schott, KG-1 and KG-4), through a colored glass filter (Schott, BG-40) and then through a series of neutral density filters. The different light irradiances were obtained by using different combinations of neutral density filters for each light exposure group, The irradiance of light was measured (as pW/cm 2) immediately prior to every light exposure with a United Detector Technology $350 photometer connected to a no. 248 sensor placed within the animal chamber. The mean of these values was reported as the irradiance for the group. The wide band spectral distribution of the stimulus had a peak (2max) of 570 nm and a half band-width (HW) of about 170
3.4 #W/cm 2. On the l l t h day of light exposure (total of 21 light exposures), these animals, as well as the animals in the control groups, were killed by ether overdose between 13.00 and 15.00 h. Blood was quickly collected by cardiac puncture and centrifuged immediately; the serum was frozen for later analysis of FSH levels by radioimmunoassay. Both testes were removed from each animal and weighed. Serum levels of FSH were determined in two assays using a NIDDK kit for measuring rat FSH. Parallelism between Djungarian hamster serum and the NIH rat standard has been documented 15'23. The radioimmunoassay allowed measurement of FSH levels down to 3.5 ng/ml. The time of activity onset for each animal was determined by inspection of the running wheel activity records. The activity data from 4 animals (out of 81) had to be omitted due to malfunctions of the equipment or animals that showed very little activity. For each animal's record, the first 3 activity onsets were excluded from the analysis to allow time for the animal to acclimate to the environment. A line was eye-fitted through the remaining 8 activity onsets. The time indicated by the midpoint of this line was taken as the time of activity onset for the animal. The group means (+ S.E.M.) were calculated from the individual onset times of each group. The mean (+ S.E.M.) time of activity offset for each group of animals was calculated in an identical manner. The FSH and paired testes weight data were analyzed by an analysis of variance followed by Scheffe's test. Significance was determined at the P < 0.05 level.
nm. At the time of light exposure, 4 animals from one group were placed into a 11.5 cm diameter, 5 cm high stimulus chamber made from white plastic. The chamber was fitted with a plastic 4 way divider so as to provide each individual with their own conpartment and prevent blockage of the light by huddling. The light was directed onto the animals from above onto an opal glass top which acted as a translucent diffuser to evenly disperse the light into the animal chamber. All light exposures occurred between 09.45-10.45 h in the morning and 17.45-18.45 h in the evening every day, with any one group being assigned to one of the four 15 min time periods within that hour so that their light exposure always occurred 8 and 16 h apart (+ 15 min). On the first day of light exposure, the 4 animals assigned to one group were removed from the group cage and placed into the light stimulus apparatus for 10 minutes during the morning exposure time and then individually placed into a cage equipped with a running wheel within a light-tight chamber, Subsequent light exposure occurred twice daily, beginning on the evening of day 1, with individual animals always exposed to the same light irradiance throughout the 10 days of exposure to light, After each 10 min light pulse, the animals were sorted (by examination of their ear punches) and returned to their individual running-wheel cages, All handling and transfer of animals between their running-wheel cages and the stimulus chamber was accomplished within a completely darkened room with the aid of a head-mounted infrared viewer (FJW Industries, Elgin, IL) and a lamp equipped with a 15 W bulb and an infrared filter, The male Djungarian hamsters with regressed testes were arranged into groups of four, marked by ear punches for individual identification, placed into individual running-wheel cages, and assigned to one of 9 groups. Two control groups remained undisturbed in 8L:16D (n = 16) or 16L:8D (n -- 9). One control group was moved twice per day into the exposure chamber, but was not exposed to light (n = 8). Six experimental groups of animals (n = 8 each) were exposed for 10 days to a 10 min light pulse twice per day at one of 6 irradiance levels: 0.0034, 0.01, 0.034, 0,10, 0.34, or
significantly larger testes t h a n a n i m a l s m a i n t a i n e d on
RESULTS
Animals
transferred
to
16L:8D
for
10 days
had
8 L : 1 6 D d u r i n g this t i m e p e r i o d (Fig. 1, top). T h e testes of animals k e p t in c o n s t a n t d a r k n e s s and t r a n s f e r r e d twice a day into t h e light e x p o s u r e c h a m b e r w e r e small and statistically similar in w e i g h t to the testes of animals in the 4 lowest i r r a d i a n c e light pulse g r o u p s (Fig. 1, top). T h e testicular r e s p o n s e of t h e animals e x p o s e d to a s k e l e t o n p h o t o p e r i o d was d e p e n d e n t u p o n t h e i r r a d i a n c e of light given d u r i n g the two 10 m i n light pulse periods. T h e testis weights of the t h r e e g r o u p s of animals e x p o s e d to the lowest i r r a d i a n c e s (0.0034, 0.01 o r 0.034 p W / c m 2) w e r e significantly less t h a n t h o s e o b s e r v e d for the g r o u p of animals e x p o s e d to 0.34 p W / c m 2. T h e m e a n testis w e i g h t of t h e animals e x p o s e d to an i n t e r m e d i a t e i r r a d i a n c e (0.1 p W / c m 2) was b e t w e e n that o b s e r v e d in the animals e x p o s e d to the t h r e e l o w e r and two h i g h e r irradiances of light, but w e r e n o t significantly different f r o m the m e a n testis weights o b s e r v e d in any of these groups. A l t h o u g h the d i f f e r e n c e was not statistically significant, the testis weights of t h e u n d i s t u r b e d animals in 8 L : 1 6 D w e r e larger t h a n t h o s e of the animals m o v e d twice a day into the light e x p o s u r e c h a m b e r w i t h o u t e x p o s u r e to light. T h i s a p p a r e n t d i f f e r e n c e in testis weight m i g h t be d u e to the stress of h a n d l i n g , since it is k n o w n that stressed animals s h o w d e c r e a s e d testicular function 4. T h e s e r u m F S H v a l u e s of t h e a n i m a l s p l a c e d in 1 6 L : 8 D for 10 days w e r e significantly g r e a t e r t h a n t h o s e of the
306
~
140
.,~
12o
~
.....
0
6
~ 0:
18
24
8L 16 :D~\'~"...........................
16L:8D
-~ tOO
Hours 12
~.. ................
, DD d i s t u r b e d , ~
~,~x~
E-. ~Ke;eton ~ \ \ \ \ \ \ \ ~ -~ 5~ I I 1 1 \ \ ' x ' x \ \ x. \ \ \ ' N \ e'notope o
.-
.-.~.~108 ~0
01 ~
"
~\~..034.
~ [ _ ~
6 4 e0
-"-~- \ \ \ \
E
16L
8L
0
0.01 0.1 1.0 Light I r r a d i a n e e (k~W/em ~)
Fig. 1. Means (___S.E.M.) of paired testes weights (top) and serum FSH levels (bottom) for groups of male Djungarian hamsters kept in full photoperiods of 16 h of light (16L; n = 9) or 8 h of light (8L; n = 16) per day (open bars), or exposed to a skeleton photoperiod consisting of two 10 min pulses of white light 8 and 16 h apart (closed circles; n = 8 each) for 10 days. A control group was transferred to the light exposure chamber twice a day, but the light was not turned on in the chamber (0; n = 8). Each group of animals exposed to a skeleton photoperiod was subjected to light of a different irradiance ranging from 0.0034 to 3.4 p W/cm: during their twice-daily exposure periods. All the animals had regressed testes due to 3 weeks of exposure to 8L:16D prior to entering the groups depicted in this figure. Serum FSH symbols without error bars indicate group means near the sensitivity level of the assay,
Fig. 2. A summary of the running wheel activity records of male Djungarian hamsters housed in various light cycles for 10 days. Open areas represent light and hatched areas represent darkness. The mean (_ S.E.M.) times of activity onset and offset for each group of hamsters is shown by the horizontal black bars. The groups of animals were housed in 16 hours of light per day (16L:8D), 8 h of light per day (8L:16D), constant darkness with transfer into a light exposure chamber for 10 rain twice a day without exposure to light (DD disturbed), or moved into the chamber and exposed to white light ranging from 0.0034 to 3.4/~W/cm 2 for 10 rain twice a day.
near that of the animals kept in 8L: 16D and the duration of activity was longer than in any of the groups of animals exposed to the skeleton p h o t o p e r i o d s . Nearly all the activity of the animals exposed to any of the 10 min skeleton p h o t o p e r i o d s occurred during the 8 h period of darkness. The shorter bars at the b o t t o m of the figure indicate that the activity p e r i o d of the animals exposed to the light pulses of higher irradiance was shorter in duration and confined between the light pulses occurring 8 h apart. In contrast, the animals exposed to the lower
animals that r e m a i n e d in 8L:16D (Fig. 1, bottom). No significant differences were observed in the serum F S H levels between the groups of animals exposed to 8L: 16D, no light or one of the 3 lowest light irradiances. In addition, the animals exposed to no light or one of the two lowest light irradiances had mean serum FSH levels below those of the animals in 16L:8D. Serum F S H levels for each of the groups of animals exposed to either no light or one of the three lower irradiances were significantly lower than the levels observed in each of the groups exposed to one of the two highest irradiances,
irradiance 10 min light pulses displayed activity patterns that were longer in duration and o v e r l a p p e d with the time of the light pulses.
Differences between the activity records of the groups of animals e x p o s e d to the different light schedules are depicted in Fig. 2. A n i m a l s in the two full p h o t o p e r i o d s were active during the dark phase of their p h o t o p e r i o d , with a longer duration of activity occurring in the group exposed to a longer d a r k period. The animals housed in constant darkness and moved to the light exposure a p p a r a t u s twice a day had a mean time of activity onset
experiment over the way animals were exposed to light in previous studies, is in the precise control over the irradiance level that could be maintained during the entire light exposure period. In previous studies (see below) animals were allowed to r o a m freely within a cage and were u n d o u b t e d l y exposed to many different light irradiances as they moved about and slept. In contrast, the animals in the present e x p e r i m e n t were exposed
DISCUSSION In o r d e r to permit a quantitative analysis of the visual pigment and p h o t o r e c e p t o r s mediating p h o t o p e r i o d i c responses, we d e v e l o p e d an e x p e r i m a n t a l protocol for assessing the response of the reproductive system to experimentally controlled light stimuli. The main advantage of the m e t h o d used for delivering light in the present
307 while awake to a very precise stimulus of light while in a chamber that did not allow the animal to move about for only 10 rain twice/day. The precise control of the light in the present study was made possible by the fact that photostimulation of neuroendocrine-gonadal activity can occur very rapidly in this species in response to short pulses of light 15'19. Our results compare quite favorably with two studies by Hoffman and colleagues examining the light irradiance threshold necessary for induction of testis growth in young golden hamsters l°'n. In these studies, groups of golden hamsters were exposed to constant darkness or a 14L:10D photoperiod at one of several low levels of illumination for up to 14 weeks and the effect on the organ weights of the animals was measured. In one of these experiments 1~, gonadal degeneration was observed in exactly half of the animals exposed for 13 weeks to a broad-band light source with an irradiance of 0.159 pW/cm 2, suggesting that this irradiance is near the threshold for stimulation of testis growth in the golden hamster. Testicular degeneration was observed in an even larger percentage of the tested animals at irradiances below this value. Hoffman's data are similar to the threshold found in the present study, where an irradiance of 0.1 to 0.34/~W/cm 2 (Fig. 1)was necessary to stimulate an increase in paired testes weight and serum FSH levels in male Djungarian hamsters exposed to two 10 min pulses of light per day for 10 days. In a series of experiments designed to examine the irradianceoflightnecessarytostimulateneuroendocrinegonadal activity, Brainard and colleagues exposed grouphoused golden hamsters to a l l L : 13D photoperiod with light during the light portion of this photoperiod (400 /~W/cm 2) extended by a 3 h period of light at varying irradiance 3. They observed significant testis growth within 12 weeks when the 3 h extended light period was 0.2 pW/cm 2 or greater. Intermediate stimulation of testis growth was achieved when the 3 h extended light period was either 0.002 or 0.02 pW/cm 2. This threshold value (0.02-0.2 /~W/cm 2) for stimulation of testis growth in male golden hamsters is about one order of magnitude less than the threshold value for stimulation of testis growth in Djungarian hamsters reported in the present experiment and for stimulation of testicular growth in golden hamsters in the studies by Hoffman and colleagues H. However, exposure to 400/,tW/cm 2 for 11 h in addition to the lower irradiance light for 3 h in this study by Brainard and colleagues complicates the interpretation of these results since it is not clear if the 11 h of exposure to bright light was playing some role in stimulating testicular growth. Furthermore, the bright lights during the prior 11 h may have altered the response of the photoreceptors to the extended period of dim light,
Another method that has been used to examine the importance of light irradiance in the photoperiodic response relies on the ability of light to suppress pineal melatonin content or pineal enzyme activity. Since pineal melatonin levels are closely linked to the prevailing light/dark cycle and the photoperiodic response of many mammalian species 13'16, measurement of the suppressive action of light on pineal melatonin content or the enzymes involved in synthesis of melatonin provide a rapid, albeit indirect, assay for the action of light on reproductive function in photoperiodic animals. A study of the ability of white light to suppress nocturnal levels of pineal melatonin content in adult male golden hamsters demonstrated that an 8 rain exposure to an irradiance of 0.186 pW/cm z led to a continued depression of the melatonin levels even after the animals were returned to darkness 2. Pineal serotonin-N-acetyltransferase (NAT) activity was suppressed by cool white fluorescent light at irradiances greater than or equal to 0.111/~W/cm 2 when golden hamsters were exposed to the light for 20 min prior to sacrifice 1. Both of these threshold values compare quite closely to the 0.1 pW/cm 2 irradiance value found in the present experiment to be the threshold for stimulation of neuroendocrine-gonadal activity in Djungarian hamsters exposed to two 10 min pulses of light per day for 10 days. In addition to stimulating neuroendocrine-gonadal activity, short pulses of light can also entrain the circadian rhythm of locomotor activity6,7,21. However, in the present study, it was not possible to determine whether the light pulses actually entrained the activity rhythm since animals were only in the light-dark cycle for 10 days. Nevertheless, from the activity records it is clear that animals in all the groups were active during the short period of darkness as were the animals exposed to a full photoperiod of 16L:8D. If the animals with small testes exposed to lower light irradiances were actually entrained to the two pulses of light, this would indicate that the threshold for photic stimulation of neuroendocrinegonadal activity is different than that for entrainment of the circadian rhythm of activity. However, further studies are necessary in order to clarify this issue. In summary, we have shown that photostimulation of reproductive function in the juvenile male Djungarian hamster can be used to explore the properties of the photoreceptive system involved in mediating photoperiodic induction of neuroendocrine-gonadal activity in mammals. Since the threshold for photoperiodic induction by light is several magnitudes greater than that necessary for vision 17'1s, the photoreceptive system involved in photoperiodic time measurement is clearly much less sensitive than that involved in vision. The sensitivity of the photoreceptors mediating the effect of light on reproduc-
308 tion and on the circadian system are similarel. It is of considerable interest to determine if the same photoreceptors mediate the effects of light on both the circadian and reproductive systems. Using D j u n g a r i a n hamsters, it should be possible to compare directly the photoreceptive responses for reproductive, pineal and circadian functions in a single species.
REFERENCES 1 Brainard, G.C., Richardson, B.A., King, T.S., Matthews, S.A. and Reiter, R.J., The suppression of pineal melatonin content and N-acetyltransferase activity by different light irradiances in the Syrian hamster: a dose-response relationship, Endocrinology, 113 (1983) 293-296. 2 Brainard, G.C., Richardson, B.A., Hurlbut, E.C., Steinlechner, S., Matthews, S.A. and Reiter, R.J., The influence of various irradiances of artificial light, twilight, and moonlight on the suppression of pineal melatonin content in the Syrian hamster, J. Pineal Res., 1 (1984) 105-119. 3 Brainard, G.C., Vaughan. M.K. and Reiter, R.J., Effect of light irradiance and wavelength on the Syrian hamster reproductive system, Endocrinology, 119 (1986) 648-654. 4 Charpenet, G., Tache, Y., Forest, M.G., Haour, E, Saez, J.M., Bernier, M., Ducharme, J.R. and Collu, R., Effects of chronic intermittent immobilization stress on rat testicular androgenic function, Endocrinology, 109 (1981) 1254-1258. 5 Darrow, J.M. and Goldman, B.D., Circadian regulation of pineal melatonin in the Djungarian hamster, J. Biol. Rhythms, 1 (1985) 39-54. 6 Earnest, D.J. and Turek, EW., Effect of one-second light pulses on testicular function and locomotor activity in the golden hamster, Biol. Reprod., 28 (1983) 557-565. 7 Elliott, J.A., Circadian rhythms, entrainment and photoperiodism in the Syrian hamster. In B.K. Follett and D.E. Follett (Eds.), Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, pp. 203-217. 8 Follett, B.K. and Follett, E.D., Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, 292 pp. 9 Heldmaier, G. and Steinlechner, S., Seasonal control of energy requirements of thermoregulation in the Djungarian hamster (Phodopus sungorus) living in natural photoperiod, J. Cornp. Physiol., 142 (1981) 429-437. 10 Hoffman, R.A. and Johnson, L.B., Effects of photic history and illuminance levels on male golden hamsters, J. Pineal Res., 2 (1985) 209-215. 11 Hoffman, R.A., Johnson, L.B. and Corth, R., The effects of spectral power distribution and illuminance levels on key parameters in the male golden hamster and rat with preliminary observations on the effects of pinealectomy, J. Pineal Res., 2
Acknowledgements. This research was supported by an NSF predoctoral fellowship to J.J.M., an NSF Presidential Young Investigator Award (DCB-8451642), NIMH Grant (MH-39595), and Searle Scholars Award (85-H-107) to J.S.T. and NIH Grants (HD-09885and HD-21921) to F.W.T. We thank Dwight Nelson for construction of the light exposure apparatus that was used in this study. A preliminary report of this work was presented at the 19th Annual Meeting of the Society for the Study of Reproduction, Ithaca, NY, July 1986.
(1985) 217-233. 12 Hoffmann, K., Photoperiodic effects in the Djungarian hamster: one minute of light during darktime mimics influence of long photoperiods on testicular recrudescence, body weight and pelage colour, Experientia, 35 (1979) 1529-1530. 13 Hoffmann, K., The role of the pineal gland in photoperiodic control of seasonal cycles in hamsters. In B.K. Follett and D.E. Follett (Eds.), Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, pp. 237-250. 14 Hoffmann, K., The effect of brief light pulses on the photoperiodic reaction in the Djungarian hamster, Phodopus sungorus, J. Comp. Physiol., 148 (1982) 529-534. 15 Milette, J.J. and Turek, EW., Circadian and photoperiodic effects of brief light pulses in male Djungarian hamsters, Biol. Reprod., 35 (1986) 327-335. 16 Reiter, R.J., The pineal gland: an intermediary between the environment and the endocrine system, Psychoneurology, 8 (1983) 31-40. 17 Ripps, H. and Weale, R.A., The visual stimulus. In H. Davson (Ed.), The Eye, Vol. 2A, Academic Press, New York, 1976, pp. 43-99. 18 Reuter, J.H., A comparison of flash evoked ERG's and ERG's evoked with sinusoidaily modulated light stimuli in a number of rodents, Pfluegers Arch., 331 (1972) 95-102. 19 Simpson, S.M., Follett, B.K. and Ellis, D.H., Modulation by photoperiod of gonadotrophin secretion in intact and castrated Djungarian hamsters, J. Reprod. Fert., 66 (1982) 243-250. 20 Steinlechner, S., Heldmaier, G. and Becker, H., The seasonal cycle of body weight in the Djungarian hamster: photoperiodic control and the influence of starvation and melatonin, Oecologia, 60 (1983) 401-405. 21 Takahashi, J.S., DeCoursey, P.J., Bauman, L. and Menaker, M., Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms, Nature (Lond.), 308 (1984) 186-188. 22 Turek, F.W. and VanCauter, E., Rhythms in reproduction. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, pp. 1789-1830. 23 Yellon, S.M. and Goldman, B.D., Photoperiod control of reproductive development in the male Djungarian hamster, (Phodopus sungorus), Endocrinology, 114 (1984) 664-670.
Brain Research. ,, 12 (1990) 304- ~ll8 Elsevier
BRES 15319
Photic threshold for stimulation of testicular growth and pituitary FSH release in male Djungarian hamsters Jill J. Milette, Joseph S. Takahashi and Fred W. Turek Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208 (U.S.A.)
(Accepted 22 August 1989) Key words. Djungarian hamster; Siberian hamster; Photoperiodic stimulation; Light; trradiance; Photoreceptor
While the effects of photoperiod on neuroendocrine-gonadal activity have been extensively studied in a number of species, surprisingly little information concerning the quantitative aspects of light regulating reproductive activity is available. In the present experiment, Djungarian hamsters were exposed to two 10 min pulses of light per day and the light irradiance was systematically varied to determine the threshold for photostimulation by white light. After 10 days testes weight and serum follicle-stimulating hormone (FSH) levels were determined. The data indicate that the irradiance threshold necessary for induction of significant increases of both serum FSH levels and testes weight lies between 0.1 and 0.34/tW/cm 2 for 10 min pulses of light. These results demonstrate a strong correlation between the effects of light on serum FSH levels and testes weight and provide the first quantitative assessment of the irradiance threshold for light involved in photoperiodic stimulation of the hypothalamic-pituitary gonadal axis of a mammalian species. INTRODUCTION Many seasonally breeding animals synchronize their reproductive cycles to the appropriate time of the year by evaluating the length of the day, or photoperiod. In many small rodent species, the long days of spring and summer are stimulatory to reproductive function while the short days of fall are inhibitory 8'22. The importance of light for stimulating reproductive function is particularly clear in young male Djungarian hamsters, a species that depends primarily upon photoperiodic information for the timing of its seasonal breeding cycle 9'2°. An increase in serum follicle-stimulating hormone (FSH) levels and initiation of testicular growth can be induced by exposing the hamsters to long day lengths. Furthermore, these responses occur very rapidly in the male Djungarian hamster. For example, within one week of photostimulation, serum FSH levels and testicular weight have increased significantly over initial control values 19. The Djungarian hamster, like other small photoperiodic rodents, relies upon a circadian time-keeping system for induction of these neuroendocrine events; even brief pulses of light presented at critical times relative to the phase of the hamster's circadian clock can induce a significant neuroendocrine response 5121415. Light reception is the first step in the cascade of physiological events involved in the initiation of neuroendocrine-gonadal function in photoperiodic animals. How-
ever, very little is known about the quantitative aspects of light responsible for initiating the photoperiodic response. In order to study the properties of the retinal photoreceptors mediating photoperiodic reproductive responses, the response of the system must first be assessed using brief pulses of light on a background of darkness in order to avoid complications that can result from light adaptation of the photoreceptors due to prolonged light exposure. We have previously shown that significant increases in both paired testes weight and serum FSH levels occur within 10 days after exposure of male Djungarian hamsters to a skeleton photoperiod consisting of two 10-min pulses of white light per day given 8 and 16 h apart 15. Because such short periods of light can stimulate neuroendocrine-gonadal activity so rapidly in the Djungarian hamster, this species is a useful model to quantify the irradiance of light necessary to stimulate reproductive function. In the present experiment, the irradiance of white light presented to groups of hamsters during two 10-min pulses of light per day was varied systematically to define the threshold irradiance necessary to induce a neuroendocrine-gonadal response. MATERIALS AND METHODS Male Djungarian hamsters (Phodopus sungorus) were born in our breeding colony under a 16 h light (L)-8 h dark (D) photoperiod
Correspondence: KW. Turek, Department of Neurobiology and Physiology, Hogan Hall, Northwestern University, Evanston, IL 60208, U.S.A.
01~6-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
305 (lights on 10.00-02.00 h). At 18 + 2 days of age the males were weaned, group housed 3-5 per cage, and transferred to a light-tight box with an 8L:16D photoperiod (lights on 22.00-06.00 h). After 21 + 3 days in the short photoperiod, testis regression was assessed by gentle palpation of the scrotal area of unanesthetized animals, Animals with palpable testes were removed from the study. The estimated paired testes weight was less than 50 mg for the remaining animals with nonpalpable testes. During the 10 day experimental period, the rhythm of locomotor activity was recorded from all animals. Each cage was equipped with a running wheel connected to an Esterline-Angus event recorder via a microswitch. All activity data from the animals was recorded as previously described 15. The animals were only exposed to light when they were placed into the photic stimulation apparatus consisting of a light-tight box with three compartments to allow separate access to the light source, the filter compartment and the animal chamber. The light was projected downward such that the animals received the light from overhead. The light source compartment was separated from the filter and animal compartments by a hand-operated metal shutter. Light from a 500-W tungsten-halogen lamp (General Electric, BCK) was condensed and projected with glass lenses through a series of infrared absorbing filters (Schott, KG-1 and KG-4), through a colored glass filter (Schott, BG-40) and then through a series of neutral density filters. The different light irradiances were obtained by using different combinations of neutral density filters for each light exposure group, The irradiance of light was measured (as pW/cm 2) immediately prior to every light exposure with a United Detector Technology $350 photometer connected to a no. 248 sensor placed within the animal chamber. The mean of these values was reported as the irradiance for the group. The wide band spectral distribution of the stimulus had a peak (2max) of 570 nm and a half band-width (HW) of about 170
3.4 #W/cm 2. On the l l t h day of light exposure (total of 21 light exposures), these animals, as well as the animals in the control groups, were killed by ether overdose between 13.00 and 15.00 h. Blood was quickly collected by cardiac puncture and centrifuged immediately; the serum was frozen for later analysis of FSH levels by radioimmunoassay. Both testes were removed from each animal and weighed. Serum levels of FSH were determined in two assays using a NIDDK kit for measuring rat FSH. Parallelism between Djungarian hamster serum and the NIH rat standard has been documented 15'23. The radioimmunoassay allowed measurement of FSH levels down to 3.5 ng/ml. The time of activity onset for each animal was determined by inspection of the running wheel activity records. The activity data from 4 animals (out of 81) had to be omitted due to malfunctions of the equipment or animals that showed very little activity. For each animal's record, the first 3 activity onsets were excluded from the analysis to allow time for the animal to acclimate to the environment. A line was eye-fitted through the remaining 8 activity onsets. The time indicated by the midpoint of this line was taken as the time of activity onset for the animal. The group means (+ S.E.M.) were calculated from the individual onset times of each group. The mean (+ S.E.M.) time of activity offset for each group of animals was calculated in an identical manner. The FSH and paired testes weight data were analyzed by an analysis of variance followed by Scheffe's test. Significance was determined at the P < 0.05 level.
nm. At the time of light exposure, 4 animals from one group were placed into a 11.5 cm diameter, 5 cm high stimulus chamber made from white plastic. The chamber was fitted with a plastic 4 way divider so as to provide each individual with their own conpartment and prevent blockage of the light by huddling. The light was directed onto the animals from above onto an opal glass top which acted as a translucent diffuser to evenly disperse the light into the animal chamber. All light exposures occurred between 09.45-10.45 h in the morning and 17.45-18.45 h in the evening every day, with any one group being assigned to one of the four 15 min time periods within that hour so that their light exposure always occurred 8 and 16 h apart (+ 15 min). On the first day of light exposure, the 4 animals assigned to one group were removed from the group cage and placed into the light stimulus apparatus for 10 minutes during the morning exposure time and then individually placed into a cage equipped with a running wheel within a light-tight chamber, Subsequent light exposure occurred twice daily, beginning on the evening of day 1, with individual animals always exposed to the same light irradiance throughout the 10 days of exposure to light, After each 10 min light pulse, the animals were sorted (by examination of their ear punches) and returned to their individual running-wheel cages, All handling and transfer of animals between their running-wheel cages and the stimulus chamber was accomplished within a completely darkened room with the aid of a head-mounted infrared viewer (FJW Industries, Elgin, IL) and a lamp equipped with a 15 W bulb and an infrared filter, The male Djungarian hamsters with regressed testes were arranged into groups of four, marked by ear punches for individual identification, placed into individual running-wheel cages, and assigned to one of 9 groups. Two control groups remained undisturbed in 8L:16D (n = 16) or 16L:8D (n -- 9). One control group was moved twice per day into the exposure chamber, but was not exposed to light (n = 8). Six experimental groups of animals (n = 8 each) were exposed for 10 days to a 10 min light pulse twice per day at one of 6 irradiance levels: 0.0034, 0.01, 0.034, 0,10, 0.34, or
significantly larger testes t h a n a n i m a l s m a i n t a i n e d on
RESULTS
Animals
transferred
to
16L:8D
for
10 days
had
8 L : 1 6 D d u r i n g this t i m e p e r i o d (Fig. 1, top). T h e testes of animals k e p t in c o n s t a n t d a r k n e s s and t r a n s f e r r e d twice a day into t h e light e x p o s u r e c h a m b e r w e r e small and statistically similar in w e i g h t to the testes of animals in the 4 lowest i r r a d i a n c e light pulse g r o u p s (Fig. 1, top). T h e testicular r e s p o n s e of t h e animals e x p o s e d to a s k e l e t o n p h o t o p e r i o d was d e p e n d e n t u p o n t h e i r r a d i a n c e of light given d u r i n g the two 10 m i n light pulse periods. T h e testis weights of the t h r e e g r o u p s of animals e x p o s e d to the lowest i r r a d i a n c e s (0.0034, 0.01 o r 0.034 p W / c m 2) w e r e significantly less t h a n t h o s e o b s e r v e d for the g r o u p of animals e x p o s e d to 0.34 p W / c m 2. T h e m e a n testis w e i g h t of t h e animals e x p o s e d to an i n t e r m e d i a t e i r r a d i a n c e (0.1 p W / c m 2) was b e t w e e n that o b s e r v e d in the animals e x p o s e d to the t h r e e l o w e r and two h i g h e r irradiances of light, but w e r e n o t significantly different f r o m the m e a n testis weights o b s e r v e d in any of these groups. A l t h o u g h the d i f f e r e n c e was not statistically significant, the testis weights of t h e u n d i s t u r b e d animals in 8 L : 1 6 D w e r e larger t h a n t h o s e of the animals m o v e d twice a day into the light e x p o s u r e c h a m b e r w i t h o u t e x p o s u r e to light. T h i s a p p a r e n t d i f f e r e n c e in testis weight m i g h t be d u e to the stress of h a n d l i n g , since it is k n o w n that stressed animals s h o w d e c r e a s e d testicular function 4. T h e s e r u m F S H v a l u e s of t h e a n i m a l s p l a c e d in 1 6 L : 8 D for 10 days w e r e significantly g r e a t e r t h a n t h o s e of the
306
~
140
.,~
12o
~
.....
0
6
~ 0:
18
24
8L 16 :D~\'~"...........................
16L:8D
-~ tOO
Hours 12
~.. ................
, DD d i s t u r b e d , ~
~,~x~
E-. ~Ke;eton ~ \ \ \ \ \ \ \ ~ -~ 5~ I I 1 1 \ \ ' x ' x \ \ x. \ \ \ ' N \ e'notope o
.-
.-.~.~108 ~0
01 ~
"
~\~..034.
~ [ _ ~
6 4 e0
-"-~- \ \ \ \
E
16L
8L
0
0.01 0.1 1.0 Light I r r a d i a n e e (k~W/em ~)
Fig. 1. Means (___S.E.M.) of paired testes weights (top) and serum FSH levels (bottom) for groups of male Djungarian hamsters kept in full photoperiods of 16 h of light (16L; n = 9) or 8 h of light (8L; n = 16) per day (open bars), or exposed to a skeleton photoperiod consisting of two 10 min pulses of white light 8 and 16 h apart (closed circles; n = 8 each) for 10 days. A control group was transferred to the light exposure chamber twice a day, but the light was not turned on in the chamber (0; n = 8). Each group of animals exposed to a skeleton photoperiod was subjected to light of a different irradiance ranging from 0.0034 to 3.4 p W/cm: during their twice-daily exposure periods. All the animals had regressed testes due to 3 weeks of exposure to 8L:16D prior to entering the groups depicted in this figure. Serum FSH symbols without error bars indicate group means near the sensitivity level of the assay,
Fig. 2. A summary of the running wheel activity records of male Djungarian hamsters housed in various light cycles for 10 days. Open areas represent light and hatched areas represent darkness. The mean (_ S.E.M.) times of activity onset and offset for each group of hamsters is shown by the horizontal black bars. The groups of animals were housed in 16 hours of light per day (16L:8D), 8 h of light per day (8L:16D), constant darkness with transfer into a light exposure chamber for 10 rain twice a day without exposure to light (DD disturbed), or moved into the chamber and exposed to white light ranging from 0.0034 to 3.4/~W/cm 2 for 10 rain twice a day.
near that of the animals kept in 8L: 16D and the duration of activity was longer than in any of the groups of animals exposed to the skeleton p h o t o p e r i o d s . Nearly all the activity of the animals exposed to any of the 10 min skeleton p h o t o p e r i o d s occurred during the 8 h period of darkness. The shorter bars at the b o t t o m of the figure indicate that the activity p e r i o d of the animals exposed to the light pulses of higher irradiance was shorter in duration and confined between the light pulses occurring 8 h apart. In contrast, the animals exposed to the lower
animals that r e m a i n e d in 8L:16D (Fig. 1, bottom). No significant differences were observed in the serum F S H levels between the groups of animals exposed to 8L: 16D, no light or one of the 3 lowest light irradiances. In addition, the animals exposed to no light or one of the two lowest light irradiances had mean serum FSH levels below those of the animals in 16L:8D. Serum F S H levels for each of the groups of animals exposed to either no light or one of the three lower irradiances were significantly lower than the levels observed in each of the groups exposed to one of the two highest irradiances,
irradiance 10 min light pulses displayed activity patterns that were longer in duration and o v e r l a p p e d with the time of the light pulses.
Differences between the activity records of the groups of animals e x p o s e d to the different light schedules are depicted in Fig. 2. A n i m a l s in the two full p h o t o p e r i o d s were active during the dark phase of their p h o t o p e r i o d , with a longer duration of activity occurring in the group exposed to a longer d a r k period. The animals housed in constant darkness and moved to the light exposure a p p a r a t u s twice a day had a mean time of activity onset
experiment over the way animals were exposed to light in previous studies, is in the precise control over the irradiance level that could be maintained during the entire light exposure period. In previous studies (see below) animals were allowed to r o a m freely within a cage and were u n d o u b t e d l y exposed to many different light irradiances as they moved about and slept. In contrast, the animals in the present e x p e r i m e n t were exposed
DISCUSSION In o r d e r to permit a quantitative analysis of the visual pigment and p h o t o r e c e p t o r s mediating p h o t o p e r i o d i c responses, we d e v e l o p e d an e x p e r i m a n t a l protocol for assessing the response of the reproductive system to experimentally controlled light stimuli. The main advantage of the m e t h o d used for delivering light in the present
307 while awake to a very precise stimulus of light while in a chamber that did not allow the animal to move about for only 10 rain twice/day. The precise control of the light in the present study was made possible by the fact that photostimulation of neuroendocrine-gonadal activity can occur very rapidly in this species in response to short pulses of light 15'19. Our results compare quite favorably with two studies by Hoffman and colleagues examining the light irradiance threshold necessary for induction of testis growth in young golden hamsters l°'n. In these studies, groups of golden hamsters were exposed to constant darkness or a 14L:10D photoperiod at one of several low levels of illumination for up to 14 weeks and the effect on the organ weights of the animals was measured. In one of these experiments 1~, gonadal degeneration was observed in exactly half of the animals exposed for 13 weeks to a broad-band light source with an irradiance of 0.159 pW/cm 2, suggesting that this irradiance is near the threshold for stimulation of testis growth in the golden hamster. Testicular degeneration was observed in an even larger percentage of the tested animals at irradiances below this value. Hoffman's data are similar to the threshold found in the present study, where an irradiance of 0.1 to 0.34/~W/cm 2 (Fig. 1)was necessary to stimulate an increase in paired testes weight and serum FSH levels in male Djungarian hamsters exposed to two 10 min pulses of light per day for 10 days. In a series of experiments designed to examine the irradianceoflightnecessarytostimulateneuroendocrinegonadal activity, Brainard and colleagues exposed grouphoused golden hamsters to a l l L : 13D photoperiod with light during the light portion of this photoperiod (400 /~W/cm 2) extended by a 3 h period of light at varying irradiance 3. They observed significant testis growth within 12 weeks when the 3 h extended light period was 0.2 pW/cm 2 or greater. Intermediate stimulation of testis growth was achieved when the 3 h extended light period was either 0.002 or 0.02 pW/cm 2. This threshold value (0.02-0.2 /~W/cm 2) for stimulation of testis growth in male golden hamsters is about one order of magnitude less than the threshold value for stimulation of testis growth in Djungarian hamsters reported in the present experiment and for stimulation of testicular growth in golden hamsters in the studies by Hoffman and colleagues H. However, exposure to 400/,tW/cm 2 for 11 h in addition to the lower irradiance light for 3 h in this study by Brainard and colleagues complicates the interpretation of these results since it is not clear if the 11 h of exposure to bright light was playing some role in stimulating testicular growth. Furthermore, the bright lights during the prior 11 h may have altered the response of the photoreceptors to the extended period of dim light,
Another method that has been used to examine the importance of light irradiance in the photoperiodic response relies on the ability of light to suppress pineal melatonin content or pineal enzyme activity. Since pineal melatonin levels are closely linked to the prevailing light/dark cycle and the photoperiodic response of many mammalian species 13'16, measurement of the suppressive action of light on pineal melatonin content or the enzymes involved in synthesis of melatonin provide a rapid, albeit indirect, assay for the action of light on reproductive function in photoperiodic animals. A study of the ability of white light to suppress nocturnal levels of pineal melatonin content in adult male golden hamsters demonstrated that an 8 rain exposure to an irradiance of 0.186 pW/cm z led to a continued depression of the melatonin levels even after the animals were returned to darkness 2. Pineal serotonin-N-acetyltransferase (NAT) activity was suppressed by cool white fluorescent light at irradiances greater than or equal to 0.111/~W/cm 2 when golden hamsters were exposed to the light for 20 min prior to sacrifice 1. Both of these threshold values compare quite closely to the 0.1 pW/cm 2 irradiance value found in the present experiment to be the threshold for stimulation of neuroendocrine-gonadal activity in Djungarian hamsters exposed to two 10 min pulses of light per day for 10 days. In addition to stimulating neuroendocrine-gonadal activity, short pulses of light can also entrain the circadian rhythm of locomotor activity6,7,21. However, in the present study, it was not possible to determine whether the light pulses actually entrained the activity rhythm since animals were only in the light-dark cycle for 10 days. Nevertheless, from the activity records it is clear that animals in all the groups were active during the short period of darkness as were the animals exposed to a full photoperiod of 16L:8D. If the animals with small testes exposed to lower light irradiances were actually entrained to the two pulses of light, this would indicate that the threshold for photic stimulation of neuroendocrinegonadal activity is different than that for entrainment of the circadian rhythm of activity. However, further studies are necessary in order to clarify this issue. In summary, we have shown that photostimulation of reproductive function in the juvenile male Djungarian hamster can be used to explore the properties of the photoreceptive system involved in mediating photoperiodic induction of neuroendocrine-gonadal activity in mammals. Since the threshold for photoperiodic induction by light is several magnitudes greater than that necessary for vision 17'1s, the photoreceptive system involved in photoperiodic time measurement is clearly much less sensitive than that involved in vision. The sensitivity of the photoreceptors mediating the effect of light on reproduc-
308 tion and on the circadian system are similarel. It is of considerable interest to determine if the same photoreceptors mediate the effects of light on both the circadian and reproductive systems. Using D j u n g a r i a n hamsters, it should be possible to compare directly the photoreceptive responses for reproductive, pineal and circadian functions in a single species.
REFERENCES 1 Brainard, G.C., Richardson, B.A., King, T.S., Matthews, S.A. and Reiter, R.J., The suppression of pineal melatonin content and N-acetyltransferase activity by different light irradiances in the Syrian hamster: a dose-response relationship, Endocrinology, 113 (1983) 293-296. 2 Brainard, G.C., Richardson, B.A., Hurlbut, E.C., Steinlechner, S., Matthews, S.A. and Reiter, R.J., The influence of various irradiances of artificial light, twilight, and moonlight on the suppression of pineal melatonin content in the Syrian hamster, J. Pineal Res., 1 (1984) 105-119. 3 Brainard, G.C., Vaughan. M.K. and Reiter, R.J., Effect of light irradiance and wavelength on the Syrian hamster reproductive system, Endocrinology, 119 (1986) 648-654. 4 Charpenet, G., Tache, Y., Forest, M.G., Haour, E, Saez, J.M., Bernier, M., Ducharme, J.R. and Collu, R., Effects of chronic intermittent immobilization stress on rat testicular androgenic function, Endocrinology, 109 (1981) 1254-1258. 5 Darrow, J.M. and Goldman, B.D., Circadian regulation of pineal melatonin in the Djungarian hamster, J. Biol. Rhythms, 1 (1985) 39-54. 6 Earnest, D.J. and Turek, EW., Effect of one-second light pulses on testicular function and locomotor activity in the golden hamster, Biol. Reprod., 28 (1983) 557-565. 7 Elliott, J.A., Circadian rhythms, entrainment and photoperiodism in the Syrian hamster. In B.K. Follett and D.E. Follett (Eds.), Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, pp. 203-217. 8 Follett, B.K. and Follett, E.D., Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, 292 pp. 9 Heldmaier, G. and Steinlechner, S., Seasonal control of energy requirements of thermoregulation in the Djungarian hamster (Phodopus sungorus) living in natural photoperiod, J. Cornp. Physiol., 142 (1981) 429-437. 10 Hoffman, R.A. and Johnson, L.B., Effects of photic history and illuminance levels on male golden hamsters, J. Pineal Res., 2 (1985) 209-215. 11 Hoffman, R.A., Johnson, L.B. and Corth, R., The effects of spectral power distribution and illuminance levels on key parameters in the male golden hamster and rat with preliminary observations on the effects of pinealectomy, J. Pineal Res., 2
Acknowledgements. This research was supported by an NSF predoctoral fellowship to J.J.M., an NSF Presidential Young Investigator Award (DCB-8451642), NIMH Grant (MH-39595), and Searle Scholars Award (85-H-107) to J.S.T. and NIH Grants (HD-09885and HD-21921) to F.W.T. We thank Dwight Nelson for construction of the light exposure apparatus that was used in this study. A preliminary report of this work was presented at the 19th Annual Meeting of the Society for the Study of Reproduction, Ithaca, NY, July 1986.
(1985) 217-233. 12 Hoffmann, K., Photoperiodic effects in the Djungarian hamster: one minute of light during darktime mimics influence of long photoperiods on testicular recrudescence, body weight and pelage colour, Experientia, 35 (1979) 1529-1530. 13 Hoffmann, K., The role of the pineal gland in photoperiodic control of seasonal cycles in hamsters. In B.K. Follett and D.E. Follett (Eds.), Biological Clocks in Seasonal Reproductive Cycles, Wright, Bristol, 1981, pp. 237-250. 14 Hoffmann, K., The effect of brief light pulses on the photoperiodic reaction in the Djungarian hamster, Phodopus sungorus, J. Comp. Physiol., 148 (1982) 529-534. 15 Milette, J.J. and Turek, EW., Circadian and photoperiodic effects of brief light pulses in male Djungarian hamsters, Biol. Reprod., 35 (1986) 327-335. 16 Reiter, R.J., The pineal gland: an intermediary between the environment and the endocrine system, Psychoneurology, 8 (1983) 31-40. 17 Ripps, H. and Weale, R.A., The visual stimulus. In H. Davson (Ed.), The Eye, Vol. 2A, Academic Press, New York, 1976, pp. 43-99. 18 Reuter, J.H., A comparison of flash evoked ERG's and ERG's evoked with sinusoidaily modulated light stimuli in a number of rodents, Pfluegers Arch., 331 (1972) 95-102. 19 Simpson, S.M., Follett, B.K. and Ellis, D.H., Modulation by photoperiod of gonadotrophin secretion in intact and castrated Djungarian hamsters, J. Reprod. Fert., 66 (1982) 243-250. 20 Steinlechner, S., Heldmaier, G. and Becker, H., The seasonal cycle of body weight in the Djungarian hamster: photoperiodic control and the influence of starvation and melatonin, Oecologia, 60 (1983) 401-405. 21 Takahashi, J.S., DeCoursey, P.J., Bauman, L. and Menaker, M., Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms, Nature (Lond.), 308 (1984) 186-188. 22 Turek, F.W. and VanCauter, E., Rhythms in reproduction. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, pp. 1789-1830. 23 Yellon, S.M. and Goldman, B.D., Photoperiod control of reproductive development in the male Djungarian hamster, (Phodopus sungorus), Endocrinology, 114 (1984) 664-670.