Photoperiodic Influences on Testicular Regression, Recrudescence and the Induction of Scotorefractoriness in Male Golden Hamsters

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1 BIOLOGY OF REPRODUCTION 17, (1977) Photoperiodic Influences on Testicular Regression, Recrudescence and the Induction of Scotorefractoriness in Male Golden Hamsters IRVING ZUCKER and LAWRENCE P. MORINi Department of Psychology, University of California, Berkeley, California ABSTRACT Hamstei testes which have undergone short photoperiod-induced atrophy become refractory to the effects of short photoperiod after spontaneous or light-induced recrudescence (Reiter, 1974). We have termed this phenomenon, scotorefractoriness. The present experiments assessed relations between photoperiod-induced testicular regression, recrudescence and scotorefractoriness. Experiment 1 established that mere testicular regression was not sufficient to induce scotorefractoriness. Testes of some animals which were regressed by 6 or 9 weeks of exposure to a LD 2:22 photoperiod (short days) and recrudesced by 9 weeks of exposure to a LD 14:10 photoperiod (long days) subsequently regressed again in response to a second 9 week exposure to LD 2:22; others appeared scotorefractory. Experiment 2 determined that animals which achieved complete testicular regression during 9 weeks of LD 2:22 were less sensitive to the stimulating effects of long days than were animals which were in LD 2:22 for weeks. The transition from long to short days initiates g#{246}nadal atrophy, permits subsequent testicular recrudescence and also induces scotorefractoriness. Short day exposure initially decreases the sensitivity of the neuroendocrinetesticular axis to the effects of long days; the subsequent recovery of sensitivity to photostimulaton may parallel processes involved in spontaneous testicular recrudescence. INTRODUCTION The golden hamster is one of a number of mammals which use seasonal changes in daylength to phase their annual reproductive rhythms. In Reiter s model (1974), the gonadal cycle is initiated during the fall when the animals experience short daylengths. After 6 to 9 weeks of exposure to short days (less than 12.5 h light/day; Gaston and Menaker, 1967) the testes undergo severe atrophy. During this time, it is assumed that hamsters retire to underground light-less burrows, remaining there until they emerge in the spring. The regressed gonads are presumed to regenerate as spring approaches so that the newly emerged animal is able to breed. Although this scheme remains to be confirmed by actual field observations it is rendered highly plausible by year-round sampling of animals under natural meteorological conditions (Vendrely et al., 1972) and by several laboratory investigations (Reiter, 1972, 1974, 1975) that more or less simulate natural photoperiodic conditions. Accepted May 20, Received January 21, Present address: Department of Psychology, Dartmouth College, Hanover, NH When hamsters are transferred from long to short days (week 0) and are maintained in short days thereafter, complete testicular regression occurs by week 9 and the state of atrophy endures until about week 20. Between weeks 20 and 30 the gonads recrudesce and subsequently do not undergo a second cycle of regression unless the animals are first exposed to many weeks of long days (Reiter, 1972). Thus, the neuroendocrine-testicular axis of a hamster that has undergone spontaneous recrudescence has seemingly become independent of the dictates of photoperiod: the testes and other reproductive apparatus remain fully functional in short days or after blinding. We have used the term scotorefractoriness to describe this condition (Morin et al., 1977) because the animals appear to be insensitive to the atrophic effects of extended daily dark periods (short days). The scotorefractory condition should be compared with photorefractoriness which has traditionally described the state in which animals (particularly birds) are insensitive to photostimulatory effects of long days (Farner and Follett, 1966; Wolfson, 1966). Regulation of the hamster reproductive cycle involves an interplay of endogenous and exogenous mechanisms. The physiological bases of many stages of this cycle have been elabo- 493

2 494 ZUCKER AND MORIN rated (Reiter, 1972, 1973, 1974; Seegal and Goldman, 1975; Turek et al., 1975). The involvement of circadian oscillations in photoperiodically mediated reproductive responses of hamsters have also been amply documented (Elliott, 1974, 1976; Elliott et al., 1972; Fitzgerald and Zucker, 1976; Morin et al., 1977; Rusak and Morin, 1976; Stetson and Watson-Whitmyre, 1976; Stetson et al., 1975; Stetson et al., 1976; Zucker, 1976). Less attention has been directed to the formal analysis of external stimuli adequate to initiate and maintain testicular regression. Nor do we understand the relation between testicular regression and recrudescence on the one hand, and scotorefractoriness on the other. The experiments described below addressed several of these issues: 1) How many weeks of short day exposure are required to produce scotorefractoriness? 2) Does photoperiodically-induced gonadal regression and recrudescence necessarily induce scotorefractoriness? 3) What are the short- and longterm consequences of photostimulation on the testicular axis when hamsters are exposed to long days at various intervals after the gonads have regressed? The latter analysis extends the work of Turek et al. (1975) which established that the neuroendocrine-gonadal axis of the male hamster increases in sensitivity to photostimulation with increasing length of prior exposure to nonstimulatory photoperiods. Housing MATERIALS AND METHODS Conditions Male hamsters (LVG-LAK) weighing between g were purchased from the Lakeview Hamster Colony, Newfield, N.J. According to information obtained from the supplier, the hamsters were exposed to an LD 16:8 photoperiod prior to shipping. From the time of arrival in our laboratory on 23 April, 1975, until the experiments commenced on 14 May, TABLE 1. Paradigm for experiment 1. Group Phase 1: weeks of LD2:22 Phase 2: weeks of LD 14:10 (week 0) the animals were maintained in a LD 14:10 photoperiod (lights on at 2200 h daily, Pacific Daylight Time). Throughout the experiments, hamsters were housed individually in wire bottom cages and provided with ad libitum access to food (Simonsen rat pellets, maintenance diet) and tap water. Room temperature was maintained at approximately 23#{176}C and light intensity during the light phase ranged between 2 and 10 ft-c depending on the position of the hamster cage in the holding racks. Laparotomy and Testis Index Laparotomies were performed under pentobarbital sodium anesthesia (80 mgfkg of body weight, injected intraperitoneally) according to procedures described elsewhere (Rusak and Morn, 1976). The maximum length and width of the right testis were measured to the nearest 0.1 mm with a micrometer and the testis index (TI) computed as follows: length X width body weight. This index is highly correlated with testicular weight/body weight (Rusak and Morin, 1976) which in turn is highly correlated with spermatogenesis and testosterone production (Berndtson and Desjardins, 1974; Desjardins et al., 1971). After the measurements were obtained, the testis was sprinkled with isotonic saline, returned to the scrotum and the wound repaired. Use of the TI as a measure of gonadal status has the advantage of permitting repeated quantitative measurements on the same animal over a period of many months encompassing different treatments. Procedures Experiment 1. Hamsters were transferred to an LD 2:22 photoperiod (lights on a 0900 h daily) at week 0. Different groups of hamsters were laparotomized at week 0 (Group 1) or after 3, 4, 6 or 9 weeks in LD 2:22 (Phase 1, Table 1). Each group was then returned to the original LD 14:10 photoperiod and 9 weeks later hamsters were subjected to a second laparotomy (Phase 2). All hamsters were next exposed to the LD 2:22 photoperiod for 9 weeks and a final laparotomy was then performed (Phase 3). A testis index was computed for each hamster for each phase of the experiment. The groups differed only with respect to the number of weeks of exposure to the LD 2:22 photoperiod during Phase 1 (Table 1). Experiment 2. Groups of hamsters were transferred to LD 2:22 at week 0. The animals in Group 1 were maintained in this photoperiod for weeks The remaining groups were housed in LD 2:22 except for a Testis index at end of Phase 2* Phase 3: weeks of LD 2: ± ± ± ± Mean ± standard error of mean.

3 PHOTOPERIODIC REGULATION OF HAMSTER TESTES 495 three week interval during which each was returned to the original LD 14:10 photoperiod. The three week intervals began at weeks 9, 12 and 15 for Groups 2, 3 and 4, respectively (see Table 2). At week 0, 27 randomly selected animals were laparotomized to obtain a baseline TI. Laparotomies were performed on all animals in Groups 2, 3 and 4 at transition points from the LD 2:22 to LD 14:10 photoperiods. After the 3 weeks in LD 14:10, all animals were laparotomized to determine the percent change in TI induced by LD 14:10. In addition, all animals were laparotomized at the end of week 18. Different animals from Group 1 were laparotomized at times corresponding to the transition points for animals in Groups 2, 3 and 4, providing control measures for comparison. Experiment 1 RESULTS Hamsters with a TI 2.0 were considered to have large fully active testes and those with an index 1.0 to have small, regressed and hypofunctional gonads (Rusak and Morin, 1976). Phase 1. Exposure to LD 2:22 eventually resulted in complete testicular regression. The results illustrated in Fig. 1 confirm previous findings (Reiter, 1968; Berndtson and Desjardins, 1974; Turek et a!., 1975) with respect to the timecourse of regression induced by blinding or short photoperiod. Three weeks of exposure to LD 2:22 did not produce a significant decrease in the TI. The decreased TI Weeks in LI) 222 during Phase I and Phase 3 FIG. 1. Mean right testis index of hamsters laparotomized after different intervals in LD 2:22 (Phase 1, hatched bars). After Phase 1 each group was placed in LD 14:10 for 9 weeks (Phase 2) and then returned to LD 2:22 for 9 additional weeks (Phase 3). At the end of Phase 3 the right testis of each animal was again measured (open bars). The extent of testicular regression during Phase 3 is a function of the amount of time animals were exposed to LD 2:22 in Phase 1. Letter symbols represent data for individual hamsters. at 4 weeks (P<0.01, 2 tailed t test) may reflect absence of photostimulation, but regression was clearly evident after 5 weeks in LD 2:22 with each hamster showing a decrease in its TI (P<0.001). In some of these animals regression was complete (Fig. 1, hamster b and c); in others, testis size had not yet reached the expected asymptote. Six weeks of exposure to the LD 2:22 photoperiod appears to be near the threshold duration for inducing regression (Reiter, 1968). After 9 weeks exposure to LD 2:2 2, gonadal atrophy seems complete (P<0.001;Fig. 1). Phase 2. After 9 weeks of exposure to LD 14:10, the TI of all groups had resumed fully mature adult status (Table 1); there were no significant group differences. The testes of each individual were also mature adult size at this time. Phase 3. Exposure to 3 or 4 weeks of short days (LD 2:22) in Phase 1 did not render the hamster neuroendocrine axis scotorefractory; that is, these animals retained sensitivity to short days and their testes appeared to regress normally after exposure to LD 2:22 for 9 weeks (Fig. 1). Exposure to 6 weeks of LD 2:22 in Phase 1 induced various amounts of testicular regression. Note that the testes of animals b and c regressed completely in Phase 1 (TI = 0.61 and 0.54, respectively) and also regressed again during Phase 3. Animal d, with intermediate testicular regression on Phase 1 (TI = 1.05), also showed near total regression during Phase 3 (TI = 0.72). Either testicular regression does not produce subsequent refractoriness to short days, or the 9 week period of LD 14:10 exposure interpolated between the two short day challenges was adequate to break refractoriness. Five of the 6 hamsters whose testes regressed in response to 9 weeks of LD 2:22 during Phase 1 were scotorefractory during Phase 3, in confirmation of earlier findings (Reiter, 1972). The sole exception was animal j, who showed near complete regression during the second short day challenge (Phase 3) as well as during Phase 1. Experiment 2 Three weeks of photostimulation (exposure to LD 14:10) induced significant testicular recrudescence in hamsters that had previously been exposed to 9, 12 or 15 weeks of nonstimulatory photoperiod (P<0.10, P<0.05, P<0.02, respectively, for comparison with respective control groups maintained in LD

4 496 ZUCKER AND MORIN 2:22). The percent increase in TI is illustrated in Fig. 2; the neuroendocrine axis of the hamster with regressed testes is apparently sensitive to the photostimulatory effect of long days throughout the 9 week interval assessed in this study. The net percent increase in TI (obtained by subtracting the percent increase attributable to spontaneous recrudescence from that due to photostimulation) averaged 34.4 percent, 59.2 percent and 52.4 percent, respectively for animals photostimulated for 3 weeks beginning after 9, 12 and 15 weeks of exposure to LD 2:22. This suggests that during the 3 week period beginning shortly after the testes have first regressed (week 9) the neuroendocrine axis is least sensitive to the stimulatory effects of long days. However, it should be noted that in the present experiment this suggestive trend fell short of statistical significance (P<0.20; cf. Turek et al., 1975). The longer term effects of photostimulation on the TI were dependent upon the duration of prior and subsequent exposure to the nonstimulatory (LD 2:22) regimen (Table 2). Thus, at week 18 animals previously exposed to LD 14:10 during weeks (Group 3) had significantly greater Tis than Group 2 hamsters photostimulated during weeks 9-12 (P<0.02). The long term benefits to be derived from photostimulation vary with the intervals elapsed since gonadal regression. Data presented by Turek et al. (1975) also suggested post-regression fluctuations in sensitivity of the neuroendocrine axis. In all experiments thus far conducted the TI appears least sensitive to photostimulation in the weeks immediately after the gonads have first regressed. For example, in the present experiment at week 18 the TIs of animals that were photostimulated x 4) I! U) en C 8) U) a 4) 5-) C C 4) 5-, I Week FIG. 2. Percent increase in right testis index (± SEM) after 3 weeks in LD 14:10, among groups previously exposed to 9, 12 or 15 weeks of LD 2:22 (open bars). Control data obtained from animals kept continuously in LD 2:22 are also shown (hatched bars). The percent increase in an experimental animal was determined relative to the testis index of that animal prior to the 3 week period of LD 14:10 exposure. The number of animals per group is indicated in parentheses. during weeks 9-12 (Group 2) were not significantly different from those of hamsters that remained in the nonstimulatory photoperiod throughout (Group 1). DISCUSSION Hamster scotorefractoriness was established by Reiter (1972) under specific experimental conditions: first, the testes were regressed during 10 weeks of exposure to short days; then testicular recrudescence was effected by exposing animals to 10 weeks of long days or spontaneously over the course of an additional 20 weeks of short day exposure. In neither TABLE 2. Stimulatory effect of 3 weeks exposure to LD 14:10 in hamsters with regressed testes (experiment 2). Group Photoperiod (weeks) N LD 2:22 LD 14:10 (3) Testis index at week 18 (M ± SEM) ± 14ab ±.lsc ±.l5ac ± significant. A common superscript indicates that group values differ significantly (P<0.02). All other differences are not

5 PHOTOPERIODIC REGULATION OF HAMSTER TESTES 497 instance did the testes undergo a second regression when challenged by 10 weeks of short days (scotorefractoriness). The present experiments attempted to characterize the effects of short days on regression, scotorefractoriness and responsiveness to subsequent photostimulation. Whether scotorefractoriness occurs may depend on the number of weeks of short day exposure which were used to induce regression; 83 percent of hamsters exposed to 9 weeks of short days were scotorefractory whereas the corresponding value for animals with testes regressed by 6 weeks of short days was 25 percent (Exp. 1). An increasing number of weeks of short day exposure may be required to produce regression and scotorefractoriness, respectively. Under natural conditions hamsters would experience many consecutive months of short days, ensuring both regression and scotorefractoriness; it is only under artificial experimental conditions that the separability of these two events is apparent. Testicular regression per se is here demonstrated as insufficient for the induction of scotorefractoriness. In addition to the data presented above, we have observed at least 20 hamsters over the course of the past 2 years whose recrudesced testes undergo.a second regression in the absence of 20 weeks exposure to long days (Bittman, Carlisle and Zucker, unpublished observations, ; B. Rusak, personal communication, 1976). These findings contrast with those of Reiter (1972) and Stetson et al. (1976); the reason for the discrepancy is presently unclear. Nor is complete testicular regression necessary for induction of scotorefractoriness: some hamsters whose testes were only minimally affected by 6 weeks exposure to short days (animal i, Fig. 1) and who were then photostimulated with long days for 9 weeks were scotorefractory during a second short day challenge. The induction of scotorefractoriness requires durations of short day treatment in excess of 4 weeks; no hamster exposed to 3 or 4 weeks of the LD 2:22 photoperiod was subsequently scotorefractory. It is possible that scotorefractoriness might have been produced during these shorter treatments and broken by 9 weeks exposure to long days. However, we favor the hypothesis that exposure to short days for 4 weeks does not induce scotorefractoriness. The implication of the latter formulation is that during the long day portion of the annual cycle (spring and summer) the fossorial hamster that fails to experience long days for as long as 4 consecutive weeks will still retain its sensitivity to short days when autumn arrives. Hamsters appear least sensitive to photostimulation at about the time the testes have first regressed. Sensitivity to long days increases as the duration of short day exposure increases (see Turek et al., 1975). The locus for this effect and the underlying mechanisms are not known. Increased sensitivity to long days may reflect altered parameters of pineal activity (e.g., decreased secretion of pineal antigonadal factor) or changes within the hypothalamus, pituitary or gonads. During the annual gonadal cycle of wild hamsters, testicular recrudescence almost certainly does not involve photostimulation, but probably occurs spontaneously during continued exposure to short days (Reiter, 1975). Nevertheless, the present observations with photostimu!ation of regressed testes lend credence to the hypothesis that endogenous, temporally coded changes within the neuroendocrine axis contribute to the recovery to mature gonadal status after gonadal atrophy has been completed. ACKNOWLEDGMENTS This study was supported by USPHS Research Grant HD-02982, by the Committee on Research of the University of California and by a postdoctoral fellowship from the National Institute of Child and Human Development to LPM. Preparation of the manuscript was aided by Biomedical Research Support from Dartmouth College. We thank Darlene Frost, Margaret Roisman and Clarence Turtle for technical assistance and Eric Birtman for criticism of an earlier version of the manuscript. REFERENCES Berndtson, W. E. and Desjardins, C. (1974). Circulating LH and FSH levels and testicular function in hamsters during light deprivation and subsequent photoperiodic stimulation. Endocrinology 95, Desjardins, C., Ewing, L. L. and Johnson, B. H. (1971). Effects of light deprivation upon spermatogenie and steroidogenic elements of hamster testes. Endocrinology 89, Elliott, J. A. (1974). Photoperiodic regulation of testis function in the golden hamster: Relation to the circadian system. Ph.D. Dissertation, University of Texas. Elliott, J. A. (1976). Circadian rhythms and photoperiodic time measurement in mammals. Fed. Proc. 35, Elliott, J. A., Stetson, M. H. and Menaker, M. (1972). Regulation of testis function in golden hamsters: A

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