Effects of morning and evening injections of pineal 5-methoxyindoles (MI), melatonin (aMT) and 5-methoxytryptamine (MT), for 60 continuous days, were observed on the testes of sham-operated (SO) and pinealectomized (Px) Indian palm squirrel, Funambulus pennanti maintained under different photoperiods during the gonad active phase. Long photoperiod (LP) of 14L:10D appeared stimulatory to the testes and caused a significant increase in the weight and seminiferous tubule diameter of both SO and Px animals, as compared to the animals under natural day-length (NDL). Short photoperiod (SP) of 10L:14D had an inhibitory influence and reduced the testes weight and its tubule diameter. aMT and MT injections during evening hours significantly reduced testes weight and tubule diameter of SO and Px animals under NDL, LP and SP. However morning injections, under all conditions, were without any significant effect. The results suggest an inhibitory effect of aMT and MT, under above photoperiodic conditions, on the testes of this tropical mammal. The time of administration of the MI is important in the expression of the effect.
Almost all animals inhabiting natural environments are exposed to changes in important climatic factors like temperature, rainfall, humidity and photoperiod. It is these factors on which they rely to cue changes in their sexual status, and an interaction between them and the sexual activity has been reported (Pevet, 1985a, b; Pevet et al., 1987; Vivien-Roles and Pevet, 1983). The pineal gland has been shown to be involved in the long term adaptation of animal to seasonal reproduction (Pevet, 1985b; Reiter, 1985). The MI synthesized by the pineal gland act on the hypothalamo-hypophysealgonadal axis and may exert a stimulatory, inhibitory or no effect on the gonads depending on their mode and time of administration (Berndtson et al., 1974; Ebels et al., 1965; Hoffmann, 1981a; Peat et al., 1971; Turek et al., 1975). The pineal gland of F. pennanti, a tropical seasonal breeder, is sensitive to environmental daylength, temperature and humidity (Haldar et al., 1988; Haldar et al., 1990). Evening injections of MI inhibit testicular activity of this rodent (Saxena et al., 1992; Saxena, 1997). Day-night variations in plasma aMT suggest the existence of a diurnal rhythmi- city in pineal activity of this mammal (Saxena et al., 1993).
There is, however, no report relating the time of MI administration to photoperiodic regulation of testicular activity in tropical species. Therefore, the present study was aimed at observing the effects of morning and evening administration of aMT and MT in an Indian tropical mammal, F. pennanti, under different photoperiodic conditions.
MATERIALS AND METHODS
The study was performed during the testicular active phase (Apr-June) of the annual testicular cycle of the animal (Haldar et al., 1990). Adult males (100–120 g body weight) of F. pennanti were obtained in the äfirst week of April and acclimatized, for two weeks, in a room fully exposed to natural environmental conditions. They were housed in wire net cages and provided with food (soaked gram seeds) and water ad libitum. They were divided into different groups of eight animals each, in accordance with the protocol in Table 1. Pinealectomy (Px) was performed following the technique of Haldar-Misra (Haldar, 1986). Besides NDL animals were exposed to LP of 14L:10D and SP of 10L:14D. The environmental temperature during the period of experimentation was recorded as a maximum of 37.02°C and a minimum of 28.11°C. The animals exposed to experimental LP and SP were maintained at 37°C. Two such sets of animals were maintained. One set. received injections in the morning at 7 hr. and another during evening at 16.30 hr. Animals under LP received morning injections 2 hr after lights on and evening injections 2 1/2 hr before light off. Animals exposed to SP received morning injections 15 min after lights on and 15 min before lights off. Solutions of aMT and MT (Sigma Chemical Co., USA) were made following the method of Pevet and Haldar-Misra (Pevet et al., 1982a). The animals were injected subcutaneously with 10μg MI/animal/day. Control animals were injected with vehicle (normal saline, 0.90% aqueous NaCl)/animal/day. After completion of 60 days the animals were sacrificed by cervical dislocation and their body weights noted. Testes were removed, weighed on a microelectrical balance and fixed in Bouin's fluid. They were processed by the routine paraffin method for histological observations. Seminiferous tubule diameter of testes was measured by an ocular micrometer. The data was statistically analysed with the help of Student's ‘t’ test and analysis of variance, ANOVA (Bruning et al., 1977).
Details of the experimental protocol. The experiment was performed during April-June (gonad active phase)
Results are presented in Figs. 1 and 2 and Tables 2 and 3. It is evident that in both morning and evening sets exposure to a LP of 14L:10D caused a significant increase in the testes weight (morning, P<0.001;. evening, P<0.001), and seminiferous tubule. diameter (morning, P<0.001; evening, P<0.001) of SO-sal, animals as compared to the saline treated animals under NDL. Exposure to a SP of 10L:14D showed a significant reduction in the testes weight (morning, P<0.001; evening,P<0.001) and seminiferous tubule diameter (morning, P<0.001, evening, P<0.001) of SO saline treated animals as compared to SO-saline animals under NDL. However, Px animals maintained under LP as well as SP and injected with saline indicated no significant effect on their testes weight and seminiferous tubule diameter when compared with the Px animals under NDL.
Effect of evening injections of melatonin (aMT) and 5- methoxytryptamine (MT), for 60 continuous days, on the seminiferous tubule diameter (μm) of testis in sham-operated (S0) and pinealectomized (Px) F. pennanti exposed to natural daylength (NDL), long photoperiod (14L:10D) and short photoperiod (10L:14D) during the gonad active phase.
Effect of morning injections of melatonin (aMT)) and 5-methoxytryptamine (MT), for 60 continuous days, on the seminiferous tubule diameter (μm) of testis in sham-operated (S0) and pinealectomized (Px) F. Pennanti exposed to natural daylength (NDL), long photoperiod (14L:10D) and short photoperiod (10L:14D) during the gonad active phase.
Evening injections of aMT and MT significantly reduced testes weight and tubule diameter of SO and Px animals maintained under NDL, LP and SP (Fig 1, Table 2). Morning injections, under all conditions, were without any significant effect on the testes (Fig. 2, Table 3).
Results of ANOVA showed a significant variation in. the testicular weight, in relation to pineal removal (F=26.14, P<0.001), time of MI administration (F=241.05, P<0.005) and different photoperiodic condition (F=10.12, P<0.001).
Although a large number of seasonally breeding rodent species are known, most studies have centred on the elucidation of pineal function in the control of reproduction in case of temperate zone animals (Pevet et al., 1987; Hoffmann, 1981b; Pevet et al., 1986).
This study shows that both aMT and MT are physiologically active compounds inhibiting the testicular activity of this tropical mammal. The present results indicate a sensitivity of the animal to change in lighting condition. This result further confirms the previous finding that the pineal and testes of F. pennanti are sensitive to daylength (Haldar et al., 1990). The results also demonstrate, for the first time, that the animal can distinguish between the time of the day when MI are administered. In other words, the inhibitory effects of aMT and MT are dependent on the period of the day when they are administered. While evening injections led to inhibition of testicular activity under both long and short photoperiods, MI administration during morning hours failed to produce any significant effect on the testes of F. pennanti under short or long photoperiod. LP was stimulatory to the testes whereas SP was inhibitory to the testes of the squirrel. Our results are similar to those on adult golden hamsters (Mesocricetus auratus) in which daily aMT injections in morning, under long days result in gonadal maintenance (Reiter et al., 1976; Stetson et al., 1983; Tamarkin et al., 1976; Tamarkin et al., 1977). Other studies indicate that morning injections of aMT under long to short days retards gonadal regression in the same animal (Pevet et al., 1982b; Turek et al., 1980).
The present results can be explained on the basis of the “hourglass mechanism” (Skopik et al., 1976; Underwood, 1981; Underwood et al., 1982) according to which photoperiodic discrimination is achieved by comparison of the environmental photoperiod to an internal rhythm of sensitivity consisting of a light-sensitive. and a light-insensitive phase. The. external coincidence model of Bunning (Bunning, 1936) explains that light coincident with the insensitive phase of the rhythm is translated as a short day, while light coincident with the photosensitive phase of the rhythm as a long day. In the external coincidence. model the environmental light cycle has two roles. It acts as a zeitgeber (entraining agent) for the circadian system and it has an inductive effect producing the long day or short day response (Pittendrigh, 1965). The resonance experiments by Nanda and Hamner (Nanda et al., 1958) for daylength discrimination consist of exposing the animals to different cycle lengths with a light phase of constant duration and dark phase of variable durations. Resonance experiments with golden hamsters indicated that the animals can discriminate daylength in different cycles with a constant light phase (Elliott et al., 1972; Stetson et al., 1975). A circadian basis for the photoperiodic reproductive response has been demonstrated with the help of resonance experiments in the case of vole, Microtus agrestis (Grocock et al., 1974) and in deer mouse, Peromyscus maniculatus (Whitsett et al., 1983). It may be opined that in F. pennanti MI injections only during evening hours are able to cause gonadal suppression as during morning hours the animals lose sensitivity to exogenous MI administration. This inhibitory effect of MI injection is evident under artificial long and short as well as natural photoperiod. Exogenous administration of aMT and MT have been found to exert a season dependent inhibitory influence on the testes of F. pennanti and evening injections of both MI inhibit the testes of the animal under natural long days and short days (Saxena, 1997). The plasma aMT content in this squirrel shows a clear circadian rhythm. This endogenous rhythm is evident during summer (April) as well as winter (December) months (Saxena et al., 1993; Saxena, 1988) and injections given 3–4 hr prior to the lights off result in testicular inhibition (Saxena et al., 1992; Saxena, 1997). In the present study also the injections given 2 1/2 hr before lights off, in LP, and 15 min before light off, in SP, causes gonadal inhibition. The effects are also found to be independent of the pineal gland since the testes of Px animals showed a similar response as those of SO animals.
It has become clear that the pineal gland is necessary for the regulation of photoperiodic responses and that pineal melatonin may be responsible for transmitting daylength information (Hoffmann, 1981b; Goldman, 1983; Hoffmann, 1985; Masson-Pevet et al., 1986).
Studies have shown that exogenous administration of MT (via injections) to golden hamsters exhibits a biphasic sensitivity. A daily injection just prior to lights on or up to 5 hr before lights out induces rapid and complete gonadal regression (Reiter et al., 1976; Tamarkin et al., 1977; Sackman et al., 1977). Injections at other times of the day are ineffective. Also pinealectomized hamster is not responsive to a single daily injection of aMT but requires three daily injections timed 3 hr or 30 min apart (Goldman et al., 1979; Tamarkin et al., 1977). Our results are different from these as in this tropical animal only a single daily injection during evening hr results in gonad inhibition. Also in F. pennanti Px animals, under the tested photoperiodic conditions, are equally responsive to the MI injections. Therefore, it can be suggested that pinealectomy does not block the circadian rhythm of aMT and MT sensitivity in this tropical species.
Pinealectomy always appeared stimulatory to the testes and pinealectomized squirrels exhibited significant increase in testes weight and seminiferous tubule diameter in comparison to SO controls. Under SP pinealectomized animals showed significantly higher testes weight and seminiferous tubule diameter as compared to SO animals as these were unable to perceive environmental information in the absence of pineal gland. Therefore, they maintained an active gonad as evident by higher testes weight and seminiferous tubule diameter.
The present study reveals, for the first time in a tropical mammalian species, that besides aMT there also exists a diurnal rhythm in the sensitivity of the animal to exogenous MT injections. Pinealecomy and constant release of aMT and MT have been reported to cause atrophy of gonad in male European hamsters kept under long photoperiod (Masson-Pevet et al., 1986). aMT implants have been reported to hasten recrudescence in male hamsters on short days (Turek et al., 1976). aMT implants cause testicular regression in long-day intact. male Peromyscus leucopus (Johnston et al., 1980; Lynch et al., 1976) but do not retard testicular regression on short days (Petterborg et al., 1981). Also varied results have been observed on the effects of photoperiod and melatonin on gonadal function of prepubertal and adult animals of Microtus and Peromyscus species. Although in the case of F. pennanti silastic capsule implants of aMT and MT lead to testicular regression in both intact and Px animals, (Saxena, 1988) the effects of this continuous mode of MI administration under different photoperiods remains to be assessed.
Thus, although MT effects on sexual axis have been studied in different animals (Vivien-Roels et al., 1983; Vevien-Roels 1983) the results have been varied (Pevet et al., 1986). Reports relating to actions of MT on the gonadal function of tropical mammals, experiencing different climatic conditions than temperate animals, are scarce. Our results of the present study provide evidence. for the role of pineal MT, besides aMT, in the photoperiodic adaptive response to reproduction in a tropical seasonally breeding mammal, F. pennanti. The results suggest the possibility of the existence of a rhythm in the gonadal response to exogenous MI administration under different photoperiods but the exact mechanisms involved in this phenomenon remain yet to be identified.