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BOR - Papers in Press, published online ahead of print December 3, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020008
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BIOLOGY OF REPRODUCTION 70, 1001–1009 (2004)
DOI: 10.1095/biolreprod.103.020008
© 2004 by the Society for the Study of Reproduction, Inc.

Environment

Reciprocal Interaction Between Seasonal Testis and Thyroid Activity in Zembra Island Wild Rabbits (Oryctolagus cuniculus): Effects of Castration, Thyroidectomy, Temperature, and Photoperiod

M.M. Ben Saad2, and D.L. Maurel1,3

Laboratoire de Physiologie Animale,2 Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia Pathologie de l'Oreille interne et Réhabilitation,3 EPI 9902 INSERM, Faculté de Médecine Nord, 13916 Marseille cedex 20, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The reproductive cycle of wild rabbits (Oryctolagus cuniculus) living in Zembra Island (North Tunisia) is dependent on an external factor, the photoperiod: the gonads are inhibited by long days and stimulated by short days or melatonin implants. Here we studied the role of an internal factor, thyroid hormones and the possible thyroid-gonadal interrelationships, in animals captured on Zembra Island and maintained in natural conditions of photoperiod and temperature. We determined the seasonal profile of the thyroid and testis cycles and investigated the effects of castration and thyroidectomy on the seasonal testosterone and thyroxine cycles. Plasma thyroxine and testosterone levels followed similar, parallel seasonal patterns, with a peak in autumn (October) and low values from January to August. In thyroidectomized animals, plasma testosterone levels, although significantly higher than those in controls (P < 0.001), remained low throughout the 13 mo of the experiment, and no testicular reactivation was observed in the fall. In castrated animals, despite the increase in thyroxine concentration in the 3 mo following castration (P < 0.01), plasma thyroxine levels remained low during the 2 yr of the study. We then investigated the combined effects of long days (16L:8D) and moderately high temperature (25°C) on these two endocrine axes. In constant gonado-inhibiting conditions (16L:8D), whether the temperature was kept constantly high or allowed to fluctuate naturally, no reactivation of the thyroid and testicular axes was observed in the fall. In control animals, the peaks of testosterone and thyroxine concentrations observed in September were larger (P < 0.001) than those in animals subjected to the same natural photoperiod conditions but with constantly high temperature. The lower level of autumnal testis stimulation (P < 0.001) in animals maintained in conditions of constant high temperature (25°C) may be attributed to the low thyroxine levels induced by high temperature. These results clearly confirm that the thyroid and testicular cycles display similar seasonal variations and show that the thyroid and gonadal axes are strictly interdependent. This study provides the first demonstration, for a given species, that the seasonal reactivation of gonad activity is controlled by the thyroid, and thyroid activity is controlled by the gonads.

male sexual function, seasonal reproduction, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Species living in temperate climates have evolved various strategies, involving physiological, ecological, and behavioral mechanisms of adaptation, to ensure the survival of both the individual and the species. One major element of these mechanisms is the existence of endocrinological variations according to season [1]. Reproduction appears to be the most important of the endocrine functions studied because it ensures the survival of the species and, in all species studied, seasonal variations provide good examples of adaptive strategies, with births programmed for the most favorable period in the year, maximizing the young's chances of survival [2, 3]. Another important endocrine axis involved in adaptive strategies is the thyroid axis. Indeed, thyroid hormones play a key role in individual survival because they participate directly in the regulation of metabolic processes, such as thermoregulatory and chemical (energy production), physical processes (molting, pelage), and the seasonal constitution/utilization of organic reserves (fat deposits). In both birds and mammals, these two endocrine functions have often been linked through positive or negative thyroid-gonadal interrelationships and, in most species studied, thyroid activity follows an annual cycle closely correlated with the sexual cycle [4]. In some mammals and birds, it has been shown that thyroid hormones are required for photoperiod to exert its effects on gonadal activity, as demonstrated in mink [5], edible dormouse [6], red deer [7, 8], and sheep [9, 10]. In the Zembra Island wild rabbit (Oryctolagus cuniculus), we have shown that seasonal testicular activity is controlled by photoperiod, with short days and melatonin stimulating reproduction and long days inhibiting it [11, 12]. Effectively, in sexually active animals, testicular activity is stimulated by experimental short days (8L:16D) and inhibited after 1 mo of experimental long days (16L:8D). If the light phase of the short-day regimen (8L:16D) is divided into two photofractions (a main photofraction of 7.5 h and a short photofraction of 0.5 h), testicular activity is stimulated if the short photofraction occurs no more than 12 h after the beginning of the main photofraction and is inhibited if it interrupts the dark phase 12.5 h or more after the beginning of the main photofraction [11, 12]. Melatonin also plays a role in this gonadostimulation by short days: after ganglionectomy, no seasonal testicular reactivation is possible; however, testicular activity is renewed in these ganglionectomized animals 2 mo after the insertion of melatonin implants [11, 12]. This pattern of photoresponse is totally different from the classical description of gonadostimulation by long days (and inhibition by melatonin) in other populations of the same species of rabbit (O. cuniculus). This insular population differs from other populations studied, essentially by this photogonadoregulation, and animals are characterized by a lower body weight (0.8–1.4 kg compared with 1.2–2.0 kg) and a reduction in the litter size (1–2 young compared with 3–5). However, as for other rabbit populations, animals become adult at 6 mo. Even if, in this insular population of rabbits, we have clearly demonstrated that reproduction is inhibited by long days, we thought that testicular inhibition might be linked not only to a decrease in melatonin concentrations when nights become shorter but also to interactions between the thyroid and testicular axes. We have previously shown that, in this species, the thyroid and testicular cycles follow similar, parallel seasonal patterns, with testosterone and thyroxine concentrations peaking in the fall [13], both for free-living animals in the natural biotope and for rabbits housed in natural outdoor conditions (semicaptivity). In this study, we aimed to investigate the effect of thyroid activity on seasonal testicular activity by studying rabbits for 1 yr after surgical thyroidectomy. We also investigated possible effects of castration on seasonal thyroid activity over a 2-yr period. A second series of experiments was conducted to determine the effects of temperature and photoperiod on seasonal activation of the thyroid and testicular axes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The animals were trapped on Zembra Island (30°50'N, 10°14'E) in the north of Tunisia during three summer trapping sessions (from early July to end August), at the end of the sexual resting period (May–September). All the animals used in this study were adult males (6–24 mo old) weighing more than 900 g and in good health (no apparent skin, fur, or eye problems). The experiments were performed in accordance with European Community Council Directive 86/609/EEC. Captured animals were transferred to the laboratory, where they were placed in individual cages (length, 70 cm; width, 50 cm; height, 30 cm). Food (rabbit chow; SNA, Tunis, Tunisia) and water were available ad libitum. Animals were handled every day for the first 2 wk of captivity and twice weekly thereafter (until the end of October), in their cage, to check their general health status (body weight, skin, eyes, pelage, absence of parasites), to clean their cages, to verify the availability of food and water, and to accustom them to being handled. After 2 or 3 wk in captivity with repeated handling, the animals exhibited no obvious signs of stress (no abnormal prostration or agitation) and it was possible to collect blood samples manually without great difficulty.

Experimental Schedule

Experiment 1 Fifteen adult male rabbits (O. cuniculus) were randomly assigned to three groups, each containing five animals: 1) controls, 2) thyroidectomized, 3) castrated. All three groups were kept in natural photoperiod conditions throughout the experiment. Thyroidectomy was performed surgically in animals heavily anesthetized with equithesin (1% pentobarbitone [Sanofi, Paris, France], 4.6% chloral hydrate, and 9.5% ethanol in saline buffer, 0.9% NaCl), in December. The skin on the trachea was cut medioventrally. The muscles were gently put aside, thyroid vessels were cut by thermocautery and each thyroid lobe was extracted after resecting the conjunctive tissues. An antibiotic powder (Penicillin-Streptomycin Diamant, Diamant Laboratories, Paris, France) was applied between the trachea and muscle tissue. The skin was then closed with clips and an antiseptic lotion (Betadine; Asta Medica, Merignac, France) was applied externally to prevent local infection.

Castration was also performed surgically, under the same anesthetic conditions, in early November when testicular activity is high. The skin and the albuginea were cut medially. Vessels were ligated just above the epididymis and each testis with epididymis was cut with a scalpel and removed. The same antibiotic powder was applied locally as for thyroidectomy before closing the skin with clips and the same antiseptic lotion was externally applied to prevent local infection.

Experiment 2 Twenty-four adult male rabbits (O. cuniculus) were randomly assigned to four groups, each containing six animals: group 1, constant high temperature (25°C) and long days (16L:8D); group 2, natural temperature and constant long days (16L:8D); group 3, constant high temperature and natural photoperiod; control group, natural conditions of temperature and photoperiod. The four groups were kept in these conditions for 14 mo, starting in February, when the thyroid and gonad axes reach their seasonal troughs. We chose to use 25°C as the constant high temperature because this is the mean annual temperature at the latitude of Tunis (range: 10°C in December–March to 30°C in June–August). For the long-day regimen, we used artificial lighting (Mazda Solara fluorescent tubes, daylight type) to ensure a light intensity of approximately 150–200 lux inside the cages. The constant high temperature groups (groups 1 and 3), were housed in rooms equipped with central heating. The temperature was regulated by a thermostat inside one room. For group 1 (constant long days), the windows were covered with black plastic and for group 3 (natural photoperiod), the windows were not covered. During the summer months, the position of the rooms used to house the constant high temperature group (north exposition and ground floor) ensured good thermal isolation, and the temperature never exceeded 28°C (even when the external temperature was over 35°C). The temperature and humidity were constantly measured with a thermohygrometer; temperature was maintained at 25 ± 2°C and humidity at 80% ± 3%.

Measurement of Endocrine Activity

To evaluate endocrine activity, ear marginal vein blood was collected during the last week of each month, between 0900 and 1100 h for all animals. In experiment 1, we monitored control and castrated animals for 26 mo and thyroidectomized animals for only 13 mo: during the first year (12–14 mo) of hypothyroidism following thyroidectomy, the animals appeared to be in good health. However, the state of health of some of these animals quickly (in 1 or 2 wk) deteriorated subsequently and these animals needed to be killed. In the second experiment, all groups were studied over a period of 14 mo. The blood collected (0.5–0.6 ml) in syringes was transferred into heparinized tubes (20 µl heparin per ml of blood), centrifuged for 15 min at 1500 x g, and the plasma stored at -20°C until assay. Plasma testosterone and thyroxine (T4) were determined by radioimmunoassay, using commercial kits (Spectria Direct Testosterone; Orion, Espoo, Finland and T4K; CisBioInternational, Gif-sur-Yvette, France). For each immunoassay, plasma samples (50 µl) were assayed in duplicate. Sensitivity, defined as the smallest quantity of hormone detectable, was 4 pg/tube for testosterone and 0.4 ng/tube for T4. The intraassay and interassay coefficients of variation were 6.5% (n = 15) and 7.8% (n = 5), respectively, for testosterone and 4.6% (n = 15) and 7.1% (n = 5), respectively, for thyroxine.

Statistical Analysis

Monthly values are presented as means ± SEM. For body weight, we used linear regression (Sigma Plot; SPSS Inc., Chicago, IL) to describe the profile linearity in the castrated and thyroidectomized groups. For testosterone and thyroxine in control or experimental groups, we calculated regressions for the two annual cycles with a nonlinear regression-type four-parameter Gaussian curve, y = y0 + ae (Sigma Plot, SPSS Inc., Chicago, IL). For body weight, monthly testosterone values and monthly thyroxine values, a two-way ANOVA with repeated measurements followed by a post hoc Fisher least significant difference test was used to compare groups. Differences between monthly values for the same group or between groups at a given time point of the annual cycle were considered statistically significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Annual Body Weight Cycles

Thirteen-month analysis in control, thyroidectomized, and castrated animals Body weight displayed no clear seasonal variations except for the control group (analyzed more precisely in the 2-yr study) (Table 1). ANOVA revealed no differences between groups (F = 3.225, P = 0.076), but revealed a combined group-month effect (F = 3.197, P < 0.001). Furthermore, monthly values were significantly higher from February to June in thyroidectomized animals than in control animals and from February to May in castrated animals compared with control animals. If we compared the initial (Dec1) and final (Dec2) weights in the three groups, a significant difference was observed only in the castrated group (P < 0.001), with a weight gain of more than 14%.


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  		TABLE 1. Monthly values (means ± SEM) of body weight, expressed in grams (g), in the three groups of rabbits maintained in natural conditions of photoperiod and temperature during the 13-mo experimental period: control (n = 5), thyroidectomized (n = 5), and castrated (n = 5)

Two-year analysis in control and castrated animals Body weights displayed a slight seasonal cycle for the control group but not for the castrated group (Fig. 1). Linear regression analysis (polynomial curve, correlation coefficient, r2: 0.8710) revealed a seasonal cycle in the control group (Fig. 1) with a minimum in March–April the first year, in December–January the second, and a maximum in August the first year and in June the second. However, the cycle was strongly attenuated in the second year and almost disappeared. The same regression revealed no such cycle in the castrated group and a linear regression (first order, y = ax + b) revealed a good linearity (r2: 0.7817), indicating that weight constantly increased through the 2-yr study period without pronounced seasonal fluctuations.



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  		FIG. 1. Seasonal variations in body weight, expressed in grams (monthly mean ± SEM), in castrated and control groups of rabbits (O. cuniculus) living in semicaptivity, outdoors, in natural conditions of photoperiod and temperature. For each group, the annual profile is fitted by an appropriate linear regression. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01) for the same month between the two groups. Shown are the theoretical minimal (m) and maximal (M) seasonal values for body weight in the control group

The two-way repeated measures ANOVA indicated a group effect (F = 15.562, P = 0.004), a time (month) effect (F = 9.226, P < 0.001), and a combined group-month effect (F = 3.054, P < 0.001). The monthly values were significantly higher in the castrated group than in the control from February (third month after gonadectomy) to May in the first year (see also Table 1), and from May (18th month after gonadectomy) to the end of experiment.

Annual Testosterone and Thyroxine Cycles in Control Animals

Plasma testosterone (Testo) and thyroxine (T4) concentrations (Fig. 2A) displayed a clearcut monophasic annual cycle, with low values from February to July–August (mean values: T4, 73.4 ± 1.3 ng/ml; Testo, 0.92 ± 0.12ng/ml). For T4, concentrations rose moderately in September, peaked in October, and then gradually decreased in November and December, remaining significantly higher (mean value October–December: 144.9 ± 15.4 ng/ml) than the low values measured in February–August, returning to these low values in January. For testosterone, concentrations peaked in September, with high values maintained throughout October and November (mean value September–November: 6.9 ± 0.5 ng/ml), followed by a decrease in December to reach low levels in January.



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  		FIG. 2. A) Seasonal variations of plasma testosterone and thyroxine concentrations (determined monthly), in a control group of rabbits (O. cuniculus) living in semicaptivity, outdoors, in natural conditions of photoperiod and temperature. B) Calculated regression curves (y = y0 + ae[-0.5(x-x0/b)2]) for plasma testosterone (Testo) and thyroxine (T4) concentrations, showing a seasonal parallel pattern with synchronous peaks

We plotted the calculated regressions (Fig. 2B) for the two annual cycles with a nonlinear regression-type four-parameter curve. Peaks occurred simultaneously, between October and November, with a negligible phase shift of 10 days between testosterone and thyroxine concentrations, demonstrating that the two hormones followed a clearly similar, parallel seasonal pattern.

Annual Testosterone Cycle in Thyroidectomized Animals

Thyroidectomy was clearly successful in all five animals, as shown by the lack of detection of thyroxine in radioimmunoassays (detection threshold 15 ng/ml), whereas the lowest thyroxine concentrations recorded for control animals were over 50 ng/ml (min: 67.0 ± 8.2 ng/ml versus max: 172.8 ± 8.7 ng/ml). In thyroidectomized animals (Fig. 3A), we observed no annual variation in plasma testosterone concentrations and no autumnal stimulation of gonad activity similar to that observed in controls. Results of two-way ANOVA indicate no group effect (F = 1.220, P = 0.301), but a month effect (F = 43.638, P < 0.001) and a combined month-group effect (F = 46.346, P < 0.001). Two months after thyroidectomy and throughout the sexual rest period in controls, plasma testosterone concentrations were significantly higher (although still low) in thyroidectomized animals than in controls (mean level February–July, thyroidectomized: 2.33 ± 0.19 ng/ml versus controls: 0.92 ± 0.12 ng/ml; P < 0.001).



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  		FIG. 3. A) Seasonal variations of plasma testosterone concentrations in control and thyroidectomized rabbits. B) Seasonal variations of plasma thyroxine concentrations in control and castrated rabbits during the 2 yr of the experiment. Note that peak thyroxine concentration was lower in the second year than in the first. Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01) for the same month between the two groups

Annual Thyroxine Cycle in Castrated Animals

For thyroxine, ANOVA analysis did not show a group effect (F = 1.684, P = 0.231) but did show a month effect (F = 10.95, P < 0.001) and a combined group-month effect (F = 11.301, P < 0.001). Three months after castration, T4 levels (Fig. 3B) were significantly higher in castrated animals than in controls (February–March: castrated versus controls, P < 0.01). However, after this short period in which T4 levels were higher in castrated animals, no seasonal activation of thyroid activity in castrated animals was observed during the 26 mo of the experiment whereas controls displayed a clear seasonal peak in T4 concentration in the fall (September–December) in both the first and second years. However, the autumnal peak was smaller in the second year than in the first (October, year 1: 172.8 ± 8.2 ng/ml versus September, year 2: 117.5 ± 8.5 ng/ml; P < 0.001).

Effects of Constant Temperature and/or Photoperiod on Plasma Thyroxine and Testosterone Cycles

When the four groups were considered, ANOVA showed a group effect (F = 9.573, P < 0.001), a month effect (F = 136.13, P < 0.001), and a combined group-month effect (F = 136.264, P < 0.001) for thyroxine and a group effect (F = 5.058, P = 0.012), a month effect (F = 22.501, P < 0.001), and a combined group-month effect (F = 20.434, P < 0.001) for testosterone. Only group 4 (control group, natural temperature and photoperiod) differed significantly from the three others for both testosterone and thyroxine values (with P always <0.01). In constant long-day conditions (16L:8D), whether the temperature was kept constant (Fig. 4A, group 1) or allowed to fluctuate naturally (Fig. 4B, group 2), no activation of the thyroid and testicular axes was observed in the fall. Plasma thyroxine and testosterone concentrations remained at basal levels whereas larger fluctuations in thyroxine concentration were observed in group 2 animals, which were subjected to natural temperature variations. A slight increase in plasma testosterone was observed in spring (April–June), but this increase was statistically significant only in April for group 2, but not for group 1. In natural photoperiod conditions (Fig. 4, C and D), slight but statistically significant increases in plasma testosterone and thyroxine concentration were observed in the fall in group 3 (Testo and T4: September versus August, P < 0.001). These concentrations gradually decreased during the fall and returned to basal levels in winter, as observed in the control group. However, in control animals (Fig. 4D), the peaks in testosterone and thyroxine concentration observed in September reached higher levels than in the animals of group 3 subjected to the same natural photoperiod conditions but with constant high temperature (Testo September: control = 8.90 + 1.58 ng/ml versus group 3 = 3.23 + 0.52 ng/ml, P < 0.001; T4 September: control = 188.5 + 7.6 ng/ml versus group 3 = 103 + 8.3 ng/ml, P < 0.001). If we applied the nonlinear regression-type four-parameter Gaussian curve to the two annual cycles as for the control group of experiment 1, no significant results were obtained for the testosterone and T4 cycles in groups 1 and 2, whereas for group 3 and the control group, all the parameters of the calculated curves were significantly determined, R and R2 (see values in Fig. 4, C and D) and a, b, x0 and y0, with P < 0.001 with a lower amplitude for group 3, maintained in constant high temperature conditions (Fig. 5). In both group 3 and the control group, testosterone and thyroxine concentrations peaked at the same time, at the end of September. These results, like those for experiment 1, demonstrate that the two hormones display similar, parallel seasonal patterns, with reactivation in the fall.



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  		FIG. 4. Seasonal variations of plasma testosterone and thyroxine concentrations (monthly mean ± SEM), in rabbits maintained in various conditions of temperature and photoperiod. A) Group 1, constant 25°C and 16L:8D; (B) group 2, natural external temperature and 16L:8D; (C) group 3, constant 25°C and natural photoperiod; (D) control group, natural external temperature and photoperiod. Asterisks indicate a significant difference (P < 0.01) for the same group between two consecutive months



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  		FIG. 5. Calculated regression curves (y = y0 + ae[-0.5(x-x0/b)2]) for plasma testosterone and thyroxine concentrations in the control group and in group 3, showing a seasonal parallel pattern with synchronous peaks


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Considering the body weight results, it is interesting to note that the difference observed between the castrated group and the control group during the early months following gonadectomy is due to the fact that castrated animals did not lose weight. This seasonal loss of body weight classically observed in male wild mammals during the period of sexual activity [14] is correlated with elevated plasma testosterone levels. The difference between castrated and control animals was still observed in the second year, indicating that gonadectomized animals constantly gained weight in a linear manner throughout the year. In control animals, the seasonal body weight cycle almost disappeared during the second year of captivity; this is due to a decrease in the amount of body weight lost in winter and spring and may be attributed to the housing conditions in captivity (regular food availability, restricted mobility in cages). In thyroidectomized rabbits, the absence of significant body weight variation during the 13 mo of the experiment, with no obvious seasonal cycle and no difference between the initial body weight compared with final may be interpreted in two ways, i) the absence of seasonal recrudescence of testicular activity in thyroidectomized rabbits may prevent the seasonal losses in body weight that occur in control animals and ii) the absence of regular weight gain may be due to the metabolic alterations, in particular, altered lipogenesis and lipolysis due to the disappearance of thyroid hormones. Although no external signs of altered health were visible during the year following thyroidectomy (no emaciation, no body weight losses, no fur or skin alterations) and even though no animals died immediately after thyroidectomy, three of the five animals exhibited health deterioration in the 13 mo following thyroidectomy. Similar health changes have been observed in the other long-term thyroidectomized wild mammals that we have studied (mink [5], badger [15]). These changes occurred after a similar delay of 12–15 mo and were attributed to the perturbation of the different seasonal cycles (hormones and metabolism) that occurred during the year without thyroid hormones.

The results of this study are consistent with the first reports on Zembra Island wild rabbits, studied both in their natural biotope and in semicaptivity, showing that seasonal cycles of testis and thyroid activity follow similar, parallel patterns, with both axes displaying activation in the fall [13]. In all species studied, the close correlations between the thyroid and gonadal cycle have been demonstrated, suggesting that each of these two functions may interfere with each other [4]. Three different patterns have been described in the various avian and mammalian species studied: i) a parallel pattern, with strictly synchronous peaks (e.g., Canada goose, Ruffed grouse, various microtinae such as Microtus arvalis and Microtus agrestis, edible dormouse, Syrian hamster), ii) an inverse pattern, with strictly opposite peaks (e.g., a great diversity of birds—essentially passerines, European badger, red fox, mink), and iii) a semi-inverse pattern, with a phase shift, pronounced to a greater or lesser degree, between peaks (e.g., European blackbird, Peking duck, teal, hedgehog) (see [4] for detailed references). The Zembra Island wild rabbit displays a parallel pattern.

In the second year, the autumnal peak of plasma thyroxine was strongly attenuated and nearly half what it had been during the first year in the control group, as was observed for the body weight. This phenomenon has often been observed in captive wild mammals (badger, fox, hedgehog, red deer, edible dormouse) for body weight, plasma testosterone, and thyroxine. In fact, this occurs despite normal, natural, external housing conditions of temperature and photoperiod in small enclosures or cages and even if the seasonal timing of renewal and end of seasonal endocrine activities are maintained for 2 or more years of captivity. We believe that these variations in amplitude (as for those observed in body weight cycle) are due to the nutritional factor (constant food supply in captivity) and also possibly a reduction in mobility due to the limited space available in captivity.

Our results suggest that, in the short term, thyroid hormones inhibit testicular endocrine activity and, in thyroidectomized animals, the plasma testosterone concentrations measured during the sexual resting period remain significantly higher than those in control animals. Such an effect in the short term is in accordance with data obtained in other wild mammals regardless of whether the seasonal pattern of gonad-thyroid activity is parallel, inverse, or semi-inverse [5, 6, 15, 16]. However, the absence of a renewal of testicular endocrine activity in thyroidectomized rabbits in the fall clearly indicates that thyroid hormones are required for the seasonal reactivation of gonadal activity. Thus, thyroidectomy performed at the end of the period of testicular activity prevents the long-term expression of the annual reproductive rhythm. In most of the species studies, in the long term (i.e., annual cycles), whatever the role of thyroid hormones (stimulating or inhibiting reproduction), these hormones are invariably required for the normal expression of seasonal reproductive activity, initiating the seasonal periods of sexual rest or reactivation.

The effects of thyroid hormones on male seasonal reproduction have been reviewed by Jannini et al. [17]. In a few species, thyroid hormones appear to act as strong inhibitors throughout the gonadal cycle, and thyroidectomy induces nonseasonal gonadal development and delayed regression or abolition of the regressive phase, as principally described in birds (see [18] for recent review). Such an inhibitory effect has recently been described in mammals, especially at the end of the reproductive period, and thyroidectomy has been shown to delay sexual regression in male mink [5]. In the ram, thyroidectomy performed at the trough in the circannual cycle of reproductive activity results in an increase in scrotal circumference and in LH and FSH concentrations [9, 19], indicating rapid stimulation of the gonadal axis and demonstrating the influence of thyroid hormones on the duration of the nonbreeding season. In contrast, many studies have demonstrated that thyroid hormones are required for stimulation of the gonadal axis, principally for sexual maturation in prepubertal animals, but also in adults (see [17] for review). Although this relationship has been clearly demonstrated in female animals (see [20]), the effects of thyroid dysfunction on the activity of the testis are less clearcut and studies have been curtailed since the demonstration in one study that testis function was unaffected by thyroid hormones [21]. However, in the last decade, many in vivo and in vitro studies, conducted principally in rats and mice but also in man (see [17, 22]), have shown that thyroid hormones (THs) act on the gonadal axis, at peripheral (testis) or central levels. In the testis, THs affect Sertoli cell activities, Leydig cell function, epididymal structure, spermatozoid maturation and motility, and testosterone production [23–30]. THs clearly play a direct role because thyroid hormone receptors (TRs) have been identified in germ cells [31] and TR mRNA and protein have been found in adult rat testis, showing that tri-iodothyronine (T3) acts directly on the metabolism of tubular cells [32]. Novel gonad-specific organic anion transporters responsible for TH transport in the brain and testis have recently been identified in rat testis (Sertoli and Leydig cells) [33] and in the human brain and testis [34]. In the brain, THs also act on GnRH neurons and LH and FSH production [17, 35]. Prendergast et al. [36] studied a long-day breeding species, the Siberian hamster (Phodopus sungorus). In this species, i) blockade of thyroid function accelerates the onset of photorefractoriness and ii) thyroid hormones are required to inhibit reproduction in short days. However, plasma T4 levels do not change whatever the photoperiod (short or long days). These authors found that the expression of genes encoding thyroid hormone-binding proteins (TBPs) was downregulated by two- or threefold in the hypothalamus. This reduces the hypothalamic concentration of T4 and regulates access of peripheral T4 to neural targets of gonadal axis. This highlights the importance of these binding proteins for hormonal action at target sites. In the Zembra Island wild rabbit, i) plasma T4 levels are regulated by the seasons and ii) thyroid hormones are required for testicular reactivation. However, we hypothesize that TBPs play a similar role in this species as in the Siberian hamster. Further studies are needed to look for seasonal variations in the expression of the TBP genes in the hypothalamus and to detect any variations in the concentrations of circulating TBPs that may modulate the availability of THs in the testis.

In wild mammals with pronounced annual variations in endocrine activities, the annual testicular rest period is characterized by a deep involution of germ, Sertoli, and Leydig cells and by a total blockade of spermatogenesis and testosterone production in numerous species. Thus, the annual reactivation of both exocrine and endocrine testicular functions is similar to puberty and the processes are similar to those that occur during testis maturation in young animals. The hormonal factors implicated in the seasonal testis reactivation in wild mammals may be identical to those found in prepubertal rats or mice. In vitro studies have shown that T3 is not involved in the regulation of short-type PB-cadherin expression (STPB-C), an additional adhesion factor implicated in contact-mediated interactions between germ and Sertoli cells. However, various studies have shown that thyroid hormones (principally T3) are involved in Sertoli and Leydig cell differentiation (see [30, 37] for reviews). We hypothesize that a lack of thyroid hormones would affect the junctions between adjacent testicular cells (Sertoli, Leydig, and germ cells) by acting on connexin (C-43)-dependent gap junctions [38], thus blocking both spermatogenesis and testosterone production.

In our study, castration prevented, in the long term, seasonal activation of the thyroid axis, which was not observed during the 2 yr of the experiment. In the short term, plasma thyroxine concentrations were significantly higher than those in controls 2 and 3 mo after castration. However, they did not peak after this short period of higher levels and remained at basal levels throughout the rest of the experiment. Fewer studies have focused on the effects of the gonads on the thyroid axis. Conflicting results have been obtained but, overall, androgens seem to have an inhibitory effect in humans, considering the thryoid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) stimulation (see [39]). Various studies on laboratory mammals, principally rats, have shown that testosterone stimulates thyroid activity at various points in the thyroid axis (thyroid gland, plasma thyroxine levels, thyroxine secretion rate, pituitary TSH values) [40–43] and that castration inhibits thyroid activity [42, 44, 45]. In wild mammals, the effects of castration or testosterone injection depend on the species considered and the season. In the short term (1–3 mo after castration) in the hedgehog [46] and the red fox [15], castration increases plasma thyroxine concentrations during the period of sexual activity but has no effect during the sexual resting period. In contrast, in the European badger [47], castration decreases plasma thyroxine concentrations during the period of sexual activity. In these three wild mammals, testosterone injection decreases plasma thyroxine concentrations during the period of sexual activity but has no effect or the opposite effect during the sexual resting period. However, in the long term, gonadectomy has no significant effect on the annual cycle of thyroid activity in the European badger, red fox [47, 48], or hedgehog [46] and seasonal cycles of plasma thyroxine concentrations are similar in control and castrated animals.

In adult rats, recent studies have shown that sex steroids (testosterone, estradiol) have opposite effects on the thyroid axis, with no effect on circulating concentrations of thyroid hormone (T3 and T4) and anterior pituitary TSH contents after orchidectomy and steroid supplementation [45, 49, 50] but transient stimulation of the TSH response to TRH in adults [50]. Sex steroids have also been described to have a stimulatory effect on hypothalamic TRH release [51], TSH mRNA expression in the pituitary gland [52, 53], and plasma concentrations of hypophysial portal TRH [54, 55], and these sexual hormones increase plasma TSH concentrations by modulating TSH binding to TSH receptors in thyrocytes [56].

Our results confirm that long days inhibit testis activation (groups 1 and 2) and that thyroid activity does not increase in the fall if testis activity is inhibited, as observed in the castrated group (experiment 1). Considering the effects of temperature, thyroid function has consistently been reported to be inhibited by heat or stimulated by cold in humans and various other mammals [57–60]. In our experiments, the high temperature conditions imposed on groups 1 and 3 seemed to inhibit thyroid function, with lower mean T4 concentrations in animals subjected to a constant temperature of 25°C than in animals subjected to natural temperature variations. On Zembra Island, mean annual temperature (20°C) is lower than that in Tunis due to the insular nature of this biotope. Although winter temperatures are identical at the two locations, monthly mean temperature in the summer reached only 25°C at Zembra Island [13] whereas it climbed to 30°C in Tunis. For this population of rabbits, 25°C may therefore be considered to be a high temperature.

In conclusion, successful completion of the annual cycles of reproductive and thyroidal activity in this population of rabbits requires the coincidence of external and internal factors: short days and high T4 levels for testis stimulation and low temperature and high testosterone levels for thyroid stimulation. Short days alone cannot induce testis stimulation if T4 levels are too low (high temperature or thyroidectomy), but a deficit in testosterone levels (castration or an inhibiting long-day regimen) prevents thyroid stimulation. Although we did not identify the endocrine level (glandular, pituitary, hypothalamic) at which androgens act on the thyroid axis and T3/T4 thyroid hormones act on the gonadal axis, this study provides the first demonstration, for a given species, that these two axes are strictly linked and that the gonads are required for the seasonal expression of thyroid activity and the thyroid is necessary for the seasonal expression of the gonadal cycle.


   FOOTNOTES
 
1 Correspondence: Daniel Maurel, Pathologie de l'Oreille interne et Réhabilitation, EPI 9902 INSERM, Faculté de Médecine Nord, Boulevard Pierre-Dramard, 13916 Marseille cedex 20, France. FAX: 33 4 91 69 87 31; maurel.d{at}jean-roche.univ-mrs.fr Back

Received: 5 June 2003.

First decision: 3 July 2003.

Accepted: 25 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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