General and Comparative Endocrinology 208 (2014) 5–11
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Photoperiodic control of testicular growth, histomorphology and serum testosterone levels in the male Eurasian tree sparrow: Involvement of circadian rhythm Anand S. Dixit ⇑, Namram S. Singh Avian Environmental Endocrinology and Chronobiology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India
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Article history: Received 3 March 2014 Revised 2 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Circadian rhythm Photoperiod Eurasian tree sparrow Testosterone
a b s t r a c t Experiments were performed on the subtropical population of male Eurasian tree sparrow (Passer montanus) to examine the mediation of the circadian rhythms in photoperiodic regulation of reproductive responses. In the first experiment, photosensitive sparrows were exposed to different resonance light dark cycles viz. 6L/6D, 6L/18D, 6L/30D, 6L/42D, 6L/54D and 6L/66D along with a control group under long day length (14L/10D) for 35 days. The birds read the cycles of 6L/6D, 6L/30D and 6L/54D as long day and exhibited significant testicular growth and increased testosterone levels while the cycles of 6L/18D, 6L/ 42D and 6L/66D were read as short day with no testicular response. In the second experiment, groups of photosensitive birds were subjected to various intermittent light dark cycles of 2L/2D, 3L/3D, 4L/4D, 6L/ 6D, 8L/8D and 12L/12D with two control groups kept under 9L/15D and 14L/10D for 35 days. The birds held under the light/dark cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D showed testicular growth and increased serum levels of testosterone while those exposed to 8L/8D did not. The responses were significantly higher in the birds exposed to 2L/2D, 3L/3D, 4L/4D and 6L/6D when compared to 12L/ 12D. Histomorphology of testes revealed different stages of spermatogenesis only under gonadostimulatory light regimes. The germinative epithelium thickness and diameter of seminiferous tubules increase while the thickness of testicular wall and area of interstitial space decrease with the increase in testicular volume. The above results indicate the involvement of an endogenous circadian rhythm in photoperiodic induction of testicular growth and functions. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Most birds exhibit well-defined seasonality in their reproductive functions such that they occur at the most appropriate time of the year when the chances of survival of their young are maximum (Wingfield and Farner, 1993; Jain and Kumar, 1995; Deviche and Small, 2001; Dawson, 2007). A precise temporal regulation of seasonal reproduction is achieved by intricate physiological processes that sense variations in environmental conditions, integrate them with internal information and regulate the reproductive state accordingly (Hau et al., 2008). As change in photoperiod is entirely predictable at given latitude, both within and between years, it is used as a stable and reliable cue, among different environmental factors, to time the physiological preparations ⇑ Corresponding author at: Avian Endocrinology and Chronobiology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, Meghalaya, India. E-mail addresses: [email protected], [email protected] (A.S. Dixit), [email protected] (N.S. Singh). http://dx.doi.org/10.1016/j.ygcen.2014.09.003 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.
for reproduction in a number of avian species (Hau et al., 2004; Bradshaw and Holzapfel, 2007; Dixit and Singh, 2011). In order to use day length as a cue for seasonal reproduction, birds would require some mechanism to measure its duration (Rani et al., 2005). In photoperiodic birds, a circadian rhythm of photoperiodic photosensitivity (CRPP) mediates photoperiodic regulation of reproductive responses (Rani and Kumar, 1999; Dawson et al., 2001). The CRPP responds to light in a phase dependent manner. Such a concept was originally formulated by Bunning (1936) and involves the operation of an external coincidence model (Pittendrigh and Minis, 1964). It predicts that photoperiodic induction occurs when the light coincides with the photosensitive phase or more precisely photoinducible phase of an entrained endogenous circadian rhythm, which occurs early in subjective night. This model attributes a dual role to light, i.e. entrainer and inducer (Pittendrigh, 1966). One of the powerful methods for testing the involvement of circadian rhythmicity in photoperiodic time measurement during seasonal responses is the resonance light– dark cycles. In these cycles, a short fixed photophase (6–8 h) is
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combined with varying durations of dark phases such that the period of light–dark cycles is lengthened by 12 h increments, such as 12-(6L/6D), 24-(6L/18D), 36-(6L/30D), 48-(6L/42D), 60-(6L/54D) and 72-(6L/66D) h. The second experiment that tests circadian rhythmicity in photoperiodic responses involves the use of intermittent light–dark cycles like 2L/2D, 3L/3D, 4L/4D, 6L/6D, 8L/8D and 12L/12D besides control groups held on short and long days (Tripathi and Tewary, 1988; Ravikumar and Tewary, 1991). Both the above experiments have been designed on the presumption that despite the light being provided at all times is qualitatively similar; its quantitative effects on gonadotropins and gonadal steroids secretion should vary depending upon where it falls in a 24 h cycle. The relationship between the day length and reproduction is ultimately mediated through the neuroendocrine system that perceives photoperiodic information and transduces signals that influence gonadal structure and increased production of estradiol and testosterone (Ball and Balthazart, 2002; Nakane and Yoshimura, 2010). In male birds, plasma testosterone titers increase with the growth and development of testes during the breeding season that regulate important reproductive processes and behaviors (Farner and Follett, 1979; Follett and Robinson, 1980; Wingfield and Kenagy, 1991; Fusani, 2008). The estimation of the reproductive status of gonads based on morphological examination of mass or size may have a significant error (Dziewulska and Domagała, 2006), which may be avoided with the simultaneous study of gonadal histology and sex steroids. Thus, this study is an important approach to investigate and correlate photoperiodically induced changes in testicular size, histology and function (testosterone secretion) together with their photoperiodic control mechanism(s). Extensive literature exists on avian breeding cycles and their control mechanisms with particular emphasis on the temperate birds and especially on the migratory species (Hau, 2001; Carey, 2009; French and Rockwell, 2011). Studies at relatively lower latitudes, particularly on local nonmigratory species, are fewer in view of the large number of avian inhabitants at these latitudes (Dixit and Singh, 2011). In spite of short photo fluctuation at these latitudes, light plays a much more significant role than has hitherto been assumed (Hau et al., 1998; Styrsky et al., 2004; Dittami and Gwinner, 1985; Dixit and Singh, 2011). Whether circadian timing mechanism is operative in all the photoperiodic birds is still inconclusive since the number of species investigated is few and mostly studies comprise temperate birds. Thus, it is reasonable and important to study more avian species especially those inhabiting tropical/subtropical regions to replicate results. It is also critical to generate data in new species to facilitate comparative studies. Therefore, in the present study we propose to investigate the involvement of circadian rhythm in photoperiodic time measurement during the induction of gonadal growth and function in the Eurasian tree sparrow (Passer montanus), a photoperiodic (Dixit and Singh, 2011) resident species, at Shillong (Latitude 25°30 N, Longitude 91°530 E).
2. Materials and methods 2.1. Birds capture, pretreatment, experimental design and maintenance
were subjected to natural variations of photoperiod, temperature and humidity. Then, they were kept under short days (9L/15D) for eight weeks to eliminate photorefractoriness if they had any in nature and to ensure their photosensitivity at the time of commencement of various experiments. Laparotomy (surgical opening of abdominal wall between the last two ribs), measurement of serum testosterone levels and histomorphometric observations (n = 4) at four weeks intervals during the pretreatment period revealed that they had maintained quiescent testis and minimal testosterone levels. These photosensitive birds were used in different experiments. In the first experiment photosensitive sparrows were divided into seven groups (n = 6 each, one control and six experimental). The experimental groups were subjected to different resonance light–dark cycles of 12-(6L/6D), 24-(6L/18D), 36-(6L/30D), 48-(6L/42D), 60-(6L/54D) and 72-(6L/66D) h for 35 days. Besides, a control group was exposed to 14L/10D (long days) for similar duration. In the second experiment, photosensitive sparrows were divided into eight groups (n = 6 each, two control and six experimental). The experimental groups were then subjected to different intermittent light dark cycles viz. 2L/2D, 3L/3D, 4L/4D, 6L/6D, 8L/8D, 12L/12D while control groups were exposed to short (9L/15D) and long (14L/10D) days for 35 days. At the end of above experiments, blood samples were collected from all the birds for testosterone assay following which they were laparotomized to record their testicular size. Thereafter, three birds from each light dark cycle in both the experiments were castrated unilaterally and their testes were fixed in Bouin’s fluid for histological examinations. All the birds were then returned to their respective aviaries and released later to the wild after wound healing. The procedures used in this study were approved by the animal care committee of the Department of Zoology, North-Eastern Hill University, Shillong, Vide code No. 1235 of 17.4.2008. Birds in both the experiments were kept in light proof wooden chambers (2.10 m 1.20 m 1.35 m) illuminated by CFL bulbs (Philips Electronics India Limited, Kolkata, India) providing light of an intensity of 400 lux at the perch level with automated control of light on and light off. Our photoperiodic chambers are well aerated through inlets and outlets connected to air circulators. Food and water were available ad libitum and were replenished only during the light phase of the cycle. 2.2. Testosterone assay Blood samples (100–150 lL) were collected from all the birds at the end of both the experiments and from six randomly selected birds at each observation during the pretreatment period by puncturing the wing vein. Such blood sampling is almost non-invasive and has no risk to bird health. Serum levels of testosterone (TL) were measured using ELISA. A highly sensitive and specific commercial ELISA kit (Dia Metra) was used for the estimation of testosterone (T: Product No. DK0002) following the protocol supplied with the kit. The validation test of this assay was not performed for our species. However, this assay has been validated and used for the measurement of testosterone in other species (Biswas et al., 2010; nee Pathak and Lal, 2010). The lower detection limit of the testosterone assay was 0.075 ng/mL. The intra- and interassay variations for testosterone were 4.6% and 7.5%, respectively. 2.3. Testicular size, histology and histomorphometric analyses
Adult male Eurasian tree sparrows were captured in and around the hills of Shillong in the fall of 2010 and kept in an outdoor aviary (size 3.0 m 2.5 m 2.5 m). This aviary is located in the vicinity of our department in an open area surrounded by natural vegetation and receiving natural light and temperature conditions. Testes at this time were completely regressed. These birds were then acclimatized to laboratory conditions for a fortnight. There they
The testicular size was recorded by performing laparotomy under local anesthesia using subcutaneous injection of 2% xylocaine (Astra-IDL Ltd., Bangalore, India) as per the procedure described in Kumar et al. (2001). Briefly, laparotomy was performed by surgical opening of the abdominal wall between the last two ribs on the left side, testis was located within the
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abdominal cavity with the help of a spatula and the length and width of the left testis were measured. Testis volume (TV) was calculated using formula 4/3pab2, where a and b denote half of the long (length) and short (width) axes, respectively. The testes obtained from castrated birds were fixed in Bouin’s fluid and processed for histological preparations following the procedure mentioned in Lillie and Fullmer (1976). Ten cross sections of seminiferous tubules with the most circular shape were measured for the achievement of the mean tubular diameter using a micrometric eyepiece 10 and 40 objectives in the light microscope. The spermatogenic activity of the testes was assessed by examining the degree of spermatogenic development in the above selected seminiferous tubules. The reproductive status of the testes was estimated as mentioned in Andrews (1968). The thickness of the testicular wall and the germinative epithelium was also measured as described above. Ten intertubular spaces were randomly selected in the cross sections and their areas were measured to calculate mean intertubular area. The histological images were observed under Motic BA310 light microscope and all the above measurements were taken using Motic images plus version 2.0 (Motic China Group Co., Ltd.) software.
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2.4. Data analyses Data are presented as mean ± SEM and were analyzed using one-way ANOVA followed by post hoc test (Newman–Keul’s Multiple range ‘t’ test) if ANOVA indicated a significance of difference. Significance was taken at 95% confidence limit. 3. Results The results are presented in Figs. 1–4. 3.1. Responses under the resonance light dark cycles Birds showed significant variations in mean testicular volume (TV: F6, 34 = 28.04, p < 0.0001; Fig. 1a) and serum level of testosterone (TL: F6, 27 = 42.48, p < 0.0001; one way ANOVA, Fig. 1b) when exposed to the resonance light dark cycles. The TL was found to be running almost parallel to the TV in birds of the control and different experimental groups (Fig. 1a and b). The birds of control groups held under 14L/10D exhibited significant testicular growth and serum level of testosterone (p < 0.001) confirming their photosensitivity at the time of exposure to various experimental light
Fig. 1. Testicular volume, serum levels of testosterone and histomorphometric aspects of the testes under resonance light cycles. g denotes significant difference when the group was compared with 14L/10D (C: control group); h denotes significant difference when the group was compared with 6L/6D; i denotes significant difference when the group was compared with 6L/54D; j denotes significant difference when the group was compared with 6L/18D or 6L/42D or 6L/66D. ⁄p < 0.01, ⁄⁄p < 0.05, ⁄⁄⁄p < 0.001.
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Fig. 2. Histological sections of the testes of Eurasian tree sparrow exposed to resonance light cycles: 14L/10D (a), 6L/6D (b), 6L/18D (c), 6L/30D (d), 6L/42D (e), 6L/54D (f) and 6L/66D (g); SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Scale bar = 15 lm.
Fig. 3. Testicular volume, serum levels of testosterone and histomorphometric aspects of the testes under intermittent light cycles. g denotes significant difference when the group was compared with 9L/15D (C1: control group1); h denotes significant difference when the group was compared with 14L/10D (C2: control group 2); i denotes significant difference when the group was compared with 12L/12D; j denotes significant difference when the group was compared with 8L/8D. ⁄p < 0.01, ⁄⁄p < 0.05, ⁄⁄⁄ p < 0.001.
dark cycles. Significant TV and consequent increase in serum TL were observed in the birds submitted to cycles of 12 (p < 0.001), 36 (p < 0.001) and 60 (TV: p < 0.001; TL: p < 0.05) h but not to cycles of 24, 48 and 72 h. Thus, the cycles of 12, 36 and 60 h acted as long days and 24, 48 and 72 h as short days, despite the fact that
each of them contained only 6 h light period per cycle. Further, clear group differences were observed in photoperiodic responses among birds under stimulatory resonance light cycles and the control group (Fig. 1b). Histological examinations of the testes revealed significant changes in cellular structures only in the birds
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Fig. 4. Histological sections of testes of the Eurasian tree sparrow under intermittent light cycles: 2L/2D (a), 3L/3D (b), 4L/4D (c), 6L/6D (d), 8L/8D (e), 9L/15D (f), 12L/12D (g), 14L/10D (h); SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Scale bar = 15 lm.
of control group and in those exposed to gonadostimulatory resonance cycles (12, 36 and 60 h). The testes in these birds were found showing different stages of spermatogenesis with actively dividing spermatogonia and spermatocytes (Fig. 2a–g). Spermatids and spermatozoa were clearly visible in the control group and 12 h cycle. Birds in the nongonadostimulatory resonance cycles exhibited testes in an inactive phase with resting spermatogonia. Further, histomorphometric analyses of the testes showed significant variations in the thickness of testicular wall (F6, 63 = 33.42, p < 0.0001; Fig. 1c) and germinal epithelium (F6, 63 = 16.88, p < 0.0001; one way ANOVA: Fig. 1d) in the birds under different light cycles. The thickness of testicular wall was observed to be significantly (p < 0.001) lesser under the gonadostimulatory cycles of 12, 36 and 60 h when compared to the nongonadostimulatory cycles of 24, 48 and 72 h (Fig. 1c). In contrast, the thickness of germinal epithelium increased (p < 0.001) with the decrease in the thickness of testicular wall and increase in TV and TL only in the birds exposed of control group and gonadostimulatory cycles of 12, 36 and 60 h (Fig. 1d). Significant changes is the tubular diameter (F6, 63 = 133.3, p < 0.0001; Fig. 1e) and the area of interstitial space (F6, 63 = 41.05, p < 0.0001; one way ANOVA: Fig. 1f) were observed only among the gonadostimulatory groups that ran almost opposite to each other. No significant variation in the body weight was observed in the birds under any light regime (F6, 34 = 0.59, p = 0.7354, One way ANOVA). 3.2. Responses under the intermittent light dark cycles One way ANOVA showed significant variations in TV (F7, 40 = 28.41, p < 0.0001; Fig. 3a) and TL (F17, 23 = 20.66, p < 0.0001; Fig. 3b) in the birds exposed to various intermittent light dark cycles. Further, significant increase in TL was observed only in the gonadostimulatary groups with the TL running almost parallel to TV suggesting the secretion of testosterone from photostimulated testes. Sparrows maintained under long days (14L/ 10D) exhibited significant (p < 0.001) increase in TV and TL while those under short days (9L/15D) did not, indicating their photosensitive at the beginning of the experiment. Significant increase in TV and TL (p < 0.001) were evidenced in the birds exposed to the intermittent cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D, whereas birds under cycle of 8L/8D maintained regressed testes. Histology and histomorphometric analyses of the testes revealed gonad under different stages of the spermatogenesis in the birds exposed
to long days and intermittent light cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D. On the other hand, resting spermatogonia were prominent in the testes of the birds under short days and 8L/8D (Fig. 4a–h). Significant differences in the thickness of testicular wall (F7, 72 = 32.92, p < 0.0001; Fig. 3c) and germinal epithelium (F7, 72 = 67.54, p < 0.0001; one way ANOVA: Fig. 3d) were observed in the birds exposed to different intermittent light cycles. Decrease in the thickness of testicular wall was observed with the increase in the thickness of germinal epithelium, TV and TL in birds under the gonadostimulatary cycles. Increase in tubular diameter (F7, 72 = 83.82, p < 0.0001; Fig. 3e) was observed linked with the decrease in the area of interstitial space (F7, 72 = 74.36, p < 0.0001; one way ANOVA: Fig. 3f) in the birds exposed to photostimulatory cycles. No significant change in the body weight was observed in the birds (F7, 47 = 0.8421, p = 0.5594) submitted to various intermittent light cycles and control groups.
4. Discussion The results obtained from resonance and intermittent light experiments (Figs. 1–4) suggest that the male Eurasian tree sparrow possesses a time measuring system that utilizes an endogenous circadian rhythmicity for reproductive functions. They can be interpreted on the basis of Bunning hypothesis and external coincidence model of photoperiodic time measurement (Bunning, 1973; Pittendrigh and Minis, 1964) which suggest that the circadian rhythm can be divided into two phases. The former phase is photoinsensitive or non-photoinducible phase (subjective day) and the latter is photosensitive or photoinducible phase (subjective night). The birds are insensitive and sensitive in these two phases, respectively, to their photoperiodic responses. Coincidence or non-coincidence of light with the photoinducible phase of an entrained circadian rhythm determines a photoperiodic response. On putting our data from the resonance experiment within the framework of external coincidence model, we find that in the cycle of 12 h (6L/6D) or control group (14L/10D) light falls daily, and in the cycles of 36 h (6L/30D) and 60 h (6L/54D) it falls at alternate cycles or after two days, respectively, in the photosensitive phase, resulting in a positive response. On the other hand, in cycles of 24 h (6L/18D), 48 h (6L/42D) and 72 h (6L/66D), light is restricted only to photoinsensitive phase of the circadian rhythm and a response fails to occur (Fig. 1a). The differences in testicular response among
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stimulatory groups are easily interpretable since no cycle (except the control group, 14L/10D) constitutes a long day treatment in the usual photoperiodic sense. However, the birds subjected to cycles of 12 h and 36 h responded well. A slower rate in testicular response in the birds under 60 h cycle is not at all surprising in view of the fact that the rate of gonadal growth decreases as a function of number of intervening short days (Follett et al., 1967). In addition, the data clearly suggest that neither the absolute duration of light or dark nor the ratio of light to dark is requisite for the initiation of photoperiodic response in the Eurasian tree sparrow. Although the duration of light in each of the resonance light cycles was the same, i.e. 6 h per cycle, the photoperiodic response was observed only in cycles of 12 h, 36 h and 60 h duration. Further, light alone is not sufficient as 6 h light period was much shorter than the threshold photoperiodic requirement (about 11 h/day) of Eurasian tree sparrow for the initiation of gonadal growth (Dixit and Singh, 2011). Our results on tree sparrow are consistent with those on initiation of gonadal growth in some photoperiodic birds (Follett and Sharp, 1969; Gwinner and Eriksson, 1977; Tewary and Kumar, 1981; Tewary et al., 1984; Dixit and Sougrakpam, 2012). The results of intermittent experiment are in conformity with those obtained from the resonance experiment suggesting the involvement of circadian rhythm in regulation of testicular growth and function. Light falling in the photoinducible phase (as in the cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D, 12L/12D and 14L/10D) of an endogenous circadian rhythm induced gonadostimulatary effects (Fig. 3a). On contrary, no gonadal response was observed in the birds of 9L/15D as the light failed to engage the photoinducible phase. The Eurasian tree sparrows did not show any response in 8L/8D (although they responded to 12L/12D) suggesting that this photoperiod possibly induces a progressive shift in the circadian phase of the cycle of photosensitivity. The non inductive effect of this photoperiod may be due to its failure to produce a phase angle difference (W) between the zeitgeber (LD) cycle and CRPP sufficiently long to enable light to coincide with the ai (photoinducible phase). In a different study Turek (1974) also reported the nongonadostimulatory effect of 8L/8D in Zonotrichia atricapilla and Zonotrichia leucophrys pugetensis. Our study involving histology and histomorphometric analyses of testes in the Eurasian tree sparrow under different resonance and night interruption cycles revealed that the increase in the volume of the testes under gonadostimulatary light cycles occurred primarily as a consequence of the enlargement of the seminiferous tubules in which sperms are produced (Figs. 1a, e and 3a, e). An increase in the diameter of seminiferous tubules with the increase in testicular volume in the Eurasian tree sparrow was in conformity with the observations in some other birds like the Whiteeye parakeet, Aratinga leucophthalma (Peixoto et al., 2012) and the Jungle babbler, Turdoides striatus (Bhavna and Geeta, 2010). The interstitial tissue of enlarged testes was found to be packed with Leydig cells, while it contained only the occasional Leydig cells with an enlarged intercellular space in the quiescent testes (Figs. 2 and 4).Testicular wall thickness decreased with the increase in testicular volume in the Eurasian tree sparrow (Figs. 1a, c and 3a, c). Similar observations were reported in the thickness of the testicular wall in the Red-winged tinamou, Rhynchotus rufescens (Baraldi-Artoni et al., 2007), exhibiting lean or thick walls in the periods of activity or spermatogenic regression, respectively. On the other hand, increase in the thickness of the germinative epithelium ran parallel with the increases in the testicular volume, testosterone level and enlargement of the seminiferous tubular diameter in our study bird (Figs. 1a, b, d, e and 3a, b, d, e). Thus, Variations in the intertubular space and seminiferous tubular diameter were found to be inversely related in the Eurasian tree sparrow.
An increase in serum level of testosterone with the increase in gonadal volume was noticed in the birds under gonadostimulatary resonance and intermittent light cycles (Figs. 1a, b and 3a, b) suggesting enhanced gonadal activity in terms of testosterone production in these cycles. This was expected, as testosterone is thought to be an imperative for sperm production and other reproductive functions such as the development and maintenance of the cloacal protuberance and song production (Smith et al., 1997). Further, increase in the serum levels of testosterone ran almost parallel to the increase in gonadal size in these cycles suggesting that the larger gonads release more hormones (Figs. 1a, b and 3a, b). Similar results were obtained with male Yellow-breasted bunting, Emberiza aureola under resonance light cycles in which serum testosterone levels corresponded with the testicular growth (Dixit and Sougrakpam, 2012). 5. Conclusions It may be concluded from the present study that the photoperiodic gonadal response in the Eurasian tree sparrow is critically controlled by a circadian cycle of photosensitivity. An entrained circadian rhythm of photosensitivity in them possess a distinct photoinducible phase, which when illuminated, leads to testicular responses. The gonadostimulatary resonance and intermittent light cycles acting as long days induce positive changes in testicular volume, serum levels of testosterone, along with various histomorphometric aspects of the testes. Furthermore, the above changes show a distinct correlation with respect to each other.
Acknowledgments Financial support through DST grant to A.S.D. from the Department of Science and Technology of the Government of India, New Delhi is gratefully acknowledged. We thank the reviewers for their suggestions and criticisms, which improved the quality of the manuscript. References Andrews, M.I., 1968. Ecology of the social system of Jungle Babbler, Turdoides striatus (Dumont) (Ph.D. thesis). Maharaja Sayajirao University of Baroda, Vadodara, India. Ball, G.F., Balthazart, J., 2002. Neuroendocrine mechanisms regulating reproductive cycles and reproductive behaviour in birds. In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrbach, S.E., Rubin, R.T., San Diego, C.A. (Eds.), Hormones, Brain and Behaviour, vol. 2. Academic Press, San Diego, CA, pp. 649–798. Baraldi-Artoni, S.M., Bottino, F., Oliveira, D., Sobue, F.V., Amoroso, L., Orsi, A.M., Cruz, C., 2007. Morphometric study of Rynchotus rufescens testis throughout the year. Braz. J. Biol. 67 (2), 363–367. Bhavna, B., Geeta, P., 2010. Histological and histomorphometric study of gametogenesis in breeders and helpers of sub-tropical, co-operative breeder jungle babbler, Turdoides striatus. JCAB 4 (5), 81–90. Biswas, A., Mohan, J., Sastry, K.V.H., 2010. Age-dependent variation in hormonal concentration and biochemical constituents in blood plasma of Indian native fowl. Vet. Med. Int. 2010, 1–5. Bradshaw, W.E., Holzapfel, C.M., 2007. Evolution of animal photoperiodism. Annu. Rev. Ecol. Evol. Syst. 38, 1–25. Bunning, E., 1936. Die Physiologische Uhr: Zweite Auflage. Springer, Berlin. Bunning, E., 1973. The Physiological Clock, 3rd ed. University Press Ltd., London; Springer-Verlag, New York; Heidelberg, Berlin. Carey, C., 2009. The impacts of climate change on the annual cycles of birds. Philos. Trans. R. Soc. B 364, 3321–3330. Dawson, A., 2007. Seasonality in a temperate zone bird can be entrained by near equatorial photoperiods. Proc. R. Soc. Lond. B 274, 721–725. Dawson, A., King, V.M., Benthly, G.E., Ball, G.F., 2001. Photoperiodic control of seasonality in birds. J. Biol. Rhythms 16, 366–381. Deviche, P., Small, T., 2001. Photoperiodic control of seasonal reproduction: neuroendocrine mechanisms and adaptation. In: Dawson, A., Chaturvedi, C.M. (Eds.), Avian Endocrinology. Narosa Publishing House, New Delhi, pp. 113–128. Dittami, J., Gwinner, E., 1985. Annual cycles in the African stonechat Saxicola torquata axillaris and their relationship to environmental factors. J. Zool. 207, 357–370.
A.S. Dixit, N.S. Singh / General and Comparative Endocrinology 208 (2014) 5–11 Dixit, A.S., Singh, N.S., 2011. Photoperiod as a proximate factor in control of seasonality in the subtropical male tree Sparrow, Passer montanus. Front. Zool. 8, 1. Dixit, A.S., Sougrakpam, R., 2012. Circadian rhythmicity in photoperiodic regulation of reproductive responses in the Yellow-breasted bunting. Biol. Rhythm Res., 1– 12, i First article. Dziewulska, K., Domagała, J., 2006. Histological assessment of early maturation in sea trout (Salmo trutta morpha trutta L.) males in Western Pomerania (Poland). J. Appl. Ichthyol. 22, 446–450. Farner, D.S., Follett, B.K., 1979. Reproductive periodicity in birds. In: Barrington, E.J.W. (Ed.), Hormones and Evolution. Academic Press, London, pp. 829–872. Follett, B.K., Robinson, J.E., 1980. Photoperiod and gonadotropin secretion in birds. Prog. Reprod. Biol. 5, 39–61. Follett, B.K., Sharp, P.J., 1969. Circadian rhythmicity in photoperiodically induced gonadotropin release and gonadal growth in the Quail. Nature 223, 968–971. Follett, B.K., Farner, D.S., Morton, M.O., 1967. The effects of alternating long and short daily photoperiods on gonadal growth and pituitary gonadotrophins in the White-crowned sparrow, Zonotrichia leucophrys gambelii. Biol. Bull. 133, 330–342. French, H.T., Rockwell, R.F., 2011. Climate change and avian population ecology in Europe. Nat. Educ. Knowl. 3 (10), 1. Fusani, L., 2008. Testosterone control of male courtship in birds. Horm. Behav. 54, 227–233. Gwinner, E., Eriksson, L., 1977. Circadiane rhythmik und photoperiodische zeitmessung beim Star (Sturnus vulgaris). J. Ornithol. 178, 60–67. Hau, M., 2001. Timing of breeding in variable environments: tropical birds as model systems. Horm. Behav. 40, 281–290. Hau, M., Perfito, N., Moore, I.T., 2008. Timing of breeding in tropical birds: mechanisms and evolutionary implications. Ornitol. Neotrop. 19, 39–59. Hau, M., Wikelski, M., Wingfield, J.C., 1998. A neotropical bird can measure changes in tropical photoperiod. Proc. R. Soc. Lond. B 265, 89–95. Hau, M., Wikelski, M., Gwinner, H., Gwinner, E., 2004. Timing of reproduction in a Darwin’s finch: temporal opportunism under spatial constraints. Oikos 106, 489–500. Jain, N., Kumar, V., 1995. Changes in food intake, body weight, gonads and plasma concentrations of thyroxine, luteinizing hormone and testosterone in captive buntings exposed to natural daylengths at 29°N. J. Biosci. 20, 417–426. Kumar, V., Singh, S., Misra, M., Malik, S., 2001. Effects of duration and time of food availability on photoperiodic responses in the migratory male blackheaded bunting (Emberiza melanocephala). J. Exp. Biol. 204, 2843–2848. Lillie, R.D., Fullmer, H.M., 1976. Histopathologic Technic and Practical Histochemistry. McGraw-Hill, New York, pp. 52–53. Nakane, Y., Yoshimura, T., 2010. Deep brain photoreceptors and a seasonal signal transduction cascade in birds. Cell Tissue Res. 342, 341–344.
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nee Pathak, N.D., Lal, B., 2010. Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: role of nitric oxide in testosterone production. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 151, 286–293. Pittendrigh, C.S., 1966. The circadian oscillation in Drosophila pseudoobscura pupae: a model for the photoperiodic clock. Z. Pflanzenphysiol. 54, 275–307. Pittendrigh, C.S., Minis, D.H., 1964. The entrainment of circadian oscillations by light and their role as photoperiodic clocks. Am. Nat. 98, 261–294. Peixoto, J.V., Paula, T.A.R., Balarini, M.K., Matta, S.L.P., Santos, J.A.D., Lima, C.B., Peixoto, G.V., 2012. Morphofunctional evaluation of the testicle and the spermatogenic process of adult White-eyed parakeets (Aratinga leucophthalma Muller, 1776) during the different seasons of the year. Anat. Histol. Embryol. 41, 248–255. Rani, S., Kumar, V., 1999. Time course of sensitivity of the photoinducible phase to light in the redheaded bunting. Biol. Rhythm Res. 30, 555–562. Rani, S., Singh, S., Mishra, M., Malik, S., Singh, B.P., Kumar, V., 2005. Daily light regulates seasonal responses in the migratory male redheaded bunting (Emberiza bruniceps). J. Exp. Zool. 303A, 541–550. Ravikumar, G., Tewary, P.D., 1991. Endogenous circadian rhythm in the photoperiodic ovarian response of the subtropical sparrow, Passer domesticus. Physiol. Behav. 50 (3), 637–639. Smith, G.T., Brenowitz, E.A., Beecher, M.D., Wingfield, J.C., 1997. Seasonal changes in testosterone, neural attributes of song control nuclei, and song structure in wild songbirds. J. Neurosci. 17 (15), 6001–6010. Styrsky, J.D., Brerthold, P., Robinson, W.D., 2004. Endogenous control of migration and calendar effects in an intratropical migrant, the yellow–green vireo. Anim. Behav. 67, 1141–1149. Tewary, P.D., Kumar, V., 1981. Circadian periodicity and the initiation of gonadal growth in male blackheaded buntings (Emberiza melanocephala). J. Comp. Physiol. 144, 201–203. Tewary, P.D., Dixit, A.S., Kumar, V., 1984. Circadian rhythmicity and initiation of reproductive functions in female passerines. Physiol. Zool. 57 (5), 563–566. Tripathi, P.M., Tewary, P.D., 1988. Circadian photoperiodicity in yellow-throated sparrow as demonstrated by intermittent light experiments. Acta Physiol. Hung. 72 (1), 79–84. Turek, F.W., 1974. Circadian rhythmicity and the initiation of gonadal growth in sparrows. J. Comp. Physiol. 92, 59–64. Wingfield, J.C., Farner, D.S., 1993. Endocrinology of reproduction in wild species. In: Farner, D.S., King, J.R., Parkes, K.C. (Eds.), Avian Biology, vol. 9. Academic Press, New York, pp. 163–277. Wingfield, J.C., Kenagy, G.J., 1991. Natural regulation of reproductive cycles. In: Pang, P., Schreibman, M. (Eds.), Vertebrate Endocrinology: Fundamental and Biomedical Implications., 4B. Academic Press, New York, pp. 303–342.
Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Photoperiodic control of testicular growth, histomorphology and serum testosterone levels in the male Eurasian tree sparrow: Involvement of circadian rhythm Anand S. Dixit ⇑, Namram S. Singh Avian Environmental Endocrinology and Chronobiology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India
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Article history: Received 3 March 2014 Revised 2 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Circadian rhythm Photoperiod Eurasian tree sparrow Testosterone
a b s t r a c t Experiments were performed on the subtropical population of male Eurasian tree sparrow (Passer montanus) to examine the mediation of the circadian rhythms in photoperiodic regulation of reproductive responses. In the first experiment, photosensitive sparrows were exposed to different resonance light dark cycles viz. 6L/6D, 6L/18D, 6L/30D, 6L/42D, 6L/54D and 6L/66D along with a control group under long day length (14L/10D) for 35 days. The birds read the cycles of 6L/6D, 6L/30D and 6L/54D as long day and exhibited significant testicular growth and increased testosterone levels while the cycles of 6L/18D, 6L/ 42D and 6L/66D were read as short day with no testicular response. In the second experiment, groups of photosensitive birds were subjected to various intermittent light dark cycles of 2L/2D, 3L/3D, 4L/4D, 6L/ 6D, 8L/8D and 12L/12D with two control groups kept under 9L/15D and 14L/10D for 35 days. The birds held under the light/dark cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D showed testicular growth and increased serum levels of testosterone while those exposed to 8L/8D did not. The responses were significantly higher in the birds exposed to 2L/2D, 3L/3D, 4L/4D and 6L/6D when compared to 12L/ 12D. Histomorphology of testes revealed different stages of spermatogenesis only under gonadostimulatory light regimes. The germinative epithelium thickness and diameter of seminiferous tubules increase while the thickness of testicular wall and area of interstitial space decrease with the increase in testicular volume. The above results indicate the involvement of an endogenous circadian rhythm in photoperiodic induction of testicular growth and functions. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Most birds exhibit well-defined seasonality in their reproductive functions such that they occur at the most appropriate time of the year when the chances of survival of their young are maximum (Wingfield and Farner, 1993; Jain and Kumar, 1995; Deviche and Small, 2001; Dawson, 2007). A precise temporal regulation of seasonal reproduction is achieved by intricate physiological processes that sense variations in environmental conditions, integrate them with internal information and regulate the reproductive state accordingly (Hau et al., 2008). As change in photoperiod is entirely predictable at given latitude, both within and between years, it is used as a stable and reliable cue, among different environmental factors, to time the physiological preparations ⇑ Corresponding author at: Avian Endocrinology and Chronobiology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, Meghalaya, India. E-mail addresses: [email protected], [email protected] (A.S. Dixit), [email protected] (N.S. Singh). http://dx.doi.org/10.1016/j.ygcen.2014.09.003 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.
for reproduction in a number of avian species (Hau et al., 2004; Bradshaw and Holzapfel, 2007; Dixit and Singh, 2011). In order to use day length as a cue for seasonal reproduction, birds would require some mechanism to measure its duration (Rani et al., 2005). In photoperiodic birds, a circadian rhythm of photoperiodic photosensitivity (CRPP) mediates photoperiodic regulation of reproductive responses (Rani and Kumar, 1999; Dawson et al., 2001). The CRPP responds to light in a phase dependent manner. Such a concept was originally formulated by Bunning (1936) and involves the operation of an external coincidence model (Pittendrigh and Minis, 1964). It predicts that photoperiodic induction occurs when the light coincides with the photosensitive phase or more precisely photoinducible phase of an entrained endogenous circadian rhythm, which occurs early in subjective night. This model attributes a dual role to light, i.e. entrainer and inducer (Pittendrigh, 1966). One of the powerful methods for testing the involvement of circadian rhythmicity in photoperiodic time measurement during seasonal responses is the resonance light– dark cycles. In these cycles, a short fixed photophase (6–8 h) is
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combined with varying durations of dark phases such that the period of light–dark cycles is lengthened by 12 h increments, such as 12-(6L/6D), 24-(6L/18D), 36-(6L/30D), 48-(6L/42D), 60-(6L/54D) and 72-(6L/66D) h. The second experiment that tests circadian rhythmicity in photoperiodic responses involves the use of intermittent light–dark cycles like 2L/2D, 3L/3D, 4L/4D, 6L/6D, 8L/8D and 12L/12D besides control groups held on short and long days (Tripathi and Tewary, 1988; Ravikumar and Tewary, 1991). Both the above experiments have been designed on the presumption that despite the light being provided at all times is qualitatively similar; its quantitative effects on gonadotropins and gonadal steroids secretion should vary depending upon where it falls in a 24 h cycle. The relationship between the day length and reproduction is ultimately mediated through the neuroendocrine system that perceives photoperiodic information and transduces signals that influence gonadal structure and increased production of estradiol and testosterone (Ball and Balthazart, 2002; Nakane and Yoshimura, 2010). In male birds, plasma testosterone titers increase with the growth and development of testes during the breeding season that regulate important reproductive processes and behaviors (Farner and Follett, 1979; Follett and Robinson, 1980; Wingfield and Kenagy, 1991; Fusani, 2008). The estimation of the reproductive status of gonads based on morphological examination of mass or size may have a significant error (Dziewulska and Domagała, 2006), which may be avoided with the simultaneous study of gonadal histology and sex steroids. Thus, this study is an important approach to investigate and correlate photoperiodically induced changes in testicular size, histology and function (testosterone secretion) together with their photoperiodic control mechanism(s). Extensive literature exists on avian breeding cycles and their control mechanisms with particular emphasis on the temperate birds and especially on the migratory species (Hau, 2001; Carey, 2009; French and Rockwell, 2011). Studies at relatively lower latitudes, particularly on local nonmigratory species, are fewer in view of the large number of avian inhabitants at these latitudes (Dixit and Singh, 2011). In spite of short photo fluctuation at these latitudes, light plays a much more significant role than has hitherto been assumed (Hau et al., 1998; Styrsky et al., 2004; Dittami and Gwinner, 1985; Dixit and Singh, 2011). Whether circadian timing mechanism is operative in all the photoperiodic birds is still inconclusive since the number of species investigated is few and mostly studies comprise temperate birds. Thus, it is reasonable and important to study more avian species especially those inhabiting tropical/subtropical regions to replicate results. It is also critical to generate data in new species to facilitate comparative studies. Therefore, in the present study we propose to investigate the involvement of circadian rhythm in photoperiodic time measurement during the induction of gonadal growth and function in the Eurasian tree sparrow (Passer montanus), a photoperiodic (Dixit and Singh, 2011) resident species, at Shillong (Latitude 25°30 N, Longitude 91°530 E).
2. Materials and methods 2.1. Birds capture, pretreatment, experimental design and maintenance
were subjected to natural variations of photoperiod, temperature and humidity. Then, they were kept under short days (9L/15D) for eight weeks to eliminate photorefractoriness if they had any in nature and to ensure their photosensitivity at the time of commencement of various experiments. Laparotomy (surgical opening of abdominal wall between the last two ribs), measurement of serum testosterone levels and histomorphometric observations (n = 4) at four weeks intervals during the pretreatment period revealed that they had maintained quiescent testis and minimal testosterone levels. These photosensitive birds were used in different experiments. In the first experiment photosensitive sparrows were divided into seven groups (n = 6 each, one control and six experimental). The experimental groups were subjected to different resonance light–dark cycles of 12-(6L/6D), 24-(6L/18D), 36-(6L/30D), 48-(6L/42D), 60-(6L/54D) and 72-(6L/66D) h for 35 days. Besides, a control group was exposed to 14L/10D (long days) for similar duration. In the second experiment, photosensitive sparrows were divided into eight groups (n = 6 each, two control and six experimental). The experimental groups were then subjected to different intermittent light dark cycles viz. 2L/2D, 3L/3D, 4L/4D, 6L/6D, 8L/8D, 12L/12D while control groups were exposed to short (9L/15D) and long (14L/10D) days for 35 days. At the end of above experiments, blood samples were collected from all the birds for testosterone assay following which they were laparotomized to record their testicular size. Thereafter, three birds from each light dark cycle in both the experiments were castrated unilaterally and their testes were fixed in Bouin’s fluid for histological examinations. All the birds were then returned to their respective aviaries and released later to the wild after wound healing. The procedures used in this study were approved by the animal care committee of the Department of Zoology, North-Eastern Hill University, Shillong, Vide code No. 1235 of 17.4.2008. Birds in both the experiments were kept in light proof wooden chambers (2.10 m 1.20 m 1.35 m) illuminated by CFL bulbs (Philips Electronics India Limited, Kolkata, India) providing light of an intensity of 400 lux at the perch level with automated control of light on and light off. Our photoperiodic chambers are well aerated through inlets and outlets connected to air circulators. Food and water were available ad libitum and were replenished only during the light phase of the cycle. 2.2. Testosterone assay Blood samples (100–150 lL) were collected from all the birds at the end of both the experiments and from six randomly selected birds at each observation during the pretreatment period by puncturing the wing vein. Such blood sampling is almost non-invasive and has no risk to bird health. Serum levels of testosterone (TL) were measured using ELISA. A highly sensitive and specific commercial ELISA kit (Dia Metra) was used for the estimation of testosterone (T: Product No. DK0002) following the protocol supplied with the kit. The validation test of this assay was not performed for our species. However, this assay has been validated and used for the measurement of testosterone in other species (Biswas et al., 2010; nee Pathak and Lal, 2010). The lower detection limit of the testosterone assay was 0.075 ng/mL. The intra- and interassay variations for testosterone were 4.6% and 7.5%, respectively. 2.3. Testicular size, histology and histomorphometric analyses
Adult male Eurasian tree sparrows were captured in and around the hills of Shillong in the fall of 2010 and kept in an outdoor aviary (size 3.0 m 2.5 m 2.5 m). This aviary is located in the vicinity of our department in an open area surrounded by natural vegetation and receiving natural light and temperature conditions. Testes at this time were completely regressed. These birds were then acclimatized to laboratory conditions for a fortnight. There they
The testicular size was recorded by performing laparotomy under local anesthesia using subcutaneous injection of 2% xylocaine (Astra-IDL Ltd., Bangalore, India) as per the procedure described in Kumar et al. (2001). Briefly, laparotomy was performed by surgical opening of the abdominal wall between the last two ribs on the left side, testis was located within the
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abdominal cavity with the help of a spatula and the length and width of the left testis were measured. Testis volume (TV) was calculated using formula 4/3pab2, where a and b denote half of the long (length) and short (width) axes, respectively. The testes obtained from castrated birds were fixed in Bouin’s fluid and processed for histological preparations following the procedure mentioned in Lillie and Fullmer (1976). Ten cross sections of seminiferous tubules with the most circular shape were measured for the achievement of the mean tubular diameter using a micrometric eyepiece 10 and 40 objectives in the light microscope. The spermatogenic activity of the testes was assessed by examining the degree of spermatogenic development in the above selected seminiferous tubules. The reproductive status of the testes was estimated as mentioned in Andrews (1968). The thickness of the testicular wall and the germinative epithelium was also measured as described above. Ten intertubular spaces were randomly selected in the cross sections and their areas were measured to calculate mean intertubular area. The histological images were observed under Motic BA310 light microscope and all the above measurements were taken using Motic images plus version 2.0 (Motic China Group Co., Ltd.) software.
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2.4. Data analyses Data are presented as mean ± SEM and were analyzed using one-way ANOVA followed by post hoc test (Newman–Keul’s Multiple range ‘t’ test) if ANOVA indicated a significance of difference. Significance was taken at 95% confidence limit. 3. Results The results are presented in Figs. 1–4. 3.1. Responses under the resonance light dark cycles Birds showed significant variations in mean testicular volume (TV: F6, 34 = 28.04, p < 0.0001; Fig. 1a) and serum level of testosterone (TL: F6, 27 = 42.48, p < 0.0001; one way ANOVA, Fig. 1b) when exposed to the resonance light dark cycles. The TL was found to be running almost parallel to the TV in birds of the control and different experimental groups (Fig. 1a and b). The birds of control groups held under 14L/10D exhibited significant testicular growth and serum level of testosterone (p < 0.001) confirming their photosensitivity at the time of exposure to various experimental light
Fig. 1. Testicular volume, serum levels of testosterone and histomorphometric aspects of the testes under resonance light cycles. g denotes significant difference when the group was compared with 14L/10D (C: control group); h denotes significant difference when the group was compared with 6L/6D; i denotes significant difference when the group was compared with 6L/54D; j denotes significant difference when the group was compared with 6L/18D or 6L/42D or 6L/66D. ⁄p < 0.01, ⁄⁄p < 0.05, ⁄⁄⁄p < 0.001.
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Fig. 2. Histological sections of the testes of Eurasian tree sparrow exposed to resonance light cycles: 14L/10D (a), 6L/6D (b), 6L/18D (c), 6L/30D (d), 6L/42D (e), 6L/54D (f) and 6L/66D (g); SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Scale bar = 15 lm.
Fig. 3. Testicular volume, serum levels of testosterone and histomorphometric aspects of the testes under intermittent light cycles. g denotes significant difference when the group was compared with 9L/15D (C1: control group1); h denotes significant difference when the group was compared with 14L/10D (C2: control group 2); i denotes significant difference when the group was compared with 12L/12D; j denotes significant difference when the group was compared with 8L/8D. ⁄p < 0.01, ⁄⁄p < 0.05, ⁄⁄⁄ p < 0.001.
dark cycles. Significant TV and consequent increase in serum TL were observed in the birds submitted to cycles of 12 (p < 0.001), 36 (p < 0.001) and 60 (TV: p < 0.001; TL: p < 0.05) h but not to cycles of 24, 48 and 72 h. Thus, the cycles of 12, 36 and 60 h acted as long days and 24, 48 and 72 h as short days, despite the fact that
each of them contained only 6 h light period per cycle. Further, clear group differences were observed in photoperiodic responses among birds under stimulatory resonance light cycles and the control group (Fig. 1b). Histological examinations of the testes revealed significant changes in cellular structures only in the birds
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Fig. 4. Histological sections of testes of the Eurasian tree sparrow under intermittent light cycles: 2L/2D (a), 3L/3D (b), 4L/4D (c), 6L/6D (d), 8L/8D (e), 9L/15D (f), 12L/12D (g), 14L/10D (h); SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Scale bar = 15 lm.
of control group and in those exposed to gonadostimulatory resonance cycles (12, 36 and 60 h). The testes in these birds were found showing different stages of spermatogenesis with actively dividing spermatogonia and spermatocytes (Fig. 2a–g). Spermatids and spermatozoa were clearly visible in the control group and 12 h cycle. Birds in the nongonadostimulatory resonance cycles exhibited testes in an inactive phase with resting spermatogonia. Further, histomorphometric analyses of the testes showed significant variations in the thickness of testicular wall (F6, 63 = 33.42, p < 0.0001; Fig. 1c) and germinal epithelium (F6, 63 = 16.88, p < 0.0001; one way ANOVA: Fig. 1d) in the birds under different light cycles. The thickness of testicular wall was observed to be significantly (p < 0.001) lesser under the gonadostimulatory cycles of 12, 36 and 60 h when compared to the nongonadostimulatory cycles of 24, 48 and 72 h (Fig. 1c). In contrast, the thickness of germinal epithelium increased (p < 0.001) with the decrease in the thickness of testicular wall and increase in TV and TL only in the birds exposed of control group and gonadostimulatory cycles of 12, 36 and 60 h (Fig. 1d). Significant changes is the tubular diameter (F6, 63 = 133.3, p < 0.0001; Fig. 1e) and the area of interstitial space (F6, 63 = 41.05, p < 0.0001; one way ANOVA: Fig. 1f) were observed only among the gonadostimulatory groups that ran almost opposite to each other. No significant variation in the body weight was observed in the birds under any light regime (F6, 34 = 0.59, p = 0.7354, One way ANOVA). 3.2. Responses under the intermittent light dark cycles One way ANOVA showed significant variations in TV (F7, 40 = 28.41, p < 0.0001; Fig. 3a) and TL (F17, 23 = 20.66, p < 0.0001; Fig. 3b) in the birds exposed to various intermittent light dark cycles. Further, significant increase in TL was observed only in the gonadostimulatary groups with the TL running almost parallel to TV suggesting the secretion of testosterone from photostimulated testes. Sparrows maintained under long days (14L/ 10D) exhibited significant (p < 0.001) increase in TV and TL while those under short days (9L/15D) did not, indicating their photosensitive at the beginning of the experiment. Significant increase in TV and TL (p < 0.001) were evidenced in the birds exposed to the intermittent cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D, whereas birds under cycle of 8L/8D maintained regressed testes. Histology and histomorphometric analyses of the testes revealed gonad under different stages of the spermatogenesis in the birds exposed
to long days and intermittent light cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D and 12L/12D. On the other hand, resting spermatogonia were prominent in the testes of the birds under short days and 8L/8D (Fig. 4a–h). Significant differences in the thickness of testicular wall (F7, 72 = 32.92, p < 0.0001; Fig. 3c) and germinal epithelium (F7, 72 = 67.54, p < 0.0001; one way ANOVA: Fig. 3d) were observed in the birds exposed to different intermittent light cycles. Decrease in the thickness of testicular wall was observed with the increase in the thickness of germinal epithelium, TV and TL in birds under the gonadostimulatary cycles. Increase in tubular diameter (F7, 72 = 83.82, p < 0.0001; Fig. 3e) was observed linked with the decrease in the area of interstitial space (F7, 72 = 74.36, p < 0.0001; one way ANOVA: Fig. 3f) in the birds exposed to photostimulatory cycles. No significant change in the body weight was observed in the birds (F7, 47 = 0.8421, p = 0.5594) submitted to various intermittent light cycles and control groups.
4. Discussion The results obtained from resonance and intermittent light experiments (Figs. 1–4) suggest that the male Eurasian tree sparrow possesses a time measuring system that utilizes an endogenous circadian rhythmicity for reproductive functions. They can be interpreted on the basis of Bunning hypothesis and external coincidence model of photoperiodic time measurement (Bunning, 1973; Pittendrigh and Minis, 1964) which suggest that the circadian rhythm can be divided into two phases. The former phase is photoinsensitive or non-photoinducible phase (subjective day) and the latter is photosensitive or photoinducible phase (subjective night). The birds are insensitive and sensitive in these two phases, respectively, to their photoperiodic responses. Coincidence or non-coincidence of light with the photoinducible phase of an entrained circadian rhythm determines a photoperiodic response. On putting our data from the resonance experiment within the framework of external coincidence model, we find that in the cycle of 12 h (6L/6D) or control group (14L/10D) light falls daily, and in the cycles of 36 h (6L/30D) and 60 h (6L/54D) it falls at alternate cycles or after two days, respectively, in the photosensitive phase, resulting in a positive response. On the other hand, in cycles of 24 h (6L/18D), 48 h (6L/42D) and 72 h (6L/66D), light is restricted only to photoinsensitive phase of the circadian rhythm and a response fails to occur (Fig. 1a). The differences in testicular response among
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stimulatory groups are easily interpretable since no cycle (except the control group, 14L/10D) constitutes a long day treatment in the usual photoperiodic sense. However, the birds subjected to cycles of 12 h and 36 h responded well. A slower rate in testicular response in the birds under 60 h cycle is not at all surprising in view of the fact that the rate of gonadal growth decreases as a function of number of intervening short days (Follett et al., 1967). In addition, the data clearly suggest that neither the absolute duration of light or dark nor the ratio of light to dark is requisite for the initiation of photoperiodic response in the Eurasian tree sparrow. Although the duration of light in each of the resonance light cycles was the same, i.e. 6 h per cycle, the photoperiodic response was observed only in cycles of 12 h, 36 h and 60 h duration. Further, light alone is not sufficient as 6 h light period was much shorter than the threshold photoperiodic requirement (about 11 h/day) of Eurasian tree sparrow for the initiation of gonadal growth (Dixit and Singh, 2011). Our results on tree sparrow are consistent with those on initiation of gonadal growth in some photoperiodic birds (Follett and Sharp, 1969; Gwinner and Eriksson, 1977; Tewary and Kumar, 1981; Tewary et al., 1984; Dixit and Sougrakpam, 2012). The results of intermittent experiment are in conformity with those obtained from the resonance experiment suggesting the involvement of circadian rhythm in regulation of testicular growth and function. Light falling in the photoinducible phase (as in the cycles of 2L/2D, 3L/3D, 4L/4D, 6L/6D, 12L/12D and 14L/10D) of an endogenous circadian rhythm induced gonadostimulatary effects (Fig. 3a). On contrary, no gonadal response was observed in the birds of 9L/15D as the light failed to engage the photoinducible phase. The Eurasian tree sparrows did not show any response in 8L/8D (although they responded to 12L/12D) suggesting that this photoperiod possibly induces a progressive shift in the circadian phase of the cycle of photosensitivity. The non inductive effect of this photoperiod may be due to its failure to produce a phase angle difference (W) between the zeitgeber (LD) cycle and CRPP sufficiently long to enable light to coincide with the ai (photoinducible phase). In a different study Turek (1974) also reported the nongonadostimulatory effect of 8L/8D in Zonotrichia atricapilla and Zonotrichia leucophrys pugetensis. Our study involving histology and histomorphometric analyses of testes in the Eurasian tree sparrow under different resonance and night interruption cycles revealed that the increase in the volume of the testes under gonadostimulatary light cycles occurred primarily as a consequence of the enlargement of the seminiferous tubules in which sperms are produced (Figs. 1a, e and 3a, e). An increase in the diameter of seminiferous tubules with the increase in testicular volume in the Eurasian tree sparrow was in conformity with the observations in some other birds like the Whiteeye parakeet, Aratinga leucophthalma (Peixoto et al., 2012) and the Jungle babbler, Turdoides striatus (Bhavna and Geeta, 2010). The interstitial tissue of enlarged testes was found to be packed with Leydig cells, while it contained only the occasional Leydig cells with an enlarged intercellular space in the quiescent testes (Figs. 2 and 4).Testicular wall thickness decreased with the increase in testicular volume in the Eurasian tree sparrow (Figs. 1a, c and 3a, c). Similar observations were reported in the thickness of the testicular wall in the Red-winged tinamou, Rhynchotus rufescens (Baraldi-Artoni et al., 2007), exhibiting lean or thick walls in the periods of activity or spermatogenic regression, respectively. On the other hand, increase in the thickness of the germinative epithelium ran parallel with the increases in the testicular volume, testosterone level and enlargement of the seminiferous tubular diameter in our study bird (Figs. 1a, b, d, e and 3a, b, d, e). Thus, Variations in the intertubular space and seminiferous tubular diameter were found to be inversely related in the Eurasian tree sparrow.
An increase in serum level of testosterone with the increase in gonadal volume was noticed in the birds under gonadostimulatary resonance and intermittent light cycles (Figs. 1a, b and 3a, b) suggesting enhanced gonadal activity in terms of testosterone production in these cycles. This was expected, as testosterone is thought to be an imperative for sperm production and other reproductive functions such as the development and maintenance of the cloacal protuberance and song production (Smith et al., 1997). Further, increase in the serum levels of testosterone ran almost parallel to the increase in gonadal size in these cycles suggesting that the larger gonads release more hormones (Figs. 1a, b and 3a, b). Similar results were obtained with male Yellow-breasted bunting, Emberiza aureola under resonance light cycles in which serum testosterone levels corresponded with the testicular growth (Dixit and Sougrakpam, 2012). 5. Conclusions It may be concluded from the present study that the photoperiodic gonadal response in the Eurasian tree sparrow is critically controlled by a circadian cycle of photosensitivity. An entrained circadian rhythm of photosensitivity in them possess a distinct photoinducible phase, which when illuminated, leads to testicular responses. The gonadostimulatary resonance and intermittent light cycles acting as long days induce positive changes in testicular volume, serum levels of testosterone, along with various histomorphometric aspects of the testes. Furthermore, the above changes show a distinct correlation with respect to each other.
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