Long-Day Suppressed Expression of Type 2 Deiodinase Gene in the Mediobasal Hypothalamus of the Saanen Goat, a Short-Day Breeder: I
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《内分泌学杂志》
Division of Biomodeling (S.Y., N.N., T.Ya., M.W., T.W., S.E., T.Yo.), Laboratory of Animal Management and Resources (S.-i.O.), Division of Applied Genetics and Physiology (K.-i.M.), Graduate School of Bioagricultural Sciences, Technical Center (S.H., A.G., H.A.)
Institute for Advanced Research (T.Yo.), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Laboratory of Neuroendocrinology (S.O., H.O.), National Institute of Agrobiological Sciences, Ikenodai, Tsukuba 305-8602, Japan
Department of Applied Biological Chemistry (M.I.), Faculty of Agriculture, Utsunomiya University, Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
Medical Research Council Human Reproductive Sciences Unit (G.A.L.), University of Edinburgh, Chancellor’s Building, Edinburgh EH16 4SB, Scotland, United Kingdom
Abstract
In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.
Introduction
IN MOST ANIMALS living in temperate regions, reproductive seasonality ensures the birth of young in spring or summer, as is appropriate for survival. Species with a short incubation or gestation period such as Japanese quail and hamsters, or species with a gestation period of nearly 1 yr such as horses, are long-day breeders, and their fertile period occurs in the springtime. On the other hand, species with a gestation period around 5–6 months such as sheep and goats are short-day breeders, and their breeding take places in autumn (1). In mammals, photoperiodic information is translated into a daily cycle of melatonin secretion from the pineal gland (2, 3). The duration of the night is reflected in the length of nocturnal melatonin secretion, and melatonin regulates GnRH secretion from the hypothalamus. An increase in melatonin secretion is associated with decreased GnRH secretion in long-day breeders and increased GnRH secretion in short-day breeders, respectively. The mechanism for the reversed effect of melatonin on GnRH secretion between long- and short-day breeders has been a profound mystery.
Melatonin is thought to act through the mediobasal hypothalamus (MBH) in both long- and short-day breeders. In Syrian hamsters, lesions of the MBH block the gonadal response to short photoperiod (4, 5). In the ewe, melatonin microimplants positioned in the MBH mimic a short-day effect on LH secretion, whereas no effect is observed in other hypothalamic areas (6, 7). Detailed analysis identified the target site of melatonin as the premammillary hypothalamic area (PMH). The PMH consists of three subdivisions: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus, and the ventral tuberomammillary nucleus, and the PMH contains 125I-melatonin binding sites (8, 9, 10). The mechanism by which melatonin regulates GnRH secretion is also currently unknown.
Using Japanese quail, an excellent animal model for studying photoperiodism, we found that expression of type 2 iodothyronine deiodinase (Dio2) was induced by long-day conditions in the MBH. Dio2 catalyzes the conversion of prohormoneT4 to bioactive T3 and controls local thyroid hormone concentration. Thyroid hormone contents in the MBH were about 10-fold higher in quail kept under long-day conditions than in those under short-day conditions, and intracerebroventricular infusion of T3 mimicked photoperiodic testicular growth under short days. These results suggested that light-induced conversion of T4 to T3 in the MBH is critical for the photoperiodic response of gonads in birds (11). Because thyroid hormones are known to be essential for the maintenance of seasonal reproductive changes in both birds and mammals, this mechanism also appears to be conserved in mammals (12, 13). Indeed, similar to quail, up-regulation of Dio2 expression under long-day conditions was observed in the ependymal cell layer lining the infralateral walls of the third ventricle (EC), the cell-clear zone overlying the tuberoinfundibular sulcus (TIS), and the arcuate nucleus (ARC) of Djungarian hamster hypothalamus, and its expression was suppressed by melatonin administration (14). In ewe, thyroid hormone is known to be involved in the transition to anestrus (15), and more recently, it was reported that microimplantation of thyroid hormone in the PMH of thyroidectomized ewes could inhibit the secretion of LH, suggesting that the PMH is the target site for thyroid hormone action (16). In the present study, therefore, we examined the effect of long days on Dio2 expression in the MBH of the Saanen goat (Capra hircus), a short-day breeder.
In ewes, thyroid hormone is known to be involved in changes in the responsiveness of the GnRH axis to estrogen-negative feedback at the transition to anestrus (17, 18). It has also been reported that thyroid hormone can affect the expression of the estrogen receptor (ER) gene (19, 20) and estrogen-sensitive gene expression (21) via estrogen and thyroid hormone receptor (TR) interactions. Although LH secretion is also under the negative feedback regulation of testicular steroids in males, the extent to which of these actions of testicular steroids result from the direct action of testosterone or its metabolite estradiol is unclear (22). Therefore, we further examined the expression of TR, TR, ER, and androgen receptor (AR) in the goat MBH and the effect of long-day stimulus on the expression of these genes. In the Saanen goat, prolactin secretion increases during spring and summer, whereas plasma LH and testosterone increases during autumn and winter (23, 24, 25) (Fig. 1A). We monitored the testicular size and plasma prolactin concentration during the experiment to determine the effect of long-day stimulus and physiological condition of goats.
Materials and Methods
Animals and housing
Male Saanen goats, approximately 4 months old, were purchased from a local dealer and kept in the Experimental Station of Highland Animal Production of Nagoya University (latitude 35° 8'N) under the influence of natural changes in photoperiod and temperature. Food and water were available ad libitum. The experimental schedule and lighting conditions are shown in Fig. 1B. Because maximum testicular regression occurs after approximately 16 wk in Soay rams (25), we exposed animals to an artificial long-day stimulus for 4 months. Eight goats were randomly divided into two groups in November 2003, and four goats were transferred to the artificial long-day conditions (16 h light, 8 h dark). The other goats were kept under natural daylengths. The measurement of testicular length was conducted once a month from September 2003 to February 2004. In March 2004, goats were killed by an overdose of pentobarbital sodium. The brains were rapidly removed and a block of hypothalamus (Fig. 2) was frozen on dry ice. The hypothalamic block was stored at –80 C until in situ hybridization. Animals were treated in accordance with the guidelines of Nagoya University.
Quantification of prolactin
Plasma samples were collected once a month from November 2003 to March 2004. Prolactin concentrations in blood plasma samples were measured using a routine RIA validated for goat plasma (26). The assay used a rabbit antisera (ASM R50) and a purified ovine prolactin standard (NIH-PRL-S13). Intraassay coefficient of variation was less than 10%. The lower limit of sensitivity was 0.5 ng NIH-PRL-S13/ml plasma.
In situ hybridization
In situ hybridization was carried out according to Yoshimura et al. (27). Antisense 45-oligomer oligonucleotide probes for goat Dio2 (antisense: 5'-tgcttgaggagaatgaccgagtcatacagcgccaggaagaggcag-3') (GenBank accession no. AB201476), ovine TR (5'-gtactgctctcctctgggtctgacccacactccaccttgcttggc-3'), ovine TR (5'-acacaggcaagccctgggcgatctgaagacatcagcaggacggcc-3'), goat ER (5'-ccatgcccacttcatagcattcgcgtagccggcagtcctggcaac-3'), and goat AR (5'-aaggaccgccagcccatggcaaacaccataagccccatccaggag-3') were labeled with [33P]deoxy-ATP (NEN Life Science Products, Boston, MA) using terminal deoxyribonucleotidyl transferase (Life Technologies, Inc., Frederick, MD). Coronal sections (20 μm thickness) of the MBH were prepared using a Cryostat. Hybridization was carried out overnight at 42 C. Two high-stringency posthybridization washes were performed at 55 C. For negative controls, sense probes were labeled and hybridized in the same way. The sections were air-dried and apposed to Biomax-MR film (Kodak, Rochester, NY) for 2 wk. 14C standards (American Radiolabeled Chemicals, St. Louis, MO) were included in each cassette, and the relative OD was measured using a computed image-analyzing system (MCID, Imaging Research, St. Catherines, Ontario, Canada) and converted into the radioactive value (nanocuries) using the 14C standard measurements. Data were normalized by subtracting the value at the ventroanterior nucleus of the thalamus, which is located in the same section and does not exhibit a hybridization signal. After exposure to x-ray film, each slide was dipped in type NTB2 autoradiography emulsion (Kodak) diluted twice with sterile distilled water and developed after 6 wk of exposure at 4 C. After development, sections were counterstained with 0.5% cresyl violet and observed with photomicroscope.
Quantification of T3 and T4
When slices for in situ hybridization were prepared, slices (200 μm thickness) of MBH were collected every 500 μm, and 13 slices per animal were pooled for the measurement of T3 and T4 concentration in the MBH (Fig. 2). Thyroid hormones in the MBH were extracted with ethanol and measured by RIA as described (28, 29). The extraction rates determined by the use of [125I]T3 or [125I]T4 (10,000 cpm/sample) were 90.6 ± 2.4 and 91.4 ± 1.7% (mean ± SEM, n = 6) for T3 and T4, respectively. The inhibition curves for the brain extracts were parallel to the curves for authentic T3 and T4 standards. The relationship between the quantity of T3 or T4 added and the quantity recovered was analyzed using linear regression analysis. Significant correlations were obtained between amounts of T3 or T4 added and recovered (T3: Y = 1.14 –0.04, r = 0.997, P < 0.001; T4: Y = 1.21 –0.02, r = 0.997, P < 0.001). Intra- and interassay coefficients of variation were 3.7 and 9.2% for T3 and 6.9 and 10.0% for T4, respectively. The lower limits of sensitivity were 6.3 pg/tube (corresponding to 68.8 pg/sample) for T3, and 2.9 pg/tube (corresponding to 31.7 pg/sample) for T4, respectively.
Results
Effect of artificial long days on testicular size and prolactin secretion
Goats were born around April 2003 and males were fertile with fully functional testes by the first autumn in our latitude (35° 8'N). Therefore, in the present study, we examined the inhibitory effect of artificial long photoperiods on the reproductive axis in the first winter (Fig. 1B). In goats kept under natural daylength, the testicular length did not change throughout the experiment [one-way ANOVA, F(5,18) = 0.29, P > 0.05], whereas that of goats kept under artificial long days significantly decreased from January 2004 [one-way ANOVA, F(5,18) = 4.33, P < 0.05] (Fig. 3A). When the animals were killed in March 2004, the paired testes weight of goats kept under artificial long days was significantly less (70%) than that of goats kept under natural daylength (Mann-Whitney U test, P < 0.05) (Fig. 3B). In goats kept under natural daylength, plasma prolactin increased from February 2004 [one-way ANOVA, F(4,15) = 11.950, P < 0.01]. In contrast, in goats kept under artificial long days, plasma prolactin increased in advance [one-way ANOVA, F(4,15) = 6.310, P < 0.01] (Fig. 3C).
Distribution of Dio2 mRNA in the MBH
We examined Dio2 mRNA expression in the MBH of Saanen goats. From the rostral to the middle region of the MBH, Dio2 mRNA expression was observed in the external zone of the median eminence (ME), especially in the outermost zone (external edge) of the ME, but not in the internal layer (Fig. 4, A and B). In the middle region of the MBH, Dio2 mRNA expression was also observed in the cell-clear zone overlying the TIS and EC, which is consistent with previous studies using the Djungarian hamster (14). From the middle to the caudal region of the MBH, cARC was labeled with a Dio2 mRNA probe (Fig. 4, C and D) (10). No signal was detected in these regions by sense probes (Fig. 4, A–D).
Photoperiodic regulation of Dio2 expression in the MBH
We compared Dio2 mRNA levels between goats kept under natural daylength and artificial long days. In the external zone of the ME, the signal was stronger under artificial long days than under natural daylength (Mann-Whitney U test, P < 0.05) (Fig. 5A). In contrast, a significant decrease in Dio2 mRNA expression under artificial long days was observed in the TIS, EC, and cARC (Mann-Whitney U test, P < 0.05) (Fig. 5, B and C).
T3 and T4 content in the MBH
To assess the functional significance of Dio2 in the MBH, we measured T3 and T4 content in the MBH under natural daylength and artificial long days. Due to the difficulty in isolating each brain region because of overlap in some regions, we measured T3 and T4 content of pooled brain slices obtained from the whole MBH (Fig. 2). Although T4 concentrations did not differ between natural daylength and artificial long days (Mann-Whitney U test, P > 0.05), changes in T3 concentration were suppressed in the MBH of goats kept under artificial long days (Table 1).
Distribution of TR, TR, ER, and AR mRNA and effect of photoperiod on their expression
Thyroid hormone actions are mediated by TRs. Therefore, we examined TR and TR expression in the goat MBH. Expression of TR was observed widely around the MBH including the cARC (30, 31) (Fig. 6A), whereas TR expression was restricted to the cARC (Fig. 6B). Expression of these genes was not different between natural daylength and artificial long days (data not shown). We next examined the expression of ER and AR in the goat MBH. ER expression was observed in the cARC, whereas AR expression was observed not only in the cARC but also in the ventral division of the premammillary nucleus (PMv) (Fig. 7, A and B). Expression levels of ER and AR were not different between natural daylength and artificial long-day conditions (Fig. 7, E and F). No signals were detected by sense probes for TR, TR, ER, and AR (Figs. 6, C and D, and 7, C and D).
Discussion
In the present study, long-day stimulus decreased testicular weight of goats to approximately 70% of that of control goats. This result was consistent with a previous report using Alpine and Saanen goats (32), confirming that the male Saanen goat is a useful animal model for studying seasonal reproduction. Plasma prolactin of goats kept under natural daylength started to increase from February, in accordance with a previous report (25), whereas that of goats kept under artificial long days increased in advance and a high level was maintained until brains were collected. These results suggest that brain collected for gene expression analysis from goats kept under natural daylength represents spring status, whereas that of goats kept under artificial long days mimics the summer status.
Dio2 expression was widely distributed in the goat MBH including the ME, the TIS, and the cARC. Among these regions, cARC is particularly noteworthy, because the T4 microimplants that were placed close to or within the posterior portion of the ARC or slightly caudal to it in thyroidectomized ewes allowed the ewes to enter neuroendocrine anestrus (16). Because T4 is a prohormone and has relatively low biological activity, Dio2 expressed in the cARC seems to generate bioactive T3 locally. Although it has also been reported that micropimplants of T4 in the ventromedial POA (preoptic area) were also effective in two of six ewes examined (16), our preliminary experiment failed to detect Dio2 expression in the goat ventromedial POA (data not shown).
In mammals, photoperiodic information is translated into a daily cycle of melatonin secretion from the pineal gland (2, 3). It is also of interest to note that 1) melatonin microimplants positioned in the MBH mimic a short-day effect on LH secretion (6, 7), 2) expression of melatonin 1A receptor mRNA shows day-night variation in the PMH including the cARC (33), and 3) the PMH is known to contain a melatonin binding site (8). In addition, lesion of the cARC disrupts the seasonal cycle of FSH secretion, and alters both the amplitude and the timing of photoperiod-induced testicular growth in the Soay ram (34). These results suggest that the cARC is the integration center for melatonin signaling. Because Dio2 expression is regulated by melatonin in the MBH of the Djungarian hamster (14), Dio2 expressed in the goat cARC may also be regulated by melatonin.
In Japanese quail, photoperiodic regulation of Dio2 expression and thyroid hormone content was observed in the MBH (11). Likewise, in the present study, we have demonstrated photoperiodic regulation of Dio2 expression and T3 content in the goat MBH. However, the effect of long-day stimulus was different among different brain regions. That is, long-day stimulus suppressed Dio2 expression in the EC, the TIS, and the cARC, whereas it increased expression in the external layer of the ME. When we measured thyroid hormone content in the MBH slices, T3 was suppressed by artificial long days. The suppressed T3 content under long-day conditions reflects the expression pattern of Dio2 in the EC, TIS, and the cARC, but not in the external zone of the ME. In Japanese quail, although strong expression of Dio2 was also observed in the ME, its expression was restricted to the inner and middle zones of the ME, and only minor expression was observed in the external zone. Because there are no cell bodies in the external layer of the ME, Dio2 mRNA appears to originate from cell bodies localized in other regions and be transported to the external layer of the ME. Dio2 protein is an endoplasmic reticulum-resident protein with a short half-life (35, 36). The short half-life of Dio2 protein results from the fact that Dio2 is a substrate for endoplasmic reticulum-associated degradation and selectively targeted for ubiquitination and subsequent proteasomal degradation (37, 38, 39). Therefore, Dio2 protein must be associated with the endoplasmic reticulum to exert an effect. However, endoplasmic reticulum cannot be observed in the external layer of the ME and mRNA observed in the external layer of the ME, does not appear to have a functional role. In addition, TR mRNA, as well as AR and ER mRNA, is not expressed in the ME, whereas all are expressed in the cARC. Taken together, these observations suggest that Dio2 expressed in the cARC appears to have a critical effect on the reproductive neuroendocrine axis in goats.
In the ewe, thyroid hormone action on the suppression of seasonal activity is known to be limited to a specific seasonal window (40, 41). From the mid to late anestrus season (i.e. summer), thyroidectomized ewes cannot enter anestrus in response to T4 treatment. Although the mechanism of this seasonal window has been a mystery, our finding of long-day suppression of Dio2 in the MBH seems to provide a potential answer. That is, we suggest that under long days (mid to late anestrus season), the lack of Dio2 protein due to long-day suppressed Dio2 expression means that T4 cannot be converted to the bioactive form T3, which results in a lack of responsiveness to T4.
The mechanism by which melatonin regulates secretion of GnRH secretion is currently unknown and the opposite effect of melatonin between long-day and short-day breeders has been a profound mystery. The suppression of Dio2 expression under long days observed in the present study is the opposite of the results for long-day breeders (11, 14). The present study may be the first demonstration of a reversed event that may be causally involved in inducing a different neuroendocrine response to a long-day melatonin signal within the brain of long- and short-day breeders. This event may also be found to provide the key to the switching mechanism for long-day breeders and short-day breeders in a future study. To test this hypothesis, it is important to examine the detailed annual expression profiles of Dio2 in the MBH and the effect of melatonin administration on Dio2 expression in this region.
The mechanism by which thyroid hormone regulates the reproductive neuroendocrine axis is of particular interest. Because thyroid hormone actions are mediated by TRs, we examined expression of TRs. Expression of TR was observed widely around the MBH, whereas that of TR was restricted to cARC. Because TR is colocalized in 47% of GnRH neurons in the ewe MBH, thyroid hormone is thought to act on the GnRH system directly in the MBH (30, 31). Although little attention has been given to TR in the MBH, TR in the cARC may also have a potential role in the regulation of the GnRH system.
In sheep, thyroid hormones are known to be involved in both changes in the responsiveness of the GnRH axis to estrogen-negative feedback at the transition to anestrus, and in the steroid-independent cycles in LH pulse frequency (17, 18). Although the underlying mechanism for these actions of thyroid hormones remains to be elucidated, the MBH, especially the cARC, appears to be very important because the cARC is known to contain ERs (10, 42) and estrogen implantation in the ARC results in the suppression of LH secretion in the ram (43). Therefore, it is possible that thyroid hormone regulates the responsiveness of the GnRH system to estradiol-negative feedback in the cARC. Indeed, the influence of thyroid hormone on estrogen actions has been convincingly demonstrated (21). TRs and ERs are members of the nuclear receptor superfamily and have a modular protein structure with high homology in the central binding domain. Furthermore, thyroid hormone can affect the expression of the ER gene (19, 20) and estrogen-sensitive gene expression (21) via estrogen and TR interactions. Because it is not clear whether the action of testicular steroid is mediated by testosterone itself or its metabolite estradiol in male (22), we further examined the expression of ER and AR in the goat MBH. As previously reported, expression of ER and AR was observed in the cARC (42, 44). Because thyroid hormone can affect steroid hormone action through receptor interactions, overlapping of TR, TR, ER, and AR mRNA in the cARC is noteworthy. Although we compared the expression level of both genes, their expression was not altered by photoperiodic treatment. Therefore, it seems that the effect of T3 on estradiol-negative feedback is not directly mediated by ER or AR gene expression, and it may be possible that thyroid hormone affects the responsiveness to estradiol-negative feedback through estrogen-sensitive genes. It is also possible that thyroid hormone action is mediated by nuclear receptor coregulators, which serve to enhance nuclear receptor-mediated transcription primarily by binding to the ligand-activated receptors to remodel the local chromatin structure (45, 46). This possibility is supported by the fact that the mRNA expression of some nuclear receptor coregulators is affected by thyroid hormones in rat pituitary cells (47), in the rat brain (48), and in Xenopus laevis tissues during metamorphosis (49). It would be interesting, therefore, to examine seasonal changes in the expression of nuclear receptor coregulators in the goat MBH in the future.
Thyroid hormones are also known to have essential roles in the development and plasticity of the central nervous system (50), and seasonal plasticity in the GnRH systems has been reported. In Japanese quail, expression of TRs is observed in the ME and the ensheathment of GnRH terminals by glial processes was increased under nonbreeding short days in the ME (51). In sheep, GnRH neurons in the POA in anestrus ewes appear to be surrounded by higher glial processes than those in breeding season ewes (52). Although both studies report changes in the morphological interaction between GnRH neurons and glia, the two studies focus on completely different locations (i.e. encasement of GnRH nerve terminals vs. synaptic input to GnRH cell bodies) and whether these two phenomena are related remains to be determined. In birds, melatonin is not required for seasonal reproduction (53) and light information seems to reach GnRH neurons directly because direct innervation of GnRH neurons by encephalic photoreceptor cells has been reported (54). In contrast, in mammals, the eye is the only organ to receive light information and melatonin is essential (2). Viewed in this light, although the fundamental mechanism for seasonal reproduction appears to be conserved in birds and mammals (14, 55), the existence of different regulatory mechanisms among birds and mammals is not surprising, particularly at the level of transmission of light information. In sheep, it is thought that melatonin acts in the PMH including the cARC and the signal is then brought to the GnRH neurons through interneurons (56). In addition, ER-expressing neural populations in the cARC project to the rostral preoptic area and diagonal band of Broca where the majority of the GnRH perikarya are found in the ewe (57, 58). Therefore, it would be interesting to examine the morphological plasticity of cells expressing the melatonin receptor (33), ER, or interneurons in the cARC.
In conclusion, we found expression of Dio2 in the cARC, which is the target site for both melatonin and thyroxine. In addition, Dio2 expression and T3 content were suppressed by the artificial long-day conditions. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to the specific seasonal window. This long-day suppression of Dio2 may provide a mechanism for the lack of responsiveness to thyroxine during mid to late anestrus. Finally, in the future, we plan to determine the mechanism of thyroid hormone action on the reproductive neuroendocrine axis.
Acknowledgments
We thank Nagoya University Radioisotope Center for use of facilities.
Footnotes
T.Yo. was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture.
First Published Online September 29, 2005
1 S.Y. and N.N. contributed equally to this work.
Abbreviations: AR, Androgen receptor; ARC, arcuate nucleus; cARC, caudal continuation of the hypothalamic ARC; Dio2, type 2 deiodinase; EC, ependymal cell layer lining the infralateral walls of the third ventricle; ER, estrogen receptor; MBH, mediobasal hypothalamus; ME, median eminence; PMH, premammillary hypothalamic area; PMv, ventral division of the premammillary nucleus; POA, preoptic area; TIS, tuberoinfundibular sulcus; TR, thyroid hormone receptor.
Accepted for publication September 19, 2005.
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Institute for Advanced Research (T.Yo.), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Laboratory of Neuroendocrinology (S.O., H.O.), National Institute of Agrobiological Sciences, Ikenodai, Tsukuba 305-8602, Japan
Department of Applied Biological Chemistry (M.I.), Faculty of Agriculture, Utsunomiya University, Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
Medical Research Council Human Reproductive Sciences Unit (G.A.L.), University of Edinburgh, Chancellor’s Building, Edinburgh EH16 4SB, Scotland, United Kingdom
Abstract
In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.
Introduction
IN MOST ANIMALS living in temperate regions, reproductive seasonality ensures the birth of young in spring or summer, as is appropriate for survival. Species with a short incubation or gestation period such as Japanese quail and hamsters, or species with a gestation period of nearly 1 yr such as horses, are long-day breeders, and their fertile period occurs in the springtime. On the other hand, species with a gestation period around 5–6 months such as sheep and goats are short-day breeders, and their breeding take places in autumn (1). In mammals, photoperiodic information is translated into a daily cycle of melatonin secretion from the pineal gland (2, 3). The duration of the night is reflected in the length of nocturnal melatonin secretion, and melatonin regulates GnRH secretion from the hypothalamus. An increase in melatonin secretion is associated with decreased GnRH secretion in long-day breeders and increased GnRH secretion in short-day breeders, respectively. The mechanism for the reversed effect of melatonin on GnRH secretion between long- and short-day breeders has been a profound mystery.
Melatonin is thought to act through the mediobasal hypothalamus (MBH) in both long- and short-day breeders. In Syrian hamsters, lesions of the MBH block the gonadal response to short photoperiod (4, 5). In the ewe, melatonin microimplants positioned in the MBH mimic a short-day effect on LH secretion, whereas no effect is observed in other hypothalamic areas (6, 7). Detailed analysis identified the target site of melatonin as the premammillary hypothalamic area (PMH). The PMH consists of three subdivisions: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus, and the ventral tuberomammillary nucleus, and the PMH contains 125I-melatonin binding sites (8, 9, 10). The mechanism by which melatonin regulates GnRH secretion is also currently unknown.
Using Japanese quail, an excellent animal model for studying photoperiodism, we found that expression of type 2 iodothyronine deiodinase (Dio2) was induced by long-day conditions in the MBH. Dio2 catalyzes the conversion of prohormoneT4 to bioactive T3 and controls local thyroid hormone concentration. Thyroid hormone contents in the MBH were about 10-fold higher in quail kept under long-day conditions than in those under short-day conditions, and intracerebroventricular infusion of T3 mimicked photoperiodic testicular growth under short days. These results suggested that light-induced conversion of T4 to T3 in the MBH is critical for the photoperiodic response of gonads in birds (11). Because thyroid hormones are known to be essential for the maintenance of seasonal reproductive changes in both birds and mammals, this mechanism also appears to be conserved in mammals (12, 13). Indeed, similar to quail, up-regulation of Dio2 expression under long-day conditions was observed in the ependymal cell layer lining the infralateral walls of the third ventricle (EC), the cell-clear zone overlying the tuberoinfundibular sulcus (TIS), and the arcuate nucleus (ARC) of Djungarian hamster hypothalamus, and its expression was suppressed by melatonin administration (14). In ewe, thyroid hormone is known to be involved in the transition to anestrus (15), and more recently, it was reported that microimplantation of thyroid hormone in the PMH of thyroidectomized ewes could inhibit the secretion of LH, suggesting that the PMH is the target site for thyroid hormone action (16). In the present study, therefore, we examined the effect of long days on Dio2 expression in the MBH of the Saanen goat (Capra hircus), a short-day breeder.
In ewes, thyroid hormone is known to be involved in changes in the responsiveness of the GnRH axis to estrogen-negative feedback at the transition to anestrus (17, 18). It has also been reported that thyroid hormone can affect the expression of the estrogen receptor (ER) gene (19, 20) and estrogen-sensitive gene expression (21) via estrogen and thyroid hormone receptor (TR) interactions. Although LH secretion is also under the negative feedback regulation of testicular steroids in males, the extent to which of these actions of testicular steroids result from the direct action of testosterone or its metabolite estradiol is unclear (22). Therefore, we further examined the expression of TR, TR, ER, and androgen receptor (AR) in the goat MBH and the effect of long-day stimulus on the expression of these genes. In the Saanen goat, prolactin secretion increases during spring and summer, whereas plasma LH and testosterone increases during autumn and winter (23, 24, 25) (Fig. 1A). We monitored the testicular size and plasma prolactin concentration during the experiment to determine the effect of long-day stimulus and physiological condition of goats.
Materials and Methods
Animals and housing
Male Saanen goats, approximately 4 months old, were purchased from a local dealer and kept in the Experimental Station of Highland Animal Production of Nagoya University (latitude 35° 8'N) under the influence of natural changes in photoperiod and temperature. Food and water were available ad libitum. The experimental schedule and lighting conditions are shown in Fig. 1B. Because maximum testicular regression occurs after approximately 16 wk in Soay rams (25), we exposed animals to an artificial long-day stimulus for 4 months. Eight goats were randomly divided into two groups in November 2003, and four goats were transferred to the artificial long-day conditions (16 h light, 8 h dark). The other goats were kept under natural daylengths. The measurement of testicular length was conducted once a month from September 2003 to February 2004. In March 2004, goats were killed by an overdose of pentobarbital sodium. The brains were rapidly removed and a block of hypothalamus (Fig. 2) was frozen on dry ice. The hypothalamic block was stored at –80 C until in situ hybridization. Animals were treated in accordance with the guidelines of Nagoya University.
Quantification of prolactin
Plasma samples were collected once a month from November 2003 to March 2004. Prolactin concentrations in blood plasma samples were measured using a routine RIA validated for goat plasma (26). The assay used a rabbit antisera (ASM R50) and a purified ovine prolactin standard (NIH-PRL-S13). Intraassay coefficient of variation was less than 10%. The lower limit of sensitivity was 0.5 ng NIH-PRL-S13/ml plasma.
In situ hybridization
In situ hybridization was carried out according to Yoshimura et al. (27). Antisense 45-oligomer oligonucleotide probes for goat Dio2 (antisense: 5'-tgcttgaggagaatgaccgagtcatacagcgccaggaagaggcag-3') (GenBank accession no. AB201476), ovine TR (5'-gtactgctctcctctgggtctgacccacactccaccttgcttggc-3'), ovine TR (5'-acacaggcaagccctgggcgatctgaagacatcagcaggacggcc-3'), goat ER (5'-ccatgcccacttcatagcattcgcgtagccggcagtcctggcaac-3'), and goat AR (5'-aaggaccgccagcccatggcaaacaccataagccccatccaggag-3') were labeled with [33P]deoxy-ATP (NEN Life Science Products, Boston, MA) using terminal deoxyribonucleotidyl transferase (Life Technologies, Inc., Frederick, MD). Coronal sections (20 μm thickness) of the MBH were prepared using a Cryostat. Hybridization was carried out overnight at 42 C. Two high-stringency posthybridization washes were performed at 55 C. For negative controls, sense probes were labeled and hybridized in the same way. The sections were air-dried and apposed to Biomax-MR film (Kodak, Rochester, NY) for 2 wk. 14C standards (American Radiolabeled Chemicals, St. Louis, MO) were included in each cassette, and the relative OD was measured using a computed image-analyzing system (MCID, Imaging Research, St. Catherines, Ontario, Canada) and converted into the radioactive value (nanocuries) using the 14C standard measurements. Data were normalized by subtracting the value at the ventroanterior nucleus of the thalamus, which is located in the same section and does not exhibit a hybridization signal. After exposure to x-ray film, each slide was dipped in type NTB2 autoradiography emulsion (Kodak) diluted twice with sterile distilled water and developed after 6 wk of exposure at 4 C. After development, sections were counterstained with 0.5% cresyl violet and observed with photomicroscope.
Quantification of T3 and T4
When slices for in situ hybridization were prepared, slices (200 μm thickness) of MBH were collected every 500 μm, and 13 slices per animal were pooled for the measurement of T3 and T4 concentration in the MBH (Fig. 2). Thyroid hormones in the MBH were extracted with ethanol and measured by RIA as described (28, 29). The extraction rates determined by the use of [125I]T3 or [125I]T4 (10,000 cpm/sample) were 90.6 ± 2.4 and 91.4 ± 1.7% (mean ± SEM, n = 6) for T3 and T4, respectively. The inhibition curves for the brain extracts were parallel to the curves for authentic T3 and T4 standards. The relationship between the quantity of T3 or T4 added and the quantity recovered was analyzed using linear regression analysis. Significant correlations were obtained between amounts of T3 or T4 added and recovered (T3: Y = 1.14 –0.04, r = 0.997, P < 0.001; T4: Y = 1.21 –0.02, r = 0.997, P < 0.001). Intra- and interassay coefficients of variation were 3.7 and 9.2% for T3 and 6.9 and 10.0% for T4, respectively. The lower limits of sensitivity were 6.3 pg/tube (corresponding to 68.8 pg/sample) for T3, and 2.9 pg/tube (corresponding to 31.7 pg/sample) for T4, respectively.
Results
Effect of artificial long days on testicular size and prolactin secretion
Goats were born around April 2003 and males were fertile with fully functional testes by the first autumn in our latitude (35° 8'N). Therefore, in the present study, we examined the inhibitory effect of artificial long photoperiods on the reproductive axis in the first winter (Fig. 1B). In goats kept under natural daylength, the testicular length did not change throughout the experiment [one-way ANOVA, F(5,18) = 0.29, P > 0.05], whereas that of goats kept under artificial long days significantly decreased from January 2004 [one-way ANOVA, F(5,18) = 4.33, P < 0.05] (Fig. 3A). When the animals were killed in March 2004, the paired testes weight of goats kept under artificial long days was significantly less (70%) than that of goats kept under natural daylength (Mann-Whitney U test, P < 0.05) (Fig. 3B). In goats kept under natural daylength, plasma prolactin increased from February 2004 [one-way ANOVA, F(4,15) = 11.950, P < 0.01]. In contrast, in goats kept under artificial long days, plasma prolactin increased in advance [one-way ANOVA, F(4,15) = 6.310, P < 0.01] (Fig. 3C).
Distribution of Dio2 mRNA in the MBH
We examined Dio2 mRNA expression in the MBH of Saanen goats. From the rostral to the middle region of the MBH, Dio2 mRNA expression was observed in the external zone of the median eminence (ME), especially in the outermost zone (external edge) of the ME, but not in the internal layer (Fig. 4, A and B). In the middle region of the MBH, Dio2 mRNA expression was also observed in the cell-clear zone overlying the TIS and EC, which is consistent with previous studies using the Djungarian hamster (14). From the middle to the caudal region of the MBH, cARC was labeled with a Dio2 mRNA probe (Fig. 4, C and D) (10). No signal was detected in these regions by sense probes (Fig. 4, A–D).
Photoperiodic regulation of Dio2 expression in the MBH
We compared Dio2 mRNA levels between goats kept under natural daylength and artificial long days. In the external zone of the ME, the signal was stronger under artificial long days than under natural daylength (Mann-Whitney U test, P < 0.05) (Fig. 5A). In contrast, a significant decrease in Dio2 mRNA expression under artificial long days was observed in the TIS, EC, and cARC (Mann-Whitney U test, P < 0.05) (Fig. 5, B and C).
T3 and T4 content in the MBH
To assess the functional significance of Dio2 in the MBH, we measured T3 and T4 content in the MBH under natural daylength and artificial long days. Due to the difficulty in isolating each brain region because of overlap in some regions, we measured T3 and T4 content of pooled brain slices obtained from the whole MBH (Fig. 2). Although T4 concentrations did not differ between natural daylength and artificial long days (Mann-Whitney U test, P > 0.05), changes in T3 concentration were suppressed in the MBH of goats kept under artificial long days (Table 1).
Distribution of TR, TR, ER, and AR mRNA and effect of photoperiod on their expression
Thyroid hormone actions are mediated by TRs. Therefore, we examined TR and TR expression in the goat MBH. Expression of TR was observed widely around the MBH including the cARC (30, 31) (Fig. 6A), whereas TR expression was restricted to the cARC (Fig. 6B). Expression of these genes was not different between natural daylength and artificial long days (data not shown). We next examined the expression of ER and AR in the goat MBH. ER expression was observed in the cARC, whereas AR expression was observed not only in the cARC but also in the ventral division of the premammillary nucleus (PMv) (Fig. 7, A and B). Expression levels of ER and AR were not different between natural daylength and artificial long-day conditions (Fig. 7, E and F). No signals were detected by sense probes for TR, TR, ER, and AR (Figs. 6, C and D, and 7, C and D).
Discussion
In the present study, long-day stimulus decreased testicular weight of goats to approximately 70% of that of control goats. This result was consistent with a previous report using Alpine and Saanen goats (32), confirming that the male Saanen goat is a useful animal model for studying seasonal reproduction. Plasma prolactin of goats kept under natural daylength started to increase from February, in accordance with a previous report (25), whereas that of goats kept under artificial long days increased in advance and a high level was maintained until brains were collected. These results suggest that brain collected for gene expression analysis from goats kept under natural daylength represents spring status, whereas that of goats kept under artificial long days mimics the summer status.
Dio2 expression was widely distributed in the goat MBH including the ME, the TIS, and the cARC. Among these regions, cARC is particularly noteworthy, because the T4 microimplants that were placed close to or within the posterior portion of the ARC or slightly caudal to it in thyroidectomized ewes allowed the ewes to enter neuroendocrine anestrus (16). Because T4 is a prohormone and has relatively low biological activity, Dio2 expressed in the cARC seems to generate bioactive T3 locally. Although it has also been reported that micropimplants of T4 in the ventromedial POA (preoptic area) were also effective in two of six ewes examined (16), our preliminary experiment failed to detect Dio2 expression in the goat ventromedial POA (data not shown).
In mammals, photoperiodic information is translated into a daily cycle of melatonin secretion from the pineal gland (2, 3). It is also of interest to note that 1) melatonin microimplants positioned in the MBH mimic a short-day effect on LH secretion (6, 7), 2) expression of melatonin 1A receptor mRNA shows day-night variation in the PMH including the cARC (33), and 3) the PMH is known to contain a melatonin binding site (8). In addition, lesion of the cARC disrupts the seasonal cycle of FSH secretion, and alters both the amplitude and the timing of photoperiod-induced testicular growth in the Soay ram (34). These results suggest that the cARC is the integration center for melatonin signaling. Because Dio2 expression is regulated by melatonin in the MBH of the Djungarian hamster (14), Dio2 expressed in the goat cARC may also be regulated by melatonin.
In Japanese quail, photoperiodic regulation of Dio2 expression and thyroid hormone content was observed in the MBH (11). Likewise, in the present study, we have demonstrated photoperiodic regulation of Dio2 expression and T3 content in the goat MBH. However, the effect of long-day stimulus was different among different brain regions. That is, long-day stimulus suppressed Dio2 expression in the EC, the TIS, and the cARC, whereas it increased expression in the external layer of the ME. When we measured thyroid hormone content in the MBH slices, T3 was suppressed by artificial long days. The suppressed T3 content under long-day conditions reflects the expression pattern of Dio2 in the EC, TIS, and the cARC, but not in the external zone of the ME. In Japanese quail, although strong expression of Dio2 was also observed in the ME, its expression was restricted to the inner and middle zones of the ME, and only minor expression was observed in the external zone. Because there are no cell bodies in the external layer of the ME, Dio2 mRNA appears to originate from cell bodies localized in other regions and be transported to the external layer of the ME. Dio2 protein is an endoplasmic reticulum-resident protein with a short half-life (35, 36). The short half-life of Dio2 protein results from the fact that Dio2 is a substrate for endoplasmic reticulum-associated degradation and selectively targeted for ubiquitination and subsequent proteasomal degradation (37, 38, 39). Therefore, Dio2 protein must be associated with the endoplasmic reticulum to exert an effect. However, endoplasmic reticulum cannot be observed in the external layer of the ME and mRNA observed in the external layer of the ME, does not appear to have a functional role. In addition, TR mRNA, as well as AR and ER mRNA, is not expressed in the ME, whereas all are expressed in the cARC. Taken together, these observations suggest that Dio2 expressed in the cARC appears to have a critical effect on the reproductive neuroendocrine axis in goats.
In the ewe, thyroid hormone action on the suppression of seasonal activity is known to be limited to a specific seasonal window (40, 41). From the mid to late anestrus season (i.e. summer), thyroidectomized ewes cannot enter anestrus in response to T4 treatment. Although the mechanism of this seasonal window has been a mystery, our finding of long-day suppression of Dio2 in the MBH seems to provide a potential answer. That is, we suggest that under long days (mid to late anestrus season), the lack of Dio2 protein due to long-day suppressed Dio2 expression means that T4 cannot be converted to the bioactive form T3, which results in a lack of responsiveness to T4.
The mechanism by which melatonin regulates secretion of GnRH secretion is currently unknown and the opposite effect of melatonin between long-day and short-day breeders has been a profound mystery. The suppression of Dio2 expression under long days observed in the present study is the opposite of the results for long-day breeders (11, 14). The present study may be the first demonstration of a reversed event that may be causally involved in inducing a different neuroendocrine response to a long-day melatonin signal within the brain of long- and short-day breeders. This event may also be found to provide the key to the switching mechanism for long-day breeders and short-day breeders in a future study. To test this hypothesis, it is important to examine the detailed annual expression profiles of Dio2 in the MBH and the effect of melatonin administration on Dio2 expression in this region.
The mechanism by which thyroid hormone regulates the reproductive neuroendocrine axis is of particular interest. Because thyroid hormone actions are mediated by TRs, we examined expression of TRs. Expression of TR was observed widely around the MBH, whereas that of TR was restricted to cARC. Because TR is colocalized in 47% of GnRH neurons in the ewe MBH, thyroid hormone is thought to act on the GnRH system directly in the MBH (30, 31). Although little attention has been given to TR in the MBH, TR in the cARC may also have a potential role in the regulation of the GnRH system.
In sheep, thyroid hormones are known to be involved in both changes in the responsiveness of the GnRH axis to estrogen-negative feedback at the transition to anestrus, and in the steroid-independent cycles in LH pulse frequency (17, 18). Although the underlying mechanism for these actions of thyroid hormones remains to be elucidated, the MBH, especially the cARC, appears to be very important because the cARC is known to contain ERs (10, 42) and estrogen implantation in the ARC results in the suppression of LH secretion in the ram (43). Therefore, it is possible that thyroid hormone regulates the responsiveness of the GnRH system to estradiol-negative feedback in the cARC. Indeed, the influence of thyroid hormone on estrogen actions has been convincingly demonstrated (21). TRs and ERs are members of the nuclear receptor superfamily and have a modular protein structure with high homology in the central binding domain. Furthermore, thyroid hormone can affect the expression of the ER gene (19, 20) and estrogen-sensitive gene expression (21) via estrogen and TR interactions. Because it is not clear whether the action of testicular steroid is mediated by testosterone itself or its metabolite estradiol in male (22), we further examined the expression of ER and AR in the goat MBH. As previously reported, expression of ER and AR was observed in the cARC (42, 44). Because thyroid hormone can affect steroid hormone action through receptor interactions, overlapping of TR, TR, ER, and AR mRNA in the cARC is noteworthy. Although we compared the expression level of both genes, their expression was not altered by photoperiodic treatment. Therefore, it seems that the effect of T3 on estradiol-negative feedback is not directly mediated by ER or AR gene expression, and it may be possible that thyroid hormone affects the responsiveness to estradiol-negative feedback through estrogen-sensitive genes. It is also possible that thyroid hormone action is mediated by nuclear receptor coregulators, which serve to enhance nuclear receptor-mediated transcription primarily by binding to the ligand-activated receptors to remodel the local chromatin structure (45, 46). This possibility is supported by the fact that the mRNA expression of some nuclear receptor coregulators is affected by thyroid hormones in rat pituitary cells (47), in the rat brain (48), and in Xenopus laevis tissues during metamorphosis (49). It would be interesting, therefore, to examine seasonal changes in the expression of nuclear receptor coregulators in the goat MBH in the future.
Thyroid hormones are also known to have essential roles in the development and plasticity of the central nervous system (50), and seasonal plasticity in the GnRH systems has been reported. In Japanese quail, expression of TRs is observed in the ME and the ensheathment of GnRH terminals by glial processes was increased under nonbreeding short days in the ME (51). In sheep, GnRH neurons in the POA in anestrus ewes appear to be surrounded by higher glial processes than those in breeding season ewes (52). Although both studies report changes in the morphological interaction between GnRH neurons and glia, the two studies focus on completely different locations (i.e. encasement of GnRH nerve terminals vs. synaptic input to GnRH cell bodies) and whether these two phenomena are related remains to be determined. In birds, melatonin is not required for seasonal reproduction (53) and light information seems to reach GnRH neurons directly because direct innervation of GnRH neurons by encephalic photoreceptor cells has been reported (54). In contrast, in mammals, the eye is the only organ to receive light information and melatonin is essential (2). Viewed in this light, although the fundamental mechanism for seasonal reproduction appears to be conserved in birds and mammals (14, 55), the existence of different regulatory mechanisms among birds and mammals is not surprising, particularly at the level of transmission of light information. In sheep, it is thought that melatonin acts in the PMH including the cARC and the signal is then brought to the GnRH neurons through interneurons (56). In addition, ER-expressing neural populations in the cARC project to the rostral preoptic area and diagonal band of Broca where the majority of the GnRH perikarya are found in the ewe (57, 58). Therefore, it would be interesting to examine the morphological plasticity of cells expressing the melatonin receptor (33), ER, or interneurons in the cARC.
In conclusion, we found expression of Dio2 in the cARC, which is the target site for both melatonin and thyroxine. In addition, Dio2 expression and T3 content were suppressed by the artificial long-day conditions. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to the specific seasonal window. This long-day suppression of Dio2 may provide a mechanism for the lack of responsiveness to thyroxine during mid to late anestrus. Finally, in the future, we plan to determine the mechanism of thyroid hormone action on the reproductive neuroendocrine axis.
Acknowledgments
We thank Nagoya University Radioisotope Center for use of facilities.
Footnotes
T.Yo. was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture.
First Published Online September 29, 2005
1 S.Y. and N.N. contributed equally to this work.
Abbreviations: AR, Androgen receptor; ARC, arcuate nucleus; cARC, caudal continuation of the hypothalamic ARC; Dio2, type 2 deiodinase; EC, ependymal cell layer lining the infralateral walls of the third ventricle; ER, estrogen receptor; MBH, mediobasal hypothalamus; ME, median eminence; PMH, premammillary hypothalamic area; PMv, ventral division of the premammillary nucleus; POA, preoptic area; TIS, tuberoinfundibular sulcus; TR, thyroid hormone receptor.
Accepted for publication September 19, 2005.
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