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Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles
Accepted Manuscript Title: Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles Author: Yuji Tomori Ken Takumi Norio Iijima Shinro Takai Hitoshi Ozawa PII: DOI: Reference:
S0168-0102(16)30183-3 http://dx.doi.org/doi:10.1016/j.neures.2016.11.005 NSR 3995
To appear in:
Neuroscience Research
Received date: Accepted date:
6-10-2016 14-11-2016
Please cite this article as: Tomori, Yuji, Takumi, Ken, Iijima, Norio, Takai, Shinro, Ozawa, Hitoshi, Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles.Neuroscience Research http://dx.doi.org/10.1016/j.neures.2016.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles
Yuji Tomori1,2, Ken Takumi1, Norio Iijima1, Shinro Takai2, Hitoshi Ozawa1*
1
Department of Anatomy and Neurobiology, Graduate School of Medicine,
Nippon Medical School, Bunkyo-ku, Tokyo, Japan 2
Department of Orthopedic Surgery, Graduate School of Medicine, Nippon
Medical School, Bunkyo-ku, Tokyo, Japan
*Corresponding author Tel: +81-3-3822-2131 ext. 5299 Fax: +81-3-5685-6640 E-mail: [email protected] Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School,1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
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Highlights ・ Irregular estrus cycles were observed in our rat model of hypothyroidism. ・ Kiss1 mRNA-expressing neurons in the ARC were decreased in hypothyroid rats. ・ Kisspeptin immunoreactive neurons in the ARC were decreased in hypothyroid rats.
Abstract Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.
Keywords: hypothyroidism; kisspeptin; propylthiouracil; thyroid hormone;
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Introduction Thyroid hormones are known to be essential for the growth, development and metabolism of various tissues (Bassett and Williams, 2016; Dezonne et al., 2015; Maran, 2003; Mullur et al., 2014). In addition to these indispensable effects, thyroid hormones are also known to play an important role in reproductive function (Castañeda Cortés et al., 2014; Dittrich et al., 2011; Krassas et al., 2010; Tohei, 2004; Tohei et al., 1998a). Hypothyroidism, which refers to an insufficiency or absence of thyroid hormones, is associated with menstrual disorders and infertility in women (Krassas, 2000; Krassas et al., 2010). Impaired gonadotropin-releasing hormone (GnRH) biosynthesis (Tamura et al., 1998; Toni et al., 2005), diminished luteinizing hormone (LH) surge (Tamura et al., 1998; Tohei, 2004; Tohei et al., 1998a), irregular estrus cycles (Hapon et al., 2010; Lutsyk and Sogomonian, 2012; Mattheij et al., 1995; Tohei, 2004; Tohei et al., 1998a) and disorganized reproductive behavior (Tohei et al., 1998b; Yu et al., 2015) have all been reported in female rat models of hypothyroidism. Moreover, in hypothyroidism, elevated serum prolactin (PRL) is also observed (Binita et al., 2009; Krassas, 2000; Krassas et al., 2010). Elevation of circulating PRL has been known to inhibit the secretion of LH by primarily decreasing GnRH secretion or signaling (Cohen-Becker et al., 1986; Fox et al., 1987; Koike et al., 1984). Despite an established relationship between hypothyroidism and suppression of reproductive functions, the mechanism of this suppression remains unclear. 3
Kisspeptin, which is encoded by the Kiss1 gene, and its receptor GPR54 have been shown to have pivotal roles in the regulation of GnRH/LH release (d'Anglemont de Tassigny and Colledge, 2010; Iijima et al., 2011; Navarro et al., 2011; Oakley et al., 2009). Data from several studies have demonstrated that kisspeptin administration increases serum LH and follicle-stimulating hormone (FSH) levels through a GnRH dependent mechanism in rodents (Matsui et al., 2004; Navarro et al., 2004; Navarro et al., 2005). Moreover, it has been shown that kisspeptin has a potent role for steroid feedback regulation of GnRH release. Substantially all kisspeptin neurons in both the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) of the female rodents hypothalamus express estrogen receptor α (ERα) (Dubois et al., 2015; Smith et al., 2005; Smith et al., 2006). Estradiol (E2) inhibits Kiss1 mRNA expression in the ARC (Smith et al., 2005; Smith et al., 2006). Conversely, E2 stimulates the expression of Kiss1 mRNA in the AVPV of female rodents (Adachi et al., 2007; Smith et al., 2005; Smith et al., 2006). These studies suggest that ARC kisspeptin neurons are linked to negative feedback and the AVPV kisspeptin neurons are linked to positive feedback. Taken together, we hypothesized that dysregulation of gonadotropin release in hypothyroidism is brought by the alteration of the activity of kisspeptin neurons. Thus, in the present study, we aimed to analyze the effects of shortage of thyroid hormones on kisspeptin expression in the hypothalamus of female rats. 4
Materials and Methods Animals and Treatment Female Wistar-Imamichi rats (6 weeks of age) were purchased from the Institute for Animal Reproduction (Kasumigaura, Ibaraki, Japan), and housed under a 14-h light/10-h dark cycle (lights on at 06:00 h) and a constant temperature of 24±2 °C with free access to food and water. Vaginal smears were examined daily to monitor the estrus cycle. Following confirmation of the presence of regular 4 day estrus cycles for at least 2 weeks, rats were divided into 2 groups. One group (control, n=5) was given normal water. In the other group (propylthiouracil (PTU)-treated rats, n=7), hypothyroidism was induced by administration of 0.02% 6-propyl-2-thiouracil (Wako, Osaka, Japan) in drinking water over a period of 8 weeks. The treatment was started at the diestrus day 2 (D2) in postnatal age of 8 weeks. Blood for hormone assays was collected from the internal jugular vein under the isoflurane anesthesia between 6:00 and 7:30 on the day before treatment started (diestrus day 1 (D1)) and on D1 at the end of treatment in postnatal age of 16 weeks. Serum was harvested by centrifugation and stored at -20 °C until used for hormone assays. All experiments were conducted according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Committee of Animal Research of the Nippon Medical School, Tolyo, Japan (approval number: 25-142, 26-177).
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Tissue preparation Under deep anesthesia with sodium pentobarbital (30 mg/kg, ip) and hydrochloric acid medetomidine (0.5 mg/kg, ip), all rats were perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. All harvesting procedures were conducted at a similar time of day, between 13:30-15:00. Pituitary and thyroid glands were rapidly removed, washed, and weighed. Brains were also removed and post-fixed in the same fixative overnight at 4°C, then immersed into 20% sucrose in PB for several days for cryoprotection, snap-frozen in liquid hexane and stored at -80°C until used. Four series of serial coronal brain sections of a 40-μm thickness, containing hypothalamus, were prepared using a cryostat (Leica CM3050, Heidelberg, Germany) and stored in cryoprotectant solution at -20°C until processed for in situ hybridization and immunohistochemistry.
In situ hybridization In situ hybridization for Kiss1 mRNA was performed upon a series of sections as described previously (Higo et al., 2015). Anti-sense RNA probes were synthesized from template cDNA of rat full-length rat Kiss1 (GeneBank accession #AY196983) (Terao et al., 2004) using a DIG-RNA labeling kit (Roche Diagnostics, Mannheim, Germany). After hybridization, sections were rinsed and treated with RNaseA (40 µg/ml) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 500 nM NaCl for 20 min at 37°C. Sections were then washed under 6
conditions of increasing stringency. DIG-labeled RNA probes were visualized using the alkaline phosphatase conjugated anti-DIG antibody (Roche Diagnostics) and chromogen solution containing nitro blue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche Diagnostics) in Tris-HCl buffered saline (pH 9.5). Images of each section ware acquired using a BX-51 microscope equipped with a DP73 digital camera (Olympus, Tokyo, Japan). Kiss1 mRNA-expressing neurons in each section (ARC: 12-15 sections/rat, AVPV: 7-9 sections/rat) were counted using NIH ImageJ software with cell counter plugin.
Immunohistochemistry A series of coronal sections were used for immunohistochemistry for kisspeptin. Sections were washed three times in 0.1M phosphate-buffered saline (PBS). After incubation in blocking buffer (5% normal goat serum, 1% bovine serum albumin, 0.3% Triton-X and 0.02% NaN3 in PBS), sections were incubated with anti-kisspeptin monoclonal antibody (1:2,000, a kind gift from Takeda Pharmaceutical Co. Ltd, Tokyo, Japan) diluted in blocking buffer for 24 h at 4 °C. The specificity of this antibody has been previously demonstrated elsewhere (Kinoshita et al., 2005). Sections were washed with PBS and incubated with biotinylated anti-mouse IgG antibody (1:150, Vector Laboratories, Burlingame, CA, USA) for 90 min at room temperature. Immunoreactivity was visualized using an ABC elite kit (Vector Laboratories) 7
and diaminobenzidine (DAB; DAKO, Inc., Carpinteria, CA, USA). Sections were mounted on glass slides. They were dried then dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with Mount-Quick mounting medium (Daido Sangyo Co., Ltd, Tokyo, Japan). Image of each section (ARC: 12-15 sections/rat, AVPV: 7-9 sections/rat) were analyzed by the same procedure with in situ hybridization.
Hormone assays Serum concentrations of thyroxine (T4), thyroid stimulating hormone (TSH), LH, E2, and PRL were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers’ instructions. T4 and TSH levels were measured using rodent ELISA kits (ERKR7014 and ERKR7015, respectively, Endocrine technologies, Newark, CA, USA). As for T4, all samples were measured in duplicate and the intraassay coefficient of variation was 5.4%. As for TSH, samples were measured in duplicate or singulate. The intra- and interassay coefficients of variation were 3.0% and 3.8%, respectively. LH levels were measured in duplicate using a rat LH ELISA kit (AKRLH-010S, Shibayagi Co., Ltd., Gunma, Japan). The intraassay coefficient of variation was 5.1%. E2 levels were measured in duplicate using an E2 ELISA kit (#582251, Cayman, An Arbor, USA). The intraassay coefficient of variation was 3.1%. PRL levels were measured in duplicate or singulate using a rat PRL ELISA Kit 8
(CSB-E06881r, CUSABIO, Hubei, China). The intraassay coefficient of variation was 4.5%.
Statistical analysis All values were expressed as the mean ± standard error for the mean (SEM). Data were statistically analyzed by two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level.
Results Induction of hypothyroidism by administration of PTU In rats exposed to PTU for 8 weeks, serum T4 levels significantly decreased compared with those of control rats (Fig. 1A). On the other hand, serum TSH levels significantly increased in rats exposed to PTU (Fig. 1B) Changes in body weight were also observed during the administration of PTU: the body weight of PTU-treated female rats became significantly lower than that of control rats after 4 weeks of exposure to PTU (Fig. 1C). All control rats exhibited regular 4-day estrus cycles (Fig. 1D), whereas the PTU-treated rats showed irregular estrus cycles. In four out of seven PTU-treated rats, proestrus-like smear was not observed (Fig. 1E), and the other three PTU-treated rats showed prolonged estrus cycles with longer diestrus phase (Fig. 1F). 9
A significant increase in the weight of the thyroid gland, but not of pituitary, was also observed in the PTU-treated rats (Fig. 1G and H). All these data indicate that the PTU-administration successfully induced a model of hypothyroidism in our experimental design.
Changes in Kiss1 mRNA expression by administration of PTU Kiss1 mRNA-expressing neurons were observed in the ARC and in the AVPV of both control and PTU-treated rats (Fig. 2A-D). A significant reduction in the number of Kiss1 mRNA-expressing neurons in the ARC of PTU-treated rats was observed in comparison with that of control rats. By contrast, the numbers of Kiss1 mRNA-expressing neurons in the AVPV were not significantly different between control and PTU-treated rats (Fig. 2E).
Changes in kisspeptin immunoreactivity by administration of PTU Kisspeptin immunoreactivity was observed in the ARC and in the AVPV of both control and PTU-treated rats, but kisspeptin-immunoreactive neurons were scarce in the AVPV (Fig. 3A-D). A statistically significant decrease in the numbers of kisspeptin-immunoreactive neurons in the ARC of PTU-treated rats was observed, although no significant differences were observed in the number of kisspeptin-immunoreactive neurons in the AVPV between control and PTU-treated rats. These observations are similar to the findings of investigation of Kiss1 mRNA expression (Fig. 3E). 10
Effect of PTU administration on serum concentrations of LH, E2 and PRL. No significant differences in serum LH, E2 and levels were observed between control and PTU-treated rats (Fig. 4A and B). Although serum PRL levels were also not significantly different between control and PTU-treated rats, a significant increase in the serum PRL level was observed after administration of PTU (Fig. 4C).
Discussion Hypothyroidism is associated with dysregulation of reproductive system in women (Castañeda Cortés et al., 2014; Dittrich et al., 2011; Krassas et al., 2010). Here, we investigated the effects of hypothyroidism on kisspeptin neurons using adult female rats. In this study, hypothyroidism was induced by the administration of PTU in drinking water. Serum T4 levels significantly decreased in PTU-treated female rats, relative to those of control rats, and the weights of thyroid glands of these animals were significantly increased. Estrus cycles were irregular in PTU-treated rats. All of these data are in agreement with previous reports (Armada-Dias et al., 2001; Hatsuta et al., 2004; Tohei et al., 1998a) and indicate that hypothyroidism was successfully induced in our experimental model. Our data revealed that the numbers of Kiss1 mRNA-expressing and 11
kisspeptin-immunoreactive neurons in the ARC, but not in the AVPV, significantly decreased in rats with experimentally induced hypothyroidism compared with control rats. This result suggests that the dysregulation of reproductive functions in hypothyroidism is related to a decrease in kisspeptin signal in the hypothalamus. Based on the fact that kisspeptin expression is suppressed by E2 in the ARC, kisspeptin neurons in the ARC are supposed to play a key role in steroid negative feedback regulation and pulsatile release of gonadotropin (d'Anglemont de Tassigny and Colledge, 2010; Navarro and Tena-Sempere, 2012; Uenoyama et al., 2009). On the other hand, because E2 stimulates kisspeptin expression in the AVPV, kisspeptin neurons in the AVPV are considered to be important for steroid positive feedback regulation, and essential for the LH surge which induces ovulation (d'Anglemont de Tassigny and Colledge, 2010; Uenoyama et al., 2009). Our observation that suppression of kisspeptin expression in hypothyroid rats is limited to the ARC is in good agreement with the finding of irregular estrus cycles with clear estrus phase. The occurrence of estrus suggests that an ovulatory LH surge, which is dependent on the function of kisspeptin neurons in the AVPV, is conserved. Moreover, the presence of irregular and/or elongated estrus cycles suggests that the pulsatile release of LH is affected by the decrease in kisspeptin expression in the ARC. Despite suppression of kisspeptin expression in the ARC, no significant changes in serum LH or E2 levels were observed in hypothyroid rats. Our 12
finding that serum E2 levels of hypothyroid rats were comparable to those of control rats suggests that the observed reduction in Kiss1 mRNA expression and kisspeptin immunoreactivity in the ARC was not caused by the negative feedback of E2. In the present study, we investigated female rats on D1, the stage of the estrus cycle at which serum E2 levels are at their lowest level, and kisspeptin expression in the AVPV is minimal. This aspect of experimental design might explain why the kisspeptin expression in the AVPV was not significantly different between control and PTU-treated rats. Whether the suppression of kisspeptin expression is a direct or an indirect consequence of a decrease in thyroid hormone levels remains unclear. Although the expression of thyroid hormone receptors in kisspeptin neurons of rodents has not yet been examined, Dufourny et al. have reported that the thyroid hormone receptor (α isoform) is expressed in kisspeptin neurons in the hypothalamus of ewes (Dufourny et al., 2016), suggesting that thyroid hormones can directly affect kisspeptin neurons through the receptors. Besides, the expression of thyroid hormone receptor mRNA in GnRH neurons has also been reported in hamsters (Jansen et al., 1997; Kalló et al., 2012). Taken together, thyroid hormone might influence reproduction by direct action on both kisspeptin and GnRH neurons. The elevation of the serum PRL levels has been reported in hypothyroidism (Krassas, 2000; Krassas et al., 2010) and such elevation of PRL has shown to inhibit LH secretion (Cohen-Becker et al., 1986; Fox et al., 1987; Koike et al., 13
1984). Here, we also observed a significant increase in serum PRL level after PTU treatment. This elevation of serum PRL level in hypothyroid rats might be caused by elevated thyrotropin-releasing hormone levels following the decrease in thyroid hormone levels (Kanasaki et al., 2015; Tashjian et al., 1971). Inhibition of hypothalamic dopamine release, which has an inhibitory effect on PRL secretion, is another possible reason for the elevation of serum prolactin levels observed in this study. Kisspeptin neurons in the ARC project to tuberoinfudibular dopaminergic (TIDA) neurons, a hypothalamic dopamine population, and are involved in the regulation of the activity of these neurons (Sawai et al., 2012; Szawka et al., 2010). This observation suggests that suppression of kisspeptin expression in the ARC of hypothyroid rats can affect the activity of TIDA neurons. However, kisspeptin administration has been reported to inhibit TIDA neurons and results in a increase in PRL secretion (Ribeiro et al., 2015; Szawka et al., 2010). These reports are not in agreement with our observation that kisspeptin expression in ARC was suppressed and serum PRL levels were elevated. Whether any changes in signaling between kisspeptin neurons and TIDA neurons are induced by hypothyroidism is still unclear. Further investigations to clarify the involvement of kisspeptin neurons in the elevation of serum PRL levels in hypothyroidism are required. Kisspeptin neurons in both the ARC and AVPV have been reported to express PRL receptor (Kokay et al., 2011) and PRL has been shown to suppress kisspeptin expression in the ARC of ovariectomized rats (Araujo-Lopes et al., 14
2014). Thus, the elevation of serum PRL levels in hypothyroidism might explain the suppression of kisspeptin expression in the ARC. On the other hand, PRL has no effect on kisspeptin expression in the AVPV of ovariectomized rats (Araujo-Lopes et al., 2014). This is consistent with our result and suggests that, at least under the low E2 condition, kisspeptin expression in the AVPV is unaffected by elevated PRL levels. In conclusion, this study demonstrated a decrease in kisspeptin expression in the ARC of female rat model of hypothyroidism. These findings suggest that the insufficiency of thyroid hormones might directly and/or indirectly affect the activity of kisspeptin neurons, resulting in dysregulation of the reproductive system. This basic scientific information provides a greater understanding of the precise mechanisms of disturbance of reproductive functions in patients with hypothyroidism, and could be applied to the development of new clinical treatments.
Competing interests The authors declare that they have no competing interests
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Acknowledgments We are grateful to Takeda Pharmaceutical Company, Ltd. for kindly providing rat Kiss1 cDNA and anti-kisspeptin antibody. This work was supported by the Grant-in-Aid (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (Grant No, 26460323 to H.O. and 26670115 to N.I.; 15K20062 to S.H.; 16K19010 to K.T.).
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References Adachi, S., Yamada, S., Takatsu, Y., Matsui, H., Kinoshita, M., Takase, K., Sugiura, H., Ohtaki, T., Matsumoto, H., Uenoyama, Y., Tsukamura, H., Inoue, K.,
Maeda,
K.,
2007.
Involvement
of
anteroventral
periventricular
metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. J Reprod Dev 53, 367-378. Araujo-Lopes, R., Crampton, J.R., Aquino, N.S., Miranda, R.M., Kokay, I.C., Reis, A.M., Franci, C.R., Grattan, D.R., Szawka, R.E., 2014. Prolactin regulates kisspeptin neurons in the arcuate nucleus to suppress LH secretion in female rats. Endocrinology 155, 1010-1020. Armada-Dias, L., Carvalho, J.J., Breitenbach, M.M., Franci, C.R., Moura, E.G., 2001. Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes? Braz J Med Biol Res 34, 1209-1215. Bassett, J.H., Williams, G.R., 2016. Role of Thyroid Hormones in Skeletal Development and Bone Maintenance. Endocr Rev 37, 135-187. Binita, G., Suprava, P., Mainak, C., Koner, B.C., Alpana, S., 2009. Correlation of prolactin and thyroid hormone concentration with menstrual patterns in infertile women. J Reprod Infertil 10, 207-212. Castañeda Cortés, D.C., Langlois, V.S., Fernandino, J.I., 2014. Crossover of the hypothalamic pituitary-adrenal/interrenal, -thyroid, and -gonadal axes in testicular development. Front Endocrinol (Lausanne) 5, 139. 17
Cohen-Becker, I.R., Selmanoff, M., Wise, P.M., 1986. Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat. Neuroendocrinology 42, 328-333. d'Anglemont de Tassigny, X., Colledge, W.H., 2010. The role of kisspeptin signaling in reproduction. Physiology (Bethesda) 25, 207-217. Dezonne, R.S., Lima, F.R., Trentin, A.G., Gomes, F.C., 2015. Thyroid hormone and astroglia: endocrine control of the neural environment. J Neuroendocrinol 27, 435-445. Dittrich, R., Beckmann, M.W., Oppelt, P.G., Hoffmann, I., Lotz, L., Kuwert, T., Mueller, A., 2011. Thyroid hormone receptors and reproduction. J Reprod Immunol 90, 58-66. Dubois, S.L., Acosta-Martínez, M., DeJoseph, M.R., Wolfe, A., Radovick, S., Boehm, U., Urban, J.H., Levine, J.E., 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156, 1111-1120. Dufourny, L., Gennetay, D., Martinet, S., Lomet, D., Caraty, A., 2016. The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent. J Neuroendocrinol 28. Fox, S.R., Hoefer, M.T., Bartke, A., Smith, M.S., 1987. Suppression of pulsatile LH secretion, pituitary GnRH receptor content and pituitary responsiveness
to
GnRH
by
hyperprolactinemia
Neuroendocrinology 46, 350-359. 18
in
the
male
rat.
Hapon, M.B., Gamarra-Luques, C., Jahn, G.A., 2010. Short term hypothyroidism affects ovarian function in the cycling rat. Reprod Biol Endocrinol 8, 14. Hatsuta, M., Abe, K., Tamura, K., Ryuno, T., Watanabe, G., Taya, K., Kogo, H., 2004. Effects of hypothyroidism on the estrous cycle and reproductive hormones in mature female rat. Eur J Pharmacol 486, 343-348. Higo, S., Aikawa, S., Iijima, N., Ozawa, H., 2015. Rapid modulation of hypothalamic Kiss1 levels by the suckling stimulus in the lactating rat. J Endocrinol 227, 105-115. Iijima, N., Takumi, K., Sawai, N., Ozawa, H., 2011. An immunohistochemical study on the expressional dynamics of kisspeptin neurons relevant to GnRH neurons using a newly developed anti-kisspeptin antibody. J Mol Neurosci 43, 146-154. Jansen, H.T., Lubbers, L.S., Macchia, E., DeGroot, L.J., Lehman, M.N., 1997. Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons. Endocrinology 138, 5039-5047. Kalló, I., Mohácsik, P., Vida, B., Zeöld, A., Bardóczi, Z., Zavacki, A.M., Farkas, E., Kádár, A., Hrabovszky, E., Arrojo E Drigo, R., Dong, L., Barna, L., Palkovits, M., Borsay, B.A., Herczeg, L., Lechan, R.M., Bianco, A.C., Liposits, Z., Fekete, C., Gereben, B., 2012. A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons. PLoS One 19
7, e37860. Kanasaki, H., Oride, A., Mijiddorj, T., Kyo, S., 2015. Role of thyrotropin-releasing
hormone
in
prolactin-producing
cell
models.
Neuropeptides 54, 73-77. Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H., Uenoyama, Y., Iwata, K., Yamada, S., Inoue, K., Ohtaki, T., Matsumoto, H., Maeda, K., 2005. Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 146, 4431-4436. Koike, K., Aono, T., Miyake, A., Tasaka, K., Chatani, F., Kurachi, K., 1984. Effect of pituitary transplants on the LH-RH concentrations in the medial basal hypothalamus and hypophyseal portal blood. Brain Res 301, 253-258. Kokay, I.C., Petersen, S.L., Grattan, D.R., 2011. Identification of prolactin-sensitive GABA and kisspeptin neurons in regions of the rat hypothalamus involved in the control of fertility. Endocrinology 152, 526-535. Krassas, G.E., 2000. Thyroid disease and female reproduction. Fertil Steril 74, 1063-1070. Krassas, G.E., Poppe, K., Glinoer, D., 2010. Thyroid function and human reproductive health. Endocr Rev 31, 702-755. Lutsyk, A., Sogomonian, E., 2012. Structural, functional, and lectin histochemical characteristics of rat ovaries and endometrium in experimental hyper- and hypothyroidism. Folia Histochem Cytobiol 50, 331-339. 20
Maran, R.R., 2003. Thyroid hormones: their role in testicular steroidogenesis. Arch Androl 49, 375-388. Matsui, H., Takatsu, Y., Kumano, S., Matsumoto, H., Ohtaki, T., 2004. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 320, 383-388. Mattheij, J.A., Swarts, J.J., Lokerse, P., van Kampen, J.T., Van der Heide, D., 1995. Effect of hypothyroidism on the pituitary-gonadal axis in the adult female rat. J Endocrinol 146, 87-94. Mullur, R., Liu, Y.Y., Brent, G.A., 2014. Thyroid hormone regulation of metabolism. Physiol Rev 94, 355-382. Navarro, V.M., Castellano, J.M., Fernández-Fernández, R., Barreiro, M.L., Roa, J., Sanchez-Criado, J.E., Aguilar, E., Dieguez, C., Pinilla, L., Tena-Sempere, M., 2004. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 145, 4565-4574. Navarro, V.M., Castellano, J.M., Fernández-Fernández, R., Tovar, S., Roa, J., Mayen, A., Nogueiras, R., Vazquez, M.J., Barreiro, M.L., Magni, P., Aguilar, E., Dieguez, C., Pinilla, L., Tena-Sempere, M., 2005. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146, 156-163. Navarro, V.M., Castellano, J.M., McConkey, S.M., Pineda, R., Ruiz-Pino, F., 21
Pinilla, L., Clifton, D.K., Tena-Sempere, M., Steiner, R.A., 2011. Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in the female rat. Am J Physiol Endocrinol Metab 300, E202-210. Navarro, V.M., Tena-Sempere, M., 2012. Neuroendocrine control by kisspeptins: role in metabolic regulation of fertility. Nat Rev Endocrinol 8, 40-53. Oakley, A.E., Clifton, D.K., Steiner, R.A., 2009. Kisspeptin signaling in the brain. Endocr Rev 30, 713-743. Ribeiro, A.B., Leite, C.M., Kalil, B., Franci, C.R., Anselmo-Franci, J.A., Szawka, R.E., 2015. Kisspeptin regulates tuberoinfundibular dopaminergic neurones and prolactin secretion in an oestradiol-dependent manner in male and female rats. J Neuroendocrinol 27, 88-99. Sawai, N., Iijima, N., Takumi, K., Matsumoto, K., Ozawa, H., 2012. Immunofluorescent histochemical and ultrastructural studies on the innervation of kisspeptin/neurokinin B neurons to tuberoinfundibular dopaminergic neurons in the arcuate nucleus of rats. Neurosci Res 74, 10-16. Smith, J.T., Cunningham, M.J., Rissman, E.F., Clifton, D.K., Steiner, R.A., 2005. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146, 3686-3692. Smith, J.T., Popa, S.M., Clifton, D.K., Hoffman, G.E., Steiner, R.A., 2006. Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci 26, 6687-6694. 22
Szawka, R.E., Ribeiro, A.B., Leite, C.M., Helena, C.V., Franci, C.R., Anderson, G.M., Hoffman, G.E., Anselmo-Franci, J.A., 2010. Kisspeptin regulates prolactin release through hypothalamic dopaminergic neurons. Endocrinology 151, 3247-3257. Tamura, K., Hatsuta, M., Watanabe, G., Taya, K., Kogo, H., 1998. Blockage of gonadotropin-induced first ovulation caused by thyroidectomy and its possible mechanisms in rats. Am J Physiol 275, E380-385. Tashjian, A.H., Barowsky, N.J., Jensen, D.K., 1971. Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem Biophys Res Commun 43, 516-523. Terao, Y., Kumano, S., Takatsu, Y., Hattori, M., Nishimura, A., Ohtaki, T., Shintani, Y., 2004. Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. Biochim Biophys Acta 1678, 102-110. Tohei, A., 2004. Studies on the functional relationship between thyroid, adrenal and gonadal hormones. J Reprod Dev 50, 9-20. Tohei, A., Imai, A., Watanabe, G., Taya, K., 1998a. Influence of thiouracil-induced hypothyroidism on adrenal and gonadal functions in adult female rats. J Vet Med Sci 60, 439-446. Tohei, A., Watanabe, G., Taya, K., 1998b. Effects of thyroidectomy or thiouracil treatment on copulatory behavior in adult male rats. J Vet Med Sci 60, 281-285. 23
Toni, R., Della Casa, C., Castorina, S., Cocchi, D., Celotti, F., 2005. Effects of hypothyroidism and endocrine disruptor-dependent non-thyroidal illness syndrome on the GnRH-gonadotroph axis of the adult male rat. J Endocrinol Invest 28, 20-27. Uenoyama, Y., Tsukamura, H., Maeda, K.I., 2009. Kisspeptin/metastin: a key molecule
controlling
two
modes
of
gonadotrophin-releasing
hormone/luteinising hormone release in female rats. J Neuroendocrinol 21, 299-304. Yu, D., Zhou, H., Yang, Y., Jiang, Y., Wang, T., Lv, L., Zhou, Q., Dong, X., He, J., Huang, X., Chen, J., Wu, K., Xu, L., Mao, R., 2015. The bidirectional effects of hypothyroidism and hyperthyroidism on anxiety- and depression-like behaviors in rats. Horm Behav 69, 106-115.
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Figure legends Fig.1. Induction of hypothyroidism by the administration of propylthiouracil (PTU). (A, B) Changes in the serum T4 and TSH levels. (C) Changes in body weight. (D-F) Estrus cycles in control and PTU-treated rats. (D) Normal 4-day estrus cycles observed in a control rat. (E) Irregular estrus cycles without proestrus and (F) elongation of the estrus cycles observed in PTU-treated rats. (G, H) The weights of thyroid glands and pituitary. All values are means ± SEM. Statistical analysis was performed using Student’s t-test or two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level. Asterisks indicate significant differences between groups. Different letters represent significant differences within groups.
Fig.2. Kiss1 mRNA expression in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). Representative photomicrographs of Kiss1 mRNA-expressing neurons in the ARC and AVPV of control (A, B) and PTU-treated rats (C, D). 3V, the third ventricle, Scale bars = 200 μm. (E) The mean number of Kiss1 mRNA-expressing neurons in the ARC and AVPV. Student’s t-test was used for statistical analysis. Asterisks indicate significant differences between groups (P < 0.05).
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Fig.3. Kisspeptin immunoreactivity in the ARC and AVPV. Representative photomicrographs of kisspeptin-immunoreactive neurons in the ARC and AVPV of control (A, B) and PTU-treated rats (C, D). 3V, the third ventricle, Scale bars = 200 μm. (E) The mean number of kisspeptin-immunoreactive neurons in the ARC and AVPV. Student’s t-test was used for statistical analysis. Asterisks indicate significant differences between groups (P < 0.05).
Fig.4. Changes in the serum levels of luteinizing hormone (LH), estradiol (E2) and prolactin (PRL) after PTU treatment. Serum levels of LH (A), E2 (B), PRL (C). All values were expressed as mean ± SEM. Data were statistically analyzed by two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level. Asterisks indicate significant differences within groups.
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S0168-0102(16)30183-3 http://dx.doi.org/doi:10.1016/j.neures.2016.11.005 NSR 3995
To appear in:
Neuroscience Research
Received date: Accepted date:
6-10-2016 14-11-2016
Please cite this article as: Tomori, Yuji, Takumi, Ken, Iijima, Norio, Takai, Shinro, Ozawa, Hitoshi, Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles.Neuroscience Research http://dx.doi.org/10.1016/j.neures.2016.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles
Yuji Tomori1,2, Ken Takumi1, Norio Iijima1, Shinro Takai2, Hitoshi Ozawa1*
1
Department of Anatomy and Neurobiology, Graduate School of Medicine,
Nippon Medical School, Bunkyo-ku, Tokyo, Japan 2
Department of Orthopedic Surgery, Graduate School of Medicine, Nippon
Medical School, Bunkyo-ku, Tokyo, Japan
*Corresponding author Tel: +81-3-3822-2131 ext. 5299 Fax: +81-3-5685-6640 E-mail: [email protected] Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School,1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
1
Highlights ・ Irregular estrus cycles were observed in our rat model of hypothyroidism. ・ Kiss1 mRNA-expressing neurons in the ARC were decreased in hypothyroid rats. ・ Kisspeptin immunoreactive neurons in the ARC were decreased in hypothyroid rats.
Abstract Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.
Keywords: hypothyroidism; kisspeptin; propylthiouracil; thyroid hormone;
2
Introduction Thyroid hormones are known to be essential for the growth, development and metabolism of various tissues (Bassett and Williams, 2016; Dezonne et al., 2015; Maran, 2003; Mullur et al., 2014). In addition to these indispensable effects, thyroid hormones are also known to play an important role in reproductive function (Castañeda Cortés et al., 2014; Dittrich et al., 2011; Krassas et al., 2010; Tohei, 2004; Tohei et al., 1998a). Hypothyroidism, which refers to an insufficiency or absence of thyroid hormones, is associated with menstrual disorders and infertility in women (Krassas, 2000; Krassas et al., 2010). Impaired gonadotropin-releasing hormone (GnRH) biosynthesis (Tamura et al., 1998; Toni et al., 2005), diminished luteinizing hormone (LH) surge (Tamura et al., 1998; Tohei, 2004; Tohei et al., 1998a), irregular estrus cycles (Hapon et al., 2010; Lutsyk and Sogomonian, 2012; Mattheij et al., 1995; Tohei, 2004; Tohei et al., 1998a) and disorganized reproductive behavior (Tohei et al., 1998b; Yu et al., 2015) have all been reported in female rat models of hypothyroidism. Moreover, in hypothyroidism, elevated serum prolactin (PRL) is also observed (Binita et al., 2009; Krassas, 2000; Krassas et al., 2010). Elevation of circulating PRL has been known to inhibit the secretion of LH by primarily decreasing GnRH secretion or signaling (Cohen-Becker et al., 1986; Fox et al., 1987; Koike et al., 1984). Despite an established relationship between hypothyroidism and suppression of reproductive functions, the mechanism of this suppression remains unclear. 3
Kisspeptin, which is encoded by the Kiss1 gene, and its receptor GPR54 have been shown to have pivotal roles in the regulation of GnRH/LH release (d'Anglemont de Tassigny and Colledge, 2010; Iijima et al., 2011; Navarro et al., 2011; Oakley et al., 2009). Data from several studies have demonstrated that kisspeptin administration increases serum LH and follicle-stimulating hormone (FSH) levels through a GnRH dependent mechanism in rodents (Matsui et al., 2004; Navarro et al., 2004; Navarro et al., 2005). Moreover, it has been shown that kisspeptin has a potent role for steroid feedback regulation of GnRH release. Substantially all kisspeptin neurons in both the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) of the female rodents hypothalamus express estrogen receptor α (ERα) (Dubois et al., 2015; Smith et al., 2005; Smith et al., 2006). Estradiol (E2) inhibits Kiss1 mRNA expression in the ARC (Smith et al., 2005; Smith et al., 2006). Conversely, E2 stimulates the expression of Kiss1 mRNA in the AVPV of female rodents (Adachi et al., 2007; Smith et al., 2005; Smith et al., 2006). These studies suggest that ARC kisspeptin neurons are linked to negative feedback and the AVPV kisspeptin neurons are linked to positive feedback. Taken together, we hypothesized that dysregulation of gonadotropin release in hypothyroidism is brought by the alteration of the activity of kisspeptin neurons. Thus, in the present study, we aimed to analyze the effects of shortage of thyroid hormones on kisspeptin expression in the hypothalamus of female rats. 4
Materials and Methods Animals and Treatment Female Wistar-Imamichi rats (6 weeks of age) were purchased from the Institute for Animal Reproduction (Kasumigaura, Ibaraki, Japan), and housed under a 14-h light/10-h dark cycle (lights on at 06:00 h) and a constant temperature of 24±2 °C with free access to food and water. Vaginal smears were examined daily to monitor the estrus cycle. Following confirmation of the presence of regular 4 day estrus cycles for at least 2 weeks, rats were divided into 2 groups. One group (control, n=5) was given normal water. In the other group (propylthiouracil (PTU)-treated rats, n=7), hypothyroidism was induced by administration of 0.02% 6-propyl-2-thiouracil (Wako, Osaka, Japan) in drinking water over a period of 8 weeks. The treatment was started at the diestrus day 2 (D2) in postnatal age of 8 weeks. Blood for hormone assays was collected from the internal jugular vein under the isoflurane anesthesia between 6:00 and 7:30 on the day before treatment started (diestrus day 1 (D1)) and on D1 at the end of treatment in postnatal age of 16 weeks. Serum was harvested by centrifugation and stored at -20 °C until used for hormone assays. All experiments were conducted according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Committee of Animal Research of the Nippon Medical School, Tolyo, Japan (approval number: 25-142, 26-177).
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Tissue preparation Under deep anesthesia with sodium pentobarbital (30 mg/kg, ip) and hydrochloric acid medetomidine (0.5 mg/kg, ip), all rats were perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. All harvesting procedures were conducted at a similar time of day, between 13:30-15:00. Pituitary and thyroid glands were rapidly removed, washed, and weighed. Brains were also removed and post-fixed in the same fixative overnight at 4°C, then immersed into 20% sucrose in PB for several days for cryoprotection, snap-frozen in liquid hexane and stored at -80°C until used. Four series of serial coronal brain sections of a 40-μm thickness, containing hypothalamus, were prepared using a cryostat (Leica CM3050, Heidelberg, Germany) and stored in cryoprotectant solution at -20°C until processed for in situ hybridization and immunohistochemistry.
In situ hybridization In situ hybridization for Kiss1 mRNA was performed upon a series of sections as described previously (Higo et al., 2015). Anti-sense RNA probes were synthesized from template cDNA of rat full-length rat Kiss1 (GeneBank accession #AY196983) (Terao et al., 2004) using a DIG-RNA labeling kit (Roche Diagnostics, Mannheim, Germany). After hybridization, sections were rinsed and treated with RNaseA (40 µg/ml) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 500 nM NaCl for 20 min at 37°C. Sections were then washed under 6
conditions of increasing stringency. DIG-labeled RNA probes were visualized using the alkaline phosphatase conjugated anti-DIG antibody (Roche Diagnostics) and chromogen solution containing nitro blue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche Diagnostics) in Tris-HCl buffered saline (pH 9.5). Images of each section ware acquired using a BX-51 microscope equipped with a DP73 digital camera (Olympus, Tokyo, Japan). Kiss1 mRNA-expressing neurons in each section (ARC: 12-15 sections/rat, AVPV: 7-9 sections/rat) were counted using NIH ImageJ software with cell counter plugin.
Immunohistochemistry A series of coronal sections were used for immunohistochemistry for kisspeptin. Sections were washed three times in 0.1M phosphate-buffered saline (PBS). After incubation in blocking buffer (5% normal goat serum, 1% bovine serum albumin, 0.3% Triton-X and 0.02% NaN3 in PBS), sections were incubated with anti-kisspeptin monoclonal antibody (1:2,000, a kind gift from Takeda Pharmaceutical Co. Ltd, Tokyo, Japan) diluted in blocking buffer for 24 h at 4 °C. The specificity of this antibody has been previously demonstrated elsewhere (Kinoshita et al., 2005). Sections were washed with PBS and incubated with biotinylated anti-mouse IgG antibody (1:150, Vector Laboratories, Burlingame, CA, USA) for 90 min at room temperature. Immunoreactivity was visualized using an ABC elite kit (Vector Laboratories) 7
and diaminobenzidine (DAB; DAKO, Inc., Carpinteria, CA, USA). Sections were mounted on glass slides. They were dried then dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with Mount-Quick mounting medium (Daido Sangyo Co., Ltd, Tokyo, Japan). Image of each section (ARC: 12-15 sections/rat, AVPV: 7-9 sections/rat) were analyzed by the same procedure with in situ hybridization.
Hormone assays Serum concentrations of thyroxine (T4), thyroid stimulating hormone (TSH), LH, E2, and PRL were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers’ instructions. T4 and TSH levels were measured using rodent ELISA kits (ERKR7014 and ERKR7015, respectively, Endocrine technologies, Newark, CA, USA). As for T4, all samples were measured in duplicate and the intraassay coefficient of variation was 5.4%. As for TSH, samples were measured in duplicate or singulate. The intra- and interassay coefficients of variation were 3.0% and 3.8%, respectively. LH levels were measured in duplicate using a rat LH ELISA kit (AKRLH-010S, Shibayagi Co., Ltd., Gunma, Japan). The intraassay coefficient of variation was 5.1%. E2 levels were measured in duplicate using an E2 ELISA kit (#582251, Cayman, An Arbor, USA). The intraassay coefficient of variation was 3.1%. PRL levels were measured in duplicate or singulate using a rat PRL ELISA Kit 8
(CSB-E06881r, CUSABIO, Hubei, China). The intraassay coefficient of variation was 4.5%.
Statistical analysis All values were expressed as the mean ± standard error for the mean (SEM). Data were statistically analyzed by two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level.
Results Induction of hypothyroidism by administration of PTU In rats exposed to PTU for 8 weeks, serum T4 levels significantly decreased compared with those of control rats (Fig. 1A). On the other hand, serum TSH levels significantly increased in rats exposed to PTU (Fig. 1B) Changes in body weight were also observed during the administration of PTU: the body weight of PTU-treated female rats became significantly lower than that of control rats after 4 weeks of exposure to PTU (Fig. 1C). All control rats exhibited regular 4-day estrus cycles (Fig. 1D), whereas the PTU-treated rats showed irregular estrus cycles. In four out of seven PTU-treated rats, proestrus-like smear was not observed (Fig. 1E), and the other three PTU-treated rats showed prolonged estrus cycles with longer diestrus phase (Fig. 1F). 9
A significant increase in the weight of the thyroid gland, but not of pituitary, was also observed in the PTU-treated rats (Fig. 1G and H). All these data indicate that the PTU-administration successfully induced a model of hypothyroidism in our experimental design.
Changes in Kiss1 mRNA expression by administration of PTU Kiss1 mRNA-expressing neurons were observed in the ARC and in the AVPV of both control and PTU-treated rats (Fig. 2A-D). A significant reduction in the number of Kiss1 mRNA-expressing neurons in the ARC of PTU-treated rats was observed in comparison with that of control rats. By contrast, the numbers of Kiss1 mRNA-expressing neurons in the AVPV were not significantly different between control and PTU-treated rats (Fig. 2E).
Changes in kisspeptin immunoreactivity by administration of PTU Kisspeptin immunoreactivity was observed in the ARC and in the AVPV of both control and PTU-treated rats, but kisspeptin-immunoreactive neurons were scarce in the AVPV (Fig. 3A-D). A statistically significant decrease in the numbers of kisspeptin-immunoreactive neurons in the ARC of PTU-treated rats was observed, although no significant differences were observed in the number of kisspeptin-immunoreactive neurons in the AVPV between control and PTU-treated rats. These observations are similar to the findings of investigation of Kiss1 mRNA expression (Fig. 3E). 10
Effect of PTU administration on serum concentrations of LH, E2 and PRL. No significant differences in serum LH, E2 and levels were observed between control and PTU-treated rats (Fig. 4A and B). Although serum PRL levels were also not significantly different between control and PTU-treated rats, a significant increase in the serum PRL level was observed after administration of PTU (Fig. 4C).
Discussion Hypothyroidism is associated with dysregulation of reproductive system in women (Castañeda Cortés et al., 2014; Dittrich et al., 2011; Krassas et al., 2010). Here, we investigated the effects of hypothyroidism on kisspeptin neurons using adult female rats. In this study, hypothyroidism was induced by the administration of PTU in drinking water. Serum T4 levels significantly decreased in PTU-treated female rats, relative to those of control rats, and the weights of thyroid glands of these animals were significantly increased. Estrus cycles were irregular in PTU-treated rats. All of these data are in agreement with previous reports (Armada-Dias et al., 2001; Hatsuta et al., 2004; Tohei et al., 1998a) and indicate that hypothyroidism was successfully induced in our experimental model. Our data revealed that the numbers of Kiss1 mRNA-expressing and 11
kisspeptin-immunoreactive neurons in the ARC, but not in the AVPV, significantly decreased in rats with experimentally induced hypothyroidism compared with control rats. This result suggests that the dysregulation of reproductive functions in hypothyroidism is related to a decrease in kisspeptin signal in the hypothalamus. Based on the fact that kisspeptin expression is suppressed by E2 in the ARC, kisspeptin neurons in the ARC are supposed to play a key role in steroid negative feedback regulation and pulsatile release of gonadotropin (d'Anglemont de Tassigny and Colledge, 2010; Navarro and Tena-Sempere, 2012; Uenoyama et al., 2009). On the other hand, because E2 stimulates kisspeptin expression in the AVPV, kisspeptin neurons in the AVPV are considered to be important for steroid positive feedback regulation, and essential for the LH surge which induces ovulation (d'Anglemont de Tassigny and Colledge, 2010; Uenoyama et al., 2009). Our observation that suppression of kisspeptin expression in hypothyroid rats is limited to the ARC is in good agreement with the finding of irregular estrus cycles with clear estrus phase. The occurrence of estrus suggests that an ovulatory LH surge, which is dependent on the function of kisspeptin neurons in the AVPV, is conserved. Moreover, the presence of irregular and/or elongated estrus cycles suggests that the pulsatile release of LH is affected by the decrease in kisspeptin expression in the ARC. Despite suppression of kisspeptin expression in the ARC, no significant changes in serum LH or E2 levels were observed in hypothyroid rats. Our 12
finding that serum E2 levels of hypothyroid rats were comparable to those of control rats suggests that the observed reduction in Kiss1 mRNA expression and kisspeptin immunoreactivity in the ARC was not caused by the negative feedback of E2. In the present study, we investigated female rats on D1, the stage of the estrus cycle at which serum E2 levels are at their lowest level, and kisspeptin expression in the AVPV is minimal. This aspect of experimental design might explain why the kisspeptin expression in the AVPV was not significantly different between control and PTU-treated rats. Whether the suppression of kisspeptin expression is a direct or an indirect consequence of a decrease in thyroid hormone levels remains unclear. Although the expression of thyroid hormone receptors in kisspeptin neurons of rodents has not yet been examined, Dufourny et al. have reported that the thyroid hormone receptor (α isoform) is expressed in kisspeptin neurons in the hypothalamus of ewes (Dufourny et al., 2016), suggesting that thyroid hormones can directly affect kisspeptin neurons through the receptors. Besides, the expression of thyroid hormone receptor mRNA in GnRH neurons has also been reported in hamsters (Jansen et al., 1997; Kalló et al., 2012). Taken together, thyroid hormone might influence reproduction by direct action on both kisspeptin and GnRH neurons. The elevation of the serum PRL levels has been reported in hypothyroidism (Krassas, 2000; Krassas et al., 2010) and such elevation of PRL has shown to inhibit LH secretion (Cohen-Becker et al., 1986; Fox et al., 1987; Koike et al., 13
1984). Here, we also observed a significant increase in serum PRL level after PTU treatment. This elevation of serum PRL level in hypothyroid rats might be caused by elevated thyrotropin-releasing hormone levels following the decrease in thyroid hormone levels (Kanasaki et al., 2015; Tashjian et al., 1971). Inhibition of hypothalamic dopamine release, which has an inhibitory effect on PRL secretion, is another possible reason for the elevation of serum prolactin levels observed in this study. Kisspeptin neurons in the ARC project to tuberoinfudibular dopaminergic (TIDA) neurons, a hypothalamic dopamine population, and are involved in the regulation of the activity of these neurons (Sawai et al., 2012; Szawka et al., 2010). This observation suggests that suppression of kisspeptin expression in the ARC of hypothyroid rats can affect the activity of TIDA neurons. However, kisspeptin administration has been reported to inhibit TIDA neurons and results in a increase in PRL secretion (Ribeiro et al., 2015; Szawka et al., 2010). These reports are not in agreement with our observation that kisspeptin expression in ARC was suppressed and serum PRL levels were elevated. Whether any changes in signaling between kisspeptin neurons and TIDA neurons are induced by hypothyroidism is still unclear. Further investigations to clarify the involvement of kisspeptin neurons in the elevation of serum PRL levels in hypothyroidism are required. Kisspeptin neurons in both the ARC and AVPV have been reported to express PRL receptor (Kokay et al., 2011) and PRL has been shown to suppress kisspeptin expression in the ARC of ovariectomized rats (Araujo-Lopes et al., 14
2014). Thus, the elevation of serum PRL levels in hypothyroidism might explain the suppression of kisspeptin expression in the ARC. On the other hand, PRL has no effect on kisspeptin expression in the AVPV of ovariectomized rats (Araujo-Lopes et al., 2014). This is consistent with our result and suggests that, at least under the low E2 condition, kisspeptin expression in the AVPV is unaffected by elevated PRL levels. In conclusion, this study demonstrated a decrease in kisspeptin expression in the ARC of female rat model of hypothyroidism. These findings suggest that the insufficiency of thyroid hormones might directly and/or indirectly affect the activity of kisspeptin neurons, resulting in dysregulation of the reproductive system. This basic scientific information provides a greater understanding of the precise mechanisms of disturbance of reproductive functions in patients with hypothyroidism, and could be applied to the development of new clinical treatments.
Competing interests The authors declare that they have no competing interests
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Acknowledgments We are grateful to Takeda Pharmaceutical Company, Ltd. for kindly providing rat Kiss1 cDNA and anti-kisspeptin antibody. This work was supported by the Grant-in-Aid (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (Grant No, 26460323 to H.O. and 26670115 to N.I.; 15K20062 to S.H.; 16K19010 to K.T.).
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References Adachi, S., Yamada, S., Takatsu, Y., Matsui, H., Kinoshita, M., Takase, K., Sugiura, H., Ohtaki, T., Matsumoto, H., Uenoyama, Y., Tsukamura, H., Inoue, K.,
Maeda,
K.,
2007.
Involvement
of
anteroventral
periventricular
metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. J Reprod Dev 53, 367-378. Araujo-Lopes, R., Crampton, J.R., Aquino, N.S., Miranda, R.M., Kokay, I.C., Reis, A.M., Franci, C.R., Grattan, D.R., Szawka, R.E., 2014. Prolactin regulates kisspeptin neurons in the arcuate nucleus to suppress LH secretion in female rats. Endocrinology 155, 1010-1020. Armada-Dias, L., Carvalho, J.J., Breitenbach, M.M., Franci, C.R., Moura, E.G., 2001. Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes? Braz J Med Biol Res 34, 1209-1215. Bassett, J.H., Williams, G.R., 2016. Role of Thyroid Hormones in Skeletal Development and Bone Maintenance. Endocr Rev 37, 135-187. Binita, G., Suprava, P., Mainak, C., Koner, B.C., Alpana, S., 2009. Correlation of prolactin and thyroid hormone concentration with menstrual patterns in infertile women. J Reprod Infertil 10, 207-212. Castañeda Cortés, D.C., Langlois, V.S., Fernandino, J.I., 2014. Crossover of the hypothalamic pituitary-adrenal/interrenal, -thyroid, and -gonadal axes in testicular development. Front Endocrinol (Lausanne) 5, 139. 17
Cohen-Becker, I.R., Selmanoff, M., Wise, P.M., 1986. Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat. Neuroendocrinology 42, 328-333. d'Anglemont de Tassigny, X., Colledge, W.H., 2010. The role of kisspeptin signaling in reproduction. Physiology (Bethesda) 25, 207-217. Dezonne, R.S., Lima, F.R., Trentin, A.G., Gomes, F.C., 2015. Thyroid hormone and astroglia: endocrine control of the neural environment. J Neuroendocrinol 27, 435-445. Dittrich, R., Beckmann, M.W., Oppelt, P.G., Hoffmann, I., Lotz, L., Kuwert, T., Mueller, A., 2011. Thyroid hormone receptors and reproduction. J Reprod Immunol 90, 58-66. Dubois, S.L., Acosta-Martínez, M., DeJoseph, M.R., Wolfe, A., Radovick, S., Boehm, U., Urban, J.H., Levine, J.E., 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156, 1111-1120. Dufourny, L., Gennetay, D., Martinet, S., Lomet, D., Caraty, A., 2016. The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent. J Neuroendocrinol 28. Fox, S.R., Hoefer, M.T., Bartke, A., Smith, M.S., 1987. Suppression of pulsatile LH secretion, pituitary GnRH receptor content and pituitary responsiveness
to
GnRH
by
hyperprolactinemia
Neuroendocrinology 46, 350-359. 18
in
the
male
rat.
Hapon, M.B., Gamarra-Luques, C., Jahn, G.A., 2010. Short term hypothyroidism affects ovarian function in the cycling rat. Reprod Biol Endocrinol 8, 14. Hatsuta, M., Abe, K., Tamura, K., Ryuno, T., Watanabe, G., Taya, K., Kogo, H., 2004. Effects of hypothyroidism on the estrous cycle and reproductive hormones in mature female rat. Eur J Pharmacol 486, 343-348. Higo, S., Aikawa, S., Iijima, N., Ozawa, H., 2015. Rapid modulation of hypothalamic Kiss1 levels by the suckling stimulus in the lactating rat. J Endocrinol 227, 105-115. Iijima, N., Takumi, K., Sawai, N., Ozawa, H., 2011. An immunohistochemical study on the expressional dynamics of kisspeptin neurons relevant to GnRH neurons using a newly developed anti-kisspeptin antibody. J Mol Neurosci 43, 146-154. Jansen, H.T., Lubbers, L.S., Macchia, E., DeGroot, L.J., Lehman, M.N., 1997. Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons. Endocrinology 138, 5039-5047. Kalló, I., Mohácsik, P., Vida, B., Zeöld, A., Bardóczi, Z., Zavacki, A.M., Farkas, E., Kádár, A., Hrabovszky, E., Arrojo E Drigo, R., Dong, L., Barna, L., Palkovits, M., Borsay, B.A., Herczeg, L., Lechan, R.M., Bianco, A.C., Liposits, Z., Fekete, C., Gereben, B., 2012. A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons. PLoS One 19
7, e37860. Kanasaki, H., Oride, A., Mijiddorj, T., Kyo, S., 2015. Role of thyrotropin-releasing
hormone
in
prolactin-producing
cell
models.
Neuropeptides 54, 73-77. Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H., Uenoyama, Y., Iwata, K., Yamada, S., Inoue, K., Ohtaki, T., Matsumoto, H., Maeda, K., 2005. Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 146, 4431-4436. Koike, K., Aono, T., Miyake, A., Tasaka, K., Chatani, F., Kurachi, K., 1984. Effect of pituitary transplants on the LH-RH concentrations in the medial basal hypothalamus and hypophyseal portal blood. Brain Res 301, 253-258. Kokay, I.C., Petersen, S.L., Grattan, D.R., 2011. Identification of prolactin-sensitive GABA and kisspeptin neurons in regions of the rat hypothalamus involved in the control of fertility. Endocrinology 152, 526-535. Krassas, G.E., 2000. Thyroid disease and female reproduction. Fertil Steril 74, 1063-1070. Krassas, G.E., Poppe, K., Glinoer, D., 2010. Thyroid function and human reproductive health. Endocr Rev 31, 702-755. Lutsyk, A., Sogomonian, E., 2012. Structural, functional, and lectin histochemical characteristics of rat ovaries and endometrium in experimental hyper- and hypothyroidism. Folia Histochem Cytobiol 50, 331-339. 20
Maran, R.R., 2003. Thyroid hormones: their role in testicular steroidogenesis. Arch Androl 49, 375-388. Matsui, H., Takatsu, Y., Kumano, S., Matsumoto, H., Ohtaki, T., 2004. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 320, 383-388. Mattheij, J.A., Swarts, J.J., Lokerse, P., van Kampen, J.T., Van der Heide, D., 1995. Effect of hypothyroidism on the pituitary-gonadal axis in the adult female rat. J Endocrinol 146, 87-94. Mullur, R., Liu, Y.Y., Brent, G.A., 2014. Thyroid hormone regulation of metabolism. Physiol Rev 94, 355-382. Navarro, V.M., Castellano, J.M., Fernández-Fernández, R., Barreiro, M.L., Roa, J., Sanchez-Criado, J.E., Aguilar, E., Dieguez, C., Pinilla, L., Tena-Sempere, M., 2004. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 145, 4565-4574. Navarro, V.M., Castellano, J.M., Fernández-Fernández, R., Tovar, S., Roa, J., Mayen, A., Nogueiras, R., Vazquez, M.J., Barreiro, M.L., Magni, P., Aguilar, E., Dieguez, C., Pinilla, L., Tena-Sempere, M., 2005. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146, 156-163. Navarro, V.M., Castellano, J.M., McConkey, S.M., Pineda, R., Ruiz-Pino, F., 21
Pinilla, L., Clifton, D.K., Tena-Sempere, M., Steiner, R.A., 2011. Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in the female rat. Am J Physiol Endocrinol Metab 300, E202-210. Navarro, V.M., Tena-Sempere, M., 2012. Neuroendocrine control by kisspeptins: role in metabolic regulation of fertility. Nat Rev Endocrinol 8, 40-53. Oakley, A.E., Clifton, D.K., Steiner, R.A., 2009. Kisspeptin signaling in the brain. Endocr Rev 30, 713-743. Ribeiro, A.B., Leite, C.M., Kalil, B., Franci, C.R., Anselmo-Franci, J.A., Szawka, R.E., 2015. Kisspeptin regulates tuberoinfundibular dopaminergic neurones and prolactin secretion in an oestradiol-dependent manner in male and female rats. J Neuroendocrinol 27, 88-99. Sawai, N., Iijima, N., Takumi, K., Matsumoto, K., Ozawa, H., 2012. Immunofluorescent histochemical and ultrastructural studies on the innervation of kisspeptin/neurokinin B neurons to tuberoinfundibular dopaminergic neurons in the arcuate nucleus of rats. Neurosci Res 74, 10-16. Smith, J.T., Cunningham, M.J., Rissman, E.F., Clifton, D.K., Steiner, R.A., 2005. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146, 3686-3692. Smith, J.T., Popa, S.M., Clifton, D.K., Hoffman, G.E., Steiner, R.A., 2006. Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci 26, 6687-6694. 22
Szawka, R.E., Ribeiro, A.B., Leite, C.M., Helena, C.V., Franci, C.R., Anderson, G.M., Hoffman, G.E., Anselmo-Franci, J.A., 2010. Kisspeptin regulates prolactin release through hypothalamic dopaminergic neurons. Endocrinology 151, 3247-3257. Tamura, K., Hatsuta, M., Watanabe, G., Taya, K., Kogo, H., 1998. Blockage of gonadotropin-induced first ovulation caused by thyroidectomy and its possible mechanisms in rats. Am J Physiol 275, E380-385. Tashjian, A.H., Barowsky, N.J., Jensen, D.K., 1971. Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem Biophys Res Commun 43, 516-523. Terao, Y., Kumano, S., Takatsu, Y., Hattori, M., Nishimura, A., Ohtaki, T., Shintani, Y., 2004. Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta. Biochim Biophys Acta 1678, 102-110. Tohei, A., 2004. Studies on the functional relationship between thyroid, adrenal and gonadal hormones. J Reprod Dev 50, 9-20. Tohei, A., Imai, A., Watanabe, G., Taya, K., 1998a. Influence of thiouracil-induced hypothyroidism on adrenal and gonadal functions in adult female rats. J Vet Med Sci 60, 439-446. Tohei, A., Watanabe, G., Taya, K., 1998b. Effects of thyroidectomy or thiouracil treatment on copulatory behavior in adult male rats. J Vet Med Sci 60, 281-285. 23
Toni, R., Della Casa, C., Castorina, S., Cocchi, D., Celotti, F., 2005. Effects of hypothyroidism and endocrine disruptor-dependent non-thyroidal illness syndrome on the GnRH-gonadotroph axis of the adult male rat. J Endocrinol Invest 28, 20-27. Uenoyama, Y., Tsukamura, H., Maeda, K.I., 2009. Kisspeptin/metastin: a key molecule
controlling
two
modes
of
gonadotrophin-releasing
hormone/luteinising hormone release in female rats. J Neuroendocrinol 21, 299-304. Yu, D., Zhou, H., Yang, Y., Jiang, Y., Wang, T., Lv, L., Zhou, Q., Dong, X., He, J., Huang, X., Chen, J., Wu, K., Xu, L., Mao, R., 2015. The bidirectional effects of hypothyroidism and hyperthyroidism on anxiety- and depression-like behaviors in rats. Horm Behav 69, 106-115.
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Figure legends Fig.1. Induction of hypothyroidism by the administration of propylthiouracil (PTU). (A, B) Changes in the serum T4 and TSH levels. (C) Changes in body weight. (D-F) Estrus cycles in control and PTU-treated rats. (D) Normal 4-day estrus cycles observed in a control rat. (E) Irregular estrus cycles without proestrus and (F) elongation of the estrus cycles observed in PTU-treated rats. (G, H) The weights of thyroid glands and pituitary. All values are means ± SEM. Statistical analysis was performed using Student’s t-test or two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level. Asterisks indicate significant differences between groups. Different letters represent significant differences within groups.
Fig.2. Kiss1 mRNA expression in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). Representative photomicrographs of Kiss1 mRNA-expressing neurons in the ARC and AVPV of control (A, B) and PTU-treated rats (C, D). 3V, the third ventricle, Scale bars = 200 μm. (E) The mean number of Kiss1 mRNA-expressing neurons in the ARC and AVPV. Student’s t-test was used for statistical analysis. Asterisks indicate significant differences between groups (P < 0.05).
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Fig.3. Kisspeptin immunoreactivity in the ARC and AVPV. Representative photomicrographs of kisspeptin-immunoreactive neurons in the ARC and AVPV of control (A, B) and PTU-treated rats (C, D). 3V, the third ventricle, Scale bars = 200 μm. (E) The mean number of kisspeptin-immunoreactive neurons in the ARC and AVPV. Student’s t-test was used for statistical analysis. Asterisks indicate significant differences between groups (P < 0.05).
Fig.4. Changes in the serum levels of luteinizing hormone (LH), estradiol (E2) and prolactin (PRL) after PTU treatment. Serum levels of LH (A), E2 (B), PRL (C). All values were expressed as mean ± SEM. Data were statistically analyzed by two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. Differences were considered significant at the P < 0.05 level. Asterisks indicate significant differences within groups.
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