The PT seemed well located to mediate photoperiodic switches in *Dio2*–*Dio3* usage. To decipher the mechanism of the photoperiodic response, Yoshimura and colleagues (91) set out an ambitious experimental set-up: hypothalamic blocks containing the MBH and PT/median eminence from quails submitted to a long-day transfer, known to activate the gonadotropic axis within 24 h, were used for hybridization on a chicken gene chip. This revealed that the expression of two genes, *Tsh*β and *Eya3*, is rapidly triggered by the transfer from SP to LP. A second wave of transcriptional changes was also observed for a handful of genes including *Dio2* and *Dio3*, which displayed acute and simultaneous induction and repression, respectively. Crucially, expression of the cognate TSH receptor (TSHR) was found in tanycytes, which express the deiodinases, providing the link between TSH output from the PT and T3 regulation within the MBH. The pathway was uncovered using an acute intracerebroventricular injection of TSH to SP-exposed quails, which induced *Dio2* expression and led to gonadal recrudescence.

In a contemporaneous study in sheep, Hanon et al. (92) suggested this mechanism to be ancestral, since their data were similar in many respects: higher *Tsh*β expression within the PT under LP than SP (see **Figure 3A**), expression of the TSHR within tanycytes and PT/median eminence, higher *Dio2* expression under LP than SP (see **Figure 3A**), and TSH-dependent induction of *Dio2* both *in vitro* and *in vivo*. The latter finding was not unexpected, since TSHR signals through a Gs protein, and *Dio2* is a cAMPresponsive gene (93). In contrast, the MT1 receptor couples to a Gi protein and the interplay between TSHR and MT1 signaling within the PT may be part of the photoperiod decoding mechanism, at least in sheep (92, 94, 95). Under LP, the PT therefore functions as an "indirect T3-generator," disconnected from both TRH and T3 feedback (see above).

Since these studies in quail and sheep, a similar TSH/deiodinases/T3 retrograde pathway (from the pituitary back to the hypothalamus, **Figure 2**) has been described not only in other photoperiodic species such as the European hamster (96), the Syrian hamster (97), the Siberian hamster (89), the common vole (98), but also in photoresponsive juvenile Fisher 344 rats (99) and in a melatonin-producing but non-photoperiodic CBA/N

hence the phase-relationship relative to Cry1 expression (a melatonininduced circadian gene) is similar irrespective of the photoperiod but Eya3 transcription increases only under LP as melatonin inhibition is relieved. **(C)** Schematics of the transcriptional control of the Tshβ gene by TEF/ SIX1/EYA3. Note that EYA3 levels are higher under LP than SP.

mouse strain (22, 100, 101). Photoperiodic variations in *Dio2* expression were however not observed in the non-photoperiodic Wistar rat (88). The use of murine knock-out strains confirmed that the MT1 melatonin receptor (22) and TSHR (100) are mandatory for the LP induction of *Dio2* expression within tanycytes. Whether this pathway is present in all vertebrates remains to be determined (102).

As mentioned before, fish species investigated thus far do not have a distinct PT, but in masu salmon a TSH/DIO2 axis implicating the *saccus vasculosus*, located below the hypothalamus and caudally to the pituitary gland, has been proposed (103). However, the *saccus vasculosus* is absent in several species of fish such as the pike (104), which is nonetheless photoperiodic (105). The other few studies on this matter in fish have yielded varied outcomes (106, 107). Regarding birds, studies in tits (108) and starlings (109) did not lend clear support to the model, but aspects of the experimental set-up prevent any conclusion to be drawn. For example, the studies of starlings were carried out in outdoor aviaries, so effects of fluctuating temperature on the peripheral thyroid axis may have obscured the photoperiodic regulation of DIO2 and DIO3 centrally. Finally, we are not aware of any study on this matter in either reptiles or amphibians.

#### **ENCODING AND DECODING THE PHOTOPERIODIC MESSAGE UPSTREAM OF THE PT**

Birds and mammals possess a similar mechanism to respond to photoperiod, but they perceive the photoperiodic message in different ways. In mammals, light is exclusively perceived by the retina, with a key role for ganglion cells expressing the photopigment melanopsin [for review, see Ref. (110)]. This information is relayed to the circadian clock of the SCN, which governs melatonin production by the pineal gland through a multi-synaptic sympathetic pathway. Melatonin is the mandatory messenger of photoperiod in mammals. In striking contrast, removing the eyes and suppressing melatonin by pinealectomy does not disrupt photoperiodism in birds [for reviews, see Ref. (44, 45, 111–113)]. In birds, light goes through the skull and acts directly upon hypothalamic deep-brain photoreceptors to control seasonal reproduction (**Figure 2**). Several photopigments expressed by different cell types, all located within the MBH and projecting to the PT/median eminence, are plausible candidates: VA-opsin (114), neuropsin [Opn5, Ref. (115, 116)], and melanopsin [Opn4, Ref. (117)]. The neurotransmitter(s) and/or neuropeptide(s) used by these cells, and how they impinge on PT thyrotropes, remain to be elucidated.

#### **WITHIN THE PT: FROM THE CIRCADIAN CLOCK TO THE SEASONAL OUTPUT**

Photoperiodic species such as quail (118) and Siberian and Syrian hamsters (119, 120) measure photoperiod length with remarkable accuracy. In these three species, reproduction switches off when the photoperiod is shorter than 12.5 h. The narrow photoperiod range over which physiological changes occur is one of the lines of evidence implicating some sort of daily timing device. The concept that circadian clock(s), clocks with a period of about 24 h, control seasonal timing is indeed not novel (120, 121).

The genetic and molecular bases and organization of circadian clocks have been recently identified (122–124). These clocks are not only located within the SCN, but are present in virtually every tissue and cell where they impact "local" physiology. The PT is no exception as it expresses a full set of clock genes and displays persistent circadian rhythmicity *in vitro* [Ref. (125–127); for review, see Ref. (28)]. The SCN and peripheral clocks share fundamental characteristics: they are cell-autonomous and self-sustained. However, individual cellular clocks within peripheral tissues rapidly become desynchronized and exhibit phase drifting in the absence of regular resetting by cues emanating, directly or indirectly, from the SCN. These cues include inputs from the autonomic nervous system, temperature cycles, and humoral factors such as glucocorticoids and melatonin.

The PT can be defined as a melatonin-dependent circadian oscillator (28, 50). Resetting of the PT clock by melatonin requires acute induction of *Cry1* expression [Ref. (128,129), see **Figure 3A**]; CRY1 being a key repressor of the circadian clock (130–132). The acute induction of *Cry1* expression involves EGR1-like factors (133) and the transcription factor NPAS4 (134, 135). In sheep, *Cry1* expression remains tightly linked to the onset of melatonin secretion and by implication night onset, irrespective of the duration of the day length [Ref. (136), see **Figure 3B**]. Interestingly, light given during the night induces *Cry1* expression within the quail PT (137), which suggests a phylogenetically conserved role for *Cry1* in the photoperiodic resetting of the PT clock.

How do we connect melatonin resetting of the PT clock with differential photoperiodic output of TSH and seasonal reproduction? The expression of the transcriptional co-activator EYA3 within the ovine PT displays large photoperiodic changes in both phase and amplitude [Ref. (39, 138); see **Figure 3A**]. Interestingly, *Eya3* was the other gene (besides *Tsh*β) immediately induced in the quail PT during the first long-day release experiment (91). We therefore investigated the transcriptional control of *Eya3* and searched for a link between inductions of both genes. The expression of *Eya3* is clock-controlled, through conserved DNA binding motifs within its promoter, and therefore phase-locked to that of the circadian clock [Ref. (138), see **Figure 3B**]. Because of this, expression peaks during the night under SP but during the day under LP. The amplitude of the peak is higher under LP than SP because melatonin suppresses *Eya3* expression, a suppression which can only occur in SP-exposed animals [Ref. (138); see **Figure 3B**]. Finally, *in vitro* data showed that induction of *Tsh*β expression is triggered by the circadian-controlled transcription factor TEF (33), which then recruits the co-activators SIX1 and EYA3. This leads to a marked increase in transcription under LP due to higher levels of EYA3 [Ref. (102, 138), **Figures 3B,C**]. A critical role for SIX1/EYA3, but not TEF, in the photoperiodic control of *Tsh*β transcription in the mouse PT has been proposed (139, 140).

#### **IS T3 OUTPUT SUFFICIENT TO ELICIT THE FULL SPECTRUM OF SEASONAL CHANGES?**

The data reviewed so far are consistent with a crucial role for the TSH output of the PT in driving seasonal changes in T3 availability within the MBH. However, swings in TSH/T3 may not be sufficient to elicit all seasonal changes. As mentioned before, since control of the lactotropic axis does not depend on T3 [for review, see Ref. (50)], complementary mechanisms are indeed expected. Neuromedin U (89, 141), histamine, and VGF secretion (82, 142, 143) may mediate seasonal effects on body weight and metabolism since their synthesis and cognate receptors display expression patterns and seasonal changes reminiscent of those seen for TSH/TSHR. However, since TSH infusion in SP-adapted Siberian hamster restores hypothalamic expression of somatostatin and body weight to LP levels (144), Neuromedin U, histamine, or VGF may be dispensable.

Retinoic acid signaling is also likely to be involved as retinoic acid receptors, transporters, and associated binding proteins display prominent photoperiodic regulation in the ependymal cell layer and posterior arcuate nucleus of Siberian hamsters and juvenile Fischer F344 rats (142, 145, 146). Interestingly, the retinoic X receptor (RXR) can heterodimerize with either the TH receptors (THRα/THRβ) or the retinoic acid related receptor (RAR). The target genes and downstream pathways governed by THR and RAR diverge, and therefore the photoperiodic regulation of RXR/RAR may fine-tune the seasonal adaptation of the metabolic status. From a more general standpoint, the notion that tanycytes coordinate a host of seasonal neuroendocrine cycles including reproduction, metabolism, and hibernation is emerging rapidly [Ref. (147, 148); for reviews, see Ref. (61, 149, 150)].

#### **PHOTOPERIODIC TIMING AND THE CIRCANNUAL CLOCK: T3 AS A UNIFYING COMPONENT?**

As mentioned earlier, whether species are classified as photoperiodic (e.g., Siberian and Syrian hamsters) or circannual (e.g., sheep), part of the seasonal cycle is generated endogenously. Hamsters and sheep maintained under constant SP do spontaneously revert to the opposite physiological state after several months. This phenomenon, referred to as "SP refractoriness," is typical of an interval timer/hourglass (5, 151). In contrast, sheep but not Siberian or Syrian hamsters, also display refractoriness to LP. Whether this species difference reflects fundamentally divergent underlying mechanisms is questionable. Indeed, Follett and Nicholls (47) proposed years ago that "it may well be that essentially identical physiological mechanisms underlie the photoperiodic responses of a wide range of vertebrates and that very minor modifications of these can cause surprisingly large (though superficial) changes in the overt responses of the animal in terms of reproduction." These authors devised a model, based on differences in threshold sensitivity, which rationalizes the LP refractoriness process (see **Figure 4**). There are indeed similarities between the photoperiodic control of the seasonal program in photoperiodic and circannual species (43, 50, 152). Siberian or Syrian hamsters and sheep might therefore exemplify "variations on a theme" rather than fundamentally different models.

Because TH is involved in many long-term life cycles events, it seems plausible that photoperiod-induced changes in T3 levels may also trigger more profound long-term changes, culminating weeks to months later. In particular, TH-induced plasticity and

cell-cycle related events have long time constants, which appear compatible with seasonal cycles (6, 153). Recent data in sheep demonstrate a photoperiodic gating of cell division within the PT and ependymal cells of the 3V and are consistent with this scheme (154–156). Nevertheless, whether photoperiodic gating of cell division depends on TH and/or is involved in seasonal transitions remains to be established.

To address a potential role for TH turn-over beyond the photoperiodic response, we investigated the expression of *Tsh*β and *Dio2*/*Dio3* within the MBH of sheep under distinct physiological states: LP, LP refractory (LPR) obtained after prolonged LP exposure, SP and SP refractory (SPR) obtained after prolonged SP exposure (157). The expressions of *Tsh*β and *Dio2* were diminished in LPR compared to LP animals but remained low in SP and SPR animals. The expression of *Dio3* was high in SP but very low in all other photoperiodic conditions, most notably under SPR; so the expression of *Dio3* under SP is transient (see **Figure 5A**).

Therefore, a diminished TSH output may cause the LPR state, while development of the SPR state would be disconnected from it. This would be consistent with the hourglass properties of the SPR mentioned before. However, changes in *Dio2*/*Dio3* may reflect an indirect effect of photoperiod: within the MBH, the local hyperthyroid state triggered by persistent LP exposure would eventually cross a certain threshold, thereby triggering *Dio3* induction and *Dio2* down-regulation (72, 74, 75). Following this, T3 levels would

hybridization autoradiograms for Tshβ, Dio2, and Dio3 in sheep under four different endocrine states: LP animals in a spring/summer-like state of reproductive arrest, LP refractory (LPR) animals showing spontaneous reproductive reactivation (late summer/autumn state), SP animals showing autumn/winter-like reproductive activation, and SP refractory (SPR) animals showing spontaneous reproductive arrest [adapted from Saenz de Miera et al. (157)]. **(B)** Schematics depicting (i) the direct effect of LP and SP on DIO2/DIO3 levels, respectively, intertwined with (ii) the possibility that their activity and the resulting TH metabolism constitutes the core of a long-term timing mechanism involved in refractoriness.

be cleared by DIO3, ultimately leading to the demise of *Dio3* expression; LP exposure would then somehow be required to induce *Dio2* once more and prime a new cycle. This LP requirement to prime the seasonal sequence may explain why circannual cycles in sheep are most obvious under constant LP (50).

Interestingly, Syrian hamsters in SPR state do not exhibit spontaneous reactivation of *Dio2* expression (88) while Siberian hamsters do (83). Furthermore, Siberian hamsters express *Dio3* upon transfer from LP to SP but its expression is not sustained through time (54, 83, 87), similar to what occurs in sheep (157). Therefore, transient *Dio3* expression under SP appears as a common feature and may explain why *Dio3* expression has not been observed in Syrian hamster (54). Even though the relative variations of *Dio2* and *Dio3* differ between species (e.g., Siberian vs. Syrian hamster) the central tenet remains the same: T3 levels are higher within the MBH under LP compared to SP (158). Furthermore, TH metabolism within the MBH may not only intervene in the photoperiodic response but may also be integral to longer-term timing processes such as circannual rhythms (see **Figure 5B**).

#### **CONCLUSION**

At this stage several outstanding questions remain: first, since the same TSH/deiodinase/T3 pathway is triggered by LP not only in long-day breeders (e.g., hamsters and quail) but also in shortday breeders (e.g., sheep) and non-photoperiodic species (e.g., mouse), how do we get opposite responses, or no response at all, of the hypothalamic–pituitary–gonadal axis? This is particularly intriguing because the increased intra-hypothalamic availability of TH is uniformly linked to an anabolic state across seasonal species. Second, through which mechanisms do local changes of T3 within the MBH ultimately impinge on gonadotropin-releasing hormone neurons? Pertinent to this second question, the MBH hosts two cell populations expressing RF-amide peptides which have attracted particular attention: neurons of the arcuate nucleus, which express *Kiss1* and neurons of the VMH/DMH, which express the *Rfrp* precursor. The concept that these RF-amide peptides are involved in seasonal breeding has been the topic of several excellent reviews (50, 158–161) and we will therefore only briefly review the most recent and salient findings.

Kisspeptin is a very potent GnRH secretagogue and governs most aspects of reproduction in mammals including sexual differentiation, steroid-dependent gonadotropin release, puberty onset, and the control of fertility by metabolic cues (162, 163). Interestingly, the annual onset of fertility in photoperiodic species had been compared to a reoccurrence of puberty, and common underlying processes were anticipated (2, 164, 165). Kisspeptin therefore appeared a prime candidate for the integration of photoperiodic and metabolic cues across the seasonal program, a prediction which has now received strong support (159–161).

In contrast to kisspeptin, the exact role(s) of peptides derived from the *Rfrp* precursor, RFRP1 and RFRP3, remain(s) unclear (160). RFRP3 may modulate feeding and various stress responses (166, 167). In the context of breeding, RFRP3 inhibits GnRH in sheep [Ref. (168), but see Ref. (169)] but inhibits or activates GnRH in Syrian and Siberian hamsters, depending on the photoperiod (170, 171). The *Rfrp* gene is orthologous to avian *GnIH*, which gives rise to gonadotropin inhibitory hormone (GnIH), a peptide with well-characterized inhibitory effects upon the gonadotropic axis in birds (172). Interestingly, there is no avian ortholog of the *Kiss1* (or *Kiss2*) gene (173), which implies that the concept of a balance between KISS1 and RFRP3 in governing GnRH secretion in mammals (174) does not apply to birds.

Both *Kiss1* and *Rfrp* expression display marked photoperiodic, melatonin-dependent, changes in mammals (159, 160, 175). Even though melatonin receptors have been localized to several hypothalamic nuclei it seems likely that the photoperiodic control over *Kiss1* and *Rfrp* is indirect [see above, Ref. (50, 145, 161)]. In this context, a role for PT-derived TSH appeared plausible. In a landmark study,Klosen et al. (144) showed that intracerebroventricular delivery of TSH in Siberian and Syrian hamsters induces *Dio2* expression within ependymal cells, restores expression of *Kiss1* and *Rfrp* to their LP levels and, most importantly, triggers reactivation of the gonadal axis. Furthermore, Henson et al. (176) showed that T3 injections to SP-adapted Siberian hamsters reactivated the gonadotropic axis, thereby confirming prior data (see Section "An Overview" and **Figure 1**), but also led to LP-like levels of RF-amide peptides within the MBH.

Therefore, even though a theoretical possibility exists that another TSH-dependent – but T3-independent pathway – leads to seasonal changes of the reproductive axis, the most parsimonious model is one in which T3 action on RF-amide neurons link the photoperiodic production of TSH within the PT to the seasonal control of GnRH secretion.

#### **ACKNOWLEDGMENTS**

Hugues Dardente is supported by a Marie Curie Career Integration Grant from the FP7-People-2012 program. Hugues Dardente wishes to thank Paul Pévet (INCI, Strasbourg, France) for continuing support and exciting discussions and Olivier Kah (Rennes, France) for sharing ideas about photoperiodism in fish.

#### **REFERENCES**

embryogenesis, defines a new class of leucine zipper proteins.*Genes Dev* (1991) **5**:1739–53. doi:10.1101/gad.5.10.1739

season in the ewe. *Endocrinology* (2003) **144**:2892–901. doi:10.1210/en.2003- 0322

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 18 December 2013; paper pending published: 03 February 2014; accepted: 10 February 2014; published online: 26 February 2014.*

*Citation: Dardente H, Hazlerigg DG and Ebling FJP (2014) Thyroid hormone and seasonal rhythmicity. Front. Endocrinol. 5:19. doi: 10.3389/fendo.2014.00019*

*This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Dardente, Hazlerigg and Ebling . This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Regulation of seasonal reproduction by hypothalamic activation of thyroid hormone

#### **Ai Shinomiya<sup>1</sup> ,Tsuyoshi Shimmura1,2,Taeko Nishiwaki-Ohkawa2,3,4 and TakashiYoshimura1,2,3,4\***

<sup>1</sup> Division of Seasonal Biology, National Institute for Basic Biology, Okazaki, Japan

<sup>2</sup> Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

<sup>4</sup> Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

#### **Edited by:**

Noriyuki Koibuchi, Gunma University Graduate School of Medicine, Japan

#### **Reviewed by:**

Maria Moreno, University of Sannio, Italy

Efisio Puxeddu, University of Perugia, Italy

#### **\*Correspondence:**

Takashi Yoshimura, Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan email<EMAIL_ADDRESS>Division of Seasonal Biology, National Institute for Basic Biology, 38 Nishigonaka Myodaiji, Okazaki 444-8585, Japan e-mail<EMAIL_ADDRESS>
Organisms living outside the tropics measure the changes in the length of the day to adapt to seasonal changes in the environment. Animals that breed during spring and summer are called long-day breeders, while those that breed during fall are called short-day breeders. Although the influence of thyroid hormone in the regulation of seasonal reproduction has been known for several decades, its precise mechanism remained unknown. Recent studies revealed that the activation of thyroid hormone within the mediobasal hypothalamus plays a key role in this phenomenon. This localized activation of the thyroid hormone is controlled by thyrotropin (thyroid-stimulating hormone) secreted from the pars tuberalis of the pituitary gland. Although seasonal reproduction is a rate-limiting factor in animal production, genes involved in photoperiodic signal transduction pathway could emerge as potential targets to facilitate domestication.

**Keywords: seasonal reproduction, mediobasal hypothalamus, ependymal cell, pars tuberalis, thyrotropin, thyroid hormone, iodothyronine deiodinase**

#### **INTRODUCTION**

Orbiting of the earth around the sun causes changing seasons. To adapt to the seasonal changes in the environment, animals alter their physiology and behavior, which is characterized by the changes in growth, metabolism, immune function, reproductive activity, migration, hibernation, and molting. Most of the organisms use the changes in the length of the day (photoperiod) as a calendar,because temperature and precipitation varies throughout each year and are unreliable when compared with the length of the day. This phenomenon is called "photoperiodism" (1). Among the various seasonally regulated phenomena, the mechanism of seasonal reproduction has been extensively studied. Small mammals and birds breed during the spring and summer. Therefore, they are called long-day (LD) breeders. The gestation or incubation period of these animals last only a few weeks and their offspring are born during the spring and summer. In contrast, larger mammals, such as goats and sheep, breed during fall. Therefore, they are called short-day (SD) breeders. These animals have a gestation period of approximately 6 months. Therefore, their offspring are also born and raised during spring and summer. Accordingly, the offspring of both LD and SD breeders grow when the climate is moderate and food is abundant (**Figure 1**).

Seasonal reproduction of vertebrate species is regulated by the hypothalamic–pituitary–gonadal (HPG) axis. The secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus induces the secretion of gonadotropins [luteinizing hormone (LH) and follicle-stimulating hormone (FSH)] from the anterior pituitary gland, which in turn activates gonadal activity. In other words, the HPG axis of seasonally breeding animals is only activated during the breeding season. Among the various vertebrate species, birds show the most dramatic changes in gonadal size (typically more than a 100-fold) (2). Therefore, birds have a highly sophisticated photoperiodic mechanism in comparison to other vertebrate species (3). In addition to the robust gonadal responses, most of the birds have very short breeding seasons, as the HPG axis is automatically switched off and their gonads start to regress even though the length of the day is still increasing. This phenomenon is known as photorefractoriness (4, 5). The length of the breeding season tends to be shorter in higher latitude due to the short benign season in higher latitude. Among mammals, hamsters and sheep are extensively studied, because they show dramatic photoperiodic responses. However, the magnitude of the seasonal gonadal development and regression is less robust in mammals than in birds, as their gonads change only by a few-folds.

#### **INFLUENCE OF THYROID HORMONE IN THE SEASONAL CHANGES**

It has been known for many decades that thyroid hormone is somehow involved in the regulation of seasonal reproductive function in various organisms including fish, birds, and mammals (2, 6, 7). In some species, thyroidectomy prevents the transition to reproductive state (i.e., seasonal testicular development and/or regression) (8–11), and thyroxine (T4) treatment mimics the effects of a long photoperiod (12–14). However, photo-stimulated gonadal

<sup>3</sup> Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan

maturation appears to have been largely unaffected by thyroidectomy in some species (2). Therefore, the reported effects of thyroidectomy on seasonal breeding are often contradictory and the role of T<sup>4</sup> is thought to be permissive. Although the requirement of T<sup>4</sup> for an appropriate response to photoperiod has been documented (15), the mechanism by which thyroid hormone regulates seasonal reproduction remained unknown for several decades.

#### **PHOTOPERIODIC CHANGES IN TYPE 2 AND TYPE 3 DEIODINASES WITHIN THE HYPOTHALAMUS**

The Japanese quail (*Coturnix japonica*) is an excellent model for studying photoperiodism, because of its rapid and robust responses to changing photoperiods (3). Local illumination of the mediobasal hypothalamus (MBH) by radioluminous-painted beads induce testicular growth (16), and lesions of MBH blocks the photoperiodic response of LH secretion and gonadal development (17, 18). In addition, expression of c-Fos, a marker of neuronal activation, is induced in the MBH by LD stimulus (19). The MBH is therefore considered central for the seasonal reproduction in quail. By using differential subtractive hybridization analysis, LDinduction of type 2 deiodinase gene (*DIO2*) and LD-suppression of type 3 deiodinase gene (*DIO3*) were observed in the ependymal cells (also known as tanycytes) that line the ventrolateral walls of the third ventricle within the MBH [Ref. (20, 21), **Figure 2**]. *DIO2* encodes the thyroid hormone-activating enzyme that converts the prohormone T<sup>4</sup> to bioactive triiodothyronine (T3) (22), while *DIO3* encodes thyroid hormone-inactivating enzyme that metabolizes T<sup>4</sup> and T<sup>3</sup> to inactive reverse T<sup>3</sup> (rT3) and 3,3<sup>0</sup> diiodothyronine (T2), respectively. The reciprocal switching of *DIO2* and *DIO3* appears to regulate the local thyroid hormone

concentration precisely within the MBH. Moreover, T<sup>3</sup> concentration within the MBH is about 10-fold higher under LD conditions than under SD conditions, even though plasma concentrations are similar to both photoperiods (20). The functional significance of this locally activated thyroid hormone has been demonstrated by pharmacological analyses. Intracerebroventricular (i.c.v.) infusion of T<sup>3</sup> in SD conditions induced testicular development while infusion of a DIO2 inhibitor (iopanoic acid) in LD conditions attenuated testicular development (20). Photoperiodic regulation of *DIO2* and/or *DIO3* has also been confirmed in a number of other avian species, such as the tree sparrow (23), chicken (24), great tits (25), and canary (26). Similarly, photoperiodic regulation of thyroid hormone metabolism in the MBH has been confirmed in various mammalian species, including LD breeders like Siberian hamsters (27–30), Syrian hamsters (31, 32), rats (33, 34), mice (35), and SD-breeding goats (36) and sheep (37). Activation of thyroid hormone within the MBH decodes the LD information. Therefore, daily T<sup>3</sup> subcutaneous injections induce testicular development (28) and chronic replacement of T<sup>3</sup> in the hypothalamus prevents the onset of testicular regression (27) in LD-breeding Siberian hamsters. In contrast, in the SD breeders, LD-induced DIO2 appears to convert T<sup>4</sup> to T<sup>3</sup> to terminate the breeding season (37). In addition, LD stimulus induces the expression of *DIO2*, and T<sup>4</sup> administration terminates the breeding season via a decrease in serum LH (38, 39).

#### **THYROID HORMONE TRANSPORT TO THE EPENDYMAL CELLS**

Due to their lipophilic nature, thyroid hormones are believed to traverse plasma membranes by passive diffusion. However,

**FIGURE 2 | Photoperiodic signal transduction pathway in mammals and birds**. In mammals, light information is received by the eye and transmitted to the pineal gland via the circadian pacemaker, the suprachiasmatic nucleus (SCN). The duration of the pineal melatonin signal encodes the length of night and regulates TSH secretion in the pars tuberalis. The pars tuberalis TSH acts on TSH receptor expressed in the ependymal cells lining ventrolateral walls of the third ventricle (VIII) to induce DIO2 and reduce DIO3. Local thyroid hormone activation within the mediobasal hypothalamus (MBH) by DIO2/DIO3 switching plays a key role in the regulation of seasonal reproduction. In contrast, light information received by deep brain photoreceptors induces TSH secretion from the pars tuberalis in birds. Nevertheless, melatonin is not involved in the seasonal reproduction of birds. The schematic is a modified version of illustration published by Ikegami and Yoshimura (40).

involvement of a membrane transport system for thyroid hormone has been reported recently and a mechanism that facilitates the transport of thyroid hormone into the ependymal cells was examined. Some members of the organic anion transporting polypeptide (Oatp) family have been shown to transport thyroid hormones in mammals (41, 42) and the involvement of a member of this family in transporting T<sup>4</sup> into the quail brain has been investigated (43). Oatp1c1, which is expressed in the ependymal cells within the MBH, has been demonstrated to be a highly specific transporter of T4. In addition to Oatp1c1, another thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), has been found in the ependymal cells within hamster MBH (29). Although MCT8 appears to be involved in the regulation of photoperiodism, its expression is upregulated under SD conditions, which does not require thyroid hormone.

#### **REGULATION OF HYPOTHALAMIC DEIODINASES BY THE PARS TUBERALIS TSH**

When quail are transferred from SD conditions to LD conditions, an increase in plasma gonadotropin (LH) is observed 22 h after the dawn of the first LD (3, 44, 45). As discussed previously, reciprocal switching of *DIO2* and *DIO3* plays a critical role in the regulation of seasonal reproduction in birds and mammals. In quail, the reciprocal switching of *DIO2* and *DIO3* precedes photoperiodic induction of gonadotropin release by roughly 4 h (21). Genomewide gene expression analysis during the transition from SD conditions to LD conditions in Japanese quail (45) identified the induction of two genes 4 h prior to *DIO2/DIO*3 switching (i.e., 14 h after dawn) in the pars tuberalis of the pituitary gland. The pars tuberalis consists of thin layers of cells surrounding the median eminence (**Figure 2**). One of these genes encode the thyroidstimulating hormone β subunit (*TSHB*) and the other encode the transcriptional co-activator eyes absent 3 (*EYA3*). Although EYA3 is a transcriptional co-activator, the expression sites of *EYA3* and *DIO2*/*DIO3* are different (i.e., *EYA3* in the pars tuberalis and *DIO2/DIO3* in the ependymal cells). Therefore, it appears that EYA3 is not involved in the regulation of *DIO2*/*DIO3* switching. On the other hand, the expression of TSH receptor (TSHR) and binding of <sup>125</sup>I-labeled thyroid-stimulating hormone (TSH) were observed in the ependymal cells where *DIO2* and *DIO3* are expressed. In addition to these, i.c.v. TSH administration induced *DIO2* expression and reduced *DIO3* expression in the ependymal cells even under SD conditions, while passive immunization against TSH attenuated LD-induction of *DIO2* expression (45). The involvement of TSHR-Gsα-cAMP signaling pathway in this TSH regulation of *DIO2* expression was demonstrated by the promoter analysis. Considering that the magnitude of testicular growth induced by i.c.v. TSH infusion was almost similar to that observed in birds exposed to LD stimulus, the LD-induced pars tuberalis TSH appears to be a major factor regulating the seasonal reproduction in birds.

In birds, eyes are not necessary for the regulation of seasonal reproduction because deep brain photoreceptors are involved in this process (46, 47). Although pineal organ is a photoreceptive organ in non-mammalian vertebrates (48, 49), pineal organ is not involved in the regulation of seasonal reproduction (50, 51). In contrast, local illumination of the septal region of the telencephalon or the MBH using radioluminous-painted beads caused testicular growth in quail, suggesting the existence of deep brain photoreceptors in these regions (16). Localization of several rhodopsin family proteins (rhodopsin; OPN4: melanopsin; OPN5: neuropsin and VA opsin: vertebrate ancient opsin) are reported in these brain regions and projections that link some of these photoreceptor cells to the pars tuberalis have also been reported (52–62). These photoreceptors are therefore thought to be involved in the seasonal regulation of reproduction in birds (**Figure 2**).

In a marked contrast to avian species, eyes are the only photoreceptive organ in mammalian species (63–69). Therefore, removal of the eyes abolishes the photoperiodic response (64, 68). Light information received by the eye is transmitted to the pineal gland through the suprachiasmatic nucleus (SCN), where the circadian pacemaker is localized (68, 70–74). The duration of night corresponds to the nocturnal secretion profile of melatonin, which plays a crucial role in the regulation of seasonal reproduction in mammalian species. For example, in both LD and SD breeders, pinealectomy abolishes seasonal responses, while melatonin administration restores them (68, 74, 75). Melatonin acts via melatonin receptors and there are two subtypes of melatonin receptors (MT1 and MT2) in mammals (76, 77). However, these melatonin receptors are not expressed in the ependymal cells where *DIO2* and *DIO*3 are expressed (78, 79). The MT1 receptor is reportedly expressed in the thyrotroph cells of the pars tuberalis (80, 81). Therefore, pars tuberalis TSH likely mediates the influence of melatonin in the *DIO2*/*DIO3* switching in mammalian species. Although it is generally considered that laboratory mice are non-seasonal breeders, many researchers noticed that mice do not breed well during the winter (e.g., small litter size) even though they are kept under standardized conditions. To determine whether pars tuberalis TSH mediates the influence of melatonin in the *DIO2/DIO3* switching, laboratory mice were analyzed as experimental models. Two key enzymes, arylalkylamine *N*-acetyltransferase (AA-NAT) and hydroxyindole-*O*-methyltransferase (HIOMT) are involved in melatonin biosynthesis from serotonin (74). However, most inbred mice genetically lack the ability to produce these enzymes, resulting in minimal melatonin generation (82, 83). Therefore, it was predicted that melatonin-producing strains would have the capacity to respond to photoperiodic changes, while melatonindeficient strains would be resilient to such changes. As expected, clear photoperiodic regulation of *TSHB*, *DIO2*, and *DIO3* was observed in the melatonin-producing CBA strain, while such responses were not observed in the melatonin-deficient C57BL strain (35). In addition, daily intraperitoneal (i.p.) melatonin injections mimicked the effect of SD conditions on the expression of these genes (35). To test the involvement of the TSH– TSHR signaling pathway in the melatonin-mediated regulation of *DIO2*/*DIO3* expression, the effects of melatonin administration were examined in TSHR-null mice (35). The TSHR-null mice failed to respond to melatonin administration. This result clearly suggested the involvement of a TSH–TSHR signaling pathway in the melatonin-mediated regulation of *DIO2*/*DIO3* in mammals. In addition, the analysis of mice that lacked the MT1 and MT2 melatonin receptors revealed the involvement of MT1 melatonin receptors in this regulation (84). It is also interesting to note that TSH is involved in the LD-induction of *DIO2* in SD-breeding sheep (37). Thus, pars tuberalis TSH appears to relay the seasonal information in both LD and SD-breeding animals and sensitize them for spring.

#### **THYROID HORMONE ACTION WITHIN THE HYPOTHALAMUS**

Thyroid hormone is involved in the development and plasticity of the central nervous system (22). The expression of thyroid hormone receptors (*THR*α, *THR*β, and *RXR*α) in the median eminence suggested that the median eminence is the target site of action for the photo-induced increase in T<sup>3</sup> in the quail MBH (20). To understand the action of thyroid hormone within the MBH, the ultrastructure of the median eminence was examined under SD and LD conditions using electron microscopy. Dynamic morphological changes were observed between the GnRH nerve

terminals and glial endfeet within the median eminence (85). In SD conditions, many GnRH nerve terminals are encased by the endfeet of glial processes and do not contact the basal lamina, while many GnRH nerve terminals are in close proximity to the basal lamina under LD conditions (**Figure 3**). It has been proposed that the nerve terminals of hypothalamic neurons are required to directly contact the pericapillary space for the secretion of the hypothalamic neurohormone from the hypothalamus into the portal capillary (86). Morphological changes between the GnRH nerve terminals and endfeet of glial processes are observed in SD quail treated with T<sup>3</sup> to stimulate testicular growth (87). Therefore, these morphological changes appear to regulate or modulate the seasonal GnRH secretion from the median eminence. It is also interesting to note that the seasonal plasticity within the GnRH system is reported in ewes (88).

#### **PHOTOPERIODIC SIGNALING PATHWAY AND DOMESTICATION**

Seasonal reproduction is a rate-limiting factor for the animal procreation. The photoperiodic signaling pathway could also be a potential target that facilitates human-driven domestication process. As discussed previously, most laboratory mice lack the enzyme activity of melatonin biosynthesis pathway (82, 83, 90, 91). In addition, occurrence of selective sweeps was found at the TSHR locus in all domestic chickens (92). This observation suggests that the TSHR may be a domestication locus in chicken (92). Although we still do not know the correlation with domestication, it is interesting to note that photoperiodic regulation of *DIO3* is absent in Syrian hamster (27). Thus, genes involved in the photoperiodic signaling pathway could emerge as useful targets for the domestication of wild animals.

#### **CONCLUSION**

Involvement of thyroid hormone in the regulation of seasonal reproduction has been suggested in the past several decades. Recent comparative studies clearly reveal that the local activation of thyroid hormone within the hypothalamus is a key factor in the regulation of seasonal reproduction in a number of mammalian and avian species. It is important to note that this mechanism is also conserved in fish (93) and is universal among various vertebrate species. Although thyroid hormone influences both LD and SD breeders, the mechanism that differentiates LD breeders from SD breeders remains unknown. Presumably, the responsiveness of pathways downstream of T<sup>3</sup> activity (e.g., responsiveness of T<sup>3</sup> target genes to LD-induced T<sup>3</sup> etc.) differs in LD and SD breeders. The switching mechanism of LD breeder and SD breeder needs to be clarified in the future studies.

#### **ACKNOWLEDGMENTS**

This work is supported by the Funding Program for Next Generation World Leading Researchers (NEXT Program) initiated by the Council for Science and Technology Policy (CSTP) (LS055). WPI-ITbM is supported by World Premier International Research Center Initiative (WPI), MEXT, Japan.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 25 December 2013; paper pending published: 28 January 2014; accepted: 31 January 2014; published online: 21 February 2014.*

*Citation: Shinomiya A, Shimmura T, Nishiwaki-Ohkawa T and Yoshimura T (2014) Regulation of seasonal reproduction by hypothalamic activation of thyroid hormone. Front. Endocrinol. 5:12. doi: 10.3389/fendo.2014.00012*

*This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Shinomiya, Shimmura, Nishiwaki-Ohkawa and Yoshimura. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Thyroid hormone upregulates hypothalamic kiss2 gene in the male Nile tilapia, Oreochromis niloticus

#### **Satoshi Ogawa<sup>1</sup> , KaiWe Ng<sup>1</sup> , Xiaoyu Xue<sup>2</sup> , Priveena Nair Ramadasan<sup>1</sup> , Mageswary Sivalingam<sup>1</sup> , Shuisheng Li <sup>2</sup> , Berta Levavi-Sivan<sup>3</sup> , Haoran Lin<sup>2</sup> , Xiaochun Liu<sup>2</sup> and Ishwar S. Parhar <sup>1</sup>\***

<sup>1</sup> Brain Research Institute, School of Medicine and Health Sciences, Monash University Malaysia, Petaling Jaya, Malaysia

<sup>2</sup> State Key Laboratory of Biocontrol, School of Life Sciences, Institute of Aquatic Economic Animals and the Guangdong Province Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou, China

<sup>3</sup> The Robert H. Smith Faculty of Agriculture, Food and Environment, Department of Animal Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

#### **Edited by:**

Noriyuki Koibuchi, Gunma University Graduate School of Medicine, Japan

#### **Reviewed by:**

Yoshitaka Oka, University of Tokyo, Japan

Ann Marie Zavacki, Brigham and Women's Hospital, USA

#### **\*Correspondence:**

Ishwar S. Parhar, Jeffrey Cheah School of Medicine and Health Sciences, Brain Research Institute, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor 46150, Malaysia e-mail<EMAIL_ADDRESS>
Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates.  In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization.The effect of thyroid hormone (triiodothyronine,T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2, and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR.The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration ofT<sup>3</sup> (5µg/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 h post injection (P < 0.001), while the treatment with an anti-thyroid, MMI (100 ppm for 6 days) significantly reduced kiss2 and gnrh1 mRNA levels (P < 0.05). gnrh2, gnrh3, and thyrotropin-releasing hormone mRNA levels were insensitive to the thyroid hormone manipulations. Furthermore, RT-PCR showed expression of thyroid hormone receptor mRNAs in laser-captured GnRH neurons but not in kiss2 neurons. This study shows that GnRH1 may be directly regulated through thyroid hormone, while the regulation of Kiss2 by T<sup>3</sup> is more likely to be indirect.

**Keywords: cichlid, in situ hybridization, hypothalamus, thyroid receptor, kisspeptin**

#### **INTRODUCTION**

Kisspeptin, encoded by *Kiss1*/*KISS1* (rodents/human) gene and its cognate receptor, GPR54 (=Kiss-R), have recently been considered the major regulator of reproductive functions, in particular the onset of puberty (1). Administration of kisspeptin stimulates gonadotropin secretion (2), either by its direct action on gonadotrophs (3) or through gonadotropin-releasing hormone (GnRH) neurons (4). Variants of *kiss1* homologous sequences (*kiss1* and *kiss2*) have been identified in several non-mammalian vertebrates including amphibian and teleosts (5, 6). In the teleosts brain, cells expressing *kiss1* mRNA are seen in the ventral habenula and/or the ventral hypothalamus, while those of *kiss2* mRNA are seen in the hypothalamic nuclei and/or the preoptic area depending on the fish species (5, 7). With multiple kisspeptin types, multiple forms of Kiss-R encoding genes (*kissr1* and *kissr2*) have been cloned and characterized in various teleosts (5, 6). Several lines of evidence have demonstrated that Kiss2 is more potent than Kiss1 in the control of reproduction in teleosts (8–11). In the sexually mature zebrafish,*Danio rerio*, administration of Kiss2 peptides significantly increases the gonadotropins β-subunit mRNA levels in the pituitary (9). Similarly in prepubertal European sea bass, *Dicentrarchus labrax*, Kiss2 but not Kiss1 injection increases

plasma gonadotropins levels (8). We have previously identified Kiss-R (*kissr2*) in the Nile tilapia, *Oreochromis niloticus* and have shown its expression in GnRH neurons (12). These results suggest the potent role of Kiss2 in the reproductive axis during prepubertal development and sexually mature stages in teleosts. In mammals, kisspeptin neurons transmit gonadal steroid feedback signals to GnRH neurons, especially the positive feedback effect of ovarian estrogen that causes the preovulatory GnRH/luteinizing hormone (LH) surge in female (13). Although the *kiss2* gene is highly conserved in non-mammalian vertebrates, a potent trigger of Kiss2 neural activity has not been identified in teleosts. In the medaka, *Oryzias latipes*, only the hypothalamic *kiss1* but not *kiss2* neurons show prominent estrogen sensitivity in their kisspeptin gene expression (14). Similarly in the goldfish, *Carassius auratus*, the preoptic but not hypothalamic *kiss2* neurons show clear estrogen sensitivity (15). In addition, these estrogen sensitive kisspeptin neuron types in the fish express estrogen receptors (ERα and ERβ) (14, 15). In the juvenile zebrafish, *kiss2* neurons are upregulated by estrogen treatment (16). These observations suggest that the hypothalamic Kiss2 neurons can be regulated by ovarian estrogen in a reproductive stage-dependent manner. However, the concept of an estrogen positive feedback mechanism that initiate the

preovulatory GnRH/LH surge is not relevant for males (17). In male aromatase knockout mice, Kiss1 expression in the hypothalamus is not reduced (18). Thus, it is possible that factors other than estrogen play an important role in the regulation of kisspeptin neurons in males (17).

Thyroid hormone is an important regulator of somatic growth, metabolism, brain development, and other vital processes in developing and adult animals (19). Additionally, thyroid hormone also plays an important role in reproductive functions during several physiological conditions (19). In fish, there are numerous studies that examined the effect of hyper- and hypo-thyroidism in sexual development, maturation, and reproductive behavior (20). Direct action of thyroid hormone on GnRH neurons as well as co-expression of thyroid hormone receptors in GnRH neurons has been previously demonstrated (21, 22). In ewe, thyroid hormones are necessary for GnRH and LH pulsatility (23, 24). Although pulsatile secretion of GnRH and kisspeptin are closely interlinked (25), the potential role of thyroid hormone in the regulation of kisspeptin system has never been studied.

In the present study, we cloned *kiss2* cDNA in the Nile tilapia. Gene expression of *kiss2* mRNA in the brain was examined by *in situ* hybridization. Furthermore, to examine the potential role of thyroid hormone in the regulation of the kisspeptin system, the effect of thyroid hormone (triiodothyronine, T3) and methimazole (MMI) on *kiss2* and GnRH types (*gnrh1*, *gnrh2*, and *gnrh3*) mRNA expression was analyzed by real-time PCR. MMI inhibits thyroperoxidase, which acts in thyroid hormone synthesis by oxidizing the anion iodide (I−) to iodine (I0), facilitating iodine's addition to tyrosine residues on the hormone precursor thyroglobulin, a necessary step in the synthesis of T<sup>3</sup> and thyroxine (T4). MMI has been shown to reduce plasma thyroid hormone levels and type III deiodinase (D3) activities (hypothyroid condition) in the brain, gill, and liver of tilapia (26). In the present study, to manipulate the plasma thyroid hormone levels in the male tilapia, we applied two different administration methodologies: for hyperthyroid condition, 24 h after intraperitoneal injection of thyroid hormone while for hypothyroid status, fish were immersed in water containing MMI for 6-days. We also measured mRNA expression levels of thyrotropin-releasing hormone (TRH) to validate the effect of thyroid hormone manipulation. Finally, to confirm the potential mechanism of thyroid hormone action on Kiss2 and GnRH neurons, the expression of thyroid hormone receptor (TR) types (*tra1*, *tra2*, and *trb*) mRNA were analyzed in laser-captured *kiss2* and GnRH neurons.

#### **MATERIALS AND METHODS**

#### **ANIMALS**

Sexually mature male Nile tilapia, *O. niloticus* (standard length: 11.6 ± 0.4 cm, body weight: 52.6 ± 5.0 g) were maintained in freshwater aquaria at 28 ± 0.5°C with a controlled natural photoregimen (14/10 h, light/dark). They were fed twice daily with commercial tilapia diets (Zeigler, USA). The fish were maintained and used in accordance with the Guidelines of the Animal Ethics Committee of Monash University (Approval Number: SOBSB/2009/58) and Sun Yat-Sen University.

#### **MOLECULAR CLONING OF kiss2 IN THE TILAPIA**

The fish were anesthetized by immersing in a 0.01% solution of tricaine methanesulfonate (MS222; Sigma, St. Louis, MO, USA) and killed by decapitation for sample collection. Total RNA from the tilapia brain (*n* = 1) was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of isolated RNA was used to synthesize the first-strand cDNA using the Rever-Tra Ace-a first-strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Partial cDNA fragments were obtained by PCR using degenerate primers or gene-specific primers designed based on the sequences of *kiss* genes of fugu, grouper, medaka, and mackerel (**Table 1**). Full-length cDNA sequences were obtained by 5<sup>0</sup> and 3<sup>0</sup> rapid amplification of cDNA ends (RACE) kit (Invitrogen). For all PCR reactions in this study, amplifications were performed with an initial denaturation step at 94°C for 3 min, followed by 40 cycles of 94°C for 15 s, 55-58°C for 15 s, and 72°C for 30 s. The reaction was ended by a further extension of 10 min at 72°C. The amplification products were purified using the E.Z.N.A. Gel Extraction Kit (Omega BioTek, GA, USA) and ligated into the pTZ57R/T vector (Fermentas, MD, USA). Three different individual positive clones were picked to confirm the sequence information using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA). Putative signal peptides and cleavage sites were predicted using SignalP 3.0<sup>1</sup> . Multiple sequence alignments of amino acids were performed with ClustalX (1.81) program. Protein phylogenetic analysis was conducted with MEGA4 using the neighbor-joining method.

Chromosomal location of tilapia *kiss2* gene was identified and its gene synteny with *kiss2* genes in other teleosts (zebrafish, medaka, *O. latipes*, and puffer fish, *Takifugu rubripes*) were examined using the Ensemble Genome Browser<sup>2</sup> .

The tilapia *kiss2* gene promoter sequence was identified *in silico* using the Ensemble Genome Browser. The 2.0-kb sequence upstream of the untranslated region was considered to be the putative promoter. The putative promoter sequence was analyzed for conserved regulatory elements using online bioinformatic tools (TESS<sup>3</sup> ; TFSearch<sup>4</sup> ; SignalScan<sup>5</sup> ).

#### **TISSUE DISTRIBUTION**

To determine the tissue distribution of *kiss2* mRNA in the tilapia, sexually mature male and female fish (*n* = 1 each) were anesthetized by immersing in a 0.01% solution of MS222 and killed by decapitation for sample collection. Tissue samples were collected and snap frozen in liquid nitrogen. Total RNA was isolated from the different brain regions (the olfactory bulb, telencephalon, optic tectum thalamus, hypothalamus, cerebellum, and medulla) and peripheral tissues (the pituitary, liver, spleen, intestine, kidney, gill, heart, muscle, testis, and ovary) with TRIzol. One microgram of total RNA from each sample was digested with deoxyribonuclease I (DNase I) and reverse-transcribed into cDNA using the ReverTra Ace-first-strand cDNA Synthesis Kit. PCR was carried

<sup>2</sup>http://asia.ensembl.org/index.html

<sup>3</sup>http://www.cbil.upenn.edu/cgi-bin/tess/tess

<sup>4</sup>http://www.cbrc.jp/research/db/TFSEARCH.html

<sup>5</sup>http://www-bimas.cit.nih.gov/molbio/signal/

<sup>1</sup>http://www.cbs.dtu.dk/services/SignalP/

#### **Table 1 | Primers for tilapia genes used in present study.**

Primer pairs used for real-time PCR and TRs localization were designed to originate in different exons to exclude false positive bands in case of potential genomic DNA contamination.

out as described above. The PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized by illumination under UV light. All PCR products were confirmed by sequencing.

#### **IN SITU HYBRIDIZATION**

Brains of sexually mature males (*n* = 3) were dissected and fixed in buffered 4% paraformaldehyde for 16 h at 4°C. The brains were then cryoprotected in 20% sucrose and embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan). Consecutive coronal sections (15µm thick) were cut on a cryostat and thaw-mounted onto 3-aminopropylsilane-coated glass slides. Sense and antisense digoxigenin (DIG)-labeled riboprobes were synthesized from partial sequence of tilapia *kiss2* (266 nt) using MAXIscript (Ambion, Austin, TX, USA) and DIG RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instruction. DIG-*in situ* hybridization was performed as described previously (9). Briefly, sections were subjected to permeabilization with 0.2M HCl for 10 min followed by proteinase K (1µg/ml) treatment for 15 min, and hybridized with the DIG-labeled riboprobes (50 ng/ml) at 58°C overnight in a humidified chamber. After hybridization, sections were washed and blocked with 2% normal sheep serum. DIG signals were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics, diluted 1:500) with 4-nitro blue tetrazolium chloride/5-bromo-4 chloro-3-indolyl-phosphate (Roche Diagnostics).

#### **THYROID HORMONE TREATMENT AND INDUCTION OF HYPOTHYROIDISM**

To induce hyperthyroidism, thyroid hormone (T3) was administered as described previously (22). Briefly, anesthetized sexually mature male fish were intraperitoneally (IP) injected with 30µl of T<sup>3</sup> (3,3<sup>0</sup> ,5-Triiodo-L-thyronine sodium salt, Sigma, US; dissolved in 100% ethanol and then diluted with sesame oil) at 5µg/g BW or sesame oil (control) through a 25-gage syringe needle (*n* = 15/group, single injection). The selected dose of T<sup>3</sup> has been reported to produce plasma T<sup>3</sup> levels of 4.6 ± 1.2 ng/ml in the male tilapia 24 h after the injection (22), which is within the physiological levels (2 ∼ 5 ng/ml) in the Mozambique tilapia, *O. mossambicus* (27). After the injection, the fish were released into the recovery tank. Twenty-four hours after the injection, the fish were anesthetized and killed by decapitation, and the brain was dissected for RNA isolation.

To induce hypothyroidism, fish were treated in water containing 100 ppm of methimazole (2-Mercapto-1-methylimidazole, MMI, Sigma; dissolved in 100% ethanol and then diluted in water) or in water containing equal volume of 100% ethanol (*n* = 5 ∼ 8/group) for 6-days. The water containing MMI was changed every day, which was required to reduce amount of endogenous T<sup>4</sup> levels from a euthyroid to hypothyroid state similar to the treatment in tilapia treated with MMI (26). The selected dose of MMI was calculated based on the concentrations that have been applied to the Nile tilapia and the sea bream, *Sparus auratus*, via diet in previous studies (26, 28). After the treatment, the brain tissue was dissected and frozen on dry ice, and stored at −80°C until use for RNA isolation.

#### **REAL-TIME PCR FOR kiss2, gnrh1, gnrh2, gnrh3, AND trh GENES**

Total RNA was extracted from the brain with TRIzol (Invitrogen) and 200 ng of total RNA were subjected to cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a final volume of 20µl reaction mixture containing 1× RT buffer, 1× dNTP mix, 1× RT Random Primers, 20U ribonuclease inhibitor, and 10U MultiScribe Reverse Transcriptase according to the manufacturer's instruction. The cDNA samples were then subjected to real-time PCR for tilapia *kiss2*, *gnrh1*, *gnrh2*, *gnrh3* (GenBank accession numbers for three GnRH types: AB104861,AB101666, and AB104863) and β-actin (*b-actin*)

mRNAs with an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). In addition, effect of thyroid hormone manipulation on TRH (also known as thyroliberin: GenBank Accession number, XM\_003438996) was also examined. The PCR reaction mixture (10µl) contained 1× POWER SYBR Green PCR Master Mix (Applied Biosystems), 0.1µM each forward and reverse primer, and 1µl of sample cDNA. Nucleotide sequences of real-time PCR primers for tilapia *kiss2*, GnRH types, *trh* and βactin are presented in **Table 1**. Reactions were carried out at 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min followed by a dissociation stage. The cycle threshold (Ct) values of all genes was determined and normalized against β-actin mRNA levels. Data was then analyzed according to relative gene expression by 2−∆∆Ct. To check PCR specificity, representative PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized by illumination under UV light. Nucleotide sequences of the PCR products were further confirmed by sequencing. Data are expressed as mean ± SEM and statistical analysis was performed by one-way ANOVA followed by *post hoc* analysis with *t*-test for parametric data or the Mann– Whitney *U* test for non-parametric data with *P* < 0.05 considered significant.

#### **EXPRESSION OF TR TYPES IN LASER-CAPTURED kiss2 AND GnRH NEURONS**

The expression of TR mRNA types (tra1, tra2 and trb) was examined in laser-captured DIG-labeled *kiss2* neurons and immunolabeled GnRH types neurons by RT-PCR. The fish were anesthetized by immersing in a 0.01% solution of MS222 and killed by decapitation for sample collection. Brains of sexually mature male fish were processed for DIG-*in situ* hybridization of *kiss2* gene and immunofluorescence labeling of three GnRH neurons types (*n* = 3 for each cell types). DIG-*in situ* hybridization for *kiss2* was performed as described above. For harvesting of GnRH neurons, the brain sections were stained with rabbit anti-tilapia GnRH antibodies against their respective GnRH associated peptide (GAP) sequence (GAP1, #ISP105; GAP2, #ISP205, and GAP3, #ISP305), which were previously generated in our lab. Dilutions (1:1000) were made in an RNase-free phosphate buffer saline (pH 7.0) containing 2% bovine serum albumin and 0.5% triton X-100, and the antiserum was applied to sections mounted on slides for 24 h in a closed moist chamber at 4°C and detected with Alexa Fluor 546 labeled anti-rabbit IgG (1:500 dilution, Invitrogen, Carlsbad, CA, USA). DIG-labeled *kiss2* and immune-fluorescently labeled GnRH neurons were laser-microdissected using an Arcturus XT system (Molecular Devices, Sunnyvale, CA, USA). Each population of the laser-microdissected cells (*kiss2*, ∼200 cells; GnRH1, ∼100 cells; GnRH2, ∼30 cells; GnRH3, ∼30 cells/fish) were placed into sterile 0.2 ml PCR tubes containing 50µl of lysis solution [1× RT buffer (Applied Biosystems, Foster City, CA, USA), 1% Nonidet P-40, and 0.05 mg/µl proteinase K] and lysed for 1 h at 50°C. After DNase I treatment, total RNA was isolated using TRIzol (Invitrogen) and dissolved in a 10-µl of DEPC-treated water. The total RNAs were subsequently subjected to cDNA synthesis as above. The cDNA samples were then subjected to RT-PCR for tilapia *tra1*, *tra2*, and *trb* (GenBank accession numbers: AF302248, AF302249, and AF302247), β-actin (*b-actin*) and *kiss2* mRNAs. The PCR mixture

(20µl) contained 1×PCR buffer,160µM of dNTP mix,250 nM of forward and reverse gene-specific primers (**Table 1**), 1U of Ampli-Taq Gold DNA polymerase (Applied Biosystems), and one 20th of a single cell's cDNA solution. Reaction conditions for PCR were 94°C for 10 min, 40 cycles at 94°C for 15 s, 60°C for 15 s, 72°C for 15 s, and 72°C for 7 min. PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized by illumination under UV light.

#### **DOUBLE-IMMUNOFLUORESCENCE OF GnRH1 FIBERS OR GnRHR WITH kiss2 NEURONS**

To confirm possible associations between GnRH1 and Kiss2 neurons, double-labeling was performed. Kiss2 neurons were detected by fluorescent *in situ* hybridization, while GnRH1 and GnRHR was detected by immunofluorescence. *kiss2* mRNA expressing cells were detected using NEN Fluorescein Tyramide Signal Amplification (TSA™) Plus kit (Perkin Elmer, Wellesley, MA, USA) according to the manufacturer's instruction. GnRH1-immunoreactive fibers were detected with the anti-tilapia GAP1 antibody (#ISP105, dilution of 1:1000) or anti-tilapia GnRHR [#ISPR3, dilution of 1:500; (29)] with Alexa Fluor 594-labeled anti-rabbit IgG (1:500 dilution, Invitrogen). Separate images were captured by using a microscope (ECLIPS 90i, Nikon Instruments) that was attached to a digital cool CCD camera (DMX1200, Nikon) with appropriate excitation for Fluorescein and Alexa Fluor 594, and a computer software (NIS Elements D3.0, Nikon) was used to superimpose the two images. The red channel was then converted to magenta, and brightness and contrast adjustments were made in Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA).

#### **RESULTS**

#### **CLONING AND SEQUENCE ANALYSIS OF TILAPIA kiss2 cDNA**

A full-length cDNA encoding the *kiss2* precursor was isolated from the tilapia, and the cDNA sequence has been deposited in the GenBank (accession number JN565693). The cDNA encoding tilapia *kiss2* is 633 base pairs (bp), containing an open reading frame of 375 bp, 35 bp of 5<sup>0</sup> -UTR, and 223 bp of 3<sup>0</sup> -UTR. The Kiss2 precursor protein has 124 amino acids (aa), with an Nterminal putative signal peptide sequence of 23 aa and a cleavage site (GKR) (**Figure 1A**). Sequence comparison of the deduced protein sequences showed that the tilapia and other vertebrate Kiss precursor proteins are poorly conserved (**Figure 1B**). However, the mature peptide (Kiss2–10) of tilapia and other species exhibit relatively conserved, differing by two amino acid at the position 6 and 7 (phenylalanine to leucine and glycine to serine) (**Figure 1B**). Phylogenetic analysis showed that kisspeptin deduced protein sequences are clustered into two separate clades: Kiss1 and Kiss2. The tilapia Kiss2 is clustered with the Kiss2 clade and shares the highest similarity with sea bass and grouper Kiss2 (**Figure 2A**).

#### **GENE SYNTENY ANALYSIS**

Tilapia Kiss2 encoding sequence was found in the chromosome, scaffold GL831328.1 (location 1,353,904–1,355,714). Chromosome synteny analysis revealed that the neighborhood genes around the tilapia *kiss2* including *ldhba* and *slc25a3* are conserved in other fish Kiss2 genes (**Figure 2B**). Some of gene loci nearby the tilapia Kiss2 including *goltlba* and *gys2* were also found on

#### **FIGURE 1 | cDNA and deduced amino acid sequence of tilapia kiss2**. **(A)** Nucleotide and deduced amino acid sequence of tilapia kiss2. Putative signal peptide (underlined) were predicted using SignalP 3.0 (http://www. cbs.dtu.dk/services/SignalP/). Putative core peptide is boxed. Potential

cleavage amidation site (GKR) is bolded. The stop codon is denoted by an

asterisk. **(B)** Comparison of amino acid sequences of kisspeptin precursors from different species. The mature peptides and potential cleavage amidation site (GKR/GK/GKK) are boxed. Sequences were aligned by the ClustalW program. Gaps (indicated by hyphens) are introduced in some sequences to maximize alignment.

**FIGURE 2 | Genomic analysis of tilapia kiss2 gene**. **(A)** Phylogenetic analysis of KISS precursors in vertebrates. The phylogenetic tree was constructed by MEGA 4.0.2 using the neighbor-joining method with 1000 bootstrap replicates. The number shown at each branch indicates the bootstrap value (%). GenBank accession numbers for KISS: human KISS1 (NP\_002247.3); Mouse KISS1 (AAI17047.1); Goat KISS1 (ACI96030.1); Clawed frog Kiss1 (NM\_001170453.1); Clawed frog Kiss2 (NM\_001162860.1); Zebrafish Kiss1 (NP\_001106961.1); Zebrafish Kiss2 (NP\_001136057.1); Grouper Kiss1 (ADF59544.1); Grouper Kiss2 (ADF59545.1); Medaka Kiss2 (NP\_001153913.1); Fugu Kiss2

human chromosome 12 and mouse chromosome 6 as reported previously (9).

#### **PUTATIVE TRANSCRIPTION FACTOR BINDING SITES ON THE PROMOTER REGION OF TILAPIA kiss2 GENE**

*In silico* analysis of putative transcription binding sites showed the presence of binding sites for several transcription factors such as AP-1, CEBP, GATA-1, Jun-D, YY1, ER, GR, and PR on the putative promoter region of tilapia *kiss2* gene (**Figure 2C**).

(ACM07423.1). Sequences predicted from Ensembl: sea lamprey Kiss2 (Contig Contig37453.1 at location 1700–6241); Lizard Kiss2 (on scaffold\_15 at location 4,601,534–4,601,935); Sea lamprey Kiss1 sequence were previously predicted by van Aerle et al. (30); Goldfish Kiss2 were obtained by Li et al. (31). **(B)** Chromosomal locations kiss2 (blue box) in various teleosts species. **(C)** Putative transcription factor binding sites (closed diamond) on the promoter region of tilapia kiss2 gene. The numbers −2000 to 0 represent distance in bp from the putative transcriptional initiation site.

(BAJ15497.1); Sea bass Kiss1 (ACM07422.1); Sea bass Kiss2

#### **TISSUE DISTRIBUTIONS AND BRAIN LOCALIZATION OF TILAPIA kiss2 mRNA**

RT-PCR analysis was performed to examine the tissue distribution patterns of the tilapia *kiss2* gene expression. In the brain, the tilapia *kiss2* mRNA was highly expressed in the hypothalamus and the pituitary in males and females (**Figure 3**). In peripheral tissues, there were sexual differences in the distribution patterns. In males, the tilapia *kiss2* mRNA was expressed in the spleen,medulla, gills, and testis, whereas in females, the tilapia *kiss2* mRNA was

expressed in the spleen, kidney, intestine, heart, medulla, gills, and ovary (**Figure 3**).

**and female tilapia**. Amplification of β-actin was used as the house-keeping gene control. OB, olfactory bulb; TE, telencephalon; OTT, optic tectum

Digoxigenin-*in situ* hybridization showed tilapia *kiss2* mRNA containing cells in the nucleus of the lateral recess [nRL, also been referred to as the dorsal zone of the periventricular hypothalamus (32)] in the brain (**Figure 4**). No DIG-labeled cells were detected in the brain using sense riboprobes (data not shown).

#### **EFFECT OF THYROID HORMONE (T3) AND HYPOTHYROIDISM ON kiss2, GnRH TYPES AND TRH GENE EXPRESSION**

Real-time PCR showed that administration of T<sup>3</sup> significantly increased the amount of *kiss2* (∼2.3-fold, *P* < 0.001) and *gnrh1* (∼3.2-fold, *P* < 0.001) mRNA levels 24 h post administration when compared with control fish (**Figure 5A**). There was no effect of T<sup>3</sup> treatment on *gnrh2* (*P* = 0.86) and *gnrh3* (*P* = 0.47) mRNA levels (**Figure 5A**).

In the fish treated with MMI, the amount of *kiss2* (∼0.1-fold, *P* < 0.05) and *gnrh1* (∼0.6-fold, *P* < 0.05) mRNA levels were significantly decreased compared with control fish (**Figure 5B**). There was no effect of MMI treatment on *gnrh2* (*P* = 0.08) and *gnrh3* (*P* = 0.14) mRNA levels (**Figure 5B**).

There was no significant effect of thyroid hormone injection and MMI treatment on TRH mRNA levels in the brain (**Figure 6**), indicating the absence of endogenous thyroid hormone feedback effect on *kiss2* mRNA levels.

#### **EXPRESSION OF TR TYPES IN LASER-CAPTURED kiss2 AND GnRH NEURON TYPES**

RT-PCR showed no expression of TR types (*tra1*, *tra2*, and *trb*) mRNA in laser-captured *kiss2* cells (**Figure 7**). In GnRH neuron types, expression of *tra1* mRNA was found in GnRH1 and GnRH2 neurons, *tra2* mRNA was found in GnRH3 neurons, and *trb* mRNA was found in GnRH1, GnRH2, and GnRH3 neurons (**Figure 7**).

#### **POSSIBLE NEURONAL ASSOCIATIONS BETWEEN GnRH1 AND kiss2 NEURONS**

Double-immunofluorescence showed neither close association of GnRH1-immunoreactive fibers with *kiss2* neurons (**Figures 8A–C**) nor co-expression of GnRHR-immunoreactivity

liver; S, spleen; K, kidney; IN, intestine; H, heart; MS, muscle; GI, gill; G,

### **DISCUSSION**

#### **Kiss2 GENE IN THE TILAPIA**

in *kiss2* neurons (**Figures 8D–F**).

gonad; NC, negative control.

The core sequence of tilapia Kiss2 showed high similarities with non-mammalian Kiss2 peptides sharing the F–F form. However, there are the two major amino acid substitutions at positions 6 and 7 (Leu-Ser instead of Phe-Gly) in the core sequence of tilapia Kiss2 decapeptides. As a result, the carboxyl half of core peptide of tilapia Kiss2 (position 6 to 10) is LSLRF, while those of all other Kiss2 identified thus far are FGLRF with complete conservation from lamprey, elephant shark through platypus that have been appeared to possess Kiss2 (7). These two amino acids substitution could be important for binding affinity to Kiss-R and calcium release activity (33). Comparison of genomic sequences showed conserved synteny between the tilapia, zebrafish, puffer fish and medaka, suggesting tilapia Kiss2 gene is ortholog. So far no *kiss1* and *kissr1* homologous sequences have been reported in the tilapia, similar to those fish that possess only one *kiss*-*kissr* gene (5,11). Nevertheless, the presence of *kiss1* in the tilapia remains to be examined.

The expression pattern of *kiss2* mRNA in various tissues in the tilapia is similar to that in other fish species (5, 7). In the brain, *kiss2* mRNA containing cells were seen only in the nRL. However, no *kiss2* cells were seen in other brain region such as the posterior tuberal nucleus or the preoptic area where *kiss2* cells exist in the medaka, zebrafish, goldfish, red seabream (*Pagrus major*) and European sea bass (*D. labrax*) (9, 15, 34–36), which could be due to species difference or because of its low expression levels in the preoptic region. Expression of kisspeptin genes in the pituitary have been reported in several species including mammals and fish (7, 36, 37). The presence of *kiss2* mRNA in the pituitary of tilapia indicates the possibility of *kiss2* mRNA being transported to the nerve terminals as seen in some neuron types (38), or being expressed locally in the pituitary similar to *kiss1* mRNA in the European sea bass and Kiss2-immunoreactive cells in the zebrafish (16, 36). The target site of tilapia Kiss2 neurons is still unknown due to the lack of specific antibody. A recent study in the zebrafish has demonstrated projections

**FIGURE 4 | kiss2 mRNA containing cells in the brain of male tilapia**. **(A)** Schematic drawing of a lateral view of the brain indicating levels of the corresponding transverse section. **(B)** Schematic drawing of transverse section of kiss2 cells (closed circles). **(C,D)** Photomicrographs of DIG-labeled kiss2 cells in the nucleus of lateral recesses (nRL) at **(C)** Lowand **(D)** high-magnification. CP, central posterior thalamic nucleus; Hd, dorsal zone of periventricular hypothalamus; Hv, ventral zone of periventricular hypothalamus; NDLI, diffuse nucleus of the inferior lobe; nRP, nucleus of posterior recess; OT, optic tectum; PTN, posterior tuberal nucleus; TLa, torus lateralis; TPp, periventricular nucleus of posterior tuberculum. Scale bars: (**C)** 200µm; **(D)** 50µm.

of Kiss2-immunoreactive fibers throughout the brain and their close association with GnRH3 (hypophysiotropic GnRH type in the zebrafish) neurons in the preoptic area (16), which suggest the primary role of Kiss2 neurons in gonadotropin secretion possibly through the stimulation of GnRH. It has been shown that electrical stimulation of the nRL in teleosts elicits feeding, gravel picking, and generally aggressive behaviors in cichlids (39). A recent study has shown significant increase in *kiss2* mRNA levels in the hypothalamus during fasting conditions in the Senegalese sole (*Solea senegalensis*) (40). These observations suggest the potential role of Kiss2 in homeostatic regulation as well as ingestive and sexual behaviors as suggested in mammals (41).

The predominant expression of *kiss2* in the brain, testis, and ovary suggests its role in reproductive functions. Specific localization of *kiss2* mRNA in the gonadal tissues has not been studied in teleost, but in the cyclic human and marmoset ovaries, kisspeptinimmunoreactive signals have been located in the theca layer of growing follicles, corpora lutea, interstitial gland, and ovarian surface epithelium (42). Similarly in teleosts, Kiss2 peptides could be locally synthesized in gonadal tissues and could regulate gonadal maturation.

#### **EFFECT OF THYROID HORMONE ON REPRODUCTIVE NEUROENDOCRINE SYSTEM**

The manipulation of thyroid hormone levels significantly altered *kiss2* mRNA levels along with *gnrh1* mRNA levels in the brain of male tilapia. Furthermore, there was no effect of thyroid hormone manipulation on *gnrh2* and *gnrh3* mRNA levels. These results indicate that thyroid hormone may act on kisspeptin-GnRH1 system which plays an important role in the reproductive neuroendocrine axis in the tilapia. A recent study in primates has proposed kisspeptin neurons as candidate action target of thyroid hormone (43). The regulation of GnRH neurons by kisspeptin is critical for the onset of puberty. During the prepubertal stage, sex steroids as well as thyroid hormone are involved in the development of the sexually mature brain. In the quail, thyroid hormone has been reported to cause seasonal change in the morphology of GnRH nerve terminals at the median eminence (44). In monkeys, hypothyroid condition with MMI treatment during the juvenile stage delays the pubertal rise in LH secretion and only 50% of the hypothyroid animals exhibit reactivation of GnRH pulse generator activity (45). In teleosts, there are limited studies that examined the role of thyroid hormone in the regulation of GnRH neurons. In the larval tilapia, the concentration of thyroid hormone levels in the whole-body peak around day 25 after hatching (46), which correspond with the period when GnRH1-immunoreactive cells are morphologically detectable in the preoptic area (47). In the zebrafish, the timing of first appearance of preoptic GnRH3 neurons and that of the increase in *gnrh3* gene expression coincides with the second peak of *kiss2* gene expression (9). However, these studies only support the potential organizational effect of thyroid hormone on the reproductive axis in juvenile or seasonal breeding animals, which is not the case for the present study that demonstrates the activational effect of thyroid hormone on kisspeptin-GnRH axis in the sexually mature fish. Nevertheless, even in non-seasonal breeding animals, thyroid hormone levels are influenced by various factors such as diurnal rhythm

vs. controls.

**FIGURE 6 | Effect of thyroid hormone manipulation on thyrotropinreleasing hormone (TRH) mRNA levels**. There was no significant effect of thyroid hormone (T3) and MMI on TRH mRNA levels in the male tilapia.

methimazole (MMI, 100 ppm for 6 days), mRNA levels of kiss2 and gnrh1

(photoperiod), metabolism, and stress, that have direct or indirect impact on the kisspeptin system (48, 49), which may alter the release of GnRH.

We previously found the significant effect of thyroid hormone on *gnrh1* mRNA levels in sexually mature but not in immature tilapia (22). This result suggests that in sexually mature fish, GnRH1 neurons may acquire sensitivity to thyroid hormone due to the presence of TR, which might be absent in sexually immature fish. Similarly in male monkeys, hypothyroidism fails to prevent the arrest of GnRH pulse generator activity during the infantjuvenile transition (43). Therefore, the action of thyroid hormone on kisspeptin-GnRH neurons could be regulated in a reproductive stage-dependent manner.

#### **EFFECT OF THYROID HORMONE ON GnRH NEURONS: DIRECT AND INDIRECT PATHWAYS**

The role of thyroid hormone in reproductive functions is important during developmental as well as in the adult stages.  A recent study in the rat has shown the presence of type II deiodinase in GnRH neuronal axons in the median eminence as well as in GT1-7 cells (53), indicating the possible synthesis of thyroid hormone within GnRH neurons and possible direct action of thyroid hormone on GnRH neurons. In the present study, we found the expression of TR mRNA types in GnRH1 neurons, which also has been reported in the sheep and hamsters (21). The promoter region of rat GnRH gene contains motifs resembling ER/TR response elements (54). Furthermore, the rat GnRH promoter contains a retinoic acid response element (54), which can interact with TRs alone or with TR/retinoic acid receptor heterodimers (21). Therefore, thyroid hormone can directly act on GnRH1 neurons to regulate the synthesis of GnRH peptides.

In the reproductive axis, pulse, and surge pattern of GnRH secretion are critical. Recent studies in mammals have suggested that kisspeptin neurons in the arcuate nucleus (Arc) are responsible for the pulsatile release of GnRH (55). In ewes, thyroid hormones are required for steroid-independent seasonal LH pulse frequency (24), in which LH pulse frequency and amplitude alters in the absence of estradiol (56). This could be mediated through TR localized in the Arc (57) that contains kisspeptin

**FIGURE 7 | Expression of thyroid hormone receptor (TR) types mRNA in laser-captured GnRH1, GnRH2, GnRH3, and kiss2 neurons**. **(A–H)** Photomicrographs of GnRH1 **(A,B)**, GnRH2 **(C,D)**, GnRH3 immunoreactive **(E,F),** and DIG-labeled kiss2 **(G,H)** neurons before **(A,C,E,G)**, and after **(B,D,F,H)** laser-capture microdissection. Scale bars, 50µm. POA,

preoptic area; MB, midbrain; TN, terminal nerve; nRL, the nucleus of lateral recesses. **(I)** RT-PCR of the tra1, tra2, trb, β-actin, and kiss2 genes in the laser-captured immune-fluorescently labeled three GnRH types neurons (G1, G2 and G3) and DIG-labeled kiss2 neurons (K2). PC, Whole-brain cDNA as a positive control; M, 100-bp DNA ladder.

neurons. Although kisspeptin has been considered a major regulator of GnRH neurons, a morphological study in the rhesus monkey has shown occasional contacts between GnRH axons and kisspeptin neurons in the Arc, indicating the possibility that GnRH could exert control over kisspeptin neuronal activity (58). However, in this study, we failed to observe any GnRH1 fibers or GnRHR in Kiss2 neurons. Therefore, it is possible that the thyroid hormone indirectly regulates Kiss2 neurons via unidentified neuronal population expressing conventional TR.

In the present study, we failed to observe the expression of TR mRNA types in Kiss2 population, which could be due to low expression levels of TR genes in *kiss2* neurons. The absence of TR does not necessarily indicate the possibility of an indirect action of thyroid hormone. Several studies have suggested the presence of a non-classical thyroid hormone signaling pathway, which is non-genomic and does not require thyroid hormone interaction with the TR (59). In addition, *kiss2* gene could also be influenced by estrogen feedback via thyroid hormone action on the hypothalamic-pituitary-gonadal axis (60). Our recent report in goldfish showed presence of ERs in *kiss1* and *kiss2* neurons as well as activation of *kiss1* and *kiss2* gene promoters by estrogen (61). Currently we have no direct evidence of steroid sensitivity of *kiss2* gene in the tilapia, but our promoter analysis showed the presence of two possible ER response elements in the upstream of tilapia *kiss2* gene. Therefore, tilapia *kiss2* gene could also be influenced by estrogen in the male tilapia.

It is well known that TRH and thyroid-stimulating hormone (TSH) genes are regulated by thyroid hormone in mammals via negative feedback mechanism (62). However, in the few fish species studied, both T<sup>4</sup> and T<sup>3</sup> have a negative feedback effect on TSH secretion by the pituitary (63). Furthermore, it is still unknown whether T<sup>4</sup> or T<sup>3</sup> influences hypothalamic release of TRH in teleosts (60). In the present study, we failed to see any change in TRH mRNA expression by thyroid hormone manipulation. Similar observation has been reported in Senegalese sole that hormonal treatments using thiourea and T<sup>4</sup> showed no regulation at transcriptional levels of TRH by thyroid hormones (64) and they suggested that TRH could not participate in the hypothalamic-pituitary-thyroid axis in teleosts. This is further supported by other studies that failed to demonstrate an induction of TSH or T<sup>4</sup> release after TRH treatments in fish (65, 66). Therefore, in the tilapia, TRH could be insensitive to thyroid hormone levels. In addition, in mammals, not all TRH expressing neurons are T<sup>3</sup> responsive (67). Therefore, it is also possible that current treatment protocol (dose and duration) in this study was not sufficient enough to alter TRH mRNA levels. We noted large variation in *gnrh3* mRNA expression in the controls in the two experiments. Such variations in *gnrh3* mRNA levels have previously been reported in teleosts (68, 69), which could be due to different social and reproductive states of fish (70–72).

In summary, we cloned *kiss2* gene in the Nile tilapia. The *kiss2* mRNA was expressed in the central and peripheral tissues. DIG-*in situ* hybridization showed *kiss2* mRNA containing cells in the nRL. Thyroid hormone (T3) treatment significantly increased *kiss2* and *gnrh1* mRNA levels, while those genes were suppressed under hypothyroid condition with MMI treatment. Presence of TR mRNA types in GnRH1 neurons and their absence in Kiss2 neurons suggest that GnRH1 may be directly regulated through thyroid hormone, while the regulation of Kiss2 by T<sup>3</sup> is more likely to be indirect.

#### **AUTHORS CONTRIBUTION**

Satoshi Ogawa and Ishwar S. Parhar designed the study; Satoshi Ogawa performed *in silico* gene sequence analysis, hormone treatments, data analysis, and wrote the manuscript; Kai We Ng performed cloning and real-time PCR; Xiaoyu Xue, Shuisheng Li, Berta Levavi-Sivan, Haoran Lin, Xiaochun Liu performed cloning, sequence analysis, RT-PCR; Priveena Nair Ramadasan performed *in situ* hybridization; Mageswary Sivalingam performed doubleimmunofluorescence and laser capture microdissection; Ishwar

S. Parhar edited the manuscript, all authors approved and commented on the manuscript.

#### **ACKNOWLEDGMENTS**

We thank to Ms. Rachel Anthonysamy for her technical assistance. This work was supported by grants from Malaysian Ministry of Higher Education, FRGS/2/2010/ST/MUSM/03/2 (to Satoshi Ogawa and Ishwar S. Parhar),Malaysian Ministry of Science, Technology, and Innovation, 02-02-10-SF0044 (to Ishwar S. Parhar and Satoshi Ogawa), and Monash University Sunway Campus, M-2-2-06 and M-2-07 (to Satoshi Ogawa), MM-2-5-06 and MM-7-07 (to Ishwar S. Parhar), Neuroscience Research Strength grant (to Ishwar S. Parhar), and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University (to Xiaochun Liu).

#### **REFERENCES**

the GnRH-gonadotroph axis of the adult male rat. *J Endocrinol Invest* (2005) **28**(Suppl 11 Proceedings):20–7.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 22 August 2013; paper pending published: 13 September 2013; accepted: 10 November 2013; published online: 25 November 2013.*

*Citation: Ogawa S, Ng KW, Xue X, Ramadasan PN, Sivalingam M, Li S, Levavi-Sivan B, Lin H, Liu X and Parhar IS (2013) Thyroid hormone upregulates hypothalamic kiss2 gene in the male Nile tilapia, Oreochromis niloticus. Front. Endocrinol. 4:184. doi: 10.3389/fendo.2013.00184*

*This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2013 Ogawa, Ng , Xue, Ramadasan, Sivalingam, Li, Levavi-Sivan, Lin, Liu and Parhar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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