Molecular and Cellular Endocrinology

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1 Molecular and Cellular Endocrinology 324 (2010) Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: Review Seasonal breeding as a neuroendocrine model for puberty in sheep Jeremy T. Smith, Iain J. Clarke Dept Physiology, PO Box 13F, Monash University, Clayton, Victoria 3800, Australia article info abstract Article history: Received 4 December 2009 Received in revised form 28 February 2010 Accepted 3 March 2010 Keywords: Kisspeptin Gonadotropin-inhibitory hormone RF-amide peptide Dopamine Reproduction Puberty is defined as the awakening of the hypothalamic-pituitary gonadal axis. Sheep are seasonal breeders, experiencing an annual period of reproductive quiescence and renaissance that can be utilized as a model for the onset of puberty. Kisspeptin and gonadotropin-inhibitory hormone appear to be important for the seasonal shift in reproductive activity and the former is mandatory for puberty. The non-breeding season is characterized by an increase in the negative feedback effect of estrogen on GnRH and gonadotropin secretion, as is the case in the pre-pubertal period. This effect of estrogen may be transmitted by kisspeptin cells. Additionally, dopaminergic A14/A15 neurons facilitate the seasonal change in estrogen negative feedback. Integrated function of these three groups of neurons appears to modulate the annual shift in photoperiod to a physiological change in fertility. This review compares and contrasts seasonal cycles of reproduction with the mechanisms that relate to the onset of puberty Elsevier Ireland Ltd. All rights reserved. Contents 1. Introduction Kisspeptins Gonadotropin-inhibitory hormone Dopaminergic A14/A15 neurons Thyroid function and type 2 and 3 deiodinases in the hypothalamus Conclusion Acknowledgements References Introduction Like most animals, sheep are seasonal breeders, with an annual cycle of reproduction that is controlled by environmental cues. These cues, primarily the annual photoperiodic cycle, cause changes in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion. Reproductive function is driven by GnRH producing neurons in the brain, which provide stimulus to pituitary gonadotropes. The pulsatile secretion of GnRH into the hypophysial portal system directly stimulates LH secretion and follicle stimulating hormone (FSH) synthesis (Clarke, 1987, 1996). These hormones then stimulate sex-steroid production and gamete formation. In turn, sex-steroids exert feedback control of GnRH and gonadotropin secretion via short-term negative feedback, long term negative feedback and a transient positive Corresponding author. Tel.: ; fax: address: iain.clarke@med.monash.edu.au (I.J. Clarke). feedback in females (essential for the preovulatory LH surge) (Clarke, 1987). It is suggested the transition from the non-breeding (anestrous) season to the breeding season represents a mechanism similar to puberty (Foster, 1988), thus providing a recurring model of transition from a sexually quiescent state to a sexually active state. This is because there are numerous similarities between the two events. In both pre-pubertal and seasonally anestrous ewes, preovulatory LH surges, which cause ovulation, do not occur. In both cases, the neuroendocrine wiring and innate ability to produce a LH surge are present (Foster and Karsch, 1975; Tran et al., 1979; Clarke, 1988) but appear to be suppressed. Prior to puberty, LH secretion is pulsatile, as is the case in the adult, but the frequency of LH pulses is markedly reduced (Foster et al., 1975). Thus, GnRH/LH pulse frequency appears to be the over-riding determinant of reproductive function. Low frequency LH pulses are unable to provide the necessary stimulus for follicular development or produce a sustained rise in estrogen concentration to activate the preovulatory surge mechanism. In the pre-pubertal state, this is due to enhanced sensitivity /$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved. doi: /j.mce

2 J.T. Smith, I.J. Clarke / Molecular and Cellular Endocrinology 324 (2010) to the negative feedback effect of estrogen, which is reversed at the onset of puberty (Foster and Ryan, 1979). The same mechanistic switch occurs in the transition from anestrous to breeding season (Legan et al., 1977; Karsch et al., 1993). The increased estrogen negative feedback response is not a direct response of GnRH neurons, as these do not express the relevant subtype of the estrogen receptor, namely ER (Shivers et al., 1983; Herbison and Theodosis, 1992; Lehman and Karsch, 1993). Regardless, this innate ability to alter feedback sensitivity, during the pubertal transition is retained postpuberty in seasonal animals to regulate seasonal reproduction. In this respect, the onset of the breeding season can be considered an annual model of puberty. The precise neuroendocrine systems underlying the seasonal shift in reproductive function and puberty are not fully elucidated. Both are known to be controlled by environmental cues primarily photoperiod, but also metabolic cues, such as nutrition and body size. Changes in day length are perceived and translated into a physiological signal by the pineal gland, through the nighttime secretion of melatonin. A number of key neuropeptide systems have been implicated in the distal arm of this pathway, sensing the pattern of melatonin secretion and controlling reproduction. 2. Kisspeptins Kisspeptins are the peptide product of the Kiss1 gene and act to stimulate GnRH secretion (Gottsch et al., 2004; Messager et al., 2005). Kisspeptins and their receptor are known to be essential for puberty onset in humans (de Roux et al., 2003; Seminara et al., 2003) and rodents (Seminara et al., 2003). In rodents, two populations of kisspeptin producing cells exist, in the arcuate nucleus (ARC) and the anteroventral periventricular area (AVPV) (Gottsch et al., 2004; Smith et al., 2005a,b). Similarly, in the ovine brain, kisspeptins are located in the ARC and the dorso-lateral preoptic area (POA) (Estrada et al., 2006; Franceschini et al., 2006; Smith et al., 2007). Whereas virtually all kisspeptin cells in the ovine ARC express ER, only 50% of those in the POA do so (Franceschini et al., 2006). A very high percentage of the kisspeptin cells in the arcuate nucleus also express the progesterone receptor (Smith et al., 2007). These ARC cells transmit sex-steroid feedback (both negative and positive) to the GnRH neurons (Smith, 2009). There is a considerable body of evidence that this neuropeptide regulates seasonal reproduction (Revel et al., 2006a; Clarke et al., 2009). Kiss1 mrna expression in the ARC is elevated at the onset of the breeding season (Fig. 1) (Wagner et al., 2008), even in the absence of gonadal feedback in ewes (Smith et al., 2007), and the number of kisspeptin immunoreactive cells is greater in the ARC during the breeding season than in the anestrous season in ovariectomized (OVX) ewes treated with estrogen implants that provide sustained levels of estrogen (Smith et al., 2008). Moreover, the inhibitory effects of chronic estrogen treatment on Kiss1 mrna and kisspeptin expression in the ARC (indicative of negative feedback) are greater during the non-breeding season (Smith et al., 2008). Thus, the seasonal change in sensitivity to estrogen, which is a major mechanism for the switch from breeding to non-breeding season, is most likely to be effected, at least in part, by changing responsiveness of the kisspeptin cells to estrogen (Fig. 1). Furthermore, kisspeptin appears to participate in both the mechanisms of seasonal breeding that are steroid-independent (Robinson et al., 1985; Barrell et al., 1992) and steroid dependent (Legan et al., 1977; Karsch et al., 1993). Importantly, it is the kisspeptin cells in the ARC, and not those in the POA that operate this switch. The seasonal changes in the ARC, but not the POA provide a clear indication that the two populations of kisspeptin cells play different roles in regulation of GnRH secretion. In the ARC of the ewe, there is a greater number of kisspeptin cells and these mediate both negative and positive feedback effects of estrogen (Smith, 2009). The transient positive feedback mechanism that causes the preovulatory LH surge is preceded by the activation of kisspeptin cells and an increase in Kiss1 expression in the ARC (Estrada et al., 2006; Smith et al., 2009a). The involvement of these cells in the positive feedback phenomenon is consistent with induction of preovulatory-like LH surges in ewes following placement of estrogen implants in the mediobasal hypothalamus (Blache et al., 1991; Caraty et al., 1998). Similarly, the negative feedback effects of chronic estrogen treatment is associated with reduced Kiss1 expression and kisspeptin levels in the ARC (Smith et al., 2007), an effect that increases during the non-breeding season (Smith et al., 2008). The function of kisspeptin cells in the POA may be different to those in the ARC, because the level of expression of ER is lower in the former group (Franceschini et al., 2006). Recent data indicate, however, that Kiss1 mrna expression in the POA region also increases immediately prior to the time of the preovulatory LH surge (Smith et al., 2009a). Kiss1 mrna and peptide expression also appear to increase with estrogen treatment (Smith et al., 2008). Whether estrogen acts directly on the subset of these cells that express ER is uncertain, the POA population may form part of an interneuronal pathway connecting kisspeptin cells in the ARC to GnRH neurons. Credence for this kisspeptin-to-kisspeptin is gained by the observation that the kisspeptin neurons of the ARC do not appear to project to GnRH neurons, but those of the POA do so (Backholer et al., 2009). In addition to the seasonal change in Kiss1 expression, the extent to which kisspeptin cells provide input to the GnRH neurons is greater during the breeding season than in the non-breeding season (Fig. 1) (Smith et al., 2008). Presumably this input arises from the preoptic kisspeptin cells although this has not been tested by determining whether the increased input is due to recruitment of cells in the arcuate nucleus. Thus, both the level of kisspeptin expression and the level of kisspeptin input to GnRH neurons are higher during the breeding season, while the negative feedback effects of estrogen on kisspeptin are lower at this time of the year. This is a strong indication that kisspeptin cells play a fundamental role in the seasonal regulation of reproduction. To ascertain whether photoperiod is a primary stimulus for changes in Kiss1 expression between breeding and non-breeding seasons, Wagner et al. (2008) placed Soay ewes on controlled photoperiods. In the ARC of these ovary-intact ewes, Kiss1 expression was 3 times higher on a photoperiod of 8 light and 16 dark (8L:16D) than that in animals on longer photoperiods. Work in seasonal rodents also provides a strong indication that the photoperiodic change in kisspeptin levels is due to alterations in photoperiod, driven by changes in the pattern of melatonin secretion (Simonneaux et al., 2009). In Syrian hamsters, Kiss1 expression is reduced in both the AVPV and ARC under inhibitory photoperiod (short days in this species) (Revel et al., 2007). This dissimilarity between sheep and hamsters in terms of Kiss1 expression and photoperiod is not yet explained. With respect to the control of puberty, original studies (de Roux et al., 2003; Seminara et al., 2003) strongly implicate a role for kisspeptin and its receptor and this is substantiated by studies in rodents, which suggest that kisspeptin cells in the AVPV (Clarkson et al., 2009) and the ARC (Kauffman et al., 2009; Takase et al., 2009) are implicated in the timing of puberty. Kisspeptin appositions to GnRH neurons also increase during pubertal development in mice (Clarkson and Herbison, 2006). To draw a parallel with the control of seasonal breeding, it is necessary to consider the way that melatonin or some other trigger of seasonal transition might act on the kisspeptin cells. In the sheep, melatonin appears to act within the premammilary nucleus of the basal hypothalamus to effect seasonal changes in breeding (Malpaux et al., 2002), but it is not known

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4 J.T. Smith, I.J. Clarke / Molecular and Cellular Endocrinology 324 (2010) how this hormonal signal is translated into a change in GnRH secretion. Kisspeptin cells are ideal candidates, since the premammilary hypothalamic area of the ovine brain overlaps the caudal region of the ARC (Sliwowska et al., 2004), the latter containing a dense population of kisspeptin cells. Melatonin treatment is reported to regulate Kiss1 expression in immortalized cell lines (Gingerich et al., 2009), but whether kisspeptin cells express melatonin receptors is unknown. There is evidence that melatonin receptors are found in this region of the ovine brain, but the level of expression is low (Bittman and Weaver, 1990; Malpaux et al., 1998). It remains possible that another signal is operative to cause a seasonal change in expression of kisspeptin cells. For example, the level of circulating prolactin is closely related to the seasonal breeding status of animals such as sheep (Lincoln et al., 2006) and it is possible that the action of this hormone causes a change in the activity of kisspeptin cells in the ARC, driving seasonal switches from breeding to non-breeding seasons. It would be informative to ascertain whether kisspeptin cells express prolactin receptors and whether prolactin changes the function of kisspeptin cells. Given that acyclicity in female sheep during the non-breeding season is associated with reduced kisspeptin function, it is not surprising that kisspeptin treatment can induce ovulation in such animals (Caraty et al., 2007). Moreover, kisspeptin treatment advances the onset of puberty in rats (Navarro et al., 2004). This lends further support to the notion that this neuropeptide system may be primarily responsible for the shift in fertility. Importantly, the data indicate that during the non-breeding season a prolonged kisspeptin infusion stimulated GnRH/LH secretion to levels comparable to the follicular phase of the estrous cycle during the breeding season. This presumably activated the ovarian production of estrogen, which then initiated positive feedback circuits in the brain. Importantly, kisspeptin treatment was able to induce LH secretion that was sustained and not overcome by the negative feedback effects of estrogen during the non-breeding season. This again points to the negative feedback effects of estrogen involving kisspeptin cells and kisspeptin treatment is distal to this. The LH response to peripherally administered kisspeptin varies between the breeding and non-breeding seasons. Surprisingly, it appears that the LH response to kisspeptin is greater during the non-breeding season in ewes (Smith et al., 2009b). This can be interpreted to mean the hypothalamic-pituitary gonadal axis is primed for a maximal response to an increase in kisspeptin levels at the onset of the breeding season, leading to the transition from nonbreeding to breeding status. Alternatively, the seasonal breeding status of numerous species does not appear to be the result of any change in GnRH synthesis (Clarke and Pompolo, 2005), but a change in the releasable pool of GnRH in the median eminence could occur. Thus, the greater LH response to kisspeptin during the non-breeding season may be interpreted to mean that the low GnRH/LH pulse frequency during this time leads to a build up in the releasable pool of GnRH in the median eminence and LH in the gonadotropes (Clarke and Cummins, 1985). Importantly, the variable effects of kisspeptin on LH appeared to be similar to the effect of GnRH treatment on LH (i.e. greater LH response during the nonbreeding season), thus the change in response could be a reflection of the GnRH effect on releasable LH. Clearly, kisspeptin is critical for the onset of puberty and appears to be similarly important for the seasonal shift in reproduction. The underlying mechanism driving pubertal and seasonal changes in kisspeptin remain to be fully elucidated. Both puberty and the shift to the breeding season involve a similar pattern of GnRH/gonadotropin response, particularly in relation to the release from enhanced sex-steroid negative feedback. The seasonal transition is most likely driven by melatonin concentrations, but puberty is not; this is an essential difference. Importantly however, melatonin concentrations can impact on pubertal development, preventing the onset of puberty during the non-breeding season (Yellon and Foster, 1986; Buchanan and Yellon, 1991). 3. Gonadotropin-inhibitory hormone Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic peptide that inhibits gonadotropins in birds (Tsutsui et al., 2000; Tsutsui, 2009) and in mammals (Kriegsfeld et al., 2006; Johnson et al., 2007; Clarke et al., 2008; Anderson et al., 2009; Ubuka et al., 2009). Mammalian GnIH has been localised to the dorsomedial nucleus of the hypothalamus and termed RF-amide related peptide-3 (RFRP-3), but there is no reason why the original nomenclature (GnIH) should not extend to all species. However, any implication that GnIH is involved in the onset of puberty is limited. In quail, GnIH content in the hypothalamus is negatively correlated with gonadotropin concentrations during development (Ubuka et al., 2003) and GnIH administration to immature birds suppresses the normal developmental rise in testosterone (Ubuka et al., 2006). RFRP-3 antisense oligonucleotide infusion into the third ventricle reduced RFRP-3 protein expression in the hypothalamus, elevates LH concentrations, but did not advance the onset of puberty in male rats (Johnson and Fraley, 2008). Similarly, intracerebroventricular infusion of RFRP-3 reduced plasma LH levels in male rats, but did not alter the timing of puberty (Johnson and Fraley, 2008). Treatments started at PND35 and puberty (pre-putial separation) occurred PND39. It is possible that the infusion of GnIH may have started too late to influence the onset of puberty in this model and the slight reduction in LH levels that were achieved suggests that a higher dose may have been effective in blocking puberty. This, the question as to whether effective doses of GnIH can block puberty in mammals remains an open question. In sheep, as in other mammalian species, RFRP-3 expressing cells are located predominantly in the dorsomedial nucleus of the hypothalamus (Clarke et al., 2008) and these cells project to GnRH cells, the median eminence (Clarke et al., 2008), and also appetite regulating cells (Qi et al., 2009). Intravenous infusion of RFRP-3 suppresses LH pulses in OVX ewes (Clarke et al., 2008) and in vitro data suggest direct action on the gonadotropes of the pituitary gland (Clarke et al., 2008). This is further substantiated by the effect to reduce gonadotropin subunit expression in GnRHstimulated gonadotropes in culture (Sari et al., 2009). RFRP-3 also appears to have direct inhibitory effects on GnRH neurons in mice (Ducret et al., 2009; Wu et al., 2009), and so the effects of this neuropeptide may be mediated at the hypothalamus and pituitary. Using immunohistochemistry, a lower number of RFRP-3 cells is detected in the breeding season than in the non-breeding season in ewes (Fig. 1), but no change was seen in RFRP gene expression assessed by in situ hybridization (Smith et al., 2008). In Soay ewes, however, RFRP mrna expression (also assessed by in situ hybridization) was higher in animals held at a long-day (16L:8D) photoperiod Fig. 1. Model for the control of seasonal breeding in the ewe involving changes in kisspeptin, GnIH and A14/A15 dopaminergic neuronal systems. Changes in photoperiod dictate the period of the breeding season (Autumn/Winter) in ewes (blue shading). This period is characterized by an increase in luteinizing hormone (LH) pulse frequency enabling the preovulatory LH surge. Kisspeptin, during the breeding season the number of identifiable kisspeptin expressing cells and appositions from kisspeptin cells to GnRH neurons increase. Alternatively, the negative feedback effects of estrogen (E) appear to decrease compared to the non-breeding anestrous season. GnIH, The number of GnIH cells and appositions to GnRH neurons decreases during the breeding season compared to the non-breeding anestrous season. A14/A15 Neurons, Dopaminergic neurons in the A14/A15 region of the hypothalamus are known to be central for the change in E negative feedback being responsive to E treatment in the non-breeding anestrous season (as indicated by the induction of the immediate early gene FOS). These cells are known to project to the mediobasal hypothalamus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

5 106 J.T. Smith, I.J. Clarke / Molecular and Cellular Endocrinology 324 (2010) than in those on short day (8L:16D) photoperiod (Dardente et al., 2008). Although, in ewes held at increasingly long daylight hours (20L:4D and 22L:2D) the effect on RFRP gene expression appears to be lost (Dardente et al., 2008). The difference between these two studies may relate to the ewes in the former being exposed to natural photoperiod conditions (Smith et al., 2008), whereas in the later artificial photoperiod was applied (Dardente et al., 2008). Moreover, after transferring ewes to a long-day photoperiod, no effect on RFRP gene expression was after 42 days (Dardente et al., 2008). This lack of a short-term effect is consistent with the notion that the transition between the breeding and non-breeding seasons requires approximately 60 days (Lincoln, 1999), so it remains possible that GnIH function is a fundamental aspect of this mechanism. The lower level of RFRP-3 producing cells that is seen during the breeding season is accompanied by a reduction in the number of GnRH cells contacted by RFRP-3 terminals (Fig. 1) (Smith et al., 2008). Given the role of RFRP-3 on the reproductive system, the net effect of these data would indicate RFRP-3 plays a role in suppressing fertility during the non-breeding season. There is also evidence that GnIH contributes to seasonality in birds. Specifically, GnIH peptide levels are highest at the end of the breeding season, when GnRH levels are lowest (Bentley et al., 2003) and the number of RFRP axons found in the POA is lower in the breeding season (Small et al., 2007). Moreover, GnIH concentration and mrna levels are increased in birds held at a short-day photoperiod, mimicking the non-breeding season, and in long-day photoperiod (breeding season) birds treated with melatonin (Ubuka et al., 2005). This is entirely consistent with a role for GnIH as an inhibitor of reproduction. Photoperiodic effects on RFRP have been observed in the hamster (Revel et al., 2008), but the expression of RFRP mrna in the dorsomedial nucleus of the hypothalamus was lower during the short-day photoperiod (during the non-breeding season) in both Syrian and Siberian hamsters (Revel et al., 2008). Contrary to this, a recent paper reports that GnIH expression is reduced in Siberian hamsters during long days (mimicking the breeding season) and is increased upon transition to a intermediate short day (Paul et al., 2009). Thus, the precise role of RFRP in the seasonal breeding of this species requires clarification. Moreover, the effects of season on RFRP appeared to be dependant on seasonal melatonin profiles as the response to photoperiod was prevented by pinealectomy and counteracted by melatonin replacement (Revel et al., 2008). These data are somewhat perplexing and not in accord with the data obtained in sheep, so it will be interesting to verify the findings in this particular seasonal mammal. As for kisspeptin cells, there are no direct data to indicate that mammalian RFRP cells possess melatonin receptors, which would provide a direct link between the pineal gland and gonadotropin secretion. Importantly, the dorsomedial nucleus of the hypothalamus, containing RFRP neurons, is a site of melatonin binding and is important for the inhibition of reproduction to short-day photoperiods in hamsters (Maywood et al., 1996). Data from birds suggest that at least some GnIH cells express melatonin 1c receptor (Ubuka et al., 2005), but similar studies are required in mammals. 4. Dopaminergic A14/A15 neurons Original work by Thiery et al. (1989) identified a region of the ovine hypothalamus that appears to be involved in the seasonal regulation of reproduction in sheep. Lesions of the A14/A15 dopaminergic cell group led to disruption of seasonal breeding cycles and seasonal change in estrogen negative feedback sensitivity (Thiery et al., 1989, 1995). Local administration of a dopaminergic neurotoxin to the A14/A15 region also increased LH pulse frequency in anestrous ewes (Havern et al., 1994). In further support for the theory that catecholaminergic cells are instrumental in effecting seasonal changes in reproductive function, systemic administration of both -adrenergic and dopaminergic D2 receptor antagonists increase LH pulse frequency in anestrous ewes, but have no effect during the breeding season (Meyer and Goodman, 1985). Importantly, these effects are estrogen-dependant (Meyer and Goodman, 1986), indicating dopaminergic cells in the brain as estrogen-sensitive neuronal system that inhibit GnRH and LH. Estrogen treatment stimulates tyrosine hydroxylase expression, the rate-limiting enzyme in the synthesis of dopamine, in the A15 region of anestrous ewes (Gayrard et al., 1994). Moreover, estrogen also induces Fos expression in A14/A15 dopaminergic neurons during the non-breeding season but not during the breeding season (Fig. 1) (Lehman et al., 1996). A14/A15 dopaminergic neurons do not, however, express ER (Lehman and Karsch, 1993), so some afferents to these cells must be responsible for the estrogen effect. Virtually all A14/A15 dopaminergic neurons are contacted by glutamatergic terminals (Singh et al., 2009), and glutamate cells of the preoptic area express ER (Pompolo et al., 2003). Importantly, the number of glutamatergic appositions onto A14/A15 dopaminergic neurons is greater during the non-breeding season (Singh et al., 2009). Finally, glutamate receptor antagonists increase pulsatile LH secretion in anestrous ewes when administered into the A14/A15 region (Singh et al., 2009). One key issue to be elucidated is the mechanism by which changes in photoperiod with season are able to regulate these neuronal systems, through changes in melatonin secretion/action. In spite of the evidence that A14/A15 dopaminergic cells are key players in the seasonal changes in breeding in sheep, these cells do not appear to project to GnRH cell bodies (Tillet et al., 1989). Dopaminergic terminals in the median eminence may, however, come into close contact to GnRH terminals (Kuljis and Advis, 1989). A14/A15 dopaminergic terminals also traverse the medial basal hypothalamus (and the ARC) where they are known to act (Havern et al., 1991). It is possible, but remains to be determine, these cells may regulate kisspeptin cell function during the anestrous season. 5. Thyroid function and type 2 and 3 deiodinases in the hypothalamus It is well established that functional thyroid glands are mandatory for the transition between breeding and non-breeding seasons. In ewes, thyroid function is essential for the estrogen-induced suppression of gonadotropin secretion in OVX ewes that occurs at the onset of the non-breeding season (Webster et al., 1991a,b; Dahl et al., 1995), but does not appear to be involved in the onset of the breeding season. In the brain, as in other parts of the body, the local concentrations of thyroid hormones are controlled by type 2 and type 3 deiodinases, the former catalysing conversion of thyroxine (T4) to, the more active triiodothyronine (T3) and the later producing inactive forms. Recently, type 2 and type 3 deiodinase have been implicated in the seasonal reproduction of birds. Specifically, the expression of type 2 deiodinase in the hypothalamus is elevated and type 3 reduced in birds transferred to a long-day photoperiod (reinstating reproduction) (Yoshimura et al., 2003). This effect is likely to increase the local concentration of active T3, and therefore it is not surprising that direct administration of T3 can also stimulate sexual activity in birds subjected to short-day photoperiod (Yoshimura et al., 2003). Similar results have also been reported in hamsters (Watanabe et al., 2004; Revel et al., 2006b; Barrett et al., 2007). In sheep, transfer to long-day photoperiod induces a rapid increase in the expression of type 2 deiodinase in the mediobasal hypothalamus (Hanon et al., 2008). Furthermore in male Saanen goats, which are also short-day breeders, the expression of type 2 deiodinase and T3 content in the hypothalamus was reduced in goats held at

6 J.T. Smith, I.J. Clarke / Molecular and Cellular Endocrinology 324 (2010) The number of kisspeptin terminal appositions to GnRH neurons increase. Kisspeptin treatment can stimulate reproductive function. A direct effect of RFRP on the onset of puberty in mammals has not yet been demonstrated, but it appears to play a role in seasonal reproduction. Equally, the role of dopaminergic A14/A15 neurons and thyroid hormone in the seasonal control of reproduction is well established, but it is unclear at this stage whether these can be extrapolated to direct mechanisms involved in the onset of puberty. Overall, the changes in two newly discovered neuropeptide system; kisspeptin and GnIH, offer new interpretations for the control of fertility that may also relate to the onset of puberty. Acknowledgements Fig. 2. The Nexus between season and puberty. Day length determines seasonal breeding patterns through a mechanism that involves the pineal production of melatonin. In both the non-breeding season and pre-pubertal state, enhanced steroid negative feedback suppresses the reproductive axis. This negative feedback sensitivity is potentially mediated by the kisspeptin/gnih/dopaminergic systems. In seasonal breeders, pubertal transition cannot occur in the non-breeding season. In the breeding season and in the post-pubertal state, release from negative feedback allows the operation of a closed-loop hypothalamo-pituitary gonadal axis. In the special case of ovulation, increased levels of estrogen lead to positive feedback and the LH surge. The switch from negative to positive feedback involves neuronal systems in the hypothalamus, notably kisspeptin (upregulation) and GnIH (downregulation). The precise pathways linking puberty and season to the kisspeptin/gnih/dopaminergic systems are not fully defined (indicated by? ). (SCN) suprachiasmatic nucleus; (SCG) superior cervical ganglion. long-day photoperiods (Yasuo et al., 2006). Thus, the net effect of these changes may be a rapid but transient induction of T3 upon transfer to long-day photoperiod. In both studies, type 2 deiodinase appeared to be regulated in the caudal portion of the ARC (Yasuo et al., 2006; Hanon et al., 2008), where thyroid hormone acts to regulate breeding season in sheep (Anderson et al., 2003), where melatonin receptors are located (Malpaux et al., 2002) and where seasonal changes in kisspeptin neurons occur (Smith et al., 2007, 2008). Clearly, the changing concentrations of thyroid hormones appear central in seasonal breeding and these hormones are known to have direct effects on the plasticity of the central nervous system (Bernal, 2002), which is known to contribute to seasonal breeding in the ewe (Adams et al., 2006). It is enticing to speculate that these hormones are directly involved in the changes in glutamatergic appositions onto A14/A15 dopaminergic neurons, and/or kisspeptin or GnIH neuronal systems. 6. Conclusion In summary, the seasonal fluctuation in fertility appears to be mediated by a number of neuropeptide systems with the net effect being upregulation of neuroendocrine stimulators and downregulation of inhibitors at the onset of the breeding season. Interestingly, these regulators originate from distinct regions of the hypothalamus, indicating that an integrated mechanism is likely to be involved. What the hierarchical relationship is, how it is arranged, and the means by which the primary stimulus, melatonin profile, is received, is not yet certain. Many of these regulators of breeding season appear similar to those neuroendocrine mechanisms that govern the onset of puberty (Fig. 2). Regarding kisspeptin, across pubertal development and at the onset of the breeding season: Cellular expression (Kiss1 mrna and kisspeptin protein) increases. This work was supported by the National Health and Medical Research Council of Australia. 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