Valérie Simonneaux

Seasonal Regulation of Reproduction in Mammals

Most mammals use photoperiod to synchronize their breeding season, so that young are raised during the spring and summer months. The pineal hormone melatonin is the major endocrine message used to relay photoperiodic information to the reproductive axis. This chapter first discusses the ecological context for seasonal breeding and the pivotal role that nocturnal melatonin secretion plays in its regulation. Melatonin acts through high affinity G protein–coupled receptors, and we discuss the emergence of the pars tuberalis of the anterior pituitary as a principal site of melatonin action. We go on to discuss recent evidence that local metabolism of thyroid hormone by tanycyte cells within the mediobasal hypothalamus is the key seasonal gateway for activation of the hypothalamic gonadotropin-releasing hormone pulse generator. Melatonin controls tanycyte function via a pathway involving a novel paracrine action of thyrotropin released by pars tuberalis cells. Downstream targets of this control pathway include RF-amide neurons implicated in hypothalamic gonadotropic signaling, suggesting an integrated model for the control of seasonal reproduction in mammals. We end by focusing on current hypotheses for mechanisms of tanycyte action and the cellular consequences of altered hypothalamic triiodothyronine metabolism.

A circannual clock drives expression of genes central for seasonal reproduction.

Animals living in temperate zones anticipate seasonal environmental changes to adapt their biological functions, especially reproduction and metabolism. Two main physiological mechanisms have evolved for this adaptation: intrinsic long-term timing mechanisms with an oscillating period of approximately 1 year, driven by a circannual clock [1], and synchronization of biological rhythms to the sidereal year using day length (photoperiod) [2]. In mammals, the pineal hormone melatonin relays photoperiodic information to the hypothalamus to control seasonal physiology through well-defined mechanisms [3-6]. In contrast, little is known about how the circannual clock drives endogenous changes in seasonal functions. The aim of this study was to determine whether genes involved in photoperiodic time measurement (TSHβ and Dio2) and central control of reproduction (Rfrp and Kiss1) display circannual rhythms in expression under constant conditions. Male European hamsters, deprived of seasonal time cues by pinealectomy and maintenance in constant photoperiod, were selected when expressing a subjective summer or subjective winter state in their circannual cycle of body weight, temperature, and testicular size. TSHβ expression in the pars tuberalis (PT) displayed a robust circannual variation with highest level in the subjective summer state, which was positively correlated with hypothalamic Dio2 and Rfrp expression. The negative sex steroid feedback was found to act specifically on arcuate Kiss1 expression. Our findings reveal TSH as a circannual output of the PT, which in turn regulates hypothalamic neurons controlling reproductive activity. Therefore, both the circannual and the melatonin signals converge on PT TSHβ expression to synchronize seasonal biological activity.

Maturation of kisspeptinergic neurons coincides with puberty onset in male rats.

Kisspeptins, derived from the Kiss1 gene play a central role in activation of the hypothalamo-pituitary gonadal (HPG) axis via stimulation of GnRH neurons. Both Kiss1 and Kiss1R (receptor) mRNA levels are found to be low in pre-pubertal rats, but whether an increase in kisspeptin and/or its receptor is the primary component in the initiation of puberty and where in the hypothalamus regulation of the kisspeptin/Kiss1R system occurs is unresolved. Using immunohistochemistry and in situ hybridization, we analyzed the level of Kiss1 mRNA and kisspeptin-immunoreactivity in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus of male rats along pubertal development. Neurons expressing Kiss1 mRNA were first detected at PND15, but increased significantly around puberty, and declined again in the adult rat. While virtually no immunoreactive cell bodies were detectable in the AVPV at any age, numerous kisspeptin-positive neurons in the arcuate nucleus were detected in the adult rat. Increasing doses of kisspeptin-54 given peripherally to male rats at PND15, 30, 45, and 60 evoked roughly similar effects, as revealed by the induction of c-Fos in the pituitary and secretion of LH and testosterone. These results show that both Kiss1 mRNA and the peptide increase in arcuate nucleus along pubertal maturation. Since kisspeptin signaling is potentially functional, even for peripheral activation, and well before the kisspeptin neuronal system is fully matured, our data support that the regulation of kisspeptin synthesis and release are key events in puberty onset in the male rat.

Circannual variation in thyroid hormone deiodinases in a short-day breeder

At temperate latitudes, many mammals and birds show internally timed, long‐term changes in seasonal physiology, synchronised to the seasons by changing day length (photoperiod). Photoperiodic control of thyroid hormone levels in the hypothalamus dictates the timing. This is effected through reciprocal regulation of thyroid hormone deiodinase gene expression. The local synthesis of type 2 deiodinase (Dio2) promotes triiodothyronine (T3) production and summer biology, whereas type 3 deiodinase (Dio3) promotes T3 degradation and winter biology. In the present study, we investigated the extent to which the hypothalamic expression of Dio2 and Dio3 is circannually regulated in the Soay sheep, a short‐day breeding mammal. Male sheep were exposed to a long photoperiod (LP; 16 : 24 h light/dark cycle) or a short photoperiod (SP; 8 : 24 h light/dark cycle), for up to 28 weeks to establish four different endocrine states: (i) LP animals in a spring/summer‐like state of reproductive arrest; (ii) LP refractory (LPR) animals showing spontaneous reproductive reactivation; (iii) SP animals showing autumn/winter‐like reproductive activation; and (iv) SP refractory (SPR) animals showing spontaneous reproductive arrest. A complex pattern of hypothalamic Dio2 and Dio3 expression was observed, revealing distinctive photoperiod‐driven and internally timed effects for both genes. The patterns of expression differed both spatially and temporally, with phases of peak Dio2 expression in the median eminence and tuberoinfundibular sulcus, as well as in the paraventricular zone (PVZ) (maximal under LP), whereas Dio3 expression was always confined to the PVZ (maximal under SP). These effects likely reflect the distinct roles of these enzymes in the localised control of hypothalamic T3 levels. The spontaneous decline in Dio2 and spontaneous increase in Dio3 in LPR animals occurred with a corresponding decline in thyroid‐stimulating hormone β expression in the neighbouring pars tuberalis (PT), although this relationship did not hold for the corresponding Dio2 increase/Dio3 decrease seen in SPR animals. We conclude that internally timed and spatially regulated changes in Dio2 and Dio3 expression may drive the cycling between breeding and nonbreeding states in long‐lived seasonal species, and may be either PT‐dependent or PT‐independent at different phases of the circannual cycle.

Photoperiodic Control of the Rat Pineal Arylalkylamine-N-Acetyltransferase and Hydroxyindole-O-Methyltransferase Gene Expression and Its Effect on Melatonin Synthesis

Photoperiodic changes of pineal melatonin (MEL) profile are accompanied by parallel changes of arylalkylamine-N-acetyltransferase (AA-NAT) activity. In the present study, the authors investigated, for the first time, whether two other important variables of pineal metabolism, AA-NAT and hydroxyindole-O-methyltransferase (HIOMT) gene expression, also may be affected by the photoperiod. Evening rises in AA-NAT and HIOMT mRNA and in circulating MEL occurred concomitantly with an increased delay from dark onset as scotophase shortened. On the opposite, the morning declines of all three variables occurred with different kinetics but were locked to light onset. These observations demonstrate that the daily rhythms in AA-NAT and HIOMT gene expression are modulated by the photoperiod and bring further evidence in favor of nor adrenaline as the possible link between the endogenous clock and MEL. Interestingly, the duration of the nocturnal peak in HIOMT mRNA was positively correlated with HIOMT activity. In conclusion, this study adds two important links to the chain of mechanisms involved in the photoperiodic control of pineal metabolism. First, photoperiodic modulation of the MEL rhythm primarily results from changes in the AA-NAT gene expression. Second, the photoperiodic regulation of HIOMT activity occurs at the transcriptional level.

Distribution and Seasonal Variation in Hypothalamic RF-amide Peptides in a Semi-Desert Rodent, the Jerboa

The jerboa is a semi‐desert rodent, in which reproductive activity depends on the seasons, being sexually active in the spring–summer. The present study aimed to determine whether the expression of two RF‐amide peptides recently described to regulate gonadotrophin‐releasing hormone neurone activity, kisspeptin (Kp) and RF‐amide‐related peptide (RFRP)‐3, displays seasonal variation in jerboa. Kp and/or RFRP‐3 immunoreactivity was investigated in the hypothalamus of jerboas captured in the field of the Middle Atlas mountain (Morocco), either in the spring or autumn. As in other rodents, the Kp‐immunoreactive (‐IR) neurones were found in the anteroventro‐periventricular and arcuate nuclei. RFRP‐3 neurones were noted within the dorso/ventromedial hypothalamus. A marked sexual dimorphism in the expression of Kp (but not RFRP‐3) was observed. The number of Kp‐IR neurones was nine‐fold higher, and the density of Kp‐IR fibres and terminal‐like elements in the median eminence was two‐fold higher in females than in males. Furthermore, a significant seasonal variation in peptide expression was obtained with an increase in both Kp‐ and RFRP‐3‐IR cell bodies in sexually active male jerboas captured in the spring compared to sexually inactive autumn animals. In the arcuate nucleus, the level of Kp‐IR cells and fibres was significant higher during the sexually active period in the spring than during the autumnal sexual quiescence. Similarly, the number of RFRP‐3‐IR neurones in the ventro/dorsomedial hypothalamus was approximately three‐fold higher in sexually active jerboa captured in the spring compared to sexually inactive autumn animals. Altogether, the present study reports the distribution of Kp and RFRP‐3 neurones in the hypothalamus of a desert species and reveals a seasonal difference in their expression that correlates with sexual activity. These findings suggest that these two RF‐amide peptides may act in concert to synchronise the gonadotrophic activity of jerboas with the seasons.

Endogenous rhythmicity of Bmal1 and Rev-erbα in the hamster pineal gland is not driven by norepinephrine

Pineal melatonin is synthesized with daily and seasonal rhythms following the hypothalamic clock‐driven release of norepinephrine (NE). The pineal gland of rats and mice, like the biological clock, expresses a number of clock genes. However, the role of pineal clock elements in pineal physiology is still unknown. We examined the expression and regulation of several clock genes (Per1, Cry2, Bmal1 and Rev‐erbα) under different lighting conditions or following adrenergic treatments in the Syrian hamster, a seasonal rodent. We found that Per1 and Cry2 genes were similarly regulated by the nocturnal release of NE: levels of Per1 and Cry2 mRNA displayed a nocturnal increase that was maintained after 2 days in constant darkness (DD) but abolished after 2 days under constant light (LL), a condition that suppresses endogenous NE release, or after an early night administration of the adrenergic antagonist propranolol. In contrast, Bmal1 and Rev‐erbα exhibited a different pattern of expression and regulation. mRNA levels of both clock genes displayed a marked daily variation, maintained in DD, with higher values at midday for Bmal1 and at day/night transition for Rev‐erbα. Remarkably, the daily variation of both Bmal1 and Rev‐erbα mRNA was maintained in LL conditions and was not affected by propranolol. This study confirms the daily regulation of Per1 and Cry2 gene expression by NE in the pineal gland of rodents and shows for the first time that a second set of clock genes, Bmal1 and Rev‐erbα are expressed with a circadian rhythm independent of the hypothalamic clock‐driven noradrenergic signal.

Photoperiodic Co-Regulation of Kisspeptin, Neurokinin B and Dynorphin in the Hypothalamus of a Seasonal Rodent

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin‐releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co‐expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod‐dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co‐express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo‐inhibitory conditions. In seasonal rodents, RFamide‐related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co‐localise with RFRP‐immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short‐day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down‐regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up‐regulated under a short photoperiod.

A Noradrenergic Sensitive Endogenous Clock Is Present in the Rat Pineal Gland

The aim of this study was to examine the occurrence of endogenous oscillations of Per1, Per2, Bmal1 and Rev-erbα genes in rat pineal explants and to investigate their regulation by adrenergic ligands. Our results show a significant and sustained rhythm of Per2,Bmal1 and Rev-erbα gene expression for up to 48 h in cultured pineal gland with a pattern similar to that observed in vivo. By contrast, the rhythms of Per1 and Aa-nat, the rate-limiting enzyme for melatonin synthesis, were strongly attenuated after 24 h in culture. Addition of the exogenous adrenergic agonist isoproterenol on cultured pineal glands induced a short-term increase in mRNA levels of Per1 and Aa-nat, but not those of Per2,Bmal1 and Rev-erbα. This study demonstrates that the rat pineal gland hosts a circadian oscillator as evidenced by the sustained, noradrenergic-independent, endogenous oscillations of Per2, Bmal1 and Rev-erbα mRNA levels in cultured tissues. Only expression of Per1 was stimulated by adrenergic ligands suggesting that, in vivo, the adrenergic input could synchronize the pineal clock by acting selectively on Per1.

Sex differences in the photoperiodic regulation of RF-Amide related peptide (RFRP) and its receptor GPR147 in the syrian hamster.

RF‐(Arg‐Phe) related peptides (RFRP‐1 and ‐3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short photoperiods to investigate the neuroanatomical basis of these differential effects. The neuroanatomical distribution of RFRP neurons and fibers, revealed using an antiserum recognizing RFRP‐1 and ‐3, as well as GPR147 mRNA, are similar in male and female Syrian hamsters. RFRP neurons are mainly found in the medial hypothalamus, whereas RFRP projections and GPR147 mRNA are observed in the preoptic area, anteroventral–periventricular nucleus, suprachiasmatic nucleus, paraventricular nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamus, habenular nucleus, and arcuate nucleus. The number of RFRP neurons is higher in females than in males, and in both sexes, the number of RFRP neurons is reduced in short photoperiods. GPR147 mRNA levels are higher in females than in males and are downregulated in short photoperiods, particularly in females. Interestingly, the number of RFRP‐positive fibers in the anteroventral–periventricular nucleus is higher only in females adjusted to a short photoperiod. Our results suggest that the RFRP system, which is strongly regulated by photoperiod in both male and female Syrian hamsters, is particularly important in females, with a distinct role in the anteroventral–periventricular nucleus, possibly in the regulation of the preovulatory luteinizing hormone surge via kisspeptin neurons. J. Comp. Neurol. 524:1825–1838, 2016.