Eric L. Bittman

Neuroendocrine basis of seasonal reproduction

Publisher Summary This chapter discusses the strategy of seasonal breeding, the role of photoperiod in timing the annual reproductive cycle, the hypothalamo-pituitary mechanisms that mediate photoperiodic regulation of estrous cyclicity, and the photoperiodic pathway to luteinizing hormone (LH) pulse generator. To understand how photic input to the LH pulse generator leads to seasonal changes in gonadal activity, the sequence of endocrine events that normally leads to ovulation during the estrous cycle of the ewe must be considered. These preovulatory events occur during a 2–3 day follicular phase and include a precipitous drop in progesterone, a progressive rise in tonic LH secretion, a sustained increase in estradiol secretion, and the LH surge. The pivotal step in this sequence is the sustained increase in tonic LH secretion. A great deal of insight has been gained into the complex interplay between the neural and endocrine response systems that underlie the seasonal reproductive process in the short-day breeding ewe. Specifically, light cues activate retinal photoreceptors and are transmitted via a monosynaptic tract to the suprachiasmatic nuclei of the hypothalamus. After interacting with the circadian system, the photic information is relayed to the pineal gland that transduces the neural message into a hormonal signal in the form of a circadian rhythm of melatonin secretion. The pattern of this melatonin signal, which is interpreted as inductive or suppressive, sets the frequency of the LH pulse generator and determines the capacity of this neural oscillator to respond to the negative feedback action of estradiol. The resulting changes in the episodic pattern of gonadotropin secretion, in turn, dictate whether or not estrous cycles can occur.

The timed infusion paradigm for melatonin delivery: what has it taught us about the melatonin signal, its reception, and the photoperiodic control of seasonal responses?

Abstract: This review summarizes the evidence showing that the duration of the nocturnal secretory profile of pineal melatonin (MEL) is critical for eliciting seasonally appropriate reproductive physiological and behavioral responses in mammals. We review experiments using the timed infusion paradigm (TIP) to deliver MEL either systemically or centrally to pinealectomized hamsters and sheep. In this paradigm, MEL is infused, usually once daily, for a specific number of hours and at a predetermined time of day. This experimental strategy tests most directly those features of the MEL signal that are necessary to trigger photoperiodic responses. The data suggest that the duration of the MEL stimulation is the critical feature of the MEL signal for both inhibitory and stimulatory effects of the hormone on the photoperiodic control of reproductive development in juvenile Siberian hamsters, and for the photoperiodic control of reproductive and metabolic responses in adult Siberian and Syrian hamsters and sheep. The use of the TIP reveals the importance of the frequency of the signal presentation of MEL and suggests the importance of a period of low‐to‐absent circulating concentrations of the hormone. The TIP also reveals that the characteristics of the MEL signal that regulate male sexual behavior are similar to those that are critical for reproductive and metabolic responses in Syrian hamsters. We summarize the locations of possible functional MEL target sites identified by combining the TIP with traditional brain lesion techniques. Evidence from such studies suggests that the integrity of the suprachiasmatic nucleus (SCN) region in Siberian hamsters and the anterior hypothalamus in Syrian hamsters is necessary for the response to short‐day MEL signals. The TIP has been used to deliver MEL to putative target sites for the hormone in the brain of juvenile and adult Siberian hamsters. The results of these preliminary experiments suggest that the regions of specific MEL binding in this species, especially the SCN, are effective sites where MEL may stimulate short‐day‐type responses. In contrast, results from intracranial application of MEL in sheep suggest the medial basal hypothalamus as a critical site of action. Finally, we also discuss potential applications of the TIP for identification of brain MEL target sites, understanding of other photoperiodic phenomena and responses, and resolution of the cellular/molecular basis underlying the reception and interpretation of MEL signals. It is our collective view that the TIP has played, and will continue to play, a pivotal role in elucidation of the function of MEL in the photoperiodic control of seasonal mammalian responses and that the duration of the MEL signal is the critical parameter of the nocturnal secretion profile of the hormone for the photoperiodic control of several seasonally adaptive responses in mammalian species as diverse as hamsters and sheep.

Suprachiasmatic Regulation of Circadian Rhythms of Gene Expression in Hamster Peripheral Organs: Effects of Transplanting the Pacemaker

Neurotransplantation of the suprachiasmatic nucleus (SCN) was used to assess communication between the central circadian pacemaker and peripheral oscillators in Syrian hamsters. Free-running rhythms of haPer1, haPer2, and Bmal1 expression were documented in liver, kidney, spleen, heart, skeletal muscle, and adrenal medulla after 3 d or 11 weeks of exposure to constant darkness. Ablation of the SCN of heterozygote tau mutants eliminated not only rhythms of locomotor activity but also rhythmic expression of these genes in all peripheral organs studied. The Per:Bmal ratio suggests that this effect was attributable not to asynchronous rhythmicity between SCN-lesioned individuals but to arrhythmicity within individuals. Grafts of wild-type SCN to heterozygous, SCN-lesioned tau mutant hamsters not only restored locomotor rhythms with the period of the donor but also led to recovery of rhythmic expression of haPer1, haPer2, and haBmal1 in liver and kidney. The phase of these rhythms most closely resembled that of intact wild-type hamsters. Rhythmic gene expression was also restored in skeletal muscle, but the phase was altered. Behaviorally effective SCN transplants failed to reinstate rhythms of clock gene expression in heart, spleen, or adrenal medulla. These findings confirm that peripheral organs differ in their response to SCN-dependent cues. Furthermore, the results indicate that conventional models of internal entrainment may need to be revised to explain control of the periphery by the pacemaker.

Pineal Melatonin Mediates Photoperiodic Control of Pulsatile Luteinizing Hormone Secretion in the Ewe

Seasonal breeding in the ewe is regulated by photoperiod through a pineal-dependent mechanism. Changes in the ability of estradiol to inhibit tonic LH secretion are critical. During anestrus, this ovarian steroid gains the ability to slow the frequency of pulsatile LH secretion through an action on the brain. Exposure of ovariectomized, estradiol-implanted ewes to short photoperiods during summer anestrus revealed that daylength can control LH pulse frequency. After removal of estradiol, LH pulse frequency still differed between long- and short-day ewes, suggesting photoperiodic modulation of LH and presumably GnRH secretion independent of gonadal steroids. Significantly, the effects of daylength expressed both in the presence and the absence of estradiol failed to occur in pinealectomized ewes. Long-term infusions of melatonin, given in physiological patterns to pinealectomized ewes, mimicked the effects of photoperiod on pineal-intact ewes. Specifically, a pattern of melatonin characteristic of that in short days (16-hour night-time rise) led to an increase in LH pulse frequency to a breeding season rate. Conversely, melatonin infusions typifying a long-day pattern (8-hour night-time rise) produced an anestrous pulse pattern. Pituitary sensitivity to GnRH was not reduced in sheep which were reproductively suppressed by photoperiod or melatonin treatments. These observations support the conclusion that day-length acts through pineal melatonin secretion to regulate a neural LH pulse generator which, by changing the frequency of GnRH pulses, determines the ewes seasonal reproductive state.

Dispersed cell suspensions of fetal SCN restore circadian rhythmicity in SCN-lesioned adult hamsters

Overt circadian rhythms are permanently disrupted following lesions of the suprachiasmatic nucleus (SCN) in hamsters. It has previously been demonstrated that whole tissue grafts which include the fetal SCN restore circadian locomotor rhythms to hamsters previously made arrhythmic by SCN lesions. In the present study, we ask whether the intrinsic peptidergic organization of the SCN is a prerequisite for functional recovery of circadian rhythms of locomotor activity. To this end, dispersed cell suspensions of [3H]thymidine-labelled fetal anterior hypothalamic tissue which contains the SCN, were injected stereotaxically into the brain of adult hamsters. Dispersed cell suspensions restored free-running locomotor rhythms, but not entrainment or gonadal regression. The period of the restored free-running rhythms following injections of SCN cell suspensions was shorter than 24 h, in contrast to intact hamsters and SCN-lesioned hamsters whose rhythms are restored by whole tissue grafts. In animals with restored rhythms, a majority of [3H]thymidine-labelled cells were located within nuclei of the midline thalamus and zona incerta. In a few individuals, donor cells were also deposited along the injection tract as far ventrally as the medial hypothalamus. Restoration of free-running locomotor rhythmicity was correlated with the presence of small numbers of isolated VIP cells along with small plexuses of VIP fibers. In animals which did not recover locomotor rhythmicity, grafts were identical in location and size to those in recovered hamsters, but did not contain peptidergic cells characteristic of the SCN. The results suggest that structural integrity of the fetal SCN is not necessary for restoration of rhythmicity after grafting.

THE SUPRACHIASMATIC AREA IN THE FEMALE HAMSTER PROJECTS TO NEURONS CONTAINING ESTROGEN-RECEPTORS AND GNRH

This study investigated whether the circadian regulation of luteinizing hormone (LH) release may be through direct input of the suprachiasmatic nucleus (SCN) to estrogen receptor (ER)- and/or gonadotropin releasing hormone (GnRH)-immunoreactive neurons. We used Phaseolus vulgaris leucoagglutinin (PHA-L) as an ante- rograde tracer of SCN efferent? and performed double label immunocytochemistry for PHA-L and ER or GnRH. Between 8 and 30% of ER cells and 11–13% of the GnRH cells showed appositions with SCN efferents. Efferent projections of the subparaventricular hypotha- lamic nucleus and the retrochiasmatic area, relay stations of the circadian system, also made appositions with these two cell types. Results suggest that the circadian system could regulate the timing of the LH surge via two pathways, through input to GnRH and to ER cells.

Role of the hypothalamic paraventricular nucleus in neuroendocrine responses to daylength in the golden hamster.

Daylength regulates reproduction in golden hamsters through a mechanism which involves the pineal indoleamine, melatonin. Retinal input to the suprachiasmatic nucleus of the hypothalamus (SCN) and sympathetic innervation of the pineal are critical to the inhibition of reproduction by short photoperiods. Since the hypothalamic paraventricular nucleus (PVN) receives extensive input from the SCN in the rat, and may influence autonomic function via its brainstem and spinal cord projections, we studied the role of this nucleus in photoperiodically induced gonadal regression in the hamster. Bilateral electrolytic destruction of either the paraventricular nucleus (PVN) or suprachiasmatic nucleus (SCN) of the hypothalamus completely blocked testicular regression induced by either blinding or exposure to short days (10L:14D). Lesions in the retrochiasmatic hypothalamus (RCA) which may have interrupted the pathway of previously identified efferents from the SCN to the PVN were also effective in preventing short day-induced gonadal regression. Pineal melatonin content was measured in intact and lesioned hamsters sacrificed 3-5 h before lights on, at the time of the expected nocturnal peak. While SCN and RCA lesions significantly reduced pineal melatonin content, PVN lesions were still more effective in this regard. We conclude that the hamsters neuroendocrine response to photoperiod is mediated by neural pathways which include retinohypothalamic input to the SCN and efferents from this nucleus to the PVN which travel dorsocaudally through the retrochiasmatic area of the hypothalamus. Effectiveness of lesions restricted to the PVN suggests that direct projections from the PVN to spinal autonomic centers convey photoperiodic information which regulates pineal, and hence gonadal, function.

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 (Per1) and Period 2 (Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

OESTROGEN RECEPTOR-ALPHA -IMMUNOREACTIVE NEURONES PROJECT TO THE SUPRACHIASMATIC NUCLEUS OF THE FEMALE SYRIAN HAMSTER

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid‐binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor‐α(ER‐α) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER‐α‐immunoreactive (ERα‐ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERα‐immunoreactivity varies between areas. As many as one‐third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERα‐ir. Very few of the afferent neurones from the septum and the central grey are ERα‐ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERα‐ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERα‐ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERα‐ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.

Regulation of prokineticin 2 expression by light and the circadian clock

BackgroundThe suprachiasmatic nucleus (SCN) contains the master circadian clock that regulates daily rhythms of many physiological and behavioural processes in mammals. Previously we have shown that prokineticin 2 (PK2) is a clock-controlled gene that may function as a critical SCN output molecule responsible for circadian locomotor rhythms. As light is the principal zeitgeber that entrains the circadian oscillator, and PK2 expression is responsive to nocturnal light pulses, we further investigated the effects of light on the molecular rhythm of PK2 in the SCN. In particular, we examined how PK2 responds to shifts of light/dark cycles and changes in photoperiod. We also investigated which photoreceptors are responsible for the light-induced PK2 expression in the SCN. To determine whether light requires an intact functional circadian pacemaker to regulate PK2, we examined PK2 expression in cryptochrome1,2-deficient (Cry1-/-Cry2-/-) mice that lack functional circadian clock under normal light/dark cycles and constant darkness.ResultsUpon abrupt shifts of the light/dark cycle, PK2 expression exhibits transients in response to phase advances but rapidly entrains to phase delays. Photoperiod studies indicate that PK2 responds differentially to changes in light period. Although the phase of PK2 expression expands as the light period increases, decreasing light period does not further condense the phase of PK2 expression. Genetic knockout studies revealed that functional melanopsin and rod-cone photoreceptive systems are required for the light-inducibility of PK2. In Cry1-/-Cry2-/- mice that lack a functional circadian clock, a low amplitude PK2 rhythm is detected under light/dark conditions, but not in constant darkness. This suggests that light can directly regulate PK2 expression in the SCN.ConclusionThese data demonstrate that the molecular rhythm of PK2 in the SCN is regulated by both the circadian clock and light. PK2 is predominantly controlled by the endogenous circadian clock, while light plays a modulatory role. The Cry1-/-Cry2-/- mice studies reveal a light-driven PK2 rhythm, indicating that light can induce PK2 expression independent of the circadian oscillator. The light inducibility of PK2 suggests that in addition to its role in clock-driven rhythms of locomotor behaviour, PK2 may also participate in the photic entrainment of circadian locomotor rhythms.