Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Photoperiodic variation in CD45-positive cells and cell proliferation in the mediobasal hypothalamus of the Soay sheep.

The Earth's solar orbit induces annual climatic changes challenging to survival. Many animals have evolved to cope with seasonal variability through compensatory annual changes in their physiology and behavior, which involve innate long-term timing and photoperiodic synchronization to anticipate the environmental seasonal cycles. Here we considered the potential involvement of cyclical histogenesis in seasonal timing mechanisms in the sheep. Adult Soay rams were established in three distinctive seasonal states by controlled photoperiod exposure. A first group, representing the condition in late spring (long-photoperiod [LP] group), was taken indoors in May and exposed to 4 wks of 16 h light/day (LP). A second group was exposed to 20 wks of LP to establish a late-summer/long-day refractory condition (LPR group). A third group of animals was brought indoors in August and exposed to 4 wks of LP followed by 4 wks of 8 h light/day (short photoperiod [SP]) to establish an autumn-like condition (SP group). At the end of these regimes, we injected 5-bromo-2-deoxyuridine (BrdU), and animals were killed 24 h or 4 wks later. When BrdU was administered 24 h before death, more BrdU-immunopositive cells were detected in the hilus of the hippocampus in LP compared with SP animals, indicative of a higher proliferation rate. When BrdU was administered 4 wks before death, more BrdU-positive cells were detected in the hippocampus under LP, compared with SP, indicating increased cell survival. These mitotic cells were occasionally seen to adopt a neuronal phenotype in the hippocampus, but not in the hypothalamus. Approximately 10% of BrdU-positive cells in the basal hypothalamus coexpressed the pan-leukocytic marker CD45, and showed morphological features and regional distribution consistent with ameboid microglia. Increased numbers of these cells were detected in the region of the median eminence and tuberoinfundibular sulcus of animals kept in SP compared with LP or LPR. These data suggest that neuroimmune mechanisms may be involved in photoperiod-dependent seasonal remodeling of the adult brain.

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 ß 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.

Evidence for RGS4 modulation of melatonin and thyrotrophin signalling pathways in the pars tuberalis.

In mammals, the pineal hormone melatonin is secreted nocturnally and acts in the pars tuberalis (PT) of the anterior pituitary to control seasonal neuroendocrine function. Melatonin signals through the type 1 Gi-protein coupled melatonin receptor (MT1), inhibiting adenylate cyclase (AC) activity and thereby reducing intracellular concentrations of the second messenger, cAMP. Because melatonin action ceases by the end of the night, this allows a daily rise in cAMP levels, which plays a key part in the photoperiodic response mechanism in the PT. In addition, melatonin receptor desensitisation and sensitisation of AC by melatonin itself appear to fine-tune this process. Opposing the actions of melatonin, thyroid-stimulating hormone (TSH), produced by PT cells, signals through its cognate Gs-protein coupled receptor (TSH-R), leading to increased cAMP production. This effect may contribute to increased TSH production by the PT during spring and summer, and is of considerable interest because TSH plays a pivotal role in seasonal neuroendocrine function. Because cAMP stands at the crossroads between melatonin and TSH signalling pathways, any protein modulating cAMP production has the potential to impact on photoperiodic readout. In the present study, we show that the regulator of G-protein signalling RGS4 is a melatonin-responsive gene, whose expression in the PT increases some 2.5-fold after melatonin treatment. Correspondingly, RGS4 expression is acutely sensitive to changing day length. In sheep acclimated to short days (SP, 8 h light/day), RGS4 expression increases sharply following dark onset, peaking in the middle of the night before declining to basal levels by dawn. Extending the day length to 16 h (LP) by an acute 8-h delay in lights off causes a corresponding delay in the evening rise of RGS4 expression, and the return to basal levels is delayed some 4 h into the next morning. To test the hypothesis that RGS4 expression modulates interactions between melatonin- and TSH-dependent cAMP signalling pathways, we used transient transfections of MT1, TSH-R and RGS4 in COS7 cells along with a cAMP-response element luciferase reporter (CRE-luc). RGS4 attenuated MT1-mediated inhibition of TSH-stimulated CRE-luc activation. We propose that RGS4 contributes to photoperiodic sensitivity in the morning induction of cAMP-dependent gene expression in the PT.

Mammalian circannual pacemakers.

Circannual clocks drive rhythms in reproduction and many other seasonal characteristics but the underlying control of these long-term oscillators remains a mystery. Now, we propose that circannual timing involves mechanisms that are integral to the ontogenetic life-history programme where annual transitions are generated by cell birth, death and tissue regeneration throughout the life cycle--the histogenesis hypothesis. The intrinsic cycle is then timed by cues from the environment. The concept is that in specific sites in the brain, pituitary and peripheral tissues, residual populations of progenitor cells (adult stem cells) synchronously initiate a phase of cell division to begin a cycle. The progeny cells then proliferate, migrate and differentiate, providing the substrate that drives physiological change over long time-spans (e.g. summer/winter); cell death may be required to trigger the next cycle. We have begun to characterise such a tissue-based timer in our Soay sheep model focusing on the pars tuberalis (PT) of the pituitary gland and the sub-ventricular zone of the mediobasal hypothalamus (MBH) as potential circannual pacemakers. The PT is of special interest because it is a melatonin-responsive tissue containing undifferentiated cells, strategically located at the gateway between the brain and pituitary gland. The PT also governs long-photoperiod activation of thyroid hormone dependant processes in the MBH required for neurogenesis. In sheep, exposure to long photoperiod markedly activates BrDU-labelled cell proliferation in the PT and MBH, and acts to entrain the circannual reproductive cycle. Variation in expression and co-ordination of multiple tissue timers may explain species differences in circannual rhythmicity. This paper is dedicated to the memory of Ebo Gwinner.

Egr1 involvement in evening gene regulation by melatonin.

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE's in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE's contribute to evening gene expression profiles in this pituitary melatonin target tissue.

RFamide-related peptide and its cognate receptor in the sheep: cDNA cloning, mRNA distribution in the hypothalamus and the effect of photoperiod.

Photoperiodic responses enable animals to adapt their physiology to predictable patterns of seasonal environmental change. In mammals, this depends on pineal melatonin secretion and effects in the hypothalamus, but the cellular and molecular substrates of its action are poorly understood. The recent identification of a mammalian orthologue of the avian gonadotrophin-inhibitory hormone gene has led to interest in its possible involvement in seasonal breeding. In long-day breeding Syrian hamsters, hypothalamic RFamide-related peptide (RFRP) expression is increased by exposure to long photoperiod. Because, opposite to hamsters, sheep are short-day breeders, we predicted that a conserved role in mammalian reproductive activation would decrease RFRP expression in sheep under a long photoperiod. We cloned the ovine RFRP cDNA and examined its expression pattern in Soay sheep acclimated to a 16 : 8 h or 8 : 16 h light /dark cycle (LP and SP, respectively). RFRP was expressed widely in the sheep hypothalamus and increased modestly overall with exposure to LP. Interestingly, RFRP expression in the ependymal cells surrounding the base of the third ventricle was highly photoperiodic, with levels being undetectable in animals held on SP but consistently high under LP. These data are inconsistent with a conserved reproductive role for RFRP across mammals. Additionally, we cloned the ovine homologue of the cognate RFRP receptor, rfr-2 (NPFF1) and found localised expression in the suprachiasmatic nuclei and in the pars tuberalis. Taken together, these data strengthen the emerging view that interplay between ependymal cells and the pars tuberalis might be important for the seasonal timing system.

Rhythmic melatonin secretion does not correlate with the expression of arylalkylamine N-acetyltransferase, inducible cyclic amp early repressor, period1 or cryptochrome1 mRNA in the sheep pineal.

The pineal gland, through nocturnal melatonin, acts as a neuroendocrine transducer of daily and seasonal time. Melatonin synthesis is driven by rhythmic activation of the rate-limiting enzyme, arylalkylamine N-acetyltransferase (AA-NAT). In ungulates, AA-NAT mRNA is constitutively high throughout the 24-h cycle, and melatonin production is primarily controlled through effects on AA-NAT enzyme activity; this is in contrast to dominant transcriptional control in rodents. To determine whether there has been a selective loss of circadian control of AA-NAT mRNA expression in the sheep pineal, we measured the expression of other genes known to be rhythmic in rodents (inducible cAMP early repressor ICER, the circadian clock genes Period1 and Cryptochrome1, as well as AA-NAT). We first assayed gene expression in pineal glands collected from Soay sheep adapted to short days (Light: dark, 8-h: 16-h), and killed at 4-h intervals through 24-h. We found no evidence for rhythmic expression of ICER, AA-NAT or Cryptochrome1 under these conditions, whilst Period1 showed a low amplitude rhythm of expression, with higher values during the dark period. In a second group of animals, lights out was delayed by 8-h during the final 24-h sampling period, a manipulation that causes an immediate shortening of the period of melatonin secretion. This did not significantly affect the expression of ICER, AA-NAT or Cryptochrome1 in the pineal, whilst a slight suppressive effect on overall Per1 levels was observed. The attenuated response to photoperiod change appears to be specific to the ovine pineal, as the first long day induced rapid changes of Period1 and ICER expression in the hypothalamic suprachiasmatic nuclei and pituitary pars tuberalis, respectively. Overall, our data suggest a general reduction of circadian control of transcript abundance in the ovine pineal gland, consistent with a marked evolutionary divergence in the mechanism regulating melatonin production between terrestrial ruminants and fossorial rodents.

Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis.

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.

Evidence that gonadotropin-releasing hormone II is not a physiological regulator of gonadotropin secretion in mammals.

Gonadotropin-releasing hormone (GnRH)-II stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion when administered at high doses in mammals, and this effect has been assumed to be mediated through the GnRH-II receptor expressed on gonadotropes. This study used two selective GnRH-I receptor antagonists to test the alternative hypothesis that GnRH-II acts through the GnRH-I receptor to elicit gonadotropin secretion. The antagonist, antide, was used to characterize the receptor-relay because it was a pure antagonist in vitro based on inositol phosphate responses in COS-7 cells transfected with either mammalian GnRH-I and GnRH-II receptors and, in vivo, potently antagonized the gonadotropin-releasing effect of a single injection of 250 ng GnRH-I in our sexually inactive sheep model. In a series of studies in sheep, antide (i). blocked the acute LH response to a single injection of GnRH-II (20 microg antide: 10 microg GnRH-II); (ii). blocked both the acute, pulsatile LH response and the FSH priming response to 2-hourly injections of GnRH-II over 36 h (100 microg antide/8 h: 4 microg GnRH-II/2 h); and (iii). chronically blocked both the pulsatile LH response and the marked FSH priming response to 4-hourly injections of GnRH-II over 10 days (75 microg antide/8 h: 4 microg GnRH-II/4 h). In two final experiments, the GnRH-I antagonist 135-18, shown previously to agonize the mammalian GnRH-II receptor, blocked the gonadotropin-releasing effects of GnRH-I (250 ng) but failed to elicit an LH response when given alone, and simultaneous administration of GnRH-II (250 ng) failed to alter the LH-releasing effect of GnRH-I (50-500 ng). These data thus support our hypothesis. Based on additional literature, it is unlikely that the GnRH-II decapeptide is a native regulator of the gonadotrope in mammals.

Prolactin cycles in sheep under constant photoperiod: evidence that photorefractoriness develops within the pituitary gland independently of the prolactin output signal.

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01-0.04 mg kg-1 day-1; 12-24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean +/- SEM, 35.3 +/- 0.6 and 37.0 +/- 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22-30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 +/- 0.4 and 37.6 +/- 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.

Clock genes and the long-term regulation of prolactin secretion: evidence for a photoperiod/circannual timer in the pars tuberalis.

Prolactin secretion is regulated by photoperiod through changes in the 24-h melatonin profile and displays circannual rhythmicity under constant photoperiod. These two processes appear to occur principally within the pituitary gland, controlled by the pars tuberalis. This is evident because: (i) hypothalamic-pituitary disconnected (HPD) sheep show marked changes in prolactin secretion in response to switches in photoperiod and manipulations of melatonin, similar to brain-intact controls; (ii) HPD sheep also show photoperiod-specific, long-term cycles in prolactin secretion under constant long or short days, with the timing maintained even when prolactin secretion is blocked for 2-3 months; and (iii) pars tuberalis cells, but not lactotrophs, express high concentrations of melatonin (MT1) receptor, and exhibit a duration-sensitive, cAMP-dependant, inhibitory response to physiological concentrations of melatonin. This suggests the existence of an intrinsic, reversible photoperiod-circannual timer in pars tuberalis cells. A full complement of clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the ovine pars tuberalis, and undergo 24-h cyclical expression as observed in a cell autonomous, circadian clock. Activation of Per genes occurs in the early day (melatonin off-set), while activation of Cry genes occurs in the early night (melatonin on-set). This temporal association is evident under both long and short days, thus the Per-Cry interval varies directly with photoperiod. Because, PER : CRY, protein : protein interactions affect stability, nuclear entry and gene transcription based on rodent data, the change in phasing of Per/Cry expression provides a potential mechanism for decoding the long day/short day melatonin signal. A speculative, but testable, extension of this hypothesis is that intrinsically regulated changes in the phase of Per/Cry rhythms, regulates both photorefractoriness and the generation of circannual rhythms in prolactin secretion.

Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth, and expression of inhibin/activin subunit mRNA and protein in adult testis.

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.

Prolactin receptor expression in human testis and accessory tissues: localization and function.

Experimental studies in animals have established prolactin (PRL) as a progonadal hormone that promotes the function of the testis and reproductive accessory glands. The present study investigated the localization of PRL receptor (PRL-R) expression in the human testis and accessory tissues. Expression of PRL-R was identified in human testis and vas deferens by RT-PCR, and further localized by immunohistochemistry to the Leydig cells and differentiating germ cells of the testis (developmental stages extending from pachytene spermatocytes to elongating spermatids). Positive staining for PRL-R was also clearly evident in the epithelium of vas deferens, epididymis, prostate and seminal vesicles. Functional activation of PRL-R was demonstrated in fresh samples of vas deferens collected at vasectomy by examination of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAP (mitogen-activated protein) kinase ERK (extracellular signal-regulated kinase) signalling pathways. Within the vas deferens, PRL induced rapid tyrosine phosphorylation of JAK 2 and STAT 5 (after 10 and 20 min respectively), and tyrosine and threonine phosphorylation of ERK 1 and 2 (after 5 min). The demonstration of function and localization of PRL-R presented here suggests multiple roles for PRL in the human male reproductive tract.

Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.

Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-gal transgene, on the other hand, displayed variable degrees of inflammatory response, with periglandular fibrosis, lymphocytic infiltrate and venulitis in almost all cases. Focal necrosis and/or apoptosis was noted in six of nine cases. In summary, we have found evidence of severe inflammatory reaction within the first seven days of adenovirus injection, amounting to significant hypophysitis. The histological extent of this reaction has not previously been recognised by studies of the efficacy of gene transfer in rodents, and was underestimated by immunocytochemical studies of hormone and transgene expression. The findings emphasise the need for careful evaluation of the safety of endocrine gene therapy, and for caution with the dose of vector used.

Noradrenaline and dopamine regulation of prolactin secretion in sheep: role in prolactin homeostasis but not photoperiodism.

The role of noradrenaline (NA) and dopamine (DA) in the hypothalamic control of prolactin (PRL) secretion was investigated in hypothalamic intact (control) and hypothalamo-pituitary disconnected (HPD) Soay rams. The animals were exposed to alternating 16-weekly periods of short (8 L : 16D) and long days (16 L : 8D) to induce marked cyclical changes in PRL secretion in both groups (as demonstrated previously). Selective NA and DA receptor antagonists (dose: 1.2 micromol/kg) were administered under short days (low endogenous PRL secretion), and agonists (dose: 0.0012-0.12 micromol/kg) were administered under long days (high endogenous PRL secretion). The acute changes in blood PRL concentrations were measured over 4 h as the index of responsiveness. Under short days, treatment with WB4101 (alpha-1 adenoceptor antagonist), and rauwolscine (alpha-2 antagonist), consistently increased PRL secretion in control, but not in HPD rams. The treatments produced similar acute, drug-specific behavioural effects in both groups. Propranolol (beta antagonist) had no effect on PRL secretion, while sulpiride (DA D-2 antagonist) induced a marked increase in blood PRL concentrations in control rams (> 4 h), and a transient effect in HPD rams (15 min). Under long days, when endogenous PRL secretion was increased, phenylephrine (alpha-1 agonist) produced no effects, while bromocriptine (DA D-2 agonist) robustly decreased PRL concentrations in both control and HPD rams, even at the lowest treatment dose. Overall, the positive responses to the antagonists in the control rams, support the view that DA (acting via D-2 receptors), and to a lesser extent NA (acting via alpha-1/alpha-2 receptors), negatively regulate PRL secretion. In contrast, the lack of responses to the antagonists in the HPD rams, support the view that neither DA, nor NA, mediate the photoperiodic control of PRL secretion.

Neuroendocrine regulation of seasonal gonadotrophin and prolactin rhythms: lessons from the Soay ram model.

Marked seasonality, responsiveness to photoperiod, diurnal behaviour, large body size, long lifespan and adaptability in captivity are characteristics that make the Soay ram a useful model for neuroendocrine research. Adult rams are routinely housed indoors under artificial lighting of alternating 16 week periods of long and short days to entrain the seasonal cycles in reproduction, growth and metabolism. The long-term cycles in individuals are monitored directly (measurements of testis diameter, androgen-dependent skin coloration, food intake, pelage moult, locomotor activity) and retrospectively (measurements of reproductive and metabolic hormone concentrations in peripheral blood). A wide spectrum of experimental procedures, including serial blood and cerebrospinal fluid (CSF) sampling with hormone or drug treatments, tissue biopsy, stereotaxic cerebral implantation and surgical lesions, not feasible in smaller species, are used to investigate the multiple interactive neuroendocrine systems regulating seasonality. The results from a recent experiment in which rams received a lesion of the caudal arcuate nucleus (caudal ARCX) or hypothalamo-pituitary disconnection (HPD) are presented to demonstrate the fidelity of long-term data derived from the Soay ram model. The results support the view that the melatonin signal that encodes photoperiod acts within the mediobasal hypothalamus to time the gonadotrophin/gonadal cycle, but acts directly within the pituitary gland to time the prolactin/pelage cycle.

Prolactin actions in the sheep testis: a test of the priming hypothesis.

This study investigated whether prolactin (PRL) plays a priming role in the testis during the nonmating season and thereby facilitates gonadal reactivation. Sexually inactive Soay rams under long days were treated as follows: 1) group C (control) received vehicle, 2) group B received bromocriptine to suppress PRL secretion, 3) group B + PRL received bromocriptine + ovine PRL to reinstate physiological levels of PRL (n = 5/group). Treatments were for 10 wk. The photoperiod was then switched to short days to reactivate the reproductive axis. Testis diameter and sex skin coloration were recorded, and routine blood samples were collected to measure concentrations of FSH, inhibin A, and testosterone (T). At the end of the treatments, blood samples were collected every 10 min for 10 h to monitor LH pulses and the T-response to exogenous LH, and a testis biopsy was collected to assess spermatogenic activity (bromodeoxyuridine [BrDU] method) and expression of PRL receptor (reverse transcription-polymerase chain reaction and immunocytochemistry). There were no significant differences between groups in spermatogenesis (BrDU index) or steroidogenesis (T-response), and no difference in the time taken to achieve full testicular redevelopment under short days. Testis diameter and inhibin A were marginally increased in group B + PRL. Overall, this thorough experiment provides minimal support for the priming hypothesis.

Hypothalamic control of photoperiod-induced cycles in food intake, body weight, and metabolic hormones in rams.

This study used a hypothalamo-pituitary disconnected (HPD) sheep model to investigate the central regulation of long-term cycles in voluntary food intake (VFI) and body weight (BW). VFI, BW, and circulating concentrations of metabolic hormones [alpha-melanocyte-stimulating hormone (alpha-MSH), insulin-like growth factor-1 (IGF-1), insulin, and leptin] were measured in HPD and control Soay rams exposed to alternating 16 weekly periods of long and short days for 80 wk. In the controls, the physiology was cyclical with a 32-wk periodicity corresponding to the lighting regimen. VFI and BW increased under long days to a maximum early into short days, and there were associated increases in blood concentrations of alpha-MSH, insulin, and leptin. In the HPD rams, there were no significant photoperiod-induced changes in any of the parameters. VFI increased after surgery for 8 wk and then gradually declined, although BW increased progressively and the HPD rams became obese. Concentrations of alpha-MSH, insulin, and leptin in peripheral blood were permanently increased (>200%), and levels of IGF-1 decreased (<55%). The HPD lesion effectively destroyed the entire median eminence [no nerve terminals immunostained for tyrosine hydroxylase (TH) and gonadotropin-releasing hormone] and the adjacent arcuate nucleus (no perikarya immunostained for proopiomelanocortin or TH, and no cells expressed neuropeptide Y mRNA). The results support the conclusion that arcuate hypothalamic systems generate long-term rhythms in VFI, BW, and energy balance.

Cell type-specific adenoviral transgene expression in the intact ovine pituitary gland after stereotaxic delivery: an in vivo system for long-term multiple parameter evaluation of human pituitary gene therapy.

Ablative therapies for pituitary tumors commonly cause irreversible damage to normal pituitary cells. Toxin gene therapy should therefore ideally be targeted to specific cell types to avoid collateral cell damage. To evaluate cell-type-specific adenoviral gene transfer in the intact pituitary gland we have used stereotaxic transcranial delivery of recombinant adenoviruses in the sheep with continuous assessment of endocrine function. Adenoviral ss-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL gene promoter. The hCMV promoter directed adenoviral ss-galactosidase expression in all pituitary cell types, but the PRL promoter restricted this exclusively to lactotropic cells, indicating that this promoter conferred appropriate cell type specificity in the context of adenoviral transduction in vivo. Serial measurements of plasma hormones showed no disruption of endocrine function over 7 days after intrapituitary injection. In summary, this work shows cell type-specific expression of an adenoviral transgene in the mixed cell population of the intact pituitary gland in vivo in a large animal model and indicates that stereotaxic intrapituitary delivery does not disrupt normal endocrine function.