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Key points Maternal shift work increases the risk of pregnancy complications, although its effects on progeny health after birth are not clear. We evaluated the impact of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on the metabolic health of sheep progeny. Simulated shift work had no effect on growth, body size, body composition or glucose tolerance in pre-pubertal or young adult progeny. Glucose-stimulated insulin secretion was reduced in adult female progeny and insulin sensitivity was increased in adult female singleton progeny. The results of the present study do not support the hypothesis that maternal shift work exposure impairs metabolic health of progeny in altricial species. Abstract Disrupted maternal circadian rhythms, such as those experienced during shift work, are associated with impaired progeny metabolism in rodents. The effects of disrupted maternal circadian rhythms on progeny metabolism have not been assessed in altricial, non-litter bearing species. We therefore assessed postnatal growth from birth to adulthood, as well as body composition, glucose tolerance, insulin secretion and insulin sensitivity, in pre-pubertal and young adult progeny of sheep exposed to control conditions (CON: 10 males, 10 females) or to a simulated shift work (SSW) protocol for the first one-third (SSW0-7: 11 males, 9 females), the first two-thirds (SSW0-14: 8 males, 11 females) or all (SSW0-21: 8 males, 13 females) of pregnancy. Progeny growth did not differ between maternal treatments. In pre-pubertal progeny (12-14 weeks of age), adiposity, glucose tolerance and insulin secretion during an i.v. glucose tolerance test and insulin sensitivity did not differ between maternal treatments. Similarly, in young adult progeny (12-14 months of age), food intake, adiposity and glucose tolerance did not differ between maternal treatments. At this age, however, insulin secretion in response to a glucose bolus was 30% lower in female progeny in the combined SSW groups compared to control females (P = 0.031), and insulin sensitivity of SSW0-21 singleton females was 236% compared to that of CON singleton female progeny (P = 0.025). At least in this model, maternal SSW does not impair progeny metabolic health, with some evidence of greater insulin action in female young adult progeny.

Introduction Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. Methods We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. Results The optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. Discussion This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Preclinical imaging studies of fetal hemodynamics require anesthesia to immobilize the animal. This may induce cardiovascular depression and confound measures under investigation. We compared the impact of four anesthetic regimes upon maternal and fetal blood gas and hemodynamics during baseline periods of normoxia, and in response to an acute hypoxic challenge in pregnant sheep. Merino ewes were surgically prepared with maternal and fetal vascular catheters and a fetal femoral artery flow probe at 105–109 days gestation. At 110–120 days gestation, ewes were anesthetized with either isoflurane (1.6%), isoflurane (0.8%) plus ketamine (3.6 mg·kg−1·h−1), ketamine (12.6 mg·kg−1·h−1) plus midazolam (0.78 mg·kg−1·h−1), propofol (30 mg·kg−1·h−1), or remained conscious. Following 60 min of baseline recording, nitrogen was administered directly into the maternal trachea to displace oxygen and induce maternal and thus fetal hypoxemia. During normoxia, maternal PaO2 was ~30 mmHg lower in anesthetized ewes compared to conscious controls, regardless of the type of anesthesia (p .05), but heart rate was 32 ± 8 bpm lower in fetuses from ewes administered isoflurane (p = .044). During maternal hypoxia, fetal MAP increased, and peripheral blood flow decreased in all fetuses except those administered propofol (p, Preclinical imaging studies of fetal haemodynamics require anaesthesia to immobilise the animal. In the presence of isoflurane, isoflurane/ketamine and ketamine/midazolam, but not propofol anaesthesia, maternal and fetal hypoxaemia increased fetal mean arterial pressure and decreased peripheral blood flow. Acute fetal hypoxaemia induced fetal tachycardia regardless of anaesthetic type. These results have implications for the design and interpretation of preclinical imaging studies where anaesthesia is required.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder affecting the reproductive, metabolic and psychological health of women. Clinic-based studies indicate that sleep disturbances and disorders including obstructive sleep apnea and excessive daytime sleepiness occur more frequently among women with PCOS compared to comparison groups without the syndrome. Evidence from the few available population-based studies is supportive. Women with PCOS tend to be overweight/obese, but this only partly accounts for their sleep problems as associations are generally upheld after adjustment for body mass index; sleep problems also occur in women with PCOS of normal weight. There are several, possibly bidirectional, pathways through which PCOS is associated with sleep disturbances. The pathophysiology of PCOS involves hyperandrogenemia, a form of insulin resistance unique to affected women, and possible changes in cortisol and melatonin secretion, arguably reflecting altered hypothalamic–pituitary–adrenal function. Psychological and behavioral pathways are also likely to play a role, as anxiety and depression, smoking, alcohol use and lack of physical activity are also common among women with PCOS, partly in response to the distressing symptoms they experience. The specific impact of sleep disturbances on the health of women with PCOS is not yet clear; however, both PCOS and sleep disturbances are associated with deterioration in cardiometabolic health in the longer term and increased risk of type 2 diabetes. Both immediate quality of life and longer-term health of women with PCOS are likely to benefit from diagnosis and management of sleep disorders as part of interdisciplinary health care. Refereed/Peer-reviewed

Implications • As a model of human pregnancy, research studies in catheterized fetal sheep have made significant contributions to our understanding of both normal and abnormal fetal development. • Owing to the invasive nature of catheterization surgery, effective postoperative analgesia is required. • Decisions regarding appropriate analgesia should consider effectiveness in reducing maternal pain and fetal well-being as well as potential impacts upon fetal physiological parameters under investigation. • The reporting of analgesic regimes in all research studies should comply with the ARRIVE guidelines Refereed/Peer-reviewed

The in utero environment is inherently rhythmic, with the fetus subjected to circadian changes in temperature, substrates, and various maternal hormones. Meanwhile, the fetus is developing an endogenous circadian timing system, preparing for life in an external environment where light, food availability, and other environmental factors change predictably and repeatedly every 24 h. In humans, there are many situations that can disrupt circadian rhythms, including shift work, international travel, insomnias, and circadian rhythm disorders (e.g., advanced/delayed sleep phase disorder), with a growing consensus that this chronodisruption can have deleterious consequences for an individual’s health and well-being. However, the impact of chronodisruption during pregnancy on the health of both the mother and fetus is not well understood. In this review, we outline circadian timing system ontogeny in mammals and examine emerging research from animal models demonstrating long-term negative implications for progeny health following maternal chronodisruption during pregnancy. Refereed/Peer-reviewed

New Findings What is the central question of this study? Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. In this study, we asked whether circadian rhythm disruption, induced by exposure of sheep to rapidly alternating photoperiods (RAPs), also disrupts metabolic homeostasis in a large diurnal animal model. What is the main finding and its importance? Exposure to RAPs disrupted central (melatonin and core body temperature) and peripheral rhythmicity (skeletal muscle clock gene expression). This led to reduced nocturnal plasma glucose concentrations, but did not affect glucose tolerance and glucose-stimulated insulin secretion. These results suggest that RAP-induced circadian rhythm disruption has minimal effect on glucose homeostasis in the sheep. Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. To determine whether these effects occur in a large diurnal animal, we assessed the impact of circadian rhythm disruption upon metabolic function in sheep. Adult ewes (n = 7) underwent 3 weeks of a control 12 h light–12 h dark photoperiod, followed by 4 weeks of rapidly alternating photoperiods (RAPs) whereby the time of light exposure was reversed twice each week. Measures of central (melatonin secretion and core body temperature) and peripheral rhythmicity (clock and metabolic gene expression in skeletal muscle) were obtained over 24 h in both conditions. Metabolic homeostasis was assessed by glucose tolerance tests and 24 h glucose and insulin profiles. Melatonin and core body temperature rhythms resynchronized within 2 days of the last photoperiod shift. High-amplitude Bmal1, Clock, Nr1d1, Cry2 and Per3 mRNA rhythms were apparent in skeletal muscle, which were phase advanced by up to 3.5 h at 2 days after the last phase shift, whereas Per1 expression was downregulated at this time. Pparα, Pgc1α and Nampt mRNA were constitutively expressed in both conditions. Nocturnal glucose concentrations were reduced following chronic phase shifts (zeitgeber time 0, −5.5%; zeitgeber time 12, −2.9%; and zeitgeber time 16, −5.7%), whereas plasma insulin, glucose tolerance and glucose-stimulated insulin secretion were not altered. These results demonstrate that clock gene expression within ovine skeletal muscle oscillates over 24 h and responds to changing photoperiods. However, metabolic genes which link circadian and metabolic clocks in rodents were arrhythmic in sheep. Differences may be due to the ruminant versus monogastric digestive organization in each species. Together, these results demonstrate that despite disruptions to central and peripheral rhythmicity following exposure to rapidly alternating photoperiods, there was minimal impact on glucose homeostasis in the sheep.

Few challenges are more critical to the survival of a species than reproduction. To ensure reproductive success, myriad aspects of physiology and behaviour need to be tightly orchestrated within the animal, as well as timed appropriately with the external environment. This is accomplished through an endogenous circadian timing system generated at the cellular level through a series of interlocked transcription/translation feedback loops, leading to the overt expression of circadian rhythms. These expression patterns are found throughout the body, and are intimately interwoven with both the timing and function of the reproductive process. In this review we highlight the many aspects of reproductive physiology in which circadian rhythms are known to play a role, including regulation of the estrus cycle, the LH surge and ovulation, the production and maturation of sperm and the timing of insemination and fertilisation. We will also describe roles for circadian rhythms in support of the preimplantation embryo in the oviduct, implantation/placentation, as well as the control of parturition and early postnatal life. There are several key differences in physiology between humans and the model systems used for the study of circadian disruption, and these challenges to interpretation will be discussed as part of this review. (C) 2013 Elsevier Ltd. All rights reserved. Refereed/Peer-reviewed

The Robinson Research Institute of the University of Adelaide convened a multidisciplinary group of n = 33 clinicians, researchers and representatives of government organisations on the 2 October 2014 for a workshop entitled “Promoting fertility and healthy conception. How do we generate greater reproductive health awareness?” The key aim of the workshop was to assess the body of knowledge that informs clinical practice and government policy, and to identify questions and additional information needed by health practitioners and government representatives working in the field of reproductive health and to frame future research and policy. The workshop identified topics that fell mostly into three categories: lifestyle-related, societal and biological factors. The lifestyle topics included nutrition and diet, exercise, obesity, shift work and other factors deemed to be modifiable at the level of the individual. The societal topics included discussions of matters that are structural, and resistant to change by individuals, including specific ethical issues, social disadvantage, government and educational policies. The biological factors are intrinsic physical states of the individual, and included many factors where there is a dense body of scientific knowledge which may not be readily accessible in less academic language. This workshop thus provided an opportunity to identify further actions that could be undertaken tomeet the needs of diverse organisations and groups of professionals with an interest in human fertility. Since so many factors in our social and biological environment can impact fertility and preconception health, it is imperative to involve many disciplines or levels of government or societal organisations that have not traditionally been involved in this area. Refereed/Peer-reviewed

The role of peripheral vs. central circadian rhythms and Clock in the maintenance of metabolic homeostasis and with aging was examined by using ClockΔ19+MEL mice. These have preserved suprachiasmatic nucleus and pineal gland rhythmicity but arrhythmic Clock gene expression in the liver and skeletal muscle. ClockΔ19+MEL mice showed fasting hypoglycemia in young-adult males, fasting hyperglycemia in older females, and substantially impaired glucose tolerance overall. ClockΔ19+MEL mice had substantially reduced plasma insulin and plasma insulin/glucose nocturnally in males and during a glucose tolerance test in females, suggesting impaired insulin secretion. ClockΔ19+MEL mice had reduced hepatic expression and loss of rhythmicity of gck, pfkfb3, and pepck mRNA, which is likely to impair glycolysis and gluconeogenesis. ClockΔ19+MEL mice also had reduced glut4 mRNA in skeletal muscle, and this may contribute to poor glucose tolerance. Whole body insulin tolerance was enhanced in ClockΔ19+MEL mice, however, suggesting enhanced insulin sensitivity. These responses occurred although the ClockΔ19mutation did not cause obesity and reduced plasma free fatty acids while increasing plasma adiponectin. These studies on clock-gene disruption in peripheral tissues and metabolic homeostasis provide compelling evidence of a relationship between circadian rhythms and the glucose/insulin and adipoinsular axes. It is, however, premature to declare that clock-gene disruption causes the full metabolic syndrome.

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 ± 1.3 and 4.5 ± 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work. Refereed/Peer-reviewed

Melatonin and wheel-running rhythmicity and the effects of acute and chronic light pulses on these rhythms were studied in ClockΔ19mutant mice selectively bred to synthesize melatonin. Homozygous melatonin-proficient ClockΔ19mutant mice ( ClockΔ19/Δ19-MEL) produced melatonin rhythmically, with peak production 2 h later than the wild-type controls (i.e., just before lights on). By contrast, the time of onset of wheel-running activity occurred within a 20-min period around lights off, irrespective of the genotype. Melatonin production in the mutants spontaneously decreased within 1 h of the expected time of lights on. On placement of the mice in continuous darkness, the melatonin rhythm persisted, and the peak occurred 2 h later in each cycle over the first two cycles, consistent with the endogenous period of the mutant. This contrasted with the onset of wheel-running activity, which did not shift for several days in constant darkness. A light pulse around the time of expected lights on followed by constant darkness reduced the expected 2-h delay of the melatonin peak of the mutants to ∼1 h and advanced the time of the melatonin peak in the wild-type mice. When the ClockΔ19/Δ19-MEL mice were maintained in a skeleton photoperiod of daily 15-min light pulses, a higher proportion entrained to the schedule (57%) than melatonin-deficient mutants (9%). These results provide compelling evidence that mice with the ClockΔ19mutation express essentially normal rhythmicity, albeit with an underlying endogenous period of 26–27 h, and they can be entrained by brief exposure to light. They also raise important questions about the role of Clock in rhythmicity and the usefulness of monitoring behavioral rhythms compared with hormonal rhythms.

There has been relatively little research conducted on pineal melatonin production in laboratory mice, in part, due to the lack of appropriate assays. We studied the pineal and plasma rhythm, response to light, adrenergic stimulation, and metabolism of melatonin in CBA mice. With the use of a sensitive and specific melatonin RIA, melatonin was detected in the pineal glands at all times of the day >21 fmol/gland in CBA mice but not in C57Bl mice. Both plasma and pineal melatonin levels peaked 2 h before dawn in a 12:12-h light-dark photoperiod (162 ± 31 pM and 1,804 ± 514 fmol/gland, respectively). A brief light pulse (200 lx/15 min), 2 h before lights on, suppressed both plasma and pineal melatonin to near basal levels within 30 min. Exposure to light pulses 4 h after lights off or 2 h before lights on resulted in delays and advances, respectively, in the early morning decline of plasma and pineal melatonin on the next cycle. Administration of the β-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold increase in plasma and pineal melatonin 2.5 to 3 h after the first injection. In the mouse, unlike the rat, melatonin was shown to be metabolized almost exclusively to 6-glucuronylmelatonin rather than 6-sulphatoxymelatonin. These studies have shown that the appropriate methodological tools are now available for studying melatonin rhythms in mice.

Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. To determine whether these effects occur in a large diurnal animal, we assessed the impact of circadian rhythm disruption upon metabolic function in sheep. Adult ewes (n = 7) underwent 3 weeks of a control 12 h light-12 h dark photoperiod, followed by 4 weeks of rapidly alternating photoperiods (RAPs) whereby the time of light exposure was reversed twice each week. Measures of central (melatonin secretion and core body temperature) and peripheral rhythmicity (clock and metabolic gene expression in skeletal muscle) were obtained over 24 h in both conditions. Metabolic homeostasis was assessed by glucose tolerance tests and 24 h glucose and insulin profiles. Melatonin and core body temperature rhythms resynchronized within 2 days of the last photoperiod shift. High-amplitude Bmal1, Clock, Nr1d1, Cry2 and Per3 mRNA rhythms were apparent in skeletal muscle, which were phase advanced by up to 3.5 h at 2 days after the last phase shift, whereas Per1 expression was downregulated at this time. Pparα, Pgc1α and Nampt mRNA were constitutively expressed in both conditions. Nocturnal glucose concentrations were reduced following chronic phase shifts (zeitgeber time 0, -5.5%; zeitgeber time 12, -2.9%; and zeitgeber time 16, -5.7%), whereas plasma insulin, glucose tolerance and glucose-stimulated insulin secretion were not altered. These results demonstrate that clock gene expression within ovine skeletal muscle oscillates over 24 h and responds to changing photoperiods. However, metabolic genes which link circadian and metabolic clocks in rodents were arrhythmic in sheep. Differences may be due to the ruminant versus monogastric digestive organization in each species. Together, these results demonstrate that despite disruptions to central and peripheral rhythmicity following exposure to rapidly alternating photoperiods, there was minimal impact on glucose homeostasis in the sheep.

Disrupting maternal circadian rhythms through exposure to chronic phase shifts of the photoperiod has lifelong consequences for the metabolic homeostasis of the fetus, such that offspring develop increased adiposity, hyperinsulinaemia and poor glucose and insulin tolerance. In an attempt to determine the mechanisms by which these poor metabolic outcomes arise, we investigated the impact of chronic phase shifts (CPS) on maternal and fetal hormonal, metabolic and circadian rhythms. We assessed weight gain and food consumption of dams exposed to either CPS or control lighting conditions throughout gestation. At day 20, dams were assessed for plasma hormone and metabolite concentrations and glucose and insulin tolerance. Additionally, the expression of a range of circadian and metabolic genes was assessed in maternal, placental and fetal tissue. Control and CPS dams consumed the same amount of food, yet CPS dams gained 70% less weight during the first week of gestation. At day 20, CPS dams had reduced retroperitoneal fat pad weight (-15%), and time-of-day dependent decreases in liver weight, whereas fetal and placental weight was not affected. Melatonin secretion was not altered, yet the timing of corticosterone, leptin, glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations were profoundly disrupted. The expression of gluconeogenic and circadian clock genes in maternal and fetal liver became either arrhythmic or were in antiphase to the controls. These results demonstrate that disruptions of the photoperiod can severely disrupt normal circadian profiles of plasma hormones and metabolites, as well as gene expression in maternal and fetal tissues. Disruptions in the timing of food consumption and the downstream metabolic processes required to utilise that food, may lead to reduced efficiency of growth such that maternal weight gain is reduced during early embryonic development. It is these perturbations that may contribute to the programming of poor metabolic homeostasis in the offspring. Refereed/Peer-reviewed

Circadian rhythms impact on a wide range of physiological systems and this impact extends to fertility, such that disruptions to timing systems can impact upon reproductive capacity. This is highlighted most obviously in mutant mouse models whereby deletion or mutation of single genes results not only in disrupted circadian rhythmicity, but also compromised male and female reproductive function. In this review, we discuss the presence of circadian clocks in female and male reproductive tissues and the role these clocks play in the generation of oestrus cycles, ovulation, sperm generation, implantation and the maintenance of pregnancy. Given the increased incidence of shiftwork and international travel which disrupt circadian rhythmicity, and the increasing prevalence of reproductive technologies whereby early embryo development occurs without external time cues, it is important for us to consider the role of circadian rhythms in fertility. Refereed/Peer-reviewed

Shift work during pregnancy is associated with an increased risk for preterm birth and low birth weight. However, the impact upon the long term health of the children is currently unknown. In this study, we used an animal model to determine the consequences of maternal shift work exposure on the health of the adult offspring. Pregnant rats were exposed to chronic phase shifts (CPS) in their photoperiod every 3-4 days throughout gestation and the first week after birth. Adult offspring were assessed for a range of metabolic, endocrine, circadian and neurobehavioural parameters. At 3 months of age, male pups exposed to the CPS schedule in utero had increased adiposity (+29%) and hyperleptinaemia (+99% at 0700h). By 12 months of age, both male and female rats displayed hyperleptinaemia (+26% and +41% respectively) and hyperinsulinaemia (+110% and +83% respectively). 12 month old female CPS rats displayed poor glucose tolerance (+18%) and increased insulin secretion (+29%) in response to an intraperitoneal glucose tolerance test. In CPS males the glucose response was unaltered, but the insulin response was reduced by 35%. The glucose response to an insulin tolerance test was decreased by 21% in CPS females but unaltered in males. Disruption of circadian rhythmicity during gestation resulted in gender dependent metabolic consequences for the adult offspring. These results highlight the need for a thorough analysis of shift work exposure in utero on the health of the adult offspring in humans. Refereed/Peer-reviewed

The light/dark cycle and suprachiasmatic nucleus rhythmicity are known to have important influences on reproductive function of rodents. We studied reproductive function in female heterozygous and homozygous brain and muscle ARNT-like protein 1 (Bmal1, also known asArntl) null mice, which lack central and peripheral cellular rhythms. HeterozygousBmal1mice developed normally and were fertile, with apparent normal pregnancy progression and litter size, although postnatal mortality up to weaning was high (1.1–1.3/litter). The genotype distribution was skewed with both heterozygous and null genotypes underrepresented (1.0:1.7:0.7;PBmal1allele may impact on postnatal survival. HomozygousBmal1null mice were 30% lighter at weaning, and while they grew at a similar rate to the wild-type mice, they never achieved a comparable body weight. They had delayed vaginal opening (4 days), disrupted estrus cyclicity, and reduced ovarian weight (30%).Bmal1null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, theBmal1mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. WhileBmal1null ovaries responded to pregnant mare serum gonadotropin/human chorionic gonadotropin stimulation, ovulation rate was reduced, and the fertilized oocytes progressed poorly to blastocysts and failed to implant. The loss ofBmal1gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation ofStar. In conclusion, it is clear that the profound infertility ofBmal1null mice is multifactorial.

The circadian rhythmicity of hormone secretion, body temperature, and sleep/wakefulness results from an endogenous rhythm of neural activity generated by clock genes in the suprachiasmatic nucleus (SCN). One of these genes, Clock, has been considered essential for the generation of cellular rhythmicity centrally and in the periphery; however, melatonin-proficient ClockΔ19 + MEL mutant mice retain melatonin rhythmicity, suggesting that their central rhythmicity is intact. Here we show that melatonin production in these mutants was rhythmic in constant darkness and could be entrained by brief single daily light pulses. Under normal light-dark conditions, per2 and prokineticin2 ( PK2) mRNA expression was rhythmic in the SCN of ClockΔ19 + MEL mice. Expression of Bmal1 and npas 2 was not altered, whereas per1 expression was arrhythmic. In contrast to the SCN, per1 and per2 expression, as well as Bmal1 expression in liver and skeletal muscle, together with plasma corticosterone, was arrhythmic in ClockΔ19 + MEL mutant mice in normal light-dark conditions. npas2 mRNA was also arrhythmic in liver but rhythmic in muscle. The ClockΔ19 mutation does not abolish central rhythmicity and light entrainment, suggesting that a functional Clock homolog, possibly npas2, exists in the SCN. Nevertheless, the SCN of ClockΔ19 + MEL mutant mice cannot maintain liver and muscle rhythmicity through rhythmic outputs, including melatonin secretion, in the absence of functional Clock expression in the tissues. Therefore, liver and muscle, but not SCN, have an absolute requirement for CLOCK, with as yet unknown Clock-independent factors able to generate the latter.

The rhythmic expression of clock and clock-controlled genes in the rat oviduct was investigated by real time RT-PCR. per1, per2, Clock, Bmal1, cry1 and cry2 were all expressed in the oviduct. With 4-hourly sampling over 24 h in a normal photoperiod, analysis of variance indicated that per2 and Bmal1 had highly significant sinusoidal-like changes with an amplitude of 3- and 10-fold respectively. Of the other clock genes, per1 and cry1 had non-significant rhythm amplitudes of 2.5- and 1.8-fold respectively. Using the same experimental approach the rhythmic expression of Bmal1, per1 and per2 mRNA in the liver was found to be highly significant with amplitudes of approximately 20-, 10- and 5-fold respectively. The expression of the clock-controlled transcription factors DBP and Rev-erb alpha showed significant rhythmicity in the oviduct with 5-fold changes in amplitude for both genes. Plasminogen activator inhibitor-1 (PAI-1), which has been implicated in oviduct function during the preimplantation period, also had a significant rhythm of expression (2.5-fold amplitude), peaking at the same time as the other clock-controlled genes, DBP and Rev-erb alpha. These results show for the first time that the female reproductive tract is inherently rhythmic and suggests that the developing embryo may be subjected to rhythmic changes in the environment created by the oviduct during transition to the uterus.

The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism. Refereed/Peer-reviewed