Progesterone receptor-dependent regulation of genes in the oviducts of female mice.

Oviducts play a critical role in gamete and embryo transport, as well as supporting early embryo development. Progesterone receptor (PGR) is a transcription factor highly expressed in oviductal cells, while its activating ligand, progesterone, surges to peak levels as ovulation approaches. Progesterone is known to regulate oviduct cilia beating and muscular contractions in vitro, but how PGR may mediate this in vivo is poorly understood. We used PGR null mice to identify genes potentially regulated by PGR in the oviducts during the periovulatory period. Histologically, oviducts from PGR null mice showed no gross structural or morphological defects compared with normal littermates. However, microarray analysis of oviducts at 8 h posthuman chorionic gonadotropin revealed >1,000 PGR-dependent genes. Using reverse-transcription polymerase chain reaction (RT-PCR) we selected 10 genes for validation based on their potential roles in oocyte/embryo transport and support. Eight genes were confirmed to be downregulated (Adamts1, Itga8, Edn3, Prlr, Ptgfr, Des, Myocd, and Actg2) and one upregulated (Agtr2) in PGR null oviducts. Expression of these genes was also assessed in oviducts of naturally cycling mice during ovulation and day 1 and day 4 of pregnancy. Adamts1, Itga8, Edn3, Prlr, and Ptgfr were significantly upregulated in oviducts at ovulation/mating. However, most genes showed basal levels of expression at other times. The exceptions were Prlr and Ptgfr, which showed pulsatile increases on day 1 and/or day 4 of pregnancy. This is the first, comprehensive study to elucidate putative PGR-regulated genes in the oviduct and reveals key downstream targets potentially mediating oocyte and embryo transport.

Rapidly alternating photoperiods disrupt central and peripheral rhythmicity and decrease plasma glucose, but do not affect glucose tolerance or insulin secretion in 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.

Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle.

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.

Reproductive biology of female Bmal1 null mice.

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 as Arntl) null mice, which lack central and peripheral cellular rhythms. Heterozygous Bmal1 mice 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; P<0.05), suggesting loss of a single Bmal1 allele may impact on postnatal survival. Homozygous Bmal1 null 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%). Bmal1 null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, the Bmal1 mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. While Bmal1 null 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 of Bmal1 gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation of Star. In conclusion, it is clear that the profound infertility of Bmal1 null mice is multifactorial.

Reproductive performance in female Clock Delta19 mutant mice.

The relationship between circadian rhythmicity and rodent reproductive cyclicity is well established, but the impact of disrupted clock gene function on reproduction has not been well established. The present study evaluated the reproductive performance of mice carrying the Clock(Delta19) mutation that were either melatonin deficient (Clock(Delta19/Delta19)) or had the capacity to synthesise melatonin reinstated (Clock(Delta19/Delta19)+MEL). The Clock(Delta19/Delta19) mice took 2-3 days longer to mate, and to subsequently deliver pups, than their control line. The melatonin-competent mutants had a smaller, but still significant (P < 0.05), delay. The Clock(Delta19) mutation resulted in smaller median litter sizes compared with control lines (seven v. eight pups; P < 0.05), whereas melatonin proficiency reversed this difference. Survival to weaning was 84% and 80% for the Clock(Delta19/Delta19) and Clock(Delta19/Delta19)+MEL lines, respectively, compared with 94-96% for the two control lines. The Clock(Delta19/Delta19) mutants became behaviourally arrhythmic in constant darkness but, despite this, seven of seven became pregnant when paired with males after at least 14 days of constant darkness (five of seven within 4 days of pairing). In the Clock(Delta19/Delta19)+MEL mice, seven of 15 became arrhythmic in constant darkness but still became pregnant. The seven mice that free ran for at least 14 days in constant darkness with a period of 27.1 h also became pregnant. The present study has demonstrated that the Clock(Delta19) mutation has significant, but subtle, effects on reproductive performance. The reintroduction of melatonin competency and/or other genes as a result of crosses with CBA mice reduced the impact of the mutation further. It would appear that redundancy in genes in the circadian system allows the reproductive cyclicity to persist in mice, albeit at a suboptimal level.

Characterisation of the maternal response to chronic phase shifts during gestation in the rat: implications for fetal metabolic programming.

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.

Circadian rhythms and fertility.

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.

Metabolic homeostasis in mice with disrupted Clock gene expression in peripheral tissues.

The role of peripheral vs. central circadian rhythms and Clock in the maintenance of metabolic homeostasis and with aging was examined by using Clock(Delta19)+MEL mice. These have preserved suprachiasmatic nucleus and pineal gland rhythmicity but arrhythmic Clock gene expression in the liver and skeletal muscle. Clock(Delta19)+MEL mice showed fasting hypoglycemia in young-adult males, fasting hyperglycemia in older females, and substantially impaired glucose tolerance overall. Clock(Delta19)+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. Clock(Delta19)+MEL mice had reduced hepatic expression and loss of rhythmicity of gck, pfkfb3, and pepck mRNA, which is likely to impair glycolysis and gluconeogenesis. Clock(Delta19)+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 Clock(Delta19)+MEL mice, however, suggesting enhanced insulin sensitivity. These responses occurred although the Clock(Delta19) mutation 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 and reproduction.

There is a growing recognition that the circadian timing system, in particular recently discovered clock genes, plays a major role in a wide range of physiological systems. Microarray studies, for example, have shown that the expression of hundreds of genes changes many fold in the suprachiasmatic nucleus, liver heart and kidney. In this review, we discuss the role of circadian rhythmicity in the control of reproductive function in animals and humans. Circadian rhythms and clock genes appear to be involved in optimal reproductive performance, but there are sufficient redundancies in their function that many of the knockout mice produced do not show overt reproductive failure. Furthermore, important strain differences have emerged from the studies especially between the various Clock (Circadian Locomotor Output Cycle Kaput) mutant strains. Nevertheless, there is emerging evidence that the primary clock genes, Clock and Bmal1 (Brain and Muscle ARNT-like protein 1, also known as Mop3), strongly influence reproductive competency. The extent to which the circadian timing system affects human reproductive performance is not known, in part, because many of the appropriate studies have not been done. With the role of Clock and Bmal1 in fertility becoming clearer, it may be time to pursue the effect of polymorphisms in these genes in relation to the various types of infertility in humans.