Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice

The hypothesis that adenosine acting on adenosine A1 receptors (A1R) regulates several renal functions and mediates tubuloglomerular feedback (TGF) was examined using A1R knockout mice. We anesthetized knockout, wild-type, and heterozygous mice and measured glomerular filtration rate, TGF response using the stop-flow pressure (P(sf)) technique, and plasma renin concentration. The A1R knockout mice had an increased blood pressure compared with wild-type and heterozygote mice. Glomerular filtration rate was similar in all genotypes. Proximal tubular P(sf) was decreased from 36.7 +/- 1.2 to 25.3 +/- 1.6 mmHg in the A1R+/+ mice and from 38.1 +/- 1.0 to 27.4 +/- 1.1 mmHg in A1R+/- mice in response to an increase in tubular flow rate from 0 to 35 nl/min. This response was abolished in the homozygous A1R-/- mice (from 39.1 +/- 4.1 to 39.2 +/- 4.5 mmHg). Plasma renin activity was significantly greater in the A1R knockout mice [74.2 +/- 14.3 milli-Goldblatt units (mGU)/ml] mice compared with the wild-type and A1R+/- mice (36.3 +/- 8.5 and 34.1 +/- 9.6 mGU/ml), respectively. The results demonstrate that adenosine acting on A1R is required for TGF and modulates renin release.     

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.     

Pancreatic neuroendocrine tumors in glucagon receptor-deficient mice

Inhibition of glucagon signaling causes hyperglucagonemia and pancreatic α cell hyperplasia in mice. We have recently demonstrated that a patient with an inactivating glucagon receptor mutation (P86S) also exhibits hyperglucagonemia and pancreatic α cell hyperplasia but further develops pancreatic neuroendocrine tumors (PNETs). To test the hypothesis that defective glucagon signaling causes PNETs, we studied the pancreata of mice deficient in glucagon receptor (Gcgr^−/−) from 2 to 12 months, using WT and heterozygous mice as controls. At 2–3 months, Gcgr^−/− mice exhibited normal islet morphology but the islets were mostly composed of α cells. At 5–7 months, dysplastic islets were evident in Gcgr^−/− mice but absent in WT or heterozygous controls. At 10–12 months, gross PNETs (≥1 mm) were detected in most Gcgr^−/− pancreata and micro-PNETs (<1 mm) were found in all (n = 14), whereas the islet morphology remained normal and no PNETs were found in any WT (n = 10) or heterozygous (n = 25) pancreata. Most PNETs in Gcgr^−/− mice were glucagonomas, but some were non-functioning. No tumors predominantly expressed insulin, pancreatic polypeptide, or somatostatin, although some harbored focal aggregates of tumor cells expressing one of those hormones. The PNETs in Gcgr^−/− mice were well differentiated and occasionally metastasized to the liver. Menin expression was aberrant in most dysplatic islets and PNETs. Vascular endothelial growth factor (VEGF) was overexpressed in PNET cells and its receptor Flk-1 was found in the abundant blood vessels or blood islands inside the tumors. We conclude that defective glucagon signaling causes PNETs in the Gcgr^−/− mice, which may be used as a model of human PNETs. Our results further suggest that completely inhibiting glucagon signaling may not be a safe approach to treat diabetes.     

Experimental Trypanosoma cruzi infection in platelet-activating factor receptor-deficient mice

The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR(-/-) mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR(-/-) mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-gamma and TNF-alpha in heart tissue of infected animals. Moreover, serum concentrations of TNF-alpha, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR(-/-) animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B(4) or MCP-1 and produced less nitric oxide when infected and activated with IFN-gamma. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.     

Sleep in Kcna2 knockout mice

Background  

Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system.     

Neuroadaptations to hyperdopaminergia in dopamine D3 receptor-deficient mice

The dopamine D3 receptor (D3R) has been implicated in schizophrenia, drug addiction, depression and Parkinson's disease. The D3R is localized post-synaptically on nucleus accumbens neurons, but is also an autoreceptor on dopaminergic neurons in the mesencephalon. Its functional role as autoreceptor is highly debated, but supported by the elevated basal extracellular dopamine levels found in D3R-deficient mice. To investigate the functional role of the D3R in vivo, we used mice with a targeted disruption of the D3R gene. We found a higher basal level of grooming in D3R-deficient mice, compared to their wild-type littermates. This behavior, which is under the control of D1R stimulation, may be related to an increased dopaminergic tone, since no changes in the gene expression of dopamine D1 and D2 receptors were noticed in the striatum of these mice. D3R-deficient mice displayed other neuroadaptive changes, including decreased tyrosine hydroxylase, increased dopamine transporter mRNAs and increased dopamine reuptake in striatum. The level of tyrosine hydroxylase protein was unchanged in the striatum, as preprodynorphin and preproenkephalin gene expressions. All the changes identified in D3R-deficient mice cannot explain hyperdopaminergia, but, on the contrary, tend to attenuate this phenotype. These results support a distinct role for D2R and D3R as autoreceptors: the D2R is the release-regulating and firing rate-regulating autoreceptor, whereas the D3R may control basal dopamine levels in the striatum, by an unknown mechanism, which does not involve regulation of dopamine transporters or tyrosine hydroxylase. This hyperdopaminergia phenotype of D3R-deficient mice may explain their hyperactivity to drug-paired environmental cues.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

Dopamine D2R DNA transfer in dopamine D2 receptor-deficient mice: effects on ethanol drinking

Dopamine (DA) signals are transmitted via specific receptors including the D2 receptors (D2R). Previous studies have shown that D2R upregulation in the nucleus accumbens (NAc) attenuated alcohol consumption. We hypothesized that upregulation of D2R in the NAc would significantly influence alcohol drinking. We tested this hypothesis by determining the effect that D2R upregulation has on alcohol intake in genetically altered mice lacking D2Rs. After a steady baseline of drinking behavior was established for all mice, a null vector or a genetically modified adenoviral vector containing the rat D2R cDNA was infused into the NAc of wild-type (Drd2+/+), heterozygous (Drd2+/-), and receptor-deficient mice (Drd2-/-). Ethanol intake and preference were then determined using the two-bottle choice paradigm. Our results indicated that Drd2+/+ mice treated with the D2R vector significantly attenuated (58 %) their ethanol intake as well as reduced preference. Drd2+/- and mutant mice showed a similar attenuation, although the change was not as marked (12 %) and did not last as long. In contrast, Drd2-/- mice treated with the D2R vector displayed a temporary but significant increase (46 %) in ethanol intake and preference (consumption). These results supported the notion that the D2R plays an important role in alcohol consumption in mice and suggest that a key threshold range of D2R levels is associated with elevated alcohol consumption. Significant deviations in D2R levels from this range could impact alcohol consumption, and could help to explain possible individual variations in alcohol response, metabolism, sensitivity and consumption.     

Genetic removal of the A2A adenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation.     

Functional striatal hypodopaminergic activity in mice lacking adenosine A(2A) receptors.

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A(2A) receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A(2A) receptor-deficient mice (A(2A)-R(-/-)). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A(2A) receptor by striatopallidal neurones. To clarify the functions of A(2A) receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60-80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three-fold increase in extracellular glutamate concentration. This was associated with an up-regulation of D(1) and D(2) dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A(1) receptors expression were indistinguishable between A(2A)-R(-/-) and wild-type mice. Altogether these results pointed out that the lack of A(2A) receptors leads to a functional hypodopaminergic state and demonstrated that A(2A) receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.     

Involvement of adenosine A1 and A2A receptors on guanosine-mediated anti-tremor effects in reserpinized mice

Parkinson’s disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A₁ and A_2A receptors (A₁R and A_2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A_2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A_2AR, thus suggesting an A_2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A₁R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A₁R dependent but A_2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.

     

Persistence of circadian variation in arterial blood pressure in beta1/beta2-adrenergic receptor-deficient mice

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.     

Vitamin D receptor-deficient mice fail to develop experimental allergic asthma

The active metabolite of vitamin D (1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))) is known to modulate the immune response in Th1 cell-directed diseases. To investigate the role of vitamin D in Th2 cell-directed diseases, experimental allergic asthma was induced in vitamin D receptor (VDR) knockout and in wild-type (WT) mice. As expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils, elevated Th2 cytokine levels, mucous production, and airway hyperresponsiveness. The administration of 1,25(OH)(2)D(3) had no effect on asthma severity. The only discernable effect of 1,25(OH)(2)D(3) on experimental allergic asthma in WT mice was an increased expression of two Th2-related genes (soluble CD23 and GATA-3) in lungs of BALB/c mice exposed to Ag through the nasal route only. By contrast, asthma-induced VDR knockout mice failed to develop airway inflammation, eosinophilia, or airway hyperresponsiveness, despite high IgE concentrations and elevated Th2 cytokines. The data suggest that although 1,25(OH)(2)D(3) induced these Th2-type genes, the treatment failed to have any affect on experimental asthma severity. However, VDR-deficient mice failed to develop experimental allergic asthma, suggesting an important role for the vitamin D endocrine system in the generation of Th2-driven inflammation in the lung.     

Immune system development and function in prolactin receptor-deficient mice.

Prolactin (PRL) is the primary lactogenic pituitary hormone that plays an essential role in many aspects of reproduction, from fertilization to mammary gland development and maternal behavior. PRL has also been reported to play a role in immunoregulation. Because initial observations indicated that hypophysectomized rats present abnormalities of the immune system, including increased thymic atrophy and lymphopenia, a number of studies have focused on the potential immunomodulatory roles of PRL. This hormone exerts its biological activities following binding to specific cell surface PRL receptors (PRLRs). In this report, we have characterized the development and function of the immune system in PRLR-deficient mice. Compared with wild-type control mice, PRLR-/- mice demonstrate no alterations in thymic or splenic cellularity or in the composition of the lymphocyte subsets present in primary (bone marrow and thymus) or secondary (spleen and lymph nodes) lymphoid organs. Lymphocytes from PRLR-/- mice are functional in vitro, as they can proliferate normally to mitogens, cytokines, and allogeneic cells. PRLR-/- splenocytes display normal NK-mediated cytotoxicity to YAC-1 target cells. In vivo studies have revealed that PRLR-/- mice are able to 1) generate normal steady-state Ig levels, 2) mount a normal specific Ig response following immunization with a T-dependent Ag, 3) eliminate injected allogeneic tumor cells, and 4) effectively control Listeria monocytogenes infection. Taken together, these results show that immune system development and function proceed normally in the absence of PRL-mediated signaling and suggest that PRLR pathways are not essential for immunomodulation in vivo.     

Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.

Growth hormone (GH) has pleiotropic effects on cholesterol and lipoprotein metabolism. Pituitary GH is important for the normal regulation of hepatic LDL receptors (LDLR), for the enzymatic activity of bile acid regulatory cholesterol 7alpha-hydroxylase (C7alphaOH), and for the maintenance of resistance to dietary cholesterol. The present study aimed to determine whether GH has beneficial effects on plasma lipids and hepatic cholesterol metabolism in mice devoid of LDLR. Compared with wild-type controls, LDLR-deficient mice had approximately 250% elevated plasma total cholesterol and approximately 50% increased hepatic cholesterol levels; hepatic HMG CoA reductase activity was reduced by 70%, whereas C7alphaOH activity was increased by 40%. In LDLR mice, GH infusion reduced plasma cholesterol and triglycerides up to 40%, whereas HMG CoA reductase and C7alphaOH activities were stimulated by approximately 50% and 110% respectively. GH also stimulated HMG CoA reductase and C7alphaOH activities in control mice, whereas hepatic LDLR and plasma lipoproteins were unchanged. The effects of cholestyramine and atorvastatin on C7alphaOH in LDLR-deficient mice were potentiated by GH, and this was associated with a further reduction in plasma cholesterol. GH treatment reduces plasma cholesterol and triglycerides and stimulates C7alphaOH activity in mice devoid of LDLR, particularly in combination with resin or statin treatment. The potential of GH therapy in patients with homozygous familial hypercholesterolemia should be evaluated.     

Structure-affinity relationships of the affinity of 2-pyrazolyl adenosine analogues for the adenosine A2A receptor

The structure-affinity relationships of two novel 2-substituted adenosine series containing a substituted pyrazole attached at the N-1 or C-4 position for the adenosine (ADO) A2A receptor are described. Compounds in the 2-(N-1-pyrazolyl) adenosine series IV provided the highest affinity for the ADO A2A receptor as compared to the 2-(C-4-pyrazolyl) series V. The main structural differences between the two series point to the N-1 nitrogen of series IV imparting more favorable binding interactions with the receptor than those of series V.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Role of prostaglandin H2 synthase 2 in murine parturition: study on ovariectomy-induced parturition in prostaglandin F receptor-deficient mice

To determine the prostaglandin (PG) H2 synthase (generally referred to as cyclooxygenase [COX]) isozyme responsible for producing uterotonic PGs during parturition, we used PGF2alpha receptor-deficient mice, which exhibit parturition failure due to impaired withdrawal of serum progesterone at term. On ovariectomy-induced parturition in these mice, uterine COX-2 mRNA expression was drastically induced in the myometrium, whereas COX-1 mRNA expression in the endometrial epithelium decreased. The concomitant administration of progesterone with ovariectomy resulted in a delay in parturition and the disappearance of both the increase in COX-2 mRNA and the decrease in COX-1 mRNA. Thus, the expression of myometrial COX-2 and the occurrence of parturition are closely associated in this model. Furthermore, administration of the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522, effectively delayed ovariectomy-induced parturition in these mice. These findings suggest that COX-2-derived PGs contribute to the onset of parturition after the decrease in serum progesterone level.