Minor Abnormalities of Testis Development in Mice Lacking the Gene Encoding the MAPK Signalling Component, MAP3K1

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans

Sustained increases in plasma C-type natriuretic peptides fail to increase concentrations in cerebrospinal fluid: Evidence from pregnant sheep

C-type natriuretic peptide (CNP) is a paracrine growth factor with high abundance in CNS tissues and cerebrospinal fluid (CSF). Consistent with findings of CNP transcripts in the cerebral microvasculature and hypothalamus, CNP increases the permeability of the blood-brain barrier and reduces food intake when administered intracerebroventricularly in rodents. Whether high concentrations of CNP in plasma can affect CSF levels is unknown. Accordingly we have studied changes (days 4, 87 and 116) in concurrent plasma and CSF concentrations of CNP peptides in pregnant sheep - a physiologically unique setting in which plasma CNP is elevated for prolonged periods. Preliminary studies in non pregnant sheep showed stable CNP levels in CSF during repetitive sampling. Compared with values in non pregnant controls, plasma concentrations of CNP peptides were markedly raised (30-fold) at days 87 and 116 in pregnant sheep, yet CSF levels in the two groups did not differ. CNP peptides in CSF decreased from day 4 to day 87 in pregnant sheep, possibly reflecting an adaptive response of the cerebral vasculature to increased hemodynamic load. We conclude that sustained high concentrations of CNP - far exceeding levels encountered in human pathophysiology - fail to affect CNP peptide levels in CSF

Nutrient restriction in early ovine pregnancy stimulates C-type natriuretic peptide production

C-type natriuretic peptide (CNP), a paracrine growth factor promoting vasodilation and angiogenesis, is upregulated in human and ovine pregnancy in response to vascular stress or nutrient restriction (NR) in late gestation. Postulating that maternal plasma CNP products are increased by modest NR (50% of metabolisable energy requirement) early in pregnancy, and further enhanced by litter size, we studied serial changes of maternal plasma CNP in pregnant ewes receiving a normal (NC, n = 12) or restricted (NR, n = 13) diet from Day 30 to Day 93 or 94 of gestation. Liveweight of NR ewes was 10 kg less than that of NC ewes at slaughter. Plasma CNP products increased progressively after Day 40 and were higher in NR (P < 0.05) ewes after Day 60; they were also enhanced by litter size (P < 0.01) and were positively associated with increased placental efficiency. In contrast, whereas fetal and placental weight were reduced by NR, fetal plasma CNP products (Day 93/94) were not affected. We conclude that increases in CNP during rapid placental growth are further enhanced by both increasing nutrient demands and by reduced supply, presumably as part of an adaptive response benefitting placental–fetal exchange

Molecular forms of C-type natriuretic peptide in cerebrospinal fluid and plasma reflect differential processing in brain and pituitary tissues

C-type natriuretic peptide (CNP) is a paracrine growth factor widely expressed within tissues of the central nervous system. Consistent with this is the high concentration of CNP in cerebrospinal fluid (CSF), exceeding levels in the systemic circulation. CNP abundance is high in hypothalamus and especially enriched in pituitary tissue where – in contrast to hypothalamus – processing to CNP-22 is minimal. Recently we have shown that dexamethasone acutely raises CNP peptides throughout the brain as well as in CSF and plasma. Postulating that molecular forms of CNP would differ in central tissues compared to forms in pituitary and plasma, we have characterized the molecular forms of CNP in tissues (hypothalamus, anterior and posterior pituitary gland) and associated fluids (CSF and plasma) using size-exclusion high performance liquid chromatography (SE-HPLC) and radioimmunoassay in control (saline-treated) and dexamethasone-treated adult sheep. Three immunoreactive-CNP components were identified which were consistent with proCNP (1-103), CNP-53 and CNP-22, but the presence and proportions of these different fragments differed among tissues. Peaks consistent with CNP-53 were the dominant form in all tissues and fluids. Peaks consistent with proCNP, conspicuous in hypothalamic extracts, were negligible in CSF whereas proportions of low molecular weight immunoreactivity (IR) consistent with CNP- 22 were similar in hypothalamus, posterior pituitary gland and CSF. In contrast, in both plasma and the anterior pituitary gland, proportions of higher molecular weight IR, consistent with CNP-53 and proCNP, predominated, and low molecular weight IR consistent with CNP-22 was very low. After dexamethasone, proCNP like material – but not other forms – was increased in all samples except CSF, consistent with increased synthesis and secretion. In conclusion, immunoreactive forms of CNP in central tissues differ from those identified in anterior pituitary tissue and plasma – suggesting that the anterior pituitary gland may contribute to systemic levels of CNP in some physiological settings

Environmental Enrichment Elicits a Transient Rise of Bioactive C-Type Natriuretic Peptide in Young but Not Aged Rats

Beneficial molecular and neuroplastic changes have been demonstrated in response to environmental enrichment (EE) in laboratory animals across the lifespan. Here, we investigated whether these effects extend to C-type Natriuretic Peptide (CNP), a widely expressed neuropeptide with putative involvement in neuroprotection, neuroplasticity, anxiety, and learning and memory. We determined the CNP response in 36 young (8–9 months) and 36 aged (22–23 months) male PVGc hooded rats that were rehoused with new cage mates in either standard laboratory cages or EE for periods of 14 or 28 days. Tissues were rapidly excised from four brain regions associated with memory formation (dorsal hippocampus, retrosplenial cortex, medial prefrontal cortex, and mammillary bodies) plus the occipital cortex and hypothalamus, and immediately frozen. Radioimmunoassay was used to measure bioactive CNP and the amino-terminal fragment of proCNP, NTproCNP. Because CNP but not NTproCNP is rapidly degraded at source, NTproCNP reflects CNP production whereas the ratio NTproCNP:CNP is a biomarker of CNP’s local degradation rate. EE increased CNP at 14 days in all brain regions in young, but not old rats; this effect in young rats was lost at 28 days in all regions of interest. NTproCNP:CNP ratio, but not NTproCNP, was reduced in all regions by EE at 14 days in young rats, but not in old rats, which suggests a period of reduced degradation or receptor mediated clearance, rather than increased production of CNP in these young EE rats. Aged rats tended to show reduced NTproCNP:CNP ratios but this did not occur in dorsal hippocampus or mammillary bodies. This is the first study demonstrating modulation of CNP protein concentrations, and the effect of age, in response to environmental stimulation. Furthermore, it is the first to show that changes in degradation rate in vivo may be an important component in determining CNP bioactivity in neural tissues

Environmental Enrichment Elicits a Transient Rise of Bioactive C-Type Natriuretic Peptide in Young but Not Aged Rats

Beneficial molecular and neuroplastic changes have been demonstrated in response to environmental enrichment (EE) in laboratory animals across the lifespan. Here, we investigated whether these effects extend to C-type Natriuretic Peptide (CNP), a widely expressed neuropeptide with putative involvement in neuroprotection, neuroplasticity, anxiety, and learning and memory. We determined the CNP response in 36 young (8-9 months) and 36 aged (22-23 months) male PVGc hooded rats that were rehoused with new cage mates in either standard laboratory cages or EE for periods of 14 or 28 days. Tissues were rapidly excised from four brain regions associated with memory formation (dorsal hippocampus, retrosplenial cortex, medial prefrontal cortex, and mammillary bodies) plus the occipital cortex and hypothalamus, and immediately frozen. Radioimmunoassay was used to measure bioactive CNP and the amino-terminal fragment of proCNP, NTproCNP. Because CNP but not NTproCNP is rapidly degraded at source, NTproCNP reflects CNP production whereas the ratio NTproCNP:CNP is a biomarker of CNP’s local degradation rate. EE increased CNP at 14 days in all brain regions in young, but not old rats; this effect in young rats was lost at 28 days in all regions of interest. NTproCNP:CNP ratio, but not NTproCNP, was reduced in all regions by EE at 14 days in young rats, but not in old rats, which suggests a period of reduced degradation or receptor mediated clearance, rather than increased production of CNP in these young EE rats. Aged rats tended to show reduced NTproCNP:CNP ratios but this did not occur in dorsal hippocampus or mammillary bodies. This is the first study demonstrating modulation of CNP protein concentrations, and the effect of age, in response to environmental stimulation. Furthermore, it is the first to show that changes in degradation rate in vivo may be an important component in determining CNP bioactivity in neural tissues

Differential response of C-type natriuretic peptide to estrogen and dexamethasone in adult bone

C-type natriuretic peptide (CNP) is crucial in promoting endochondral bone growth in mammals including humans but whether this paracrine hormone participates in maintaining bone integrity in the mature skeleton is unknown. Accordingly we studied changes in plasma and bone tissue CNP in anoestrus adult ewes receiving short term anabolic (estrogen) or catabolic (dexamethasone) treatment for 7 days. CNP and the aminoterminal fragment of the CNP prohormone (NTproCNP) were measured in plasma and extracts of cancellous bone excised from vertebral, iliac, tibial and marrow tissues. Concentrations of CNP peptides were much higher in vertebral and iliac extracts than those of tibial or marrow. Both plasma CNP and NTproCNP increased rapidly after estrogen followed by a later rise in bone alkaline phosphatase. Vertebral and iliac (but not tibial or marrow) CNP peptide content were significantly increased by estrogen. Consistent with a skeletal source, plasma NTproCNP was significantly associated with vertebral tissue CNP. In contrast, bone tissue CNP peptide content was unaffected by dexamethasone despite suppression of plasma CNP peptides and bone alkaline phosphatase. We postulate that increases in trabecular bone CNP reflect new endosteal bone formation in these estrogen responsive tissues whereas reduced plasma CNP peptides after dexamethasone, without change in cancellous bone content, reflects reductions in cortical bone turnover. © 2014 Elsevier Inc. All rights reserved