Jane C. Whitley

The pregnant ovine endometrium constitutively expresses and secretes a highly stable bombesin-like peptide, which shares C-terminal sequence but differs structurally from gastrin-releasing peptide

Previous studies have shown that a peptide closely related to gastrin releasing peptide (GRP) is expressed by the pregnant ovine endometrium throughout gestation, however its molecular form and the mode of its secretion have not been defined. We have partially purified the endometrial GRP-like peptide and characterised it chromatographically. In contrast to other tissues, the main molecular form of endometrial GRP is larger (6-8 kDa versus 1-3 kDa), and based on the increased hydrophobicity of its circulating form after reduction, contains at least one disulfide bond. Reduction and treatment with chaotropic agents showed that the protein is not a cleavage product of pro-GRP bound to a binding protein. Tryptic cleavage demonstrated that the C-terminus of the peptide is closely related to GRP18-27 suggesting that bioactivity is likely. The partially purified peptide remained intact after incubation in ovine plasma for 16 h indicating that it is extremely stable and consistent with an hormonal role during pregnancy. Quantification of peptide from monolayer cultures of ovine endometrial cells showed that the GRP-like peptide was secreted constitutively. These data show that a stable, GRP-like peptide, distinct from the known processing products of pre-pro-GRP is constitutively expressed by the gravid ovine endometrium. Since endometrial GRP has an intact bioactive C-terminus and is mitogenic for numerous tissues including the uterus, then it is likely to play an important regulatory role in ovine pregnancy.

Taxonomic significance of differences in DNA methylation within the Mycoplasma mycoides cluster detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ‘Mycoplasma mycoides cluster’ was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI, which is inhibited by the methylation, or DpnI, which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum, plus the F38‐like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.

Active immunoneutralization of somatostatin in the sheep : effects on gastrointestinal somatostatin expression, storage and secretion

In the absence of somatostatin antagonists, somatostatin antisera administered acutely or animals chronically immunized against somatostatin have been used to define the functions of somatostatin. However, the circulating immunoglobulins from immunized animals may contain substantial quantities of endogenous hormones. This has not been examined for somatostatin. We have measured the amount of free somatostatin bound to circulating immunoglobulins in somatostatin-immunized animals and the effect of this sequestering of the free peptide on somatostatin secretion and gastric somatostatin synthesis and storage. The average concentration of somatostatin bound to the antisera was 6.9 nmol/l, about 1000-fold higher than normal circulating levels. Compared to control animals, there was a doubling of somatostatin mRNA in the fundus and a 4-fold increase in fundic somatostatin peptide. Similar increases were seen in pancreas, but the antrum was not significantly affected providing further evidence of distinct regulatory mechanisms between the antrum and fundus. We suggest that withdrawal of active somatostatin activates a regulatory loop to increase fundic somatostatin biosynthesis and storage. The data support the concept that somatostatin autoregulates its own expression at both the RNA and peptide level.

FOETAL METABOLISM, PLACENTAL TRANSFER AND ORIGIN OF GASTRIN RELEASING PEPTIDE IN THE SHEEP

1. Plasma gastrin relesing peptide (GRP) is elevated in the foetal and maternal circulations of pregnant sheep. To determine the mechanisms for this increase the synthesis, secretion rate, metabolism and placental transfer of GRP were measued.

Gene expression in the mammary gland of the tammar wallaby during the lactation cycle reveals conserved mechanisms regulating mammalian lactation

The tammar wallaby (Macropus eugenii), an Australian marsupial, has evolved a different lactation strategy compared with eutherian mammals, making it a valuable comparative model for lactation studies. The tammar mammary gland was investigated for changes in gene expression during key stages of the lactation cycle using microarrays. Differentially regulated genes were identified, annotated and subsequent gene ontologies, pathways and molecular networks analysed. Major milk-protein gene expression changes during lactation were in accord with changes in milk-protein secretion. However, other gene expression changes included changes in genes affecting mRNA stability, hormone and cytokine signalling and genes for transport and metabolism of amino acids and lipids. Some genes with large changes in expression have poorly known roles in lactation. For instance, SIM2 was upregulated at lactation initiation and may inhibit proliferation and involution of mammary epithelial cells, while FUT8 was upregulated in Phase 3 of lactation and may support the large increase in milk volume that occurs at this point in the lactation cycle. This pattern of regulation has not previously been reported and suggests that these genes may play a crucial regulatory role in marsupial milk production and are likely to play a related role in other mammals.

Phase Contrast Imaging Reveals Low Lung Volumes and Surface Areas in the Developing Marsupial

Marsupials are born with immature lungs when compared to eutherian mammals and rely, to various extents, on cutaneous gas exchange in order to meet metabolic requirements. Indeed, the fat-tailed dunnart is born with lungs in the canalicular stage of development and relies almost entirely on the skin for gas exchange at birth; consequently undergoing the majority of lung development in air. Plane radiographs and computed tomography data sets were acquired using phase contrast imaging with a synchrotron radiation source for two marsupial species, the fat-tailed dunnart and the larger tammar wallaby, during the first weeks of postnatal life. Phase contrast imaging revealed that only two lung sacs contain air after the first hour of life in the fat-tailed dunnart. While the lung of the tammar wallaby was comparatively more developed, both species demonstrated massive increases in air sac number and architectural complexity during the postnatal period. In addition, both the tammar wallaby and fat-tailed dunnart had lower lung volumes and parenchymal surface areas than were expected from morphometrically determined allometric equations relating these variables to body mass during the neonatal period. However, lung volume is predicted to scale with mass as expected after the neonatal marsupial reaches a body mass of ∼1 g and no longer relies on the skin for gas exchange. Decreased lung volume in the marsupial neonate further supports the maxim that cutaneous gas exchange occurs in the marsupial neonate because the respiratory apparatus is not yet capable of meeting the gas exchange requirements of the newborn.