It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.
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- Author: A López Bernal x
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B Rodriguez-Linares, S Phaneuf, A Lopez Bernal, and EA Linton
G N Europe-Finner, S Phaneuf, E Cartwright, H J Mardon, and A López Bernal
ABSTRACT
We have shown previously that expression of 46 and 54 kDa human myometrial Gαs protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by Gαs-Small (with a serine residue at position 72) and Gαs-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a Gαs cDNA template to generate [32P]cytidine cRNA ribo-probes for use in RNase protection assays, so as to measure total myometrial Gαs mRNA levels in relation to the pattern of expression of Gαs mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial Gαs mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of Gαs precursor mRNA has a primary role in regulating expression of Gαs protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in Gαs mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.
G N Europe-Finner, E Cartwright, J Bellinger, H J Mardon, D H Barlow, and A López Bernal
ABSTRACT
Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.
