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Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine required for blastocyst implantation in mice. Uterine expression of LIF and that of its receptors has been demonstrated in a number of mammalian species indicating that LIF may have widespread importance in the establishment of pregnancy. The variations in the reaction of the uterus in preparation for and during implantation are considerable between species and understanding the differences and similarities assists in the interpretation of how this cytokine functions. Recent studies suggest that reduced endometrial LIF contributes to human infertility. Studies also demonstrate a potential role in placentation and fetal development. Thus, LIF has become an important cytokine warranting further investigation in the human. It is anticipated that when the mechanisms underlying normal embryonic and endometrial development are elucidated, fertility and infertility will be more precisely understood and hence able to be effectively controlled.
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Synthesis of both mRNA and peptide for gastrin-releasing peptide (GRP) has been demonstrated in the pregnant endometrium of sheep and women. However, it is not known whether GRP is synthesized in the sheep uterus during the oestrous cycle. Furthermore the cellular site of GRP mRNA synthesis in the uterus has not been determined. Therefore we examined the synthesis of GRP and determined the cellular location of GRP peptide and mRNA in sheep uterus taken at different times during the oestrous cycle (duration 17 days) and pregnancy (duration 145 days). Northern blot analysis of RNA isolated from ovine endometrium revealed low or no GRP mRNA at days 4, 10, 12 and 14 of the oestrous cycle and a 24-fold rise in GRP mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA) between days 14 and 16. A similar pattern was observed during early pregnancy, with a 12-fold rise in GRP mRNA:GAPDH mRNA between days 17 and 20 of pregnancy. Levels of GRP peptide were determined by RIA and found to be low in endometrium isolated at days 4, 10, 12 and 14 of the oestrous cycle (1.0-1.6 pmol/g) and 4 to 5-fold higher at day 16. In situ hybridization localized GRP synthesis to the epithelial cells of the uterine glands at day 16 of the oestrous cycle and at days 17, 20, 40 and 50 of pregnancy. At day 140 of pregnancy diffuse hybridization to cells of the myometrium was also observed. Immunohistochemistry localized GRP peptide to the apical cytoplasm of uterine glandular epithelial cells at day 16 of the oestrous cycle. For samples obtained at day 20 of pregnancy, the area surrounding the glands also showed moderately strong staining. Further staining in the glandular lumen and the stromal tissue surrounding the glands was apparent at day 140 of pregnancy. No GRP immunoreactivity could be detected in the peripheral plasma during the oestrous cycle or the first 20 days of pregnancy. Sizing chromatography of GRP immunoreactivity extracted from endometrial tissue taken at day 10 of the oestrous cycle revealed two peaks that co-eluted with GRP(1-27) and GRP(18-27). However, during luteolysis and oestrus the major peak of GRP immunoreactivity extracted from endometrial tissue was larger than GRP(1-27) and similar to that seen previously in the gravid ovine endometrium. These studies demonstrate that a peptide similar to, but larger than, GRP is a major product of the glandular epithelium of the ovine uterus during the luteal regression phase of the oestrous cycle and post-blastocyst implantation in pregnancy and provide further evidence that GRP-related peptides have important regulatory roles in uterine function.
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ABSTRACT
Ovine endometrial cells were isolated from ovariectomized oestrogen and progesterone-treated ewes and maintained in primary culture. In-vitro treatment with human interferon-α2 (Roferon, Hoffman La Roche) (5, 50 IU/ml) or purified ovine trophoblast protein 1 (oTP-1, 30 ng/ml) significantly attenuated PGF2α (25±17, 29±17, 28±9%±SEM of control [no in-vitro treatment = 100%] respectively, N=4 ewes) and PGE (11±4, 16±4, 16±5% of control) release from the cultured cells. Fluorography of two dimensional polyacrylamide gel electrophoretic analyses of proteins secreted by the cells following 35S-methionine incorporation, revealed that synthesis and secretion of the same "pregnancy-related" proteins was stimulated by both interferon-α2 and oTP-1. Thus, interferon-α2 (which has sequence homology with oTP-1) acts on the ovine endometrium, eliciting similar biological responses to those of oTP-1.
