The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.

The presence of prolactin (PRL) mRNA in the mammary gland of lactating goats and sheep was demonstrated by Northern analysis and RT-PCR. This provides evidence that the PRL gene is transcribed in this tissue. This ectopic expression is not restricted to the lactational period, as PRL transcripts were also found during the last third of pregnancy. Comparison of mammary and pituitary PRL mRNAs showed that they are similar in size but less abundant in mammary gland. In addition, an 847-bp cDNA fragment amplified from mammary retrotranscripts, containing the entire coding region and the major part of the 5’ and 3’ untranslated regions (UTRs), was found to be identical in sequence to its pituitary counterpart. Primer extension analysis, performed to obtain further information on the structure of the mammary PRL mRNA, has shown that the 5’ UTR is 56 nucleotides (nt) long for both species. This is comparable with the size (53 nt) found using the caprine pituitary RNA as template. These results strongly suggest that the PRL gene is not transcribed from a different promoter in mammary gland, as has been demonstrated for placental and lymphocyte cells, but is more likely transcribed from the pituitary-specific promoter. Finally, the presence of PRL mRNA in polysomal fractions suggests that PRL is synthesized in mammary cells.

The two non-allelic forms of as2-casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, including both cysteine residues at positions 34 and 42 in the mature chain. Sequencing of several crs2-casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons + 34 to + 42 affecting 30-40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5' untranslated region, was found. S1-nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in-vitro-amplified genomic DNA demonstrated the presence of the 44-nucleotide stretch in the as2-casein transcriptional unit, thus ruling out the possibility of a cloning artefact.The different a,,-casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5' and 3' ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44-nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3' and 5' ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy as2-casein gene/haploid genome. During the course of evolution, the as2-casein-encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre-mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse as2-casein mRNA. If so, the overall organization of the as2-casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.In ruminants, caseins comprise the major fraction of secretory proteins synthesized in mammary epithelial cells. Primary structures of the four bovine caseins (reviewed in [l]) share little similarity, except for the well-conserved multiple phosphorylation sites and signal peptides [2] of the calciumsensitive caseins, asl, as2 and j?. The proposal that they have a common origin [2] was further substantiated by the similarity in organization of the relevant genes in the region spanning the promoter and the 5' end of the transcription unit [3].The striking similarity (40%) between the N-terminal and C-terminal halves of bovine crs2-casein [4] is partially known for the rat [lo] and bovine [l 13 species. It may contain at least nine introns, but sequence data are only available for the region upstream from exon 11.Previous SDS/PAGE studies of ovine as2-casein, synthesized in both cell-free translation systems and mammary gland explants, revealed the occurrence of two polypeptide chains differing by an internal deletion...

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Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.