The diversity of the PRL gene family has been demonstrated in mouse, rat, cattle and goat, but functional information about these proteins is limited except for PRL, PL and some rodent prolactin-like proteins (Prlps) [2, 19, 25–29]. In particular, ruminants commonly have various genes of this family, but there is no information regarding sheep, even though anatomical evidence shows that placentae are similar among bovidae [1, 20, 21, 30, 31]. Thirteen varieties of PRP paralogs have been reported in bovines and two in goat [1, 5, 20, 21]. In the present study, we have identified novel PRPs in sheep and compared the properties of sheep and cattle/goat.
The novel oPRP1 and oPRP2 genes were detected in and cloned from ovine placenta and oPRP1 was deduced to have a shortened amino acid sequence. Phylogenetic analysis suggests that PRP molecules evolved as ruminant species diverged, because these ovine PRPs are phylogenetically adjacent to caprine and bovine PRPs (Fig. 2).
We confirmed the short variant form of PRP in ovine placenta (Figs. 3 and 4). Although there is no report that short variant forms result from alternative splicing or proteolytic cleavage in non-classical members of the PRL family, namely bovine PRP, caprine PRP, mouse Prlp and rat Prlp [1, 2, 5, 25, 26], short variants of bPL and PRL have been reported [32, 33]. Two types of short bPL variant are formed by alternative splicing . One has a premature stop codon because of a shift in the reading frame. Although the defective position in the bPL variant sequence was similar to that in oPRP1, bPL lacked only 23 bp in this region. It is not yet known whether the short bPL variant exists or what function it may have. Some structural variants of PRL proteins have been confirmed in various mammalian species . A short fragment of PRL protein has also been confirmed in rat, mouse and human [35–37]. Although these short PRL (16 kDa-PRL) molecules may result from alternative splicing, they could also be generated by proteolytic cleavage. The cleavage site (positions 145–149 in the mature region) almost coincides with the position of the oPRP1 stop codon. These short PRLs in mouse and human are known to have potential as inhibitors of capillary endothelial cell proliferation [38, 39]. The short PRL in rat produces an anti-angiogenic effect via an unique, high-affinity, saturable receptor that is different from the PRL receptor . Whether oPRP1 corresponds to the short PRL receptor is not certain, because full-length PRL binds to the PRL receptor and some ruminant PRPs do not. However, one hypothesis may be that oPRP1 has an inhibitory effect on the proliferation of vascular endothelial cells, because the sequence length and the mature protein region are similar to those of the short PRL variants in human and rodents. Members of the rodent Prl superfamily, namely prolactin-like proteins (Prlp), proliferins (Plf) and proliferin-related protein (Plfr), are non-classical and have several specific activities such as angiogenesis [41–43], hematopoiesis [44–46] and immunomodulation [47–50]. A possible hypothetical function for oPRP1 may be in the regulation of angiogenesis, but its actual function remains to be clarified.
Primary expression of oPRP1 and oPRP2 mRNA was observed in BNC (Fig. 6). The localization of oPRP mRNAs is similar to that of PRP family members in other ruminants [5, 20, 21, 30, 31, 51]. The mRNAs of both oPRPs were detected in the PTM and ICOT tissues throughout pregnancy (Fig. 7). However, the expression patterns were different; oPRP1 expression declined with the progress of the pregnancy in PTM and ICOT, but in contrast, oPRP2 expression remained constant throughout pregnancy. However, there seems to be a discrepancy between the localization and quantitative expression of oPRP2: quantitative real-time RT-PCR (QPCR) data suggest that oPRP2 may be expressed not only in BNC but also in conventional trophoblast cells, as in bovine [20, 21]. In bPRP expression profiles, four types of expression pattern were found during pregnancy in PTM: (i) genes expressed around the window; (ii) genes with peak expression around mid-gestation; (iii) genes that show increasing expression during the progression of gestation and peak late in gestation; (iv) genes with approximately constant expression throughout gestation . In the present study, although expression was not determined during the implantation period, oPRP1 expression may correspond to the type (ii) pattern. In contrast, oPRP2 expression might be type (iv) but without lactogenic activity. The functional significance of short PRPs and various other kinds of PRP in ruminant placenta is still unclear. We could produce recombinant oPRP2 protein as well as proteins of other ruminant species (cattle and goat) [5, 20, 21]. Only the absence of lactogenic activity is clearly confirmed for recombinant oPRP2 and bPRP1 by a bioassay using Nb2 lymphoma cells.