Identification of novel pregnancy-associated glycoproteins (PAG) expressed by the peri-implantation conceptus of domestic ruminants

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Abstract

Pregnancy-associated glycoproteins (PAG) are a multigene family, related to aspartic proteinases, expressed in the placenta of artiodactyls. Ruminant PAG expression varies spatially as well as temporally during gestation. The aim of this study was the identification of novel PAG expressed around implantation in domestic ruminants and to study their phylogenetic relationships. Day 12, 14, 17 (ovine); day 15, 18 (bovine); and day 12, 14, 15, 16, 18 (caprine) conceptuses were used for reverse transcription-polymerase chain reaction (RT-PCR) performed on RNA extracted from conceptuses pooled on each day of recovery. In ovine, a single PAG was detected on day 14, ovPAG2, from the PAG II group. On day 17, three more PAG were identified. One of them similar to ovPAG1 and two unknown named as ovPAG10 and ovPAG11, apparently from the PAG I group. In bovine, PAG expression was detected by day 15 and two transcripts were identified on day 18. One, boPAG2, from the PAG II group and other was previously unknown, boPAG22, closest to boPAG2. In goats, PAG expression was not detected on non-elongated conceptuses (days 12, 14 and 15). Two different transcripts were identified in both day 16 and 18 elongated conceptuses, one similar to caPAG2, from the PAG II group, whereas the other, caPAG12, apparently from the PAG I group, was previously unknown. Interestingly, the three new early PAG identified in sheep and goats are clustered together within the PAG I group. In summary, a complement of new PAGs expressed at implantation in domestic ruminants has been cloned.

Introduction

Pregnancy-associated glycoproteins (PAG) are a multigene family expressed in the placenta of Artiodactyla (Ruminants and Suidae). They were discovered during attempts to develop an early pregnancy test in cattle (Butler et al., 1982, Zoli et al., 1991, Mialon et al., 1993). Cross-reacting antigens were also isolated from sheep placentas (Zoli et al., 1990, Willard et al., 1995). They proved as useful markers of gestation since they were detected in maternal bloodstream soon after onset of implantation and its concentration rose until parturition (Ruder et al., 1988, Zoli et al., 1992, Ranilla et al., 1994). Molecular cloning of bovine and ovine PAG (hereafter referred to as boPAG1 and ovPAG1) showed that they belonged to the aspartic proteinase family of digestive enzymes, and most closely resembled pepsinogen, although they are not believed to act as proteinases since they posses mutations of the active site that probably prevents catalytic activity (Xie et al., 1991).

The ruminant PAGs have been classified into two main groups, both in terms of sequence identity as well as their tissue expression as determined by in situ hybridisation (Garbayo et al., 2000, Green et al., 2000). One group comprises many recently duplicated PAG genes where molecules from different species are intermixed. Within this group are boPAG1 and ovPAG1 (Xie et al., 1991) as well as caPAG1 (Garbayo et al., 2000). Therefore, this group was referred to as the PAG I group (Xie et al., 1997b). PAG I group expression is predominantly localized to binucleate cells of the trophectoderm (Xie et al., 1991, Garbayo et al., 2000, Green et al., 2000). Natural selection seems to have acted to diversify PAG I group molecules at the amino acid level within the variable regions. This is shown by a high rate of non-synonymous substitutions within these regions which, in turn, suggest that PAG I group molecules have undergone functional diversification, probably associated with the synepitheliochorial placenta typical of ruminants (Hughes et al., 2000).

Although PAG I group molecules are not believed to be proteolytically active as many possess nucleotide substitutions within the catalytic center (Xie et al., 1997b), they have retained the characteristic bilobed structure of aspartic proteinases (Guruprasad et al., 1996) and are able to bind small peptides (Landon et al., 1999). Thus, it has been suggested that functional diversification relates to the ability to bind different peptides (Xie et al., 1997b, Hughes et al., 2000).

Several PAG proteins have been isolated and end-sequenced from bovine (Zoli et al., 1991, Sousa et al., 2002, Klisch et al., 2005), ovine (Atkinson et al., 1993, Xie et al., 1997a, El Amiri et al., 2003, El Amiri et al., 2004) and caprine (Garbayo et al., 1998) all of them belonging to the PAG I group. Every PAG protein had been processed post-translationally by removal of a propeptide as in aspartic proteinases (Davies, 1990). A conserved motif for propeptide removal is the sequence ISF↓RGS (with the arrow representing the cleavage site). This or a similar sequence is present in all members of the PAG I group in contrast to members of the PAG II group, where the site of propeptide removal is difficult to ascertain.

The second group of ruminant PAG, hereafter referred to as the PAG II group, is less numerous and composed of more ancient PAG. Within this group are boPAG2 (Xie et al., 1994), ovPAG2 (Nagel et al., 1993) and caPAG2 (Garbayo et al., 2000). Members of the PAG II group are expressed throughout trophectoderm (Garbayo et al., 2000, Green et al., 2000). This group is intermediate between the PAG I group and porcine PAG, and its members are closer to active aspartic proteinases. Within this group, a cluster of closely related PAG: boPAG11, ovPAG2 and caPAG2 have been suggested to be functional homologues (Xie et al., 1997b, Garbayo et al., 2000).

Expression of PAG varies in a temporal manner over pregnancy as well. Individual PAG are expressed at certain stages and absent at others (Green et al., 2000). In general, PAG II group molecules expression starts earlier and stays longer than PAG I group transcripts. Implantation is a critical event in the establishment of a successful pregnancy. Therefore, the aim of this study was to identify novel PAG expressed at the beginning of implantation in three species of domestic ruminants and to establish phylogenetic relationship between them as well as with other PAG.

Section snippets

Tissue collection and RNA isolation

Twenty Rasa Aragonesa ewes were synchronized for estrus with intravaginal sponges of 40 mg fluorogestone acetate (Chrono-gest, Intervet, Salamanca, Spain) for 12 days and received 440 IU PMSG i.m. (Foligon, Intervet) the day of the sponge withdrawal. Estrus was observed 24 h later and the ewes were hand-mated on that day (day 0). Conceptuses were collected 12 days (n = 4), 14 days (n = 4) or 17 days (n = 12) after breeding under epidural anaesthesia by mid-ventral laparotomy and flushing the uterine

Ovine

Five, 10 and an undetermined number of conceptuses were flushed from 2, 3 and 11 ewes, respectively, on days 12, 14 and 17 after breeding, respectively. Conceptuses recovered on day 12 after breeding were still non-elongated in contrast to conceptuses recovered on both days 14 and 17 after breeding.

RT-PCR products were generated from all stage conceptuses. RT-PCR products were generated by using the boPAG1-5′ oligonucleotide in combination with either the ovPAG2-3′ (Fig. 1A) or the boPAG2-3′

Discussion

A main goal of this work was to identify novel PAG expressed by the peri-implantation ruminant conceptus. Comparison of three species of ruminants would enable to see whether there are common features that would give insight into the complement of PAG being expressed at implantation. In order to detect as many different PAG as possible, RT-PCR was performed with a combination of a well-conserved 5′ oligonucleotide (Xie et al., 1991) along with 3′ oligonucleotides specific of PAG I (boPAG1-3′;

Conclusions

Novel PAGs have been identified from peri-implantational conceptuses of domestic ruminants. While in cattle the new PAG was closer to boPAG2, and expected to be expressed throughout trophectoderm (PAG II group) new PAGs from goats and sheep were expected to be predominantly expressed in the binucleate cells of the trophectoderm (PAG I group). Moreover, phylogenetic relationships between domestic ruminants have been established showing a cluster of most related PAGs expressed included within the

Acknowledgements

This work was supported by a INIA grant (RTA-02-094) to Juana M. Garbayo. The authors thank to E. Echegoyen for his assistance in collecting conceptuses and the farm staff of the Department of Animal Production of the CITA of Zaragoza for caring the animals used in this study as well as to the farm “Ramaderia Mari” for providing the cows.

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