Identification of pregnancy-associated glycoprotein family (PAG) in the brown bear (Ursus arctos L.)
Introduction
The Pregnancy-Associated Glycoproteins (PAG) gene family, classified for aspartic proteinases (EC 3.4.23), encode multiple secretory placental proteins belonging to two subfamilies: catalytically active and potentially inactive (Szafranska et al., 2006; Wallace et al., 2015). To date, complementary DNA (cDNA) of PAG genes have been identified in various mammalian orders: Artiodactyla and Perissodactyla (Szafranska et al., 1995, 2006; Green et al., 1999, 2010; Panasiewicz et al., 2004; Brandt et al., 2007; Jerome et al., 2011; Silvia et al., 2012; Moley et al., 2014; Wilsher et al., 2014), Rodentia (Chen et al., 2001; Lipka et al., 2018), Primates (Majewska et al., 2017) and only one representative of the Carnivora – cat (Green et al., 1999).
The PAG mRNAs expression profile is conditioned by the stage of placental development (Green et al., 2000; Szafranska et al., 2006) and starts in the trophoblast (TRF) during the peri-implantation period and continues in the trophectoderm (TRD – chorionic epithelium) in different animals with various placenta types (Szafranska et al., 1995, 2006; Xie et al., 1995, 1997; Roberts et al., 2004). To date, specific placental PAG mRNA expression has been identified in: cattle and sheep (Xie et al., 1997; Green et al., 2000; Telugu et al., 2009), pigs (Szafranska et al., 1995; Szafranska and Panasiewicz, 2002; Panasiewicz et al., 2004), horses (Green et al., 1999), goats (Garbayo et al., 2000, 2008), the European bison (Szafranska et al., 2005), white-tail deer (Brandt et al., 2007) and some camelids, including the alpaca, dromedary and Bactrian camel (Majewska et al., 2009), but not in wild Carnivora species. Placental (TRF/TRD) transcription of PAGs occurs mainly in mononucleate cells (MNC) and/or binucleate cells (BNC), which are moderately invasive (Xie et al., 1997; Green et al., 2000; Telugu et al., 2010). The PAG proteins were localized within various cells (MNC/BNC) throughout placenta development in some domestic and wild life species: cattle (Wooding et al., 2005), pig, European bison, alpaca, camels (Majewska et al., 2005, 2006, 2008, 2011, 2013) and European moose (Lipka et al., 2016). Other studies have revealed various PAG isoforms (35–76 kDa) purified from placental tissue obtained from pregnant cows, goats, sheep (Xie et al., 1997; Zoli et al., 1991; Garbayo et al., 1998; El Amiri et al., 2004), zebu (Sousa et al., 2002), water buffalo (Barbato et al., 2008) and American and European bison (Kiewisz et al., 2008, 2009). PAGs produced by both cell types (MNC and BNC) are released as secretory granules into maternal blood, which allowed the PAGs to be used for various diagnostic tests (Szafranska et al., 2006). Detection of the PAGs in peripheral blood or milk (RIA and ELISA) of various domestic or wild ruminants has been applied for prenatal tests to diagnose early pregnancy with accuracy rates of 99% (González et al., 1999; Green et al., 2005; Friedrich and Holtz, 2010; Takahashi et al., 2013; Abdulkareem et al., 2011; Karen et al., 2015; Ricci et al., 2015; Roberts et al., 2015; Ropstad et al., 2005; Savela et al., 2009). Furthermore, differences in PAG concentration are useful for identification of fetal sex, single or multiple gestations, as well as to forecast miscarriages following embryo transfer and to detect pathological pregnancies (Lopes-da-Costa et al., 2011; Breukelman et al., 2012). Only a few studies have indicated the potential physiological importance of the PAGs, as embryo-originated “signal molecules” interacting with different maternal gonadotropin receptors (gonadal and extra-gonadal) in cyclic pigs and cows (Szafranska et al., 2007) or pregnant pigs (Panasiewicz et al., 2007), and thus involved in the luteoprotective mechanism and the regulation of pregnancy maintenance.
Brown bear (Ursus arctos L. – Ua) is the largest terrestrial carnivorous mammal of the Urisidae family seen across northern Eurasia and North America. The total world population Ua is estimated to exceed 200,000 (Garshelis, 2009). These estimates seem to be underestimated because only in Russia, according to official estimates (Lomanova, 2011), the current population of brown bear is over 180,000 and the annual hunting quota exceeds 12,000 individuals.
Determination of the pregnancy length in wild brown bears is very problematic. The length of pregnancy (174–257 days) is affected by a relatively long mating season (approximately 2.5 months), from early May and lasts until July (Mano and Tsubota, 2002; Schwartz et al., 2003). Moreover, the phenomenon of delayed implantation occurs in Ua pregnant females. The fertilized ova remain dormant in the uterus for about five-six months, prior to the implantation. After the end of delayed implantation (November–December) a gestation period starts lasting approximately 8 weeks. The babies are born during the winter sleep from December to March (White et al., 1998; Dahle and Swenson, 2003; Schwartz et al., 2003; Spady et al., 2007). Interestingly, the mature allantoic placenta of bears differs from that of most carnivores, being discoidal (placenta discoidalis) with a distinct circular marginal hematoma. Histologically, this placenta belongs to the endotheliochorial placentas type and is characterized by a placental labyrinth and transitional zone (Wimsatt, 1974; Michel, 1984). Despite all available information concerning the Ua pregnancy and placenta, there is no information on the PAG proteins. Therefore, thus study was conducted to identify the presence of the PAGs in the Ua: (1) expression within the placental proteome and (2) cellular localization in the discoidal placenta of the brown bear.
Section snippets
Animals and tissue harvesting
Uterine tissue (myometrium, endometrium and uterine lumen with trophoblast) samples were harvested post mortem from pregnant females (5–6 years old) of the Ua (N = 3). Based on the body weight, dental data and morphological measurements all Ua females were in the first pregnancy (Mano and Tsubota, 2002; Spady et al., 2007; Steyaert et al., 2012). Every Ua was bagged according to official local agreements/permissions for animal hunting for scientific purposes issued by the Department of
Identification of placental UaPAG-L isoforms
Electrophoretic separation (SDS-PAGE) and CBB staining allowed for the identification of the protein profile of Ua placenta during early pregnancy (Fig. 1). The placental proteome of all analyzed females was characterized with dominant 55 kDa proteins. CBB staining also revealed 72 and 66 kDa proteins in the placenta of Ua females No. 2 and 3, while these forms were not detected within proteins from placenta of Ua female No. 1. (Fig. 1a). In addition, several placental fractions of 36–45 kDa
Discussion
This is the first study reporting the identification of the PAG-like protein family within discoidal-type placenta of the brown bear (Ursus arctos L.), in which the PAG family was named ‘UaPAG-L’. Diversified profiles of the cellular UaPAG-L forms (kDa) were immuno-detected. Placental expression was localized within trophoblastic and trophectodermal cells. To date, this is the first study describing the component of molecular mechanisms involved in proper implantation and placenta development
Conflict of interest
The authors declare no conflict of interest.
Funding
This study was supported by the University of Warmia and Mazury in Olsztyn (12.610.005-300).
Author contributions
GP coordinated the experimental work, prepared of figures, analysis of the results and wrote the final version of the manuscript; AL, MM and MB-K carried out Western blots and immunohistochemistry; AS collected tissues and participated in the discussion of the results; BS conceived and participated in the research.
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