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Claudin-5, a tight junctional protein associated with ion and size selectivity, has been found in the uterus of skinks. This study has generated critical information about the molecular assembly of the tight junction at various stages of the reproductive cycle in the skink uterus. Recent studies looking at tight junctional proteins found occludin expression in the tight junction region of uterine epithelial cells in the skink uterus; however, occludin did not disclose any further information about the ions and size of ions permeating across the paracellular pathway. A ∼22-kDa claudin-5 band was detected in the uterus of the skinks present in this study and immunohistochemistry revealed that claudin-5 redistributes to the tight junction region of the lateral plasma membrane of uterine epithelial cells in late stage pregnancy/gravidity. This finding indicates that the tight junction becomes more assembled to precisely regulate ion and solute permeation in late stage pregnancy/gravidity. Claudin-5 with its functional role as a molecular sieve due to the formation of ion and size selective pores suggests that permeation of ions smaller than 0.8 kDa are restricted when claudin-5 is redistributed to the tight junction region of the later plasma membrane. This report is the first description of the molecular mechanisms that may be involved in regulating nutrient provision in the reptilian uterus. Anat Rec, 291:547–556, 2008. © 2008 Wiley-Liss, Inc.
Viviparity, or the birth of live young, occurs in many animal groups (Kaye,1971) and has evolved over 120 independent times in vertebrates. Over 100 of these origins have occurred in the Order Squamata (lizards and snakes) and viviparity is particularly common in the family Scincidae (Blackburn,2006), including the Eugongylus and Sphenomorphus group. In addition to a reduction in eggshell thickness and prolonged egg retention (Blackburn,1982; Shine,1983), viviparity involves the transfer of water, oxygen, and in many cases, nutrients (Guillette,1993). Thus, the uterus ultimately transforms from a passive environment to a nourishing chamber, in which the developing embryo relies on placentotrophy (Blackburn,1993; Stewart and Thompson,1993). Skinks are an ideal model to study the physiological and morphological changes taking place in the uterine epithelium and, thus, the evolution of placentation and viviparity, because some species exhibit oviparity (egg laying), whereas others are viviparous (live bearing) with chorioallantoic placentae ranging from simple (Weekes,1935) to complex (Thompson et al.,2002).
Viviparous skinks and some mammals (e.g., ungulates) have an epitheliochorial placenta (Luckett,1977). Epitheliochorial placentation involves close apposition of fetal and maternal tissue, but there is no breaching of the uterine epithelial layer (Grosser,1927; Amoroso,1952; Friess et al.,1980). The persisting barrier associated with epitheliochorial placentation illustrates the importance of the uterine epithelium to maximize nutrient provision and gas exchange between the mother and embryo. To cross the epithelium, molecules must pass through cells (transcellular pathway) or go between cells (paracellular pathway; Citi and Cordenonsi,1998; Anderson,2001). High molecular weight tracers can freely diffuse along the paracellular pathway until they reach the tight junction (TJ), the most apical structure of the epithelial junctional complex (Farquhar and Palade,1963), which seals the paracellular route (Anderson and Van Itallie,1995; Balda and Matter,1998). The TJ makes up a barrier that is an essential feature of epithelial and endothelial cells for the regulation of nonspecific passive diffusion of water, solutes, and immune cells driven by electro-osmotic gradients (Anderson and Van Itallie,1995; Nusrat et al.,2000; Van Itallie and Anderson,2004). In freeze fracture replicas, the TJ shows an anastomosing meshwork of strands (Winterhager et al.,1987; Claude and Goodenough,1973; Staehelin,1973; Murphy et al.,1982) which associate laterally with other TJ strands in the opposing membrane of adjacent cells (Tsukita et al.,2001). Recent studies carried out to identify TJ proteins in the uterine epithelium of skinks revealed that occludin, a 60-kDa membrane protein directly incorporated into individual TJ strands (Furuse et al.,1993; Ando-Akatsuka et al.,1996) was present in some lineages of Australian skinks but not in others (Biazik et al.,2007). The discovery of occludin suggests that the normally passive paracellular pathway becomes more regulated to prevent free diffusion of ions and solutes as the embryo develops in utero; however, this barrier is relatively nonspecific. Suggestions have also been made that the number of TJ strands does not determine the properties of the TJ barrier and changes in permeability result from changes in the quality of the TJ strands, such as their molecular composition, rather than the quantity of the TJ strands (Kojima,2002; Saitou et al.,1998). This finding led to the discovery of two ∼22-kDa novel TJ integral proteins named claudin-1 and claudin-2 (Furuse et al.,1998a).
Claudins have relevance in embryonic development and organogenesis by influencing epithelia–mesenchymal transitions (Bello et al.,2007) and are associated with forming ion selective pores (Tsukita and Furuse,2000; Heiskala et al.,2001). When opposing uterine epithelial cells express different claudins, a mismatch or a heteropolymeric interaction occurs (Furuse et al.,1999; Coyne et al.,2003; Wang et al.,2003a), thus, resulting in a pore formation that increases ion and solute flow (Furuse et al.,1999). Because the discovery of occludin in the uterus of skinks did not disclose information about the ion selectivity or size of solutes passing across the paracellular pathway, determining the presence or absence of claudins in the uterine epithelial cells in the skinks uterus may indeed reveal the possible mechanism for transepithelial nutrient permeation.
The claudin genome shows conservation between species. Mammals have 24 claudin protein members (Van Itallie and Anderson,2004), zebrafish 15 (Hardison et al.,2005), the puffer fish 56 (Loh et al.,2004), and six claudin species have also been described in Drosophila (Behr et al.,2003). Additionally, the expansion of the claudin gene family that exhibits tissue- and cell-type restricted expression in mammals (Furuse et al.,1993; Morita et al.,1999a; Heiskala et al.,2001) has resulted in the evolution of increasingly complex tissues and organs (Loh et al.,2004). Claudin-1, -2, -3, -4, and -5 are the most commonly studied claudins and have generated the most interest in research. Claudin-1 prevails in mouse epithelial liver and kidney TJ (Furuse et al.,1998b) and is a barrier to fluid loss (Furuse et al.,2002). Claudin-2 occurs in the lung (Mitic et al.,2000) and forms aqueous pores in epithelia (Furuse et al.,2001). Claudin-3 is present in lung and liver (Wolburg and Lippoldt,2002) and is a constituent of TJ strands (Tsukita et al.,2001) and claudin-4 occurs in mouse rat and kidney (Mitic et al.,2000) and influences paracellular ion selectivity (Van Itallie et al.,2001). Claudin-5 occurs in the TJ region of uterine epithelial cells in diestrus and proestrus rat uterus (Mendoza-Rodriguez et al.,2005), in retinal pigment epithelium (Kojima et al.,2002), airway epithelium (Coyne et al.,2003), and colonic epithelium (Amasheh et al.,2005). Claudin-5 is also a major component of the TJ in brain endothelial cells and fundamentally organizes the blood–brain barrier (Morita et al.,1999b). The deletion of the claudin-5 gene from brain endothelial cells led to an incremental increase in the size of tracers allowed to exit from the vascular space into the brain, resulting in the blood–brain barrier becoming a molecular sieve that is more permeable to small molecules (<0.8 kDa), but not larger molecules (Nitta et al.,2003).
Each claudin plays a unique role in epithelia. Examining the presence or absence and distribution of claudin-1, -2, -3, -4, and -5 in the uterine epithelium of skinks at different stages of the reproductive cycle and with differing parity modes will describe the molecular composition and function of the TJ in the uterus of skinks. Understanding the permeation property of the paracellular pathway in the skink uterus may determine whether this pathway is associated with possible nutrient provision in the skink uterus.
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- LITERATURE CITED
The discovery of claudin-5 in the skink uterus as seen in this study describes the specificity of the tight junctional regulation of the paracellular pathway in uterine epithelial cells. Additionally, differential expression of claudin-5 at various stages of the reproductive cycle suggests that possible changes in ion and solute permeation occurs depending on the stage of embryonic development in the skink uterus. The uterine epithelium of nonreproductive skinks showed claudin-5 expression along the apical, lateral, and basal regions of the cytoplasm. In the uterine epithelium of late stage pregnant/gravid skinks, claudin-5 redistributed to the TJ region of the cells and suggests that paracellular permeability is changed to precisely regulate ion and molecule diffusion. Without these changes to paracellular barriers in late stage pregnancy/gravidity, close regulation of paracellular diffusion would not be possible. Expression of claudin-5 in endothelial cells of the blood–brain barrier (Nitta et al.,2003) is indicative of a completely suppressed paracellular pathway and suggests that the barrier that is established in the uterus of skinks in late stage pregnancy/gravidity has similar attributes.
Claudin-5 was detected in the uterine epithelium of all the skinks studied, and was also confirmed in skink control tissue in the TJ region between alveolar and endothelial cells in the lung. In nonreproductive skinks, the uterine wall is thick and large numbers of uterine glands are present. Claudin-5 was expressed not only in the TJ region of the lateral plasma membrane, but also along the apical, lateral, and basal region of the cytoplasm of uterine epithelial and glandular epithelial cells. In various cells, it has been documented that claudins are distributed not only in the TJs, but also at the lateral membranes without forming TJ strands (Furuse et al.,2002; Li et al.,2004) or it can occur as diffuse labeling in endothelial cell cytoplasm as in the human brain (Virgintino et al.,2004). Similarly, in the estrus phase in rats, claudin-5 is detected in the basolateral region of the plasma membrane and in the cytosol (Mendoza-Rodriguez et al.,2005), and is thus not associated with a TJ seal. At this point, the TJ has not been assembled and fluid fills the uterine lumen to provide an important microenvironment for sperm capacitation (Wang et al.,2003b) and an energy source for the blastocyst (Magnuson et al.,1978). This finding suggests that, in the nonreproductive skink uterus, the TJ is not yet established and paracellular diffusion is not regulated, therefore allowing ions and solutes to freely enter the luminal space.
In the uterus of late stage pregnant/gravid skinks, the thickness of the uterine wall is reduced from that of the nonreproductive uterus and consequently the uterine glands are reduced or completely lost. Claudin-5 expression dramatically alters, and there is a redistribution and confinement of claudin-5 only to the TJ region of the lateral plasma membrane of uterine epithelial cells. No other immunofluorescence is detected elsewhere in the uterus. During the diestrus and proestrus phase in rats, claudin-5 and occludin shifts to the TJ region of the lateral plasma membrane, therefore assembling a strict paracellular barrier (Mendoza-Rodriguez et al.,2005) associated with regulation of the luminal fluid to prepare the uterus for implantation (de Jesus et al.,1972). The redistribution of claudin-5 from the cytosol to the region of the TJ in endothelial cells has also been detected during fetal development of the brain (Virgintino et al.,2004). This finding suggests that, as claudin-5 redistributes to the TJ region of the lateral plasma membrane in the uterine epithelium of skinks, free diffusion of solutes across the paracellular pathway is greatly reduced.
Claudin-5 expression is associated with the formation of size selective and ion selective pores, and claudin-5 knockout manipulation suggests that smaller molecules are able to diffuse across the normally “tight” barrier (Nitta et al.,2003). The expression of claudin-5 in the TJ of uterine epithelial cells in the skink uterus suggests that the size of the diffusional pore formed by claudin-5 expression allows for molecules that are larger than 0.8 kDa to pass across the paracellular space. This pathway is, therefore, impermeable to ions such as Ca2+ and Mg2+ because they are all smaller than 0.8 kDa and must utilize the transcellular pathway instead. Additionally, there is a limit to the size of the molecule that can diffuse across these pores. Histotrophy is another mechanism where macromolecules and lipids are transported to the developing embryo by means of vesicle secretion in reptiles (Corso et al.,2000; Blackburn and Lorenz,2003; Adams et al.,2005), and indeed occurs at the time when claudin-5 is present in the TJ region of the lateral plasma membrane. It may be that smaller ions and molecules are transported by means of the TJ pores, which are formed by claudin-5 and transcellular channels and larger molecules are transported by means of histotrophy.
The redistribution of claudin-5 in late stage pregnancy/gravidity to the TJ region of the lateral plasma membrane in oviparous, viviparous, and bimodally reproductive skinks suggests that TJ regulation is not only important in viviparous species, but also in egg layers. The significance of this finding reveals that, even in oviparous species in which most nutrients needed for development are confined to the egg yolk, a maternal nutrient contribution still exists before calcification of the egg in utero. This explanation is also suggested by studies carried out on egg yolk calcium levels, which revealed that calcium levels were insufficient to sustain development (Tuan et al.,1991) and our recent work carried out in the uterus of oviparous skinks, indeed show the presence of active Ca2+ATPase channels in the uterine epithelium of the skink uterus during the egg shelling phase of the reproductive cycle (Herbert et al.,2006). It may also be that, as transcellular and histotrophic activity imports fluid and ions into the luminal space between the maternal and fetal epithelium, the redistribution of claudin-5 and occludin to the tight junction may be associated with confining histotrophic and transcellular products to the fetal and maternal interface to allow for a more effective fetal absorption of those products.
Claudins -1, -2, and -3 were not detected in the uterine epithelium at any stage of the reproductive cycle in this present study and were not detected in the skink control tissue either. This finding suggests that these claudins are not associated with ion and solute regulation in the skink uterus or that the antibodies for claudins-1, -2, and -3 do not cross react in the skink. Claudin-4 was detected in the skink kidney control tissue with immunofluorescence in the TJ region of epithelium lining the duct system and with Western blotting detection as a single ∼22-kDa band from whole skink kidney homogenate. Claudin-4, however, was absent in the skink uterine epithelium at all stages of the reproductive cycle, and this finding suggests that claudin-4 is tissue specific in the skink.
In summary, this is the first documentation of the presence and distribution of claudin-5 in the TJ region of uterine epithelium in the skink uterus. Due to the uniqueness and ionic selectivity of claudin-5, the presence of this protein in the skink uterus provides evidence of a precise mechanism that regulates ion and solute permeation. The claudin-5 pore essentially forms an impermeable barrier to ions and molecules that are smaller than 0.8 kDa and, therefore, signifies the importance of transcellular and histotrophic activity in the uterus of late stage pregnant/gravid skinks. Because the paracellular pathway is slowly being understood in the uterus of skinks, future work on the transcellular pathway will further provide a molecular mechanism for the transepithelial nutrient provision and, thus, the evolution of placentotrophy and viviparity in reptiles.