Human Reproduction Update Advance Access originally published online on July 13, 2006
Human Reproduction Update 2006 12(6):785-795; doi:10.1093/humupd/dml035
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Function of aquaporins in female and male reproductive systems
Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
1 To whom correspondence should be addressed at: Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, China. E-mail: huanghefg{at}hotmail.com.
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Abstract |
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The flow of water and some other small molecules across cell membranes is important in many of the processes underlying reproduction. The fluid movement is strongly associated with the presence of aquaporins (AQPs) in the female and male reproductive systems. It has been suggested that AQPs mediate water movement into the antral follicle and play important roles in follicle development. AQPs are known to be involved in the early stage of spermatogenesis, in the secretion of tubule liquid and in the concentration and storage of spermatozoa. Fluid reabsorption in some regions of the male reproductive tract is under steroid hormone control and could be mediated by various AQPs. Also AQPs take part in the processes of fertilization, blastocyst formation (as the pathway for transtrophoectodermal water movement during cavitation) and implantation. Alterations in the expression and function or regulation of AQPs have already been demonstrated in disorders of the male reproductive system, such as abnormal sperm motility, the abnormal epididymis and infertility seen in cystic fibrosis, and varicocele. This article extensively reviews the distribution of AQPs in mammalian reproductive tissues and discusses their possible physiological and pathophysiological roles.
Key words: aquaporins / endocrinology / female tract / male tract / reproduction
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Introduction |
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TOPAbstractIntroductionFunction of AQPs in...Function of AQPs in...Role of AQPs in...ConclusionReferences |
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Water is the major component of cells and tissues, and water movement across cell membrane is a fundamental property of life. It was long assumed that the transport of water was due to simple diffusion through the lipid bilayer membrane that encloses cells. Because lipid bilayer is hydrophobic, it was hard to explain high water permeability of some cells, such as red blood cells and renal tubular epithelial cells, by simple diffusion before aquaporin (AQP)-1, the first water channel, was identified in human erythrocytes in 1992 (Preston et al., 1992
). The AQPs are a family of small (25–34 kDa), hydrophobic, integral membrane channel proteins that facilitate rapid passive movement of water. To date, 13 isoforms of AQPs (AQP0–AQP12) have been identified in mammals. In mammals, various AQPs have been identified in numerous tissues. AQPs are located in a wide variety of cells, such as kidney, lung, pancreas, brain, gastrointestinal tract, eye, ear, immune system, skin, adipose, muscles, uterus and testis (Li et al., 1994; Ishibashi et al., 1997a,b; Frigeri et al., 1998; Page et al., 1998; Beitz et al., 1999; Shanahan et al., 1999). Based on sequence homology data, phylogenetic comparisons and permeability properties, AQPs of AQP0–AQP10 are now subdivided into two major groups: orthodox AQPs and aquaglyceroporins (Agre et al., 2002; Agre and Kozono, 2003; Zardoya, 2005). The group of orthodox AQPs is composed of six members: AQP0, AQP1, AQP2, AQP4, AQP5 and AQP6. They are water-selective channels and permeable to water but not to small organic and inorganic molecules. AQP6 presents unusual gating properties and anion conductance that may transport chloride at low pH (Yasui et al., 1999; Engel et al., 2000). The group of aquaglyceroporins includes four members: AQP3, AQP7, AQP9 and AQP10. They are non-selective water channels which are permeable to glycerol, urea and other small non-electrolytes as well as to water (Agre et al., 2002; Borgnia et al., 1999; Ishibashi et al., 2002). Previous studies showed that mouse AQP8, but not human and rat AQP8, was permeable to urea (Ishibashi et al., 1997b; Ma et al., 1997; Koyama et al., 1998), suggesting that AQP8 might represent another phylogenic branch. Recently, Agre’s laboratory examined AQP8 from human, rat and mouse (hAQP8, rAQP8 and mAQP8) and they found that hAQP8 and rAQP8 were not permeable to urea or glycerol, and hAQP8 was permeable to ammonium analogues (formamide and methylammonium) (Liu et al., 2006). AQP11 and AQP12 are identified as novel AQPs whose functions remain undetermined (Morishita et al., 2004; Itoh et al., 2005).
Mutagenetic, topological and crystallographic analysis of AQP1 has revealed the basic structure of AQPs (Figure 1). AQP1 monomers contain water pores but associate in the membrane as tetramers. Each AQP1 monomer typically contains six membrane-spanning 
helices, with the N- and C-termini both located on the cytoplasmic side of the membrane. The N- and C-terminal halves of the monomer show significant sequence similarity to each other and are arranged as a tandem repeat; each half has a smaller hydrophobic loop (loop B or loop E) that includes a highly conserved asparagine–proline–alanine (NPA) motif, in which the asparagine residue is a key to the formation of the pore water-selectivity filter. The site of mercurial inhibition was demonstrated at Cys-189 proximal to the NPA motif in loop E (Preston et al., 1993).
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It is well established that heavy metals can directly interact with AQPs thereby affecting their activity. Mercury was found to inhibit rapid transcellular water transport in epithelial cells before the water channels were discovered. The activities of most mammalian AQPs have now been shown to be inhibited by mercury, such as AQP1, AQP2, but not AQP4, because of their special structure (Hasegawa et al., 1994). Copper and nickel were recently reported to inhibit the activity of human AQP3 (Zelenina et al., 2003, 2004). Three amino acid residues in loop C and loop E, Trp128, Ser152 and His241, were identified to be involved in both the copper- and nickel-mediated AQP3 inhibition (Zelenina et al., 2003, 2004). On the other hand, gold and silver were reported to inhibit human AQP1, but a molecular basis for this inhibition has not been revealed (Niemietz and Tyerman, 2002). Some AQPs are sensitive to the change in extracellular pH, including AQP0 (Németh-Cahalan and Hall, 2000), AQP3 (Zeuthen and Klaerke, 1999; Zelenina et al., 2003) and AQP6 (Yasui et al., 1999). Bovine AQP0 has been shown to be modulated by intracellular Ca2+ (Németh-Cahalan and Hall, 2000). Study on human AQP3 reveals that the amino acid residues, His53, Tyr124, Ser152 and His154, are involved in regulation of AQP3 by extracellular pH (Zelenina et al., 2003).
The importance of AQPs in mammalian physiology has been proposed based on the pattern and regulation of their tissue expression. Because there are no selective AQP inhibitors suitable for in vivo use, the physiological significance of individual AQP has been addressed by analysing the phenotype of specific AQP knockout mice generated by targeted gene disruption. The availability of AQP-knockout mice models provides a method to test the hypotheses regarding AQP function and has contributed to the appreciation of the importance of AQPs in fluid transport (Schnermann et al., 1998; Ma et al., 2000).
To date, 11 isoforms of AQPs have been reported to be expressed in the female and male reproductive systems (Table I). In this review, we survey the expression and distribution of AQPs in the female and male reproductive systems and discuss their functions in reproduction.
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Function of AQPs in the female reproductive system |
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The first reported confirmation of AQP in the female reproductive system was achieved by isolating the complementary DNA (cDNA) encoding a water channel from a human uterus cDNA library template more than 10 years ago. The cloned cDNA had high (99.8%) homology to the 28 kDa human erythrocyte CHIP28 (channel-forming integral membrane protein) water channel gene (Li et al., 1994
). The reading frames of both cDNAs were of same length, showing 99% identity, only with valine in place of alanine at position 45 of the human erythrocyte CHIP28 sequence (Li et al., 1994). This variant might be unique to the human uterus (Li et al., 1994). Subsequently, the presence of AQP1 messenger RNA (mRNA) was observed in rat uterus (Li et al., 1997). To date, at least nine AQP isoforms (AQP1–AQP9) have been confirmed to be expressed in the female reproductive tract or in cells involved in assisted reproductive technology procedures. Their specific expression pattern suggests that they play a role in water movement between the intraluminal, interstitial, and capillary compartments. In addition, there is evidence that the expression of various AQPs can be regulated by steroid sex hormones (Jablonski et al., 2003; Richard et al., 2003; He et al., 2006; Lindsay and Murphy, 2006). Together, these results provide indirect evidence of a role for AQPs in reproductive physiology.
Dramatic changes in the uterus occur in response to ovarian steroid hormones, including hyperemia (Finlay et al., 1983), increased capillary permeability (Cullinan-Bove and Koos, 1993), recurrent stromal oedema (Okada et al., 2001) and fluctuation of uterine fluid volume (Maier and Kuslis, 1988). In ovariectomized rats, estradiol-17 (E2) causes secretion of sodium, potassium and water into the lumen of the uterine horn and progesterone causes reabsorption of those substances (Clemetson et al., 1977). Although the effect of sex hormones on water transport into and within the mammalian uterus is well known, the mechanisms responsible for balancing water concentration within the uterus have not yet been identified. To gain insight into this mechanism, Jablonski et al. (2003) explored the expression profile and functionality of AQPs in the ovariectomized mouse uterus treated with ovarian steroid hormones. AQP1 is in the myometrium and may be slightly regulated by ovarian steroid hormones. AQP2 was absent in animals treated with vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus (Jablonski et al., 2003). AQP3 was also detected in the uterine epithelial cells. The distribution of AQP2 and AQP3 suggests that both of them contribute to water movement into the lumen of the uterus. AQP8 is present in the stroma and myometrium, where it may shuttle water and play an important role in limiting water flow into the stroma and protecting the myometrial layer from oedema (Jablonski et al., 2003). These four isoforms, including AQP1, AQP2, AQP3 and AQP8, might participate in water movement during uterine imbibition.
Our recent work demonstrates that human uterine endometrium expresses AQP2 (He et al., 2006). The expression of human endometrial AQP2 is menstrual cycle-dependent and reaches a high level at the mid-secretory phase, the time of embryo implantation (He et al., 2006). The endometrial AQP2 levels correlate to serum E2 and progesterone concentrations (He et al., 2006). Our results demonstrate that AQP2 in human endometrium may be regulated by ovarian steroid hormones. On the other hand, AQP5 in rat uterus was shown to be up-regulated by progesterone (Lindsay and Murphy, 2006). The expression of AQPs in uterus is regulated by ovarian steroid hormones, suggesting that these AQPs may play important roles in hormone-mediated water and other small molecules transport in uterine imbibition.
Moreover, the presence of AQPs (AQP3, AQP4, AQP5 and AQP8) in the mouse cervix may contribute to the biochemical changes that occur in the cervical connective tissue during gestation to allow for cervical dilatation during labor (Anderson et al., 2006). The changes include progressive disorganization of the collagen network and increased water content. Altered expression of AQPs in models of preterm and delayed cervical ripening could also shed light on distinct aspects of cervical water balance during pregnancy and parturition (Anderson et al., 2006). The presence of AQP1 in vaginal smooth muscle suggests that it is also a place for rapid movement of water either into or out of the muscle cells (Gannon et al., 2000).
Ovum transport and oviductal fluid balance
In mammals, the oviduct not only transports the ovum towards the uterus but is the site of fertilization and early embryonic development as well. The mechanisms controlling ovum transport from the isthmus of the fallopian tube to the uterus remain unclear. According to one theory (‘tube locking’), the ovum is delayed at the ampullary–isthmic junction, possibly as a result of muscle contraction (Pauerstein and Eddy, 1979), isthmic oedema (Black and Asdell, 1959; Johns et al., 1982) or vascular distention (Verco, 1994).
There was a report on AQP1 in frog oviduct 10 years ago, but the study failed to establish its localization (Abrami et al., 1995). Another study discovered that AQP1 labelling was most pronounced in the innermost longitudinal layer and the inner cells of the circular muscle layer of the myosalpinx. It could therefore play an important role in regulating ovum transport in the fallopian tube by altering luminal diameter: increased water movement into the smooth muscle cells through AQP1 water channels could lead to muscle swelling that ultimately shuts down the lumen, thus ‘locking’ the tube (Gannon et al., 2000).
AQPs in the oviduct could also influence the epithelial cells’ production of oviductal fluid, which provides the physiological medium for fertilization and early embryonic development. The oviductal fluid changes in quality and quantity in response to fluctuations of the serum levels of sex hormones during the estrous and menstrual cycle (Leese et al., 2001). Apparently, ovarian hormones regulate the flow of water from epithelial cells towards the oviductal lumen. Immunohistochemistry revealed that AQP5, AQP8 and AQP9 are localized in epithelial cells (Branes et al., 2005), which provides a new insight into the characteristics of oviductal fluid and the role of hormones in regulating the success of fertilization and early embryonic development.
Follicle maturation and oocyte cryopreservation
The formation of follicular fluid is the prominent feature of the antrum; however, the exact mechanisms by which large amounts of fluid pass into the antral cavity of the follicle are unknown. McConnell et al. (2002) demonstrated that the rate of water movement into the antral cavity of the follicle was 3.5-fold greater than that of inulin (a complex carbohydrate restricted to the extracellular compartment). Further study confirmed the presence of AQPs in granulosa cells, thus suggesting that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by AQP7, AQP8 and AQP9 in granulosa cells (McConnell et al., 2002). Notably, AQP7 and AQP9 are two members of the aquaglyceroporin group, which are permeable to glycerol, urea and other small non-electrolytes as well as to water. The expression of these two isoforms of AQPs in the granulosa cells suggests that rapid transport of small neutral molecules might also be important in follicle development.
Development of mammalian female germ cells in vivo is dependent on the surrounding follicular environment in the ovary and passes through several phases (Hirshfield, 1991; Erickson and Danforth, 1995; Rankin et al., 2000; Richards, 2001). In a primordial follicle, the immature oocyte is surrounded by a monolayer of undifferentiated epithelial cells. After recruitment, the primordial follicle develops into a preantral follicle, in which the oocyte is surrounded by a distinct layer of proliferated and differentiated granulosa cells (Hirshfield, 1991). At the antral stage, granulosa cell proliferation lends an insignificant contribution to the increase in follicle size (Hirshfield, 1991), and overexpression of AQP activity can enhance the rate of apoptosis of granulosa cells (Jablonski et al., 2004). Development into an antral follicle is marked by the formation of the antrum, a fluid-filled cavity adjacent to the oocyte within the follicle. Further increase in the size of the follicle results mainly from an increase in the size of the antral cavity (Hirshfield, 1991). Antral expansion, which occurs rapidly under gonadotropin stimulation, requires a quick and massive transport of water. Three isoforms of AQPs (AQP7, AQP8 and AQP9), which are known to be expressed in granulosa cells (McConnell et al., 2002), and two isoforms (AQP3 and AQP7), which are known to be expressed in mouse oocytes (Edashige et al., 2000; Meng et al., 2005), can meet this kind of need.
Understanding of the activities of AQPs in oocytes could lead to improvements in the methods used for oocyte cryopreservation. The methods that have been successful in preserving sperm and embryos have shown poor results for preserving oocytes, and difficulties in oocyte cryopreservation are an important limiting factor in reproductive technologies, such as fertility treatment and gene banking. High concentrations of cryoprotectants may damage the oocytes (Hong et al., 1999; Fabbri et al., 2000), possibly because AQP3 and AQP7, which have been shown to be expressed in the mouse oocytes (Edashige et al., 2000), are permeable not only to water but also to small linear sugar molecules, as well as to glycerol and urea (Borgnia et al., 1999; Agre et al., 2002). This is important in cryopreservation of oocytes because water transport between oocytes and cryoprotectants may be mediated by AQP3 and AQP7. Induced expression of AQP3 in mouse oocytes has been shown to improve water and glycerol permeability as well as oocyte survival after cryopreservation (Edashige et al., 2003). So change of AQP expression in oocytes may affect the survival of mouse oocytes after cryopreservation. Our laboratory also found that controlled ovarian stimulation significantly decreased the expression of AQP3 mRNA in mouse Metaphase II oocytes. Maybe this was one of the reasons for the low survival of oocytes after cryopreservation (Meng et al., 2005).
Preimplantation development comprises a tightly controlled programme of gene expression and cell division, which starts with the fertilized egg and ends with the implantation of the blastocyst roughly 5 days later. The blastocyst consists of a spherical shell of epithelial cells (trophectoderm) surrounding a fluid-filled cavity and a small group of cells that are the progenitors of the embryo proper (Wiley, 1987; Biggers et al., 1988; Watson, 1992). Gene families that take an essential role in controlling this developmental interval include genes that code for products involved in cell polarity, cell junctions, cytoskeleton, ion transporters and water channels (Watson and Barcroft, 2001; Stanton et al., 2003). Development of the trophectoderm begins with onset of cell-to-cell adhesion (compaction) and finalizes during cavitation (blastocyst formation). Because the trophectoderm initiates contacts with the maternal uterine endometrium and promotes embryo invasion into the endometrium during implantation in invasive species, its development is very important to blastocyst formation.
AQPs move water under the physiological conditions of a nearly iso-osmolar gradient (Deen and van Os, 1998). The osmotic gradient across the rabbit trophectoderm is reported to be as low as 8 mOsm, so the water movement that occurs across the trophectoderm during blastocyst formation is an example of nearly iso-osmolar water movement (Borland et al., 1977). To clarify the hypothesis that AQPs are present in the trophectoderm cells of the preimplantation embryo, Offenberg et al. (2000) determined the full complements of mRNA encoding AQPs in preimplantation mouse embryos and found that up to seven members of this gene family are present during the first week of mammalian development: mRNAs encoding AQP1, AQP3, AQP5, AQP6, AQP7 and AQP9 were detected in murine embryos from one-cell stage up to the blastocyst stage. AQP8 mRNA was not detected in early cleavage stages but was present in morula and blastocyst-stage embryos. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development.
Three AQP polypeptides are localized to murine trophectoderm membranes. The presence of apical and basolateral AQPs in the trophectoderm (Barcroft et al., 2003) have been established. More importantly, Barcroft et al. (2003) have confirmed that these AQPs are functional in the early embryo by measuring variations in water movement across this epithelium following exposure to a hyperosmotic gradient in the presence or absence of p-chloromercuriphenylsulfonic acid, a mercuric inhibitor of AQP transport (Barcroft et al., 2003). Na/K-ATPase, located in the basolateral membrane of the trophectoderm cells, was demonstrated to play a role in water movement across the epithelium in the process of cavitation (Betts et al., 1998; Watson and Barcroft, 2001). However, the presence of functional AQPs in the trophectoderm membrane, coupled with the likelihood that the osmotic gradient across the trophectoderm is not all that steep and the survival of the Na/K-ATPase–1 murine null mutants to the blastocyst stage, all suggest that the Na/K-ATPase may not be an indispensable regulator of blastocyst formation. The model of blastocyst formation now incorporates AQP-mediated water movement across the trophectoderm, coupled with the establishment of a tight junctional seal to block the leakage of water, as the primary mechanism that promotes blastocyst formation in the preimplantation embryo.
The mammalian uterus undergoes great morphologic changes, including periodic stromal oedema in preparation for embryo implantation. To better understand the mechanisms that underlie uterine oedema during implantation, Richard et al. (2003) studied AQP1, AQP4, AQP5 and AQP8 expression during the peri-implantation period. AQP1 is expressed in the myometrial smooth muscle and can be induced by estrogen in the uterine stromal vasculature, which supports a role for this AQP in peri-implantation uterine oedema. AQP4 signals are localized in the luminal epithelium and reach a maximum at the time of insemination. AQP5 expression is restricted to the glandular epithelium after blastocyst attachment and is dependent on estrogen stimulation of the progesterone-primed uterus. These findings suggest that a subset of AQPs is involved in peri-implantation fluid homeostasis.
The usual final position of embryo implantation is into the antimesometrial side of the uterine lumen, with the inner cell mass facing the mesometrial side (Finn, 1989). A study on the mechanisms controlling this phenomenon found that an increase in AQP1 in the mesometrial muscle might contribute to the antimesometrial positioning of the embryo within the uterine lumen (Lindsay and Murphy, 2004). Another well-known phenomenon is the dramatic reduction in uterine luminal fluid at the time of implantation (Png and Murphy, 2000). The question is how this fluid could be removed in such a short time. Removal of water is most important, because water constitutes a major proportion of the fluid. The paracellular pathway seems impossible, because of the existence of even tighter junctions between epithelial cells of the uterus during implantation (Murphy, 2000, 2004). Maybe AQPs, which are intrinsic membrane proteins and exhibit a great capacity for water movement, contribute to that process. The presence of AQP1, AQP4 and AQP5 in pregnant rat uterus, and most importantly the redistribution of AQP5 from completely cytoplasmic in the early days of pregnancy to a predominant localization on the apical plasma membrane at the time of implantation may account for that (Lindsay and Murphy, 2006). On day 6 and 7 of pregnancy, redistribution of AQP 5 was most striking at the mesometrial pole of the uterus, which implied that the water removed at the time of implantation might pass through apically located AQP5 water channels in uterine epithelial cells. AQP5 may play an important role in reabsorption of luminal fluid and the antimesometrial positioning of the blastocyst (Murphy, 2000, 2004; Lindsay and Murphy, 2006). Our laboratory found a high level of endometrial AQP2 expression at the mid-secretory phase, suggesting that AQP2 might play a physiological role in the receptivity of the human uterus (He et al., 2006).
Uterine oedema is a general feature of implantation in rodents, non-human primates and humans (Lobel et al., 1967; Tarara et al., 1987; Ghosh et al., 1993; Okada et al., 2001). An initial phase of generalized uterine swelling facilitates closure of the uterine lumen around the free-floating blastocyst (Lobel et al., 1967; Nilsson, 1974), thus indicating that AQPs might regulate tissue fluid balance during implantation. By RT-PCR and in situ hybridization, Richard et al. (2003) found that among the ten known murine AQPs, only AQP1, AQP4 and AQP5 are significantly expressed in the peri-implantation uterus and AQP8 and AQP9 mRNAs were expressed in the implanting blastocyst in a mouse pregnancy model. The different AQPs have distinct responses to ovarian steroid hormones, and their contribution to uterine imbibition was also clarified. Those findings suggest that a subset of AQPs is involved in peri-implantation fluid homeostasis. A previous study by microarray analysis detected a decreased expression of uterine AQP1 gene in the endometrial implantation window (Reese et al., 2001), suggesting a possible role of AQPs in the implantation window. However, there are no data available to confirm this hypothesis.
Amniotic fluid (AF) provides the fluid-filled compartment that is essential for normal fetal growth, movement and development. Disorders of AF volume, either in excessive or deficient amounts, are associated with significant perinatal morbidity and mortality. In sheep, one of the best-studied species in terms of AF regulation, the transcellular route has been recognized as a critical regulatory path for amniotic fluid reabsorption (Hedriana et al., 1995). Transcellular water movement is facilitated by a family of water-selective channels: AQPs that increase plasma membrane permeability and provide a route for rapid fluid movement (Verkman et al., 1996; Verkman and Mitra, 2000). To date, among the 13 mammalian AQPs identified, the expression of only four AQPs (AQP1, AQP3, AQP8 and AQP9) has been demonstrated in mammalian chorioamniotic membranes and placenta.
AQP1 is expressed in murine syncytiotrophoblasts and placental and chorionic endothelium (Hasegawa et al., 1994; Johnston et al., 2000). In human, AQP1 was found in amnion and chorion from both membrane locations, but no AQP1 was seen in the trophoblast cells of the chorion (Mann et al., 2002). Moreover, AQP1 was localized in the amniotic epithelium of both the chorionic plate and reflected region of the fetal membranes. The strongest signal was detected in the epithelium of the chorionic plate amnion (Mann et al., 2002). These observations suggest that the epithelium AQPs rapidly facilitate water transport between the amniotic cavity and the fetal circulation (Mann et al., 2002). To determine what effect the absence of this water channel would have on amniotic fluid volume and composition, the authors utilized an AQP1 knockout mouse model (Mann et al., 2005). Indeed, with complete knockout of the water channel, the fetal mice showed increases in amniotic fluid volume and decreases in osmolality, further confirming that the AQP water channels are important in the regulation of amniotic fluid volume.
AQP3 is expressed in the epithelia of ovine placenta and chorion, but not in amnion (Johnston et al., 2000). AQP3 is also expressed in fibroblasts of the amnion and allantois, but not in the epithelia of these membranes (Johnston et al., 2000). In human, AQP3 protein was not found in the fetal membranes, while AQP3 mRNA was faintly detected in the chorion from both membrane regions but not the amnion (Mann et al., 2002). Another study reported the presence of AQP3 in the apical membranes of syncytiotrophoblast of human term placenta (Damiano et al., 2001). AQP3 is the most highly expressed AQP quantitatively, thus revealing that AQP3 may facilitate water transport across the trophoblast barrier. The similar expression of AQP8 in the ovine trophoblast and membrane epithelial cells was reported (Liu et al., 2004).
Wang et al. (2001) have demonstrated that AQP8 is expressed in human epithelial cells of chorion and amnion and of the syncytiotrophoblasts and outer layer trophoblasts of placenta. That is the first study demonstrating the expression of the AQP8 water channel in human chorioamniotic membranes. Furthermore, AQP8 expression is rapidly up-regulated by second messenger cyclic adenosine monophosphate (cAMP) in human Wistar Institute Susan Hayflick (WISH) cells, demonstrating a relatively short biologic half-life in vitro (Wang et al., 2003). Using RT-PCR, immunoblotting and immunohistochemistry, Damiano et al. (2001) found that AQP9 was present in the apical membranes of syncytiotrophoblast of human term placenta. Wang et al. (2005) reported AQP9 mRNA expression in ovine amnion and allantois and AQP9 protein localization in epithelia of amnion and allantois, indicating that AQP9 may be a major water channel for intramembranous amniotic fluid reabsorption in sheep. Thus, the presence of AQP3, AQP8 and AQP9 represents a molecular mechanism of amniotic water absorption through intramembranous pathways, contributing to the high water permeability of the placenta. Although the functional importance of AQPs in amniotic fluid transport remains to be determined, its presence in human fetal membranes suggests potential therapeutic strategies for the treatment of amniotic fluid volume disorders and inhibitors of AQP water channels could also be useful for managing oligohydramnios and polyhydramnios.
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Function of AQPs in the male reproductive physiology |
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Water and solute movement across the epithelium of the male reproductive tract is responsible for balancing the luminal environment for spermatogenesis; for the maturation, storage, transport and liberation of sperm; and for increasing sperm concentration. At present, multiple AQPs have been recognized in the testis (AQP0, AQP1, AQP7, AQP8 and AQP9), efferent ducts (AQP1, AQP9 and AQP10), epididymis (AQP1, AQP3, AQP9 and AQP10), vas deferens (AQP1, AQP2 and AQP9) and accessory glands (AQP1 and AQP9) of adult mammals. Some of them in specific tissues are regulated by sex steroid hormones. But only a few studies on AQPs in the human male reproductive tract, as well as in birds, (Zaniboni and Bakst, 2004
; Zaniboni et al., 2004) have been reported. The heterogeneous, segment-specific and developing expression of AQPs along the male reproductive system suggests that fluid transport in these tissues could be locally modulated by physiological regulation of the expression and/or function of AQPs. The pathological expression and regulation of AQPs might cause several disorders of male reproductive system. Spermatogenesis, the maturation of spermatozoa, and sperm concentration are associated with considerable fluid secretion and/or absorption in the testis. The presence of multiple AQPs in germ cells and other tissues is consistent with the phenomenon of fluid movement in testis. AQPs could be involved in the early stages of spermatogenesis and in the secretion of tubule liquid. Alterations in the expression and function and/or regulation of AQPs have already been demonstrated to be at the basis of some forms of male sub-fertility and infertility.
During spermatogenesis, and especially in the metamorphosis of round spermatids into elongated spermatids, one of the most distinct morphological changes is a striking reduction of germ cell volume, largely because of the osmotically driven fluid efflux. Several studies have shown that spermatozoa of humans, rams, fowl and bulls exhibit a high water permeability that is mercury-resistant with low activation energy (Watson, 1992; Curry et al., 1994; Liu et al., 1995), but the molecular mechanism and its physiological significance in reproductive biology are poorly elucidated. AQPs as water channels are usually found in the selected tissues and cells where water movement is abundant. In earlier studies, AQP1 (Brown et al., 1993; Curry et al., 1994; Liu et al., 1995) was not detected in spermatozoa and AQP2 (Fushimi et al., 1993), AQP3 and AQP4 (Frigeri et al., 1995) were all absent from testis. Two AQPs (AQP7 and AQP8) have been identified in rat testis and have been shown by in situ hybridization to be abundantly expressed in germ cells (Ishibashi et al., 1997a,b). This intriguing finding suggests that AQP7 and AQP8 may play an important role in spermatogenesis. Although they share a similar transmembrane structure, AQP7 and AQP8 are different in function and distribution pattern in testis. AQP7 mRNA is transiently expressed at a late phase of spermatogenesis, and AQP7 protein cellular and subcellular localization varies, depending on different stages of spermatogenesis (Suzuki-Toyota et al., 1999; Calamita et al., 2001a,b). However, AQP8 mRNA was found to be present uniformly and constantly in every seminiferous tubule, consistent with the expression of AQP8 at the protein level (Elkjaer et al., 2001), which was found intracellularly as well as over the plasma membrane of all germ cells (Calamita et al., 2001b). Further studies of the two AQPs in the developing rat testis in accordance with the maturation of germ cells support the roles of AQPs in spermatogenesis (Calamita et al., 2001a; Kageyama et al., 2001). Interestingly, in contrast to the abundant AQP8 in rat testis, AQP8 mRNA was absent in human testis (Koyama et al., 1998). AQP9 mRNA was also detected in spermatocytes at early developmental stages in rats (Tsukaguchi et al., 1998).
The complexity and differences of the expression of AQP7 and AQP8 in germ cells in adult rats and developing rats reflect their distinct significance in spermatogenesis, which may relate to their different functional properties. AQP7 was observed in epididymal spermatozoa (Calamita et al., 2001a), thus indicating its possible role in sperm maturation and storage as well as its function in spermatogenesis (Suzuki-Toyota et al., 1999). Since AQP7 is permeable not only to water but also to urea and glycerol, which is a universally effective cryoprotectant, the role of AQP7 in the glycerol permeability of sperm during cryopreservation deserves investigation. Besides, AQP7 and AQP8 might both contribute in generating the seminiferous tubule fluid, which is thought to be from both Sertoli cell secretion and water efflux of germ cells (Russell et al., 1989).
Among their many functions, Sertoli cells in spermatogenic epithelium are known to secrete fluid to form a fluid-filled tubular lumen, serving as the vehicle for transporting sperm from the testis to the epididymis (Setchell et al., 1969; Hinton and Setchell, 1993). A semicircular pattern of AQP0 expression is reported in the Sertoli cells (Hermo et al., 2004). Furthermore, the expression is variable in different stages during spermatogenesis (Hermo et al., 2004). AQP8 is another water channel localized to Sertoli cells (Tani et al., 2001; Badran and Hermo, 2002).
Transgenic mice lacking AQPs have been useful to study the physiological function of AQPs in fluid absorption/secretion in many tissues. Surprisingly, a recent study on phenotype analysis of AQP8 null mice showed few and only mild phenotype differences between wild-type and AQP8-deficient mice (Yang et al., 2005). Although the weight and size of the testes in AQP8-null mice were increased, no impaired fertility or abnormalities in sperm count or morphology were found. The greater ratio of spermatogenic cells to Sertoli cells in seminiferous tubules and reduced water permeability in plasma membranes of testis indicate the possibility of an abnormality in sperm development. The unanticipated results of rare impaired fertility might relate to the complicated systems for sperm maturation or the compensatory changes in the expression of other AQPs.
Efferent ductules are very important reabsorptive segments of the male reproductive tract, where between 50 and 90% of the luminal fluid secreted by seminiferous tubules is reabsorbed (Levine and Marsh, 1971; Hohlbrugger, 1980; Clulow et al., 1994, 1998). The high rate of reabsorption of luminal fluid requires various water and ion transporters in absorptive epithelial cells of efferent ductules. Besides ion transporters—including basolateral Na+/K+-ATPase (Ilio and Hess, 1992, 1994), the apical Na+/H+ exchanger-3 (Hansen et al., 1999; Lee et al., 2001; Leung et al., 2001), Cl–/HCO3– exchanger (Lee et al., 2001) and chloride channel cystic fibrosis transmembrane regulator (CFTR) (Lee et al., 2001; Leung et al., 2001)—water channels (AQP1, AQP9 and AQP10) may contribute to the transepithelial water movement.
AQP1 was expressed intensely [to a degree comparable with that for estrogen receptor (ER)] in efferent ductules of rats and marmoset monkeys at all ages from late fetal life through puberty to adulthood (Fisher et al., 1998). Once the epithelial cells had differentiated, AQP1 was mainly localized in the brush-border and, to a lesser extent, the basolateral membranes and apical endosomes of non-ciliated cells and the cilia of ciliated cells (Brown et al., 1993; Fisher et al., 1998; Badran and Hermo, 2002; Oliveira et al., 2005). However, no impaired fluid reabsorption in efferent ductules was observed in AQP1-null mouse (Zhou et al., 2001), suggesting the existence of other AQPs in this epithelium, proven later. AQP9 was enriched on the microvilli of non-ciliated cells (Pastor-Soler et al., 2001; Badran and Hermo, 2002; Oliveira et al., 2005). Recently, another AQP (AQP10) was found to be expressed over the epithelium in the efferent ductules (Hermo et al., 2004). AQP1 and AQP10 were both intensely detected at the endothelial cells of vascular channels in this region (Badran and Hermo, 2002; Hermo et al., 2004). The expression of AQP1 and AQP9 in the efferent ductules might be regulated by estrogen because the expression of these two isoforms of AQPs was significantly reduced in the efferent ductules of mice deficient in ER (Ruz et al., 2006).
In addition to the efferent ductules, considerable fluid reabsorption also occurs in the distal segments of the male excurrent duct system, notably in epididymis, where concentration of sperm increases significantly (Wong and Yeung, 1978) and a hypertonic luminal fluid is established (Levine and Marsh, 1971; Johnson and Howards, 1977; Turner and Cesarini, 1983). Several members of AQP family expressed in a region-specific pattern in the epididymal epithelial cells may be of importance in the transepithelial water movement and in postnatal development of this organ as well.
AQP9 is the first and main AQP detected in the epididymis. In rat, the stereocilia of the principal cells, not the basal cells, exhibit strong AQP9 protein labelling (Badran and Hermo, 2002; Elkjaer et al., 2000; Pastor-Soler et al., 2001, 2002; Oliveira et al., 2005; Da Silva et al., 2006). Human epididymis (Pastor-Soler et al., 2001) has an expression pattern similar to that found in rats. As a broadly selective neutral solute and water channel, AQP9 probably enables the rapid cellular movement of metabolites required by fast-growing spermatocytes. As for other AQPs, mRNA for AQP2, AQP5, AQP7 and AQP11 are also detected in epididymal epithelium (Da Silva et al., 2006). However, AQP5 co-localized with AQP9 in the apical membrane of a subpopulation of principal cells in the corpus and cauda regions. Surprisingly, AQP2 mRNA is present throughout the development of rats up to adulthood, while the protein was detected in the distal region of the epididymis only in young rats, not in adult ones. Maybe a post-transcriptional mechanism is involved in the regulation of AQP2 expression.
As a basolateral water channel, AQP3 is localized exclusively and intensely in basal cells of epididymis (Hermo et al., 2004). The AQP8 expression in epididymis is controversial. Several studies showed that neither AQP8 transcript nor protein was detected (Calamita et al., 2001b; Da Silva et al., 2006), but others interestingly demonstrated the localization of AQP8 in the basal cells in the ductus epididymis (Elkjaer et al., 2001). Basolateral expression of AQPs leading to transepithelial flow remains unknown. Maybe they are involved in the differentiation from basal cells to more differentiated cell types in these epithelia. Moreover, AQP1 is absent from the epithelium of the epididymis, but it was expressed over the endothelial cells of the vascular channels of epididymis (Badran and Hermo, 2002; Oliveira et al., 2005) as well as AQP10 (Hermo et al., 2004).
The vas deferens also plays a significant role in transepithelial water reabsorption of the reproductive tract (Wong and Yeung, 1978; Wong et al., 1978; Hohlbrugger and Pfaller, 1983) and is responsible for maintaining the luminal environment during maturation of sperm. AQP1 has been reported to be present at plasma membranes of epithelial cells in the ampulla of the vas deferens, not the cells in proximal parts, in rats (Brown et al., 1993). Unexpectedly, AQP2 was found to be present at the epithelial cells of the vas deferens by a transgenic mouse approach, which was used to examine the mechanism of the principal cell-specific expression of AQP2 within the renal collecting duct. The AQP2 expression in vas deferens was confirmed in normal rats (Nelson et al., 1998). AQP2 (Stevens et al., 2000) began to appear in some epithelial cells of the middle vas deferens and was concentrated on the apical plasma membrane of principal cells in the distal vas deferens (ampulla), where AQP1 (Brown et al., 1993) was highly expressed. No expression was detected in the proximal region, and very little was observed on intracellular vesicles. The variable expression of AQP1 and AQP2 in different regions of the vas deferens may be due to the marked structural differences of principal cells along the vas deferens (Andonian and Hermo, 1999), reflective of diverse functional activities. In contrast, AQP9 is present throughout the entire length of the vas deferens (Pastor-Soler et al., 2001). Therefore, AQP9 is the only AQP detected in the proximal vas deferens.
Seminal vesicles and the prostate secrete fluids that are rich in nutrients that are required for sperm to survive and fertilize eggs. Several AQPs are localized to the plasma membranes of epithelial cells of prostate (AQP1), seminal vesicle (AQP1) and coagulating gland (AQP9), all of which show both secretory and reabsorptive functions (Brown et al., 1993; Pastor-Soler et al., 2001). AQP9 is also located abundantly on intracellular structures in the prostate. All of the AQPs might contribute to fluid secretion in these organs.
As important endocrine cells in testis, Leydig cells synthesize and secrete the male sex hormone testosterone. In rats, AQP9 mRNA was detected in the interstitial Leydig cells (Tsukaguchi et al., 1998), while AQP9 protein was conspicuously expressed at the plasma and intracellular membrane in this cell (Elkjaer et al., 2000; Badran and Hermo, 2002). AQP0 was also expressed in Leydig cells (Hermo et al., 2004). The role of AQP0 and AQP9 in Leydig cells is obscure. Whether AQP0 and AQP9 are involved in endocrine functions is interesting.
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Role of AQPs in reproductive pathophysiology |
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As described above, there are multiple studies on physical expression and function of AQPs in the female and male reproductive systems. However, only a few studies have been undertaken to elucidate the roles of AQPs in reproductive disorders in males and, so far, no report on AQPs in female reproductive pathophysiology has been published. So we focus our review on AQPs in male reproductive pathophysiology.
The first report (Saito et al., 2004) on AQP7 expression in the human testis and ejaculated sperm from fertile men and from infertile patients suggests the possible roles of AQP7 in male infertility. The spatial expression of AQP7 in normal human testes was the same as that found in earlier studies in rats (Suzuki-Toyota et al., 1999; Calamita et al., 2001a; Kageyama et al., 2001). Some of the infertile patients who lacked AQP7 expression in ejaculated sperm had lower sperm motility than did any of the fertile men with AQP7-positive sperm. AQP7 may be involved in the maintenance of sperm motility, and a lack of AQP7 expression in sperm may be an underlying mechanism of male infertility.
CFTR, functioning as a cAMP-activated chloride channel (Gong et al., 2002), and AQP9 (Pastor-Soler et al., 2001) are co-expressed in the luminal membrane of the principal cells of the rat and human epididymis (Cheung et al., 2003; Ruz et al., 2004). They play an essential role in formation of the luminal fluid for sperm maturation and storage, especially in the cauda epididymis, where secretion is important to prevent luminal dehydration because of the totally reabsorbed testicular fluid. An additional role of CFTR in the regulation of other membrane transport proteins has been reported (Kunzelmann, 2001). Evidence is accumulating that CFTR could potentiate the water permeability of AQP9 in epididymal epithelium, and this potentiation was markedly reduced by phloretin and lonidamine (inhibitors of AQP9 and CFTR, respectively), both in vivo and in vitro (Cheung et al., 2003). These findings may provide the possibility that the abnormal epididymis and infertility of men with the genetic disease cystic fibrosis may be caused by defective CFTR and abnormal AQPs.
Varicocele is known to imply an increased vascular permeability in the testis, which absolutely causes microenvironmental changes for germ cells, including the unbalanced transmembrane water flow resulting in dysfunction of spermatogenesis. In normal testes, AQP1 is expressed only in the microvessel endothelial cells. In contrast, a diffuse AQP1 overexpression occurred at venular endothelial cell membranes (along with unexpected positive expression at the cell membranes of the Sertoli cells, immature germ cells and Leydig cells) in the testis of adolescents with varicocele (Nicotina et al., 2005). Such findings suggest the involvement of AQP1 in reducing the excessive endotubular and extratubular fluid caused by varicocele.
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As described above, AQPs have specific expression patterns in both the female and the male reproductive systems. These expression patterns suggest that AQPs play a role in water movement between the reproductive tract space and parenchyma. Direct investigation of AQPs in reproductive physiology requires the development of specific AQP blockers or phenotype analysis of animal models with defined AQP deficiencies. To date, the lack of AQP inhibitors suitable for use in vivo has precluded direct investigation of their function.
Unfortunately, there is no study on in vivo AQP function by the generation and phenotype analysis of transgenic knockout mice deficient in specific AQPs in the reproductive system. Phenotype studies support the predicted roles of AQPs in kidney tubule and microvessel fluid transport for urinary concentrating function (Yang et al., 2001a,b; Verkman, 2005) and in fluid-secreting glandular epithelia (Verkman, 2005). The phenotype studies have also shown unexpected roles of AQPs in brain and corneal swelling (Kuang et al., 2004; Bloch et al., 2005), in neural signal transduction (Binder et al., 2004; Li and Verkman, 2001; Mhatre et al., 2002), in regulation of intracranial and intraocular pressure (Zhang et al., 2002), and in tumor angiogenesis and cell migration (Saadoun et al., 2005). However, many phenotype studies were negative, despite AQP expression (Song et al., 2000; Yang et al., 2000), indicating that tissue-specific AQP expression does not indicate physiological significance (Moore et al., 2000). From these and additional examples, we conclude that tissue-specific AQP expression does not imply physiological significance. Therefore, AQP function in organ physiology needs to be evaluated on a case-by-case basis. Animal models may provide some clues about the functions of AQPs in human. The role of AQPs in the human reproductive system deserves further investigation. Understanding of the involvement of AQPs in human germ cell cryopreservation may lead to improved protocols in assisted procreation.
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References |
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