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Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis
Abstract
Cell volume reduction is one of the most distinct morphological changes during spermiogenesis and may be largely attributable to water efflux from the cell. A strong candidate for a water efflux route, aquaporin 7 (AQP7), which is a water channel, was studied immunohistochemically in the rat testis. Immunoreactivity was restricted within the elongated spermatids, testicular spermatozoa, and residual bodies remaining in the seminiferous epithelium. Weak but distinct immunoreactivity was first observed in the cytoplasmic mass of the spermatid at step 8 of spermiogenesis. The Golgi-like apparatus became steadily immunoreactive at step 10. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity after step 16. At this step, the middle piece of the tail also showed immunoreactivity at the portion protruding into the lumen. The whole head and distal tail, where the elongated spermatid had only a limited amount of cytoplasm, showed no immunoreactivity throughout spermiogenesis. After spermiation, the immunoreactivity of AQP7 remained at the middle piece and in the cytoplasmic droplet in the testicular spermatozoon. The present observations suggest that AQP7 contributes to the volume reduction of spermatids, since this water channel protein is localized on the plasma membrane covering the condensing cytoplasmic mass of the elongated spermatid, and since the seminiferous tubule fluid is hypertonic.
... AQP7 and AQP8 have been identified in rodent testicles and are abundantly expressed in germ cells [171]. AQP7 mRNA is expressed transiently in the later phases of spermatogenesis, with the cellular and subcellular localization of this AQP varying according with the stages of spermatogenesis [172][173][174]. AQP7 is expressed in the round and elongated spermatids [175] and is also localized to the rat sperm tail [171,172,176]. ...
... AQP7 mRNA is expressed transiently in the later phases of spermatogenesis, with the cellular and subcellular localization of this AQP varying according with the stages of spermatogenesis [172][173][174]. AQP7 is expressed in the round and elongated spermatids [175] and is also localized to the rat sperm tail [171,172,176]. It has been suggested that AQP7 may be involved in the maintenance of sperm motility since human spermatozoa lacking AQP7 have lower motility and those individuals are infertile [177]. ...
... In this event round spermatids change from a spherical form into an elongated and, eventually, into a flagellar shape, suffering a profound reduction of volume due to a loss of cytoplasmic mass. AQP7 and AQP8 are expressed in rat testis with a remarkably similar distribution to CFTR [150,172]. These findings suggest the involvement of CFTR in volume reduction causing water efflux [29]. ...
(1)CICS - UBI - Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal, 2Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS) and Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, 4050-313 Porto, Portugal Abstract: The formation of competent spermatozoa is associated with the movement of large quantities of water and electrolytes in the various tissues and luminal fluids of the male reproductive tract. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- and HCO3- membrane transporter. CFTR gene mutations cause cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Of note, one hallmark in CF is male infertility. Indeed, mutations of CFTR gene cause abnormal production of germ cells and a reduction in germ cell quality and number. Compelling evidence illustrates that CFTR is involved in several pivotal processes for male fertility, including spermatogenesis and sperm capacitation. Recent studies show that CFTR acts as a molecular partner of specific water channels, known as aquaporins, in somatic testicular cells. Aquaporins are water-selective channels that enable high permeability fluxes of water across plasma membranes. In the male reproductive tract, water movements and ion concentrations are determinants for the male reproductive function. Therefore, aquaporins expression and function play a key role in male fertility. Herein we present an overview of the expression and function of CFTR in the male reproductive tract, highlighting the reproductive outcomes in male carriers of CFTR mutations and CF couples. We also present an up-to-date discussion on the expression and role of aquaporins in the male reproductive tract. Finally, we discuss the regulation of aquaporin-mediated water transport by CFTR in the male reproductive tract and its implication for male fertility.
... AQP1 is localized to the plasma membrane of nonciliated efferent duct cells and on the basolateral membrane of epithelial cells in the ampulla of the vas deferens, seminal vesicles, and prostate [14]. AQP2 is constitutively expressed in the apical membrane of the ampulla of the vas deferens [15], AQP7 is expressed in spermiogenesis [16,17], AQP9 is found in the Leydig cells [18] and ciliated epithelial cells of the epididymal duct [19]. The mRNAs of AQP8 and AQP10 have been detected in the seminiferous tubules [20] and in the Sertoli cells [21], respectively. ...
... The mRNAs of AQP8 and AQP10 have been detected in the seminiferous tubules [20] and in the Sertoli cells [21], respectively. While appealing physiological roles have already been hypothesized for AQP1, AQP2, and AQP7 in the male reproductive tract [14,15,17] the functional significance of AQP8, AQP9, and AQP10 remains to be elucidated. ...
... AQP7 staining was restricted to the cytoplasmic mass of differentiating spermatids and became steadily more intense over the plasma membrane of elongated spermatids at the luminal rim of the seminiferous epithelium prior to spermiation (Fig. 4bЈ, inset). This pattern confirmed previous immunohistochemical studies [17]. The plasma membrane and cytoplasmic portion of the residual bodies remaining in the adluminal compartment were clearly immunoreactive (Fig. 4cЈ, arrows). ...
... AQP1 is localized to the plasma membrane of nonciliated efferent duct cells and on the basolateral membrane of epithelial cells in the ampulla of the vas deferens, seminal vesicles, and prostate [14]. AQP2 is constitutively expressed in the apical membrane of the ampulla of the vas deferens [15], AQP7 is expressed in spermiogenesis [16,17], AQP9 is found in the Leydig cells [18] and ciliated epithelial cells of the epididymal duct [19]. The mRNAs of AQP8 and AQP10 have been detected in the seminiferous tubules [20] and in the Sertoli cells [21], respectively. ...
... The mRNAs of AQP8 and AQP10 have been detected in the seminiferous tubules [20] and in the Sertoli cells [21], respectively. While appealing physiological roles have already been hypothesized for AQP1, AQP2, and AQP7 in the male reproductive tract [14,15,17] the functional significance of AQP8, AQP9, and AQP10 remains to be elucidated. ...
... AQP7 staining was restricted to the cytoplasmic mass of differentiating spermatids and became steadily more intense over the plasma membrane of elongated spermatids at the luminal rim of the seminiferous epithelium prior to spermiation (Fig. 4bЈ, inset). This pattern confirmed previous immunohistochemical studies [17]. The plasma membrane and cytoplasmic portion of the residual bodies remaining in the adluminal compartment were clearly immunoreactive (Fig. 4cЈ, arrows). ...
Spermatogenesis and sperm maturation and storage are accompanied by significant movements of water, and multiple aquaporin transmembrane water channels (AQPs) have been recognized in the male reproductive tract. Nevertheless, the involvement of aquaporins in male reproductive physiology is mostly unknown. Here the expression and localization of AQP8 in rat spermatogenesis is defined and compared to that of AQP7, another aquaporin expressed in male germ cells. AQP8 mRNA was found in testis but not in epididymis, whereas the AQP7 transcript was present in both locations. By immunoblotting, the AQP8 protein was detected as a 25-kDa band and a 32-to 40-kDa diffuse component corresponding to the core and glyco-sylated protein, respectively. Membrane fractionation revealed AQP8 both in microsomal and plasma membrane-enriched fractions of rat testis while no apparent bands were detected in epididymis. AQP7 appeared as a 23-to 24-kDa band and was found both in testis and epididymis. By immunofluorescence, AQP8 labeling was found intracellularly as well as over the plasma membrane of germ cells throughout spermatogenesis. AQP7 was present in spermatids and spermatozoa and was predominant over the plasma membrane. AQP8 may be involved in the cytoplasmic condensation occurring during differentiation of spermatids into spermatozoa and in the generation of seminif-erous tubule fluid.
... AQP7 has been localized in germ cells and is mostly expressed in post meiotic male germ cells. In particular, it has been identified in the plasma membrane of elongated spermatids and maturing spermatozoa from rat testis [Ishibashi et al. 1997; Suzuki-Toyota et al. 1999; Calamita et al. 2001; Kageyama et al. 2001] and even in the residual bodies remaining in the seminiferous epithelium [Suzuki-Toyota et al. 1999; Calamita et al. 2001]. In addition, Hermo and Smith [2011] observed an AQP7 immunoreaction over the whole cytoplasm of late elongating spermatids located near the lumen of rat seminiferous epithelium. ...
... AQP7 has been localized in germ cells and is mostly expressed in post meiotic male germ cells. In particular, it has been identified in the plasma membrane of elongated spermatids and maturing spermatozoa from rat testis [Ishibashi et al. 1997; Suzuki-Toyota et al. 1999; Calamita et al. 2001; Kageyama et al. 2001] and even in the residual bodies remaining in the seminiferous epithelium [Suzuki-Toyota et al. 1999; Calamita et al. 2001]. In addition, Hermo and Smith [2011] observed an AQP7 immunoreaction over the whole cytoplasm of late elongating spermatids located near the lumen of rat seminiferous epithelium. ...
... Among the sperm alterations observed during evaluation of PAP stained slides, we noted the percentage of cytoplasmic residues, since AQP7 has been localized in this cell structure [Hermo and Smith 2011] and coiled tails, as they are often concomitant with the presence of cytoplasmic residue. Perhaps AQP7 is involved in the reduction of spermatid volume during spermiogenesis by mediating the efflux of water [Suzuki-Toyota et al. 1999; Calamita et al. 2001; Kageyama et al. 2001] and it may persist in this diffuse localization in the membrane of cytoplasmic residue of abnormal sperm. The population studied is rather small, however it should be considered that the relationship between the distribution of AQP7 and sperm morphology is supported by statistical analysis of many variables obtained by light and electron microscopy. ...
Aquaporins (AQPs) are a family of 13 small hydrophobic trans-membrane proteins expressed in numerous tissues and cells. Some AQPs work as strict water channels, others are permeable to a range of substances, including glycerol. In the male reproductive system their localization in testis, efferent ducts, epididymis, and spermatozoa has been described. We studied the distribution of AQP7 in ejaculated human sperm and the relationship between AQP7 labeling and sperm characteristics. Semen samples from 33 men were examined by light and transmission electron microscopy (TEM). TEM data were quantified using a mathematical formula that calculates a fertility index (FI) and the percentages of sperm apoptosis, immaturity, and necrosis. Immunocytochemistry with a polyclonal antibody anti-AQP7 was performed on the sperm samples. Normal sperm were labeled in the pericentriolar area, midpiece, equatorial segment, and weakly in the tail (grade 1). Abnormal sperm showed a diffuse low intensity of fluorescence evident in the cytoplasmic residues, coiled tails, in the entire head, and acrosome (grade 2). A high number of motile sperm obtained by swim up were labeled in a dotted manner in the mitochondria. A significant positive correlation was found between the spermatozoa with AQP7 grade 1 labeling and the percentage of normal form (P<0.008), progressive motility and FI (P<0.005); a negative correlation was noted with the percentages of cytoplasmic residues (P<0.010) and immaturity (P<0.006) and coiled tails (P<0.012). The link between AQP7 distribution and sperm morphology and the particular dotted labeling in swim up selected motile sperm are novel and deserve additional studies.
... It consists of 16.25 kilobase pairs and encodes the AQP7 protein of 269 amino acids. The AQP7 gene is expressed in the tail of spermatids, spermatozoa and the anterior tail portion of ejaculated sperm (SUZUKI-TOYOTA et al., 1999). Immuno-histochemical analysis demonstrated that the AQP7 protein exists in elongated spermatids, testicular spermatozoa, and residual bodies (CALAMITA et al., 2001). ...
... Immuno-histochemical analysis demonstrated that the AQP7 protein exists in elongated spermatids, testicular spermatozoa, and residual bodies (CALAMITA et al., 2001). The differential expression pattern of the AQP7 in infertile and fertile ejaculated human sperm, as well as the different motility rates between AQP7 positive or negative sperms, proves its role in male fertility and the transient expression of mRNA in the late phase of spermatogenesis (SUZUKI-TOYOTA et al., 1999;SAITO et al., 2004). In addition, it also contributes to generating the seminiferous tubule fluid (RUSSELL et al., 1989). ...
The Aquaporin 7 (AQP7) gene, a member of the aqua-glyceroporins, was characterised using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, in order to investigate the association of genetic variants of the AQP7 gene with semen quality in Murrah bulls. In this study, genomic DNA was extracted from the blood of Murrah bulls (n = 69) using the high salt method. Sequence data were analyzed using Bio Edit software (version 7.0.5) for detecting single nucleotide polymorphisms (SNPs). Statistical analysis was carried out using repeated GLM procedure measures (SAS 9.3). Analysis revealed 17 different SSCP variants in the AQP7 gene in Murrah buffaloes. Three unique SSCP band patterns were observed in exons 1 and 4. Exons 5, 7 and 8 showed five, four and two unique SSCP band patterns, respectively. Sequence analysis revealed a total of 20 SNPs (8 in exonic and 12 in intronic region). PCR-SSCP variants of exon 5 were significantly associated (P<0.01) with sperm concentration. SSCP variants of exons 5 and 8 were associated (P<0.05) with sperm viability and post thaw motility (PTM), respectively. The study revealed a high degree of genetic variability in the AQP7 gene in Murrah bulls. The SSCP variants observed are associated with semen parameters which suggests the possibility of using the AQP7 gene as a candidate gene for identification of markers for semen quality traits in buffaloes. © 2018, University of Zagreb, Facultty of Veterinary Medicine. All rights reserved.
... Aquaporin 7 (AQP7) gene is member of aqua-glyceroporins which is non-selective water channels and permeable to glycerol, urea, small non-electrolytes and water (Borgnia et al., 1999;Agre et al., 2002;Ishibashi et al., 2002). Its protein is reported to be present in elongated spermatids, testicular spermatozoa, residual bodies, middle piece and the anterior tail portion of ejaculated sperm (Calamita et al., 2001;Suzuki-Toyota et al., 1999). AQP7 gene has been mapped on Bos taurus autosome 8 and spans nearly 16.25 kilobase pairs comprising of 8 exons out of which seven are coding exon and one is non coding (exon 8). ...
... AQP7 gene has been mapped on Bos taurus autosome 8 and spans nearly 16.25 kilobase pairs comprising of 8 exons out of which seven are coding exon and one is non coding (exon 8). Since, AQP7 gene is expressed at the tail of spermatids and spermatozoa in the human testis which helps in differential expression pattern and different motility rates between infertile and fertile ejaculated human sperms (Suzuki-Toyota et al., 1999;Saito et al., 2004). Suzuki-Toyota et al. ...
... Aquaporin 7 was first identified in the seminiferous epithelium of the rat testis by Ishibashi et al. (1997) and was suggested to be involved Table 1). After spermiation, AQP7 staining is mainly confined to the sperm mid-piece and cytoplasmic droplet (Suzuki-Toyota, Ishibashi, & Yuasa, 1999), and its localization in ejaculated sperm will be discussed in the next section. In the dog, AQP7 is present in the epididymis and vas deferens (Domeniconi et al., 2008). ...
... Aquaporins appear to play a crucial role in the regulation of sperm volume, as these cells encounter media of different osmolality along their journey, from the testis and epididymis to entering the uterus and the oviduct. In effect, AQP7 is involved in the reduction of spermatid volume during spermiogenesis, as the environment in the seminiferous tubule is hypertonic (Suzuki-Toyota et al., 1999). AQP7 is present in epididymal spermatozoa and is involved in shedding the cytoplasmic droplet during sperm maturation. ...
Contents
Aquaporins ( AQP s) play a vital role for the transport of water and solutes across cell membranes. Classification of these ubiquitous proteins into three categories (orthodox AQP s, aquaglyceroporins and superaquaporins) is based on their sequence similarity and substrate selectivity. In the male reproductive tract of mammals, most AQP s (except AQP 6 and AQP 12) are found in different organs (including testis, efferent ducts and epididymis). AQP 1 and AQP 9 are the most abundant AQP s in the efferent ducts and epididymis and play a crucial role for the secretion/reabsorption dynamics of luminal fluid during sperm transport and maturation. AQP 3, AQP 7, AQP 8 and AQP 11 are the most abundant AQP s in sperm and are involved in the regulation of their volume, which is required for the differentiation of spermatids into spermatozoa during spermatogenesis, as well as in sperm transit along environments of different osmolality (male and female reproductive tracts). While different studies conducted in oocytes and embryos have demonstrated that AQP s are important for cryotolerance, data in sperm are scarce. At present, mounting evidence indicates that AQP 3, AQP 7 and AQP 11 are involved in the sperm response to variations of osmolality and to freeze‐thawing procedures. All these studies contribute to understand the physiology of both male reproductive tract and sperm, and open up new research ventures on the improvement of sperm cryopreservation protocols.
... Immunohistochemical analysis demonstrated that the AQP7 protein exists in elongated spermatids, testicular spermatozoa, and residual bodies (Calamita et al., 2001). The AQP7 protein is also detected at the middle piece and the anterior tail portion of ejaculated sperm (Suzuki-Toyota et al., 1999). AQP7 was observed in epididymal spermatozoa (Calamita et al., 2001), indicating its possible role in sperm maturation, storage as well as in spermatogenesis (Suzuki-Toyota et al., 1999). ...
... The AQP7 protein is also detected at the middle piece and the anterior tail portion of ejaculated sperm (Suzuki-Toyota et al., 1999). AQP7 was observed in epididymal spermatozoa (Calamita et al., 2001), indicating its possible role in sperm maturation, storage as well as in spermatogenesis (Suzuki-Toyota et al., 1999). Polymorphisms in AQP7 gene has association with semen quality parameters was reported in cattle (Rui-feng et al., 2009;Ma et al., 2011). ...
Aquaporin 7 (AQP7) gene is a member of aqua-glyceroporins which transports glycerol and water to spermatids. The present study aimed to investigate the polymorphisms within exons 2, 3, 4, 5 and their flanking intronic regions in AQP7 gene of Murrah bulls. Genomic DNA was extracted from 69 Murrah bulls blood samples and was subjected to polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP analysis revealed a total of eight different variants for amplicons of exons 4 and 5. Amplicon of exon 4 revealed three different patterns viz. E4P1, E4P2 and E4P3 with the frequency of 0.30, 0.22 and 0.48, respectively. Analysis of exon 5 revealed five unique SSCP patterns viz. E5P1, E5P2, E5P3, E5P4 and E5P5, with the frequency of 0.10, 0.37, 0.20, 0.20 and 0.13, respectively. Sequence analysis showed 16 single nucleotide polymorphisms, 7 of which were observed in coding region. Amplicons of exons 2 and 3 showed monomorphic patterns. However, compared to the reference sequence of taurine cattle one transition (C6878T) in exon 3 and 4 transitions (G2099A, C2116T, A2117G, G6848C) in intron 2 were observed for all the bulls under study. The genetic variability identified in the AQP7 gene may serve as potential genetic marker(s) for semen quality traits in buffalo.
... Besides, other members of AQP family interact with CFTR to regulate osmotic water permeability in various cellular systems, including in the epididymis [18]. Considering that AQP7 and AQP8 are expressed in tar testis with a remarkable similar distribution of CFTR [19,20], several studies attempted to describe their molecular interaction. The interaction between AQP4 and CFTR occurs in vivo on condition of a fully intact blood-testis barrier [21,22]. ...
Water transport through membrane is so intricate that there are still some debates. AQPs are entirely accepted to allow water transmembrane movement depending on osmotic gradient. Cotransporters and uniporters, however, are also concerned in water homeostasis. UT-B has a single-channel water permeability that is similar to AQP1. CFTR was initially thought as a water channel but now not believed to transport water directly. By cotransporters, such as KCC4, NKCC1, SGLT1, GAT1, EAAT1, and MCT1, water is transported by water osmosis coupling with substrates, which explains how water is transported across the isolated small intestine. This chapter provides information about water transport mediated by other membrane proteins except AQPs.
... Immunolocalization experiments have determined its presence in the sperm mid-piece in pig and cattle, whereas in human and mouse sperm it has been identified in the principal piece. Aquaporin 7 (AQP7) has been identified in pig [42,43], cattle [36,37], horse, human [39,[44][45][46], mouse [47] and rat [48][49][50] sperm [41]. It is present in the tail of ejaculated sperm, and has also been identified in certain regions of the head of human sperm [39,46]. ...
The interaction between cells and the extracellular medium is of great importance, and drastic changes in extracellular solute concentrations drive water movement across the plasma membrane. Aquaporins (AQPs) are a family of transmembrane channels that allow the transport of water and small solutes across cell membranes. Different members of this family have been identified in gametes. In sperm, they are relevant to osmoadaptation after entering the female reproductive tract, which is crucial for sperm motility activation and capacitation and, thus, for their fertilizing ability. In addition, they are relevant during the cryopreservation process, since some members of this family are also permeable to glycerol, one of the most frequently used cryoprotective agents in livestock. Regarding oocytes, AQPs are very important in their maturation but also during cryopreservation. Further research to define the exact sets of AQPs that are present in oocytes from different species is needed, since the available literature envisages certain AQPs and their roles but does not provide complete information on the whole set of AQPs. This is of considerable importance because, in sperm, specific AQPs are known to compensate the role of non-functional members.
... Solutes such as glycerol can be used as an energy source for sperm cells [136] but also to regulate sperm volume in hypertonic solutions. It is also worth mentioning the role of AQP7 on the spermatogenic process of rat and seabream sperm cells [85,137] AQP8 is an orthodox aquaporin that is also found in mice and dog spermatozoa and rat spermatogenic cells [124,125,138]. However, its localization is not yet specified. ...
Cryopreservation is globally used as a method for long-term preservation, although freezethawing procedures may strongly impair the gamete function. The correct cryopreservation procedure is characterized by the balance between freezing rate and cryoprotective agents (CPAs), which minimizes cellular dehydration and intracellular ice formation. For this purpose, osmoregulation is a central process in cryopreservation. During cryopreservation, water and small solutes, including penetrating cryoprotective agents, cross the plasma membrane. Aquaporins (AQPs) constitute a family of channel proteins responsible for the transport of water, small solutes, and certain gases across biological membranes. Thirteen homologs of AQPs (AQP0-12) have been described. AQPs are widely distributed throughout the male and female reproductive systems, including the sperm and oocyte membrane. The composition of the male and female gamete membrane is of special interest for assisted reproductive techniques (ART), including cryopreservation. In this review, we detail the mechanisms involved in gamete cryopreservation, including the most used techniques and CPAs. In addition, the expression and function of AQPs in the male and female gametes are explored,
highlighting the potential protective role of AQPs against damage induced during cryopreservation.
... AQP-5 is expressed in sweet, lacrimal, and airway submucosal glands and in the lung and corneal epithelium [94,96,97]. AQP-7 is a 269-amino acid aquaglyceroporin and facilitates glycerol transport in adipose tissues [98,99]. AQP-9 is present in the liver and testis and facilitates the passage of water, glycerol, and urea [94,100]. ...
Cardiac surgeries have been improved by accompanying developing cardioplegia solutions. However, the cardioplegia application presents an ongoing challenge with a view of a sufficiently restored cardiac function. In this review, we focus on the cardioplegia-induced mechanism and summarize the findings of studies undertaken to improve cardioprotective strategies. Currently, and somewhat surprisingly, relatively little is known about cardiac electrolyte regulation through channel physiology. We hope that an improved understanding of the electrolyte transport through ion channels/transporters and modulations of water channel aquaporins will provide an insight into cardiac channel physiology and a channel-based cardiac pathology of a cardiochannelopathy.
... The AQP7 and AQP8 proteins, located in the seminiferous epithelium, are probably involved in the cytoplasm volume reduction during spermatid differentiation into spermatozoa, because the fluid in the seminiferous tubule represents a hypertonic environment (Suzuki-Toyota et al., 1999;Calamita et al., 2001). Results from studies using the Aqp8-knockout mouse model indicated the lack of AQP8 resulted in a larger testis weight and size, and also a greater ratio of spermatogenic cells to Sertoli cells in seminiferous tubules compared to the wild type. ...
Improving the methods utilized to facilitate reproduction is associated with a constant need to search for new factors that not only significantly affect reproductive processes, but also create new possibilities when assessing male reproductive potential. Aquaporins (AQPs) belong to a family of small (28−30 kDa) proteins that facilitate the transport of water and other small molecules. There have been 13 AQPs (AQP0-AQP12) discovered in mammals, and these proteins are present in a wide range of cell types. Almost all AQPs, except AQP6 and AQP12 are present in the male reproductive organs and sperm of mammals and birds. Increasing evidence suggests that these proteins are involved in a number of processes responsible for the optimal functioning of the male reproductive system. This review presents the current state of knowledge regarding the abundance and distribution of AQPs in the male reproductive organs and sperm of various livestock and poultry species, including buffalo, cattle, sheep, horses, pigs, turkeys and goose. Furthermore, the possible physiological and pathophysiological significance of AQPs in male reproduction, as well as hormonal regulation of quantities are discussed. It can be concluded from the studies analyzed in this paper that abundance patterns of AQPs may be considered in the future as specific and universal biomarkers of male fertility and infertility in animal husbandry.
... Therefore, this link with sperm morphology indicates that defective spermatogenesis and epididymal maturation lead to an abnormal localization and expression of AQP7. In light of all the aforementioned, current evidence supports the idea that AQP7 is crucial during spermatogenesis, differentiation into spermatozoa (spermiation) and epididymal maturation, glycerol metabolism in sperm and volume reduction of spermatids [36,56]. ...
Aquaporins (AQPs) are a family of channel proteins that facilitate the transport of water and small solutes across biological membranes. They are widely distributed throughout the organism, having a number of key functions, some of them unexpected, both in health and disease. Among the various diseases in which AQPs are involved, infertility has been overlooked. According to the World Health Organization (WHO) infertility is a global public health problem with one third of the couples suffering from subfertility or even infertility due to male or female factors alone or combined. Thus, there is an urgent need to unveil the molecular mechanisms that control gametes production, maturation and fertilization-related events, to more specifically determine infertility causes. In addition, as more couples seek for fertility treatment through assisted reproductive technologies (ART), it is pivotal to understand how these techniques can be improved. AQPs are heterogeneously expressed throughout the male and female reproductive tracts, highlighting a possible regulatory role for these proteins in conception. In fact, their function, far beyond water transport, highlights potential intervention points to enhance ART. In this review we discuss AQPs distribution and structural organization, functions, and modulation throughout the male and female reproductive tracts and their relevance to the reproductive success. We also highlight the most recent advances and research trends regarding how the different AQPs are involved and regulated in specific mechanisms underlying (in)fertility. Finally, we discuss the involvement of AQPs in ART-related processes and how their handling can lead to improvement of infertility treatment.
... Thus, it has been suggested that CFTR may potentiate water permeability of AQP9 in the epididymal epithelium, which is essential for proper sperm maturation and movement. On a side note, it was also described that AQP7 and AQP8 are expressed in a highly similar pattern with CFTR in the testis, highlighting a possible interaction [103,120]. In support of this hypothesis, CFTR was co-localized with several other AQPs in different tissues, such as AQP1 and AQP5 in the pancreas [121]. ...
Aquaporins (AQPs) are a family of transmembrane channel proteins responsible for the transport of water and small uncharged molecules. Thirteen distinct isoforms of AQPs have been identified in mammals (AQP0-12). Throughout the male reproductive tract, AQPs greatly enhance water transport across all biological barriers, providing a constant and expeditious movement of water and playing an active role in the regulation of water and ion homeostasis. This regulation of fluids is particularly important in the male reproductive tract, where proper fluid composition is directly linked with a healthy and competent spermatozoa production. For instance, in the testis, fluid regulation is essential for spermatogenesis and posterior spermatozoa transport into the epididymal ducts, while maintaining proper ionic conditions for their maturation and storage. Alterations in the expression pattern of AQPs or their dysfunction is linked with male subfertility/infertility. Thus, AQPs are important for male reproductive health. In this review, we will discuss the most recent data on the expression and function of the AQPs isoforms in the human, mouse and rat male reproductive tract. In addition, the regulation of AQPs expression and dysfunction linked with male infertility will be discussed.
... Hence, it is possible for CFTR to interact with AQPs in spermatozoa, although the role of both channels in these cells needs further investigation. On a side note, it was also described that AQP7 and AQP8 are expressed with in a highly similar pattern with CFTR in the testis, highlighting a possible interaction (Huang et al., 2006;Suzuki-Toyota, Ishibashi, & Yuasa, 1999). In support of this hypothesis, CFTR was co-localized with several other AQPs in different tissues, such as AQP1 and AQP5 in the pancreas (Burghardt et al., 2003). ...
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel responsible for the direct transport of bicarbonate and chloride. CFTR-dependent ionic transport is crucial for pH regulation and fluid homeodynamics among epithelial surfaces. Particularly, CFTR performs an essential role in the male reproductive tract, which requires a tight regulation of water and electrolytes in order to produce healthy spermatozoa. The absence or malfunction of CFTR results in cystic fibrosis, the most common lethal disease among Caucasians, that is characterized by an impaired fluid and ionic homeostasis in the whole organism. Due to the wide expression and importance of CFTR, the male reproductive tract is highly affected by cystic fibrosis, resulting in male infertility. Although CFTR is not permeable to water, this protein acts as a regulator of other protein channels, such as aquaporins. In fact, CFTR acts as a molecular partner of aquaporins in epithelial cells, regulating fluid homeodynamics. Herein, up-to-date data concerning the regulation of aquaporin-mediated water transport by CFTR will be discussed, highlighting the role of both channels in the male reproductive tract.
https://www.sciencedirect.com/science/article/pii/S0083672919300640
... Besides, other members of AQP family interact with CFTR to regulate osmotic water permeability in various cellular systems, including in the epididymis [17]. Considering that AQP7 and AQP8 are expressed in tar testis with a remarkable similar distribution of CFTR [18,19], several studies attempted to describe their molecular interaction. The interaction between AQP4 and CFTR occurs in vivo on condition of a fully intact blood-testis barrier [20,21]. ...
Water
transport
through membrane is so intricate that there are still some debates. (Aquaporins) AQPs
are entirely accepted to allow water
transmembrane movement depending on osmotic gradient. Cotransporters and uniporters
, however, are also concerned in water homeotatsis. Urea transporter B (UT-B)
has a single-channel water permeability that is similar to AQP1. Cystic fibrosis transmembrane conductance regulator (CFTR
) was initially thought as a water channel
but now not believed to transport water directly. By cotranporters, water is transported by water osmosis coupling with substrates, which explains how water is transported across the isolated small intestine. This chapter provides information about water transport
mediated by other membrane proteins except AQPs
.
... During sperm cryopreservation, AQPs will also regulate glycerol flow (Chen and Duan 2011). The bidirectional water movement through the AQP-7 channel suggests it modulates water flow during cryopreservation, since it allows water to permeate beyond the levels permeated through the plasma membrane owing to its lipid composition (Suzuki-Toyota et al. 1999). The present results clearly indicate that AQP-7 and AQP-9 are present in boar spermatozoa. ...
Contents
Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.
... In humans, it is found in primary spermatocytes and maturing spermatids, but only in spermatocytes in the rat, and exclusively in spermatids in stallion. The presence of AQP8, -9, and -11 in the plasma membrame of late germ cells, and particularly of AQP7, which is up-regulated specifically in spermatids, suggests that these channels might be involved in mediating the water loss and cytoplasmic condensation that occurs during spermatozoa differentiation (Suzuki-Toyota et al., 1999;Calamita et al., 2001a, b;Yeung et al., 2009aSohara et al., 2009). For AQP11, it has recently been shown that siRNA-mediated AQP11 knockdown in the hamster testis affects the expression of genes involved in spermatogenesis and androgen production, suggesting a role for this intracellular channel in the coordinated regulation of signaling pathways during testis development (Shannonhouse et al., 2014). ...
Fluid homeostasis is recognized as a critical factor during the development, maturation, and function of vertebrate male germ cells. These processes have been associated with the presence of multiple members of the aquaporin superfamily of water and solute channels in different cell types along the reproductive tract as well as in spermatozoa. We present a comparative analysis of the existing knowledge of aquaporin biology in the male reproductive tissues of mammals and teleosts. Current data suggest that in both vertebrate groups, aquaporins may have similar functions during differentiation of spermatozoa in the germinal epithelium, in the concentration and maturation of sperm in the testicular ducts, and in the regulation of osmotically induced volume changes in ejaculated spermatozoa. Recent studies have also provided insight into the possible function of aquaporins beyond water transport, such as in signaling pathways during spermatogenesis or the sensing of cell swelling and mitochondrial peroxide transport in activated sperm. However, an understanding of the specific physiological functions of the various aquaporins during germ cell development and sperm motility, as well as the molecular mechanisms involved, remains elusive. Novel experimental approaches need to be developed to elucidate these processes and to dissect the regulatory intracellular pathways implicated, which will greatly help to uncover the molecular basis of sperm physiology and male fertility in vertebrates.
© 2015 Marine Biological Laboratory.
... E-mail: drrajibdeb@gmail.com. to the sperm tail in rat (Ishibashi et al. 1997;Suzuki-Toyota et al. 1999;Kageyama et al. 2001), mouse (Skowronski et al. 2007) and human (Saito et al. 2004). The plausible role of AQP7 in male reproductive system is unclear as AQP7 gene knockout mice are fertile producing normal functional spermatozoa (Sohara et al. 2007), whereas an association with sperm motility has been suggested in infertile men (Saito et al. 2004). ...
... In this process round spermatids change from a spherical form into an elongated and, ultimately, into a flagellar shape by reduction of volume, mainly due to a loss of cytoplasmic mass. These facts suggest the involvement of CFTR in volume reduction causing water efflux [158] , being that Aquaporin 7 and Aquaporin 8 are expressed in rat testis with a distribution remarkably similar to CFTR [162, 163]. Thus, it's possible that CFTR and these water channels could be functionally linked. ...
Bicarbonate (HCO3-) membrane transport systems are crucial players in the physiology of several tissues. The molecular basis of HCO3- membrane transport is of major physiological relevance since this ion is involved in the establishment of intracellular and extracellular ionic composition, osmolariy and pH. The membrane HCO3- transporters are divided in two main families: solute carrier 4 (SLC4) and solute carrier 26 (SLC26), although HCO3- concentration can also be regulated by the cystic fibrosis transmembrane regulator (CFTR). In most tissues the SLC4 family represents the majority of HCO3- transporters members, which can be divided in two subgroups: the Na+-dependent and the Na+-independent transporters. The SLC26 family consists of ten members that can transport diverse ions besides HCO3-. In the male reproductive tract, HCO3- transport occurs in several processes in order to assure a correct pursuance of the spermatogenetic event and spermatozoa capacitation, being also necessary for egg fertilization. Indeed, the formation of competent spermatozoa, the maintenance of an adequate ductal luminal milieu and spermatozoa capacitation are highly dependent of ionic balance and pH. Perturbations in these processes result in reduced male reproductive health and consequently male subfertility and/or infertility. Thus, it is imperative to understand HCO3- transport dynamics in order to identify and counteract possible alterations related with reduced male fertility caused by pathological conditions. Herein, we will review the major families and subfamilies of HCO3- membrane transport, discussing the molecular basis of HCO3- transport in the male reproductive tract and its role in male-associated subfertility and/or infertility.
... In addition, the Western blot analyses indicated that in nonactivated spermatozoa the amount of Aqp1ab and -10b in the plasma membrane seems to be lower than that of Aqp1aa, -7, or -8b. The spermatozoa of mammals also express the AQP7 and -8 orthologs, but their localization differs from that in the seabream, since AQP7 is localized in the cytoplasmic droplet and/or the middle piece of the tail and AQP8 is present as a punctuated pattern in the cytoplasmic droplet and along the tail [21,24,26,77]. The expression of Aqp10b in the head and flagella of seabream spermatozoa agrees with our previous report [7], but this is not the case for Aqp1aa, which was previously detected in the head and tail. ...
In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.
... In rat testis, AQP7 expression is reported to localize to germ cells, predominantly spermatids, whereas somatic cells such as Sertoli cells or Leydig cells reportedly do not express AQP7 (Calamita et al., 2001;Kageyama et al., 2001). Suzuki-Toyate et al. suggested that AQP7 contributed to volume reduction of spermatids, since its expression localized to the condensing cytoplasmic mass of the elongated spermatid (Suzuki-Toyota et al., 1999). Immunolocalization for AQP7 was not done in the current study, due to a lack of cross-reactivity of commercially available antibodies with the equine protein. ...
The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, -3, -7, and -9). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, -5, -7, and -8). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, -2, -5, and -9) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.
... As shown inFigure 5A, strong staining was observed at the seminiferous tubules, and not at the interstitial tissue surrounding the tubules. At higher magnifications (Fig 5B) , the stain was localized to mature spermatids, in agreement with previous studies (Suzuki et al., 1999). By immunoblot (Fig. 5C ), affinitypurified antibodies recognize a strong protein band of 24 –25 kDa in testis homogenate. ...
In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl2 insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation. GLIA 31:29–38, 2000. © 2000 Wiley-Liss, Inc.
... As shown in Figure 5A, strong staining was observed at the seminiferous tubules, and not at the interstitial tissue surrounding the tubules. At higher magnifications (Fig 5B), the stain was localized to mature spermatids, in agreement with previous studies (Suzuki et al., 1999). By immunoblot (Fig. 5C), affinitypurified antibodies recognize a strong protein band of 24 -25 kDa in testis homogenate. ...
Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.
... These differences in the AQP1b distribution , especially that of testis, may reflect differences between species and/or sexual maturation stages, although the exact reasons are unclear, as detailed physiological information about the stage of fish was not provided in the previous report [32]. In mammals, AQPs are involved in the regulation of fluid resorption in the efferent duct [35,36] and also in the volume reduction of spermatids [37]. Therefore, in teleosts, further investigation of the physiological significance of AQP in testis is warranted. ...
To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica) novel-water selective aquaporin 1 (AQP1b) that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.
... AQP 7 has been identified in germ cells, where it is expressed mainly in elongating spermatids as well as in epididymal spermatozoa (Ishibashi et al, 1997;Suzuki-Toyota et al, 1999;Calamita et al, 2001;Kageyama et al, 2001). We also found AQP 7 in late spermatids with immunoreactions appearing over cytoplasm (Figure 1A). ...
Water content within the male reproductive tract is stringently regulated in order to promote sperm differentiation and maturation. Aquaporins (AQP) are a family of integral membrane proteins allowing the transcellular transport of water, gases, urea, glycerol, and ions. Past studies from our lab have revealed the following. In the testis, Sertoli cells express AQP 8, whereas germ cells express AQP 7. In the efferent ducts (ED), AQP 1, 9, and 10 localize to microvilli of nonciliated cells, in addition to a basolateral staining for AQP 1, whereas AQP 1 and 10 localize to ciliated cells. AQP 7 and 11 are expressed in the ED epithelium of young but not adult rats, suggesting suppression of translation as rats age. In the adult epididymis, AQP 1 appears in endothelial cells of vascular channels and myoid cells, whereas AQP 3 delineates basal cells. In principal cells, AQP 9 and 11 appear on microvilli, whereas AQP 7 localizes to lateral then to basal plasma membranes in a region-specific manner; AQP 7 also associates with myoid cells. AQP 5 is expressed in corpus and cauda regions. Additionally, several AQPs are expressed by some but not all basal (AQP 7, 11), clear (AQP 7, 9), and halo (AQP 7, 11) cells. Regulation studies reveal a role for estrogen, androgens, and lumicrine factors. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the EDs and epididymis, as well as complex regulation patterns of expression.
... When AQP7 was first identified and localized in round and elongated spermatids, it was natural for the authors to propose a role in the reduction of cell volume in the development of round spermatids into testicular spermatozoa [70,71]. However, this assigned significance in spermiogenesis cannot be substantiated by the Aqp7 −/− mice, which show only a mild phenotype with normal testicular and epididymal morphology [72], normal testicular weight, normal daily sperm production, normal sperm morphological and kinematic characteristics, as well as in vitro and in vivo fertility [73]. ...
Mammalian spermatozoa have relatively high water permeability and swell readily, as in the hypo-osmotic swelling test used in the andrology clinic. Physiologically, spermatozoa experience changes in the osmolality of the surrounding fluids in both the male and the female tracts on their journey from the testis to the ovum. Sperm volume regulation in response to such osmotic challenges is important to maintain a stable cell size for the normal shape and function of the sperm tail. Alongside ion channels for the fluxes of osmolytes, water channels would be crucial for sperm volume regulation. In contrast to the deep knowledge and numerous studies on somatic cell aquaporins (AQPs), the understanding of sperm AQPs is limited. Among the 13 AQPs, convincing evidence for their presence in spermatozoa has been confined to AQP7, AQP8 and AQP11. Overall, current findings indicate a major role of AQP8 in water influx and efflux for sperm volume regulation, which is required for natural fertilization. The preliminary data suggestive of a role for AQP7 in sperm glycerol metabolism needs further substantiation. The association of AQP11 with the residual cytoplasm of elongated spermatids and the distal tail of spermatozoa supports the hypothesis of more than just a role in conferring water permeability and also in the turnover and recycling of surplus cellular components made redundant during spermiogenesis and spermiation. This would be crucial for the maintenance of a germinal epithelium functioning efficiently in the production of spermatozoa.
... In young rats, AQP 7 is expressed in early spermatocytes (Fig. 3C). In adult rats, AQP 7 [ID: T4-717] is expressed at a late phase of spermatogenesis where it is highly expressed in elongating spermatids ( Fig. 3D) as well as testicular and epididymal sperm (Calamita et al., 2001b;Hermo et al., 2008;Ishibashi et al., 1997;Saito et al., 2004;Suzuki-Toyota et al., 1999). No testicular morphological differences are seen in Aqp7 KO versus wild-type mice; they have similar numbers of offsprings, similar daily sperm production and motility and rate of in vitro fertilization Sohara et al., 2007). ...
As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic.
... AQP10 in the mouse is a pseudogene (Morinaga et al., 2002) and AQP10 is not expressed in the rat testis (Hermo et al., 2004). The only aquaporin so far consistently identified at the protein level in mammalian spermatozoa is the aquaglyceroporin AQP7 (Suzuki-Toyota et al., 1999;Saito et al., 2004;Skowronski et al., 2007). AQP8 has also been reported on rat (Calamita et al., 2001) and murine spermatozoa (Yeung et al., 2009a). ...
Despite the high water-permeability of human spermatozoa, little is known about the identity and the role of aquaporins (AQP) in them or germ cells. Using ejaculates from donors, sperm AQPs were identified by western blotting followed by the analysis of mRNA with RT-PCR. Protein expression in the testis and spermatozoa was localized by immunocytochemistry. Inhibitors were used to investigate the involvement of aquaporins in water transport when ejaculated spermatozoa were swollen in medium mimicking uterine hypo-osmolality by quinine that blocks volume regulation. Sperm AQP7 and AQP8 in 39 infertile patients and 11 healthy donors were quantified by flow cytometry. AQP1 was absent from spermatozoa. Sperm and testicular AQP7-9 had nucleotide sequences identical to those of somatic cells but AQP8 mRNA also showed shorter variants. AQP7 was expressed abundantly by round and elongated spermatids and ejaculated spermatozoa, AQP8 by all germ cells and spermatozoa, and AQP9 rarely by spermatocytes or Sertoli cells. Protein bands showed specificity by western blotting for AQP7 and AQP8 but not AQP9. The absence of sperm AQP9 was further suggested by the ineffectiveness of its inhibitor phloretin in blocking quinine-induced swelling, but HgCl(2,) which inhibits AQP8, was effective. Sperm AQP7 expression was correlated with progressive motility and was lower in patients than in donors. Sperm AQP8 expression was inversely correlated with the extent of sperm coiling, which is a swelling phenomenon, but showed no difference between patients and donors. In conclusion, AQP7 and AQP8 were identified in human spermatozoa and could play a role in glycerol metabolism and water transport respectively.
In order to accomplish their primary goal, mammalian spermatozoa must undergo a series of physiological, biochemical, and functional changes crucial for the acquisition of fertilization ability. Spermatozoa are highly polarized cells, which must swiftly respond to ionic changes on their passage through the female reproductive tract, and which are necessary for male gametes to acquire their functional competence. This review summarizes the current knowledge about specific ion channels and transporters located in the mammalian sperm plasma membrane, which are intricately involved in the initiation of changes within the ionic milieu of the sperm cell, leading to variations in the sperm membrane potential, membrane depolarization and hyperpolarization, changes in sperm motility and capacitation to further lead to the acrosome reaction and sperm-egg fusion. We also discuss the functionality of selected ion channels in male reproductive health and/or disease since these may become promising targets for clinical management of infertility in the future.
AQP0-12, a total of 13 aquaporins are expressed in the mammalian reproductive system. These aquaporins mediate the transport of water and small solutes across biofilms for maintaining reproductive tract water balance and germ cell water homeostasis. These aquaporins play important roles in the regulation of sperm and egg cell production, maturation, and fertilization processes. Impaired AQP function may lead to diminished male and female fertility. This review focuses on the distribution, function, and regulation of AQPs throughout the male and female reproductive organs and tracts. Their correlation with reproductive success, revealing recent advances in the physiological and pathophysiological roles of aquaporins in the reproductive system.
Spermatids eliminate excess cytoplasm to form streamlined sperm during spermiogenesis, which mechanism is insufficiently elucidated in fish. In this study, we investigated the cytoplasmic elimination procedure in spermatid during spermiogenesis in the large yellow croaker (Larimichthys crocea) using transmission electron microscopy. The early spermatid is subrotund with a centrally located nucleus. With further development, nucleus polarizes into one side of the cell while the cytoplasm with numerous vesicles near the membrane migrates to the caudal region. Furthermore, exocytosis-like structures were detected in middle spermatid. In late spermatid, the vesicles are reduced and rarely observed. These findings indicate that vesicles may be involved in cytoplasmic elimination possibly via exocytosis. In the later spermatid, a double-membrane, autophagosome-like structure envelopes the cytoplasm, which may develop into a single-membrane structure, and gets discarded from the cell as a residual body from the caudal region. This suggests its potential functions in the formation of residual body and cytoplasmic elimination. Overall, our results revealed that polarized development of spermatid causes polarized distribution of cytoplasm necessary for cytoplasmic elimination. Moreover, they provide ultrastructure evidence for vesicles and double-membrane structures involved in discarding spermatid cytoplasm in large yellow croaker, thus offering novel insights into cytoplasmic elimination during spermiogenesis in fish.
Aquaporins (AQPs) are transmembrane water channels and some of them are permeable in addition to water to other small solutes including hydrogen peroxide. The sperm cells of mammals and fishes express different AQPs, although there is no agreement in the literature on their localization. In humans, AQP3 and AQP11 are expressed mainly in the tail, AQP7 in the head and AQP8 in the midpiece. Thanks to the results of experiments with KO mice and to data obtained by comparing sub-fertile patients with normospermic subjects, the importance of AQPs for the normal functioning of sperms to ensure normal fertility emerged. AQP3, AQP7 and AQP11 appeared involved in the sperm volume regulation, a key role for fertility because osmoadaptation protect the sperm against a swelling and tail bending that could affect sperm motility. AQP8 seems to have a fundamental role in regulating the elimination of hydrogen peroxide, the most abundant reactive oxygen species (ROS), and therefore in the response to oxidative stress. In this review, the human AQPs expression, their localization and functions, as well as their relevance in normal fertility are discussed. To understand better the AQPs role in human sperm functionality, the results of studies obtained in other animal species were also considered.
The elimination of the spermatid cytoplasm during spermiogenesis enables the sperm to acquire a streamlined architecture, which allows for unhindered swimming. While this process has been well described in vertebrates, it has rarely been reported in invertebrates. In this study, we observed the process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei (Mollusca, Cephalopoda) using light microscopy, transmission electron microscopy, and immunofluorescence. In the early spermatid, the cell is circular, and the nucleus is centrally located. With spermatid development, the cell becomes polarized. The nucleus gradually elongates and moves toward the end of the cell where the tail is forming. As a result, the cytoplasm moves past the nucleus at the anterior region of the future sperm head (the foreside of the acrosome). Following this, during the late stage of spermiogenesis, the cytoplasm condenses and collects on the foreside of the acrosome until finally the residual body is discarded from the top of the sperm head. This represents a distinct directionality for the development of cytoplasmic polarity and discarding of residual body compared with that reported for vertebrates (in which the cytoplasm of the elongating spermatids is polarized toward the caudal region). The fact that the cytoplasm also becomes concentrated suggests that water pumps may be involved in the elimination of water from the cytoplasm before the residual body is discarded. Furthermore, we found that microtubules, forming a manchette‐like structure, are involved not only in reshaping of the nucleus but also in the transport of mitochondria and vesicles to the foreside of the acrosome, subsequently allowing them to be discarded with the residual body. This study broadens our understanding of the development of polarization and elimination of cytoplasm from spermatids in animals.
Specific aquaporins (AQP), called peroxyporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasting during oxidative stress. Another target involved in oxidative stress is the Sigma1 Receptor (S1R), since its activation is triggered by oxidative or endoplasmic reticulum stress. Herein we evaluated the effect of S1R modulators on AQP-dependent water permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in Hela cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. All compounds except one were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together, our results support the hypothesis that the investigated compounds act as dual aquaporin and Sigma1 receptor (DAS) modulators. The finding that small molecules can modulate both AQP and S1R opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, like cancer and degenerative diseases.
Testicular transcriptomes were analyzed to characterize the
differentially expressed genes between mulard and Pekin ducks, which will
help establish gene expression datasets to assist in further determination
of the mechanisms of genetic sterility in mulard ducks. Paraffin sections
were made to compare the developmental differences in testis tissue between
mulard and Pekin ducks. Comparative transcriptome sequencing of testis
tissues was performed, and the expression of candidate genes was verified by
quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In
mulard ducks, spermatogonia and spermatocytes were arranged in a disordered
manner, and no mature sperm were observed in the testis tissue. However,
different stages of development of sperm were observed in seminiferous
tubules in the testis tissue of Pekin ducks. A total of 43.84 Gb of clean
reads were assembled into 193 535 UniGenes. Of these, 2131 transcripts
exhibited differential expression (false discover rate <0.001 and
fold change ≥2), including 997 upregulated and 1134 downregulated
transcripts in mulard ducks as compared to those in Pekin duck testis
tissues. Several upregulated genes were related to reproductive functions,
including ryanodine receptor 2 (RYR2), calmodulin (CALM), argininosuccinate
synthase and delta-1-pyrroline-5-carboxylate synthetase ALDH18A1 (P5CS).
Downregulated transcripts included the testis-specific
serine/threonine-protein kinase 3, aquaporin-7 (AQP7) and glycerol kinase
GlpK (GK). The 10 related transcripts involved in the developmental biological
process were identified by GO (Gene Ontology) annotation. The KEGG (Kyoto
Encyclopedia of Genes and Genomes) pathways indicated that peroxisome
proliferator-activated receptors (PPARs) and calcium signaling pathways were
significantly (P<0.001) associated with normal testis physiology.
The differential expression of select genes implicated in reproductive
processes was verified by qRT-PCR, which was consistent with the expression
trend of transcriptome sequencing (RNA-seq). Differentially expressed candidate genes RYR2, CALM, P5CS,
AQP7 and GK were identified by transcriptional analysis in mulard and Pekin
duck testes. These were important for the normal development of the male
duck reproductive system. These data provide a framework for the further
exploration of the molecular and genetic mechanisms of sterility in mulard
ducks.
Highlights. The mulard duck is an intergeneric sterile hybrid
offspring resulting from mating between Muscovy and Pekin ducks. The
transcriptomes of testis tissue from mulard and Pekin ducks were
systematically characterized, and differentially expressed genes were screened, in
order to gain insights into potential gonad gene expression mechanisms
contributing to genetic sterility in mulard ducks.
The aim of the present study was to investigate aquaporin (AQP)8 and AQP9 expression in patients with polycystic ovary syndrome (PCOS) and its association with in vitro fertilization-embryo transfer (IVF-ET) outcomes. A total of 45 patients with PCOS undergoing IVF-ET (test group) and 50 patients with oviduct obstruction or ovarian cyst (control group) were assessed for the mRNA expression of AQP8 and AQP9 in ovarian tissues by reverse transcription-quantitative (RT-q)PCR. The levels of luteinizing hormone, anti-mullerian hormone and testosterone were determined, which were revealed to be significantly different between the two groups (P<0.05). The RT-qPCR results indicated that AQP8 expression in the control group was lower than that in the test group (t=37.75, P<0.01), whereas AQP9 expression in the control group was higher than that in the test group (t=19.59, P<0.01). The number of eggs obtained in the group with high AQP8 expression was significantly lower than that in the group with low AQP8 expression (t=2.64, P<0.01). The number of high-quality embryos in the high AQP8 expression group was not significantly different from that in the low AQP8 expression group (t=1.02, P>0.05). The pregnancy rate in patients with high AQP9 expression was higher than that in the low AQP9 expression group (P<0.05) and the abortion rate in the former was lower than that in the latter (P<0.05). In conclusion, AQP8 and AQP9 are differentially expressed in ovarian tissues of patients with PCOS vs. normal control subjects. The expression of AQP8 is closely associated with the occurrence and development of oocytes, whereas the expression of AQP9 is associated with the success rate of pregnancy in patients with PCOS.
Mammalian sperm are exposed to a natural hypoosmotic environment during male-to-female reproductive tract transition; although this activates sperm motility in vivo, excessive swelling can harm sperm structure and function. Aquaporins (AQPs) is a family of membrane-channel proteins implicated in sperm osmoregulation. The objective was to determine associations among relative sperm volume shift, hypoosmotic swelling test (HOST), sperm aquaporin (AQP) 7 mRNA abundances, and sire conception rate (SCR; fertility estimate) in Holstein bulls at a commercial artificial insemination center. Three or four sires for each full point SCR score from −4 to +4 were included. Each SCR estimate for study bulls (N = 30) was based on > 500 services (mean ± SEM) of 725 ± 13 services/sire). Sperm from a single collection day (two ejaculates) from these commercial Holstein bulls were used. Relative mRNA expression of AQP7 in sperm was determined by polymerase chain reaction. Mean relative sperm volume shift and percentage of sperm reacted in a HOST (% HOST) were determined (400 sperm per bull) after incubating in isoosmotic (300 mOsm/kg) and hypoosmotic (100 mOsm/kg) solutions for 30 min. There was no correlation between %HOST and SCR (r = 0.28 P > 0.1). However, there was a positive correlation between relative sperm volume shift and SCR (r = 0.65, P < 0.05). Furthermore, AQP7 mRNA abundance was positively correlated to both relative volume shift (r = 0.73; P < 0.05) and to SCR (r = 0.67; P < 0.05). The mRNA expressions of AQP7 and relative sperm volume shift differed (P < 0.05) among low- (<2 SCR), average- (-2 to +2) and high- (>2) fertility sire groups. In conclusion, bulls with higher SCR had significantly greater AQP7 mRNA abundance in frozen-thawed sperm. This plausibly contributed to greater regulation of sperm volume shift, which apparently conferred protection from detrimental swelling and impaired functions.
From their humble beginnings as spermatogonia and throughout their development, spermatozoa are continually exposed to a specialized luminal fluid microenvironment. The composition of this fluid is unique to the ducts of the male reproductive tract and is distinctly different from blood plasma. Moreover, the composition of the testicular and epididymal luminal fluid is not constant but changes dramatically from one region to the next. Remarkably, the changes in the luminal microenvironment occur even along very small distances in the reproductive tract. These changes have been assumed to reflect the needs of spermatozoa as they undergo spermatogenesis in the testes and maturation in the epididymis. However, with the exception of some proteins, few components of the microenvironment have been shown to directly play a role in either testicular or epididymal function. This chapter will review some of the key components of the testicular and epididymal luminal fluid milieu and briefly discuss how this microenvironment is formed. Special attention will be given to the role of transport proteins in regulating the luminal fluid microenvironment.
Efferent ductules and epididymis are involved in water and solute transport, which is indispensable for storage and maintenance of the sperm viability. The reabsorption process involves proteins such as aquaporins (AQP), which has been described in the male genital system of limited species, including primate, rodents, cats and dogs. To contribute with information about AQPs in the male system, here we investigated the distribution of AQP1 and AQP9 in the tropical bat Artibeus lituratus, along the annual reproductive cycle. A.lituratus is a seasonal breeder with natural variation in components of the androgen and estrogen responsive system, thus being a good model for exploring the AQPs modulation. AQP1 was found restricted to differentiating spermatids, efferent ductules epithelium and venular endothelia along the male tract. AQP9 was detected throughout the epididymis being more abundant in the cauda and ductus deferens, but was not found in testis, rete testis and efferent ductules. Contrasting with AQP1 which appear to be constitutively expressed, there was seasonal variation in AQP9 expression, which was reduced in regressed epididymis. The AQP9 does not appear to be modulated by estradiol or androgens, but possibly by other factor related to luminal sperm. The establishment of specific function for aquaporins in the male tract remains undetermined; however, the cellular distribution presently found are compatible with the main function of AQP1, as a selective water channel, and AQP9, which is a conduct for water and a plethora of neutral solutes present in the epididymis milieu such as glycerol and urea.
Cystic fibrosis is the most frequent autosomal recessive disease in the Caucasian population, with an incidence of 1:2500 newborn and a frequency of 1:25. The associated gene is Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and it encodes the CFTR protein that functions as a chloride (Cl(-)) channel. It is found in the apical membrane of exocrine epithelial cells, responsible for the regulation of the movement of water and solutes through biological membranes. To our knowledge, there are no studies on protein localization in the different cell types of the seminiferous epithelium with different pathologies. The aim of the present study was to analyze the expression of the CFTR protein in the human seminiferous epithelium of infertile males with different pathologies. CFTR protein expression was studied by immunohistochemistry in paraffin sections of testicular biopsies of six infertile men: Sertoli cell only syndrome, maturation arrest, secondary obstructive azoospermia, primary obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD), severe oligozoospermia, and retrograde ejaculation. All cell types of the seminiferous epithelium were studied: Sertoli cells, spermatogonia, primary spermatocytes at the leptotene/zygotene and at the pachytene stages, secondary spermatocytes, round, elongating and elongated spermatids, and spermatozoa. With the exception of sperm, all cells were labeled in the cytoplasm and in the cytoplasmic membrane. In the patient with CBAVD labeling was light at the cell membrane and absent in the cytoplasm of Sertoli cells and diploid germ cells. Generally, labeling was stronger after the diploid stage, which is probably related to cell volume reduction during spermiogenesis. The results obtained also suggest that the CFTR protein may impact CBAVD spermatogenesis and other pathologies.
Aquaporin 7 (AQP7) gene as a candidate Antifreeze gene was investigated and associated with fresh and frozen semen quality traits in 45 Simmental and Charolais bulls. PCR-SSCP, PCR-RFLP, and sequencing method were employed to detect the single nucleotide polymorphisms (SNPs) of the AQP7 gene. Two mutations, A264G and G371C, were identified. Th e allele frequencies were A (0.97, 0.96) to B (0.03, 0.04) and C (0.97, 0.91) to D (0.03, 0.09) in Simmental and Charolais bulls, respectively. A264G substitution was significantly associated with acrosome integrity ratio (P < 0.01) and motility (P < 0.05) in frozen semen. Meanwhile, G371C substitution was significantly associated with acrosome integrity ratio (P < 0.05), percentages of viable sperm (P < 0.05), and motility (P < 0.01) in frozen semen. Th e results prove that AQP7 gene SNPs are useful for marker-assisted selection in early seed selection in the bull industry.
Twelve water channels (aquaporins) are expressed in mammalian reproductive systems, and play very important roles in maintaining water homeostasis in reproductive cells. Impairment of their functions can result in attenuated male and female fertility. Alteration of AQPs expression is also found in reproductive tissues of the patients with polycystic ovarian syndrome, endometriosis or endometrium carcinoma. A lot of data have increased understanding of the functions and mechanisms of regulation of aquaporins at both the molecular and the clinical level. Researches have also focused on aquaporins as therapeutic targets. This review discusses recent advances in uncovering the physiological and pathophysiological roles of aquaporins in the reproductive systems.
We have previously reported the expression of aquaporin-7 (AQP7) in the rat testis and cloned human AQP7 from the testicular cDNA library. However, to our knowledge the spatial expression pattern and biological roles of AQP7 remain to be elucidated in humans.
We investigated AQP7 expression in the human testis and ejaculated sperm from fertile men and from infertile patients.
AQP7 was expressed at the tail of spermatids and spermatozoa in the human testis. AQP7 protein was also detected at the middle piece and the anterior tale portion of ejaculated sperm. However, some infertile patients lacked AQP7 expression in ejaculated sperm, although all fertile men expressed AQP7 protein. The motility rate of AQP7 negative sperm was significantly lower than that of AQP7 positive sperm, while the sperm concentration was not different between AQP7 positive and negative subjects.
AQP7 shows a spatial expression pattern in the human testis. AQP7 may be involved in the maintenance of sperm motility. Furthermore, a lack of AQP7 expression in sperm may be an underlying mechanism of male infertility.
The transcription factor p53 functions not only to suppress tumorigenesis but also to maintain normal development and homeostasis.
Although p53 was implicated in different aspects of fertility, including spermatogenesis and implantation, the mechanism underlying
p53 involvement in spermatogenesis is poorly resolved. In this study we describe the identification of a spermatogenesis-associated
gene, SPATA18, as a novel p53 transcriptional target and show that SPATA18 transcription is induced by p53 in a variety of cell types of both human and mouse origin. p53 binds a consensus DNA motif
that resides within the first intron of SPATA18. We describe the spatiotemporal expression patterns of SPATA18 in mouse seminiferous tubules and suggest that SPATA18 transcription is regulated in vivo by p53. We also demonstrate the induction of SPATA18 by p63 and suggest that p63 can compensate for the loss of p53 activity in vivo. Our data not only enrich the known collection of p53 targets but may also provide insights on spermatogenesis defects that
are associated with p53 deficiency.
High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8+/-11.0 nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9+/-1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.
The in vitro incorporation of tritiated uridine into RNA by the spermatogenic cells of the rat has been analyzed by high-resolution autoradiography. Special attention has been focused on the unique cytoplasmic organelle, the chromatoid body. After a short labeling time (2 h), this organelle remains unlabeled in the vast majority of the early spermatids although the nuclei are labeled. When the 2-h incubation with (3H)uridine is followed by a 14-h chase, the chromatoid body is seen distinctly labeled in all spermatids during early spermiogenesis from step 1 to step 8. Very few grains are seen elsewhere in the cytoplasm of these cells. When RNA synthesis in the spermatid ceases, the chromatoid body also remains unlabeled. It is likely that the chromatoid body contains RNA which is synthesized in the nuclei of the spermatids. The function of this RNA as a stable messenger RNA needed for the regulation of late spermiogenesis is discussed.
Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.
The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.
A new member of the aquaporin (AQP) family has been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins ( approximately 30%), and it is highest with AQP3 (48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport by 5- and 9-fold, respectively. The activation energy for glycerol was also low (5.3 kcal/mol after the correction of the endogenous glycerol permeability of oocytes). Northern blot analysis revealed a 1.5-kilobase pair transcript expressed abundantly in testis. In situ hybridization of testis revealed the expression of AQP7 at late spermatids in seminiferous tubules. The immunohistochemistry of testis localized the AQP7 expression at late spermatids and at maturing sperms. AQP7 may play an important role in sperm function.
A novel integral membrane protein with an apparent molecular mass of 28 kDa (CHIP28) was first isolated from human erythrocytes and is now recognized as a water channel protein. The expression of this protein has been found in several other cell types that all require high water permeability for their functions. Recent studies have shown that the water permeability (Lp) of human spermatozoa is among the highest reported for mammalian cells. Together with the low activation energy of human spermatozoa for Lp, this suggests that CHIP28 water channel may be present in the plasma membrane of human spermatozoa. However, our current studies do not support this hypothesis. Results from Western blot analysis on human sperm plasma membrane proteins, performed through use of an antibody against human erythrocyte CHIP28 protein, indicated that human spermatozoa do not express CHIP28 protein on their cell surface (n = 10). Consistent with the Western blot finding, mercuric chloride (HgCl2), a known water channel blocker, failed to reduce the osmotic water permeability of human spermatozoa. The calculated Lp values were 1.30 +/- 0.29 micron/min/atm (n = 16; mean +/- SEM) for the control group and 1.31 +/- 0.29 (n = 9; mean +/- SEM), 1.04 +/- 0.27 (n = 11; mean +/- SEM), and 1.34 +/- 0.19 (n = 6; mean +/- SEM), respectively, for the 10 microM, 30 microM, and 50 microM HgCl2-treated groups. These Lp values are not different (p > 0.05). In contrast, the same concentration of HgCl2 significantly blocked the osmotic water transport across the membrane of human erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Ten aquaporins have been cloned from various mammalian tissues. They are grouped according to their structure and function.
The first group consists of 7 aquaporins; AQP0, 1, 2, 4, 5, 6, and 8. These channel molecules selectively transport water
and do not transport glycerol and urea. The second group consists of 3 aquaporins; AQP3, 7, and 9. They transport not only
water, but also small nonionic molecules such as glycerol and urea. The extensive tissue distribution and physiologic regulation
by dehydration and hormones of these aquaporins suggest that aquaporins have important functions in water and solute transport
in the body. However, the recent studies of knockout animals and humans with defective mutations of aquaporins showed unexpectedly
small phenotypic effects. It is possible that other, unidentified aquaporins may compensate for these deficiencies. The future
challenge of research in aquaporins should be the identification of their physiologic significance, and the discovery of new
members, which will expand the research area of water metabolism and deepen our understanding of the physiology and pathophysiology
of water transport in our body.
A new member (AQP9) of the aquaporin family was identified from human leukocytes by homology cloning using PCR. A full length clone was obtained by screening human liver cDNA library. AQP9 encodes a 295-amino-acid protein with the amino acid sequence identity with AQP3 (48%), AQP7 (45%), and other aquaporins (approximately 30%), suggesting that AQP3, AQP7, and AQP9 belong to a subfamily of the aquaporin family. Injection of AQP9-cRNA into Xenopus oocytes stimulated the osmotic water permeability 7-folds with a low activation energy (4.2 kcal/mol) which was inhibited by 0.3 mM mercury chloride by 48%. AQP9 also facilitated urea transport 4-folds. However, in contrast to AQP3 and AQP7, AQP9 did not stimulate the glycerol permeability, suggesting a unique permeability character. Northern blot analysis revealed the high expression of 3.5-kb messages in peripheral leukocytes > liver > lung = spleen, but not in thymus. The possible role of AQP9 in the immunological function of leukocytes is intriguing and the identification of AQP9 with unique permeability profile may expand our understanding of water and small solute transport in the body.
The traditional and generally accepted mode of elimination of excess spermatid cytoplasm, known to take place at the time of sperm release, occurs as a result of the disengagement of the residual cytoplasmic mass. The present study suggests that cytoplasm is also eliminated from the head region of the spermatid in the period prior to sperm release. Data presented herein indicates that the amount of cytoplasm eliminated in this manner is substantial since the volume of spermatid cytoplasm diminishes by about 70% in the period of formation and resorption of tubulobulbar complexes. Reports from this laboratory (Russell and Clermont, '76; Russell, '79) indicated that characteristic evaginations of spermatid cytoplasm, termed tubulobulbar complexes , are resorbed by Sertoli cells in the form of numerous small pockets of cytoplasm. These pockets of cytoplasm are organelle‐free and show a “watery” consistency. They are subsequently degraded by the Sertoli cell prior to sperm release.
During the period of turnover of tubulobular complexes the organelles of the cytoplasmic lobe, which at first are loosely organized, become clustered and tightly packed. As a consequence, the cytoplasmic lobe appears electron dense and is stained intensely with toluidine blue. This feature is interpreted, although not proven, to be the result of elimination of the watery component of the cytoplasm via resorption of tubulobulbar complexes by the Sertoli cell. During the period in which tubulobulbar complexes form, there is an open channel in which cytoplasm may flow from the flagellum to the head and out the head into tubulobulbar complexes.
Intraluminal fluids from hamster seminiferous tubules, rete testis, and caput, corpus and cauda epididymidis were obtained in vivo by micropuncture. Rete testis fluid is isosmolar with serum. Fluids from the seminiferous tubules and all sites in the epididymis are hyperosmolar.
Near the end of spermiogensis, the late spermatids remain attached to the superficial layer of the seminiferous epithelium for an appreciable period of time (i.e., 3 to 4 days). Ths sickle-shaped heads of the spermatids are embedded in an apical process of Sertoli cell cytoplasm which is connected to the rest of the cell by a narrow stalk. In the concavity of the head several long (2-3 mum) and very narrow (50 nm) tubular projections of the spermatid's plasma membrane invaginate the Sertoli cell cytoplasm. These tubular processes terminate by a bulbous swelling which may measure up to 1 mum in diameter. Along the process the plasma membrane of the Sertoli cell is closely apposed to the spermatid's membrane, the intracellular space being only 6-8 nm wide. In the Sertoli cytoplasm immediately surrounding the tubular portion of the structure there is an accumulation of filamentous material, while next to the bulbous extremity there are, at a shrot distance, smooth surfaced cisternae of endoplasmic reticulum. The whole structure was referred to as a tubulobulbar complex. These complexes, of which there are up to 24 per spermatid, appear as these cells complete their migration toward the apex of the Sertoli cells. They disappear just before the release of the spermatids in the lumen of the seminiferous tubule as a result of the fragmentation of the spermatid's plasma membrane followed by a resorption of the Sertoli plasma membrane. Morphological evidence suggests that the Tubulobulbar complexes serve as anchoring devices that retain the spermatids at the surface of the seminiferous epithelium while their dissolution contributes in part to the process of spermiation. Similar tubulobulbar complexes were also formed by the plasma membranes of two adjacent Sertoli cells close to the Sertoli-Sertoli tight junctions near the tubular limiting membrane.
Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although
selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane
protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions.
Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited
by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane
water channels.
In the transformation of a spermatid into a spermatozoon, cytoplasmic loss occurs, allowing the sperm to swim unhindered. Cytoplasmic loss takes place via elimination of the residual body and through structures known as tubulobulbar complexes. A determination of cytoplasmic loss in several species was undertaken by using high-resolution electron microscopic morphometric techniques. During the period that tubulobulbar complexes are present, an average 53% cytoplasmic loss was recorded for five species (guinea pig, 48.8 +/- 6.2% (SEM); monkey, 60.3 +/- 4.3%; opossum, 54.5 +/- 4.4%; rabbit, 46.9 +/- 2.7%; and rat, 55.7 +/- 4.9%), whereas there was essentially no loss or gain in cytoplasm during the same period for round spermatids. Surprisingly, during spermatid elongation an approximate 36% loss of cytoplasm was also recorded for five species (guinea pig, 50.1 +/- 6.3%; monkey, 30.0 +/- 15.4%; opossum, 25.4 +/- 9.0%; rabbit, 42.4 +/- 8.6%; and rat, 34.9 +/- 11.9%), which is only partially (approximately 60%) accounted for by fluid pumping from the nucleus during nuclear condensation. A densification of the cytoplasm of elongate spermatids, as compared with round spermatids, suggests fluid is also pumped from the elongating spermatid cytoplasm. Fluid loss from germ cells may contribute to the seminiferous tubule fluid, a fluid previously thought to be solely of Sertoli-cell origin.
We have discussed cell to cell junctions revealed by freeze-fracture in the seminiferous tubules and the lining cells of the excurrent duct. Sealing elements of these lining cells have a significant function in maintaining the specific microenvironments playing an important role in spermatogenesis, transportation, maturation, and storage of the spermatozoa. Unique features of the Sertoli cell junctions are emphasized. Instead of the conventional method of freeze-fracture, the techniques of rapid freezing and deep etching without use of aldehyde fixative and cryoprotectants will be a powerful tool to reveal the 'new' fine structure of membrane and cytoplasm. Plausible explanations of the unique particle distribution in the Sertoli cell junctions both in fixed and 'unfixed' conditions are expected in further studies. Concomitantly, it remains to be understood how the different configurations of the tight junctions along the excurrent duct reflect the results obtained by micropuncture analysis of the fluid components in different segments of the duct system. More comparative, experimental, and clinicopathological studies on the junctional complexes would be necessary to understand the blood-testis barrier and the environments of the excurrent duct system.
The surface of mammalian sperm is known to exhibit regional heterogeneity. Using monoclonal antibodies, we have analyzed the nature of this regional variation at the molecular level. A map of the surface of guinea pig sperm has been constructed that defines a number of regions in which surface antigens are localized and analyzes the diversity of antigens found in each region. In an initial screening of 117 hybridoma antibodies to a sperm membrane preparation, a remarkable result was obtained: all the antibodies bound to a localized region of the cell. From the initial hybrid lines, we established a collection of 56 stable hybridomas producing antibodies to surface antigens. These antibodies detect antigens localized in five surface regions: anterior head (AH), posterior head (PH), whole head (WH), posterior tail (PT), and whole tail (WT). At least 12 distinct surface antigens are recognized that bind antibodies in one of the localized regions (five AH antigens, three PH, two WH, one PT, and one WT). Some of the recognized antigens have been identified as proteins, comprised of either one or several 125I-labeled polypeptides. The identified AH antigens have labeled polypeptides of molecular weights (Mr) 52,000 (52K); 70K, 62K, 46K, 25K, and 18K; 62K, 52K, and 38K; 16K; and 38K. Identified PH antigens have polypeptides of Mr 60K; 66K, 48K, and 41K; and 58K and 48K. Identified WH antigens have polypeptides of Mr 89K and 45K; and 42K. We conclude that the sperm cell can maintain contiguous membrane domains which have quite different compositions. Its surface is a mosaic consisting of multiple regions and each region can contain several localized antigens.
We used immunoperoxidase histochemistry and confocal immunofluorescence microscopy to examine the events involved in the compartmentalization of CE9 to the posterior-tail plasma membrane domain during spermatogenesis in the rat. We identified two major episodes of spermatogenesis during which CE9 appeared to accumulate in relatively large amounts intracellularly within elements of the secretory pathway. The first episode encompassed cells from preleptotene through early pachytene primary spermatocytes and was evident as intense intracellular labeling of the endoplasmic reticulum and the Golgi complex. The second episode encompassed spermatids in steps 8-12 of spermiogenesis and was evident as intense intracellular labeling of the Golgi complex and smaller vesicular structures observed within the cytoplasm of the spermatid. Between these two episodes, CE9 was detected in considerably reduced amounts. Although present within the Golgi complex and the acrosomic system throughout much of the first half of spermiogenesis, CE9 was not detected on the tail of the spermatid until steps 8-9 of spermiogenesis. Although detected initially in relatively small amounts along the entire length of the tail beginning at steps 8-9, there was no evidence for the presence of relatively large amounts of CE9 on the tail or anywhere else on the surface of the spermatid until after step 11 of spermiogenesis. Between step 11 and steps 13-14 of spermiogenesis, CE9 was observed to accumulate in relatively large amounts on the whole tail coincident with its apparent loss from the Golgi complex. CE9 was observed to then undergo further compartmentalization to the posterior-tail domain sometime between steps 13-14 of spermiogenesis and spermiation. Our results suggest that CE9 is synthesized and enters the secretory pathway throughout much of spermatogenesis, but that the site of accumulation of CE9 varies considerably as a function of development. With respect to the biogenesis of the posterior-tail plasma membrane domain, our results suggest that CE9 is targeted from the Golgi complex to the plasma membrane of the whole tail during mid to late spermiogenesis and then redistributes laterally into the posterior-tail domain coincident with the caudal migration of the annulus late in spermiogenesis. This proposed pathway has a number of important implications for the logistical capabilities of the mammalian spermatid.
During mammalian spermiogenesis a spherical spermatid is transformed into a highly asymmetric sperm cell. Concurrently, the plasma membrane of the cell develops into a mosaic of discrete membrane regions, with each region containing a unique set of proteins. Biogenesis of these surface domains was studied by following the surface expression and localization of nine different antigens during spermiogenesis. Each of these antigens exhibits one of four distinct patterns of localization on testicular sperm (whole cell, whole head, anterior tail, and posterior tail), indicating that there are at least three distinct surface domains on testicular sperm. Our results on the timing of antigen localization suggest that the generation of surface domains in mammalian sperm is a complex process. This process involves temporal and spatial regulation of surface expression of the antigens, as well as the specific removal of antigens from inappropriate domains after they have reached the cell surface.
The aquaporin water channels are expressed in various fluid-transporting epithelia. Physiological and genetic investigations have revealed that aquaporin channel-like intrinsic protein is expressed in numerous tissues, but its significance in water transport physiology is unclear. It has been shown that aquaporin-collecting duct is a vasopressin-responsive water channel, and that it is regulated by a membrane shuttle mechanism. Three unique models for a water pore have been presented but further studies will be required to verify them. New aquaporin members have been isolated and their discrete localization may reflect their specific physiological roles.
Discovery of aquaporin water channel proteins has provided insight into the molecular mechanism of membrane water permeability. The distribution of known mammalian aquaporins predicts roles in physiology and disease. Aquaporin-1 mediates proximal tubule fluid reabsorption, secretion of aqueous humor and cerebrospinal fluid, and lung water homeostasis. Aquaporin-2 mediates vasopressin-dependent renal collecting duct water permeability; mutations or downregulation can cause nephrogenic diabetes insipidus. Aquaporin-3 in the basolateral membrane of the collecting duct provides an exit pathway for reabsorbed water. Aquaporin-4 is abundant in brain and probably participates in reabsorption of cerebrospinal fluid, osmoregulation, and regulation of brain edema. Aquaporin-5 mediates fluid secretion in salivary and lacrimal glands and is abundant in alveolar epithelium of the lung. Specific regulation of membrane water permeability will likely prove important to understanding edema formation and fluid balance in both normal physiology and disease.
The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity for water and/or neutral solutes. We have identified 84 fully or partially sequenced members of this family, have multiply aligned over 50 representative, divergent, fully sequenced members, have used the resultant multiple alignment to derive current MIP family-specific signature sequences, and have constructed a phylogenetic tree. The tree reveals novel features relevant to the evolutionary history of this protein family. These features plus an evaluation of functional studies lead to the postulates: (i) that all current MIP family proteins derived from two divergent bacterial paralogues, one a glycerol facilitator, the other an aquaporin, and (ii) that most or all current members of the family have retained these or closely related physiological functions.
The electron crystallographic structure of the aquaporin-1 water channel, determined at approximately 6A, reveals that the protein has six transmembrane alpha-helices forming a trapezoid-like cylinder. There is a branched rod-like structure within the cylinder that traverses the membrane and likely contains at least one alpha-helix.
A new member of water channels has been identified from rat testis. This gene, termed aquaporin 8 (AQP8), encoded a 263-amino-acid protein that contained the conserved NPA motifs of MIP family proteins. AQP8 has amino acid sequence identity with other aquaporins (approximately 35%) and highest with a plant water channel, AQP-gamma TIP (39%), suggesting that AQP8 is a unique member in mammalian aquaporins. The expression of AQP8 in Xenopus oocytes stimulated the osmotic water permeability (Pr) 8.5 folds. The increase of Pr was inhibited with 0.3 mM mercury chloride by 55%, which was reversed with mercaptoethanol. The Arrhenius activation energy for the stimulated water permeability was low (5.1 kcal/mol). AQP8 did not facilitate glycerol transport. Northern blot analysis revealed a 1.5-kb transcript of AQP8 abundantly in testis and slightly in liver. In situ hybridization of testis revealed the expression of AQP8 mRNA in all stages of spermatogenesis from primary spermatocytes to spermatids in seminiferous tubules. Together with previously cloned AQP7, AQP8 may also play an important role in spermatogenesis. The unexpected complexity of the presence of two aquaporins in testis may call for the further analysis of the role of aquaporins in the reproduction biology.
MIP family proteins can be divided into two groups according to their primary sequences. The CHIP group is predominant in the plant and animal kingdoms and functions primarily as water channels. The GLP group is a minor group with limited prevalence and functions primarily as glycerol transporters. Both prototypes are present in bacteria and may have evolved separately.
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