Showing papers on "Sperm plasma membrane published in 2013"

Sperm capacitation: a distant landscape glimpsed but unexplored.

Abstract: Capacitation is a remarkable process whereby spermatozoa prepare themselves for engagement with the oocyte. Although the existence of this process has been appreciated as a biological phenomenon for more than half a century, its molecular underpinnings still await clarification. We know that some of the major changes involve sterol oxidation and efflux from the plasma membrane, the anterior movement of lipid rafts, changes in the surface expression of a variety of proteins including hyaluronidase and receptors for the zona pellucida, an increase in intracellular cyclic adenosine monophosphate (cAMP), the induction of tyrosine phosphorylation and the expression of hyperactivated motility. These changes are dependent on the presence of bicarbonate, to facilitate cAMP generation, maintain an alkaline intracellular pH and support an optimal level of reactive oxygen species generation and are enhanced by the presence of albumin to provide antioxidant protection to the plasma membrane and promote cholesterol efflux. In vivo, the rate at which sperm cells capacitate is carefully controlled in order to ensure that the release of capacitated spermatozoa from a post-insemination reservoir in the isthmic region of the oviduct is synchronized with ovulation. The factors that control these critical events are now being resolved, aided by proteomic studies that are providing critical definitive information on the range of receptors that exist in the sperm plasma membrane and define the manner in which these exquisitely complex cells interact with their environment. Progress in this area has been enhanced by IVF technology pioneered by Bob Edwards and will ultimately facilitate the design of safe, effective culture conditions for optimization of this revolutionary therapy.

Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa

Abstract: Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

Seminal characteristics, sperm fatty acids, and blood biochemical attributes in breeder roosters orally administered with sage (Salvia officinalis) extract

Abstract: Seminal characteristics and blood biochemical attributes were studied in breeder roosters orally administered with sage extract (SG), an herbal extract well known to have potent antioxidant activities. Sixty roosters (34 weeks old) were randomly allotted to five treatment groups to receive no SG, or orally administered with 110, 210, 320, or 420 mg SG/kg liveweight for 8 weeks. Semen samples were evaluated weekly. Blood samples were taken fortnightly and a total of 21 biochemical indices were measured to unmask the effects of SG (especially the adverse ones) on the clinical profile. Excluding the sperm concentration and seminal content of thiobarbituric acid reactive substances (sperm membrane lipid peroxidation index), other seminal traits exhibited one of the linear, quadratic, or cubic responses to the various levels of SG. The most improvements in total live sperm number and sperm membrane integrity (as determined by the hypoosmotic swelling test) were observed in birds receiving 210 and 320 mg SG/kg liveweight, respectively. Serum testosterone level was generally higher (cubically, P = 0.015), but serum copper was lower (linearly, P = 0.014) in SG-administered birds. Birds receiving 320 and 420 mg SG showed a decreased content of C18 : 2(n-6) in sperm plasma membrane. Other biochemical attributes or sperm fatty acids were not affected. It seems that most improvement in the seminal characteristics could be achieved 5–6 weeks following the administration of 210 and 320 mg SG/kg liveweight without any apparent adverse effect on the blood biochemical indices. The improvements, however, could not be attributed to the antioxidative effect of SG. Although it is hypothesised that an increased serum testosterone might have been involved, the underlying mechanism(s) remain(s) to be clarified.

Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation

Abstract: STUDY QUESTION Can selenium (Se) independent, epididymal-specific glutathione peroxidase 5 (GPX5) protect CHO-K1 cells from oxidative damage and, more specifically, from lipid peroxidation and DNA mutation? SUMMARY ANSWER CHO-K1 cells expressing GPX5 have increased resistance to oxidative challenge and, more specifically, decreased levels of lipid peroxidation and decreased levels of the downstream DNA lesion 8-oxo-7,8-dihydroguanine (8-oxodG) compared with control cells. WHAT IS KNOWN ALREADY GPX5 associates with sperm during transit of the epididymis, and has been postulated to protect sperm from peroxide-mediated attack. However, its function as an active glutathione peroxidase has been questioned due to substitution of the classical selenocysteine residue at its active site. Indirect evidence for a functional role for GPX5 has been provided by in vivo studies, in particular from the GPX5 knockout mouse whereby offspring sired by GPX5(-/-) males have a higher rate of spontaneous abortion and developmental defects, attributed to increased oxidative injury (8-oxodG) to sperm DNA, but only when the GPX5(-/-) males are over 1 year of age. Interestingly, we have previously shown severely reduced levels of GPX5 in humans. STUDY DESIGN, SIZE, DURATION To look more directly at its role in protection against oxidative damage, we have used an in vitro system, generating a CHO-K1 mammalian cell line expressing recombinant rat GPX5. PARTICIPANTS/MATERIALS, SETTING, METHODS We have used the recombinant CHO-K1 cells to determine whether GPX5 is able to protect these cells from an administered oxidative challenge, using a range of approaches. We compared the viability of GPX5-expressing cells with control cells by both MTT and trypan blue exclusion assays. We next investigated whether GPX5 protects the cells specifically from lipid peroxidation, by using the fluorescent reporter molecule C11-BODIPY(581/591), and thus from downstream DNA mutation, by comparing levels of the DNA lesion 8-oxodG. We also investigated whether GPX5 can be transferred to rat sperm via epididymosomes. MAIN RESULTS AND THE ROLE OF CHANCE GPX5-expressing CHO-K1 cells had increased viability compared with control cells following oxidative challenge (P < 0.005). We also found that GPX5-expressing CHO-K1 cells had significantly lower levels of C11-BODIPY(581/591) oxidation, and hence lipid peroxidation, compared with control cells. Levels of 8-oxodG DNA damage were also markedly lower in the nuclei of GPX5-expressing cells than in control cells. Finally, we showed that GPX5 can be transferred to rat sperm via epididymosomes. LIMITATIONS, REASONS FOR CAUTION GPX5 is not active in glutathione peroxidase assays using H₂O₂ as the substrate. However, the related non-mammalian Se-independent GPXs show preference for electron donors other than glutathione, with a number utilizing thioredoxin as a reducing equivalent. Hence, the in vitro activity of GPX5 needs to be assessed using a range of alternative substrates and electron donors. GPX5 is secreted by the epididymis and associates with the sperm plasma membrane. We showed that this transfer can occur via epididymosomes; however, the mechanism for transfer and the identity of a potential binding partner in the sperm membrane needs to be determined. Finally, our study utilized an in vitro system that needs to be translated to human sperm. WIDER IMPLICATIONS OF THE FINDINGS Our study supports an important role for GPX5 as an antioxidant, possibly acting as a phospholipid hydroperoxidase and participating in the maintenance of cell and DNA integrity.

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Abstract: Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.

Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5.

Abstract: Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

Sperm patch-clamp.

Abstract: Sperm intracellular pH and calcium concentration ([Ca(2+)]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca(2+)] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels.

Evaluation of the Spermicidal and Contraceptive Activity of Platycodin D, a Saponin from Platycodon grandiflorum

Abstract: BACKGROUND: The extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity as well as the safety of Platycodin D (PD) a major saponin in Platycodon grandiflorum. METHODS: Using the computer-aided sperm analysis (CASA) test criteria the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation. RESULTS: A dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats was non-DNA damaging and was not harmful to the vaginal tissue in the rats. CONCLUSION: PD has significant spermicidal activity that should be explored in further studies.

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Abstract: Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.

The Boar Spermatozoon

Abstract: The microscopic appearance of the boar spermatozoon allows us to appreciate both its inner and outer structural complexity. Both light and electron microscopy may be used to study the structure and ultrastructure of this highly specialized cell and the way it probably works to achieve successful fertilization. Compartmentalization of the spermatozoon is a critically important feature of its structure as it enables this cell to perform the variety of tasks needed to fulfill its role. Different sperm malformations usually affect some cellular components essential for the correct development of the spermatozoon–oocyte interaction in the fertility process. Careful assessment of sperm morphology may sometimes indicate the possible cause of sperm quality and fertility decrease. Moreover, regional specialization of the plasma membrane, related to lipid/protein composition and distribution, allows the underlying cellular molecules to interact independently with their external environment, thereby enabling the efficient performance of the various tasks necessary for successful fertilization.

Original research article Search for a potent microbicidal spermicide from the isolates of Shorea robusta resin

Abstract: Background: An alarming increase in global population is the root cause of poverty, malnutrition, sexually transmitted infections (STIs) and many other social problems. Microbicidal spermicides possessing dual function of contraception and STI protection can effectively combat this problem, and their development is of utmost importance at present. Study Design: A major metabolite isolated from Shorea robusta resin was spectroscopically characterized as asiatic acid. Spermicidal efficacy of the isolate was evaluated in vitro by a modified Sander–Cramer test. The mode of spermicidal action was assessed by (a) double fluoroprobe staining, (b) hypoosmotic swelling test and (c) scanning electron microscopy. Antimicrobial efficacy was assessed by disc diffusion and broth dilution methods using human isolates of bacteria (Escherichia coli ATCC 25938 and Pseudomonas aeruginosa 71) and fungus (Candida tropicalis). Results: The minimum effective concentration of asiatic acid that induced instantaneous immobilization of rat spermatozoa in vitro was 125 mcg/mL. The mechanism of action involved disruption of sperm plasma membrane. The microbicidal efficacy was found to be moderate for vaginal pathogens, with no effect on normal vaginal flora. Conclusion: Asiatic acid possesses appreciable spermicidal and microbicidal potential and may be explored as an effective microbicidal spermicide.

Sperm cyropreservation and oxidative damage. What does it mean

Abstract: About 15% of couples that desire children are affected by infertility. Oxidative stress has been involved in unexplained subfertility with a male factor [1]. Although the excessive production of reactive oxygen species (ROS) is detrimental to human spermatozoa, there is a growing body of evidence that suggests that ROS are also involved in the physiological control of some sperm functions. Under physiological conditions, small amounts of ROS are produced by spermatozoids. These ROS are necessary for efficiency, acrosomal reaction, and finally fertilization [2], but its excessive levels can negatively affect sperm quality. There is a current presumption that the most prolific source of ROS in sperm suspensions is an NADPH oxidase located in leukocytes or in spermatozoa that produces superoxide, which is further converted to peroxide by the action of superoxide dismutase (SOD). Since antioxidants suppress the action of ROS, these compounds have been used in the medical treatment of male infertility (they are beneficial in terms of improving sperm function and DNA integrity) or have been added to the culture medium during sperm separation techniques. Antioxidants have demonstrated their impact on sperm improvement in several studies, in particular in men with high levels of ROS in their sperm. However, in many cases, no beneficial effect was obtained after antioxidant supplementation. Negative effects could be observed in long–term treatment or with excessive doses. This medical therapy should not be used in patients with known genetic factors such as karyotype anomalies or Y chromosome deletion. Therefore, it is essential to perform a complete diagnostic workup of the man before deciding which men will respond to medical therapy and which will need to be referred to assisted reproduction. Treatment of oxidative stress should first involve strategies to reduce or eliminate stress–provoking conditions including smoking, varicocele (increases ROS levels in testes and semen), genital infection, gonadotoxins, and hyperthermia. In recent years, interest has increased in the role of antioxidants and B vitamins as modulators of fertility outcome. The antioxidants – alpha–tocopherol (vitamin E), ascorbic acid (vitamin C), and the retinoids (vitamin A) – are potent scavengers of ROS. Deficient vitamin B concentrations cause elevated homocysteine concentrations and impair the remethylation cycle of phospholipids, proteins, DNA, and RNA. These processes are essential in spermatogenesis. Treatment with oral antioxidants has generally been associated with improvement in sperm DNA integrity and in some cases pregnancy rates after assisted reproduction. Actually, antioxidants are provided in diet or can be found in enriched food. It is possible that a subset of infertile men with specific lifestyles (e.g., smoking, increased alcohol intake, and dieting) may be at risk for antioxidant deficiency, particularly vitamin C deficiency. A low intake of antioxidant nutrients was associated with a poor semen quality [3]. Overall, the data published suggest that no single antioxidant is able to enhance the fertilization capability of infertile men, whereas a combination of them seems to provide a better approach [4]. Few studies have shown that the incidence of ROS caused DNA fragmentation in ejaculated spermatozoa can be reduced by oral antioxidant treatment. Oral antioxidant treatment appears to improve ICSI (intracytoplasmic sperm injection) outcomes in those patients with sperm DNA damage, in whom this treatment reduces the percentage of damaged spermatozoa [5]. It is not clear why some men responded to antioxidants by reducing the extent of sperm DNA fragmentation while others did not. Greco et al. [6] suggest that the increased percentage of DNA–damaged spermatozoa may be a sequela of different pathophysiological mechanisms in different patients and only some of these conditions may be responsive to antioxidant treatment. This would also explain the discrepancies in the literature concerning the clinical usefulness of antioxidants in the treatment of male infertility (reviewed in Agarwal et al., 2004) [7]. Sperm cryopreservation is a widely used procedure in the context of assisted reproductive techniques. Cryopreservation and thawing is a procedure that inflicts irreversible injury on human spermatozoa. One of the possible mechanisms involved in sperm cryoinjury is apoptosis upon exposure to oxidative stress. Cryopreservation can also result in increased lipid peroxidation in human spermatozoa [8] and has been shown to reduce antioxidant defenses. Thus, the observed protective effect of vitamin E addition on post–thaw motility might be due to vitamin E suppression of lipid peroxidation via the sperm plasma membrane. The positive effect of vitamin E on motility is greater in semen samples from men over 40 years of age. The spermatozoa from older males had increased ROS and lipid peroxidation, suggesting a reduced capacity to cope with oxidative stress [9]. During in vitro fertilization the seminal plasma is removed during semen processing and the toxic oxygen metabolites (generated by immature spermatozoa and leukocytes) are able to attack spermatozoa without being protected by seminal plasma antioxidants. In addition, the detrimental effect of oxidative stress on sperm functional competence can be exaggerated by the in vitro sperm processing techniques (centrifugation and prolonged incubation) that usually precede assisted reproductive techniques. The addition of an antioxidant to the cryopreservation medium (vitamin E, both ascorbate and catalase) significantly reduces ROS concentrations in post–thaw spermatozoa [10]. Taking into account the pros and the cons of antioxidant treatment of male infertility, the potential advantages that it offers cannot be ignored.

Analyses of spermatozoa surface proteins using different separation techniques

Abstract: Passage of spermatozoa through the female reproductive tract is essential for the regulation of fertilization, ensuring that healthy sperm reach the oocyte. Previous studies were devoted to morphological selection of sperm cells by the cervical mucus. However, research prove that the loss of integrity of the sperm plasma membrane is associated with infertile men, irrespective of their normal semen parameters. This indicates that the sperm plasma membrane plays an important role in fertilization. Further studies indicated that sperm surface proteins assist penetration through the female reproductive tract and would therefore provide useful insight in understanding other factors associated with male infertility. The aim of this project was to determine if there are any differences between sperm surface proteins of fertile donor samples in relation to infertile patient samples using different separation techniques and different detergents. Three different sperm separation techniques were employed, including wash, swim-up (SU) and Percoll density gradient centrifugation (DGC).Parallel to this, the deoxy-ribose nucleic acid (DNA) fragmentation of these cells were analysed for comparison of the extent of DNA damage induced due to different separation techniques used. This provided evidence that the best separation technique is the DGC as it minimises the amount of DNA fragmentation caused. Four different detergents were used in the process of extracting the membrane proteins from spermatozoa, namely sodium dodecyl sulphate (SDS), saponin,cetyl-trimethyl-ammonium bromide (CTAB), and TWEEN-20. The membrane proteins were then separated on a12% SDS poly-acrylamide gel electrophoresis (PAGE), and analysed by Coomassie blue and silver staining techniques as well as densitometry. Due to the different chemical nature of the detergents that extracted different surface proteins, CTAB (cationic) and SDS (anionic) extracted the most because of its strong solubilising abilities as non-ionic detergents. Common proteins that were extracted in donor samples included; 115, 92.5, 89, 61, 55.5, 51.5, 47, 44.5, 43, 38.5, 34 and 28 kDa proteins. In patients, commonly occurring proteins included; 92.5, 74.5, 70, 60.5, 51.5, 50, 44.5, 43, 36, 29.5, and 25.5 kDa proteins. Marked differences were found between membrane proteins extracted from donor samples in comparison to patient samples. Identification of these proteins was done using the SwissProt database and a literature search. Mostly non-genomic progesterone receptors were identified; others included oestrogen receptor, a phosphotyrosyl protein, P34H, equatorial segment protein, mannose lectin receptor, human guanylylcyclase receptor, epididymal protease inhibitor receptor, PH30 and estradiol binding protein. The function of the membrane surface proteins identified in this study plays a vital role in fertilization. A few of these functions include sperm attachment and binding to the oocyte as well as penetration thereof. Others play a role in signalling events such as capacitation,…

Level of extra and intracellular enzymes during preservation of good and poor quality ejaculates in crossbred bulls

Abstract: Activities of superoxide dismutase (SOD), catalase (CAT) as extracellular and glutamate pyruvate transaminase (GPT), lactic dehydrogenase (LDH) as well as glutamate oxaloacetate transaminase (GOT) as intracellular enzyme were estimated in seminal plasma of good and poor quality ejaculates of crossbred bull semen. SOD, LDH and GOT activities differed significantly in both types of ejaculates at pre-freeze and post- thaw levels, whereas no difference was observed in catalase activity. The GPT activity was also higher in seminal plasma at post-thaw than prefreeze level in both types of ejaculates but it was less compared to other enzymes. Although there was loss in SOD activity during freezing thawing in both type of ejaculates but it was more in poor quality (62.5%) than good quality (50.4%), which may be due to the higher percentage of dead spermatozoa during freeze thaw in former than later. LDH and GOT enzyme activity also increased in seminal plasma in both types of ejaculates after post-thaw but it was higher in poor quality, which indicated that damage in sperm plasma membrane was more in poor quality during freezing thawing.

Survival of Drowning Sperm: Do Spermatozoa from External Fertilizers Adapt to Differing Osmotic Environments Through the Use of Aquaporins?

Abstract: In spawning animals, external fertilization usually requires spermatozoa to become activated (motile) in the environmental medium and then quickly locate and contact the egg. In most teleost fish, sperm are usually quiescent within the testes and seminal fluid but then initiate vigorous motility upon dilution into the external medium in which spawning occurs [1]. In teleost fish sperm, an increase in intracellular calcium occurs upon motility initiation by way of different signaling mechanisms (reviewed by [2]) [3–5]. Following dilution into medium, four different triggers of sperm motility in teleosts have been identified: 1) a reduction in external K þ upon dilution of semen initiates motility in salmonids [6, 7], 2) hypotonic exposure after dilution into freshwater is the trigger in nonsalmonid freshwater fishes [8–11], 3) hypertonic exposure initiates sperm motility in marine teleosts [12, 13], and 4) egg-associated molecules activate sperm motility [14– 17]. While there are many examples in the literature of specific ions and ion channels playing roles in sperm motility initiation and maintenance, it is known that the osmotic pressure of the medium alone can affect both motility initiation (reviewed by [5]) [18] and survival of sperm following dilution. In some of these studies, osmotic changes may stimulate stretch-activated channels that trigger motility initiation [19]. Sperm volume changes are known to occur and are associated with the osmotic change upon dilution in hypotonic medium [20]. These volume changes may, by themselves, activate stretchactivated channels and/or water channel proteins, termed aquaporins, which play a role in the activation of sperm motility in seabream spermatozoa [21]. In this case, the dilution of seabream spermatozoa into seawater results in a hyperosmotic change, and the water efflux through aquaporins that results creates an increase in intracellular ions; this is believed to be responsible for activation of adenylyl cyclase and the cAMP-signaling pathway causing phosphorylation of flagellar proteins [22]. The proposed model for aquaporin-mediated motility initiation is shown in Figure 1 [21]. A new study in this issue of Biology of Reproduction by Chauvigne et al. [23] has now shown that multiple aquaporins are associated with water and fluid homeostasis during spermatogenesis and in the male reproductive tract, as well as in mature motile spermatozoa. This study demonstrates that the water-specific aquaporin Aqp1aa and the glycerol/urea/ water-transporting channel Aqp10b are both present (with others) in the plasma membrane of both the head and flagellum of ejaculated sperm. Inhibitors of aquaporin channels are shown to inhibit motility, indicating a direct role for aquaporins in sperm motility initiation in this marine teleost. The Chauvigne et al. study [23] isolated and functionally characterized five additional members of the aquaporin superfamily using a comprehensive approach of immunolocalization and biochemical fractionation of sperm. They have shown, for the first time, that a nonmammalian vertebrate possesses up to seven classes of aquaporins that are differentially expressed during spermatogenesis and spermiogenesis, with several of these being redistributed in the plasma membrane upon motility initiation. During hyperosmotic shock upon dilution in seawater, Aqp1aa may mediate water efflux, whereas Aqp10b might play a role in maintenance of motility [21, 23]. The data in this new study clearly show the conservation of aquaporin paralogs in the male reproductive tract of a nonmammalian vertebrate and highlight the similarities in the pattern and localization of aquaporins both in the testes and in mature sperm, as has been described in mammals [24, 25, 26]. One of the most intriguing findings of the current Chauvigne et al. study [23] is the change in intensity of immunolabeling of Aqp1ab as motility is initiated, with the head becoming more intense and the midpiece becoming strongly labeled. Aqp8b did not change intensity or labeling pattern in the head after activation, but did relocate to the midpiece. Aqp10b also showed a strong increase in intensity in the head upon motility activation. These changes in intensity and spatial patterns of sperm plasma membrane aquaporins during motility initiation in a hypertonic medium strongly suggest that they are regulating water/fluid efflux and have a direct role in motility initiation and maintenance. Such a direct role for aquaporins in motility regulation in external spawning species needs serious consideration in other species and across phyla. Species that reproduce in environments of varying salinities [3, 27] would be of particular interest to study, as one might hypothesize that at higher salinities aquaporins may play a direct role in motility initiation; however, at lower salinities (iososmotic or hypoosmotic), other mechanisms may be responsible. Now that we know the potential importance of aquaporins in sperm motility initiation in a marine teleost from this study, the functionality and redistribution of these waterregulating permeability membrane proteins would be of great Correspondence: Gary N. Cherr, University of California Davis Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923. E-mail: gncherr@ucdavis.edu