Novel Insights Into the Mechanisms of Pregnancy Establishment
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REPRODUCTIONRESEARCH Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase Yuki Katoh1,2, Kohsuke Takebayashi1, Akihiko Kikuchi1, Ayumi Iki1, Kazuhiro Kikuchi3, Michiko Tamba1, Akihiro Kawashima1, Manabu Matsuda1 and Naomichi Okamura1,4 1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan, 2Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Ami 4669-2, Ami, Ibaraki 300-0394, Japan, 3Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan and 4Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan Correspondence should be addressed to N Okamura; Email: [email protected] Abstract Mammalian sperm must be activated in the tubal isthmus through capacitation to induce the acrosome reaction and subsequent fertilization. Although the molecular mechanisms involved in capacitation have yet to be fully elucidated, increased concentrations of reactive oxygen species (ROS) and the extent of tyrosine phosphorylation of proteins have been suggested to play central roles in the completion of capacitation. In this study, aldose reductase was for the first time identified as one of the tyrosine-phosphorylated proteins involved in the capacitation of porcine cauda epididymal sperm. Both tyrosine phosphorylation and activity of aldose reductase associated with the particulate fraction of sperm cells were significantly enhanced during capacitation. Alrestatin, a membrane- permeable and specific inhibitor of aldose reductase, plays a role in the inhibition of aldose reductase activity, elevation of intracellular levels of ROS, and induction of hyperactivated motility, all at similar dose dependencies. Alrestatin canceled both the increase in the tyrosine phosphorylation of aldose reductase and the decrease in the glutathione levels in sperm-induced during capacitation. The hyperactivated motility was induced to a higher extent in the presence of glucose than in the presence of fructose. These results indicate that aldose reductase plays an important role in induction of hyperactivation and capacitation of sperm through the elevation of ROS in sperm cells. Furthermore, aldose reductase was shown to be added to sperm during transit through the epididymis, suggesting that aldose reductase is one of the key proteins that support the functional maturation of sperm. Reproduction (2014) 148 389–401 Introduction O’Flaherty 2008). NO% is produced by NO synthases, which have been found in both the head and the flagellar Mammalian sperm require time-dependent changes in regions of sperm (Hou et al. 2008, de Lamirande et al. the female reproductive tract to acquire the capacity to %K 2009). On the other hand, the source of O2 fertilize oocytes; this acquisition is termed as ‘capaci- production has yet to be fully elucidated, although tation’ (Yanagimachi 1994, Fraser 2010, Aitken & Nixon NAD(P)H oxidase has been indicated as the enzyme 2013). Capacitation involves modulation of cell mem- responsible for its production in sperm during capaci- brane properties and signaling cascades, which results tation (Bedard & Krause 2007, Sabeur & Ball 2007, in the hyperactivation of motility and the acquisition of de Lamirande & Lamothe 2009). capacity to undergo the acrosome reaction (Gadella & The polyol pathway contributes to diabetes-induced Harrison 2000, Harrison & Gadella 2005, Salicioni et al. oxidative stress (Chung et al. 2003, Giacco & Brownlee 2007). Several studies have suggested that both gener- 2010). In particular, aldose reductase, the first and ation of reactive oxygen species (ROS) and tyrosine rate-limiting enzyme of the polyol pathway, has been phosphorylation of proteins play essential roles in the suggested to be the major contributor to hyper- process of capacitation (Visconti et al. 1995, 2002, glycemia-induced oxidative stress in nerve cells Leclerc et al. 1996, Lewis & Aitken 2001, O’Flaherty (Vikramadithyan et al. 2005). As the oviductal fluids, et al. 2006a,b, de Lamirande & O’Flaherty 2008). where sperm undergo capacitation, contain higher levels %K ROS such as superoxide anion (O2 ) and nitric oxide of glucose than of fructose (Larose et al. 2012), glucose % (NO ) are produced at the initial step of capacitation and flux through the polyol pathway is likely to increase regulate the downstream events leading to hyperactiva- in capacitating sperm, resulting in the induction of tion and the acrosome reaction (de Lamirande & oxidative stress. In preliminary experiments, aldose q 2014 Society for Reproduction and Fertility DOI: 10.1530/REP-14-0199 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/24/2021 08:30:06AM via free access 390 Y Katoh and others reductase in porcine sperm was found to be tyrosine- Sperm motility was observed under the microscope, and the phosphorylated during capacitation. This finding percentages of sperm with hyperactivated motion in all of prompted us to analyze the effects of aldose reductase the motile sperm were calculated. Sperm suspensions of 20 ml activity on capacitation. Herein, we report that tyrosine (1!105 cells/ml) prepared before and after capacitation were phosphorylation and activation of aldose reductase are placed on a prewarmed (37 8C) slide and analysis of sperm involved in porcine sperm capacitation in vitro, which motility was done using a computer-assisted sperm analysis are in turn inhibited by a specific and membrane- (CASA) system (HTM-CEROS; Hamilton Thorne Research, permeable aldose reductase inhibitor, alrestatin. Beverly, MA, USA) (Fig. 1a). Motion parameters such as curvilinear velocity (VCL; mm/s), straight-line velocity (VSL; mm/s), average path velocity (VAP; mm/s), linearity of the Materials and methods curvilinear trajectory (LIN; VSL!100/VCL), straightness (STR; VSL!100/VAP), amplitude of lateral head displacement (ALH; Animals and chemicals mm), and beat cross-frequency (Hz) were determined. For All animal experiments were approved by the Animal flagellar movement analyses, sperm motility was recorded Experiment Committee of the University of Tsukuba. by Nikon-NY-D5100 Super System (Microscope Network, Fresh porcine testes and epididymes were purchased from the Saitama, Japan) on the slide glass with 0.02 mm depth local slaughter house. Alrestatin was obtained from Enzo Life (Hamilton Thorne Research), and both the flagellar beat angle Sciences (Farmingdale, NY,USA). Lysyl endopeptidase, NADPH, (FBA) and the flagellar curvature ratio (FCR) were determined DTNB, glutathione (GSH) reductase, and 6-carboxy-20,70- according to the method of Schmidt & Kamp (2004), as shown dichlorodihydrofluorescein diacetate, diacetoxymethyl ester in Supplementary videos 1 and 2, see section on supplementary (H2DCFDA) were purchased from Wako Pure Chemical data given at the end of this article and Fig. 1b. Industries (Osaka, Japan). GSH was purchased from Tokyo Chemical Industry (Tokyo, Japan). Chlortetracycline (CTC), peroxidase-conjugated anti-mouse IgG antibody, peroxidase- Western blotting analysis of tyrosine-phosphorylated conjugated anti-rabbit IgG, Freund’s complete adjuvant, and proteins Freund’s incomplete adjuvant were obtained from Sigma– Total sperm proteins were extracted with 1% Triton X-100 in Aldrich. Protein A Sepharose 4 Fast Flow, ECL plus, and homogenizing buffer (20 mM Tris–HCl, pH 7.4, containing PreScission Protease were purchased from GE Healthcare 2.5% Protease Inhibitor Cocktail and 0.25% Phosphatase (Chalfont St Giles, Buckinghamshire, UK). Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail, and Chemi-Lumi One L (a) were obtained from Nacalai Tesque (Kyoto, Japan). A sorbitol dehydrogenase (SORD) inhibitor, CP-470,711, was kindly donated by Pfizer. All other chemicals were reagent grade. Preparation of cauda epididymal sperm and induction of capacitation Spermatozoa were collected from the porcine cauda epididy- (b) mis by air perfusion through the ductus epididymidis and washed at 20 8C with a noncapacitating medium (Non-cap medium) containing 4.8 mM KCl, 1.2 mM KH2PO4,95mM NaCl, 2 mM sodium pyruvate, and 5.56 mM glucose (pH 7.4) by centrifugation at 400 g for 5 min. The sperm pellets were washed twice more with the Non-cap medium by centrifu- gation as mentioned above. When capacitation was induced, the Cap medium: 2 mM CaCl2,0.4%BSA,and25mM NaHCO3 in the Non-cap medium, was used for washing the FCR = 0.64 y sperm pellets. The sperm forward motility was immediately y x ° determined under the microscope by the addition of Non-cap FCR = 0.91 FBA = 32 x ° medium containing 25 mM NaHCO3. Only sperm samples FBA = 117 with over 95% of the sperm showing progressive motility were used for the experiments. Sperm capacitation was induced Noncapacitated sperm Capacitated sperm according to the method reported by of Tardif et al. (2001), with slight modifications. The washed sperm were incubated either Figure 1 Analysis of sperm motility. (a) Comparison of swimming ! 6 trajectories obtained with CASA (frame rate 30 Hz, frame acquired 80) in 15 ml of Non-cap medium or in Cap medium (5 10 cells/ml) between noncapacitated (left) and capacitated sperm (right). at 37 8C for 3 h in a 5% CO2 atmosphere. Capacitated sperm (b) Representative flagellar movement of noncapacitated (left) and were assessed on the basis of the changes in the pattern of capacitated sperm (right) and determination of FBA