Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis)

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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis)

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Kuerban Tulake a,b , Xuguang Wang b , Yong Chen c , Chucai Yu b , Binyu Jing d , Heping Li a,∗ a

College of Wildlife Resources, Northeast Forestry University, Harbin, China Laboratory of Animal Reproduction Biology and Technology, College of Animal Science, Xinjiang Agricultural University, Urumqi, Xinjiang, China c Xinjiang Key Laboratory of Herbivore Nutrition for Meat & Milk Production, Xinjiang Agricultural University, Urumqi, Xinjiang, China d Xinjiang Houshi Biotechnology Co., Ltd., Korla, Xinjiang, China

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Article history: Received 16 December 2013 Received in revised form 26 November 2014 Accepted 28 November 2014 Available online xxx

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1. Introduction

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High efficiency of in vitro capacitation of deer sperm has not yet been achieved as low sperm penetration rates were reported in in vitro fertilization studies. Our main goal in this study was to identify the changes of frozen-thawed sperm of the rare red deer Tarim wapiti (Cervus elaphus yarkandensis) and detect the effect of bovine serum albumin (BSA), serum, and heparin on the protein tyrosine phosphorylation of frozen-thawed sperm. The frozen-thawed sperm of Tarim wapiti was suspended in improved modified tyrode–albumin–lactate–pyruvate medium and cultured in 5% CO2 at 38.5 ◦ C, and the status of protein tyrosine phosphorylation of sperm was detected by Western blotting. Although the results showed that the type number and expression of protein tyrosine phosphorylation of frozen-thawed wapiti sperm were decreased, the tyrosine-phosphorylated proteins such as 10, 14, 40, 47, and 55 kDa were increased significantly during the process of capacitation culture (1–2 h). In addition, tyrosine-phosphorylated proteins were promoted by BSA rather than serum, and estrus sheep serum (ESS) rather than estrus deer serum. When ESS and heparin were used together at 4 h after capacitation, four main tyrosine phosphorylation proteins (10 ± 2, 14 ± 2, 25 ± 3, and 47 ± 3 kDa) had a significantly higher expression than that at 2 h after capacitation. We demonstrated that these proteins were involved in wapiti sperm in vitro capacitation, heparin in the incubation media was necessary for the capacitation and tyrosine phosphorylation protein was promoted by ESS. © 2015 Published by Elsevier B.V.

To be able to fertilize the oocyte, sperm undergo capacitation and phosphorylation of tyrosine residues on certain sperm proteins is an essential intracellular mechanism

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∗ Corresponding author. Tel.: +86 45188245782. E-mail address: [email protected] (H. Li).

for capacitation (Yanagimachi, 1994). However, the capacitation of sperm has been described as a complex of poorly defined cellular events that occur in situ within the female reproductive tract and that are obligatory for the acrosome reaction and fertilization (Yanagimachi, 1994; Kopf and Gerton, 1991; Visconti et al., 1998). Capacitation involves sperm plasma membrane events that lead to an increased efflux of cholesterol, cellular calcium influx, fusion and vesiculation of the plasma and outer acrosomal

http://dx.doi.org/10.1016/j.anireprosci.2014.11.020 0378-4320/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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membranes, and loss of the acrosomal protein matrix in the acrosome reaction, an exocytotic event (Yanagimachi, 1994). This process is generally observed in vitro in defined 36 NaHCO3 − buffered media (Lee and Storey, 1986; Boatman 37 and Robbins, 1991; Visconti et al., 1995a, 1999) and 38 has been shown to be associated with changes in cAMP 39 metabolism (Visconti et al., 1995b, 1997) and protein tyro40 sine phosphorylation (Visconti et al., 1995b; Arcelay et al., 41 2008). 42 Protein tyrosine phosphorylation is very important to 43 the physiological process as maintenance of protein tyro44 sine phosphorylation to sperm motility force, capacitation 45 of sperm, hyperactivated motility, and acrosome reaction. 46 It has been reported in mice, humans, and many other 47 48Q4 species (Visconti and Kopf, 1998; Beverley and Aitken, 2001). Since Leyton and Saling (1989) used phosphoryla49 tion specific antibody for the first time to prove that there 50 existed tyrosine-phosphorylated proteins in the sperm of 51 mice, great progress has been made in the research on pro52 tein tyrosine phosphorylation during sperm capacitation 53 (Ficarro et al., 2003; Roy and Atreja, 2008; Signorelli et al., 54 2012). 55 There is substantial evidence that cryopreservation pro56 motes the capacitation of sperm (Watson, 2000; Bailey 57 et al., 2000) and this cryocapacitation is frequently cited 58 as one factor associated with the reduced longevity of 59 cryopreserved sperm in the female reproductive tract 60 (Bailey et al., 2000). Induction of cryocapacitation has been 61 attributed to an increase in intracellular calcium associ62 ated with membrane damage (Bailey and Buhr, 1994). The 63 reduced longevity of sperm in the female reproductive tract 64 is a major barrier to the application of frozen sperm in the 65 deer (Asher et al., 2000). Preserving sperm function follow66 ing cooled or frozen storage is essential to maintain the 67 optimum fertility. More and more researchers believe that 68 serum is an essential factor in the system of in vitro fertil69 ization (IVF), and many have found a stable and high level 70 of sheep embryos cleavage, frozen-thawed sperm capacita71 tion, and fertilization after addition of estrus sheep serum 72 (ESS) (Huneau et al., 1994; Dattena et al., 2000; Berg et al., 73 2002). 74 Tarim wapiti (Cervus elaphus yarkandensis) is a unique 75 subspecies of red deer in Xinjiang Uygur Autonomous 76 Region in China and mainly distributed in Tarim River and 77 its coastal tributaries. It is the only subspecies among many 78 red deer subspecies living in desert landscape and was 79 included in the Red List (1994) of the International Union 80 for Conservation of Nature and Natural Resources (IUCN) 81 in the category of an endangered species and classified as 82 a second class protected animal in China (Mahmut et al., 83 2001). 84 In various mammalian species, rather than Tarim 85 wapiti, it has been well documented that capacitation 86 appears to be associated with tyrosine phosphorylation of 87 sperm proteins of rats (Visconti and Kopf, 1998) and mice 88 89Q5 (Beverley and Aitken, 2001). In this study, frozen-thawed sperm of Tarim wapiti were cultured and analyzed by 90 sodium dodecyl sulfate polyacrylamide gel electrophoresis 91 (SDS–PAGE). The in vitro sperm capacitation was analyzed 92 by Western blotting to detect the expression of tyrosine 93 phosphorylation for studying the molecular mechanism 94 34 35

and the relationship of the protein tyrosine phosphorylation and wapiti sperm capacitation.

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2. Materials and methods

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2.1. Ethics statement

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Animal experiments were approved by Animal Ethics Committee of Xinjiang Agricultural University, China and performed in accordance with animal ethical guidelines and approved protocols. 2.2. Chemical reagents and equipment The reagents included andromed® medium (egg yolkfree concentrated medium for bull semen, Minitube, Tiefenbach, Germany), Heparin (Cat. H3149; Sigma, St. Louis, MO, USA), bovine serum albumin (BSA; Cat. A7030; Sigma, St. Louis, MO, USA), sodium pyruvate (Cat. P5280; Sigma, St. Louis, MO, USA), BCA Protein Assay Kit (Cat. 23225; Pierce, Rockford, IL, USA), BioTraceTM NT Nitrocellulose Transfer Membrane (Cat. 66485; Pall, Pensacola, USA), PM Western Midview (Cat. CW0021; Beijing ComWin Biotech Co., Lt, Beijing, China), Phospho-tyrosine Mouse mAb (P-Tyr-100) (Cat. 9411; Cell Signaling, New England Biolabs, MA, USA), DyLightTM 800-labeled antibody to mouse IgG (H+L) (Cat. 042-07-18-06; KPL, Gaithersburg, MD, USA). All other biochemical reagents used were analytically pure. The main instruments included a CO2 incubator (WH-4500, Germany), an inverted microscope (Olympus IX71), an upright fluorescence microscope (Olympus BH2), a vertical electrophoresis tank and transfer instrument (Bio-rad), Li-Cor’s Odyssey Infrared Imaging System (LICOR Biosciences, Lincoln, NE, USA), and a low temperature high speed centrifuge (Eppendorf-5810 R). 2.3. Experimental animals, semen collection, and cryopreservation The experimental sperm was derived from five healthy Tarim wapiti (5–8 years of age), which were maintained at the Xinjiang Houshi Biotechnology Co., Ltd. according to the Tarim wapiti breeding standards. They grazed and drank water freely. Semen was collected as described previously (MartínezPastor et al., 2006). The average semen volume per ejaculate was 1.5 mL. Percentage of individual motile sperm (motility) was observed and the quality of motility was assessed using a scale of 0–5. Sperm Motility Index [SMI = % individual motility + (quality of motility × 20) × 0.5] was calculated using the method by Comizzoli et al. (2001). Sperm concentration was estimated using a hemocytometer. Total sperm number was calculated using the actual volume of sperm collected. After assessment of sperm concentration, the sperm-rich fraction was adjusted to 50–100 × 106 sperm/mL in the appropriate pre-warmed semen diluent (Andromed® medium, Minitüb, Germany). Diluted semen samples in sterile 10 mL centrifuge tubes were wrapped with eight layers of gauze and transported to the laboratory in an icebox within 5 h.

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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washed with TBST four times for 10 min, incubated with DyLightTM 800-labeled anti-mouse IgG (H+L) at room temperature (25 ◦ C) for 1 h, and washed 4× 10 min with TBST and 2× 5 min with PBS. The NC membrane was dried at room temperature and scanned using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The tyrosine phosphorylated protein conversion was quantified using Odyssey Imaging System.

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The samples were divided into two groups, one for fresh samples and the other for storage in the liquid nitrogen. Diluted samples at 4 ◦ C were loaded into 0.25 mL straws (50 × 106 sperm/mL) and semen in Styrofoam box (200 mm × 265 mm × 350 mm) was frozen at a distance of 3.5 cm above the liquid nitrogen surface for 10 min. The straws were placed in liquid nitrogen vapor and then transferred to liquid nitrogen containers. Sperm frozen in the liquid nitrogen for more than 2 weeks was thawed at 37 ◦ C for 30 s for further experiment.

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2.4. Preparations of animal sera

Experiment 1: Comparison of the membrane protein tyrosine phosphorylation of fresh and frozen-thawed wapiti sperm The semen sample was subjected to a swim-up technique to separate the progressively motile sperm from non-motile sperm. The fresh and frozen-thawed sperm samples were transferred into 5 mL round bottom centrifuge tubes, 1:3 with sp-TALP (100 mM NaCl, 3.10 mM KCl, 0.29 mM NaH2 PO4 , 21.6 mM sodium lactate, 2.1 mM CaCl2 , 0.4 mM MgCl2 , 10.0 mM HEPES, 25 mM NaHCO3 , and 1.0 mM pyruvate) containing 6 mg/mL BSA was slowly added along the wall, and incubated for 40 min in a humidified atmosphere of 5% CO2 at 38.5 ◦ C. The precipitate was collected after centrifugation at room temperature at 275 × g for 5 min and subjected to washes by resuspension with 3 mL of sp-TALP and centrifugation at 275 × g for 5 min. Sperm were diluted to 50 × 106 cells/mL with the fert-TALP (114 mM NaCl, 3.15 mM KCl, 0.4 mM NaH2 PO4 , 9.0 mM sodium lactate, 2.1 mM CaCl2 , 0.5 mM MgCl2 , 25 mM NaHCO3 , and 0.09 mM pyruvate) containing 6 mg/mL BSA and 20 ␮g/mL heparin, and incubated in the CO2 incubator. The sperm membrane proteins were extracted and stored at −20 ◦ C for use in Western blotting.

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ESS: Blood was sampled from the jugular vein of the sheep during the natural estrus on the second afternoon after mating. NSS: Blood from the non-estrus sheep was sampled from the jugular vein in the breeding season. EDS: In the breeding season blood was collected from the jugular vein of each Tarim wapiti. After collection blood was allowed to stand at 4 ◦ C for 4 h,and the supernatant was collected, filter sterilized, packed, and stored at −20 ◦ C.

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2.5. Extraction of sperm membrane proteins

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Sperm membrane proteins were extracted by using non-ionic detergent NP-40 (Beyotime Institute of Biotechnology, Beyotime, Jiangsu, China) lysis method. At different processing time points (0, 1, 2, and 4 h) 1.8 mL (50 × 106 cells/mL) the extracted sperm suspension was centrifugated at 420 × g at 4 ◦ C for 10 min, washed with 0.01 M PBS by centrifugation 3 times at 420 × g at 4 ◦ C for 10 min, washed with 0.01 M Tris–HCL by centrifugation 3 times at 420 × g at 4 ◦ C for 10 min, and then resuspended in 120 ␮L of suspension solution containing 1% NP-40 membrane protein lysis buffer (0.1 M Tris, 1% NP-40, 1% SDS, 119.8 mM NaCl, 0.32 mM MgCl2 , 1 mM EDTA-Na2 , 1 mM phenylmethylsulfonyl fluoride, pH 8.0). After being lysed in the incubator (at 0–4 ◦ C) with a shaker for 90 min, the sperm lysates were centrifuged at 10,000 × g at 4 ◦ C for 15 min. The supernatant was transferred to a new tube containing 45 ␮L of 5× SDS buffer solution, incubated for 5 min in boiling water, and stored at −20 ◦ C. The sperm membrane protein samples were thawed at 4 ◦ C. The protein amount was measured using a BCA protein analysis kit (Pierce Biotechnology Inc., Rockford, IL, USA). 2.6. Western blotting A total of about 25 ␮L (about 10 ␮g) of sperm membrane protein sample was thawed at 4 ◦ C and loaded onto an SDS–PAGE on 12% of separation gel and PM Western Midview (Beijing Cowin Biotech Co., Ltd., Beijing, China) was used as the protein marker with molecular weights of 22, 40, 60, 85, and 120 kDa. The proteins on the gel were transferred onto the BioTraceTM NT Nitrocellulose Transfer Membrane (Cat. 66485; PALL Corporation, Pensacola, FL, USA) at 60 V at 4 ◦ C for 2 h. The membrane was blocked with 1% BSA/TPBS at room temperature for 1 h. The first antibody Phospho-tyrosine Mouse mAb (P-Tyr-100) (Cat. 9411; Cell Signaling Technology, Boston, MA, USA) was added and incubated at 4 ◦ C overnight. The membrane was

2.7. Experimental design and in vitro sperm capacitation

Experiment 2: The effects of the BSA and serum on the membrane protein tyrosine phosphorylation of frozen-thawed wapiti sperm The frozen-thawed sperm was washed with 1:3 sp-TALP (BSA free), centrifugated at room temperature at 275 × g for 5 min twice, and resuspended with Fert-TALP containing 20% different serum (ESS, NSS, and EDS) and 0, 3, 6, and 10 mg/mL BSA, respectively. Sperm concentration was adjusted to 50 × 106 cells/mL and incubated in the CO2 incubator for 0 (without incubation) and 2 h. The sperm membrane proteins were extracted and stored at −20 ◦ C for use in Western blotting. Experiment 3: The effects of the BSA and serum with heparin on the membrane protein tyrosine phosphorylation of frozen-thawed wapiti sperm Frozen-thawed sperm was washed with 1:3 sp-TALP (BSA free), centrifugated at room temperature at 275 × g for 5 min twice, and resuspended in Fert-TALP containing 6 mg/mL BSA and 20% ESS as well as 20 ␮g/mL heparin. Sperm were adjusted to 50 × 106 cells/mL and incubated in the CO2 incubator for 0 (without incubation), 2, and 4 h. Sperm membrane proteins were extracted and stored at −20 ◦ C for use in Western blotting to quantify tyrosine phosphorylated protein conversion as described above.

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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2.8. Assessment of sperm capacitation

The methods used to prepare spermatozoa for the chlortetracycline (CTC) assessment were essentially the 262 Q6 same as those described for bull spermatozoa by Fraser and 263 Abeydeera, 1995 and for boar spermatozoa by Wang et al. 264 (1995). 265 The three patterns observed were: uniform fluorescence 266 over the whole head (F); fluorescence-free band in the 267 post acrosomal region (B); almost no fluorescence over the 268 whole head except for a thin band of fluorescence in the 269 equatorial segment (AR). 260 261

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Data were presented as mean ± SEM. All data were analyzed using the SPSS 13.0 package software. (SPSS Inc., Chicago, IL). Data were analyzed by 2 (chi-square test) and statistical differences between the various treatment groups were compared. A difference with a value P < 0.05 was considered statistically significant.

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3. Result

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3.1. Experiment 1

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Comparison of the changes of sperm motility and capacitation rate in fresh and frozen-thawed Tarim wapiti sperm capacitation The motility of the fresh sperm was significantly higher than that of the frozen-thawed sperm before capacitation (0 h) (2 = 8.2, P < 0.05). After swim-up the motility of sperm in both groups significantly increased (2 = 6.5,

Fig. 1. Changes of sperm motility in fresh and frozen-thawed Tarim wapiti sperm during capacitation. () Fresh sperm, () frozen-thawed sperm. Bars with different letters indicate significant differences (P < 0.05).

P < 0.05), but decreased with the culture time. At 4 h after capacitation, the motility of the fresh sperm was significantly higher than that of the frozen-thawed sperm (0 h) (2 = 4.2, P < 0.05) (Fig. 1). The percentage of the frozen-thawed sperm with the B pattern was significantly higher than that of the fresh sperm before capacitation (0 h) (2 = 4.1, P < 0.05). After swim-up and capacitation, the percentage of the sperm with AR pattern in both groups gradually increased, and peaked at 2 and 4 h in the frozen-thawed group and in the fresh group, respectively. At 4 h after capacitation, the percentage of the sperm with AR in the frozen-thawed group was significantly higher than that of the fresh group (2 = 5.8, P < 0.05) (Fig. 2).

Fig. 2. Changes of capacitation rate percentages of the fresh and frozen-thawed Tarim wapiti sperm during capacitation. () Fresh sperm, () frozenthawed sperm. B pattern: capacitated sperm, AR pattern: acrosome-reacted sperm. The same letters in the same column indicate no significant differences (P > 0.05) whereas the different letters indicate significant differences (P < 0.05).

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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Fig. 3. Western blot analysis of tyrosine phosphorylated proteins during in vitro capacitation of fresh (A) and frozen-thawed (B) Tarim wapiti sperm. Sperm were washed and swim-up conducted in a modified sp-TALP containing 6 mg/mL BSA. Sperm capacitation was conducted in an modified fert-TALP containing 6 mg/mL BSA and 20 ug/mL Heparin in 5% CO2 incubator at 38.5 ◦ C for 1, 2, and 4 h, respectively. Sperm membrane protein samples were subjected to SDS–PAGE followed by transfer onto a nitrocellulose transfer membrane. Blots were first incubated with Phospho-tyrosine Mouse mAb (P-Tyr-100), and then with DyLightTM 800-labeled anti-mouse IgG (H+L). Mr, molecular mass.

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Comparison of the changes of protein tyrosine phosphorylation in fresh and frozen-thawed Tarim wapiti sperm capacitation As shown from the Western blot analysis (Fig. 3), three main tyrosine phosphorylated proteins (10, 40, and 47 kDa) were detected in the fresh sperm without incubation, whereas two main tyrosine phosphorylated proteins (40 and 47 kDa) were detected in the frozen-thawed sperm without incubation. Five main tyrosine phosphorylated proteins (10, 14, 40, 47, and 55 kDa) were detected during capacitation, and the levels of tyrosine phosphorylated proteins in the fresh sperm were higher than those of the frozen-thawed sperm during the first 1–2 h of capacitation (2 = 4.5, P < 0.05).

3.2. Experiment 2 Effects of different BSA concentrations on capacitation rates of the frozen-thawed Tarim wapiti sperm There was no significant difference between the percentages of the sperm with motility and AR pattern (2 = 0.31, P > 0.05) among the groups before capacitation (0 h). At 2 h after capacitation, the percentages of sperm with AR pattern in the treatment groups with 6 and 10 mg/mL of BSA were significantly higher than those in the groups with 0 and 3 mg/mL of BSA (2 = 5.1, P < 0.05), but there was no significant difference between the two groups with 0 and 3 mg/mL of BSA (2 = 0.25, P > 0.05). The percentages of the sperm with AR pattern in the treatment

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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were detected before capacitation, and four main tyrosine phosphorylated proteins (14, 40, 47, and 55 kDa) were detected at the end of capacitation. The expressions of the four proteins after capacitation (2 h) increased significantly (2 = 8.89, P < 0.05) compared with those before capacitation (0 h). The expression of 55 kDa tyrosine phosphorylated protein in two groups (the 6 and 10 mg/mL BSA) was significantly higher than that of 3 mg/mL BSA group and control group (without BSA) (2 = 5.58, P < 0.05). There was no significant difference of expression between the 6 mg/mL BSA group and the 10 mg/mL BSA group or between the 3 mg/mL BSA group and the control group (without BSA) (2 = 0.818, P > 0.05).

Fig. 4. Effects of different BSA concentrations on the capacitation rates of frozen-thawed Tarim wapiti sperm. The same letters in the same column indicate no significant differences (P > 0.05) whereas the different letters indicate significant differences (P < 0.05) (B pattern, capacitated sperm; AR pattern, acrosome-reacted sperm). 327 328 329 330

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groups were significantly higher than that of the control group (2 = 4.8, P < 0.05), but there were no significant differences among the treatment groups (2 = 0.21, P > 0.05) (Fig. 4). Effects of different BSA concentrations on protein tyrosine phosphorylation of frozen-thawed Tarim wapiti sperm As shown in the Western blot analysis (Fig. 5), two main tyrosine phosphorylated proteins (40 and 47 kDa)

Effect of different serum on capacitation rate of frozen-thawed Tarim wapiti sperm There was no significant difference between the percentages of the sperm with motility and AR pattern (2 = 0.24, P > 0.05) before capacitation (0 h). At 2 h after capacitation, the percentages of sperm with motility and AR pattern in the serum groups were significantly higher than that of the control (2 = 7.8, P < 0.05); The ESS group had the highest capacitated sperm percentage, which was significantly higher than those of NSS and EDS groups; There was no significant difference between the NSS and the EDS groups (2 = 0.26, P > 0.05) and there was also no significant difference in the percentage of sperm with AR pattern among the serum treatment groups (2 = 0.14, P > 0.05) (Fig. 6).

Fig. 5. Effects of different BSA concentrations (0, 3, 6, and 10 mg/mL) on protein tyrosine phosphorylation of the frozen-thawed Tarim wapiti sperm. Sperm were washed in a modified sp-TALP (devoid BSA) and sperm capacitation was conducted in a modified fert-TALP with 0, 3, 6, and 10 mg/mL of BSA in 5% CO2 incubator at 38.5 ◦ C for 0 h (without incubation) and 2 h. Sperm membrane protein samples were subjected to SDS–PAGE followed by transfer onto a nitrocellulose transfer membrane. Blots were first incubated with Phospho-tyrosine Mouse mAb (P-Tyr-100), and then with DyLightTM 800-labeled anti-mouse IgG (H+L). Mr, molecular mass.

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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higher than those of the EDS group and the control (without serum) group (0 h) (2 = 6.69, P < 0.05), but there were no significant differences between the ESS group and the NSS group or between the EDS group and the control group (2 = 0.156, P > 0.05). 3.3. Experiment 3

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Fig. 6. Effects of different sera on the capacitation rates of frozen-thawed Tarim wapiti sperm. The same letters in the same column indicate no significant differences (P > 0.05) whereas the different letters indicate significant differences (P < 0.05) (B pattern, capacitated sperm; AR pattern, acrosome-reacted sperm).

Effects of BSA, ESS, and heparin (Hep) on capacitation rate of frozen-thawed Tarim wapiti sperm The percentages of the capacitated sperm and sperm with AR pattern at 2 h after capacitation were significantly higher than those before capacitation (0 h) (2 = 3.6, P < 0.05) when either BSA or ESS was used, or was combined with hepparin. There was no significant difference between the treatment groups (2 = 0.26, P > 0.05). At 4 h after capacitation, the percentages of the capacitated sperm in the ESS group and in the ESS + Hep group were significantly higher than those in the BSA group and in the ESS + Hep group (2 = 3.4, P < 0.05) (Fig. 8).

Effect of different serum on tyrosine protein phosphorylation of frozen-thawed Tarim wapiti sperm As shown in the Western blot analysis (Fig. 7), two main tyrosine phosphorylated proteins (40 and 47 kDa) were detected before capacitation (0 h), and four main tyrosine phosphorylated proteins (14, 40, 47, and 55 kDa) were detected at the end of capacitation (2 h). The expressions of these four main proteins increased significantly (2 = 8.5, P < 0.05) compared with those before capacitation (0 h). The expression of these tyrosine phosphorylated proteins in the ESS group and the NSS group were significantly

Effects of BSA, ESS, and heparin (Hep) on protein tyrosine phosphorylation of frozen-thawed Tarim wapiti sperm As shown in the Western blot (Fig. 9), the expression of tyrosine phosphorylated proteins in every single group increased during the process of capacitation. When only BSA was added, the expression of 10 ± 2 kDa tyrosine phosphorylated protein at 4 h after capacitated increased significantly than that at 2 h after capacitated (2 = 4.65, P < 0.05). There were no significant changes in the expression of other tyrosine phosphorylated proteins (2 = 0.096, P > 0.05). When only ESS was added, expressions of the

Fig. 7. Effects of different sera (estrus sheep serum: ESS; non-estrus sheep serum: NSS; and estrus Deer serum: EDS) on protein tyrosine phosphorylation of frozen-thawed Tarim wapiti sperm. Sperm was washed in a modified sp-TALP (devoid BSA) and sperm capacitation was conducted in a modified fert-TALP with different serum (ESS, NSS, and EDS) in a 5% CO2 incubator at 38.5 ◦ C for 0 h (without incubation) and 2 h (2 h after capacitation). Sperm membrane protein samples were subjected to SDS–PAGE followed by transfer onto a nitrocellulose transfer membrane. Blots were first incubated with Phospho-tyrosine Mouse mAb (P-Tyr-100), and then with DyLightTM 800-labeled anti-mouse IgG (H+L). Mr, molecular mass.

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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four main tyrosine phosphorylated proteins (10 ± 2, 14 ± 2, 25 ± 3, and 47 ± 3 kDa) were significantly higher at 4 h after capacitation than those at 2 h after capacitation (2 = 7.45, P < 0.05), but there was no significant difference between the combined use of BSA and heparin, or the ESS only. When ESS and heparin were used together at 4 h after capacitation, four main tyrosine phosphorylated proteins (10 ± 2, 14 ± 2, 25 ± 3, and 47 ± 3 kDa) had a significantly higher expression than that at 2 h after capacitated (2 = 9.15, P < 0.05). Moreover, the high molecular weight proteins of 85–120 kDa were also detected.

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4. Discussion

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4.1. Sperm motility and capacitation in fresh and frozen-thawed sperm Sperm swim-up is widely used for effectively collecting highly dynamic sperm in the in vitro sperm capacitation system. Sperm capacitation process accelerates sperm metabolism, hyperactivates the motion, capacitates sperm, acrosome reaction eventually occurs, sperm motility decreases, and then dies (Harrison et al., 1996). Sperm capacitation can be time-dependent (Nimmo and Cross, 2003), and the final outcome is acrosome reaction. Acrosome reaction is induced when sperm contact the egg zona pellucida in vivo. However, sperm acrosome reaction during in vitro fertilization process also occurs in the absence of the zona pellucida conditions even though the mechanism of this spontaneous acrosome reaction is not clear yet. In this study, the motility of Tarim red deer fresh and frozen-thawed sperm through swim-up significantly increased. Sperm motility peaked during 1–2 h of capacitation, and then gradually decreased with the motility of the frozen-thawed sperm dropping faster. Based on

Fig. 8. Effects of BSA, ESS, and heparin (Hep) on the capacitation rates of frozen-thawed Tarim wapiti sperm. The same letters in the same column indicate no significant differences (P > 0.05) whereas the different letters indicate significant differences (P < 0.05) (B pattern, capacitated sperm; AR pattern, acrosome-reacted sperm).

the percentage changes of the sperm capacitation and acrosome reaction, Tarim red deer sperm capacitation is time-dependent. The capacitation rate of the frozenthawed sperm peaked between 1 and 2 h of capacitation while the fresh sperm achieved the best state at 4 h or more of capacitation. The frozen-thawed sperm achieved capacitation at least 2 h ahead of the fresh sperm, which may be because the membrane of the frozen-thawed sperm was damaged and therefore spontaneous acrosome reactions were induced. In addition, the BSA and heparin in the in vitro culture system promote the loss of sperm membrane cholesterol, increase intracellular calcium, cAMP concentration and protein phosphorylation, and increase intracellular pH value, which further validates the above findings.

Fig. 9. Effects of BSA, estrus sheep serum (ESS), and heparin (Hep) on protein tyrosine phosphorylation of frozen-thawed Tarim wapiti sperm. Sperm was washed in a modified sp-TALP (devoid BSA) and sperm capacitation was conducted in a modified fert-TALP containing BSA, estrus sheep serum, and heparin in 5% CO2 incubator at 38.5 ◦ C at 0 (without incubation), 2, and 4 h (after capacitation). Sperm membrane protein samples were subjected to SDS–PAGE followed by transfer onto a nitrocellulose transfer membrane. Blots were first incubated with Phospho-tyrosine Mouse mAb (P-Tyr-100), and then with DyLightTM 800-labeled anti-mouse IgG (H+L). Mr, molecular mass.

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4.2. Effects of BSA and sera on capacitation of frozen-thawed wapiti sperm

BSA is an important macromolecule affecting sperm capacitation and fertilization and is widely used in sperm 454 capacitation. Previous studies have shown that many 455 species require BSA for capacitation (Go and Wolf, 1985; 456 Langlais and Roberts, 1985), but Fraser (1985) believed that 457 BSA is mainly needed in the acrosome reaction and not 458 in the process of capacitation. In this study, a low con459 centration (3 mg/mL) of BSA had little effect on Tarim red 460 deer sperm capacitation, but at certain concentrations (6, 461 10 mg/mL) of BSA prompted the sperm capacitation rate. 462 Different concentrations of BSA prompted the sperm acro463 some reaction rates, which is consistent with the findings 464 by Fraser (1985). 465 In the fertilization studies, even though serum is added 466 into sperm capacitation solutions and semen to enable bet467 ter in vitro embryo development, there are inconsistent 468 reports on the serum added and serum collected at differ469 ent stages of the estrous cycle. By adding 20% estrus sheep 470 serum into the in vitro fertilization system, 20% blastocyst 471 rates were obtained in red deer and sika deer (Comizzoli 472 et al., 2001), and 30% blastocyst rates obtained in sika deer 473 Q7 (Locatelli et al., 2012). In vitro fertilization rate after adding 474 20% sheep serum collected 1.5 days of the estrus cycle was 475 significantly higher than those after adding 20% deer serum 476 collected on the day of estrus and 1.5 days of the estrus cycle 477 (Berg et al., 1995). 478 In this study, by adding ESS for 1.5 days, NSS, and 479 EDS into the capitation solutions, the capitation rates 480 and AR percentages increased significantly compared to 481 the control group, with ESS inducing the highest per482 centage of sperm capitation. This is in agreement with 483 Berg’s results (Berg et al., 1995, 2002). This is probably 484 because sheep serum contained rich cell growth factor, 485 estrogen, FSH, and other substances. These substances 486 may be involved in regulating sperm capacitation and 487 sperm protein tyrosine phosphorylation process. Huneau 488 et al. (1994) demonstrated that estrus sheep serum was 489 beneficial to sperm-egg interactions and maintaining the 490 “trans-activation motion” sperm after the acrosome reac491 tion, to prevent hardening of the zona pellucida of oocytes, 492 which is advantageously supported by the results of this 493 study. The reason why the effect of estrus deer serum was 494 not as good as estrus sheep serum and its mechanism are 495 unknown. 496 453

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4.3. Effects of BSA, ESS, and heparin on capacitation of the frozen-thawed sperm After Parrish et al. (1984) first reported successful in vitro capacitation of bovine sperm using heparin, heparin has been used to successfully induce in vitro sperm capacitation in goats (Younis et al., 1991), sheep (Slavik et al., 1992), and deer (Pollard et al., 1995; Berg et al., 2002). There are no reports on combined application of estrus sheep serum and heparin in sperm capacitation even though capacitation was induced with 20 ␮g/mL of heparin (Berg et al., 2002). In this study, we investigated the effects of the BSA and estrus sheep serum on in vitro sperm

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capacitation with or without heparin. These results showed that estrus sheep serum alone or in combination with heparin induced significantly higher sperm capacitation than BSA alone or in combination with heparin, and estrus sheep serum used in combination with heparin induced better sperm capacitation than either of them. Therefore, estrus sheep serum used in combination with heparin induced effective in vitro sperm capacitation in Tarim red deer. 4.4. Changes of protein tyrosine phosphorylation of fresh and frozen-thawed sperm Freezing/thawing can cause damage to the sperm proteins and harm the sperm vitality. Preserved semen often has decreased sperm motility and fertility that can vary widely between individual stallions (Bedford et al., 1995). It was found that bull sperm protein changes before and after freezing and thawing (Lessard et al., 2000; Martin et al., 2007). In this study, after freeze–thaw cycles, the number of tyrosine phosphorylated proteins increased, and five main tyrosine phosphorylated proteins (10, 14, 40, 47, and 55 kDa) were detected during capacitation as well as the tyrosine phosphorylated proteins (14, 40, 47, and 55 kDa) expressed in swine (Flesch et al., 1999; Bucci et al., 2012; Gonza´ılez-Ferna´ındez et al., 2009), buffalo (Kumar and Atreja, 2012), bovine (Bucci et al., 2012), and sheep (Grasa et al., 2009; Leahya et al., 2011). These plasma membrane proteins are thought to play an important role in inducing capacitation and acrosome reaction. The number and expression of tyrosine phosphorylated proteins of frozen-thawed wapiti sperm decreased. The tyrosine phosphorylated proteins such as 10, 14, 40, 47, and 55 kDa were detected during the capacitation, especially the 55 kDa protein tyrosine phosphorylation level increased significantly. The samples used in this experiment were not very fresh after 5 h transportation and sperm cryocapacitation might have partly occurred during the 5 h transportation to the laboratory at 0–4◦ C. The dramatic changes of the way sperm moves and sharply irregular movement of the flagella led to speculation that the protein of molecular weight 55 kDa may be involved in sperm activation and movement regulation (Vijayaraghavan et al., 1997). The 55 kDa tyrosine phosphorylated protein on the capacitation of bovine sperm was also found and its phosphorylation is associated with the fibrous sheath of sperm (Dube et al., 2005). Further research may be needed to prove whether the 55 kDa protein of wapiti sperm is related to A-kinase anchoring proteins (AKAPs). 4.5. Effects of BSA and serum on tyrosine phosphorylation of frozen-thawed sperm BSA is a cholesterol acceptor. Removal of the cholesterol results in elevated fluidity of the membrane, allows changes in intracellular calcium concentration and pH etc, promotes cAMP and protein tyrosine phosphorylation, and ultimately the sperm is capacitated (Huang et al., 2005). In this study addition of BSA in the capacitation solution promoted the sperm protein tyrosine phosphorylation. The expression of 55 kDa tyrosine phosphorylated protein was

Please cite this article in press as: Tulake, K., et al., Protein tyrosine phosphorylation during capacitation in sperm of a rare red deer, Tarim wapiti (Cervus elaphus yarkandensis). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.11.020

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significantly higher when BSA concentration was 6 mg/mL or more. Serum is frequently used in in vitro fertilization system in mammals. It has been reported that the sheep serum plays an important role in the process of IVF (Huneau et al., 1994; Dattena et al., 2000). Berg et al. (2002) argued that in vitro fertilization rate of ESS was obviously higher than EDS, but the mechanism is unclear. From the results in this study, addition of serum increased protein tyrosine phosphorylation of wapiti sperm, which is similar to the function of BSA, but the effect of BSA was not obvious when serum was present. In addition, similar results were obtained when either BSA, heparin, or ESS was used alone. However, at 4 h after capacitation, some tyrosine phosphorylated proteins with low molecular weight (10 ± 2, 14 ± 2, 25 ± 3, and 47 ± 3 kDa) and with high molecular weight (85–120 kDa) were also detected and increased significantly when ESS and heparin were used compared with the control group. In summary, both BSA and heparin in the incubation media are necessary for the capacitation-associated protein tyrosine phosphorylation, which is promoted by ESS. This may be because sheep serum is rich in pregnenolone and estrous estrogen. These substances may be involved in the process of sperm capacitation and the regulation of protein tyrosine phosphorylation, which may need further investigation. Conflict of interest The authors declare no conflict of interest. Acknowledgments

This work was partially supported by a grant from Q8 Scientific Research Program of the Xinjiang Uygur 597 Autonomous Region Natural Science Foundation (No. 598 2013211A029) and Higher Education Institution of Xin599 Jiang “FSRPHEXJ” (No. XJEDU2010I24). We thank Wapiti 600 Domesticating & Breeding Base of Xinjiang Houshi Biotech601 nology Co., Ltd., Xinjiang Changji Shenghua Trading Co., 602 Ltd., and related personnel who gave great support and help 603 to this project. 604 596

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