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Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation
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Theriogenology 69 (2008) 925–931 www.theriojournal.com
Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation§ S. Imam a, M.R. Ansari a, N. Ahmed b, A. Kumaresan c,* a
Artificial Insemination Laboratory, Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India b Protein Research Laboratory, Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India c Division of Animal Production, ICAR Research complex for NEH Region, Barapani, Umiam, Meghalaya 793 103, India Received 30 September 2006; received in revised form 24 May 2007; accepted 28 May 2007
Abstract The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS–PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen–thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P < 0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P < 0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity. # 2008 Elsevier Inc. All rights reserved. Keywords: Oviductal proteins; Heparin-Sapharose fractionation: Sperm functions; Cryopreservation; Buffalo
1. Introduction Sperm survive and remain functionally active for >20 h in oviduct [1], whereas there is substantial
§
Part of the thesis submitted by the first author to the Deemed University, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India. * Corresponding author. Tel.: +91 9436143226; fax: +91 364 2570362. E-mail address: [email protected] (A. Kumaresan). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.066
deterioration of sperm quality after as little as 1 h incubation in vitro at 37 8C [2]. Therefore, the oviduct may secrete factors that protect sperm functional integrity. The epithelial lining of the oviduct and its secretory products may influence the functions of sperm, ovum and embryo [3]. Oviductal fluid consists of serum transudate [4] and a secretory product of the oviductal epithelium [5,6]. Several workers have reported that a number of proteins are secreted from the oviduct, which promote viability of sperm [7], prolong sperm motility [8], and protect membrane
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integrity [9,10]. Oviductal proteins were also reported to induce sperm capacitation [7,11,12]. Attempts have been made to purify proteins of oviductal fluid of mammals; an estrus-associated protein (85–95 kDa) was purified by salt fractionation and ion-exchange chromatography [13]. Variations in the effects of oviductal proteins on sperm functions in relation to the stage of the estrous cycle at which the oviductal proteins were obtained have already been reported [10]. Proteins obtained from oviducts collected during the nonluteal phase improved sperm function during cryopreservation, whereas proteins from luteal oviducts depressed sperm motility [10,12]. However, most of the above-mentioned studies utilized whole oviductal proteins. Despite the potential importance of oviductal fluid proteins in reproductive processes, there are limited data regarding fractionated oviductal fluid proteins and their effects on motility, viability and membrane integrity of sperm during cryopreservation, especially in buffaloes. Perhaps different fractions of the oviductal proteins exhibit different actions on sperm characteristics. As most of the proteins secreted by the oviduct are glycoprotein in nature [14], they can be fractionated based on their binding property with heparin. Heparin is a sulphated glycosaminoglycon, consisting mainly of repeating diasaccharide sequences of a-L-idopyranuronic acid 2sulphate and 2-deoxy-2-sulphamino-a-D-glucopyranose-6-phosphate, linked through Positions 1 and 4, which support its binding with glycoproteins [15]. Therefore, the objective of the present study was to determine the effect of Heparin-Sepharose bound and unbound oviductal proteins on sperm functions during cryopreservation. 2. Materials and methods 2.1. Chemicals and reagents D-fructose, sodium chloride, sodium dihydrogen orthophosphate, sodium phosphate dibasic, trisodium citrate, sodium azide, isopropanol, glycine, methanol, acetic acid, HCl and bromophenol blue (GR grade) were purchased from Merck, New Delhi, India. Tris, EDTA, acrylamide (3 crystalised), agarose, ammonium persulfate, and sodium lauryl sulphate (molecular biology grade) were purchased from Sisco Research Laboratory, Mumbai, India. Electrophoresis chemicals (bis-acrylamide, coomassie Brillant Blue R-250 and TEMED), Protease inhibitor (PMSF) and HeparinSepharose column were purchased from Sigma– Aldrich, St. Louis, MO, USA. Protein molecular
markers were purchased from Bangalore Genei. Minisart filters (0.20 mm) were obtained from Sartorius, Bangalore, India, and dialysis tubing were from Gibco BRL, New Delhi, India. Glass triple-distilled water was used throughout the study. 2.2. Preparation of oviductal fluid proteins Buffalo oviducts and ovaries were collected from apparently healthy animals slaughtered at the National Buffalo Abbatoir, Bareilly, India and brought to the laboratory on ice immediately after slaughter. Upon arrival, ovarian morphology was examined and the phase of the estrous cycle was determined. Only oviducts from buffalo that were not in a luteal phase (n = 190) were utilized for the present study [10]. The oviducts were dissected free of the surrounding connective tissues and fimbria and rinsed with chilled phosphate buffer saline (PBS) containing phosphate buffer (10 mM, pH 7.2), sodium azide (0.03%), EDTA (5 mM), PMSF (2 mM), penicillin (120 mg/mL) and streptomycin (200 mg/mL). Flushing was done at 4 8C from the isthmic end of the oviduct with PBS containing sodium azide, EDTA, and PMSF. The collected fluid was centrifuged at 10,000 g for 20 min at 4 8C and dialyzed extensively against PB containing EDTA and sodium azide. The dialyzed fluid was clarified by centrifugation at 10,000 g for 20 min at 4 8C, and the supernatant obtained was stored at 20 8C pending further use. 2.3. Heparin-Sepharose affinity chromatography Dialyzed and clarified oviductal fluid was applied on to the Heparin-Sepharose affinity column (0.96 cm 1.4 cm; bed volume, 1 mL) pre-equilibrated with phosphate buffer. The unbound fractions were collected and monitored spectrophotometrically at 280 nm. The column was washed thoroughly with the same buffer; bound proteins were eluted with elution buffer (10 mM phosphate buffer, pH 7.2, containing 0.5 mM NaCl, 5 mM EDTA, 0.03% sodium azide and 2 mM PMSF) and monitored (at 280 nm) for protein concentration. The column was regenerated using citrate buffer and Tris buffer successively (5 mL of each) and the media was stored in 0.5 M NaCl containing 0.03% sodium azide. Protein concentration was estimated spectrophotometrically at 280 nm [16]. During the fractionation of oviductal fluid proteins, EDTA and PMSF were used to prevent degradation of the oviductal proteins from metalloprotease and serine protease present in the oviductal fluid.
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2.4. Linear acrylamide gradient electrophoresis Buffalo blood plasma proteins were run concurrently with the oviductal proteins. Protein samples were prepared by mixing protein solution with sample buffer (0.312 M Tris–HCl buffer, pH 6.8, containing 0.125% (w/v) bromophenol blue, 10% (w/v) SDS and 50% (v/v) glycerol). The protein samples were kept in water bath (60–70 8C) for 2–3 min. Approximately 10–30 mg of proteins were loaded into the wells with a Hamilton syringe. Samples for electrophoresis under reducing conditions contained 2–5% marcaptoethanol. The modified procedure of Hames [17] was followed using the discontinuous buffer system of Laemmli [18]. The gradient gel was prepared by mixing equal volumes of higher and lower concentrations of acrylamide solution (5–10%), which were pre-cooled at 4 8C for 15 min. Electrophoresis was carried out at room temperature in constant voltage mode, at 50 V for 1 h, and thereafter at 100–110 V (until the end). When the dye front approached the bottom of gel, the power supply was disconnected. The gel was removed, immersed in fixing solution for 1–2 h, and then stained overnight with Coomassie blue. The gel was subsequently destained with destaining solution and stored in 7% acetic acid. 2.5. Effect of oviductal proteins on semen Sixteen good quality ejaculates from four buffalo bulls (four ejaculates each) were used. Mass activity of the freshly collected semen was graded on a scale of 0–5, based on waves and swirls [19]. The proportion of progressively motile sperm was subjectively estimated at 400 magnification and sperm concentration in neat semen was determined with a hemocytometer [19]. After fresh semen analysis, each ejaculate was divided into three parts and extended with a Tris-egg yolk-citrate extender, as described [20]. The final dilution yielded 7% glycerol and approximately 60 106 sperm/mL. Semen was frozen in 0.5 mL French straws (30 106 sperm/ straw), with an equilibration period of 4 h. The straws were frozen as per the standard protocol, after treatment with fractions of oviductal proteins, as follows: Part 1, treated with heparin binding oviductal proteins (0.326 mg/straw); Part 2, treated with non-heparin binding oviductal proteins (1 mg/straw); and Part 3, control (no protein treatment).
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viability, and acrosomal integrity before freezing as well as after thawing. Sperm motility was evaluated by putting a drop of semen on a pre-warmed slide and subjectively estimating the percentage of sperm possessing progressive motility at 400 magnification. All motility estimations were carried out by a single, well-experienced person, without the knowledge of treatments (blind evaluation). The proportion of live sperm were evaluated using Eosin–Nigrosin staining [21] (partly stained cells were considered dead). Acrosomal integrity percentage was evaluated under 1000 magnification by a Giemsa staining method, as described [22]. For assessing sperm viability and acrosomal integrity, at least 100 sperm cells/smear were evaluated. 2.7. Cervical mucus penetration test Bovine cervical mucus penetration test (BCMPT) was performed (in duplicate) in accordance with a previous protocol [23]. Cervical mucus was collected from 12 buffaloes in estrus [24]; only samples with a typical fern pattern (n = 10) were selected for experiment. White side test [25] was performed to detect infection in the mucus; only samples that were negative for infection (n = 9) were used. Capillary tubes (8 cm long) were loaded with mucus and one end was sealed with haemoseal, whereas the other end was left open. An aliquot (0.5 mL) of semen was put into a small test tube; the loaded capillary tubes (in duplicate) were placed so that the free end remained at the bottom of the tubes and incubated for 60 min at 37 8C. After incubation, the capillary tubes were removed from the test tubes, wiped clean and placed on a graduated glass slide and the distance travelled (mm) by the vanguard sperm was measured (magnification, 400). 2.8. Hypo-osmotic sperm swelling test This test was conducted as described [26]. An aliquot (0.1 mL) of semen was added to 2 mL of hypo-osmotic solution in a small test tube, mixed, and incubated at 37 8C for 60 min. After incubation, a small drop from the suspension was placed on a clean, dry, grease-free glass slide, and covered with a cover slip. The slide was examined under a phase-contrast microscope (400). For each slide, at least 100 sperm were assessed.
2.6. Semen analysis
2.9. Statistical analysis
Randomly selected semen straws from each group and each ejaculate were assessed for sperm motility,
Two semen straws from each treatment and each ejaculate were examined for sperm characteristics.
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Proportional data were subjected to an arc sine transformation and transformed data was analyzed. We used ANOVA for a randomized block design; replicate was considered as fixed variable and treatments were random variables blocked within replicate. For P < 0.05, differences were located with a Fisher’s least significant difference test [27]. All analyses were done with SPSS software (SPSS, Chicago, IL, USA). 3. Results 3.1. Fractionation and analysis of oviductal fluid proteins The average protein concentration of diluted oviductal fluid recovered by flushing was 2.3 mg/ mL and the total protein content was 3.5 mg/oviduct. Approximately 65 mL of oviductal fluid (150 mg protein) was applied to the Heparin-Sepharose column. The bound and unbound proteins fractions were collected and the protein content of the fractions was determined. Several cycles were performed until no further binding of proteins was detected. The profile of the first elution is shown (Fig. 1). The amount of protein bound to the heparin-Sepharose column was approximately 50% of the unbound fraction of the oviductal fluid. Electrophoretic profile of proteins not bound to the Heparin-Sepharose column (H-unbound) had eight discrete protein bands (12–177 kDa); proteins of molecular size 68, 55, 31, 26, and 12 kDa were prominent (Fig. 2: lanes A and B). The Heparin-Sepharose bound proteins (H-bound) had 15 discrete protein bands (12–165 kDa). Nine proteins of lower molecular weight present in heparin-bound and heparin-unbound fractions were absent in blood plasma (Fig. 2, lanes E and F), suggesting that these proteins were of oviductal origin.
Fig. 2. SDS–PAGE analysis of buffalo oviductal proteins (fractionated by a Heparin-Sepharose affinity column) and buffalo blood plasma (the gel was stained with Coomassie Blue). Lanes A and B, unbound proteins (53 and 107 mg, respectively). Lanes C and D, bound proteins (80 and 120 mg). Lanes E and F, plasma proteins (65 and 130 mg).
3.2. Effect of fractionated oviductal fluid proteins on sperm characteristics The quality of neat semen in all bulls was acceptable (Table 1). After equilibration and post thaw, sperm treated with the H-bound oviductal fluid proteins had higher (P < 0.05) progressive motility than sperm treated with H-unbound oviductal fluid proteins treated or control sperm (Table 2). Addition of H-bound or unbound oviductal proteins improved (P < 0.05) sperm viability and acrosomal integrity (relative to the Control) for both pre-freeze and post-thaw sperm. 3.3. Sperm penetration distance in cervical mucus and hypo osmotic sperm swelling test Both pre-freeze and post-thaw sperm exposed to Hbound oviductal fluid proteins migrated further (P < 0.05) in the BCMPT than sperm in the other two groups (Table 3). In the HOST, the percentage of pre-freeze sperm with a viable plasma membrane was greater (P < 0.05) in the group with H-bound oviductal fluid proteins than in the other two groups; for post-thaw sperm, the groups exposed to either bound or unbound proteins were not significantly different, but both were greater than for the control group. 4. Discussion
Fig. 1. Fractionation of buffalo oviductal fluid proteins on a HeparinSepharose column.
In the female reproductive tract, oviductal fluid interacts with sperm to modulate sperm functions; recent studies have confirmed that the factor responsible
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Table 1 Mean (S.E.M.) characteristics of fresh buffalo semen Bull no.
Volume (mL) b
4.0 0.0 3.9 0.3 3.8 0.3 3.8 0.3
1.5 0.1 1.6 0.1 ab 1.9 0.1 a 1.6 0.1 ab
1 2 3 4
Mass activity (1–5)
Progressivemotility (%)
Concentration (106/mL) a
72.5 2.5 75.0 5.4 67.5 2.5 77.5 2.5
Viability (%) a
1007.5 31.2 1055.0 83.8a 1030.0 80.6a 770.0 78.5b
90.5 0.5 90.8 0.8a 86.5 1.2b 85.0 2.0b
Acrosomal integrity (%) 91.3 0.8a 90.8 0.5a 89.3 1.5a 81.8 1.2b
a,b
Within a column, means without a common superscript differ (P < 0.05).
Table 2 Mean (S.E.M.) effect of different fractions of oviductal proteins on motility, viability, and intact acrosomes of buffalo sperm Group*
Motility (%)
Viability (%)
Pre-freeze Group I Group II Group III a–c *
Post-thaw a
Pre-freeze a
45.3 1.7 29.4 2.1 b 34.7 2.4 b
65.9 1.2 59.4 1.5 b 56.6 1.6 b
Post-thaw a
79.6 1.3 77.3 1.5 a 70.3 1.7 b
Pre-freeze a
67.3 1.1 63.9 1.1 a 52.4 1.5 b
Post-thaw a
86.0 0.8 80.7 0.9 b 73.1 1.3 c
75.4 1.5 a 68.8 1.2 b 48.3 3.5 c
Within a column, means without a common superscript differ (P < 0.05). Group I, H-bound oviductal protein; Group II, H-unbound oviductal protein; and Group III, Control.
is proteinaceous [13]. In previous studies, oviductal fluid proteins were fractionated by gel filtration, immunity affinity chromatography, and ion-exchange chromatography [28–30]. In the present study, HeparinSepharose affinity chromatography was used; approximately one-third of the oviductal fluid proteins were bound to the column. Electrophoteric analysis of the oviductal fluid proteins revealed that many of these proteins had their counterpart in blood serum proteins, suggesting that were simply transudates of blood serum. However, proteins of molecular size 135, 84, 75, 60, 55, 45, 31, 29, and 26 kDa were present exclusively in oviductal fluid and may be of oviductal origin. A 68 kDa protein, apparently serum albumin, appeared to be the major component of H-unbound proteins. It may have a capacitating effect on sperm, as it acts as a steroid acceptor and removes sperm membrane cholesterol during capacitation [31]. Table 3 Mean (S.E.M.) effect of different fractions of oviductal proteins on bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST) Group*
BCMPT (mm) Pre-freeze
Group I Group II Group III a,b
Acrosomal integrity (%)
HOST (%) Post-thaw
a
32.1 2.0 21.8 1.4b 24.7 1.5b
Pre-freeze a
27.5 2.1 18.3 1.5b 20.9 1.5b
Post-thaw a
67.8 1.7 59.1 2.1b 54.8 1.8b
35.9 1.8 a 32.4 1.8 a 27.0 1.6 b
Within a column, means without a common superscript differ (P < 0.05). * Group I, H-bound oviductal protein; Group II, H-unbound oviductal protein; and Group III, Control.
The addition of H-bound oviductal fluid fractions had a significant beneficial effect on pre-freeze as well as post-thaw sperm motility, viability and acrosomal integrity. In contrast, H-unbound oviductal fluid fractions did not significantly increase sperm motility. Pre-freeze and post-thaw viability of sperm were significantly higher in both treatment groups as compared to control. It was recently reported that addition of oviductal fluid or oviductal proteins before freezing enhanced post-thaw sperm characteristics [9,10]. Oviductal fluid proteins increased sperm motility, viability and acrosomal integrity in cattle [7,9,32] and buffalo [33]. Other studies have also reported an increase in viability of bull sperm incubated with bovine oviductal flushings [32], fluid [7] and cellculture-conditioned media [34,35]. On the contrary, Boquest et al. [36] reported that treatment of semen with oviductal fluid proteins of cattle decreased sperm motility. However, the previous studies used whole oviductal proteins and not different fractions. We speculated that different fractions of oviductal proteins may exhibit different actions on sperm functions. Both pre-freeze and post-thaw sperm in the H-bound protein treated group had a higher percentage of intact acrosomes than the other two groups; the H-unbound proteins also improved acrosomal integrity, but to a lesser extent than H-bound proteins. It has been reported that oviductal proteins stabilized the sperm membrane and increased the percentage of acrosomal integrity [37]. Perhaps the H-bound fractions of oviductal proteins protected the sperm during cryopreservation
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from oxidative and proteolytic damage by providing a protective covering on the acrosome [29,36]. In the present study, the pre-freeze and post thaw sperm penetration distance were significantly higher in the H-bound oviductal proteins treated-group compared to the other groups; this may have been due to higher sperm motility in the H-bound treated group. It is postulated that the H-bound oviductal proteins stimulated sperm motility and provided the membrane strength that enabled the sperm to travel further in the cervical mucus, whereas some protein(s) in the Hunbound fractions suppressed sperm motility and, in turn, led to decreased sperm penetration distance. A significantly higher percentage of sperm tail swelling (intact plasma membrane) was observed in both treatment groups; perhaps proteins in both fractions maintained the selective permeability of the sperm, strengthened the sperm membrane and thus maintained their membrane integrity. Oviductal proteins protected sperm during extension [36] and freezing [33]. Hunter [38] suggested that the oviductal proteins might protect sperm in dilution suspension from the leaching influence of oviductal fluid. It has been also reported that oviductal catalase binds to the acrosome and protects the sperm membrane from oxidative damage and maintained higher viability [29] and acrosomal integrity, thus maintaining a higher percentage of HOST [10]. The exact mechanism of protective action of oviductal proteins is not clearly understood. A 60 kDa protein obtained from the cattle oviduct, which could be catalase, eliminated peroxides by interrupting with free radical production that prevents lipid peroxidation [29]. The binding of this protein to the acrosome could confer protection against oxidative damage, thus improving membrane integrity [29] and intactness [37]. There are also reports citing a role of oviductal proteins in maintaining integrity by delaying capacitation and by neutralizing toxic byproducts of metabolism [36]. In conclusion, we inferred from the present study that the H-bound protein of oviductal fluid played a role in maintaining the motility, viability and membrane integrity of buffalo sperm during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity. Acknowledgements The authors thank The Director, Joint Director, IVRI and The Head, Animal Reproduction Division for providing the necessary facilities for conducting the study.
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Theriogenology 69 (2008) 925–931 www.theriojournal.com
Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation§ S. Imam a, M.R. Ansari a, N. Ahmed b, A. Kumaresan c,* a
Artificial Insemination Laboratory, Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India b Protein Research Laboratory, Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India c Division of Animal Production, ICAR Research complex for NEH Region, Barapani, Umiam, Meghalaya 793 103, India Received 30 September 2006; received in revised form 24 May 2007; accepted 28 May 2007
Abstract The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS–PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen–thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P < 0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P < 0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity. # 2008 Elsevier Inc. All rights reserved. Keywords: Oviductal proteins; Heparin-Sapharose fractionation: Sperm functions; Cryopreservation; Buffalo
1. Introduction Sperm survive and remain functionally active for >20 h in oviduct [1], whereas there is substantial
§
Part of the thesis submitted by the first author to the Deemed University, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India. * Corresponding author. Tel.: +91 9436143226; fax: +91 364 2570362. E-mail address: [email protected] (A. Kumaresan). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.066
deterioration of sperm quality after as little as 1 h incubation in vitro at 37 8C [2]. Therefore, the oviduct may secrete factors that protect sperm functional integrity. The epithelial lining of the oviduct and its secretory products may influence the functions of sperm, ovum and embryo [3]. Oviductal fluid consists of serum transudate [4] and a secretory product of the oviductal epithelium [5,6]. Several workers have reported that a number of proteins are secreted from the oviduct, which promote viability of sperm [7], prolong sperm motility [8], and protect membrane
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integrity [9,10]. Oviductal proteins were also reported to induce sperm capacitation [7,11,12]. Attempts have been made to purify proteins of oviductal fluid of mammals; an estrus-associated protein (85–95 kDa) was purified by salt fractionation and ion-exchange chromatography [13]. Variations in the effects of oviductal proteins on sperm functions in relation to the stage of the estrous cycle at which the oviductal proteins were obtained have already been reported [10]. Proteins obtained from oviducts collected during the nonluteal phase improved sperm function during cryopreservation, whereas proteins from luteal oviducts depressed sperm motility [10,12]. However, most of the above-mentioned studies utilized whole oviductal proteins. Despite the potential importance of oviductal fluid proteins in reproductive processes, there are limited data regarding fractionated oviductal fluid proteins and their effects on motility, viability and membrane integrity of sperm during cryopreservation, especially in buffaloes. Perhaps different fractions of the oviductal proteins exhibit different actions on sperm characteristics. As most of the proteins secreted by the oviduct are glycoprotein in nature [14], they can be fractionated based on their binding property with heparin. Heparin is a sulphated glycosaminoglycon, consisting mainly of repeating diasaccharide sequences of a-L-idopyranuronic acid 2sulphate and 2-deoxy-2-sulphamino-a-D-glucopyranose-6-phosphate, linked through Positions 1 and 4, which support its binding with glycoproteins [15]. Therefore, the objective of the present study was to determine the effect of Heparin-Sepharose bound and unbound oviductal proteins on sperm functions during cryopreservation. 2. Materials and methods 2.1. Chemicals and reagents D-fructose, sodium chloride, sodium dihydrogen orthophosphate, sodium phosphate dibasic, trisodium citrate, sodium azide, isopropanol, glycine, methanol, acetic acid, HCl and bromophenol blue (GR grade) were purchased from Merck, New Delhi, India. Tris, EDTA, acrylamide (3 crystalised), agarose, ammonium persulfate, and sodium lauryl sulphate (molecular biology grade) were purchased from Sisco Research Laboratory, Mumbai, India. Electrophoresis chemicals (bis-acrylamide, coomassie Brillant Blue R-250 and TEMED), Protease inhibitor (PMSF) and HeparinSepharose column were purchased from Sigma– Aldrich, St. Louis, MO, USA. Protein molecular
markers were purchased from Bangalore Genei. Minisart filters (0.20 mm) were obtained from Sartorius, Bangalore, India, and dialysis tubing were from Gibco BRL, New Delhi, India. Glass triple-distilled water was used throughout the study. 2.2. Preparation of oviductal fluid proteins Buffalo oviducts and ovaries were collected from apparently healthy animals slaughtered at the National Buffalo Abbatoir, Bareilly, India and brought to the laboratory on ice immediately after slaughter. Upon arrival, ovarian morphology was examined and the phase of the estrous cycle was determined. Only oviducts from buffalo that were not in a luteal phase (n = 190) were utilized for the present study [10]. The oviducts were dissected free of the surrounding connective tissues and fimbria and rinsed with chilled phosphate buffer saline (PBS) containing phosphate buffer (10 mM, pH 7.2), sodium azide (0.03%), EDTA (5 mM), PMSF (2 mM), penicillin (120 mg/mL) and streptomycin (200 mg/mL). Flushing was done at 4 8C from the isthmic end of the oviduct with PBS containing sodium azide, EDTA, and PMSF. The collected fluid was centrifuged at 10,000 g for 20 min at 4 8C and dialyzed extensively against PB containing EDTA and sodium azide. The dialyzed fluid was clarified by centrifugation at 10,000 g for 20 min at 4 8C, and the supernatant obtained was stored at 20 8C pending further use. 2.3. Heparin-Sepharose affinity chromatography Dialyzed and clarified oviductal fluid was applied on to the Heparin-Sepharose affinity column (0.96 cm 1.4 cm; bed volume, 1 mL) pre-equilibrated with phosphate buffer. The unbound fractions were collected and monitored spectrophotometrically at 280 nm. The column was washed thoroughly with the same buffer; bound proteins were eluted with elution buffer (10 mM phosphate buffer, pH 7.2, containing 0.5 mM NaCl, 5 mM EDTA, 0.03% sodium azide and 2 mM PMSF) and monitored (at 280 nm) for protein concentration. The column was regenerated using citrate buffer and Tris buffer successively (5 mL of each) and the media was stored in 0.5 M NaCl containing 0.03% sodium azide. Protein concentration was estimated spectrophotometrically at 280 nm [16]. During the fractionation of oviductal fluid proteins, EDTA and PMSF were used to prevent degradation of the oviductal proteins from metalloprotease and serine protease present in the oviductal fluid.
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2.4. Linear acrylamide gradient electrophoresis Buffalo blood plasma proteins were run concurrently with the oviductal proteins. Protein samples were prepared by mixing protein solution with sample buffer (0.312 M Tris–HCl buffer, pH 6.8, containing 0.125% (w/v) bromophenol blue, 10% (w/v) SDS and 50% (v/v) glycerol). The protein samples were kept in water bath (60–70 8C) for 2–3 min. Approximately 10–30 mg of proteins were loaded into the wells with a Hamilton syringe. Samples for electrophoresis under reducing conditions contained 2–5% marcaptoethanol. The modified procedure of Hames [17] was followed using the discontinuous buffer system of Laemmli [18]. The gradient gel was prepared by mixing equal volumes of higher and lower concentrations of acrylamide solution (5–10%), which were pre-cooled at 4 8C for 15 min. Electrophoresis was carried out at room temperature in constant voltage mode, at 50 V for 1 h, and thereafter at 100–110 V (until the end). When the dye front approached the bottom of gel, the power supply was disconnected. The gel was removed, immersed in fixing solution for 1–2 h, and then stained overnight with Coomassie blue. The gel was subsequently destained with destaining solution and stored in 7% acetic acid. 2.5. Effect of oviductal proteins on semen Sixteen good quality ejaculates from four buffalo bulls (four ejaculates each) were used. Mass activity of the freshly collected semen was graded on a scale of 0–5, based on waves and swirls [19]. The proportion of progressively motile sperm was subjectively estimated at 400 magnification and sperm concentration in neat semen was determined with a hemocytometer [19]. After fresh semen analysis, each ejaculate was divided into three parts and extended with a Tris-egg yolk-citrate extender, as described [20]. The final dilution yielded 7% glycerol and approximately 60 106 sperm/mL. Semen was frozen in 0.5 mL French straws (30 106 sperm/ straw), with an equilibration period of 4 h. The straws were frozen as per the standard protocol, after treatment with fractions of oviductal proteins, as follows: Part 1, treated with heparin binding oviductal proteins (0.326 mg/straw); Part 2, treated with non-heparin binding oviductal proteins (1 mg/straw); and Part 3, control (no protein treatment).
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viability, and acrosomal integrity before freezing as well as after thawing. Sperm motility was evaluated by putting a drop of semen on a pre-warmed slide and subjectively estimating the percentage of sperm possessing progressive motility at 400 magnification. All motility estimations were carried out by a single, well-experienced person, without the knowledge of treatments (blind evaluation). The proportion of live sperm were evaluated using Eosin–Nigrosin staining [21] (partly stained cells were considered dead). Acrosomal integrity percentage was evaluated under 1000 magnification by a Giemsa staining method, as described [22]. For assessing sperm viability and acrosomal integrity, at least 100 sperm cells/smear were evaluated. 2.7. Cervical mucus penetration test Bovine cervical mucus penetration test (BCMPT) was performed (in duplicate) in accordance with a previous protocol [23]. Cervical mucus was collected from 12 buffaloes in estrus [24]; only samples with a typical fern pattern (n = 10) were selected for experiment. White side test [25] was performed to detect infection in the mucus; only samples that were negative for infection (n = 9) were used. Capillary tubes (8 cm long) were loaded with mucus and one end was sealed with haemoseal, whereas the other end was left open. An aliquot (0.5 mL) of semen was put into a small test tube; the loaded capillary tubes (in duplicate) were placed so that the free end remained at the bottom of the tubes and incubated for 60 min at 37 8C. After incubation, the capillary tubes were removed from the test tubes, wiped clean and placed on a graduated glass slide and the distance travelled (mm) by the vanguard sperm was measured (magnification, 400). 2.8. Hypo-osmotic sperm swelling test This test was conducted as described [26]. An aliquot (0.1 mL) of semen was added to 2 mL of hypo-osmotic solution in a small test tube, mixed, and incubated at 37 8C for 60 min. After incubation, a small drop from the suspension was placed on a clean, dry, grease-free glass slide, and covered with a cover slip. The slide was examined under a phase-contrast microscope (400). For each slide, at least 100 sperm were assessed.
2.6. Semen analysis
2.9. Statistical analysis
Randomly selected semen straws from each group and each ejaculate were assessed for sperm motility,
Two semen straws from each treatment and each ejaculate were examined for sperm characteristics.
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Proportional data were subjected to an arc sine transformation and transformed data was analyzed. We used ANOVA for a randomized block design; replicate was considered as fixed variable and treatments were random variables blocked within replicate. For P < 0.05, differences were located with a Fisher’s least significant difference test [27]. All analyses were done with SPSS software (SPSS, Chicago, IL, USA). 3. Results 3.1. Fractionation and analysis of oviductal fluid proteins The average protein concentration of diluted oviductal fluid recovered by flushing was 2.3 mg/ mL and the total protein content was 3.5 mg/oviduct. Approximately 65 mL of oviductal fluid (150 mg protein) was applied to the Heparin-Sepharose column. The bound and unbound proteins fractions were collected and the protein content of the fractions was determined. Several cycles were performed until no further binding of proteins was detected. The profile of the first elution is shown (Fig. 1). The amount of protein bound to the heparin-Sepharose column was approximately 50% of the unbound fraction of the oviductal fluid. Electrophoretic profile of proteins not bound to the Heparin-Sepharose column (H-unbound) had eight discrete protein bands (12–177 kDa); proteins of molecular size 68, 55, 31, 26, and 12 kDa were prominent (Fig. 2: lanes A and B). The Heparin-Sepharose bound proteins (H-bound) had 15 discrete protein bands (12–165 kDa). Nine proteins of lower molecular weight present in heparin-bound and heparin-unbound fractions were absent in blood plasma (Fig. 2, lanes E and F), suggesting that these proteins were of oviductal origin.
Fig. 2. SDS–PAGE analysis of buffalo oviductal proteins (fractionated by a Heparin-Sepharose affinity column) and buffalo blood plasma (the gel was stained with Coomassie Blue). Lanes A and B, unbound proteins (53 and 107 mg, respectively). Lanes C and D, bound proteins (80 and 120 mg). Lanes E and F, plasma proteins (65 and 130 mg).
3.2. Effect of fractionated oviductal fluid proteins on sperm characteristics The quality of neat semen in all bulls was acceptable (Table 1). After equilibration and post thaw, sperm treated with the H-bound oviductal fluid proteins had higher (P < 0.05) progressive motility than sperm treated with H-unbound oviductal fluid proteins treated or control sperm (Table 2). Addition of H-bound or unbound oviductal proteins improved (P < 0.05) sperm viability and acrosomal integrity (relative to the Control) for both pre-freeze and post-thaw sperm. 3.3. Sperm penetration distance in cervical mucus and hypo osmotic sperm swelling test Both pre-freeze and post-thaw sperm exposed to Hbound oviductal fluid proteins migrated further (P < 0.05) in the BCMPT than sperm in the other two groups (Table 3). In the HOST, the percentage of pre-freeze sperm with a viable plasma membrane was greater (P < 0.05) in the group with H-bound oviductal fluid proteins than in the other two groups; for post-thaw sperm, the groups exposed to either bound or unbound proteins were not significantly different, but both were greater than for the control group. 4. Discussion
Fig. 1. Fractionation of buffalo oviductal fluid proteins on a HeparinSepharose column.
In the female reproductive tract, oviductal fluid interacts with sperm to modulate sperm functions; recent studies have confirmed that the factor responsible
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Table 1 Mean (S.E.M.) characteristics of fresh buffalo semen Bull no.
Volume (mL) b
4.0 0.0 3.9 0.3 3.8 0.3 3.8 0.3
1.5 0.1 1.6 0.1 ab 1.9 0.1 a 1.6 0.1 ab
1 2 3 4
Mass activity (1–5)
Progressivemotility (%)
Concentration (106/mL) a
72.5 2.5 75.0 5.4 67.5 2.5 77.5 2.5
Viability (%) a
1007.5 31.2 1055.0 83.8a 1030.0 80.6a 770.0 78.5b
90.5 0.5 90.8 0.8a 86.5 1.2b 85.0 2.0b
Acrosomal integrity (%) 91.3 0.8a 90.8 0.5a 89.3 1.5a 81.8 1.2b
a,b
Within a column, means without a common superscript differ (P < 0.05).
Table 2 Mean (S.E.M.) effect of different fractions of oviductal proteins on motility, viability, and intact acrosomes of buffalo sperm Group*
Motility (%)
Viability (%)
Pre-freeze Group I Group II Group III a–c *
Post-thaw a
Pre-freeze a
45.3 1.7 29.4 2.1 b 34.7 2.4 b
65.9 1.2 59.4 1.5 b 56.6 1.6 b
Post-thaw a
79.6 1.3 77.3 1.5 a 70.3 1.7 b
Pre-freeze a
67.3 1.1 63.9 1.1 a 52.4 1.5 b
Post-thaw a
86.0 0.8 80.7 0.9 b 73.1 1.3 c
75.4 1.5 a 68.8 1.2 b 48.3 3.5 c
Within a column, means without a common superscript differ (P < 0.05). Group I, H-bound oviductal protein; Group II, H-unbound oviductal protein; and Group III, Control.
is proteinaceous [13]. In previous studies, oviductal fluid proteins were fractionated by gel filtration, immunity affinity chromatography, and ion-exchange chromatography [28–30]. In the present study, HeparinSepharose affinity chromatography was used; approximately one-third of the oviductal fluid proteins were bound to the column. Electrophoteric analysis of the oviductal fluid proteins revealed that many of these proteins had their counterpart in blood serum proteins, suggesting that were simply transudates of blood serum. However, proteins of molecular size 135, 84, 75, 60, 55, 45, 31, 29, and 26 kDa were present exclusively in oviductal fluid and may be of oviductal origin. A 68 kDa protein, apparently serum albumin, appeared to be the major component of H-unbound proteins. It may have a capacitating effect on sperm, as it acts as a steroid acceptor and removes sperm membrane cholesterol during capacitation [31]. Table 3 Mean (S.E.M.) effect of different fractions of oviductal proteins on bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST) Group*
BCMPT (mm) Pre-freeze
Group I Group II Group III a,b
Acrosomal integrity (%)
HOST (%) Post-thaw
a
32.1 2.0 21.8 1.4b 24.7 1.5b
Pre-freeze a
27.5 2.1 18.3 1.5b 20.9 1.5b
Post-thaw a
67.8 1.7 59.1 2.1b 54.8 1.8b
35.9 1.8 a 32.4 1.8 a 27.0 1.6 b
Within a column, means without a common superscript differ (P < 0.05). * Group I, H-bound oviductal protein; Group II, H-unbound oviductal protein; and Group III, Control.
The addition of H-bound oviductal fluid fractions had a significant beneficial effect on pre-freeze as well as post-thaw sperm motility, viability and acrosomal integrity. In contrast, H-unbound oviductal fluid fractions did not significantly increase sperm motility. Pre-freeze and post-thaw viability of sperm were significantly higher in both treatment groups as compared to control. It was recently reported that addition of oviductal fluid or oviductal proteins before freezing enhanced post-thaw sperm characteristics [9,10]. Oviductal fluid proteins increased sperm motility, viability and acrosomal integrity in cattle [7,9,32] and buffalo [33]. Other studies have also reported an increase in viability of bull sperm incubated with bovine oviductal flushings [32], fluid [7] and cellculture-conditioned media [34,35]. On the contrary, Boquest et al. [36] reported that treatment of semen with oviductal fluid proteins of cattle decreased sperm motility. However, the previous studies used whole oviductal proteins and not different fractions. We speculated that different fractions of oviductal proteins may exhibit different actions on sperm functions. Both pre-freeze and post-thaw sperm in the H-bound protein treated group had a higher percentage of intact acrosomes than the other two groups; the H-unbound proteins also improved acrosomal integrity, but to a lesser extent than H-bound proteins. It has been reported that oviductal proteins stabilized the sperm membrane and increased the percentage of acrosomal integrity [37]. Perhaps the H-bound fractions of oviductal proteins protected the sperm during cryopreservation
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from oxidative and proteolytic damage by providing a protective covering on the acrosome [29,36]. In the present study, the pre-freeze and post thaw sperm penetration distance were significantly higher in the H-bound oviductal proteins treated-group compared to the other groups; this may have been due to higher sperm motility in the H-bound treated group. It is postulated that the H-bound oviductal proteins stimulated sperm motility and provided the membrane strength that enabled the sperm to travel further in the cervical mucus, whereas some protein(s) in the Hunbound fractions suppressed sperm motility and, in turn, led to decreased sperm penetration distance. A significantly higher percentage of sperm tail swelling (intact plasma membrane) was observed in both treatment groups; perhaps proteins in both fractions maintained the selective permeability of the sperm, strengthened the sperm membrane and thus maintained their membrane integrity. Oviductal proteins protected sperm during extension [36] and freezing [33]. Hunter [38] suggested that the oviductal proteins might protect sperm in dilution suspension from the leaching influence of oviductal fluid. It has been also reported that oviductal catalase binds to the acrosome and protects the sperm membrane from oxidative damage and maintained higher viability [29] and acrosomal integrity, thus maintaining a higher percentage of HOST [10]. The exact mechanism of protective action of oviductal proteins is not clearly understood. A 60 kDa protein obtained from the cattle oviduct, which could be catalase, eliminated peroxides by interrupting with free radical production that prevents lipid peroxidation [29]. The binding of this protein to the acrosome could confer protection against oxidative damage, thus improving membrane integrity [29] and intactness [37]. There are also reports citing a role of oviductal proteins in maintaining integrity by delaying capacitation and by neutralizing toxic byproducts of metabolism [36]. In conclusion, we inferred from the present study that the H-bound protein of oviductal fluid played a role in maintaining the motility, viability and membrane integrity of buffalo sperm during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity. Acknowledgements The authors thank The Director, Joint Director, IVRI and The Head, Animal Reproduction Division for providing the necessary facilities for conducting the study.
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