K. H. El-Shahat, Taysser M. I., M. R. Badr, K. A. Zaki

1Department of Theriogenology, Faculty of Veterinary Medicine , Cairo University

2Department of Animal Reproduction and Artificial Insemination, Animal Reproduction Research Institute, Egypt

3El Shieh Zaied Vet. Clinic, Egypt



Effect of heparin, caffeine and calcium ionophore A 23187 on in vitro induction of the acrosome reaction of fresh ram spermatozoa

K. H. El-Shahat1*, Taysser M. I.1, M. R. Badr2, K. A. Zaki3

1Department of Theriogenology, Faculty of Veterinary Medicine , Cairo University

2Department of Animal Reproduction and Artificial Insemination, Animal Reproduction Research Institute, Egypt

3El Shieh Zaied Vet. Clinic, Egypt

ARTICLE INFO

Article history:

Ram sperm

Capacitation

Hyperactivation

Acrosome reaction

Hyper activity

Oocyte

Heparin

Caffeine

Ca ionophore

IVF

ABSTRACT

Objective: To determine the effect of different concentrations of heparin, caffeine or calcium ionosphore and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) of ram sperm in vitro. Methods: Semen samples were collected by artificial vagina from three mature ram. Split fractions (0.1 mL) of the pooled semen were layered under 1 mL of S-TALP medium supplemented with either either heparin (10, 25, 50, 75 and 100 µg/mL), caffeine (0.5, 0.75, 1.00, 2.00 mg/mL), or calcium ionophore A23187 (0.50, 0.75, 1, 1.55 mM/mL). Individual motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 hours post-incubation for all treatments and control. Moreover, they were examined for ability to fertilize sheep oocyte in vitro. Results: Heparin, caffeiene and calcium ionophore A23187 at a concentration of 75 µg/mL, 1 mg/mL, and 1.55 mM/mL respectively can be used as a protocol to provide the best results for in vitro cap citation and acrosome reaction in ram. The penetration rates of rate of oocytes inseminated with spermatozoa treated with calcium ionophore A23187, and heparin were higher as compared with caffeine. Moreover, heparin achieved higher fertilization rates but without significant difference with others. Conclusion: The best concentration of heparin, caffeine and ionophore A23187 are 75 µg/mL, 1 mg/mL, 1.55 mM/mL for 3, 1, 4 hours incubation respectively and can be used for in vitro fertilization in sheep.

Document heading doi: 10.1016/j.apjr.2016.01.012

1. Introduction

Development of successful in vitro fertilization (IVF) techniques is essential for the study of the basic aspects of fertilization process. Freshly ejaculated mammalian spermatozoa are not immediately capable of achieving fertilization [1]. During residence in the female tract, the sperm cell undergoes a complex and poorly understood set of modifications which confer fertilization competence, a process collectively called capacitation [2-5]. Capacitation is believed primarily to involve membrane modifications, including changes in lipid composition, surface properties, fluidity, permeability to calcium and lowered concentration of cholesterol in membranes[6]. Most of these alterations are related to changes in the plasma membrane of spermatozoa and have led to the contention that capacitation is a process of membrane maturation [7, 8]. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction and resulting in loss of sperm fertilization capacity [5]. Fresh ram spermatozoa will spontaneously undergo the acrosome reaction when incubated at 39 曟 over a period of 4 h in the absence of any inducing agent [9]. The acrosome reaction occurs in response to natural inducers, i.e., the zona pllucida and oviductal fluids, but it can also be artificially induced by a variety of substances such as Ca2+ionophore A23187[10], heparin[11], caffeine [12]. Many researches are concerned with method of recovery and maturation of sheep oocytes, however, few studies are concerned with the process of IVF, particularly, preparation of the ram spermatozoa prior to insemination of oocytes. The effects of many capacitating factors have been studiedin separate researches, and under different conditions. Very few data is available about comparing the effect of different capacitating active compounds on capacitation and acrosome reaction of ram spermatozoa. Therefore, the present study was aimed to investigate the effect of different concentrations and incubation times of heparin, caffeine or calcium ionophore A23187on the motility, hyperactivity, and acrosome reaction of ram spermatozoa and ability of them on in vitro fertilization of ram oocytes.

2. Material and methods

2.1. Semen collection

Semen was collected by artificial vagina from 3 ram of proven fertility, aged 2-3 years and housed indoors at farm of Animal Reproduction Research Institute. All semen sampled were evaluated immediately for percentage of individual motility (IM %). Samples with IM% not less than 60% were pooled.

2.2. Sperm capacitation

Split fractions (0.1 mL) of the pooled semen were layered under 1 mL of S-TALP medium, according the method described by[13], supplemented with either heparin (10, 25, 50, 75 and 100 µg/mL), caffeine (0.5, 0.75, 1, 2 mg/mL) or calcium ionophore A23187. (0.5, 0.75, 1, 1.55 mM/mL) according to experimental procedure. Semen diluted in S-TALP medium without treatment was used as a control. This technique (swim up technique) was performed in 15 mL centrifuge tubes, held at an angle 45oand incubated in an atmosphere of 5% CO2incubator at 39 曟. Individual motility percentage (IM%), hyperactivity percentage (HA%) and acrosomal status were recorded at 0, 1, 2, 3 and 4 hours post-incubation for all treatments.

2.3. Evaluation of sperm individual motility

Percentage of individual motility (IM%) was subjectively estimated in a small drop of the sperm suspension from the most supernatant and covered by a cover slip, and examined under phase contrast microscope (400伊) equipped with a heated (37 曟). Only progressive forward motility was considered among different treatments and control.

2.4. Evaluation of sperm hyperactivity

Percentage of hyperactivated motility (HA %) were determined by recording the percentage of sperm cells with flagellar beating vigor and circular movement[14,15]. Hyperactivated motility percentage was considered from the percentage of IM% and expressed by“pluses” [15], where (+) means HA% <20%, ++ means HA% 20% –40%, +++ means HA% 40% -60%, ++++ means HA% 60% – 80% and +++++ means HA% >80%.

2.5. Evaluation of sperm acrosomal status

Percentage of incomplete and complete acrosome reacted spermatozoa were determined by silver nitrate staining technique according to[16]. A total of 100 spermatozoa were checked in randomly selected fields under oil immersion lens (1 000伊) of phase contrast microscope. The spermatozoa were classified into three groups: (1). Spermatozoa with an intact plasma and outer acrosomal membrane; (2). Spermatozoa with incomplete AR showing fenestrations, vesiculation and loosening between plasma membrane and outer acrosomal membrane; (3). Spermatozoa with complete AR showing complete loss of the outer acrosomal membrane leaving cup-shaped appearance. Both incomplete and complete AR percentages were considered collectively as total AR.

2.6. Evaluation of the fertilizing capacity of the treated ram spermatozoa

2.6.1. Collection of ovaries

The ovaries of native breed of sheep of unknown reproductive history (1-4 years old) were collected from El-Moneib abattoir. The ovaries were separated shortly after slaughter of animals, dissecting away from the surrounding tissues and maintained in a thermo box containing a pre-warmed sterile saline (30 曟) supplemented with antibiotic (100 IU/mL penicillin and 100 µg/mL streptomycin). The average transport time of the ovaries to the laboratory was 2 hours. At the laboratory, the ovaries were then washed with warm sterile saline to remove adhering blood [17].

2.6.2. Oocytes recovery and selection

Oocytes were harvested from the ovaries by aspiration technique using 21 G needle fitted to a 10 mL disposable syringe containing a small volume of maturation medium. Follicular fluids from all visible surface follicles ranged from 2–5 mm in diameter were aspirated. The contents were pooled in a sterile 15 mL centrifuge tubes and allowed to settle for 5 minutes in laminar flow hood. The clear fluid was then discarded while the bottom portion was poured in Petri dish containing hormone free Ham’s F-10 media examined under stereomicroscope under low magnification (10伊-20伊) for the presence of oocytes [18]. Oocytes were evaluated on the bases of the characteristics of surrounding cumulus investment, homogeneity of ooplasm mass and regularity of the peripheral shape. Oocytes with a complete corona layered and at least two compact cumulus cell layers and with a homogenous granulated ooplasm were used in this study. Oocytes with no cumulus cell layers (completely denuded) or those dark scattered or irregular ooplasm were discarded. Selected cumulus oocyte complexes (COCs) were washed three times with maturation medium [19].

2.6.3. In vitro oocyte maturation and fertilization

After preparation of Ham’s F-10, minidrops of 50 µL were tittered in a polystyrene culture dish (35 mm 伊10 mm) and covered with equilibrated sterile. Millipore filtered (0.45 µm) light-weight paraffin oil and incubated in CO2incubator in 5% CO2, 39 曟 and at maximum humidity for at least 1 hours before culturing. Group of 10-15 selected oocytes were transported to each minidrop then the culture dish was incubated statically for 24 hour in the previously mentioned incubation conditions. Then the oocytes were assessed for maturation signs. The oocytes showing expanded cumulus cells, perivitelline space or extruded first polar body were selected for IVF [20]. Selected in vitro matured oocytes were washed three times in S–TALP medium. The surrounding cumulus cells were partially removed by gentle pipetting. Five to 10 oocytes were then allocated to each minidrop, containing F-TALP medium, in polystyrene culture dish covered with warm sterile paraffin oil and incubated for 1 hour until spermatozoa were added. Pre-treated semen with optimal concentration of the different capacitating compound andoptimal time of incubation (determined on the bases of the best motility, hyperactivity, percentage of incomplete AR with high tendency to undergo complete AR, in addition to total AR) was used for insemination. Sperm cell concentration of semen was estimated after swim up and a total of 2 伊 106sperm cell were added to the minidrops so that the final volume of each minidrop is 50 µL. Oocytes and spermatozoa were incubated together for 6 hours at 39曟 under 5% CO2in air and at maximum humidity. At the end of the incubation period, oocytes/zygotes were examined for the evidence of fertilization. Oocytes with male and female pronuclei and/or secondary polar bodies were considered to be normally fertilized. The penetration and fertilization rates were recorded [21].

2.7. Statistical analysis

The obtained data for motility, hyperactivity and acrosome reaction were analyzed statistically using Costat computer program; version 3.03, Copyright Cottort Software. Data from 10 replicates were subjected to analysis of variance (two way ANOVA) to clarify the effect of different capacitating agents concentrations and incubation times. Moreover, unpaired t test was used to evaluate the differences between means. On the other hand in vitro penetration and fertilization rates were analyzed by using Chi-square analysis (氈2).

3. Results

3.1. Effect of different concentrations of heparin on ram sperm functions

3.1.1. Effect on sperm motility (IM)

The effect of different heparin concentrations and different incubation times on motility of ram sperm is shown in Table 1. Irrespective of incubation time, there was a highly significant (P<0.05) increase in motility percentage of spermatozoa treated with 10, 25, 50, 75 and 100 µg/mL heparin as compared to control one. Immediately after dilution sperm motility was significantly higher in semen sample with 100 mg /mL heparin than concentrations of 10, 25, 50 µg/mL. However, there were no significant differences between the treatments for motility after one hour of incubation and all over the incubation period. Meanwhile a significant decrease in motility started from the second hour of incubation period in semen samples diluted with 75 and 100 mg/mL heparin.

Table 1 Effect of different concentrations of heparin (µg/mL) and incubation time (hours) on motility of ram spermatozoa.

3.1.2. Effect on sperm hyperactivity (HA)

The first hour after treatment, the highest HA% was observed at 100 µg/mL heparin (2.80±0.32) followed by 50 µg/mL (2.60±0.80) and 75 µg/mL (1.80±0.24) but showing no significant differences among the three concentrations (Table 2). Irrespective of heparin concentration, incubation of ram spermatozoa for 4 hours resulted in a significant (P<0.05) increase in HA% as compared to 0 time.

Table 2 Effect of different heparin concentrations (µg/mL) and incubation time (hours) on hyperactivity of ram spermatozoa.

3.1.3. Effect on total acrosome reaction percentage (AR %)

A significant (P<0.05) increase in total AR% was concomitant to the increase in incubation time, and a highly significant (P<0.05) increase was achieved after 3 and 4 hour incubation as shown in Table 3. It was clear that 50 µg/mL and 75 µg/mL heparin achieved a highly significant (P<0.01) increase in total AR% (89.00±1.97 and 88.00±2.00, respectively) as compared to control (0.00±0.00).

Table 3 Effect of different heparin concentrations (µg/mL) and incubation time (hours) on total acrosome reaction of ram sperm.

3.2. Effect of different concentrations of caffeine on ram sperm functions

3.2.1. Effect on sperm motility

Table 4 shows the influence of different concentrations of caffeine and increasing incubation time on individual motility percentage of ram spermatozoa. Higher concentration (2 mg/mL) resulted in a significant (P<0.05) decrease in IM% immediately after dilution and during 1st and 2nd hours of incubation. However, low concentrationsof caffeine (0.5, 0.75 and 1 mg/mL) increased IM% without any significant differences between them and control. Maximum improvement in IM% was noticed after 1 hour incubation and maintained up to 2 hours followed by a significant (P<0.05) decrease when incubation time was prolonged.

3.2.2. Effect on sperm hyperactivity

The effect of caffeine and incubation time on HA of ram spermatozoa are presented in Table 5. A highly significant (P<0.05) increase in HA% was achieved by treatment of ram spermatozoa with different concentrations of caffeine (0.5, 0.75, 1 and 2 mg/mL) as compared to untreated control one. Irrespective to caffeine concentration, maximum increase in HA% was noticed after 4 hours incubation in control semen samples. Meanwhile a highly significant (P<0.01) increase in HA% was observed in all caffeine treated samples after 2 hours of incubation.

Table 5 Effect of different caffeine concentrations (mg/mL) and incubation time (hours) on hyperactivity of ram spermatozoa.

3.2.3. Effect on total acrosome reaction

Data regarding the effect of caffeine and incubation time on TAR% of ram spermatozoa are presented in Table 6. Irrespective to incubation time, when ram spermatozoa were treated with 1 mg/ mL caffeine, a highly significant (P<0.01) increase in total AR% of (19.90±2.11) was noticed after 1 h of incubation. After 2 h of incubation, the total AR% was significantly higher (P<0.01) in semen samples treated with 0.75, 1 and 2 mg/mL of caffeine than 0.5 mg/mL and control. The highest effect of treatment of caffeine on total AR% was observed during the last two hours of incubation period with 1 and 2 mg of caffeine concentrations. On the other hand, time dependent improvement in total AR% was detected. A maximum total AR% was noticed after 3 and 4 hours incubation.

Table 6 Effect of different caffeine concentrations (mg/mL) and incubation time (hours) on total acrosome reaction of ram spermatozoa.

3.3. Effect of different concentrations of calcium ionophore A23187 on ram sperm functions

3.3.1. Effect on sperm motility

Immediately after dilution different concentrations of calcium ionophore A23187 (0.5, 0.75, 1, 15.5 mM/mL) showed a significant (P<0.05) increase in IM when compared to control (Table 7). There was no significant differences could be detected in IM between control sample and different concentrations of calcium ionophore A23187 treated samples during different hours of incubation period. After 3 h of incubation, IM% was significantly (P<0.05) higher in semen samples treated with 1.55 mM/mL calcium ionophore than control. With respect to time factor, there was a significant (P<0.05) decrease in motility simultaneous to the increase in incubation time.

Table 7 Effect of different Ca2+ionophoreA23187 concentrations (mM/mL) and incubation time (hours) on motility of ram spermatozoa.

3.3.2. Effect on sperm hyperactivity

Table 8 presents the effect of different concentrations of calcium ionophore A23187 and different incubation times on HA% of ram spermatozoa. There was significant increase in HA% for different concentrations of calcium ionophore A23187 as compared to control one without any significant difference between them. Irrespective toconcentration, maximum HA% was observed in semen samples after 2 hours incubation.

Table 8 Effect of different Ca2+ionophoreA23187 concentrations (mM/mL) and incubation time (hours) on hyperactivity of Ram spermatozoa.

3.3.3. Effect on total acrosome reaction percentage

Table 9 describes the effect of calcium ionophore A23187 and incubation time on percentage of total AR% of ram spermatozoa. A significant (P<0.05) increase of total AR% was noticed in all calcium ionophore A23187 treated spermatozoa. The maximum effect was detected in samples treated with 1.55 mM/mLas compared to control. Likewise, a significant (P<0.05) increase in total AR% was noticed throughout the incubation period with maximum effect after 3 and 4 hours.

Table 9 Effect of different Ca2+ionophore A23187 concentrations (mM/mL)and incubation time (hours)on total acrosome reaction of ram spermatozoa.

3.4. Effect of best concentration of heparin, caffeine or calcium ionophore A23187 on in vitro fertilization of ewe oocytes

Table 10 showed that penetration rates of rate of oocytes inseminated with spermatozoa treated with calcium ionophore A23187, and heparin were higher as compared with caffeine. Moreover, heparin achieved higher fertilization rates but without significant difference with others.

Table 10 Effect of best concentration of different capacitating active compounds on in vitro fertilization of ovine oocytes.

4. Discussion

The in vivo sperm capacitation occurs during migration in the reproductive tract of the female, whereas in vitro capacitation requires the exposition of fresh or freeze semen to specific capacitating agents [22]. Capacitation process is a prerequisite step for sperm to bind to the zona pellucida, it tested acrosomic reaction in response to natural agonistics and express hyper- motility, a special movement that allow spermatozoa to move in the viscose fluid of the oviduct and get into the zona pellucida [23].

In the present study, different concentrations of heparin were used. Regarding the effect of heparin on motility, treatment of ram spermatozoa with 10 and 75 µg/mL resulted in improved individual motility percentage. In addition, 100 µg/mL heparin achieved best HA% after 4 hours incubation. In this respect, [24] indicated that heparin at dose 10 µg/mL potentially induces the capacitation of ram fresh ejaculated and frozen- thawed spermatozoa in vitro. However, some other authors utilized 100 µg heparin with one hour of incubation in ram [25] or two hours of incubation to promote sperm capacitation[26] in bull semen. Furthermore, the capacitation of epididymial ram spermatozoa after incubation in TALP media with 100 µg /mL heparin, resulted in the best result for sperm maturation and capacitating in vitro [27]. Heparin is a potent glycosaminoglycan required for bovine sperm capacitation [28] that has been shown to improve the fertilizing ability of goat and ram spermatozoa when used in addition to ESS [29]. This increase in motility and HA% is due to the ability of heparin to induce increased calcium uptake, increased intracellular free calcium and decreased calmodulin concentration in spermatozoa [30-32]. The present finding revealed that, ram spermatozoa treated with 100 µg/mL heparin showed maximum increase in the incomplete AR% after 2 and 3 hours incubation. In addition, the tendency of spermatozoa treated with 50 or 75 µg/mL heparin to show complete AR after longer incubation time was very high if compared to other concentrations. Moreover, 50 and 75 µg/mL resulted in a maximum total AR%. The data obtained by us are better than 9.6% and16.5% results obtained by[33, 34], respectively in sheep. This is difference may be attributed to use different concentration of heparin (2.5 µg/mL) and short incubation period (2 hours).

With respect to the effect of caffeine on ram sperm functions,the current study showed that, supplementation of ram semen with low concentration (up to 1 mg/mL) of caffeine during swim up revealed a significant (P<0.05) improvement in sperm motility and hyperactivity in a dose dependent manner, with maximum motility after 1 hours incubation. Similar tendency was observed for hyperactivity after 2hours. Higher caffeine concentration or longer incubation time resulted in a reverse effect in sperm motility. Similar finding was obtained by [35, 36]. The reduction of the motility of in vitro capacitated spermatozoa treated with heparin or caffeine may be due to reduction of the glycosylable substrates like glucose and/or fructose, and also to a reduction in the availability of pyruvate and lactate, substrates that during the cell metabolism are primordial to produce ATPs [37]. Caffeine was reported to induce an increase in intracellular calcium and an immediate hyperactivation of ram sperm [38]. The essential role of calcium on sperm capacitation has been proved in several mammalian species, and an increase in intracellular Ca2+concentration has been shown in hyperactivated flagella. Thus, under our experimental conditions treatment of ram spermatozoa with 1 mg/mL of caffeine for 4 hours was considered the best concentration of caffeine to be used for IVF. However, other study[39] indicates that concentration of 4 mmol has beneficial effects on ram sperm motility. They observed positive impact of this concentration on sperm motility at all time points. This concentration of caffeine may be a threshold value for stimulating ram sperm.

Regarding the effect of the calcium ionophore A23187, the current work suggested that significant effect could be detected in all treated spermatozoa with different concentrations as compared to control one. On the other hand, higher concentrations (1.55 mM/mL) resulted in a significant (P<0.01) increase in motility percent if compared to control after 2h of incubation. With respect to time factor, there was a significant (P<0.05) decrease in motility simultaneous to the increase in incubation time. The reverse trend in the percent of individual motility of ram spermatozoa was also noticed in by [40, 41] who reported that maximum response was achieved at an ionophore concentration of 1 µM or above, whereas during two hours’ incubation ionophore concentrations of 0.01 uM or less had no observable. They added that, calcium ionophore treatment had a dramatic effect on sperm motility. In the presence of Ca2+, levels of A23187 at or above 0.1 µM abolished motility. The decline of motility simultaneous to the increase in incubation time may be due to reduction in cellular ATPases, which fail to maintain normal intracellular calcium concentrations not only in head region, but also in midpiece where it affects mitochondrial function[42]. Hyperactivated motility in the current study was significantly (P<0.01) increased in samples treated with different concentrations of calcium ionophore A23187 as compared to control one without any significant difference between them). Irrespective to concentration, maximum HA% was detected in spermatozoa after 2 hour incubation. Calcium ionophore A23187 induced an immediate calcium uptake into both fresh and frozen thawed bull spermatozoa[43].

Regarding the effect on acrosome reaction, our results revealed that incomplete AR% was significantly increased corresponding to different concentrations of calcium ionophore A23187 with higher tendency to undergo complete and total AR% in spermatozoa treated with 1.55 mM/mL (over 90%) after 3 hour of incubation at 37 曟. These results are however, less comparable with the findings of [9, 44] who reported that the proportion of the population which had undergone a spontaneous acrosome reaction increased over 3-8 h, indicating a measure of asynchrony, whereas over 90% of the cells undergoing a reaction within 30min of exposure to calcium ionophore. On other hand, there was a marked interaction between concentration of A23187 and incubation time upon the number of spermatozoa having undergone AR. As the concentration of A23187 increased, the percentage of spermatozoa with intact (non-reacted) acrosomes decreased over the incubation time [41]. In conclusion, under our experimental conditions, the best concentration of heparin, caffeine and ionophore A23187 are 75 µg/mL, 1 mg/mL, 1.55 mM/mL for 3, 1,4 hours incubation respectively and can be used for in vitro fertilization in sheep.

Conflict of interest statement

The authors declare that there is no conflict of interests.

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1 August 2015

K.H.El-Shahat, Faculty of Veterinary Medicine, Giza, 12211Egypt.

Tel: 0201064688386

Fax: 0020235725240

E-mail: attiakh@yahoo.com

Received in revised form 10 October 2015 Accepted 12 November 2015

Available online 1 March 2016