Influences of dibutyryl cyclic adenosine monophosphate and forskolin on human sperm motility in vitro

Asian J Androl 2003 Jun; 5: 113-115             

Keywords: dibutyryl cyclic AMP; forskolin; sperm motility

Abstract

Aim: To study the influences of dibutyryl cyclic adenosine monophosphate (dbcAMP) and forskolin on human sperm motility in vitro. Methods: Semen samples, aseptically obtained by masturbation and prepared by swim-up technique from 20 fertile men, were incubated with different concentrations of dbcAMP and forskolin at 37 . Measurements were carried out after 10 min, 20 min, 30 min and 60 min incubation. Motility parameters were estimated by using an automatic analyzing system. Results: Treatment with dbcAMP or forskolin resulted in a significant increase in sperm motility and progressive motility. The larger the concentrations of dbcAMP or forskolin, the greater the effect appeared. The straight linear velocity and curvilinear velocity were not affected by both agents. Conclusion: dbcAMP and forskolin increase the motility and progressive motility of human sperm in vitro.

1 Introduction

Mammalian spermatozoa exhibit characteristic motility patterns after completion of capacitation. This whiplash or hyperactivated motility, crucial for both oviductal passage and zona pellucida penetration [1], was characterized by vigorous and high-amplitude flagellar beats [2].

Cyclic AMP (cAMP) stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. It had been proposed that elevated cAMP stimulated the phosphorylation of sperm proteins by protein kinase A (PKA), leading to increased motility[3, 4]. PK A-dependent phosphorylation was essential for mammalian sperm motility during epididymal maturation and for the maintenance of motility in mature sperm [5]. PKA activity had been shown to be increased during capacitation [6]. Pharmacological and biochemical studies have shown that the signal conduction mediated by PKA played a central role in regulating sperm motility [7]. cAMP analogues and phosphodiesterase inhibitors increased the motility of mature spermatozoa [8, 9]. Dibutyryl cyclic AMP (dbcAMP), an activator of PKA, promoted the motility and capacitation of macaque sperm [10]. Forskolin, a neutral adenylate cyclase activator, initiated the motility of bovine caput spermatozoa by elevating the cAMP level [11].

In the present study, the influences of different concentrations of dbcAMP and forskolin (a diterpene) on human sperm motility was studied by means of the computer-assisted semen analysis (CASA) technique [12, 13].

2 Materials and methods

2.1 Sperm suspension preparation

Healthy fertile men (n=20, 10 for dbcAMP and 10 for forskolin study), aged 20~35 (average 27.8) years, were enrolled. Ejaculates were obtained by masturbation after 5 days of abstinence into sterile plastic containers. The routine semen parameters were all within the normal range according to World Health Organization criteria [13].

Semen specimens were preprocessed by swim-up optimization technique described by Diemer et al.[14] to obtain highly motile spermatozoa, which were then diluted with the Ham's F-10 medium (Sigma, USA) to obtain a sperm suspension with a concentration of 4010⁶ mL^-1. The percentage of progressively motile spermatozoa (level a+b) should be >35 % and sperm motility, >65 %.

Sperm suspensions of 10 subjects were incubated with dbcAMP and the other 10 with forskolin (both drugs from Calbiochem Biochemicals, USA) in a 37 water bath for 1 h. Sperm suspension (100 mL) of every subject was placed into each of 4 sterile tubes marked A, B, C and D. In the dbcAMP experiment, tube A was added 100 mL of Ham's F-10 medium and served as the control (0 drug concentration). In tubes B, C and D, 100 mL of dbcAMP solution at 0.1 mmol/L, 0.5 mmol/L and 1.0 mmol/L, respectively were added. In the forskolin experiment, the control tube was similarly added the Ham's F-10 medium and the concentrations of forskolin solution (100 mL) added to tubes B, C and D were 0.01 mmol/L, 0.1 mmol/L and 1.0 mmol/L, respectively.

After 10 min, 20 min, 30 min and 60 min of incubation, the sperm motility parameters were estimated with a CASA system, a computer-analyzed multiple-exposure photography instrument (Madi Corp. Zhejiang, China). A 10-mL aliquot of sperm suspension was loaded into the counting chamber at 37 for analysis.

The following motion characteristics were recorded: curvilinear velocity (VCL, derived from 30 head positions), straight linear velocity (VSL, based on the first and last head positions), motility and progressive motility (grades a+b). Video recordings were made from eight different fields using a 20 magnification objective. Approximately 150 sperm were evaluated per drug concentration.

Data were expressed in meanSE. Multi variable analysis of variance using SAS statistical software was employed for data analysis. P<0.05 was considered significant.

3 Results

The effects of dbcAMP and forskolin on sperm motility after 20 min incubation were shown in Tables 1 and 2. It could be seen that compared with the controls, significant increases in the motility and progressive motility were observed in all the dbcAMP and forskolin concentrations (P<0.01). Analysis of linear correlation showed that with both drugs, the motility and the progressive motility had a positive correlation with the drug concentration. However, no significant difference in VSL and VCL was observed in all the groups compared with the controls (P>0.05).

Table 1. Effect of 20 min incubation with dbcAMP on sperm parameters (n=10). ^cP<0.01, compared with the controls.

Table 2. Effect of 20 min incubation with forskolin on sperm parameters (n=10). ^cP<0.01, compared with the controls.

The effects of 1.0 mmol/L of dbcAMP and forskolin on sperm parameters were presented in Tables 3 and 4. In both drugs, there was no significant difference (P> 0.05) in all these data as compared with the relevant controls regardless of the time of incubation.

Table 3. Effect of 1.0 mmol/L dbcAMP on sperm motility (n=10).

Table 4. Effect of 1.0 mmol/L forskolin on sperm motility (n=10).

4 Discussion

The cAMP-stimulated protein phosphorylation is essential for the initiation or maintenance of sperm motility[10] and the cAMP-dependent protein kinase (PKA) is a key enzyme in the control of human sperm activity. PKA-dependent protein phosphorylation is essential for the development of sperm motility during epididymal maturation and the maintenance of motility in mature sperm[15]. Thus, elucidation of the proteins involved in sperm cAMP-dependent control pathways is a major goal of current research in reproductive biology.

Recently, dibutyryl cAMP, an analogue of cAMP, has been employed in the study of second messenger and signal transduction in the regulation of sperm motility. Forskolin, a neutral adenylate cyclase activator, was indicated to reversibly stimulate adenylate cyclase. In the present study, the motility and the progressive motility, particularly in Group D, was significantly increased compared with the controls. Nevertheless, no expected increase in VSL and VCL was observed in all the experimental groups. Mahony et al. [16] demonstrated that in cynomolgus monkey (Macaca fascicularis) spermatozoa, capacitation and capacitation-related hyperactivated motility did not occur spontaneously; instead, both had an absolute requirement for exogenous stimulation with caffeine and dbcAMP and treatment with these agents resulted in an increase in VCL and flagella bending amplitude. Mahony et al.[17] studied an in vitro system in baboon that allowed the transport of sperm from a treatment facility to an off-site location for subsequent evaluation of functional capacity; they showed that treatment with the sperm activators, caffeine and dbcAMP, resulted in significant increases in sperm progressive motility, VSL, lateral head displacement (P<0.05) and VCL (P<0.01).

The effect of cAMP analogues on sperm intracellular cAMP level is not known and the sperm of different species may have different responses. The increase in intracellular cAMP level by forskolin and the effect of more potential cAMP analogues, e.g., 8-Br-cAMP and pCPT-cAMP, on human sperm motility may be worthy of further research. In conclusion, both dbcAMP and forskolin increased the motility and progressive motility of sperm in vitro, indicating that cAMP-dependent signal conduction may play an important role in the regulation of human sperm motility.

References

[1] Katz DF, Yanagimachi R. Movement characteristics of hamster and guinea pig spermatozoa upon attachment to the zona pellucida. Biol Reprod 1981; 25: 785-91.
[2] Yanagimachi R. In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod Fertil 1969; 18: 275-86.
[3] Ishiguro K, Murofushi H, Sakai H. Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa. J Cell Biol 1982; 92: 777-82.
[4] Lindemann CB. A cAMP-induced increase in the motility of demembranated bull sperm models. Cell 1978; 13: 9-18.
[5] Yeung CH, Weinbauer GF, Cooper TG. Responses of monkey epididymal sperm of different maturational status to second messengers mediating protein tyrosine phosphorylation, acrosome reaction, and mobility. Mol Reprod Dev 1999; 54: 194-202.
[6] Visconti PE, Johnson LR, Oyaski M, Fornes M, Moss SB, Gerton GL, et al. Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev Biol 1997; 192: 351-63.
[7] Vijayaraghavan S, Goueli SA, Davey MP, Carr DW. Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility. J Biol Chem 1997; 272: 4747-52.
[8] Tash JS, Means AR. Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod 1983; 28: 75-104.
[9] Lindemann CB, Kanous KS. Regulation of mammalian sperm motility. Arch Androl 1989; 23: 1-22.
[10] VandeVoort CA, Tollner TL, Overstreet JW. Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida. Mol Reprod Dev 1994; 37: 299-304.
[11] Vijayaraghavan S, Critchlow LM, Hoskins DD. Evidence for a role for cellular alkalinization in the cyclic adenosine 3?5?monophosphate-mediated initiation of motility in bovine caput spermatozoa. Biol Reprod 1985; 32: 489-500.
[12] Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH, et al. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Human Reprod 2000; 15: 1562-7.
[13] Liu JH, Li HY, Cao ZG, Duan YF, Li Y, Ye ZQ. Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro. Asian J Androl 2002; 4: 179-82.
[14] Diemer T, Weidner W, Michelmann HW, Schiefer HG, Rovan E, Mayer F. Influence of Escherichia coli on motility parameters of human spermatozoa in vitro. Int J Androl 1996; 19: 271-7.
[15] Jaiswal BS, Majumder GC. Cyclic AMP phosphodiesterase: a regulator of forward motility initiation during epididymal sperm maturation. Biochem Cell Biol 1996; 74: 669-74.
[16] Mahony MC, Gwathmey T. Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa. Biol Reprod 1999; 60: 1239-43.
[17] Mahony MC, Rice K, Goldberg E, Doncel G. Baboon spermatozoa-zona pellucida binding assay. Contraception 2000; 61:235-40.

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Correspondence to: Ji-Hong LIU, Ph.D., Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,China.
Tel: +86-27-8366 2278, Fax: +86-27-83662591
E-mail: jhliu888@hotmail.com
Received 2002-09-02      Accepted 2003-02-20