Human sperm motility stimulating activity of a sulfono glycolipid isolated from Sri Lankan marine red alga Gelidiella acerosa

¹Natural Products Development Group, Industrial Technology Institute (CISIR), Colombo  7, Sri Lanka
²Department of Zoology & ³Chemistry, University of Colombo, Colombo  03, Sri Lanka
⁴HEJ Research Institute of Chemistry, University of Karachi, Karachi 32, Pakistan

Asian J Androl  2001 Mar; 3: 27-31

Keywords: sperm motility;  stimulants; reproduction; sulfono glycolipids; red alga; Gelidiella acerosa

Abstract

Aim: To evaluate the sperm motility stimulating activity of a sulfono glycolipid (S-ACT-1) isolated from Gelidiella acerosa, a Sri Lankan marine red algae. Methods: S-ACT-1, a white amorphous powder was separated from more polar fractions of the hexane soluble of 1:1 CH₂Cl₂/MeOH extract and subjected to ¹H, ¹³C NMR and IR Spectroscopy after reverse phase HPLC for identification. Effects of S-ACT-1 on human sperm motility was assessed in vitro at 10, 100 and 1000 g/mL concentrations at 37 for 0, 5, 15, 30 and 60 min. Results: S-ACT-1 was identified as a glycolipid sulfate. The lower dose increased the sperm motility slightly, whilst the medium dose significantly increased the motility (P<0.05) from 5 min of incubation reaching a peak at 15 min and  the stimulant effect was sustained throughout the experimental period. Furthermore, the medium dose rendered 80% of the immotile viable sperm motile. In contrast, the highest dose impaired the sperm motility.  The sperm stimulating activity of S-ACT-1 was dose-dependent and had a bell-shaped dose response curve for all the 5 incubation periods. Conclusion: S-ACT-1 of Gelidiella acerosa is a Sulfono glycolipid. S-ACT-1 has a potent sperm motility stimulating activity in vitro and has the potential to be developed into a sperm stimulant.

1 Introduction

The potential of natural marine products for the development of  bio-active agents is enormous^[1].  Red algae (Rodophyta), with over 555 genera^[2] is virtually an untapped resource for bio-active substances.  So far, we have reported several biological activities of different red algal extracts: the antihypertensive^[3] and antiimplantation^[4] activity of Gracilaria corticata,  the gastroprotective activity of Jania sp^[5] and the contraceptive activity of Gelidiella acerosa^[4].

2 Materials and methods

2.1 Collection and extraction of algae

Fresh specimen of Gelidiella acerosa (family Gelidiaceae) were collected from the coastal rocky reef of Beruwala in southern Sri Lanka.  The identity of algae was authenticated by Ms. P.M.A. Jayasooriya, National Aquatic Resources Agency, Sri Lanka and the Late Professor S. Balasubramaniam, Department of Botany, University of Peradeniya, Sri Lanka.  A voucher specimen is on deposit at the museum, Department of Zoology, University of Colombo, Sri Lanka, under the registration number of RA7.  The collected samples were carefully separated from other associated organisms (polychaets, small crustaceans and other macroscopic algae) and debris, shade dried for 24 h and stored in 1:1 CH₂Cl₂/MeOH solvent mixture (Petroleum Corporation, Colombo, Sri Lanka).  After 14 days, the solvent was decanted, filtered and concentrated under vacuo at 301 to obtain reddish-brown gummy crude extract (CE).

2.2 Isolation of Bio-active compound S-ACT-1

CE 100 g was dissolved in 10% H₂O/MeOH and partitioned into hexane (Petroleum corporation, Colombo, Sri Lanka).  Hexane soluble fraction (50 g) was then subjected to gel filtration column chromatography over Sephadex LH₂₀ (Pharmacia, Piscataway, Sweden) eluting with 1:1 CH₂Cl₂/MeOH.  Fractions collected were pooled according to their similar TLC (on pre-coated silica gel ⁶⁰HF₂₅₄ plates; Merk, Darmatadt, Germany) patterns to yield 6 fractions (F₁-F₆) (see scheme 1).  Fraction F₅ was further fractionated over Bio-Sil (200-400 mesh, Bio-Rad, Richmond, USA) column eluting with a gradient of MeOH/CH₂Cl₂  (0%30%) to result in 4 different fractions (F₁-F₄) according to similar TLC patterns. S-ACT-1 containing fraction F₄ (single bluish green spot for vanillin-sulfuric in HPTLC, Merk, Darmatadt, Germany) was chromatographed on HPLC (Waters model 510, Milford, USA) over reverse phase C₁₈ -Bonda pack column (Waters, USA) eluting with 15% H₂O/MeOH (BDH, Poole, England) (flow rate=2.5 mL/min) to afford S-ACT-1 (major peak).  Solvents of the major peak were evaporated to yield S-ACT-1 as a white amorphous powder.

Scheme 1.  Isolation scheme of S-ACT-1 from crude extract of Gelidiella acerosa.

2.3 Spectroscopy

¹H and ¹³C NMR for S-ACT-1 were recorded with a 500 MHz Bruker NMR machine (Bruker, Falladen, Switzerland) in CD₃OD. IR spectrums were recorded in KBr with a JASCO, FT/IR 5300 spectrophotometer  (JASCO, Tokyo, Japan).

2.4 Semen samples

Semen samples were obtained from healthy adult donors through masturbation and kept in sterilized containers.  The samples were allowed to liquefy for 15-30 min and those liquefied within this period of time were subjected to standard semen analysis^[6].  Ejaculates (n=6) with a volume of>1.5 mL, sperm concentration >4010⁶ spermatozoa/mL, >40% motile sperm and >60% normal morphology sperm were used.

2.5 Evaluation of sperm motility stimulating activity

Dissolved 1 mg of S-ACT-1 in 500 L normal saline (0.9% NaCl, w/v) and stored at 4 until use.  The semen samples were diluted with normal saline to obtain the desired concentration (4010⁶ spermatozoa/mL). Three concentrations (10, 100, 1000 g/mL) of S-ACT-1 were prepared from the stock solution immediately before use.  One hundred L aliquots of semen were placed in clean glass vials and equal volumes of test solutions or vehicle (normal saline) were added, mixed well. Immediately after mixing (0 min) and at 5, 15, 30 and 60 min of incubation, 10 L aliquots were transferred onto clean slides, covered with 2222 mm cover glass. The number of motile cells was counted under phase contrast optics (400 magnification) at 37 using squared grid eye piece graticule.  Results were expressed as number of motile cells per 100 cells.

2.6 Evaluation of sperm viability

The viability of spermatozoa was assessed using 0.5% Eosin-Y (Sigma, St. Louis, USA) immediately before mixing with S-ACT-1 and after 15 min of incubation with 100 g/mL S-ACT-1, according to the method described by Jeyendran  et al^[7].

2.7 Statistical analysis

3 Results

3.1 Isolation

Yield of the CE was 2.71 g/kg. Fraction F₅ resulted from column chromatography over Sephadex LH₂₀ was a yellowish white solid.  HPTLC of F₅ developed in 15% MeOH/CH₂Cl₂ had a cluster of spots within Rf=0.0-0.3.  Bluish green spot for vanillin sulfuric at Rf-0.2 was the major component.  This was concentrated to fraction F₄ which resulted from gradient elution of fraction F₅ with 0-30% MeOH/CH₂Cl₂ over Biosil.  Reverse phase HPLC of F₄ had one major peak (S-ACT-1) and a minor peak.

3.2 Partial characterization

For S-ACT-1, ¹H NMR had 6H triplet at 1.3 (ca. 48 H; for long alkyl chains) and mass of signals between 2.5 & 5.0 (for sugar protons). ¹³C NMR had 1 signal at 99.9 (for anomeric carbon) and 2 signals at  174.85 & 175.04 (for ester carbonyl groups).  IR spectrum had prominent bands at 1178 & 1035 cm^-1 (for S=O stretchings).  These spectral evidences reveal that S-ACT-1 is a sulfated glycolipid.

3.3 Sperm motility

Results of the sperm motility experiment are depicted in Figure 1.  S-ACT-1 increased the number of motile cells significantly at 100 g/mL concentration level.  This increment in motility was significant from the 5th min and lasted throughout the incubation period.  Although not quantified, a marked increment in forward velocity was also evident at this concentration. A similar trend was seen at the 10 g/mL concentration, however, the stimulation was statistically insignificant.  In contrast, with the 1000 g/mL concentration, an apparent but insignificant reduction in motility could be observed.  The dose response curve for all 5 incubation periods had a bell shape distribution (Figure 2).  Irrespective of the initial value, all six sperm samples reached their maximum motility by 15 min of incubation at the 100 g/mL concentration.   Furthermore, motility increment from 0 to 5 min was rapid compared to that  from 5 to 15 min in all the samples tested.

Figure 1.  Effect of S-ACT-1 on motility of human spermatozoa in vitro (time-response curve). ♦=Control; =10 g/mL; =100 g/mL; =1000 g/mL (meanSEM, n=6). ^bP<0.05.
Figure 2.  Effect of S-ACT-1 on motility of human spermatozoa in vitro (concentration-response curve). ◊=0 min; =5 min; =15 min; ♦=30 min; =60 min (meanSEM, n=6).

3.4 Sperm viability

4 Discussion

The results of this study demonstrated that S-ACT-1, isolated from Sri Lankan red alga, Gelidiella acerosa, is a sulfated glycolipid and this compound markedly increased the motility of human spermatozoa in vitro. This is an important finding that may have therapeutic and pharmaceutical potentials.

The stimulatory effect on sperm motility was evident within 5 min of incubation.  This effect appeared to be concentration-dependent and displayed a bell shape distribution. An obvious increment in the forward velocity was also evident at the intermediate concentration.

Incubation of sperm with S-ACT-1 induced 80% of immotile viable sperm motile.  Similar triggering of sperm motility has been demonstrated with caffeine (65-75% by Ratnasooriya  et al^[8]) a known sperm motility stimulant^[9].  However, with caffeine the maximum sperm motility stimulation is achieved instantaneously whereas with S-ACT-1, it is achieved after 15 minutes of incubation.  It is worthwhile to note that the effective concentration of caffeine and pentoxifylline (the currently used motility stimulant) required for maximal motility stimulation is 2.5 mmol/L^[9] and 3.6 mmol/L^[10], respectively, whereas that of S-ACT-1 is only 0.123 mmol/L (molecular weight of S-ACT-1: 816)^[11]. Accordingly, as a human sperm motility stimulant, S-ACT-1 is approximately 20 and 28 times more potent than caffeine and pentoxifylline, respectively.              

The precise mode of action of the sperm stimulating effect of S-ACT-1 is unknown. However, it appears to be receptor mediated, due to a rapid onset of action^[12] and a bell-shaped dose-response curve^[13].  Glycosaminoglycans (GAGs) are natural sperm motility promoters and capacitating agents found in the mammalian reproductive systems^[14]. They are highly sulfated glycoconjugates^[14].  GAGs promote motility through increment in intracellular cAMP concentration via enhancement of adenylate cyclase activity^[14,15]. Since S-ACT-1 is also a sulfated glycoconjugate, its sperm motility promoting activity could also be mediated via a similar mechanism.

Acknowledgements

Financial assistance from grants AP/3/ii/88/S/12 of University of Colombo and NRC-99.31 of National Research Council of Sri Lanka. UNESCO/ICSU for short-term fellowship and NARESA/SAREC for travel grant to Dr.G.A.S. Premakumara.

References

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[4] Ratnasooriya WD, Premakumara GAS, Tillekeratne LMV. Post-coital contraceptive activity of crude extract of Sri Lankan marine red algae. Contraception 1994; 50: 291-9.
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[19] Ricker DD, Minhas BS, Kumar R, Robertson JL, Dodson MG. The effects of platelet-activating factor on the motility of human spermatozoa. Fertil Steril 1989;  4: 399-410.
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Correspondence to: Professor W.D.Ratnasooriya, Department of Zoology, University of Colombo, Colombo 03, Sri Lanka. 
Tel: +94-1-503 399        Fax: +94-1-503148
e-mail: gasp@iti.lk
Received 2000-10-08     Accepted 2001-02-20