Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.

Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 µM VRP treatment and at 350 µM, all kinds of sperm motility ceased within 20 s as assessed by the Sander-Cramer assay. The spermicidal effect was confirmed by eosin-nigrosin assay and HOS test. VRP treatment (100 µM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 µM VRP. MTT assay showed after treatments with 100 and 350 µM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality.

A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO-cGMP pathway.

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 µM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility.

A novel membrane protein-specific serine/threonine kinase: tissue distribution and role in sperm maturation.

Our recent studies have described for the first time the purification of an ectoprotein kinase to apparent homogeneity using caprine sperm as the model. Purified ectokinase (CIK) is a novel membrane protein-specific kinase that phosphorylates serine and threonine residues of ectophosphoproteins. This study, using ELISA based on ecto-CIK antibody demonstrates that ecto-CIK level is remarkably higher in the sperm membrane than in the cytosol. The epididymal sperm maturational event as well as sperm vertical velocity is associated with a significant increase in the ecto-CIK level. Ecto-CIK, the membrane protein-specific kinase, is also present in all the tissues tested and is predominantly localized in the cell membrane. Ubiquitous localization of the novel kinase on the mammalian cell membrane suggests that the kinase may play pivotal role in gamete as well as somatic cell regulation by modulating membrane biology through serine/threonine phosphorylation of specific membrane proteins located in the ectodomains.

Sperm ecto-protein kinase and its protein substrate: novel regulators of membrane fusion during acrosome reaction.

Previously we have purified and characterized a unique plasma membrane-specific cyclic AMP-independent ecto-protein kinase (ecto-CIK) as well as its ecto-phosphoprotein substrate (MPS) using caprine sperm model. This study reports for the first time the role of the sperm external surface protein phosphorylation system on sperm acrosome reaction, which is essential for fertilization. Calcium ionophore A23187 has been used to trigger the sperm acrosome reaction in vitro. Treatment of sperm cells with CIK antibody (dil: 1:500) causes a significant decrease (approx. 50%) in percentage of acrosome reacted sperm. Onset of the acrosome reaction causes a remarkable increase in the rate of acrosin release from the cells in the medium. However, CIK antibody inhibits significantly (approx. 50%) the acrosin release. The level of membrane-bound MPS as estimated by ELISA and the degree of its phosphorylation catalyzed by the endogenous ecto-CIK, increase significantly with the progress of the acrosome reaction. Both the parameters increase by approximately 100% during the 20 min of the reaction. MPS antibody (1:100 dilution) markedly decreases (approx. 75%) percentage of acrosome-reacted sperm. MPS antibody as well shows high efficacy to inhibit acrosin release from spermatozoa. The results demonstrate that the cell-surface protein kinase and its protein substrate are essential for membrane fusion component of acrosome reaction. The data are consistent with the view that MPS regulates acrosomal membrane fusion with the overlying plasma membrane by the mechanism of its phosphorylation and dephosphorylation.

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2-ATPase isolated from goat spermatozoa.

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg2+ -independent Ca2+-ATPase without any significant effect on Mg2+-dependent Ca2+-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: a tribal medicine.

The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 micro/g/ml) and one n-butanol fraction (Fraction A; 100 microg/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50-60% of the spermatozoa lost their motility when treated with 600 microg/ml of methanol extract or 100 microg/ml of Fraction A. The Fraction A at 400 microg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 100 degrees C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains beta-sitosterol, a common phytosterol. The results demonstrate that Fraction A (beta-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive.

A potent sperm motility-inhibiting activity of bioflavonoids from an ethnomedicine of Onge, Alstonia macrophylla Wall ex A. DC, leaf extract.

The methanol extract (ME) and the n-butanol fractions of methanolic extract of Alstonia macrophylla Wall ex A. DC leaves were investigated on the forward motility (FM) of mammalian (goat and human) spermatozoa. The ME at 600 microg mL-1 as well as fraction B at 100 microg mL-1 concentrations showed marked inhibition of sperm FM in both goat and human species when tested by microscopic and spectrophotometric methods. Approximately 60-80% of the goat spermatozoa lost their FM when treated with 600 microg mL-1 of ME and 100 microg mL-1 of fraction B. At 100 microg mL-1 concentration, fraction B showed 90% loss of FM in human spermatozoa, while fraction B at 400 microg mL-1 concentration showed complete inhibition of sperm FM at 0 min. The inhibitory activity of fraction B increases with increasing concentration in a dose-dependent manner. Phytochemical study of the extract revealed that the leaf contains tannins, flavonoids, sterols, triterpenes, alkaloids and reducing sugars. Further fractionation and purification of the bioactive n-butanol part of ME showed the presence of ursolic acid (fraction B), beta-sitosterol (fraction A), beta-sitosterol glucoside and a mixture of minor compounds (fraction C, detected on thin-layer chromatography). The results reveal that fraction B (ursolic acid), a pentacyclic triterpene, has the potential of sperm motility inhibition and can serve as a topical vaginal contraceptive.