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.

Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.

Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4µmol/mL following incubation for 1.5h. Notably, all parameters were potently inhibited by 10µmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10µmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.

2-APB-potentiated channels amplify CatSper-induced Ca(2+) signals in human sperm.

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pre-treatment with 5 µM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 µM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50-100 µM 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50-100 µM 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.

Secretory phospholipase A2 group IID Is involved in progesterone-induced acrosomal exocytosis of human spermatozoa.

Phospholipase A2 (PLA(2)) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA(2) subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA(2) Group IID (sPLA(2)-IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA(2)-IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography-mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer-assisted sperm analysis. sPLA(2)-IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16-kd protein band was detected by Western blotting in sperm extracts. Progesterone-induced AE was significantly inhibited in a concentration-dependent manner using a sPLA(2)-IID neutralizing antibody. The increase in AA levels seen during progesterone-stimulated exocytosis was significantly abrogated by the antibody. The sPLA(2)-IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA(2)-IID is present at the head and midpiece in the human sperm, and activation of such sPLA(2)-IID seems to be involved in AE. Therefore, sPLA(2)-IID isoform plays a functional role during the AE in human sperm.

ATP-activated P2X2 current in mouse spermatozoa.

Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca(2+) entry via cation channel of sperm (CatSper) Ca(2+)-selective ion channels in the sperm tail. Ca(2+) entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K(+) channel also present in the sperm tail. To date, no transmitter-mediated currents have been reported in sperm and no currents have been detected in the head or midpiece of mature spermatozoa. We screened a number of neurotransmitters and biomolecules to examine their ability to induce ion channel currents in the whole spermatozoa. Surprisingly, we find that none of the previously reported neurotransmitter receptors detected by antibodies alone are functional in mouse spermatozoa. Instead, we find that mouse spermatozoa have a cation-nonselective current in the midpiece of spermatozoa that is activated by external ATP, consistent with an ATP-mediated increase in intracellular Ca(2+) as previously reported. The ATP-dependent current is not detected in mice lacking the P2X2 receptor gene (P2rx2(-/-)). Furthermore, the slowly desensitizing and strongly outwardly rectifying ATP-gated current has the biophysical and pharmacological properties that mimic heterologously expressed mouse P2X2. We conclude that the ATP-induced current on mouse spermatozoa is mediated by the P2X2 purinergic receptor/channel. Despite the loss of ATP-gated current, P2rx2(-/-) spermatozoa have normal progressive motility, hyperactivated motility, and acrosome reactions. However, fertility of P2rx2(-/-) males declines with frequent mating over days, suggesting that P2X2 receptor adds a selection advantage under these conditions.

Functional deficiencies and a reduced response to calcium in the flagellum of mouse sperm lacking SPAG16L.

The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action. In addition, the passive flagella, inhibited by sodium metavanadate to disable the dyneins, were examined for mechanical abnormalities. Live Spag16L-null sperm exhibited much less bending of the flagellum during the beat. The amount of microtubule sliding in the R-bend direction of the beat was selectively restricted, which suggests that there is limited activation of the dyneins on one side of the axoneme in the live cells. This is corroborated by the results on detergent-extracted sperm models. The flagellar response to calcium is greatly reduced. The calcium response requires the activation of the dyneins on outer doublets 1, 2, 3, and 4. These are the same dyneins required for R-bend formation. In axonemes prepared to disintegrate by microtubule sliding, we observed little or no extrusion of doublets 1 and 2, consistent with a reduced activity of their dyneins. This deficit in motor function, and an increased rigidity of the midpiece region which we detected in the passive flagella, together can explain the observed motility characteristics of the Spag16L-null sperm.