Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium–hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent K m for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.
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- Author: Maria Elena Dell’Aquila x
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Stefania Muzzachi, Lorenzo Guerra, Nicola Antonio Martino, Maria Favia, Giuseppe Punzi, Fabio Silvestre, Antonio Ciro Guaricci, Maria Teresa Roscino, Ciro Leonardo Pierri, Maria Elena Dell’Aquila, Valeria Casavola, Giovanni Michele Lacalandra, and Elena Ciani
Teresa De Santis, Valeria Casavola, Stephan Joel Reshkin, Lorenzo Guerra, Barbara Ambruosi, Nadia Fiandanese, Rozenn Dalbies-Tran, Ghylene Goudet, and Maria Elena Dell'Aquila
The extracellular calcium-sensing receptor (CASR) plays an important role in cells involved in calcium (Ca2 +) homeostasis by directly sensing changes in the extracellular Ca2 + ion concentration. We previously reported the localization and quantitative expression of CASR protein in human oocytes. In this study, we examined the expression and the functional role of CASR during oocyte meiotic maturation in a large mammal animal model, the horse. As in humans, CASR protein was found to be expressed in equine oocytes and cumulus cells. Western-blot analysis revealed a single 130 kDa band in denuded oocytes and a doublet of 130–120 kDa in cumulus cells. CASR labeling was observed by confocal microscopy in cumulus cells and in oocytes on the plasma membrane and within the cytoplasm at all examined stages of meiosis. Functionally, the CASR allosteric effector NPS R-467, in the presence of 2.92 mM external Ca2 +, increased oocyte maturation rate in a dose-dependent manner and its stimulatory effect was attenuated by pre-treatment with the CASR antagonist NPS 2390. NPS R-467 had no effect in suboptimal external Ca2 + (0.5 mM), indicating that it requires higher external Ca2 + to promote oocyte maturation. In oocytes treated with NPS R-467, CASR staining increased at the plasmalemma and was reduced in the cytosol. Moreover, NPS R-467 increased the activity of MAPK, also called ERK, in cumulus cells and oocytes. These results provide evidence of a novel signal transduction pathway modulating oocyte meiotic maturation in mammals in addition to the well-known systemic hormones.
Maria Albrizio, Antonio Ciro Guaricci, Filippo Maritato, Raffaele Luigi Sciorsci, Gaetano Mari, Giuseppe Calamita, Giovanni Michele Lacalandra, Giulio Guido Aiudi, Rosa Minoia, Maria Elena Dell’Aquila, and Paolo Minoia
The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the μ-opioid receptossr in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds μ agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, μ-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computer-assisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10−3 M naloxone (P < 0.05), whereas it increased significantly after 5 h in 10−8 M naloxone (P < 0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10−3 M naloxone (P < 0.05), but increased significantly in the presence of 10−8 M (P < 0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10−8 M naloxone significantly stimulated capacitation (P < 0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the μ-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.