Expression and subcellular localization of the mu-opioid receptor in equine spermatozoa: evidence for its functional role.
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 mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu 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, mu-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 mu-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.
Albrizio
Maria
M
Department of Animal Production, University of Bari, Str. Prov. Casamassima Km 3, 70010 Valenzano, Bari, Italy. m.albrizio@veterinaria.uniba.it
Guaricci
Antonio Ciro
AC
Maritato
Filippo
F
Sciorsci
Raffaele Luigi
RL
Mari
Gaetano
G
Calamita
Giuseppe
G
Lacalandra
Giovanni Michele
GM
Aiudi
Giulio Guido
GG
Minoia
Rosa
R
Dell'Aquila
Maria Elena
ME
Minoia
Paolo
P
D020101:Acrosome Reaction; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique; D006736:Horses / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D051381:Rats; D017450:Receptors, Opioid, mu / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013075:Sperm Capacitation / Q000187:drug effects; D013077:Sperm Head / Q000737:chemistry / Q000378:metabolism; D013081:Sperm Motility / Q000187:drug effects; D013082:Sperm Tail / Q000737:chemistry / Q000378:metabolism; D013083:Sperm Transport / Q000187:drug effects; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling
D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D009270:Naloxone