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Analysis of Epididymal Protein Synthesis and Secretion
Published on: August 25, 2018
Expression and subcellular localization of the mu-opioid receptor in equine spermatozoa: evidence for its functional
Maria Albrizio1, Antonio Ciro Guaricci, Filippo Maritato
1Department of Animal Production, University of Bari, Str. Prov. Casamassima Km 3, 70010 Valenzano, Bari, Italy. m.albrizio@veterinaria.uniba.it
Abstract:
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.
Insights
This study reveals the mu-opioid receptor in equine sperm, showing naloxone impacts sperm motility and capacitation. These findings offer new insights into sperm function and fertilization.
Area of Science:
- Reproductive Biology
- Molecular Endocrinology
- Sperm Physiology
Background:
- Sperm fertilizing ability involves plasma membrane changes, but receptor and signal pathways are unclear.
- The role of opioid receptors in sperm function remains largely unexplored.
Purpose of the Study:
- To investigate the expression and localization of the mu-opioid receptor in equine spermatozoa.
- To determine the effects of the opioid antagonist naloxone on equine sperm motility, viability, and capacitation.
Main Methods:
- Mu-opioid receptor transcript amplification, sequencing, and nucleotide comparison.
- Immunofluorescence for receptor localization and immunoblotting for protein identification.
- Computer-assisted sperm analysis and Hoechst 33258/chlortetracycline staining for functional assays.
Main Results:
- A conserved mu-opioid receptor transcript was identified in equine sperm.
- Immunolabeling showed mu-opioid receptor on sperm head and tail, absent in acrosome-reacted sperm.
- Naloxone affected sperm motility and significantly stimulated capacitation at a low concentration (10(-8) M), without impacting viability.
Conclusions:
- This study provides the first evidence of mu-opioid receptor expression in equine spermatozoa.
- Naloxone significantly influences equine sperm motility and capacitation, suggesting a role for mu-opioid signaling in fertilization.
