Dep. of Animal Production, University of Bari, Bari; 1Dep. of Surgery, Physiopathology and Clinic of Domestic Animals Reproduction, University of Messina
Many physiological and pathological conditions are controlled by the fluctuation of endogenous opioid peptides whose action accomplishes by their link to opioid receptors. The present study was carried out to evaluate the presence of the µ-opioid receptor on cat epididymal sperm cells.
Materials & Methods
Sperm cells collected from epididymis obtained from orchiectomized toms were normal for motility and morphology. Spermatozoa were fixed in 4% (v/v) paraformaldehyde, incubated in PBS-1%BSA and let to react in a 1:2500 dilution of primary rabbit polyclonal antibody against rat 3rd extracellular loop of µ-opioid receptor. The antibody was omitted in control samples. The FITC-conjugated-IgG-secondary antibody diluted 1:200 was added and after incubation, samples were smeared on slides. Duplicate slides were prepared, assembled with mounting medium and observed under a fluorescent microscope (1000x magnification).
µ-opioid receptors were evidenced as a light green stain on the surface of the anterior region of the head, on the neck and on the final segment of the sperm tail. No staining was detected in negative controls incubated with the FITC labeled secondary antibody.
The immunocytofluorescence method is specific and reliable to study distribution and expression of opioid receptors on cats spermatozoa and may be useful to detect abnormal distribution or absence of the µ-opioid receptor on spermatozoa.