Fig 2: Real-time polymerase chain reaction (qPCR) analysis of (a) MT1 and (b) MT2 melatonin receptors from mRNA obtained from various tissues of the ram reproductive tract: Testis (1), caput (2), corpus (3) and cauda epididymis (4), ampulla (5), prostate (6), seminal vesicles (7), bulbourethral glands (8), and ductus deferens (9) . Ovine granulosa cells and ovine retina were used as positive control (+) for (a) MT1 and (b) MT2, respectively. Values of mRNA relative expression are shown as mean ± SEM of two males analyzed twice (n = 4). Each qPCR run was performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actine genes were used as housekeeping genes. Different letters a, b mean statistical differences of p < 0.05 (positive controls were only used for representative purposes, and were not included in the statistical analyses).

Fig 3: Immunohistochemical localization using the avidin-biotin complex technique of MT1 (arrows in panels a, d, g, j and m) and MT2 (arrows in panels b, e, h, k, and n) in testis (a–c), cauda epididymis (d–f), ampulla (g–i), seminal vesicles (j–l), and ductus deferens (m–o). Magnification 1000×. A 20 µm measurement bar is displayed in panel o. Negative controls are shown (panels c, f, i, l, and o).

Fig 4: Percentages of the different immunotypes, assessed by indirect immunofluorescence (IIF) against MT1 (a) and MT2 (b) melatonin receptors in spermatozoa obtained from the testis and the caput, corpus, and cauda epididymis. For the MT1 receptor, type I (labelling all over the head and tail), type II (equatorial and post-acrosomal regions, neck, and tail), type III (equatorial zone and tail), and type IV (staining only on the tail) were detected. For the MT2 melatonin receptor, type A (more intense staining at the acrosome than the post-acrosome), type P (more intense staining at the post-acrosome region than the acrosome), type AP (the same intensity of immunostaining at both acrosome and post-acrosome), and type N (staining only in the neck) spermatozoa were identified. Additionally, spermatozoa without immunoreactivity were recorded for both receptors. Results are shown as mean ± SEM, n = 5. Different letters (a, b, c) mean statistical differences of p < 0.05.