Melatonin in Male Dromedary Camel (Camelus dromedarius) Seminal Plasma and Its Specific MT1 and MT2 Receptors on Sperm Membranes

Abstract

Camels (Camelus dromedarius) are seasonal short-day breeders, regulated by photoperiod and melatonin secretion. However, no studies have explored melatonin levels in camel seminal plasma or their relationship with testosterone, age, or climatic factors, nor is it known whether melatonin receptors exist in camel spermatozoa to respond to seminal melatonin. This study aimed to analyze melatonin levels in camel seminal plasma and its specific receptors in spermatozoa. Semen samples were obtained from November to March (breeding season). Testosterone and melatonin levels were measured in seminal plasma by ELISA. Melatonin receptors were localized in spermatozoa using immunofluorescence, and their presence was confirmed by Western Blot. Melatonin levels were higher from November to January and decreased in February and March. No correlation between testosterone and melatonin levels was found, but both hormones were negatively correlated with daylength (p = 0.0089 and p = 0.0688, respectively). Testosterone, but not melatonin, levels were affected by age. Two melatonin receptors (MT1, MT2) were detected on camel spermatozoa, with several immunotypes labeled mainly in the tail and post-acrosome region, but also in the acrosome and neck. Western Blot analysis confirmed the presence of these receptors, showing a 39 kDa band for MT1 and a 36 kDa band for MT2. Understanding melatonin's effects on sperm could help ejaculates' processing procedures, semen handling, and infertility issues in camels.

Keywords: camel; humidity; melatonin membrane receptors; spermatozoa; testosterone.

Figure 1

Effects of age (A), month (B) and season of collection (C) on Log (melatonin) in camel seminal plasma. Values are shown as LSM ± SE of n =118. a, b indicates p < 0.05. Y: Young males; O: Old males; N–D: November–December; J: January; F: February; M: March; S1: Season 1 (2017–2018); S2: Season 2 (2018–2019); S3: Season 3 (2019–2020).

Figure 2

Effects of age (A), month (B) and season of collection (C) on testosterone in camel seminal plasma. Values are shown as LSM ± SD of n = 118. a, b indicates p < 0.05. Y: Young males; O: Old males; N–D: November–December; J: January; F: February; M: March; S1: Season 1 (2017–2018); S2: Season 2 (2018–2019); S3: Season 3 (2019–2020).

Figure 3

Distribution of MT1 melatonin receptors in camel spermatozoa, evaluated by the indirect immunofluorescence method. Immunostaining in the head (H), post-acrosome (PA), neck (N), tail (T) and cytoplasmic droplet (CD) was evidenced. Magnification 1000×. MT1 receptors (A,D,G,J), Hoechst staining (B,E,H,K), and bright field (C,F,I,L) are shown.

Figure 4

Distribution of MT2 melatonin receptors in camel spermatozoa, evaluated by the indirect immunoassay method. Immunostaining in head (H), acrosome (A), post-acrosome (PA), neck (N), tail (T) and apical edge (AE) was evidenced. Magnification 1000×. MT2 receptors (A,C,E,G) and bright field (B,D,F,H) are shown.

Figure 5

Indirect immunofluorescence controls. Samples were incubated with only the MT1 (A) or MT2 (C) primary or secondary antibody (E) and (G) for Alexa Fluor 594 anti-mouse antibody and Alexa Fluor 488 anti-rabbit antibody, respectively). Magnification 1000×. Fluorescence after 30 s exposition (A,C,E,G) and bright field (B,D,F,H) are shown.

Figure 6

Comparison among age classes of males at different melatonin receptors localizations (MT1 (A) and MT2 (B)) in acrosome (A), post-acrosome (PA), head (H), neck (N), tail (T), cytoplasmic droplet (CD) and apical edge (AE). Values are shown as percentage of localization of n = 439 spermatozoa. * indicates p < 0.05.

Figure 7

Representative Western blot images showing the presence of MT1 (A) and MT2 (B) melatonin receptors in protein extracts from camel spermatozoa (M: Molecular weight marker (kDa), C: Camel sperm proteins, (+): Positive ram control).