INTRODUCTION

Mimosine (MiMo), a major component of Leucaena leucocephala (Lamk.) de Wit (LL), has been demonstrated to have inhibitory property of many cancers, cell divisions, and cell proliferations (^{Adeneye, 1991}; ^{Hughes & Cook, 1996}; ^{Krude, 1999}; ^{Kubota et al., 2014}). Additionally, previous studies showed that MiMo has the herbicidal, insecticide, and nematicide activities (^{Tawata, 1990}; ^{Nguyen & Tawata, 2015}). Such activities could be explained that MiMo has ability to arrest the cell cycle progression to inhibit cell proliferation and differentiation (^{Chung et al., 2012}; Park et al., 2012; ^{Bottini-Luzardo et al., 2015}; ^{Fallon, 2015}; Nguyen & Tawata). Therefore, LL plant containing MiMo has been reported to be toxic to cattle and poultry animals in growth retardation, sub or infertility, and morbidity (^{Wayman et al., 1970}; ^{Hammond, 1995}; ^{Anderson et al., 2001}). Indeed, ^{Burawat et al. (2016}) have reported the presence of MiMo in crude LL leave extract and demonstrated its adverse effects on male reproductive organs including seminal vesicle (SV). Moreover, a previous study reported that acute treatment with purified MiMo for 7 consecutive days affected testicular, epididymal, and SV morphologies with significant reduction of sperm count (^{Kanla et al., 2018}). Such results suggested that MiMo could directly inhibit spermatogenesis and testosterone production. It is known that male infertility is associated with SV secretion impairments especially magnesium and fructosamine deficiencies (^{Abou-Shakra et al., 1989}; ^{Tomaszewski et al., 1992}; ^{Omu et al., 2001}; ^{Wong et al., 2001}). Although atrophy of SV treated with MiMo was observed (Burawat et al., 2016; Kanla et al.), its function such as electrolyte and protein secretion has never been reported. Essential secretions of SV may include various functional proteins including tyrosine phosphorylated (TyrPho) proteins (^{Camargo et al., 2018}; ^{Druart & de Graaf, 2018}). Particularly, TyrPho proteins have been localized in testicular and epididymal tissues (^{Chaichun et al., 2017}; ^{Sawatpanich et al., 2018}) but in SV have never been reported. Moreover, TyrPho proteins have been reviewed to play important roles in sperm capacitation and acrosome reaction (^{Seshagiri et al., 2007}; ^{Ickowicz et al., 2012}). Taken together, this study attempted to investigate the effects of purified mimosine on secretion of seminal vesicle in male mice.

MATERIAL AND METHOD

Mimosine preparation. Purified L-Mimosine powders were purchased from Sigma-Aldrich, Lot# 077K7007V, USA. The mimosine powder (1 g) was mixed with 0.1 N HCl (pH 7) to prepare stock solution.

Animals and treatments. ICR male mice (8 weeks) were purchased from the Animal Laboratory Unit, Faculty of Medicine, Khon Kaen University, Thailand. Animals were housed in plastic cages under a 12 h light/dark cycle at room temperature. This study was approved by the Animal Ethics Committee of KKU, based on the Ethics of Animal Experimentation of the National Research Council of Thailand (ref. No.0514.1.12.2/93). All mice were divided into 4 groups (n =8); in control group, mice were i.p. injected with 0.1 N HCl while experimental groups (mimosine 15, 30, and 60), animals were injected (i.p.) with mimosine at 15, 30, and 60 mg/KgBW, for 35 consecutive days. At the end of experiment, all mice were euthanized and sacrificed to collect seminal vesicles.

Seminal weight analysis and gross morphology. The seminal vesicles (SV) from control and minosine treatedanimals were collected. The fat pads surrounding SV were gentle removed before weighing as absolute organ weight. Then, SVs were calculated as individual relative organ weights using a formulation of one hundred multiplied by the absolute weight of SV and divided by body weight (g of SV/100 gBw). After weighing the SV, the representative SV of control and mimosine treated groups were grossly observed for their sizes and morphology before captured by digital camera (Nikon Coolpix S2600, Japan).

Assay of magnesium and fructosamine levels in seminal fluid. To prepare seminal fluid, the fresh seminal vesicle was punctured using a micropipette tip and transferred into an eppendorf tube. Then, PBS was added into the seminal fluid and sonicated by ultrasonic homogenizer on ice. Subsequently, the diluted seminal fluid was centrifuged at 15,000 rpm for 15 min at 4 °C to separate the seminal supernatant fluid from the pellet. The magnesium and fructosamine levels in seminal supernatant were measured using the colorimetric method. Phosphorus was measured with molybdate UV. PSA was measured by electrochemiluminescence immunoassay (ECLIA).

Western blot. Total protein concentration of seminal vesicle lysate was measured by using NanoDrop 2000 Spectrophotometers (Thermo Scientific) at an absorbance of 280 nm. Eighty micrograms of seminal vesicle lysate was separated on a 12 % acrylamide gel by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDSPAGE) and transferred onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The transferred protein membrane was incubated with 3 % skim milk in 0.1 PBST (phosphate buffer solution, pH 7.4, 0.1 % [v/v] Tween 20) for 1 hr. Then, the seminal membranes were incubated with antiphosphotyrosine (1:1,000 (v/v); Millipore Sigma) at 4 °C overnight. After washing the non-binding primary antibody with 0.05 % PBST, the membranes were incubated with HRP-conjugated secondary antibody (goat anti-mouse IgG, Santa Cruz) at 25 °C for 1 h and washed with 0.05 % PBST for 5 min (three times). The positive protein expressions on the membrane were detected by enhanced chemiluminescence (ECL) substrate reagent kits (GE Healthcare, Chicago, Il, USA) under an Amersham Imager 600 (GE Healthcare). To confirm the real reactivity of anti TyrPho antibody, the epidermal growth factor stimulated A413 cell lysate (EGF; Millipore CO., USA) and bovine serum albumin (BSA; Millipore CO., USA) were used as positive and negative controls, respectively.

Statistical analysis. All data are expressed as mean± standard deviation (S.D.). The one way analysis of variance (ANOVA) was performed to examine the significant differences among sets of data using SPSS statistics 19.0 software. P<0.05 was considered for significant difference.

RESULTS

Effect of mimosine on weight of seminal vesicle (SV). After injection with purified mimosine for 35 consecutive days, the results showed that the absolute weights of mouse SV treated with mimosine (15, 30, or 60 mg/KgBw) were significantly decreased in dose dependent manner from that of control mice (Fig. 1A, left panel). However, the SV relative weights in only 30 and 60 mg/KgBw groups were significantly decreased as compared to the control (Fig. 1A, right panel). This result is corroborated with its representative morphology as shown in Figure 1B.

Fig. 1 Comparisons of seminal vesicles (SV) absolute and relative weights of control and mimosine-treated groups (15, 30, and 60 mg/KgBw, respectively) in 35 consecutive days (A). Representative SV morphology of control and mimosine groups (B). Each data point represented as means ± S.D (n = 8). * Significant difference (p < 0.05) compared to the control. 

Magnesium and fructosamine levels in SV fluid. The results showed that the magnesium and fructosamine levels in SV fluid in mimosine treated mice in 3 different doses were not significantly different when compared to the control (Figs. 2 A,B)

Fig. 2 Showing the levels of magnesium (A) and fructosamine (B) in seminal fluid of control and mimosine-treated mice (15, 30, and 60 mg/KgBw, respectively). Each data point represented as means ± S.D. (n = 8). 

Effects of mimosine (MiMo) on SV tyrosine phosphorylated (TyrPho) protein expression. The total protein profiles revealed by SDS-PAGE and expression of TyrPho proteins detected by western blot of SV protein lysate compared among control and MiMo treated groups were shown in Figure 3. The result showed that the protein profiles of all groups were relatively similar (Fig. 3A). However, it was obviously found that only a 130 kDa protein in SV lysate of the high-dose MiMo group (60 mg/KgBw) was undetectable as compared to the rest groups (Fig. 3A). Moreover, the 72 and 55 kDas TyrPho proteins were expressed in control and MiMo- treated seminal vesicles (Fig. 3B). Obviously, the expression of such TyrPho proteins in SV lysate were increased with dose dependent manner treated with purified MiMo (15, 30, and 60 mg/KgBw, respectively) as demonstrated in Figure 3B.

Fig. 3 Showing total protein profiles by SDS-PAGE (A) and expression of tyrosine phosphorylated (TyrPho) proteins detected by western blot in SV lysate (B) of control and mimosine-treated mice (15, 30, and 60 mg/KgBw, respectively) the epidermal growth factor (EGF) and bovine serum albumin (BSA) were used as positive and negative controls, respectively. 

DISCUSSION

Mimosine (MiMo) has been proven to have activities against many cancers, cell divisions, cell proliferations and differentiations (^{Wang et al., 1995}; Hughes & Cook; Krude). Indeed, MiMo is a toxic substance in Leucaena leucocephala (LL) (^{Zayed et al., 2014}; ^{Burawat, 2017}; Burawat et al., 2016, 2018). By using Thin Layer Chromatography (TLC) with method validation, Burawat et al. (2018) have demonstrated for the first time that MiMo levels in aqueous crude extract of LL shoot tips plus young leaves cultured in Northeastern, Thailand were approximately 17.35 % dry crude extract. Particularly, both aqueous crude extract of LL shoot tips plus young leaves and purified MiMo have previously reported to have adverse effects on male reproductive mouse organs such as testis, epididymis, and seminal vesicle (Burawat et al., 2018; Kanla et al.). It has been shown that acute effect of MiMo treatment for 7 days could damage testicular tissue which may affect the sperm production resulted in significant decreasing of epididymal sperm concentration (Kanla et al.). Herein, our study has demonstrated the effect of MiMo treatment for 35 days on seminal vesicle for the first time. We have hypothesized that the levels of magnesium and fructosamine in MiMo treated mice might be decreased and associated with adverse reproductive parameters as previously reported (Burawat et al., 2018; Kanla et al.). Moreover, such levels have been reported to relate with male infertility (AbouShakra et al.; Tomaszewski et al.; Omu et al.; Wong et al.). Unfortunately, our results showed no difference in those levels among control and MiMo mice. In contrast, we found that MiMo at a dose of 60 mg/KgBw affected the decreased expression of a 130 kDa protein in SV lysate as compared to the two lower groups. Moreover, the unknown TyrPho proteins of the 72 and 55 kDas were investigated in seminal lysate for the first time but their expression were dose-dependently increased in purified MiMo groups (15, 30, and 60 mg/KgBw, respectively). Since seminal proteins including TyrPho proteins have been reported to be important in sperm capacitation and acrosome reaction before fertilization process (^{Aitken et al., 2007}; Seshagiri et al.; Ickowicz et al.), the changes of protein expressions in MiMo treated SV may be involved in some sperm physiology in female tract. However, the characterization and functional roles of the 72 and 55 kDas TyrPho proteins in MiMo treated SV need to be further elucidated. In conclusion, purified MiMo decreased the SV weights and increased the expressions of TyrPho proteins in seminal fluid. This finding may support the effects of mimosine in Leucaena leucocephala on adverse male reproductive parameters of domesticated animals.