Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: After the blocking of non-specific sites on the membrane with SuperBlock Blocking Buffer (ThermoFisher Scientific, Waltham, MA, USA) for 10 min with shaking, the melatonin receptors were immunodetected by incubating for 1 h at room temperature with the primary antibody Mel-1A-R rabbit polyclonal antibody against the MT1 receptor (GeneTex Inc., Irvine, CA, USA; Cat# GTX100003, RRID: AB_1241048) or rabbit polyclonal antibody against the MT2 receptor (Acris Antibodies, GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198), both diluted 1:500 in the Primary Antibody Diluent (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Immunostaining

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: After the blocking of non-specific sites on the membrane with SuperBlock Blocking Buffer (ThermoFisher Scientific, Waltham, MA, USA) for 10 min with shaking, the melatonin receptors were immunodetected by incubating for 1 h at room temperature with the primary antibody Mel-1A-R rabbit polyclonal antibody against the MT1 receptor (GeneTex Inc., Irvine, CA, USA; Cat# GTX100003, RRID: AB_1241048) or rabbit polyclonal antibody against the MT2 receptor (Acris Antibodies, GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198), both diluted 1:500 in the Primary Antibody Diluent (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Immunofluorescence, Incubation, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: After the blocking of non-specific sites on the membrane with SuperBlock Blocking Buffer (ThermoFisher Scientific, Waltham, MA, USA) for 10 min with shaking, the melatonin receptors were immunodetected by incubating for 1 h at room temperature with the primary antibody Mel-1A-R rabbit polyclonal antibody against the MT1 receptor (GeneTex Inc., Irvine, CA, USA; Cat# GTX100003, RRID: AB_1241048) or rabbit polyclonal antibody against the MT2 receptor (Acris Antibodies, GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198), both diluted 1:500 in the Primary Antibody Diluent (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Comparison

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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).

Article Snippet: After the blocking of non-specific sites on the membrane with SuperBlock Blocking Buffer (ThermoFisher Scientific, Waltham, MA, USA) for 10 min with shaking, the melatonin receptors were immunodetected by incubating for 1 h at room temperature with the primary antibody Mel-1A-R rabbit polyclonal antibody against the MT1 receptor (GeneTex Inc., Irvine, CA, USA; Cat# GTX100003, RRID: AB_1241048) or rabbit polyclonal antibody against the MT2 receptor (Acris Antibodies, GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198), both diluted 1:500 in the Primary Antibody Diluent (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Western Blot, Molecular Weight, Marker, Control

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Immunostaining

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Immunofluorescence, Incubation, Fluorescence

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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.

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Comparison

Journal: Animals : an Open Access Journal from MDPI

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

doi: 10.3390/ani15010083

Figure Lengend Snippet: 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).

Article Snippet: Following three additional PBS washes, the spermatozoa were incubated with the primary antibody for melatonin receptor MT1 (MTNR1A mouse polyclonal antibody; Abnova, Taipei, Taiwan; Cat# H00004543-A01, RRID: AB_462681) diluted 1:10 in PBS with 1% BSA, or the primary antibody for melatonin receptor MT2 (MTNR1B Rabbit Polyclonal Antibody, Acris Antibodies GmbH, Herford, Germany; Cat# AP01322PU-N, RRID: AB_1619198) diluted 1:20 in PBS with 1% BSA overnight at 4 °C in a wet chamber.

Techniques: Western Blot, Molecular Weight, Marker, Control

Journal: Acta Endocrinologica (Bucharest)

Article Title: MT2 INHIBITS OSTEOCLASTOGENESIS BY SCAVENGING ROS

doi: 10.4183/aeb.2023.447

Figure Lengend Snippet: Bone marrow OCPs in OVX mice had higher ROS level and lower MT2 expression. OVX-operated female mice were molded for 45 days. (A) Representative 3D Micro-CT reconstructed images of the tibiae from each group. Scale bar, 1 mm. (B) Representative H&E-stained tibial sections from each group. Scale bar, 20 μm. (C) The indicated bone marrow Ly6C+CD11b- cells sorted by FACS were incubated with DCFH-DA fluorescent probes, and cellular fluorescent intensities were detected (representative images shown). Scale bar, 20 μm. (D) MT2 fluorescent expression in bone marrow Ly6C+CD11b- cells was evaluated via immunofluorescence staining. Scale bar, 50 μm. (E-J) Micro-CT analyses showing trabecular bone parameters, including BMD, BV/TV, Tb.N, Tb.Th, BS/BV and Tb.Sp (N=6/group). (K) The trabecular bone area (%Tb.Ar) was analysed by H&E staining and using the IPP system (N=6/group). (L-M) The serum levels of TRAP-5b and CTX were detected via ELISA kits. (N) The histogram represents the quantitative results of ROS levels represented as the fold induction by normalizing the fluorescence value of OVX group to that of SHAM group. (O) NADPH oxidase activity in bone marrow Ly6C+CD11b- cells. (P) The histogram represents the quantitative results of MT2-positive cells in D (100 cells per field, n=2). These experiments were replicated at least three times. The data are expressed as the mean±SEM from three independent experiments. ***P<0.001 by Student's T Test. SHAM, sham operated mice; OVX, ovariectomized mice.

Article Snippet: For the immunofluorescence staining, the perforated OCPs were incubated with rabbit MT2 (PA5-76652, 1:200; ThermoFisher Scientific) at 4°C overnight, and then stained with fluorochrome-labelled secondary antibody for 1 hour.

Techniques: Expressing, Micro-CT, Staining, Incubation, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Fluorescence, Activity Assay

Journal: Acta Endocrinologica (Bucharest)

Article Title: MT2 INHIBITS OSTEOCLASTOGENESIS BY SCAVENGING ROS

doi: 10.4183/aeb.2023.447

Figure Lengend Snippet: MT2 overexpression inhibited osteoclastic differentiation while MT2 knockdown was contrary. (A) The protein expression of MT2 in OCPs transduced with corresponding lentiviruses. (B-D) After osteoclastic induction for 4 days in the presence of RANKL and M-CSF, the formation of differentiated osteoclasts (TRAP+ multinucleate cells) in OCPs transduced with corresponding lentiviruses was assessed via TRAP staining. The histogram in C represents the quantitative results of differentiated osteoclasts in B, and the histogram in D represents the quantitative results of large osteoclasts over 5 nuclei in B. (E-G) After osteoclastic induction for 4 days in the presence of RANKL and M-CSF, mRNA expression of osteoclastic genes (TRAP, CTSK and MMP9) were measured via qRT-PCR assays. These experiments were replicated at least three times. The data are expressed as the mean±SEM from three independent experiments. ***P<0.001 by Student's T Test. ns, not significant; LV-sh-Cont, lentivirus encoding control shRNA; LV-sh-MT2, lentivirus encoding shRNA targeting MT2; LV-Cont, lentivirus encoding control cDNA; LV-MT2, lentivirus encoding MT2-cDNA.

Article Snippet: For the immunofluorescence staining, the perforated OCPs were incubated with rabbit MT2 (PA5-76652, 1:200; ThermoFisher Scientific) at 4°C overnight, and then stained with fluorochrome-labelled secondary antibody for 1 hour.

Techniques: Over Expression, Knockdown, Expressing, Transduction, Staining, Quantitative RT-PCR, Control, shRNA

Journal: Acta Endocrinologica (Bucharest)

Article Title: MT2 INHIBITS OSTEOCLASTOGENESIS BY SCAVENGING ROS

doi: 10.4183/aeb.2023.447

Figure Lengend Snippet: MT2 overexpression inhibited ROS production in OCPs while MT2 knockdown was contrary. (A-B) The fluorescence intensity of lentiviruses-transduced OCPs incubated with DCFH-DA fluorescent probe (representative images shown) for 2 days under osteoclastic induction. Scale bar, 20 μm. ROS levels represented as the fold induction by normalizing the fluorescence value of other groups to that of LV-sh-Cont group. (C) NADPH oxidase activity in corresponding OCPs incubated for 2 days under osteoclastic induction. (D-G) The protein levels of Catalase, p47phox and p67phox in corresponding OCPs incubated for 2 days under osteoclastic induction. These experiments were replicated at least three times. The data are expressed as the mean±SEM from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 by Student's T Test. LV-sh-Cont, lentivirus encoding control shRNA; LV-sh-MT2, lentivirus encoding shRNA targeting MT2; LV-Cont, lentivirus encoding control cDNA; LV-MT2, lentivirus encoding MT2-cDNA.

Article Snippet: For the immunofluorescence staining, the perforated OCPs were incubated with rabbit MT2 (PA5-76652, 1:200; ThermoFisher Scientific) at 4°C overnight, and then stained with fluorochrome-labelled secondary antibody for 1 hour.

Techniques: Over Expression, Knockdown, Fluorescence, Incubation, Activity Assay, Control, shRNA

Journal: Acta Endocrinologica (Bucharest)

Article Title: MT2 INHIBITS OSTEOCLASTOGENESIS BY SCAVENGING ROS

doi: 10.4183/aeb.2023.447

Figure Lengend Snippet: The addition of H2O2 reversed the efficacy of MT2 overexpression on osteoclastic differentiation. (A-B) The fluorescence intensity of lentiviruses-transduced OCPs incubated with DCFH-DA fluorescent probe (representative images shown) following treatment with H2O2for 2 days under osteoclastic induction. Scale bar, 20 μm. ROS levels represented as the fold induction by normalizing the fluorescence value of other groups to that of LV-Cont group. (C) NADPH oxidase activity in corresponding OCPs treated with H2O2 for 2 days under osteoclastic induction. (D-F) After osteoclastic induction for 4 days in the presence of RANKL and M-CSF, the formation of differentiated osteoclasts (TRAP+ multinucleate cells) in corresponding OCPs treated with H2O2 was assessed via TRAP staining. The histogram in E represents the quantitative results of differentiated osteoclasts in D, and the histogram in F represents the quantitative results of large osteoclasts over 5 nuclei in D. These experiments were replicated at least three times. The data are expressed as the mean±SEM from three independent experiments. ***P<0.001 by one-way ANOVA and Bonferroni Post-Hoc multiple comparisons. LV-Cont, lentivirus encoding control cDNA; LV-MT2, lentivirus encoding MT2-cDNA.

Article Snippet: For the immunofluorescence staining, the perforated OCPs were incubated with rabbit MT2 (PA5-76652, 1:200; ThermoFisher Scientific) at 4°C overnight, and then stained with fluorochrome-labelled secondary antibody for 1 hour.

Techniques: Over Expression, Fluorescence, Incubation, Activity Assay, Staining, Control

Journal: Acta Endocrinologica (Bucharest)

Article Title: MT2 INHIBITS OSTEOCLASTOGENESIS BY SCAVENGING ROS

doi: 10.4183/aeb.2023.447

Figure Lengend Snippet: The working model regarding the role of MT2 in osteoclastogenesis. In brief, ROS is conducive to the differentiation of OCPs into mature osteoclasts. MT2 can scavenge ROS, which inhibits the promoting effect of ROS on osteoclastic differentiation.

Article Snippet: For the immunofluorescence staining, the perforated OCPs were incubated with rabbit MT2 (PA5-76652, 1:200; ThermoFisher Scientific) at 4°C overnight, and then stained with fluorochrome-labelled secondary antibody for 1 hour.

Techniques: