Hamsters
Thirty-five adult hamsters (Mesocricetus auratus) were housed under 12 h/12 h light/dark cycle conditions with water and fed ad libitum (male = 15 and female = 20). The animals were divided into seven groups: intact males (n = 5), males castrated seven days ago (n = 5), males castrated seven days ago and intra-muscularly injected (10 µg) daily with dihydrotestosterone (DHT) or the vehicle alone (50 µl corn oil) (n = 5). The four stages of the estrous cycle [proestrus (P; n = 5), estrus (E; n = 5), metestrus (M; n = 5), and diestrus (D; n = 5)] were determined in female animals using vaginal smears; groups of 20 animals in each stage were defined. The male hamsters were anaesthetized with ketamine:xylazine (80 mg/kg:8 mg/kg, intramuscularly) prior to gonadectomy and decapitated 24 h after the last injection. A 1.5-cm incision was made at the scrotum, and the testes were exposed, ligated, extracted, and then the wound was sutured closed. Several tissues were immediately removed, frozen on dry ice, and stored at − 70°C until the experiments were performed. I used the HGs of the males and females under different endocrine conditions to determine sex steroid-dependent effect on MafB expression. I collected the animals’ HG, lungs, livers, epididymis, hearts, uterus, brains, hypothalamus, guts, spleens, testes, ovaries, adrenals, and pancreas. The Ethics Committee for Research in Animals at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ, BRE-1930-18-19-1) approved the research for the care and use of animals.
RNA isolation and cDNA synthesis
I used TRIzol reagent (Invitrogen, Carlsbad, CA, USA), as recommended by supplier, to extract total RNA. The concentration and purity of the total RNA was evaluated using a Beckmann (DU 650, Fullerton, CA, USA) spectrophotometer (at 260/280 nm, optical density: 1.8). The integrity of the isolated RNA was assessed by running the RNA samples directly on denaturing formaldehyde-agarose gel (1.2%) stained with ethidium bromide; the RNA was verified by the presence of large and small ribosomal RNA. The first-strand cDNA was synthesized from (1 µg) total RNA using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Indianapolis, IN, USA) and a Maxima First Strand cDNA Synthesis kit for RT-qPCR (ThermoScientific, Vilnius, Lithuania), according to the manufacturer’s guidelines. All the cDNA samples were stored at − 20°C until analysis.
Molecular cloning
I designed two primers (forward 5′-gactcatctcgaggacctgta-3′ and reverse 5′-cgttctccaggtgatgtttct-3′) to obtain via Polymerase Chain Reaction (PCR) for a partial sequence of hamster MafB cDNA. Primers were designed from the nucleotide sequences of mouse (NM_010658.3), rat (NM_019316.2), and human (NM_005461.5) MafB cDNA. The partial fragment was sequenced. Based on the partial cDNA sequence obtained by PCR sequencing, we designed two gene-specific primers (reverse 5′-cctcagggttcatctgctggtagtt-3′ for 5′-end and forward 5′-cccagtcgtgcaggtataaaacgcgt-3′ for 3′-end) to amplify the 5′-end and 3′-end of MafB using a SMART Rapid Amplification of cDNA Ends (RACE) kit (Clontech, Mountain View, CA, USA), in accordance with the manufacturer’s guidelines. These fragments were sequenced and two specific primers (forward 5′-cgttggctccgcgagt-3′ and reverse 5′-acaggacagggagtcagg-3′) for amplifying of complete MafB cDNA were synthesized. The amplified PCR product was purified using electroelution/Amicon ultra-4 10k centrifugal filter devices (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co., Cork, IRL) and cloned using a TOPO-TA Cloning Kit for Sequencing (Invitrogen/ThermoFisher, Waltham, MA, USA). Plasmid cDNA was isolated using a GenElute Five-Minute Plasmid Miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) and a PureYield Plasmid Maxiprep kit (Promega, Woods Hollow, Madison, WI, USA), according to the manufacturer’s guidelines.
Sequencing and bioinformatic analysis
The nucleotide sequence of full-length cDNA and partial fragments of hamster MafB were determined using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Austin, TX, USA). The thermal cycling conditions included 1 min at 96°C followed by 35 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min (Veriti 96 well Thermal Cycler, Applied Biosystems). The resulting material was then purified using a BigDye XTerminator Purification kit (Applied Biosystems) and run on an ABI-PRISM 310 genetic analyzer Applied Biosystems, Foster City, CA, USA), following the manufacturer’s guidelines. The electrophoresis conditions were as follows: temperature: 50°C; injection voltage: 15 kV; injection time: 5–7 s; 5–8 µA; the run module was KB_310POP6_BDTv3_36Rapid. The sequencing reactions were performed in the forward and reverse directions in two independent experiments. The amino acid MafB sequence was determined using an Expert Protein Analysis System (https://web.expasy.org/translate/). The molecular weight and theoretical isoelectric point of the hamster MafB were predicted using ExPASy ProtParam (https://www.web.expasy.org/protparam/). The multiple sequence alignments with other mammalian MafB proteins were determined using the CLUSTALW program (http://www.genome.jp/tools/clustalw/), and the identity between the amino acid MafB sequences were performed using the Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The subcellular localization of hamster MafB was identified using DeepLoc-1.0, an eukaryotic protein subcellular localization predictor (http://www.cbs.dtu.dk/services/DeepLoc/).
MafB phylogenetic tree
One hundred and twenty-five amino acid sequences of mammalian MafB were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/protein) database and aligned with the multiple sequence alignment tool CLUSTALW (https://www.genome.jp/tools-bin/clustalw). The multiple alignment formats were obtained in FASTA file format using the protein database for MafB (https://www.ncbi.nlm.nih.gov/protein/). The molecular evolutionary analyses were performed using Molecular Evolutionary Genetics Analysis (MEGA X) software. The evolutionary history was inferred using the Maximum Likelihood method and a Jones-Taylor-Thornton (JTT) matrix-based model. The tree with the highest log likelihood (-10399.30) is shown in Fig. 4.
Three-dimensional structure prediction
I used the Robetta software package (http://robetta.bakerlab.org) to determine the 3D structure of hamster MafB. I additionally visualized the 3D structure using PyMOL version 2.3 (http://www.pymol.org/).
Gene-expression analysis
The isolation of total RNA was carried out using TRIzol reagent (Invitrogen), according to the manufacturer’s guidelines. The concentration and purity of the total RNA was assessed using a Beckmann spectrophotometer (at 260/280 nm; optical density: 1.8). The integrity of the all of the isolated total RNAs was determined in formaldehyde/MOPS/agarose gels by looking for the presence of ribosomal RNAs. Two µg of total RNA was used for reverse transcription with the Maxima First Strand cDNA Synthesis kit for qPCR (ThermoScientific). Reverse transcription was performed according to the manufacturer’s guidelines. qPCR was carried out in a LightCycler 2.0 system from Roche (Applied Science) with LightCycler TaqMan Master Mix and pre-validated TaqMan hydrolysis probes (Roche Diagnostics, Mannheim, Germany). The relative level of MafB mRNA (sense: 5′-acgctgcagagcttcgac-3′ and antisense 5′-ctgggtacccgtggtgag-3′, 82 bp) was normalized based on the level of hamster β-actin mRNA (sense: 5′-agctatgagctgcctgatgg-3′ and antisense: 5′- caggaaggaaggctggaaa-3′; 82 bp). Transcripts of MafB and β-actin were detected using the universal fluorogenic probes #77 (04–689–003–001) and #9 (04–685–075–001), respectively. The cycling conditions were 95°C for 10 min, 40 cycles of amplification at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s, and a final cycle of cooling at 40°C. The qPCR data were analyzed using the relative quantification method provided by LightCycler software, and they are expressed in arbitrary mRNA units as the mean ± standard deviation (SD) of five biological independent replicates for each group.
Statistical analysis
Differences in MafB mRNA levels assessed using one-way ANOVA. A P value less than 0.05 was considered to indicate statistical significance.