C57/BL6 mice were housed in a specific pathogen-free (SPF) environment, given sufficient water and food, and followed the circadian rhythm of the mice. We carried out animal breeding and experiments following the requirements of the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1601117).
To generate Trim69 mutant mice, the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, USA, AM1345) and a RNeasy Mini Kit (Qiagen, Germany, 74104) were used to produce Cas9 mRNA by in vitro transcription and purifying it. The sgRNAs sequences are as follow: 5′-TCAGGTTCTCTCCATGCTCTGGG-3′ and 5′-GCTTTCAATGCAAGGATGCACGG-3′ which the PAM sequences are GGG and CGG. SgRNA oligonucleotides were ligated with pUC57-T7-sgRNA plasmid which has been Bsa I (NEB, USA, R0535S)-digested. The plasmid was digested with Dra I (NEB, R0129S) and purified using MinElute PCR Purification Kit (Qiagen, 28004). SgRNAs were produced using the MEGAshortscript Kit (Ambion, AM1354) and purified using the MEGAclear Kit (Ambion, AM1908). We collected 128 fertilized eggs from C57/BL6 mice, which were subjected to Cas9 mRNA and sgRNA injection, and transferred into the ampullary-isthmic junction of the oviducts of 5 adult female recipient mice. The recipient mice were placed on a heated table to maintain body temperature at 37 °C. Ten pups were finally born, and all of them survived.
C57/BL6 mice were used for all the experiments. The mice with frameshift deletion were backcrossed to wild-type C57/BL6 mice for at least 5 generations. And heterozygous mice were mated to produce homozygous mice, which were used for phenotype analysis.
The fertility test was performed by mating each male with two wild-type C57/BL6 females. All the mice used for the fertility test were 8 weeks old. The number of pups and the sex ratio were recorded.
We performed a sperm analysis to determine sperm quality. Cauda epididymis was cut into pieces with scissors in a cell culture dish containing 100 μL of modified human tubal fluid medium (mHTF) (Irvine Scientific, USA, 90126) supplemented with 10% fetal bovine serum at 37 °C, filtered through 200 mesh nylon membrane, and washed by additional 200 μL of mHTF into a 1.5 mL EP tube to collect the sperm. After incubated at 37 °C for 5 minutes, sperm motility and number were analyzed with Hamilton Thorne IVOS II system (Hamilton Thorne, USA).
Total wild type and homozygous testicular RNA were extracted with Trizol Reagent (Invitrogen, USA, 15596-018). We obtained cDNA by reversing transcription of total RNA (2 μg) using Prime Script RT Master Mix (TaKaRa, Japan, RR047A). We used AceQ qPCR SYBR Green Master Mix (Low ROX Premixed) (Vazyme, USA, Q131-02) to perform quantitative reverse transcription PCR (qRT-PCR) in a StepOne Real-Time PCR System (Applied Biosystems). The qRT-PCR reaction was carried out following the steps on the reagent instructions and 18S was used as an internal control. The primers used in the experiment were arranged in Table 1.
Primer name Sequence (5′ to 3′) Trim69-WT-F GCCACAACTTCTGCCAAGACTG Trim69-WT-R GAGGACTCTGAGCGTTCTCGTTAT Trim69-RT-F TTCTCTCTCCAGGCCCATCTC Trim69-RT-R GAGGTGAAGCCTTTTGAGCC Trim5-RT-F CCATCGCCAGGGAACAAAGAAAGT Trim5-RT-R CACCTGAACCCAGTAGCGTTGAG Trim7-RT-F CAGGAGGGTCGGCTTCTAAG Trim7-RT-R GAGACTGTGGTTGGCTTGGA Trim10-RT-F GGAACACGGGGAGAAAATCTAC Trim10-RT-R AGACACACGAGACACTTCTGT Trim11-RT-F CCTGGATTACTTCACCGACCC Trim11-RT-R AGGCGTCGTGCCATTTCTG Trim15-RT-F CAGGTGGAGGAGCTAGAGGAGA Trim15-RT-R GTGGAGGTTTCGGATCTTCTTGAC Trim17-RT-F CTTGCCAGACGGTTACAAGAG Trim17-RT-R CTCAGCCACTTTTGTCAGGAG Trim21-RT-F GGAGAGGAGGATTCGTGGTTCAGA Trim21-RT-R GCGGTGTTGCGATCCAGAGTAAT Trim25-RT-F ATGGCTCAGGTAACAAGGGAG Trim25-RT-R GGGAGCAACAGGGGTTTTCTT Trim26-RT-F GAAAGCCATCCCTCACATGGTTAA Trim26-RT-R GCCAGTATAGGTCACACACTTCC Trim27-RT-F GGAGCAAATCCAGAACCGACT Trim27-RT-R GCCCCGTTGATGCTGTTATAG Trim34a-RT-F CTCAGGAGCATCGTGGTCAC Trim34a-RT-R CCTTGTCCAGGATTCTTCTAAGC Trim34b-RT-F GCGGCGTGAGTGGTCAGATA Trim34b-RT-R AAATGTGAAGTTGTCCCAGTAGGC Trim35-RT-F TTCCGGGCCAAGTGTAAGAAC Trim35-RT-R CCAAGTCGTTTGCACCTCA Trim38-RT-F ATGGGCTCAGACTTTAGCACG Trim38-RT-R CTGTTTTTGGGCTGACATTGC Trim39-RT-F GCAAAGCCTCCGAAGAGAAGAA Trim39-RT-R GAAGTCGCTGTAGAATGTCCTGTT Trim41-RT-F ATGAGCCGCATGTTTTGTCAG Trim41-RT-R GCCCCTAGTACACAGCAGT Trim43a-RT-F TGGTGCAGGACTTGGATGACTTGT Trim43a-RT-R GGTCTGACTGAGGTGTAGCCGAAC Trim43c-RT-F TGAAGCTACGATAGTCCACGAGAG Trim43c-RT-R GGCACGGAGTCTTGGTATCATAC
Table 1. List of primer sequences
Primer name Sequence (5′ to 3′) Trim47-RT-F GAGGACCGCATGGATGAACT Trim47-RT-R AACTCTGAAGGGTGGCTGTG Trim50-RT-F CCCATTTGCCTGGAGGTCTTC Trim50-RT-R CAGGACAGCATAGCTCGGAG Trim58-RT-F CTGGCCCAACAGAACAAGG Trim58-RT-R CTCACATCGTGTCAAGACTCC Trim60-RT-F CTGCCTCCGTTTGCTCTGG Trim60-RT-R GAGCCTAATGGTTTCAGTCATGT Trim62-RT-F TGCGAGCACTACTTCTGCC Trim62-RT-R CTTGACCTTGTCGTGAGCC Trim65-RT-F GAGGACGTGGTGACTTGCTC Trim65-RT-R ACGCCATGAATCCTGGATGC Trim68-RT-F TCCCAGAACTTGAGCTACACC Trim68-RT-R AGACGGACCTTGTCTACAACA Trim72-RT-F CCGCAGGCTCTAAGCACTAAC Trim72-RT-R CTGCTCGCAGTAGATGCTCA Trim75-RT-F ATAGCAGCATTAGAACACCACG Trim75-RT-R ATGAGCTGACGCCTACAATTC Trim6-RT-F CGGATTCCAGATTGGATTCTTTT Trim6-RT-R TGATGCAGACCTGGCAGAAG Triml1-RT-F TGGAGAACCTCAGGGAAGAGC Triml1-RT-R GAGGAGGCACATCAGACAAAA Trim69-exon1-3-RT-F CTCCTCCTCTCATGGGCTCT Trim69-exon1-3-RT-R GCTCCTTCAGGGTTGTCTGA 18s-RT-F TAACGAACGAGACTCTGGCAT 18s-RT-R CGGACATCTAAGGGCATCACAG
Table 1. List of primer sequences (continued)
We used the STA-PUT method to sort different spermatogenic cells in mouse testes, including spermatogonia, pachytene spermatocytes, round spermatids, and elongated spermatids. Mice between postnatal day 6 to 8 were selected for spermatogonia sorting because the percentage of spermatogonia in this period was high. Other cell types were obtained from adult mice. First, the testicular tissue was digested into small tubes by collagenase (1 mg/mL) (Invitrogen, 17104-019), and then the small tubes were further digested into a single cell suspension with 0.25% Trypsin containing DNase I (1 mg/mL) (Ruibio, China, TD3212).
After the single cell suspension flowed into the sedimentation tank, the single cell suspension was lifted up with a 2% to 4% bovine serum albumin (BSA) (Beyotime, China, ST023) gradient. After about 2 hours of sedimentation, cells of different types would distribute in different positions of the BSA gradient.
Freshly taken testicles were placed in a dish containing PBS to rinse the blood, and then RIPA lysate with protease inhibitor cocktail tablets (Roche, Switzerland, 11873580001) was added. In order to thoroughly lysis, tissues were cut into small pieces with scissors and sonicated to homogenate and then centrifuged at 4 degrees for 40 minutes to obtain supernatant. Using the bicinchoninic acid (BCA) (Beyotime, P0012) kit to measure protein concentration and determine sample loading amount. Equal protein amounts (30 μg) per lane were separated by 12% SDS-PAGE in electrophoresis buffer and were transferred onto polyvinylidene difluoride membranes (Bio-Rad, USA, 1620177) in transfer buffer at 90 mA at 4 °C for 2 hours. The membrane was blocked in 5% nonfat dry milk in TBS for 2 hours and then incubated overnight at 4 °C. The next day, we conducted secondary antibody incubation (1:1000 dilution) and exposure. Primary antibodies used were listed in Table 2.
Antibodies Source Catalog number Dilution Rabbit Polyclonal Anti-TRIM69 Proteintech Group 12951-1-AP 1:500 Rabbit Polyclonal Anti-Lin28 Abcam ab46020 1:200 Mouse Monoclonal Anti-gamma H2A.X Abcam ab26350 1:200 Rabbit Polyclonal Anti-SCP3 Abcam ab15093 1:150 Rabbit polyclonal Anti-Sox9 EMD Millipore Corp AB5535 1:200 Mouse Monoclonal Anti-Tubulin, Acetylated Sigma T6793 1:200 Rabbit Polyclonal Anti-bax Cell signaling technology 2772 1:1000 Rabbit Polyclonal Anti-Bcl2 Proteintech Group 26593-1-AP 1:500 Mouse Monoclonal Anti-Caspase 3/p17/p19 Proteintech Group 66470-2-Ig 1:1000 Rabbit Polyclonal Anti-beta Tubulin Abways AB0039 1:5000 Lectin PNA Conjugate (Alexa Fluor 568) Thermo Fisher L-32458 1:1000 Goat Anti-Rabbit IgG H&L (HRP) Thermo Fisher 31460 1:1000 Goat Anti-Mouse IgG H&L (HRP) Thermo Fisher 31430 1:1000 Donkey Anti-Rabbit IgG H&L (Alexa Fluor 555) Thermo Fisher A-31572 1:1000 Donkey Anti-Mouse IgG H&L (Alexa Fluor 488) Thermo Fisher A-21202 1:1000 Donkey Anti-Mouse IgG H&L (Cy3-AffiniPure） Jackson 715-165-150 1:1000
Table 2. List of antibodies
The testicular tissue was fixed with modified Davidson's fluid (MDF) for 24 hours, cut in half and fixed with MDF for an additional 24 hours. After dehydration and entrapment, sections were cut at 5 μm thickness. After dewaxed with xylene and hydrated with gradient alcohol, testicular paraffin section was washed with PBS. Subsequent acidic antigen retrieval of the sections was conducted in the microwave oven in 10 mm citrate buffer (pH 6.0) with high fire for 3 minutes and low fire for 7 minutes. We blocked the slides with 5% BSA at room temperature for 2 hours, then diluted the antibodies according to the ratio of antibody instructions and incubated slides at 4 °C overnight. The next day, secondary antibody and Hoechst 33342 were incubated at room temperature for 2 hours and 5 minutes, respectively. Finally, confocal photographs were taken after slides sealing with glycerol (Carl Zeiss, LSM800, Germany).
Spermatozoa obtained from cauda were air-dried and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The other operation steps were consistent with the paraffin section. Antibodies were listed in Table 2.
The testicular and epididymal tissues were fixed with MDF for 24 hours, cut in half and fixed with MDF for an additional 24 hours. After dehydration entrapment of mouse testes or epididymis, sections were cut at a 5 μm thickness. After deparaffinization, slides were stained with Hematoxylin and Eosin (H&E) for histological analysis.
The paraffin section of the testis can be used for detecting the apoptosis signal by using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A113-03) according to the manufacturer's instructions.
The process of prophase Ⅰ spermatocytes was observed by chromosome spreads. Primary antibodies were as follows: anti-SYCP3 (1∶150; Proteintech, USA, 23024-1-AP) and anti-γH2AX (1∶200; Abcam, UK, ab26350). Mounted the slides with glycerol after staining, then took pictures with a confocal microscope.
Cauda epididymides were dissected and fixed in Gluta solution (Sigma, USA, P1126). Post-fixation with 2% (wt/vol) OsO4, tissue blocks were embedded in epoxy resin. Transmission electron microscope (TEM) sections were shot after ultra-thin sectioning (FEI Tecnai G2 Spirit Bio TWIN, FEI Company, USA).
SPSS 19.0 statistical software was used for data analysis. Chi-square test was used for statistics of counting data, and one-way ANOVA was used for group comparison. The measurement data were expressed in the form of mean±SD. The comparison between the two groups was analyzed by the Student's t-test. P values less than 0.05 were considered statistically significant.
The testis-specifically expressed gene Trim69 is not essential for fertility in mice
- Received Date: 2020-05-12
- Accepted Date: 2020-06-19
- Rev Recd Date: 2020-06-18
- Available Online: 2020-08-21
Abstract: Protein ubiquitination is essential for diverse cellular functions including spermatogenesis. The tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activity, are highly conserved in mammals. They are involved in important cellular processes such as embryonic development, immunity, and fertility. Our previous studies indicated that Trim69, a testis-specific expressed TRIM family gene, potentially participates in the spermatogenesis by mediating testicular cells apoptosis. In this study, we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology. Here, we reported that the male Trim69 knockout mice had normal fertility. The adult knockout mice have shown that the appearance of testes, testis/body weight ratios, testicular histomorphology, and the number and quality of sperm were consistent with wild-type mice. These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility, and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis. This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.
|Citation:||Xi He, Wenxiu Xie, Huiling Li, Yiqiang Cui, Ya Wang, Xuejiang Guo, Jiahao Sha. The testis-specifically expressed gene Trim69 is not essential for fertility in mice[J]. The Journal of Biomedical Research. doi: 10.7555/JBR.34.20200069|