The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8-30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5-18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.Reproduction (2017) 153 [785][786][787][788][789][790][791][792][793][794][795][796]

Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

Myostatin (MSTN) is an important gene involved in the regulation of embryonic muscle cells and adult muscle development; it has a good application prospect in transgenic animal production by improving the yield of muscle. The purpose of this study is to construct MSTN gene knockout vector using clustered regularly interspaced short palindromic repeats ( CRISPR)/CRISPR‐associated protein 9 ( Cas9). The knockout efficiency was evaluated in sheep ear fibroblasts (SEFs) by cleavage activity of transcription of guide RNA ( gRNA), luciferase‐single‐strand annealing assay, T7 endonuclease I assay (T7E1), and TA clone sequence (10/38); and above all, detection showed that the cleavage activity of CRISPR/Cas9‐mediated MSTN reached 29%. MSTN‐Cas9/gRNA4 was transfected into sheep skeletal muscle satellite cell (sSMSC) to confirm the function of MSTN in myotomes formation induced by starvation in low‐serum medium. The results showed that myotubes formation efficiency were 11.2 ± 1.3% and 19.5 ± 2.1% in the control group and knockout group, respectively. The average length of myotomes was 22 ± 5.3 and 47 ± 3.6 μm, displaying that MSTN knockout can promote sSMSC differentiation in number and length. The unlabeled MSTN‐Cas9/gRNA4 was transfected into SEFs and monoclonal positive cells was obtained after 48 hours transfection. The MSTN‐positive cells were used as donor cells to perform somatic cell nuclear transplantation to produce transgenic sheep. A total of 20 embryos were transplanted into surrogate mothers, four of them normally produce offspring. The genomic DNA of surviving lambs were used as a template, three positive individuals were identified by T7E1 digestion. All the results demonstrated that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in animal breeding.

The highly efficient novel methods to produce transgenic chickens were established by directly injecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach, four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection, the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 microg/mL group, respectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F(1) and F(2) transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detection of polymerase chain reaction (PCR) of F(1) and F(2) transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F(1) and 52.9% (9/17) of F(2) were positive. Southern blot showed GFP DNA was inserted in their genomic DNAs. (4) Frozen whole mount tissue sections of F(1) and F(2) transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP expression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.

In this study we investigated the methylome of chickens immunized with Infectious laryngotracheitis (ILT) vaccine derived from chicken embryos. Methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) method was employed in the detection of the 1,155 differentially methylated regions (DMRs) across the entire genome. After validation, we ascertained the genomic DMRs distribution and annotated them regarding genes, transcription start sites (TSS) and CpG islands. We found that global DNA methylation decreased in vaccinated birds, presenting 704 hypomethylated and 451 hypermethylated DMRs, respectively. Additionally, we performed an enrichment analysis detecting gene networks, in which cancer and RNA post-transcriptional modification appeared in the first place, followed by humoral immune response, immunological disease and inflammatory disease. The top four identified canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, axonal guidance signaling and mTOR signaling, providing new insight regarding the mechanisms of ILT etiology. Lastly, the association between DNA methylation and differentially expressed genes was examined, and detected negative correlation in seventeen of the eighteen genes.