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Objective To establish a mouse model treated with busulfan and to investigate its effects on the metabolism of spermatogonial stem cells (SSCs) of mouse testis. Methods C57BL/6J male mice with age of 8 weeks were injected with 10 mg/kg of busulfan intraperitoneally, then Thy1 positive cells were selected by immunomagnetic beads on day 0, day 5 and day 10 and followed by identification for purity and metabolomic analysis. ResultsThe testis weight ratio decreased and the tissue structure of testis was damaged (P＜0.05). Based on the results of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) , there were significant metabolic differences between the sample groups treated for 0 d, 5 d and 10 d. A total of 89 differential metabolites were identified including glutathione (GSH), arginine and unsaturatedfatty acids (UFAs), and their important metabolic pathways involved glycerophospholipid metabolism, arginine and proline metabolism. Conclusions Affecting the specific metabolic pathway may result in obvious reproductive toxicity and lead to decrease of testicular weight as well as tissue structure damage in mice. Metabolomic analysis showed that the potential reproductive toxicity mechanism of SSCs may be related to the metabolic pathways such as lipid metabolism, arginine and proline metabolism.

Objective To explore the physiological function of gene for cell devision cycle associated 2 (Cdca2), a highly expressed testis gene, in spermatogenesis and fertility in mice. Methods The expression of Cdca2 in different tissues of mice was detected by Q-PCR and Western blot; Using CRISPR/Cas9 gene editing technology and Cre-loxP mediated recombination system, a mouse strain with Cdca2 tissue specific knockout was constructed; By hematoxylin-eosin(HE) staining and morphological analysis, the effect of CDCA2 deletion on spermatogenic cell morphology at different stages of spermatogenesis was studied; The effects of CDCA2 deletion on sperm motility and motility parameters were detected by an automatic sperm detection and analysis system; The effect of CDCA2 deletion on the fertility of male mice was examined by fertility test. Results Cdca2 was a highly expressed gene in testis; Cdca2 germ cell specific knockout mice were successfully obtained by CRISPR/Cas9 technology and reproductive system; CDCA2 deletion in mice had no statistically significant effect on spermatogenic cells, sperm motility parameters and male fertility at all stages of spermatogenesis. Conclusions Cdca2 is probably not necessary for spermatogenesis and male fertility maintenance in mice.

Objective To investigate the role of cytoplasmic poly (A) binding protein-6 (PABPC6) in spermatogenesis of male mice. Methods The Pabpc6 in C57BL/6J mice was knocked out by CRISPR/Cas9 targeting, and the Pabpc6 was identified by PCR. Three types of mice with different genotypes of Pabpc6+/+, Pabpc6+/- and Pabpc6-/- were obtained by mating and breeding. Testis was weighed;Morphological observation and sperm counting were carried out. The mRNA and protein expression levels of Pabpc6 in different tissues were detected by real-time quantitative PCR and Western blot. Immunofluorescence and hematoxylin-eosin staining were used to observe the location of the gene and the morphological changes in the convoluted spermatic tubule of testis. Results The Pabpc6 knockout mice were successfully identified. Pabpc6 was highly expressed in testicular tissue(P＜0.001). Immunofluorescence showed that Pabpc6 was mainly expressed in spermatocyte at early sperm stage. Compared with wild-type mice, knockout mice showed no significant differences in testicular shape, sperm morphology, sperm count and the morphology of convoluted spermatic ducts. Conclusions Pabpc6 is found to be highly expressed in the testis tissue of mice. Pabpc6 has no obvious effect on spermatogenesis of male mice after knockout of Pabpc6. The results indicate that the protein encoded by Pabpc6 is not necessary for spermatogenesis of mice.

[Objective] Ecological restoration has become the main component and important content of ecological civilization construction. However, with the continuous expansion and in-depth development of ecological restoration work, the issue of misalignment of ecological restoration practices has emerged. This urgently requires attention and effective solutions to ensure the sustainable development of ecological restoration practices in China. [Methods] Based on the theoretical research on ecological restoration and by clarifying its connotation and goals, this study defined the connotation of misalignment of ecological restoration practices. It also examined the basis for the identification of ecological restoration misalignment, summarized the main types of ecological restoration misalignment in China, analyzed the reasons for the misalignment of ecological restoration, and put forward suggestions for correcting the misalignment of ecological restoration. [Results] The results show that the main types of ecological restoration misalignment in China include violation of the basic theory of ecological restoration, deviation from the connotation of ecological restoration goals, and inappropriate planning and implementation of ecological restoration projects. The main reasons for the misalignment of ecological restoration are incorrect conceptual understanding, insufficient grasp of patterns and mechanisms, insufficient integration of theory and practice, and inappropriate implementation in practice. [Conclusion] In the future, the misalignment of ecological restoration should be dealt with from the following four aspects: establishing correct ecological civilization concepts, understanding the patterns and mechanisms of the ecosystem, integrating theory and practice, and improving the implementation of ecological restoration projects. Promoting the ecological restoration of the national territorial space also needs to adopt diversified strategies.

Objective To investigate the application of CRISPR/CasRx mediated gene knockdown at the RNA level in mouse spermatogonia cell line GC1-spg. Methods The EF1a core promoter. CasRx.SV40.U6.DRs expression plasmid (CasRx-gRNA) and three fluorescent protein of EF1a.EGFP, EF1a.mCherry, EF1a.tdToamto expression plasmids were constructed by homologous recombination. Three fluorescent protein expression plasmids and CasRx-gRNA expression plasmids were transiently transfected into human embryonic kidney cell line HEK-293T, and the knockdown of exogenous gene (the fluorescent proteins,EGFP and mCherry) was used to detect by fluorescence intensity and Western blot. Additionally, the expression levels of endogenous gene (mRNA and lncRNA) were measured by q-PCR in HEK-293T cells transfected with CasRx-gRNA. The exogenous gene egfp, mCherry and CasRx-gRNA expression plasmids were transiently transfected into the mouse spermatogonia cell line GC1, and the knockdown efficiency of exogenous gene (the fluorescent proteins) was detected by the above method. Results CasRx-gRNA and three kinds of fluorescent protein expression plasmids were constructed successfully. After CRISPR/CasRx mediated RNA interference of exogenous genes (egfp, mCherry) and endogenous genes (mRNA, lncRNA) in HEK-293T cells, the expression level was significantly reduced (P＜0.01). The CRISPR/CasRx system was verified in the mouse spermatogonia cell line GC1, which could effectively and specifically knock down exogenous genes egfp and mCherry. Conclusions CRISPR/CasRx system exerts an effective and specific knock-down function in GC1-spg, providing a new gene silencing tool potentially used in the study of male reproduction.

In this study, the ancient doctors' concentrated prescriptions for arthralgia syndrome were collected and Lantern 3.2 software was used to construct and analyze the latent structure model of ancient arthralgia syndrome. A total of 10 syndrome types of ancient arthralgia syndrome were obtained, namely, cold-coagulation meridian syndrome(partial Yang deficiency), cold-dampness obstruction syndrome(cold weight), damp-heat obstruction syndrome, liver and kidney deficiency syndrome, essence deficiency syndrome, phlegm and blood stasis syndrome, spleen and stomach Qi deficiency syndrome, dampness evil accumulation syndrome, arthralgia meridian syndrome, cold-dampness binding lung syndrome, meridian obstruction syndrome, and wind-cold-dampness arthralgia syndrome(both wind-cold and dampness). This syndrome differentiation method further strengthened the thought on eight-class syndrome differentiation, and showed the characteristics of different syndrome differentiation types of the core drugs in the latent structure model, which was mainly reflected in paying attention to removing blood stasis and eliminating phlegm with use of pungent taste products having dispersing effect, emphasizing the effect of nourishing Yin and generating marrow to supplement congenital deficiency, invigorating spleen and replenishing Qi to regulate the spleen and stomach functions, nourishing vital energy and expelling evil. In addition, Python 3.7 software was used to mine the frequent item sets and association rules for the frequently used drugs for ancient arthralgia syndrome. The drug compatibility law is characterized by making good use of blood-nourishing drugs. Blood-activating drugs are often compatible with cold-dispelling drugs, liver and kidney tonifying drugs, and wind-dampness-dispelling drugs, following the compatibility principle of dispelling wind and dispelling cold as well as tonifying liver and kidney. The induction of syndrome differentiation types of ancient arthralgia syndrome and the discussion on the characteristics of drug use and compatibility law provide a new way for clinical syndrome differentiation and treatment, with certain reference value for the research and development of new Chinese patent medicines.