IGF2/IGFBP4 reduces apoptosis and increases free cholesterol of chicken granulosa cells in vitro.

Follicle selection, a crucial step in maintaining continuous egg production in chickens, is a process that relies on granulosa cells (GCs). In this study, we aimed to identify the key genes that are involved in follicle selection from our previous single-cell transcriptomic data. We used a combination of techniques and assays, including quantitative real-time PCR, immunofluorescence, Oil Red O staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), monodansylcadaverine (MDC) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cell counting Kit-8 (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Multiple indices, such as cell proliferation, cell differentiation, progesterone synthesis, lipid droplet production, total and free cholesterol content, apoptosis, and autophagy, were measured to determine the states of GCs in vitro. The results demonstrated that overexpression of genes related to insulin-like growth factor 2 (IGF2) or IGF-binding protein 4 (IGFBP4) increases intracellular free cholesterol (progesterone precursors) and lipid droplet production, inhibits apoptosis through increased autophagy, and inhibits cell proliferation. This indicates that IGF2 or IGFBP4 can maintain the survival state and improve differentiation tendency of chicken granulosa cells in vitro. Therefore, this study provides new evidence on the functions of IGFs and IGFBPs in chickens, establishing a crucial experimental foundation for understanding the regulatory mechanisms of follicle selection. In addition, our study contributes to understanding follicular development and improves the egg-laying performance of chickens.

Comparative and phylogenetic analyses of plastid genomes of the medicinally important genus Alisma (Alismataceae).

Alisma L. is a medicinally important genus of aquatic and wetland plants consisting of c. 10 recognized species. However, largely due to polyploidy and limited taxon and gene sampling, the phylogenomic relationships of Alisma remain challenging. In this study, we sequenced 34 accessions of Alismataceae, including eight of the ten species of Alisma, one species of Echinodorus and one species of Luronium, to perform comparative analyses of plastid genomes and phylogenetic analyses. Comparative analysis of plastid genomes revealed high sequence similarity among species within the genus. Our study analyzed structural changes and variations in the plastomes of Alisma, including IR expansion or contraction, and gene duplication or loss. Phylogenetic results suggest that Alisma is monophyletic, and constitutes four groups: (1) A. lanceolatum and A. canaliculatum; (2) the North American clade of A. subcordatum and A. triviale; (3) A. wahlenbergii and A. gramineum; and (4) A. plantago-aquatica from Eurasia and northern Africa with the eastern Asian A. orientale nested within it. Hence the results challenge the recognition of A. orientale as a distinct species and raise the possibility of treating it as a synonym of the widespread A. plantago-aquatica. The well-known Alismatis Rhizoma (Zexie) in Chinese medicine was likely derived from the morphologically variable Alisma plantago-aquatica throughout its long history of cultivation in Asia. The plastome phylogenetic results also support the tetraploid A. lanceolatum as the likely maternal parent of the hexaploid eastern Asian A. canaliculatum.

Analysis of the Molecular Mechanism of Energy Metabolism in the Sex Differentiation of Chickens Based on Transcriptome Sequencing.

The determination of sex in mammals is established and controlled by various complex mechanisms. In contrast, sex control in poultry remains an unresolved issue. In this study, RNA-sequencing was conducted for male gonads and ovarian tissues in chicken embryos of up to 18.5 days to identify metabolic factors influencing male and female sex differentiation, as well as gonadal development. Our results reveal that PKM2, a critical glycolysis-related protein, plays a significant role in chicken sex differentiation via PPARG, a crucial hormone gene. We propose that our discoveries bolster the notion that glycolysis and oxidative phosphorylation function as antecedent contributors to sexual phenotypic development and preservation.

Rapid discrimination of Alismatis Rhizoma and quantitative analysis of triterpenoids based on near-infrared spectroscopy.

This study developed a rapid, accurate, objective and economic method to identify and evaluate the quality of Alismatis Rhizoma (AR) commodities. Traditionally, the identification of plant species and geographical origins of AR commodities mainly relied on experienced staff. However, the subjectivity and inaccuracy of human identification negatively impacted the trade of AR. Besides, liquid chromatographic methods such as ultra-high-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC), the major approach for the determination of triterpenoid contents in AR was time-consuming, expensive, and highly demanded in manoeuvre specialists. In this study, the combination of near-infrared (NIR) spectroscopy and chemometrics as the method was developed and utilised to address the two common issues of identifying the quality of AR commodities. Through the discriminant analysis (DA), the raw NIR spectroscopy data on 119 batches samples from two species and four origins in China were processed to the best pre-processed data. Subsequently, orthogonal partial least squares-discriminant analysis (OPLS-DA) and random forest (RF) as the major chemometrics were used to analyse the best pre-processed data. The accuracy rates by OPLS-DA and RF were respectively 100% and 97.2% for the two species of AR, and respectively100% and 94.4% for the four origins of AR. Meanwhile, a quantitative correction model was established to rapidly and economically predict the seven triterpenoid contents of AR through combining the partial least squares (PLS) method and NIR spectroscopy, and taking the triterpenoid contents measured by UPLC as the reference value, and carry out spectral pre-processing methods and band selection. The final quantitative model correlation coefficients of the seven triterpenoid contents of AR ranged from 0.9000 to 0.9999, indicating that prediction ability of this model had good stability and applicability.

The complete chloroplast genomes of three Alismataceae species, including the medicinally important Alisma orientale.

Alismataceae is one of the early diverged families of monocotyledonous plants. We report the complete chloroplast genomes of three Alisma species, including Alisma orientale (Sam.) Juzep. 1934, A. subcordatum Raf. 1908, and A. triviale Pursh 1813, of which A. orientale is a traditional Chinese medical plant used widely to treat diuretics, diabetes, hepatitis, and inflammation. We sequenced the complete chloroplast genomes with the Illumina Nova-Seq 6000 platform using herbarium collections. The chloroplast genomes of A. orientale, A. subcordatum and A. triviale are 159,861 bp, 160,180 bp, and 159,727 bp in length, respectively. The three chloroplast genomes each contain 113 genes, including four rRNAs, 30 tRNAs genes, and 79 protein-coding genes, and the average GC content is 36.0%. Based on the whole chloroplast genomes of 19 species of Alismataceae and the close allies, the medicinally important A. orientale was found to be closely related to another medicinal plant Alisma plantago-aquatica L. 1753 in the phylogenetic analysis. The genus Alisma was supported to be monophyletic.

Analysis of the Promoter Regions of gga-miR-31 and Its Regulation by RA and C-jun in Chicken.

The role of gga-miR-31 in chicken germ cell differentiation and spermatogenesis is of significant importance. The transcriptional properties of gga-miR-31 are crucial in establishing the foundation for the formation of chicken spermatogonia stem cells and spermatogenesis. In this study, a series of recombinant vectors including varying lengths of the gga-miR-31 promoter were predicted and constructed. Through the utilization of the dual luciferase reporting system, the upstream -2180~0 bp region of gga-miR-31 was identified as its promoter region. Furthermore, it was predicted and confirmed that the activity of the gga-miR-31 promoter is increased by retinoic acid (RA). The binding of RA to the gga-miR-31 and Stra8 promoter regions was found to be competitive. Through the deletion of C-jun binding sites and the manipulation of C-jun expression levels, it was determined that C-jun inhibits the activity of the gga-miR-31 promoter. Furthermore, the combined treatment of C-jun and RA demonstrated that the positive regulatory effect of RA on the gga-miR-31 promoter is attenuated in the presence of high levels of C-jun. Overall, this study establishes a foundation for further investigation into the regulatory mechanisms of gga-miR-31 action, and provides a new avenue for inducing chicken embryonic stem cells (ESC) to differentiate into spermatogonial stem cells (SSC), and sperm formation.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.

In Ovo Intravascular Injection in Chicken Embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

Retinoic acid promotes formation of chicken (Gallus gallus) spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinß1, the specific marker protein of SSCs cells, and the integrinß1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinß1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Taxonomy of the Quedius mukuensis group (Coleoptera: Staphylinidae: Staphylinini: Quediina) with descriptions of four new species from China.

Four new species of the subgenus Microsaurus Dejean, 1833 of the genus Quedius Stephens, 1829 are described based on specimens collected from China: Q. (Microsaurus) indigestus sp. nov., Q. (Microsaurus) purus sp. nov. and Q. (Microsaurus) rectus sp. nov. from Sichuan, and Q. (Microsaurus) medius sp. nov. from Hubei. The new species all belong to the Quedius mukuensis group, which is now increased to a total of 16 species. Line drawings and color illustrations of the adults, and the genitalia of the new species and some other ones are given. A key to all the species of this group is provided.

Phylogeny and taxonomic revision of the subgenus Velleius Leach (Coleoptera: Staphylinidae: Staphylininae).

The subgenus Velleius Leach, 1819 of the genus Quedius Stephens, 1829 is a small and very distinctive group in the subtribe Quediina (Staphylinidae: Staphylininae) with pectinate antennal segments and larvae living in nests of Vespa species. This paper reviews the taxonomy of all Velleius species and analyzes the phylogeny of this group. Two new species, Quedius (Velleius) sagittalis sp. nov. from Shaanxi, China and Q. (V.) rectilatus sp. nov. from Guangdong, China, are described. Q. (V.) simillimus (Fairmaire,1891) syn. nov. is proposed as a new synonym of Q. (V.) pectinatus (Sharp, 1874). Phylogenetic result shows that all nine Velleius species can be divided into two clades and both have strong tree supports of Bremer/Bootstrap/Jackknife values.

Taxonomy on Quedius euryalus group (Coleoptera: Staphylinidae: Staphylinini: Quediina) from China with description of eight new species.

Eight new species of the subgenus Microsaurus Dejean, 1833 of the genus Quedius Stephens, 1829 are described based on specimens collected in China, namely Q. aculeatus sp. nov., Q. acutulus sp. nov., Q. altus sp. nov., Q. capillus sp. nov., Q. perlucidus sp. nov., Q. postangulus sp. nov., Q. subwrasei sp. nov. from Sichuan and Q. arcus sp. nov. from Ningxia. The female of Q. songpanoides Smetana, 2009 is described for the first time. These new species belong to the Quedius euryalus group and the fauna of this species group is thus increased to 53 species. In addition, this paper reviews the taxonomic history of the Quedius euryalus group and reports the sexual dimorphism in the hind wing length, i.e. male with longer hind wings in many species of the Quedius euryalus group. Line drawings and color illustrations of adults and genitalia of the new species and some other ones are provided, along with a key to all the known species of the euryalus group.

Three new species of the genus Quedius (subgenus Microsaurus) from China (Coleoptera: Staphylinidae: Staphylinini: Quediina).

Three new species of the subgenus Microsaurus Dejean, 1833 of the genus Quedius Stephens, 1829 are described based on specimens collected from China: Q. (Microsaurus) bilobus sp. nov. and Q. (Microsaurus) varius sp. nov. from Sichuan, and Q. (Microsaurus) cornutus sp. nov. from Yunnan. Line drawings and color illustrations of adults and genitalia of the new species are provided.

Taxonomy of the genus Bolitogyrus Chevrolat (Coleoptera: Staphylinidae: Staphylinini: Quediina) from China with description of seven new species.

Seven new species of the genus Bolitogyrus Chevrolat, 1842 are described based on specimens collected in China, namely B. depressus sp. nov. from Guangdong, B. hainanensis sp. nov. and B. magnimaculosus sp. nov. from Hainan, B. metallicus sp. nov. from Hubei, and B. loculus sp. nov., B. profundus sp. nov. and B. uncus sp. nov. from Yunnan. The number of Bolitogyrus species is thus increased to 59. Line drawings and color illustrations of adults and genitalia of the new species and some others are given. A key to species known from China is provided, with the exception of B. fukienensis Scheerpeltz, 1974, which is only known from females. A geographical distribution map of all Chinese species is also compiled.