Optimization of recovery and maturation methods for cumulus-oocyte complexes in jennies.

Embryo production in donkeys is inefficient compared with that in other livestock. Obtaining a sufficient number of MII oocytes is the first step to solving this problem. In this study, the number, morphology and maturation rates of cumulus-oocyte complexes (COCs) obtained from abattoir-derived ovaries or live jennies were compared. The diameter of follicles from abattoir-derived ovaries was measured and divided into group 1 (2-6 mm), group 2 (6-10 mm), group 3 (10-20 mm), group 4 (20-28 mm) and group 5 (>28 mm). The results showed that the number of follicles per ovary in group 2 (3.6 ± 0.28) and 3 (4.2 ± 0.90) was higher than that in the other groups (p < .05). The recovery rate in group 3 was higher than group 1 (48.8% vs. 26.8%, p = .00), but lower than group 5 (48.8% vs. 76.5%, p = .025). The percentage of grade A COCs in group 3 was higher than group 2 (59.3% vs. 39.5%, p = .00) and group 1 (59.3% vs. 26.7%, p = .00). Moreover, the percentage of grade A COCs in group 4 (55.0%, p = .710) and group 5 (46.2%, p = .351) was reduced compared with that in group 3. From the above results, the developing follicles (group ovum pick-up [OPU], 10-20 mm) and preovulation follicles (group OPU-Preov, >35 mm) were aspirated from live jennies using OPU. Although there was no difference in the recovery rates of COCs between group 3 and OPU (48.8% vs. 43.0%, p = .184), the percentage of grades A COCs in group OPU was higher than group 3 (72.5% vs. 59.3%, p = .036). There was no difference in the maturation rate between group 3 and OPU (60.3% vs. 69.3%, p = .171) after the COCs matured in vitro. The rates of recovery (72.2%) and maturation (92.3%) in group OPU-Preov were higher than those in other groups (p < .05). Moreover, the effects of maturation time and serum type on maturation rates were evaluated in groups B44 (44 h, FBS), B36 (36 h, FBS) and D44 (44 h, foetal donkey serum, FDS). These results indicated that the maturation rate in group B36 was lower than group B44 (13.1% vs. 47.0%, p = .00) and group D44 (13.1% vs. 53.3%, p = .00). In conclusion, the quality of donkey COCs from OPU was higher than that from abattoir-derived ovaries, the suitable time of donkey in vitro maturation (IVM) was 44 h, and FBS could be replaced with FDS in donkey IVM medium.

Overexpression of Tfap2a in Mouse Oocytes Impaired Spindle and Chromosome Organization.

Transcription factor AP-2-alpha (Tfap2a) is an important sequence-specific DNA-binding protein that can regulate the transcription of multiple genes by collaborating with inducible viral and cellular enhancer elements. In this experiment, the expression, localization, and functions of Tfap2a were investigated in mouse oocytes during maturation. Overexpression via microinjection of Myc-Tfap2a mRNA into the ooplasm, immunofluorescence, and immunoblotting were used to study the role of Tfap2a in mouse oocyte meiosis. According to our results, Tfap2a plays a vital role in mouse oocyte maturation. Levels of Tfap2a in GV oocytes of mice suffering from type 2 diabetes increased considerably. Tfap2a was distributed in both the ooplasm and nucleoplasm, and its level gradually increased as meiosis resumption progressed. The overexpression of Tfap2a loosened the chromatin, accelerated germinal vesicle breakdown (GVBD), and blocked the first polar body extrusion 14 h after maturation in vitro. The width of the metaphase plate at metaphase I stage increased, and the spindle and chromosome organization at metaphase II stage were disrupted in the oocytes by overexpressed Tfap2a. Furthermore, Tfap2a overexpression dramatically boosted the expression of p300 in mouse GV oocytes. Additionally, the levels of pan histone lysine acetylation (Pan Kac), histone H4 lysine 12 acetylation (H4K12ac), and H4 lysine 16 acetylation (H4K16ac), as well as pan histone lysine lactylation (Pan Kla), histone H3 lysine18 lactylation (H3K18la), and H4 lysine12 lactylation (H4K12la), were all increased in GV oocytes after Tfap2a overexpression. Collectively, Tfap2a overexpression upregulated p300, increased the levels of histone acetylation and lactylation, impeded spindle assembly and chromosome alignment, and ultimately hindered mouse oocyte meiosis.

Expression Profiling and Functional Analysis of Circular RNAs in Inner Mongolian Cashmere Goat Hair Follicles.

BACKGROUND: Inner Mongolian cashmere goats have hair of excellent quality and high economic value, and the skin hair follicle traits of cashmere goats have a direct and important effect on cashmere yield and quality. Circular RNA has been studied in a variety of tissues and cells. RESULT: In this study, high-throughput sequencing was used to obtain the expression profiles of circular RNA (circRNA) in the hair follicles of Inner Mongolian cashmere goats at different embryonic stages (45, 55, 65, and 75 days). A total of 21,784 circRNAs were identified. At the same time, the differentially expressed circRNA in the six comparison groups formed in the four stages were: d75vsd45, 59 upregulated and 33 downregulated DE circRNAs; d75vsd55, 61 upregulated and 102 downregulated DE circRNAs; d75vsd65, 32 upregulated and 33 downregulated DE circRNAs; d65vsd55, 67 upregulated and 169 downregulated DE circRNAs; d65vsd45, 96 upregulated and 63 downregulated DE circRNAs; and d55vsd45, 76 upregulated and 42 downregulated DE circRNAs. Six DE circRNA were randomly selected to verify the reliability of the sequencing results by quantitative RT-PCR. Subsequently, the circRNA corresponding host genes were analyzed by the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The results showed that the biological processes related to hair follicle growth and development enriched by GO mainly included hair follicle morphogenesis and cell development, and the signaling pathways related to hair follicle development included the Notch signaling pathway and NF-κB signaling pathway. We combined the DE circRNA of d75vsd45 with miRNA and mRNA databases (unpublished) to construct the regulatory network of circRNA-miRNA-mRNA, and formed a total of 102 pairs of circRNA-miRNA and 126 pairs of miRNA-mRNA interactions. The binding relationship of circRNA3236-chi-miR-27b-3p and circRNA3236-chi-miR-16b-3p was further verified by dual-luciferase reporter assays, and the results showed that circRNA3236 and chi-miR-27b-3p, and circRNA3236 and chi-miR-16b-3p have a targeted binding relationship. CONCLUSION: To summarize, we established the expression profiling of circRNA in the fetal skin hair follicles of cashmere goats, and found that the host gene of circRNA may be involved in the development of hair follicles of cashmere goats. The regulatory network of circRNA-miRNA-mRNA was constructed and preliminarily verified using DE circRNAs.

Chi-miR-130b-3p regulates Inner Mongolia cashmere goat skin hair follicles in fetuses by targeting Wnt family member 10A.

The development of hair follicles (HFs) is dependent on interactions between epithelial cells and dermal fibroblasts, which may play an important role in maintaining the structure of HFs during their development and maturation. Wnt family member 10 (WNT10A) is a hub gene during HF development and maturation that may regulate the proliferation of dermal fibroblasts and epithelial cells through microRNAs (miRNAs) and messenger RNAs (mRNAs) to maintain the structural stability of HFs. In the present study, we confirmed that WNT10A is the target gene of chi-miR-130b-3p by real-time quantitative PCR, western blotting, and a dual-luciferase reporter gene assay. We successfully cultured fetal epithelial cells and dermal fibroblasts using the tissue block attachment method, and Cell Counting Kit-8 (CCK8) results showed that chi-miR-130b-3p regulates epithelial cell and dermal fibroblast proliferation by targeting WNT10A.

Using WGCNA (weighted gene co-expression network analysis) to identify the hub genes of skin hair follicle development in fetus stage of Inner Mongolia cashmere goat.

OBJECTIVE: Mature hair follicles represent an important stage of hair follicle development, which determines the stability of hair follicle structure and its ability to enter the hair cycle. Here, we used weighted gene co-expression network analysis (WGCNA) to identify hub genes of mature skin and hair follicles in Inner Mongolian cashmere goats. METHODS: We used transcriptome sequencing data for the skin of Inner Mongolian cashmere goats from fetal days 45-135 days, and divided the co expressed genes into different modules by WGCNA. Characteristic values were used to screen out modules that were highly expressed in mature skin follicles. Module hub genes were then selected based on the correlation coefficients between the gene and module eigenvalue, gene connectivity, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results were confirmed by quantitative polymerase chain reaction (qPCR). RESULTS: Ten modules were successfully defined, of which one, with a total of 3166 genes, was selected as a specific module through sample and gene expression pattern analyses. A total of 584 candidate hub genes in the module were screened by the correlation coefficients between the genes and module eigenvalue and gene connectivity. Finally, GO/KEGG functional enrichment analyses detected WNT10A as a key gene in the development and maturation of skin hair follicles in fetal Inner Mongolian cashmere goats. qPCR showed that the expression trends of 13 genes from seven fetal skin samples were consistent with the sequencing results, indicating that the sequencing results were reliable.n.

EDA and EDAR expression at different stages of hair follicle development in cashmere goats and effects on expression of related genes.

This study is focused on the detection of ectodysplasin A (EDA) and ectodysplasin A receptor (EDAR) mRNA expression levels and protein positions in seven stages of cashmere goat fetus development (45, 55, 65, 75 95, 115, and 135 d), with the main goal of investigating the effect of EDA and EDAR on genes related to hair follicle development. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to measure EDA and EDAR expression levels in seven stages of cashmere goat fetus development. Immunohistochemistry (IHC) was used to locate EDA and EDAR in the critical stage of fetal hair follicle development (45-135 d). EDA and EDAR expression in fetal fibroblasts and epithelial cells was interfered with by short hairpin RNA (sh-RNA). The results indicated that EDA and EDAR were both expressed in the skin tissue in the seven cashmere goat embryo stages. Moreover, EDA and EDAR play an important role in the formation of embryonic placode (Pc). After interfering with EDA and EDAR, the expression of BMP2, BMP4, noggin, ß -catenin, TGF- ß 2, Wnt-10b, and NOTCH1 in fibroblasts and epithelial cells changed significantly. This study provides a theoretical and experimental basis for further studying the molecular regulation mechanism of hair follicle development.