| Spermatogenesis includes a classic stem cell system. Continuous production of highly differentiated, short-lived sperm is maintained throught reproductive life by a small, dedicated population of male germ line stem cells (GSCs). Male GSCs are unipotent, devoted solely to the generation of sperm. As precursors of the spermatogonial lineage, male GSCs must maintain a balance between the production of mature sperm and the self-renewal of stem cell potential. Male GSCs is a kind of single performance cell, which is only for the maintainance of sperm production in biological body. Therefore, male GSCs usually considered to be the initial form of spermatogenesis, and the generation, migration and colonization of primordial germ cells (PGCs) considered to be the origin of male GSCs. At present, researchers have studied the generation and differentiation of PGCs or SSCs in multiple ways, such as isolation and culture, induction in vitro, transgenic cells and so on. But we still know little about the regulation signaling network of male germ cells generation, which is becoming the focus of scientists in recent years. The process of male germ cells differentiation controlled by many endogenous factors, as well as some external factors such as extracellular matrix and so on. Those varieties of factors constitute a complex regulation network through multiple signaling pathways, which could directly or indirectly regulate the generation of male germ cells.With the development of modern high throughput sequencing technology, the transcriptome sequencing (RNA-Seq) has now becoming an important technique in genome-wide gene expression study. Based on the chicken male ESCs, male PGCs and SSCs RNA-Seq results done previously by our lab, we made comprehensive analysis of the whole genome transcriptome level during chicken male germ cells generation. We hope to make contribution to the overall systematic gene dynamic expression analysis, finding signaling pathways involved in the process, building control network, and providing theoretical biases for the generation mechanisms of chicken male germ cells. Through the RNA-Seq data analysis, our lab has found that Notch and TGF-βsignaling pathway involved in the generation of chicken male germ cells generation. However, due to the complex regulation network, only from these two signaling pathways is not enough to make full understanding of chicken male germ cells regulation mechanism. There must be some other signaling pathway involved in this process, which needs our further study.To further understanding the regulation mechanism of chicken male germ cells generation, this study made deep analysis of the RNA-Seq results of chicken male ESCs, PGCs and SSCs. During the analysis, we found another important signaling pathway——JAK-STAT signaling pathway which involved in this process. To verify the role of JAK-STAT signaling pathway, we made in vivo and in vitro experiments. In vivo and in vitro experiments all confirmed that JAK-STAT signaling pathway played an important role in chicken germ cells generation. Moreover, to solve the low efficiency of male germ cells differentiation, we groped for the optimum condition for embryoid body (EB) formation from chicken ESCs through hanging-drop culture; and we also made comparison of different inducers, so as to select the best induction system for germ cells differentiation from ESCs, and to provide theoretical and experimental basis for male germ cells regulation mechanism.The results addressed as follows:(1) Base on the RNA-Seq results of chicken male ESCs, PGCs and SSCs, we made whole transcriptome analysis to discover gene regulation mechanism during male germ cells generation. By screening the differentially expressed genes (DEGs), potential key genes expressed differently among these three cells were analyzed. Go analysis and pathway analysis were used to find key signaling pathways involved in the regulation of chicken male germ cells generation. We found that JAK-STAT signaling pathway could be involved in this process by RNA-Seq results. To verify the reliability of this RNA-Seq analysis results, we used qRT-PCR and Microarray technology to do two-way test. The results showed that, although the genes’ expression levels were different among RNA-Seq, qRT-PCR and Microarray, but the trend of these genes were match. This verified the reliability of RNA-Seq results, and confirmed the key genes and signaling pathways that we screened from RNA-Seq were also reliable and accurate.(2) Collect and digest the pure clones from the third generation of chicken ESCs, re-suspension the cells in to three cell concentrations:1×104/mL,3×104/mL and 6×104/mL to make suspension culture. Morphology changes of EB were observed, qRT-PCR and immunofluorescence methods were used to detect the marker genes’ expression, self-differentiation and karyotype analysis were made to make full tests of EB. The results showed that the amount of EB with 3×104/mL cell concentration was higher than the other groups, and the EB almost shared the same spherical shape. The stem cell marker genes remained expression with 48h of EB formation, and stem cell surface specific antigen detection was positive. After self-differentiation of EB, three embryonic germ layers specific antigen detection all showed positive. Karyotype analysis showed that the formatted EB maintained normal karyotype. The results indicated that the suitable concentration for chicken ESCs form the EB through suspension culture is 3×104/mL, and the formatted EB have viability to provide experimental foundation for chicken ESCs induction in vitro.(3) 2 passaged maleness ESCs were cultured in media contained ATRA, Am80 and E2 to differentiate toward a germ cell lineage. The induced effects were detected by morphology, QRT-PCR and immunofluorescence. According to the changes in cell morphology, during ATRA induction, embryoid bodies (EB) began to appear in 2-4d and gradually increased in both size and number, subsequently some of EBs began to disintegrate into small round cells in 6-8d, then a small number of germ cell-like cells could be observed in 12d, the expression of Cvh, C-kit, integrina.6 and integrinβ1 were up-to 0.85,0.40,1.01 and 0.98 respectively; during Am80 induction, EB could also be observed during 4-6d, and germ cell-like cells in 12d; during E2 induction, EB could also be observed during 2-4d, and germ cell-like cells in 10d, the expression of Cvh, C-kit, integrina.6 and integrinβ1 were up-to 0.85,0.40,1.01 and 0.98 respectively; during E2 induction, embryoid bodies (EB) began to appear in 2-4d and gradually increased in both size and number until 8d, subsequently some of EBs began to disintegrate into small round cells in 10d, and a small number of germ cell-like cells could be observed, germ cell-like cells got more in 12d, the expression of Cvh, C-kit, integrina.6 and integrinβ1 were up-to 0.48,0.50,1.10 and 1.20 respectively. These indicated that ATRA, Am80 and E2 could promote the expression of the corresponding genes of the germ cells, and had effect on differentiation of ESCs to male germ cells. The result provides references for optimization of induction system for differentiation on male germ cells in vitro and theoretical foundation for studying regulatory mechanism of male germ cells.(4) JAK-STAT signaling pathway inhibitor Cucurbitacin I and agonist IL6 were directly injected into the albumen at the pointed end of the fresh fertilized eggs (E0). Injected eggs were heat-sealed with ParafilmM and incubated at normal condition. Genital ridges in 4.5d, testiculars in 19d at developmental stages were collected for the further detection. The expression of downstream signaling molecular involved in this signaling pathway were observed by qRT-PCR and Western blot. qRT-PCR, Western blot, immunocytochemistry, PAS staining and flow cytometry were used to detect the differentiation of germ cells in vivo. The results showed that:with the inhibitor Cucurbitacin I injection, Cvh (12.34) and C-kit (14.83) expression of 4.5d incubation chicken embryo were significantly lower than control group; integrina.6 (30.03) and integrinβ1 expression of 4.5d incubation chicken embryo were also lower than control group. Immunocytochemistry results showed Cvh, C-kit, integrina6 and integrinfβ1 positive cells down-regulated to 7.3%,12.8%,5.6% and 5.3% respectively. PAS staining results showed that the inhibitor group of chicken had less PGCs number during 4.5d incubation. With contrary, the agonist group of chicken had higher rate of Cvh and C-kit positive cells; PAS and immunohistochemical staining results showed that the PGCs number were significantly higher than control group. These results indicated that JAK-STAT signaling pathway involved in chicken male germ cells generation, especially in the PGCs generation process.(5) 2 passaged maleness ESCs were cultured in media contained RA to differentiate toward a germ cell lineage, and the above inhibitor and agonist were added into induce media. QRT-PCR and immunofluorescence methods were used to detect the differentiation of germ cells in vitro. The results showed that, RA could successfully increase the expression of germ cells specific genes:C-kit, Cvh, integrina6 and intergrinβ1. With JAK-STAT signaling pathway inhibitor Cucurbitacin I, the above genes expression were depressed in varying degrees, C-kit lower as 0.32 and Cvh lower as 0.41; immunofluorescence also showed the number of C-kit and Cvh positived cells were lower than RA induction. While with JAK-STAT signaling pathway agonist IL6, the expression of C-kit and Cvh were extremely increased around 6d of induction, up to 1.60 and 1.65 respectively; and immunofluorescence showed that the number of integrina6 and intergrinβ1 positived cells were higher than RA induction. The results indicated that, the activated JAK-STAT signaling pathway could accelerate RA differentiate ESCs to male germ cells. |
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