Abstract：Abstract Primordial germ cells (PGCs) are the progenitor cells of gametes involving in the development of embryonic germ cells (ESCs). 3β-hydroxysteroid dehydrogenase 2 (Hsd3β2) can promote cell differentiation in the steroid hormone pathway, but its specific regulation mechanism on the formation of PGCs is still unknown. The study aims to construct a short hairpin RNA (shRNA) interfering vector of Hsd3β2, a key gene in the process of steroid hormone synthesis and to observe its effect on the differentiation of chicken (Gallus gallus) ESCs into PGCs. Hsd3β2 was sythesized, amplified, and ligated into the lentiviral plasmid pGMLV-SC5. Gene sequencing was done to identify the successful construction of the vector. DF1 was transfected by 3 vectors and a control vector to choose the best one for the next lentiviral package. On the basis of retinoic acid (RA) induction, ESCs were transfected by sh-Hsd3β2 and the cell morphology was observed at 2, 4 and 6 d, respectively. Cell samples were collected for further experiments. qRT-PCR was used to detect the expression of Hsd3β2, marker genes chicken KIT proto-oncogene receptor tyrosine kinase (Cvh) and chicken KIT proto-oncogene receptor tyrosine kinase (C-kit), downstream genes cytochrome P450 subenzyme (Cyp1a1), UDP glucuronosyltransferase family 1 member A1 (Ugt1a1) and 17beta-hydroxysteroid dehydrogenase7 (Hsd17b7). Flow cytometry in vitro showed the number of the positive cells. After injecting the vector in vivo, genital ridges were collected in 4.5 d. qRT-PCR was used to detect the expression of Hsd3β2, marker genes and downstream genes. Flow cytometry was used to analyze the number of PGCs. PGCs were observed by optical microscopy and periodic acid-schiff stain (PAS). The target gene was successfully ligated into the lentiviral vector. The vector had a high efficiency and shRNA-2 has a better interference efficiency in DF1. qRT-PCR showed that after transfecting sh-Hsd3β2 and pc-Hsd3β2, the expression of Hsd3β2 increased. After packaging with lentiviral, the viral titer was 5×106 TU/ mL. qRT-PCR showed that after interfering Hsd3β2 in vitro/in vivo, the expression of downstream genes Cyp1a1, Ugt1a1 and Hsd17b7 in the steroid hormone signaling pathway was significantly decreased (P＜0.01). After inhibiting Hsd3β2 during the induction experiment, the number of embryoid body decreased and embryoid body appeared later. qRT-PCR showed that marker genes Cvh, C-kit significantly decreased (P＜0.01). Flow cytometry indicated that after inhibiting Hsd3β2, the number of positive cells (3.27±0.19)% compared with control group (7.39±0.09)% significantly decreased in vitro. In vivo, after inhibiting Hsd3β2, PAS staining showed that the number of PGCs had been descreased. qRT-PCR indicated that marker genes decreased (P＜0.01). Flow cytometry showed that after inhibiting the expression of Hsd3β2, DDX4+ positive cells decreased.Hsd3β2 could regulate the steroid hormone synthesis pathway to positively affect the differentiation of ESCs to PGCs.
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