A New Method for Isolating Viable Gonadal Germ Cells from 7-day-old Chick Embryos

Abstract

A novel and simple method for isolating viable gonadal germ cells (GGCs) was developed in the domestic chicken, Developing gonads were isolated from-day-old chick embryos and cultured for up to hours at. in phosphate bu ered saline without Ca and Mg (PBS [ ]). A discharge of GGCs from the gonad was observed within minutes after introducing the embryonic gonad into PBS [ ], and the number of discharged GGCs increased until hours of incubation. The purity of the GGCs (number of discharged GGCs/total number of discharged cells) was approximately for the initial. hours of incubation and decreased thereafter. The number of discharged GGCs from the left gonad was significantly higher than that from the right gonads in females. Fifty discharged GGCs in PBS [ ] were injected into the blood stream of-day-old chick embryos after staining with PHK fluorescent dye. GGCs exhibiting a fluorescent signal were detected after incubating recipient embryos for days. These results indicate that high-purity, viable GGCs can be collected by simply introducing isolated developing gonads into PBS [ ]. : chicken, gonadal germ cells, isolation, PBS [ ]

Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

“…Results under microscopic observations showed that gonadal PGCs that were released from the gonads reached a high number in 1 h after incubation of the gonads (Figure 2). This results show that gonadal PGCs can be isolated by incubating embryonic gonads of chicks in PBS [-] at 38 o C for about 1 h (Nakajima & Tajima, 2013). …”

“…The primordial germ cells were then collected using PBS [-] solution. The gonads were incubated using PBS [-] for one hour (Nakajima et al, 2011). The morphological characteristics and the total number of gonadal PGCs of KUB chicken were observed microscopically.…”

“…The cells leave the capillary system and migrate to the form of genital ridge, where they colonize the developing gonad (Minematsu et al, 2004;Glover & McGrew, 2012). After migrating to the gonadal anlage, the PGCs are called gonadal germ cells (GGCs) (Nakajima et al, 2011). Gonadal germ cells or gonadal primordial germ cells (gonadal PGCs) in the gonad, ultimately differentiate into spermatogonia, which takes place in the testes or into oogonia, which takes place in the ovaries.…”

“…Primordial germ cells can be isolated from X-stage blastoderm, blood at 2.5-3-day-old embryos (stages 13-17 HH), or gonads at 5.5-6-day-old embryos (stages 26-28 HH) (Chojbacka-Puchta et al, 2012;Tajima, 2013). Numerous studies had reported the isolation and collection of PGCs from gonadal embryos (Nakamura et al, 2011;Nakajima et al, 2011;Nakajima et al, 2016). Moreover, germline chimeras had been produced in chicken by transferring PGCs or GGCs into the bloodstream at 2-day-old recipient embryos (Furuta et al, 2007(Furuta et al, , 2008 so that PGCs could be used as a genetic resource to produce germ line chimera (Furuta, 2012).…”

“…There are several methods used to isolate avian gonadal PGCs, one is by using proteinases, such as trypsin solution Kohara et al, 2008) and the other is by using a phosphate-buffered saline without Ca 2+ and Mg 2+ solution (PBS [-]) (Nakajima et al, 2011). The present study was conducted to apply this method to isolate gonadal PGCs from KUB chicken embryos by using phosphatebuffered saline without Ca 2+ and Mg 2+ solution (PBS [-]) (Nakajima et al, 2011). The main objective of this study was to determine the number of gonadal PGCs of KUB chicken at different developmental stages of embryos.…”