Production of quail (Coturnix japonica) germline chimeras by transfer of Ficoll-enriched spermatogonial stem cells

Highlights

• Enriched SSCs are derived from quail testis using density gradients separation manner.

• Ficoll-enriched cells are more expressed SSC-specific genes than other methods.

• Ficoll-enriched cells are exhibit well-known SSCs ultrastructural characteristics.

• Germline chimeras are efficiently produced from Ficoll-enriched SSCs transplantation.

Abstract

Due to the absence of long-term in vitro germline competent stem cell maintenance systems and efficient methods for germline transmission, efforts to develop an effective transgenic system in quail has remained limited. To overcome this limitation, here we produced germline chimeric quails through transplantation of spermatogonial stem cells (SSCs) enriched by density gradient methods utilizing Ficoll-Paque PLUS (Ficoll), Percoll and sucrose solution as a practical strategy for germline transmission in quail. For all gradient methods, testicular cells were separated as two fractions, and the expression levels of SSC-specific genes (GFRA1, ITGA6, ITGB1) and pluripotency genes (NANOG, POUV) were examined. As a result, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA probe hybridization analysis revealed that the upper fraction that was separated by Ficoll showed the highest expression of SSC-specific and pluripotency genes among all fractions. Cells in the upper Ficoll gradient fraction also displayed reduced heterochromatin distribution, as observed in differentiated spermatogonia using transmission electron microscopy (TEM). These results indicate that SSCs were enriched in the upper fraction by Ficoll density gradient centrifugation. Subsequent transplantation experiments revealed that the efficiency of germline transmission to donor-derived gametes in the germline chimeras with transplanted SSCs and whole testicular cells was 0–13.2% and 0–4.4%, respectively. Collectively, these results demonstrate that quail SSCs were easily enriched with a density gradient method and that this method is a feasible and practical way to preserve the germplasm of quail. Furthermore, we can expect to apply this method in research examining the production of transgenic quail and preservation of avian species.

Introduction

Quail (Coturnix japonica) has been considered as an appropriate model for developmental studies because it is easy to manipulate quail eggs during all developmental stages of embryos [1]. In addition, their small body size, low maintenance cost and short generation period make quail a suitable experimental model organism for transgenic research [[2], [3], [4], [5]]. Particularly, the unique route of germ cell migration through the bloodstream enables exogenous germ cell transplantation in birds [6]. However, unlike mammals, it is difficult to produce transgenic animals by embryo transfer or nuclear transfer methods in avian species because of their oviparous characteristics. Therefore, the use of germline competent stem cells, such as primordial germ cells (PGCs) or spermatogonial stem cells (SSCs), for germline chimera production is regarded as an alternative way to produce transgenic birds [7,8].

To date, several studies have examined germline chimeric quails generated through PGC transfer into recipient embryos [[9], [10], [11]]. The percentage of germline transmission to donor-derived gametes was 1.8–63.0% from noncultured blood PGC (bPGC) [11], 2.2–4.7% from noncultured gonadal PGC (gPGC) [9] and 2.4–2.5% from liquid nitrogen preserved gPGC [12]. In 2008, transgenic quail was produced by transferring gPGC, but the transgenic quail production efficiency was still low (1.9%) [5]. Moreover, the establishment of an in vitro cultivation system of quail gPGC and production of germline chimeric quail by transferring cells was attempted. However, in vitro cultivation of quail gPGC has remained limited because of its survival duration, proliferation rate and germline transmission efficiency [10,13]. Therefore, the development of another way to produce germline chimeric quails is needed for practical transgenic research.

SSCs are germline stem cells from testis and, are also an important source for transgenic research [14]. SSCs have unique characteristics, including self-renewal properties and differentiation ability into mature spermatozoa. Based on these properties, they have been regarded as a useful cell source for transgenic animal production and regenerative therapies for humans [15]. Transplantation of SSCs was first suggested in 1994 and actively studied in mouse and human [16]. In other mammalian species such as sheep and goat, donor-derived progeny were successfully produced by SSC transplantation [17]. However, because of the low proportion of SSCs in adult testis (0.02–0.03% of whole testicular cells in mice), enrichment and purification of SSCs from testicular cells are required for practical applications [18].

Several methods have been reported for enriching and purifying SSCs from testicular cells, including differential plating [19], density gradient centrifugation [20,21], and antibody-mediated purification methods such as fluorescence-activated cell sorting (FACS) [22] and magnetic cell sorting (MACS) [23]. Though the differential plating of quail SSCs and the production of germline chimeric quails have been reported in previous studies [24,25], enrichment of quail SSCs is still a challenge. Antibody-mediated SSC purification using surface markers such as ITGA6, ITGB1 and GFRA1 was successfully established in mammalian species, but specific antibodies for SSCs in quail and other birds have not yet been developed. In contrast, density gradient centrifugation has been widely used as an SSC enrichment method because of its relatively low density compared with other mature germ and somatic cells [26]. Therefore, we purposed to apply density gradient centrifugation methods for SSC enrichment and enhancement of germline transmission efficiency in quail, and it could be an alternative way for the efficient production of germline chimeras and transgenic quails.