DDX4-EGFP transgenic rat model for the study of germline development and spermatogenesis †

Abstract

Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.

Hormone induced differential transcriptome analysis of Sertoli cells during postnatal maturation of rat testes

“…Rat is considered a better model (than mice) for understanding the regulation of testicular functions [ 36 ]. Due to larger body size and higher blood volume and litter size, rats are more favored for surgical, physiological and pharmacological experiments [ 37 , 38 ].…”

Germline development in rat revealed by visualization and deletion of Prdm14

“…Histological analysis to identify rat PGCs by morphology (Beaumont and Mandl, 1963), alkaline phosphatase activity (Kemper and Peters, 1987) and immunostaining of some representative markers (Encinas et al, 2012;Leitch et al, 2010;Northrup et al, 2011) have provided a rough timeline for rat germline development. In a recent report, to visualize gonadal to adult germ cells in vivo, Ddx4-EGFP transgenic rats were generated (Gassei et al, 2017). However, owing to the absence of Ddx4 expression in earlier PGCs, the specification and migration stages of rat PGCs have not yet been documented.…”

Comparison of the effects of buffalo LIF and mouse LIF on the in vitro culture of buffalo spermatogonia

“…We identified nonbuffalo antibodies by immunofluorescence staining of juvenile buffalo testis tissues. DDX4 (VASA) is a member of the DEAD‐box family, and the results showed that GATA4‐positive Sertoli cells and Leydig cells were not immunolabeled with VASA; VASA can be utilized as a molecular marker of germ cells, which has been reported in several mammals, including humans, cattle, pigs, and goats (Gassei et al, 2017; J. Kim et al, 2015; Sada et al, 2012; Xu et al, 2021). NANOS2 is an RNA‐binding protein that is mostly expressed in A S and A pr spermatogonia, is required for the self‐renewal of SSCs and is generally considered a marker of SSCs; when NANOS2 was knocked out, many undifferentiated A‐type spermatogonial cells were lost; however, overexpression of NANOS2 resulted in strong accumulation of undifferentiated A‐type spermatogonial cells (Codino et al, 2021; Sada et al, 2012).…”