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.

Keywords: DDX4; VASA; germline; rats; spermatogenesis; spermatogonial stem cells; transgenesis.

Figure 1.

Egfp mRNA expression in testis and ovary compared to somatic tissues in DDX4-EGFP transgenic rats. Fold changes of Egfp mRNA levels (2^−ΔΔCt) in selected organs are shown. The deltaCt (Egfp Ct – Actb (β-actin) Ct) was computed for each tissue on each animal. Repeated-measures ANOVA was used to determine whether the deltaCt values were significantly different across tissues. All tissues were compared to the testis (reference group) by calculating delta-delta Ct values (dCt(tissue) –dCt(testis)). Egfp expression in testis and ovary was compared between wildtype and transgenic animals using two-tailed Student's t-test. P-values less than 0.05 were considered statistically significant and are denoted by different letters.

Figure 2.

Live imaging evaluation of endogenous EGFP expression in selected organs of DDX4-EGFP transgenic rats. Representative bright-field (left) and fluorescent (right) images of selected organs from DDX4-EGFP transgenic rats are shown. Wildtype testes and ovaries served as negative controls. Strong endogenous EGFP expression was observed in transgenic testes. In transgenic ovaries, EGFP was observed in oocytes that are visible as small round green fluorescent dots. Somatic tissues did not express EGFP. Scale bar = 1 mm.

Figure 3.

EGFP immunohistochemistry of various tissues from F3 offspring. Paraffin embedded tissue sections were stained with anti-EGFP antibody. Only germ cells in DDX4-EGFP testes (A) and ovaries (C) were EGFP positive. No staining was evident in wildtype testis (B) and ovary (D) sections, or any of the other organs that were evaluated (E–L). Sections were not counterstained with DAPI to allow observation of the different levels of autofluorescence in intestine, spleen, lung, and kidney tissue. Scale bars = 10 μm.

Figure 4.

Mixed EGFP expression in oocytes of DDX4-EGFP transgenic rats. EGFP expression in ovaries was determined by coimmunofluorescence staining for DDX4 and EGFP in paraffin-embedded ovarian sections. (A–C) DDX4-positive, EGFP-positive rat oocyte. (D–F) Not all oocytes in DDX4-EGFP ovaries expressed EGFP. Panel E shows the lack of EGFP expression in an oocytes marked by DDX4 staining. (G–I) DDX4 expression is not consistent in all oocytes from 4-week-old rats as shown in panel G. EGFP was expressed in a DDX4-negative oocyte. Scale bars = 10 μm. (J–L) EGFP was not detectable in the majority of early (primordial/primary) DDX4-positive follicles (Arrows). Scale bar = 30 μm. Quantification of the DDX4 and EGFP costaining is represented in M.

Figure 5.

EGFP is expressed in germ cells throughout germ lineage development and spermatogenesis in postnatal transgenic DDX4-EGFP testes. Germ cells are identified by positive DDX4 staining at 1, 8, 14, and 28 dpp, and in adult testes (left column top to bottom). EGFP was consistently expressed in all DDX4-positive germ cells during postnatal development and during spermatogenesis (center column top to bottom), as confirmed in merged images (right column top to bottom). Scale bar = 50 μm.

Figure 6.

Transplanted SSCs from DDX4-EGFP rats produce green fluorescent colonies of spermatogenesis. (A) Single-cell suspensions from 14-day-old DDX4-EGFP testes were stained with anti-DDX4 antibody to identify germ cells, and anti-EGFP antibody to detect transgene expressing germ cells. All germ cells expressed EGFP. (B) The same single-cell suspension was costained with anti-SALL4 antibody to mark undifferentiated spermatogonia including SSCs. While most SALL4-positive spermatogonia at this age expressed EGFP, SALL4-negative/EGFP-positive germ cells were also observed (white arrowheads). Scale bars = 10 μm. (C) The same single-cell suspension as shown in A and B was transplanted into busulfan-treated infertile nude mouse recipient testes. Three months after transplantation, EGFP-positive colonies of donor-derived spermatogenesis could be observed, confirming the expression of the reporter gene in SSCs.

Figure 7.

FACS isolation of DDX4-EGFP-positive spermatogonia for long-term rat SSC culture. (A) Sorting gates for separating EGFP-positive cells from EGFP-negative cells. Sorting of PND 10 testicular cells is shown as a representative example. (B) Transplantation of negative fractions into infertile recipient mouse testes did not result in colonies of spermatogenesis. (C) Abundant green fluorescent colonies of spermatogenesis were obtained when transplanting the same cell number of EGFP-positive sorted fractions into infertile recipient testes. Scale bar in B and C is 1 mm. (D–F) EGFP-positive germ cells isolated from PND 10 rat testes were cultured on STO feeder cells and maintained endogenous EGFP expression. Scale bar = 50 μm. (G–I) Long-term culture of rSSCs for 12 weeks was established that maintains DDX4 expression (germ cell marker), and SALL4 and ZBTB16 (two established spermatogonia marker). Scale bars = 50 μm.