Abundance of Early Embryonic Primordial Germ Cells Promotes Zebrafish Female Differentiation as Revealed by Lifetime Labeling of Germline

Abstract

Teleost sex differentiation largely depends on the number of undifferentiated germ cells. Here, we describe the generation and characterization of a novel transgenic zebrafish line, Tg(piwil1:egfp-UTRnanos3)^ihb327Tg, which specifically labels the whole lifetime of germ cells, i.e., from primordial germ cells (PGCs) at shield stage to the oogonia and early stage of oocytes in the ovary and to the early stage of spermatogonia, spermatocyte, and spermatid in the testis. By using this transgenic line, we carefully observed the numbers of PGCs from early embryonic stage to juvenile stage and the differentiation process of ovary and testis. The numbers of PGCs became variable at as early as 1 day post-fertilization (dpf). Interestingly, the embryos with a high amount of PGCs mainly developed into females and the ones with a low amount of PGCs mainly developed into males. By using transient overexpression and transgenic induction of PGC-specific bucky ball (buc), we further proved that induction of abundant PGCs at embryonic stage promoted later ovary differentiation and female development. Taken together, we generate an ideal transgenic line Tg(piwil1:egfp-UTRnanos3)^ihb327Tg which can visualize zebrafish germline for a lifetime, and we have utilized this line to study germ cell development and gonad differentiation of teleost and to demonstrate that the increase of PGC number at embryonic stage promotes female differentiation.

Keywords: Oogenesis; Oogonia; Primordial germ cell; Spermatogenesis; Spermatogonia; piwil1.

Fig. 1

Generation and characterization of Tg(piwil1:egfp-UTRnos3)^ihb327Tg. a The structure of the transcriptional regulatory region of piwil1 locus of zebrafish and the sketch map of transgenic elements. b–h Germ cell-specific expression at various developmental stages as indicated in the figure (arrows indicate the EGFP-positive germ cells; outlines indicate the swim bladder). g Dissected adult testis. h Dissected adult ovary (arrows indicate EGFP-positive oogonia or early stage of oocytes). i–k Immunofluorescence of Vasa in the transgenic embryo at 1 dpf. The selected region for imaging is indicated by a red box. i EGFP; j anti-Vasa; k merged channel

Fig. 2

Using live tracing to study the correlation between the initial PGC numbers and sexual development. a Representative images of PGC-less and PGC-rich embryos at 1 dpf (a1, a5), 5 dpf (a2, a6), and 7 dpf (a3, a7). b Representative images of PGC-less and PGC-rich larva fish at 11 dpf (b1, b4), 14 dpf (b2, b5), and 20 dpf (b3, b6). c Frequency distribution of PGC number at 1 dpf; two boxes with dashed frame label the selected population of “PGC-less” and “PGC-rich.” The lateral areas of gonads were calculated and were shown at the lower right corner of images in a and b. d The sex ratio in the population of PGC-less and PGC-rich embryos. e Confocal microscopy of small and big gonads at 20 dpf with Tg(piwil1:egfp-UTRnos3)^ihb327Tg transgenic fish. e1 Confocal image of small gonads. e2 Higher magnification of a representative image of small gonad displaying combined channels of DAPI, F-actin, and EGFP. e3 Magnification image showing the nuclei of gonocyte in a small gonad. e4 Confocal image of big gonads. e5, e6 Higher magnification of representative images of big gonads displaying combined channels of DAPI, F-actin, and EGFP. e5 shows an example in which the germ cells have an irregular shape of nuclei; e6 shows an example in which chromatin nucleolus-stage oocytes exist. e7 Magnification image showing cells with irregular shape of nuclei. e8 Magnification image showing that the nuclei of gonocyte were similar to the one in a small gonad

Fig. 3

The abundance of early PGCs promotes female development. a Representative images showing PGCs in wildtype and buc-overexpressed embryos at shield stage (PGCS was visualized by WISH). b Percentage of embryos with increased PGCs or normal number of PGCs in wildtype and buc-overexpressed embryos. c Representative images showing PGCs in wildtype and buc-overexpressed embryos at 1 dpf (PGCS was visualized using Tg(piwil1:egfp-UTRnanos3)^ihb327Tg). d The sex ratio in population of wildtype and buc-overexpressed embryos. e RT-qPCR showing that buc was overexpressed in Tg(kop:KalTA4-UTRnanos3,CMV:EGFP)^ihb8Tg/Tg(UAS:buc-UTRnanos3)^ihb120Tg double-transgenic fish (dual-Tg) and Tg(UAS:buc-UTRnanos3)^ihb120Tg. f The sex ratio in the populations of sibling control (Tg(kop:KalTA4-UTRnanos3,CMV:EGFP)^ihb8Tg and wildtype fish), Tg(UAS:buc-UTRnanos3)^ihb120Tg and double transgenics

Fig. 4

Confocal microscopy of ovary development. a–h Confocal images of developing ovaries at 25 dpf (a), 30 dpf (b), 35 dpf (c), 40 dpf (d), 45 dpf (e), 50 dpf, and 3 mpf (f). a1, b1, c1, d1, and h1 are the magnified regions of interest in a, b, c, d, and h respectively. e The graph showing the percentage of oogonia, stage IA oocyte, and stage IB oocyte in ovaries of 26 dpf, 30 dpf, 35 dpf, and 40 dpf. Arrows in a1 indicate the oogonia (Og), stage IA oocyte (IA), and stage IB oocyte (IB); asterisk in c1 and d1 indicates the Balbiani body; arrow in h1 indicates the oogonia

Fig. 5

Confocal microscopy of testis development. a–d Confocal images of developing testes at 25 dpf (a), 30 dpf (b), 35 dpf (c), and 40 dpf (d). Arrows in b, c, and d indicate the degenerating oocytes. a1–a3 are the magnified regions of interest labeling 1 to 3, respectively, in a; b1 is the magnified region of interest labeling 1 in b. a1–a3 Representative image showing EGFP-positive early spermatogonia (a1), EGFP-positive early spermatogonia of Pachytene stage (a2), and weak EGFP in late spermatogonia (a3) in a. b1 Representative image showing degenerating oocyte in b. e–f Confocal images of developing testes at 45 dpf (e) and 50 dpf (f). e1–e8 are the magnified regions of interest labeling 1 to 8, respectively, in e. e1–e8 Representative images showing early spermatogonia (e1), late spermatogonia (e2), primary spermatocyte at leptotene/zygotene stage (e3), primary spermatocyte at pachytene stage (e4), secondary spermatocyte (e5), early spermatid (e6), late spermatid (e7), and primary spermatocyte at metaphase I (e8). g Graph showing EGFP intensity of various types of spermatogenic cells in e. h Graph showing the nuclear diameter of various types of spermatogenic cells in e. The left, middle, and right images in a1–a3 and b1 showed DAPI, F-actin, and EGFP, respectively; upper row in e1–e8: DAPI; lower row in e1–e8: EGFP; Asterisk indicates P value < 0.01

Fig. 6

Confocal microscopy of adult testis. a Confocal image of adult testis. b–j The magnified regions of interest in a; b–j Representative images showing early spermatogonia (b), late spermatogonia (c), primary spermatocyte at leptotene/zygotene stage (d), primary spermatocyte at late leptotene/zygotene stage based on the EGFP intensity (e), primary spermatocyte at pachytene stage (f), primary spermatocyte at metaphase I (g), secondary spermatocyte (h), spermatid (i), and spermatozoa (j). Upper row in b–j: DAPI; lower row in b–j: EGFP