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First published online 2 December 2004
doi: 10.1242/dev.01555

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Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

¹ Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
² Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
³ The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki 305-0074, Japan
⁴ Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

View larger version (133K): [in a new window]   Fig. 1. Spermatogenesis and teratogenesis from fetal germ cells and epiblast cells. (A) Embryos at 6.5 and 8.5 dpc, a mid-part of 10.5 dpc embryo, and a male gonad and mesonephros at 12.5 dpc. Dotted lines demarcate regions used for transplantation. At 10.5 dpc, the urogenital ridges (asterisk) and mesentery with gut (arrow) were dissected separately. (B) A section of a W male testis (control recipient) stained with HE. Spermatogenesis is absent. (C) W mouse testis after transplantation of 8.5 dpc PGCs. Spermatozoa (arrow) are present in the center of the seminiferous tubule. (D) Anti-SCP3 immunostaining (green) of W testis after transplantation of 8.5 dpc PGCs, counterstained with Hoechst 33258 dye (blue). Inset, higher magnification view of the same sample. (E) Transplantation of epiblast cells at 6.5 dpc. Spermatocyte (arrow) and round spermatid (arrow head) were found. Inset shows acrosomes stained with PAS (red) in round spermatids. (F) W mouse testis transplanted with 8.5 dpc PGCs from Green mice embryos. Colonization of the recipient seminiferous tubule by EGFP (+) donor cells (green) was observed (arrow). (G) A section of the same testis as in (F), stained with Rhodamine-PNA (red) for acrosomes and with Hoechst 33258 dye (blue) for nuclei. Spermatogenenic colonies derived from EGFP (+) donor cells are present (arrow). (H) Higher magnification view of (G), showing spermatogonia (arrowhead) residing at the base of the seminiferous tubule, and elongated spermatids (arrow) with acrosomes (red), shedding the EGFP (+) cytoplasm. (I) Teratoma from epiblast cells at 6.5 dpc. Muscle, dermoid cyst and neuronal tissue are observed. (J) Spermatozoa (arrow) clustered around a Sertoli cell, released from a recipient testis of 8.5 dpc PGCs. (K) An offspring developed from an oocyte injected with a sperm derived from 8.5 dpc PGCs. (L) DNA sequences of the Kit gene around the W (left panels) and Wv (right panels) point mutations of an offspring derived from transplantation of 8.5 dpc PGCs (upper panels) and a W/Wv mouse as a control heterozygote (lower panels). Scale bars: 1 mm in F; 10 µm in H; 20 µm in J; 50 µm in others.

View larger version (90K): [in a new window]   Fig. 2. DMR methylation of the Igf2r and H19 genes in offspring derived from transplantation of 8.5 and 12.5 dpc PGCs. DNA methylation was analyzed by bisulfite genomic sequencing. Both male and female offspring of each stage of donor PGCs were analyzed. Individual lines represent sequenced clones. Black ovals indicate methylated cytosine-guanine sites (CpGs) and white ovals indicate unmethylated CpGs.