Expression and functional characterization of zebrafish gonadal soma-derived growth factor.

摘要

Gonadal soma-derived factor (Gsdf) is a transforming growth factor-beta (TGF-β)-related peptide that is found in fish and has been shown to promote the growth of primordial germ cells (PGCs) and spermatogonia in rainbow trout. To examine the biological function of Gsdf in zebrafish, I studied the expression pattern and biological activity of Gsdf in embryos and adult fish. Temporal expression analysis by RT-PCR revealed that gsdf transcripts were first detected at 1dpf and the level remained stable through 15dpf. Analysis of gonads by in-situ hybridization in wild type zebrafish showed that gsdf was predominantly expressed in granulosa cells surrounding early stage oocytes and Sertoli cells of adult testis. Inhibition of Gsdf translation by an antisense morpholino suppressed PGC number in the embryo while introduction of gsdf mRNA resulted in an increased number of germ cells. Cell culture studies revealed for the first time that recombinant zebrafish Gsdf promoted the proliferation of zebrafish ovarian germ cells (OGCs) in vitro and enhanced the expression of the germ cell markers nanos3 and dnd. The recombinant Gsdf activated a MAPK/ERK signaling pathway to control ovarian germ cell (OGC) proliferation and upregulates nanos3 expression. These results demonstrate that Gsdf plays a significant role in PGC development and OGC proliferation. To further investigate Gsdf function, I generated a zebrafish transgenic line, Tg(gsdf:neo-gsdf), in which the expression of a neomycin-resistant gene (neo) is driven by the gsdf promoter, and the 3'UTR. Immunohistochemistry analysis of this transgenic line revealed that the expression of the neomycin gene is detected in the granulosa cells that surround early-stage oocytes and Sertoli cells of adult testis. The use of this transgenic line made it possible to isolate homogenous populations of cells that express Gsdf from a four month old testis and ovary by G-418 selection. Use of the drug selected, Gsdf-expressing cells as a feeder layer for cell culture experiments resulted in significantly greater proliferation of spermatogonial and OGCs and enhanced expression of the germ cell markers, vasa and dnd. Use of this transgenic feeder cell line was an important step in optimizing the conditions required for establishment of long-term germ cell cultures. The OGC and spermatogonial cell cultures resulting from this work will provide an in vitro system for studies of testicular and ovarian germ cell biology.