engleski

Specific transgene expression in mouse spermatogonial stem cells

Spermatogenesis is the process that lasts throught adult life. Spermatogonia, especially spermatogonial stem cells, are the cells essential for the continued maintenance of spermatogenesis. Sperm output and the integrity of the male genome relies on their continous &laquo ; ; ; ; error free&raquo ; ; ; ; proliferation and diffentiation (Meachem et al., 2001). Stem cells are defined only in terms of their function. They are self renewing precursor cells that can generate differentiating cell populations to replenish the lost of cells. Stem cells are rare in in most system making it difficult to isolate them and study their function. Estimation is about two stem cells per 104 total testis cells (Shinohara et al., 1999). Many studies of spermatogonia have been performed with morphological methods but the true nature of spermatogonia still remains unknown. Limitation in understanding their control is also the fact that no specific markers are still available for their selection (Ogawa, 2001). The aim of this study was to identify a promoter which direct trangene expression to the earlier stages of male germ line differentiation and to use the promoter to drive the expression of a reporter gene and a neutral surface marker. The Stra8 gene expressed in spermatogonia appeared to be hopeful candidate (Oulad-Abdelghani et al., 1996). The 400 pb fragment encompassing the transcription start was sufficient for the expression of a luciferase reporter gene in the testis of Stra8/luc transgenic mice (Giuili et al., 2000). In two independent families of transgenic mice expressing the reporter, spermatogenesis proceeded normally. Determination of testis section and establishment of developmental patterns of expression in the prepubertal testis indicated that the cloned promoter was active in spermatogonia, but unlike the endogenous gene, only in a fraction of the adult spermatogonia. In order to isolate and identify the cells which express the transgene, Stra8/HAglo transgenic families were generated in which the same promoter drived the expression of a neutral surface marker (HAgloCD4) constituted by two domains of the human CD4 protein fused with the transmembrane and intracytoplasmic regions of the influenza hemagglutinin. Magnetic sorting of CD4+ cells yielded a morphologically homogenous cell fraction which expressed spermatogonial markers: RBM and cyclin A2. All recovered cells were also positive for integrin-b1 and Ep-CAM, which are known as stem cell markers. Isolated cells were negative for c-kit which is expressed in differentiated spermatogonia. Their identity as stem cells was demonstrated by a high efficiency in re-establishing spermatogenesis in a germ cell-depleted testis. The isolation of spermatogonial stem cells is neccessary for innovative research into stem cell biology and for the understanding of spermatogonial physiology. Understanding of spermatogonia could have important clinical implications in: infertility treatments, protections of spermatogonial stem cells in oncological patients, development of contraceptive strategies, the field of animal husbandry, generations of transgenic animals, in preservations of valuable animals etc. This work is a contribution to this aims.

Spermatogonia; marker; transgene expression

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