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J. Biol. Chem., Vol. 282, Issue 1, 721-730, January 5, 2007

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Identification of Neuregulin as a Factor Required for Formation of Aligned Spermatogonia^*

F. Kent Hamra¹, Karen M. Chapman^¶, Derek Nguyen^¶, and David L. Garbers^¶

From the Cecil H. and Ida Green Center for Reproductive Biology Sciences, ^¶Howard Hughes Medical Institute and the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

In the absence of somatic cells, medium conditioned by the SNL fibroblast line (SNL-CM) is able to stimulate primary cultures of rat type-A single spermatogonia to develop into chains of aligned spermatogonia at the 8-, 16-, and 32-cell stages. By comparison, medium conditioned by an MSC-1 Sertoli cell line is ineffective. Glial cell line-derived neurotrophic factor (GDNF)-like molecules were identified in SNL-CM and recombinant forms of GDNF, neurturin, and artemin were shown to stimulate formation of aligned spermatogonia, but principally to only the 4- and 8-cell stages. Because SNL-CM and GDNF-like molecules stimulated the formation of spermatogonial chain length differently, we purified components of SNL-CM to identify the additional contributing factor(s). A fraction was isolated that was dependent on GDNF, but required for effective formation of 16- and 32-cell chain lengths. Sequence analysis identified the factor as mouse neuregulin-1. At picomolar concentrations, recombinant neuregulin-1 in combination with GDNF effectively stimulated formation of aligned spermatogonia up to the 32-cell stage. Neuregulin in the absence of GDNF was relatively ineffective. Soluble receptors for neuregulins blocked the effects of GDNF and SNL-CM, suggesting that both neuregulin and GDNF are required for effective formation of long spermatogonial chains. Addition of neuregulin-1 to cultures on MSC-1 feeder layers resulted in spermatogonial behavior similar to that seen on feeder layers of SNL fibroblasts. In fact, SNL cells were found to express 100-fold higher levels of neuregulin-1 transcripts than MSC-1 cells. Thus, we identify neuregulin as a factor required for spermatogonial amplification and differentiation in culture.

Received for publication, August 31, 2006 , and in revised form, November 10, 2006.

^* This work was supported by the Cecil H. & Ida Green Center for Reproductive Biology Sciences and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Tables S1 and S2.

¹ To whom correspondence should be addressed: 6001 Forest Park Rd., Dallas, TX 75390-9051. Tel.: 214-645-6279; Fax: 214-645-6276; E-mail: kent.hamra{at}utsouthwestern.edu.

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