title: Identification of neuregulin as a factor required for formation of aligned spermatogonia.
abstract: 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.
authors.affiliation: Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. kent.hamra@utsouthwestern.edu
authors.forename: F Kent
authors.initials: FK
authors.lastname: Hamra
authors.forename: Karen M
authors.initials: KM
authors.lastname: Chapman
authors.forename: Derek
authors.initials: D
authors.lastname: Nguyen
authors.forename: David L
authors.initials: DL
authors.lastname: Garbers
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D003094:Collagen / Q000737:chemistry; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D008297:Male; D051379:Mice; D020890:Neuregulin-1 / Q000737:chemistry* / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology; D010455:Peptides / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013093:Spermatogonia / Q000378:metabolism*; D013997:Time Factors
chemical_list: D017077:Culture Media, Conditioned; D051100:Glial Cell Line-Derived Neurotrophic Factor; D020890:Neuregulin-1; D010455:Peptides; D003094:Collagen