Follicle-Stimulating Hormone Regulates igfbp Gene Expression Directly or via Downstream Effectors to Modulate Igf3 Effects on Zebrafish Spermatogenesis

Abstract

Previous work showed that pharmacological inactivation of Igf-binding proteins (Igfbps), modulators of Igf activity, resulted in an excessive differentiation of type A undifferentiated (A_und) spermatogonia in zebrafish testis in tissue culture when Fsh was present in the incubation medium. Using this testis tissue culture system, we studied here the regulation of <i>igfbp</i> transcript levels by Fsh and two of its downstream effectors, Igf3 and 11-ketotestosterone (11-KT). We also explored how Fsh-modulated <i>igfbp</i> expression affected spermatogonial proliferation by adding or removing the Igfbp inhibitor NBI-31772 at different times. Fsh (100 ng/mL) decreased the transcript levels of <i>igfbp1a, -3</i>, and <i>-6a</i> after 1 or 3 days, while increasing <i>igfbp2a</i> and <i>-5b</i> expression, but only after 5 days of incubation. Igf3 down-regulated the same <i>igfbp</i> transcripts as Fsh but with a delay of at least 4 days. 11-KT increased the transcripts (<i>igfbp2a</i> and <i>5b</i>) that were elevated by Fsh and decreased those of <i>igfbp6a</i>, as did Fsh, while 11-KT did not change <i>igfbp1a</i> or <i>-3</i> transcript levels. To evaluate Igfbps effects on spermatogenesis, we quantified under different conditions the mitotic indices and relative section areas occupied by the different spermatogonial generations (type A_und, type A differentiating (A_diff), or type B (B) spermatogonia). Igf3 (100 ng/mL) increased the area occupied by A_diff and B while decreasing the one for A_und. Interestingly, a concentration of Igf3 that was inactive by itself (25 ng/mL) became active in the presence of the Igfbp inhibitor NBI-31772 and mimicked the effect of 100 ng/mL Igf3 on spermatogonia. Studies exploiting the different dynamics of <i>igfbp</i> expression in response to Fsh and adding or removing NBI-31772 at different times showed that the quick downregulation of three <i>igfbp</i> as well as the delayed upregulated of two <i>igfbps</i> all support Igf3 bioactivity, namely the stimulation of spermatogonial differentiation. We conclude that Fsh modulates, directly or <i>via</i> androgens and Igf3, <i>igfbp</i> gene expression, supporting Igf3 bioactivity either by decreasing <i>igfbp1a, -3, -6a</i> or by increasing <i>igfbp2a</i> and -<i>5b</i> gene expression.