Suppression of Insulin-Like 3 by Androgen Depletion in Hormone-Treated Irradiated Rat Testis.

Abstract

Irradiation causes spermatogenic arrest in adult rats and this arrest can be reversed by reduction of intratesticular testosterone by GnRH-antagonist, whereas further testosterone treatment attenuates the spermatogenesis recovery. To identify hormonally regulated genes correlating with the spermatogenic differentiation status, gene expression profiles were investigated by microarray with Rat 230 2.0 GeneChips (Affymetrix), comparing normal control rats, irradiated rats with no treatment, irradiated rats treated with GnRH-antagonist and either flutamide (androgen receptor antagonist) or testosterone, for 2 weeks, at which time there were minimal changes in number of germ cells. Insulin-like 3 (Insl3), a member of the relaxin-insulin hormone family was the second most strongly down-regulated gene by testosterone suppression. Real-time RT-PCR confirmed microarray observations: after irradiation, relative Insl3 mRNA level increased about 9-fold above the normal control; suppression of testosterone by GnRH-antagonist and flutamide repressed Insl3 level by 20-fold compared to the irradiated-only group; adding testosterone together with GnRH antagonist elevated Insl3 level so that it was only 6-fold reduced from the irradiated only group. Characterization of the changes in INSL3 protein levels by immunoblot supported the mRNA results: irradiation caused over 10-fold increase in relative prepro-(or pro-)INSL3 protein levels compared to that of normal controls; treatment of GnRH-antagonist and flutamide down-regulated INSL3 protein to an undetectable level; supplementation with testosterone restored INSL3 protein levels that were 7-fold reduced from the irradiated-only group. As a first step to determining if the down-regulation of Insl3 mRNA level by androgen suppression is direct or indirect, irradiated rats were treated with GnRH-antagonist + flutamide for 1, 3, and 7 days, respectively. One-day androgen suppression induced only a 1.3-fold reduction in Insl3 mRNA level compared to irradiated-only rats; 3 days of androgen suppression resulted in 4-fold Insl3 mRNA reduction; and 7 days androgen depletion caused a 10-fold reduction in Insl3 mRNA level. Meanwhile, one day GnRH-antagonist + flutamide reduced INSL3 protein 1.7-fold, and both 3 days and 7 days androgen depletion suppressed INSL3 protein to undetectable levels. Measurement of intratesticular testosterone levels will give us more insight on whether Insl3 is directly or indirectly regulated by androgen. Similar regulation of Insl3 mRNA by androgen has been observed in germ cell depleted (e.g. jsd) mice treated with GnRH-antagonist plus flutamide or testosterone. Thus INSL3 levels are associated with spermatogenesis arrest/recovery in at least two different pathogenic models. Although INSL3 plays a major role in testicular descent during embryonic development by acting on the gubernaculum, little is known about its function in the adult. Further characterization of INSL3 regulation will help us understand the role of INSL3 in the recovery of spermatogenesis after toxicant treatment of adult mammals. (Supported by Grant ES-08075 from NIEHS and HD-40397 from NICHD)