213. Regulation of TGFβ superfamily-related genes in the newborn mouse testis by activin

Abstract

Several members of the transforming growth factor-β (TGFβ) superfamily of ligands are known to influence development of male germ cells. In our ongoing investigations of genetically modified mice with different bioactive levels of activin, we noted that testicular germ cell numbers at birth are increased in the absence of activin.1 In this study we compared expression levels of TGFβ-related genes in mice with 0, 1 or 2 copies of the activin βA subunit gene. Candidate genes were identified through hybridisation of total testis RNA from newborn (Day 0) wild type (WT; 2 copies) and activin βA null mice (KO; 0 copies) to a TGFβ SuperArray Simplicity™ membrane. Densitometric analysis of resulting signals revealed several genes that appeared to differ between genotypes. To validate these findings, pooled testes from newborn WT (2 independent groups), heterozygous (het; 2 groups) and activin βA KO (3 groups) mice were collected for RNA extraction. Quantitative real-time PCR analysis was performed using the Roche Light Cycler, with β-actin mRNA level used as the housekeeping reference. The mRNAs selected for analysis were: inhibin α, p15INK4b, Smad5, insulin-like growth factor 1 (IGF-1), ALK6 (BMPRI-B), and tissue inhibitor of metalloproteinase 1 (Timp1). The inhibin α transcript level was significantly decreased in the activin βA KO animal compared with WT, while the het sample showed an intermediate effect. Both Smad5 and IGF-1 transcripts appeared to increase in the KO animals compared with WT; however, the cyclin-dependent kinase inhibitor, p15INK4b, did not change significantly across the genotypes. Similarly, ALK-6 and Timp1 mRNA levels were also unaffected by genotype. These findings illustrate the impact of graded levels of activin A on specific genes in the fetal testis. (1)Mendis et al. (2004). Reprod. Fertil. Dev. 16(Suppl.), 103.