Expression Patterns of Mouse Follistatin Variants Fst288 and Fst315, Fstl3, Activin A and Its Receptors and the Inhibin Subunits, Throughout the Male Mouse Reproductive Tract.

Activin A and its binding protein, follistatin (FST), play critical roles in the development and function of the male reproductive tract. During differentiation of the Wolffian duct into the male reproductive tract, activin A stimulates coiling of the epididymal duct, but the roles of activin A and FST in the adult tract are not well established. In humans and rats, alternative splicing leads to two forms of follistatin: FST288, which is predominantly tissue-bound and the circulating form, FST315. The sequence for mouse Fst288 mRNA was unknown at the outset of these studies. This sequence was established by RT-PCR of FVB mouse ovary and uterus cDNA, using primers based on a RIKEN project sequence containing partial mouse Fst288 and the mouse genome sequence of the 5th intron of Fst315. Sequencing of the mRNA for mouse Fst288 indicated that the 3'UTR of mouse Fst288 in mouse ovary and uterus was much shorter than that of human or rat FST288, and did not overlap with any part of the sequence of the 3'UTR of mouse Fst315. Using the difference in the 3'UTR of the mouse Fst288 and Fst315, it was possible to establish a QRT-PCR screen to measure the expression of each variant. When this screen was used to detect mouse Fst288 and Fst315 in the mouse ovary and uterus, only one product was amplified for each variant, and sequencing of these products confirmed the appropriate identity of the products. Expression of Inhba (the gene encoding the activin A subunits), Inha and Inhbb (the genes encoding the inhibin B subunits, and follistatin-like 3 (Fstl3), and the activin receptor subunits (Acvr1, Acvr2a and Acvr2b) were also measured by QRT-PCR. Activin A protein was measured by ELISA, and total follistatin was measured by RIA. In adult male C57Bl6 mice, expression of activin A mRNA was highest in the epididymal caput and cauda, while receptor subunit expression was highest in the corpus. There was an increasing gradient of both Fst forms passing from the cauda epididymis to the distal vas deferens. In fact, Fst288 mRNA in the vas deferens was significantly higher than in 22 different adult mouse tissues. Fst protein levels were also highest in the vas deferens, followed by the prostate and seminal vesicles, but Fst protein levels in the accessory organs was not matched by elevated mRNA expression. Skin also had high levels of FST protein, and showed the highest Fst315 mRNA levels. The expression patterns of the two Fst transcripts throughout the male reproductive system were very similar. Expression of activin A and the Fst forms was relatively low in the testis, but inhibin alpha-subunit mRNA and inhibin betaB-subunit was expressed at highest levels in the testis, as expected. In contrast to Fst, Fstl3 was expressed at constant levels throughout the reproductive tract. These data indicate that activin A and Fst play a role in paracrine communication along the male reproductive tract in the adult. High levels of Fst288 in the vas deferens suggests that activin bioactivity is very low in this tissue, and may explain the lack of coiling of the vas deferens. (platform)