Forms Of Follistatin Research Articles

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin βA subunit (Inhba) and the inhibin α subunit (Inha) in uterine tissues during early pregnancy (days 1–4, preimplantation) and in response to exogenous 17β-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17β-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus.Follistatin and activin are proteins with important functions in female reproduction. There are two forms of follistatin (FST288 and FST315), which interact with activin in different ways and subsequently may have different functions. We are interested in the function of these two forms of follistatin in the uterus. In this study, we examined how the expression of the two follistatin variants and activin changed in the uterus during early pregnancy in mice, as well as in response to the female hormones oestrogen and progesterone. We saw an increase in the expression of the genes for FST288, FST315 and activin in the uterus during early pregnancy. We also saw an increase in these proteins in the mouse uterus in response to progesterone treatment. We concluded that the hormone progesterone regulates expression of both follistatin variants, as well as activin, during early pregnancy in the mouse uterus.

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Identification of activins and follistatin proteins in human follicular fluid and placenta.

Follistatin, an activin-binding protein, is able to neutralize the various activities of activin by forming an inactive complex with it. The widespread tissue localization of follistatin is very similar to that of activin, which suggests that it plays a local modulatory role in the various paracrine/autocrine actions of activin. We detected significant activin-binding activities in human follicular fluid and placental homogenates, although they were much lower than those in porcine and bovine follicular fluids, which raised the possibility that follistatin is present in human follicular fluid and placenta. Therefore, we attempted to identify the protein molecules responsible for this activin-binding activity in human follicular fluid and placental homogenates. Human follicular fluid, collected from in vitro fertilization patients, was processed by affinity chromatography successive steps on sulfated gel matrices and reverse phase high performance liquid chromatography (HPLC). The final HPLC yielded abundant follistatin and almost equimolar amounts of activin-A, -AB, and -B. The follistatin protein showed characteristic multiple bands when analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ability of each band to bind activin specifically was determined by activin binding assay and ligand blotting analysis. Several pieces of evidence, including the immunoblotting analysis and functional assay results, demonstrated the presence of three activin isoforms, A, AB, and B, in the follicular fluid. In contrast, human placental homogenates were found to contain follistatin and activin-A proteins only. Activin-AB and -B were not detected in any HPLC fraction, indicating that activin-A is the major form of activin in the human placenta. The present data indicate that the three activin isoforms and multiple forms of follistatin exist in human follicular fluid, and the activin-A isoform and follistatin exist in human placenta. They suggest that the physiological functions of the activin isoforms during embryonic development differ and that follistatin plays a functional role in the local control system(s) that regulates human reproduction.

Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins

Using in situ hybridization we have studied the localization of the messenger RNAs encoding the inhibin/activin subunits (alpha, beta A, beta B), the activin-binding protein follistatin and activin receptors (IIA, IIB) in mouse embryos during postimplantation development. From 6.5- to 9.5-days post coitum (p.c.) activin beta A and beta B subunit expression was restricted to the decidua, while activin receptor type IIB messages were exclusively detected in the embryo. Expression of activin receptor type IIA was apparent in the embryo as early as 9.5 days p.c. In contrast, follistatin transcripts were present in both the decidua and the embryo at the early postimplantation stages. In particular, the primitive streak region, specific rhombomeres in the developing hindbrain, somites, paraxial mesoderm and parietal endoderm cells attached to the Reichert's membrane showed strong expression of follistatin. In 10.5- and 12.5-day embryos expression of the beta A subunit message was abundant in mesenchymal tissue, in particular in the developing face, the body wall, the heart, precartilage condensations in the limb and in the mesenchyme of structures that show both epithelial and mesenchymal components, including tissues of the embryonic digestive, respiratory and genital tracts. The distribution of beta B transcripts was quite different from that observed for beta A. beta B is strongly expressed in selected regions of the brain, in particular the fore- and hindbrain, and in the spinal cord. Specific hybridization signals were also present in the epithelium of the stomach and oesophagus. Common sites of beta A and beta B expression are blood vessels, intervertebral disc anlagen, mesenchymal condensations in the flank region and the gonad primordium. The latter organ is the only site in the embryo where the alpha subunit is expressed, and thus where inhibit activity may be present. During the period of organogenesis the sites of expression of activin receptors type IIA and IIB messenger RNA (mRNA) generally coincide with or are adjacent to the sites of beta subunit expression. Differences in the expression patterns of the receptor RNAs are the whisker follicles, where type IIA is expressed, and the metanephros and the forebrain where type IIB transcripts are present. Taken together, the present data suggest that follistatin, but not one of the known activin forms (A,B,AB) is involved in early postimplantation development.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat follistatin: Gonadal and extragonadal expression and evidence for alternative splicing

Follistatin (FS) or FSH-suppressing protein is a polypeptide which exists in multiple forms and has inhibin-like activity. We investigated the distribution of tissues expressing FS mRNA. A segment of the rat FS mRNA corresponding to the last 157 nucleotides of exon 5 and the first 71 nucleotides of exon 6 was prepared by PCR and subcloned in plasmid GEM4Z (pGEM4Z) to produce a radiolabeled RNA probe. S1-Nuclease analysis of RNA extracted from rat tissues indicated alternative splicing of the rat FS gene and gonadal as well as extragonadal expression. In addition to the ovary and testis, the FS mRNA was detected in cerebral cortex, pituitary, adrenal, thymus, pancreas, gut, kidney, heart, uterus, skeletal muscle and lung. Treatment of female rats with combined Pregnant Mare Serum Gonadotropin (PMSG) and human Chorionic Gonadotropin (hCG) increased expression of FS mRNA in the ovary. Our results show that the mRNA for the precursor of the larger known form of FS (FS 344) is much more abundant than the mRNA for the smaller molecule (less than 5%) and that stimulation of FS transcription by PMSG and hCG in the ovary does not change this relationship.

Follistatin specifically inhibits pituitary follicle stimulating hormone release [formula omitted

Two forms of purified follistatin, a single-chain polypeptide of mol wt 35,000 (35 Kd) protein, and a related molecule of mol wt 32,000 (32 Kd), which differs from the 35 Kd form in glycosylation or carboxyl terminal truncation, specifically inhibit the release of immunoreactive FSH by primary cultures of rat pituitary cells. Both forms of follistatin and inhibin-A give similar dose-response curves, with identical slopes and maximal effects, suggesting that they may all act through the same mechanism on the pituitary cells. The median effective dose (ED50) of each of the follistatins is 6.2–7.3 ng/ml (1.8 × 10−10 M), which corresponds to ∼13 of the potency of inhibin. The effect of 35 Kd or 32 Kd follistatin is highly specific for suppressing the release of immunoreactive FSH since there is no demonstrable concomitant effect on the secretion of other pituitary hormones. The effect of follistatins, like that of inhibins, is different from that of the hypothalamic hypophysiotropic factors, requiring ≧ 18 h of incubation in a pituitary monolayer culture system to demonstrate. Coincubation of inhibin and follistatin shows an additive effect in the suppression of FSH release. Pituitary cells exposed to follistatin have significantly less depletion of intracellular FSH (0.01) than those treated with inhibin, indicating that follistatin may act primarily on the suppression of FSH release rather than on both release and synthesis of FSH, as is the case with inhibin.