Expression of activin A and follistatin core proteins by human prostate tumor cell lines.

Activin and follistatin (FS) messenger RNA and protein are expressed and localized to human prostate tissue from men with high grade cancer and to human prostate tumor cell lines LNCaP, DU145, and PC3. Although activin A induces apoptosis and inhibits cell proliferation in LNCaP cells, PC3 cells are insensitive to the effect of exogenous addition of activin A. The results of this study show that activin A and FS are produced and can be measured by specific enzyme-linked immunosorbent assays in PC3 cells and media but are not detectable in LNCaP cells. Over 10 days in culture, the production of activin A by PC3 cells declines and is inversely correlated (r = -0.779) to FS288 production, which steadily increases and is significantly elevated compared with Day 1 of culture. The presence of FS288 and FS315 proteins was confirmed by immunocytochemistry and showed that only PC3 cells produced the FS288 isoform. Western blotting of PC3 cell media confirmed the presence of the FS288 isoform. Blockade of FS288 activity with a neutralizing antibody rendered PC3 cells responsive to activin A, as measured by inhibition of proliferation. Collectively, these results suggest that PC3 tumor cells are insensitive to activin A because they produce measurable amounts of activin ligand and FS288 protein, which is capable of blocking the autocrine response of these cells to activin A.

Expression and localization of activin subunits and follistatins in tissues from men with high grade prostate cancer.

Activins are growth and differentiation factors that have growth inhibitory effects on LNCaP and DU145, but not PC3, human prostate tumor cell lines. Activin-binding proteins, follistatins, block the inhibitory actions of exogenously added activins on LNCaP and DU145 tumor cell lines. Based on these in vitro observations using human prostate tumor cell lines, the aims of this study were to determine whether activins and follistatins are expressed in the human prostate in tissues from men with high grade prostate cancer. The expression and cellular localization of these proteins in malignant and nonmalignant regions of these tissues were compared to determine whether any changes occur with progression to malignancy. The results demonstrate that activins and follistatins are synthesized in tissues from men with high grade prostate cancer, and that messenger ribonucleic acid (mRNA) and protein for the activin beta A- and beta B-subunits and follistatin is expressed and localized to poorly differentiated tumor cells. In the nonmalignant regions, activin beta A and beta B subunit mRNA and proteins are predominantly localized to the epithelium. Follistatin mRNA was expressed in the basal epithelial cells and in the fibroblastic stroma; however, the localization of follistatin proteins using two specific antisera demonstrated a difference between the follistatin isoforms expressed in basal cells and the stroma. In the progression to malignancy, the colocalization of follistatin and activins to the tumor cells in vivo implies that resistance to the growth inhibitory effects of activin may be conferred by follistatins.

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin.

Recent studies have found follistatin to be an important regulator of activin bioactivity. Whilst a number of assay formats have been described, all are of limited sensitivity and require the use of isotopes. Many use polyclonal antibodies. Furthermore, a wide range of follistatin preparations have been used as standards, complicating inter-laboratory comparison. We now describe an ultra-sensitive two-site enzyme immunoassay using a pair of mouse monoclonal antibodies raised against follistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follistatin. The assay had a detection limit of <19 pg/ml and recovery of spiked follistatin 288 from amniotic fluid, serum seminal plasma, human follicular fluid and granulosa cell conditioned medium averaged 100.7 +/- 7.5%, 89.1 +/- 5.5%, 98 +/- 4.9%, 96 +/- 7.2% and 123.9 +/- 11% respectively. The intra- and interplate coefficients of variation were < 5%. An excess of activin-A (50 ng/ml) prior to assay did not affect follistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and activin-AB had minimal cross-reactivity (<0.3%). However, follistatin 315 had a significant cross-reaction (9.9%). Serially diluted human samples gave dose-response curves parallel to the standard. Pooled human follicular fluid contained high concentrations of follistatin (approximately 242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester approximately 0.8 ng/ml, third trimester approximately 2.8 ng/ml), normal male serum (approximately 0.45 ng/ml), amniotic fluid (sixteen week approximately 3.63 ng/ml, term approximately 0.89 ng/ml), seminal plasma (2.4-30 ng/ml) and human granulosa cell conditioned media (approximately 0.44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (approximately 0.62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women appeared to have higher follistatin levels than any of the normal women (approximately 1.4 ng/ml). The possible presence in certain samples of mixtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follistatin immunoassays. Further work is needed to identify the major immunoreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prove a useful tool for various clinical and physiological studies.

Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB.

Monoclonal antibodies, specific for the beta A and beta B subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0.19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentration of activin-AB were between 1.3% and 2.67%. The between-plate coefficient of variation was 5.5%. Cross-reactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting < 0.2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M(r) of > 700000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an M(r) of approximately 25000 consistent with the complete dissociation from binding proteins. Activin-A was detected in relatively high concentrations in human FF (approximately 5 ng/ml), homogenized placental extracts (4.35-95.5 ng/g), sera from pregnant women (> 4 ng/ml) and amniotic fluid (3-13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ ml), normal cycle serum (100-200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml), and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (< 0.19 ng/ml) in all of these samples with the exception of human FF (approximately 7 ng/ml). In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Increased maternal serum activin A but not follistatin levels in pregnant women with hypertensive disorders.

Activin A levels are elevated in maternal serum of pregnant women with hypertensive disturbances. Because follistatin is a circulating binding protein for activin A, the present study was designed to evaluate whether serum follistatin and activin A levels also change in patients with hypertensive disorders in the last gestational trimester. The study design was a controlled survey performed in the setting of an academic prenatal care unit. Healthy pregnant women (controls, n=38) were compared with patients suffering from pregnancy-induced hypertension (PIH, n=18) or pre-eclampsia (n=16). In addition, the study included a subset of patients with pre-eclampsia associated with intrauterine growth restriction (IUGR, n=5). Maternal blood samples were withdrawn at the time of diagnosis (patients) or in a random prenatal visit (controls), and serum was assayed for follistatin and activin A levels using specific enzyme immunoassays. Hormone concentrations were corrected for gestational age by conversion to multiples of median (MoM) of the healthy controls of the same gestational age. Follistatin levels were not different between controls and patients, while activin A levels were significantly increased in patients with PIH (1.8 MoM), pre-eclampsia (4.6 MoM), and pre-eclampsia+IUGR (3.2 MoM, P<0.01, ANOVA). The ratio between activin A and follistatin was significantly increased in patients with PIH (1.5 MoM) and was further increased in patients with pre-eclampsia (4.5 MoM) and in the group with pre-eclampsia+IUGR (2.6 MoM). Follistatin levels were positively correlated with gestational age in control subjects (r=0. 36, P<0.05) and in patients with PIH (r=0.46, P<0.05) or pre-eclampsia (r=0.61, P<0.01), while activin A correlated with gestational age only in the healthy control group (r=0.69, P<0.0001). The finding of apparently normal follistatin and high activin A levels in patients with PIH and pre-eclampsia suggests that unbound, biologically active, activin A is increased in women with these gestational diseases.

Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n = 6) and control (CON; n = 8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles > or = 3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had approximately 3-fold more follicles > or = 3 mm (P < 0.0001) and approximately 3-fold more oestrogenic follicle (P < 0.001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (approximately 6-fold; P < 0.001) and inhibin A (approximately 3-fold; P < 0.001) but only slightly more follistatin (approximately 1.4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (approximately 1:1) was significantly higher (P < 0.001) than in follicles from CON ewes (approximately 0.3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.

Concentrations of activin A, inhibin A and follistatin in human amnion, choriodecidual and placental tissues at term and preterm.

To investigate labour-associated changes in production of activin and related hormones by gestational tissues we prepared extracts from amnion, choriodecidual and placental tissues delivered at term before labour (TNL; n=15), at term after spontaneous labour (TSL; n=15) or preterm (PTD; n=31) and measured concentrations of inhibin A, activin A and follistatin by ELISA. Activin concentrations in placental tissues were significantly (Mann-Whitney U-test; P<0.05) elevated with term labour (pg/mg protein, median; 1313 vs 2591), but in the PTD tissues concentrations were lower than those delivered spontaneously at term (3650 vs 2649). Inhibin concentrations also increased with term labour in the placenta (480 vs 686), but paradoxically decreased in amnion (188 vs 64) and choriodecidua (657 vs 358). Little or no significant changes in follistatin concentrations were observed. Concentrations of all three proteins were significantly correlated between amnion and choriodecidual tissues, and were significantly correlated with each other in most tissues (Spearman's ranked correlation; P<0.05). The activin:inhibin ratio in term amnion and choriodecidual tissues was increased 2 to 3-fold (P<0.0005 by Mann-Whitney U-test) after term labour, with similar trends also observed in the activin:follistatin ratio in placental tissue. These data suggest that a modest increase in placental activin and inhibin production may occur with labour at term. In addition, an increase in activin bioactivity may occur with labour, potentiating any paracrine effects of activin during parturition. The data, however, do not support an association between increased intrauterine activin biosynthesis and preterm delivery.

Amniotic fluid concentrations of dimeric inhibins, activin A and follistatin in pregnancy.

OBJECTIVE: The feto-placental unit is the major source of circulating concentrations of inhibin A and activin A in human pregnancy. The aim of this study was to measure the amniotic fluid concentrations of inhibin A, inhibin B, activin A and follistatin in pregnancies bearing male and female fetuses. DESIGN AND METHOD: Amniotic fluid samples collected by amniocentesis were stored at -20 degrees C. Dimeric inhibins, 'total' activin A and 'total' follistatin were measured using specific two-site enzyme immunoassays. Samples were assayed blindly and the information on fetal sex was obtained from the cytogenetics laboratory. RESULTS: Data show that amniotic fluid concentrations of inhibin A, inhibin B and activin A gradually increase with gestation whilst concentrations of follistatin are similar between weeks 15 and 20 of pregnancy. Mean amniotic fluid levels of inhibin A and inhibin B at 16 and 17 weeks gestation and mean activin A levels at 15 and 16 weeks gestation are considerably lower in pregnancies with male (n=24) compared with female (n=28) fetuses. Levels of follistatin are not different in the male and female fetal pregnancies at any studied gestation. CONCLUSIONS: The results indicate that amniotic fluid contains high concentrations of inhibins (A and B), activin A and follistatin in early pregnancy suggesting that these hormones are produced by the fetal membranes and may be involved in the development of the fetus.

Adrenergic mechanisms in the hindlimb circulation of baboons.

The effects of the adrenergic agonists norepinephrine, epinephrine, isoproterenol, and phenylephrine upon femoral arterial blood flow were measured in baboons before and after alpha (phenoxybenzamine) and beta (propranolol) adrenergic receptor blockade. Flow was measured with an electromagnetic flowmeter. Arterial and venous pressures were recorded simultaneously. Femoral vascular resistance was calculated from these data. Catecholamines were injected intra-arterially (10(-3)--10(0) mug, base, kg.(-1) and intravenously (1.0 mug kg.(-1) in a randomized sequence. All four adrenergic amines were vasodilators at low dose (10(-3) mug kg.(-1), intra-arterially) and this effect was abolished during beta adrenergic receptor blockade. Intra-arterial isoproterenol elicited dose-dependent increases in femoral flow; the other amines were vasoconstrictors at high doses. Alpha adrenergic blockade "reversed" the vasoconstrictor effects of these three amines. At the same dose isoproterenol increased flow more through the intra-arterial than the intravenous route. Conversely, norepinephrine and epinephrine were potent femoral vasodilators when injected intravenously. The findings indicate that the classical adrenergic amines are all vasodilators of the subhuman primate hindlimb at low doses due to their interaction with beta receptor sites. The fact that epinephrine and norepinephrine exert a greater increase in flow when given intravenously than when given intra-arterially is presumably secondary to increased arterial pressure, in turn due to the vasoconstrictor effects of these agents on other regional circulations.

Regulation of activin A, inhibin A, and follistatin production in human amnion and choriodecidual explants by inflammatory mediators.

OBJECTIVE: To determine the effects of inflammatory mediators on the production of activin A, inhibin A, and the binding protein follistatin in term amnion and choriodecidual tissues. METHODS: The effects of interleukin-1 beta (IL-1 beta; 1 ng/mL), tumor necrosis factor-alpha (TNF-alpha; 10 ng/mL), and bacterial lipopolysaccharide (LPS; 5 microg/mL) on production rates of activin A, inhibin A, and follistatin by term choriodecidual and amnion membranes in explant culture were determined using specific enzyme-linked immunoabsorbent assays. RESULTS: All explants (n = 6 placentas) produced detectable amounts of activin A, inhibin A, and follistatin under basal conditions; choriodecidual production rates were more than tenfold higher than amnion rates. In amnion explants, activin A production was stimulated by IL-1 beta and TNF-alpha to 450 +/- 155.4% and 531 +/- 170.8% of control, respectively (mean +/- standard error of the mean; P <.05 by analysis of variance), whereas production of inhibin and follistatin was stimulated to a much more modest extent. Similar responses were observed in the choriodecidual explants. Lipopolysaccharide had no significant effect on amnion activin A production, but stimulated choriodecidual production to 290 +/- 34% of control. Lipopolysaccharide exerted only limited effects on inhibin A and follistatin production. CONCLUSIONS: Treatment with proinflammatory mediators resulted in a preferential increase in activin A production compared with that of inhibin A or follistatin. These findings suggest that inflammation of the gestational membranes could result in increased local activin A production and bioactivity.

Contractility of renal cortex following complete ureteral obstruction.

Contractility of strips of renal cortex obtained from rabbit kidneys obstructed by complete ureteral ligation for 8 and 32 days was tested and compared with control tissue. Concentration-response data were obtained for norepinephrine, angiotensin, and 5-hydroxytrypamine, and were expressed as mg tension/cm of tissue. Control tissue tested simultaneously revealed minimal responses, but obstructed tissue containing modified interstital cells revealed statistically significant evidence of contraction in response to all three drugs studied. The response to norepinephrine could be blocked by the alpha adrenergic antagonist, phenoxybenzamine, but this drug did not significantly affect the response to angiotensin or 5-hydroxytryptamine. The findings are related to similar observations reported in stimulated fibroblasts of granulation tissue and are discussed in terms of the possible role this phenomenon may play in the pathogenesis of renal interstitial injury.

Transforming growth factor-beta 1, activin and follistatin in patients with hepatocellular carcinoma and patients with alcoholic cirrhosis.

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta1) exerts an inhibitory effect on DNA synthesis in hepatocytes. Activin, through different mechanisms, also exhibits an apoptotic effect on hepatocytes. Follistatin antagonizes the actions of activin. METHODS: Patients with hepatocellular carcinoma (HCC, n = 20), patients with alcoholic cirrhosis (n = 12), patients with cirrhosis due to other causes (n = 5) and normal controls (n = 19) were studied. TGF-beta1, activin and follistatin concentrations in blood and ascites were measured by ELISA. RESULTS: All three groups of patients had significantly higher serum levels of total TGF-beta1, activin and follistatin compared to those of controls. In patients with HCC, the total TGF-beta1 level correlated negatively with tumour size (r = -0.644, P = 0.001). The activin level correlated with alkaline phosphatase (ALP) level (r = 0.374, P = 0.046). The follistatin level correlated with the ALP level (r = 0.404, P = 0.026), and the glutamyl transpeptidase level (r = 0.457, P = 0.01). In patients with alcoholic cirrhosis, serum activin correlated with the Child-Pugh score (r = 0.601, P = 0.01). The levels of the cytokines in ascites (n = 16) did not correlate with the corresponding levels in serum. CONCLUSIONS: Serum levels of total TGF-beta1, activin and follistatin were elevated in patients with hepatocellular carcinoma and in patients with alcoholic cirrhosis. Apoptosis of tumour cells may be reduced by a subsequent decrease in serum TGF-beta1 levels when the tumours expand in size. Activin and follistatin were associated with tumour activity, as both correlated with ALP and/or GGT levels. Further studies are required to define the exact relationships between these cytokines, the dynamics of tumour growth and their significance in cirrhosis.

Maternal serum activin A and follistatin levels in pregnancies with Down syndrome.

Inhibin A is now an established second-trimester maternal serum marker of Down syndrome. Since activin A has a common beta-subunit to inhibin A we evaluated this substance and its binding protein, follistatin, as potential markers. We studied 30 affected and 199 unaffected pregnancies at 13-16 weeks' gestation. There was a statistically significant increase in activin A level among the cases with 8 (27 per cent) exceeding the 90th centile in the controls, and 6 (20 per cent) above the 95th centile. However, the extent of overlap was too great to be of value in screening. There was a small decrease in follistatin levels among cases but it did not reach statistical significance.

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy.

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.

Characterization of serum activin-A and follistatin and their relation to virological and histological determinants in chronic viral hepatitis.

BACKGROUND/METHODS: Hepatocyte proliferation in viral hepatitis is regulated by a number of growth factors. Activin-A inhibits hepatocyte DNA synthesis while follistatin, a potent activin-A antagonist, promotes liver regeneration. We report the first study of activin-A and follistatin in human viral hepatitis. Sera from 15 normal subjects, 22 hepatitis B and 47 hepatitis C patients were analysed for activin-A and follistatin and correlated with serological and histological markers of liver injury and with specific immunohistochemistry. RESULTS: All groups showed immunoreactivity for activin with hepatocyte localisation. Serum activin-A was significantly increased in viral hepatitis patients compared to controls, was greater in hepatitis B compared to hepatitis C, and correlated with serum aminotransferase and hepatitis B viral replication. A concurrent rise in serum follistatin was not observed in either group, but serum follistatin correlated inversely with hepatitis B DNA levels. Although hepatocyte apoptosis in hepatitis C and proliferation in both groups was significantly elevated compared to controls, there was no correlation with serum activin-A or follistatin. CONCLUSIONS: Activin-A and follistatin are constitutively expressed in human liver and serum concentrations are increased in viral hepatitis. Dysregulation of the activin/follistatin axis may be linked to hepatitis B replication but does not correlate with hepatocyte apoptosis.

Changes in serum inhibin, activin and follistatin concentrations during puberty in girls.

Serum concentrations of inhibin A, inhibin B, activin A and follistatin were determined using two-site enzyme-linked immunosorbent assays (ELISA) during pubertal ovarian development in 28 girls and five follicular phase women. Blood obtained every 15 to 20 min overnight was pooled for peptide determination. Serum inhibin A concentrations increased in mid puberty, exhibiting positive correlations with bone age (r = 0.527, P = 0.0016) and oestradiol concentrations (r = 0.581, P = 0.0005). Inhibin B concentrations peaked in mid puberty and declined thereafter, but remained greater than concentrations seen in prepubertal girls, and correlating positively with oestradiol (r = 0.362, P = 0.046) and follicle stimulating hormone (FSH) concentrations (r = 0.369, P = 0.038). Total activin A concentrations did not vary significantly across pubertal stages. Total follistatin concentrations, determined by radioimmunoassay, decreased with advancing puberty, exhibiting negative correlations with bone age (r = -0.634, P = 0.0001) and oestradiol concentration (r = -0.687, P = 0.0001). Follistatin concentrations determined by an ELISA specific for follistatin 288 were greatest in mid-pubertal girls, but concentrations in late puberty were less than those in early puberty. The free follistatin assay indicated that all circulating follistatin was activin-bound. These results suggest that significant changes in serum concentrations of FSH-regulatory peptides accompany the onset of puberty.

Follistatin and activin A production by the male reproductive tract.

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the betaA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B.

Serum inhibin, activin and follistatin in postmenopausal women with epithelial ovarian carcinoma.

OBJECTIVE: To investigate the role of serum inhibin A, inhibin pro-alphaC immunoreactivity, activin A, and follistatin in postmenopausal women with epithelial ovarian cancer. DESIGN: Case-control study. SAMPLE: e Serum samples from 27 postmenopausal women with epithelial ovarian cancer and 54 controls from the general population participating in an ovarian cancer screening trial. RESULTS: Women with epithelial ovarian cancer had significantly higher serum levels of pro-alphaC immunoreactivity (P = 0.03), activin A (P = 0.004) and follistatin (P = 0.04), but not inhibin A (P = 0.13). Using the 90th centile in the control group as the cut off, pro-alphaC levels were elevated in 41% of women with epithelial ovarian cancer, while inhibin A was elevated in only 15%. Using the 95th centile as the cut off, serum pro-alphaC was elevated in only 11% of women with epithelial ovarian cancer (3/27), while activin A was elevated in 48% (11/23). Follicle stimulating hormone levels were significantly lower in women with epithelial ovarian cancer (P = 0.01). Although, inhibin-related peptides can modulate follicle stimulating hormone levels, there was no correlation between inhibin A, pro-alphaC immunoreactivity, activin A or follistatin and follicle stimulating hormone. CONCLUSION: These data demonstrate that though there is preferential secretion of precursor forms of the alpha subunit rather than dimeric inhibin A by epithelial ovarian cancer, pro-alphaC is unlikely to be a useful tumour marker. Activin A is more commonly elevated in postmenopausal women with epithelial ovarian cancer and its role as a tumour marker in the diagnosis and screening of epithelial ovarian cancer warrants further evaluation.

Maternal serum total activin A and follistatin in pregnancy and parturition.

OBJECTIVE: To examine changes in maternal serum levels of activin A and follistatin during pregnancy and labour. DESIGN: In three cross sectional and three longitudinal studies venous blood was collected from women during pregnancy, spontaneous labour, labour induction and prior to elective caesarean section for the measurement of activin A and follistatin. SETTING: Monash Medical Centre, Clayton, Victoria, Australia. POPULATION: One hundred and twenty-three women participated in a cross sectional study in pregnancy, 18 women in two longitudinal pregnancy studies, 36 women in a cross sectional labour study, nine women in a longitudinal study of labour induction. Ten women undergoing elective caesarean section were also studied. METHODS: Activin A and follistatin were measured using two sensitive and specific enzyme-linked immunosorbent assays. RESULTS: In the cross sectional study of pregnancy, mean (SEM) maternal serum activin A and follistatin levels increased towards term (2.4 ng/mL (0.3) and 1.8 ng/mL (0.3) in first trimester to 18.9 ng/mL (3.8) and 5.3 ng/mL (0.9) at term, respectively), but the longitudinal study revealed that levels plateau in the last three weeks of pregnancy (16.0 ng/mL (2.6) and 6.2 ng/mL (1.4) at 37 weeks and 16.6 ng/mL (3.5) and 6.2 ng/mL (0.5) before labour for activin A and follistatin, respectively). There was no difference in levels of activin A and follistatin between women delivered by caesarean section and labouring women at term (14.9 ng/mL (2.8) vs 11.0 ng/mL (0.93) and 5.95 ng/mL (0.67) vs 5.71 ng/mL (0.63), respectively) and levels of both proteins did not alter throughout spontaneous or induced labour. CONCLUSIONS: We believe that these data argue against activin A playing an acute role in the initiation or regulation of human parturition.