Inhibin/activin beta-subunit monomer: isolation and characterization.

The release of activin A in response to intravenous injection of the bacterial cell-wall component lipopolysaccharide (LPS) was investigated in an ovine model of acute inflammatory challenge in newborn and adult sheep, and in non-pregnant and pregnant ewes. Neonatal lambs (<20 days of age) showed a quantitatively similar response in terms of circulating concentrations of activin A, its binding protein follistatin and the cytokine interleukin-6 compared with adult ewes challenged with an equivalent dose (300 ng/kg bodyweight) of LPS. The fever response and plasma tumour necrosis factor-release in response to LPS, however, were significantly (P<0·01) less in lambs than in the adult group. Pregnant ewes in the last trimester of gestation had similar responses to LPS, in all aspects measured, compared with their non-pregnant counterparts, apart from an ablated fever response. Although the adult and neonatal sheep responded to LPS, a similar response was not apparent in the fetal circulation, possibly due to a protective effect of the placenta. A 10-fold increase in the dose of LPS (from 300 ng to 3 µg/kg bodyweight) given to neonatal lambs elicited an increase in several cytokine responses measured, with a significant (P<0·05) increase in follistatin release. In contrast, the amount of activin released by the increased dose of LPS was similar to that invoked by the lower dose. The effect of tolerance to LPS was investigated by giving a second challenge of LPS 5 days after the initial injection. In all animals studied, there was an ablated (P<0·05) response to the subsequent LPS injection, apart from a similar temperature-response profile. These data provide further evidence that activin A concentrations in the bloodstream are acutely responsive to inflammatory challenge in postnatal life and suggest that the response forms a significant component of the innate immune system.

Section: Assaysmentioning

confidence: 99%

Effects of age and pregnancy on the circulatory activin response of sheep to acute inflammatory challenge by lipopolysaccharide

McClure

O’Connor

Hayward

et al. 2005

“…The standard used was human recombinant activin A, as described previously (Robertson et al 1992). Standards and samples were diluted in unconditioned culture medium containing the same additives as were used in the culture.…”

Section: Activin a Elisamentioning

confidence: 99%

Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro

Okuma

Saito

O’Connor

et al. 2005

Self Cite

In several biological systems, the inhibin A homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1 and IL-1 ) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1 or IL-1 , human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of A -subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin B -subunit and, to a lesser extent, -subunit mRNA expression, thereby reducing basal and FSHstimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.

“…Culture flasks and 96-well plates were supplied by Becton Dickinson (Franklin Lakes, NJ, USA). Human recombinant (hr) activin A, hr-inhibin A and bovine follistatin were as described previously (Robertson et al 1992), activin B was from conditioned medium of 293 cells transfected with an activin B subunit expression plasmid (Mason et al 1989), ovine luteinizing hormone (LH) was generously supplied by the National Hormone and Pituitary Program (NHPP, Torrance, CA, USA) and hr-interleukin-1 (IL-1 ), hr-TGF 1, -2 and -3 were purchased from R&D Systems (Minneapolis, MN, USA). Human sera were obtained from the Melbourne Blood Bank from normal volunteers and pooled serum from women in week 39 of pregnancy was generously provided by Dr Euan Wallace, Department of Obstetrics and Gynaecology, Monash University, with the appropriate ethical approval and informed consent of the patients.…”

Section: Reagentsmentioning

confidence: 99%

“…Activin, other test reagents and sera (100 µl/well) were added to the cultures diluted in phosphate buffer (pH 7·4) containing 6·5 mM Na 2 HPO 4 , 1·5 mM KH 2 PO 4 , 0·14 M NaCl, 0·01% BSA and Pen-strep. Cells were cultured in the presence of test reagents for two days; on the third day 25 µl [ 3 H]thymidine (0·25 µCi/25 µl, 6·7 Ci/mmol; NEN, Wilmington, DA, USA) were added to each well and 24 h later the cells harvested and thymidine incorporation assessed using standard methodologies (Robertson et al 1992). All test preparations, including activin standards and sera, were assayed in quadruplicate, and experiments were repeated at least twice.…”

Section: Cell Culture Proceduresmentioning

confidence: 99%

“…In vitro bioassays for activin have been developed using the production of FSH from cultured pituitary cells (Robertson et al 1992), the accumulation of haemoglobin in K562 erythroleukaemia cells (Schwall & Lai 1991), and the ability to induce mesoderm tissue in animal cap explants of Xenopus (de Winter et al 1992). These assays normally are responsive to both activin A and activin B, which are formed from homodimers of inhibin A and B subunits respectively; these two subunits share around 64% sequence homology.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11

Phillips¹,

Brauman²,

Aj³

et al. 1999

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-1, -2 and -3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0·4 ng/ml, an ED 50 response of 3·5 ng/ml, and an intraassay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.