Alterations in activin A-myostatin-follistatin system associate with disease activity in inflammatory myopathies.

OBJECTIVES: The aim of this study was to investigate the systemic and skeletal muscle levels of atrophy-associated myokines in patients with idiopathic inflammatory myopathies (IIM) and their association with clinical characteristics of myositis. METHODS: A total of 94 IIM patients and 162 healthy controls were recruited. Of those, 20 IIM patients and 28 healthy controls underwent a muscle biopsy. Circulating concentrations of myostatin, follistatin, activin A and TGF-ß1 were assessed by ELISA. The expression of myokines and associated genes involved in the myostatin signalling pathway in muscle tissue was determined by real-time PCR. RESULTS: We report decreased levels of circulating myostatin (median 1817 vs 2659 pg/ml; P = 0.003) and increased follistatin (1319 vs 1055 pg/ml; P = 0.028) in IIM compared with healthy controls. Activin A levels were also higher in IIM (414 vs 309 pg/ml; P = 0.0005) compared with controls. Myostatin was negatively correlated to muscle disease activity assessed by physician on visual analogue scale (MDA) (r = -0.289, P = 0.015) and positively to manual muscle testing of eight muscles (r = 0.366, P = 0.002). On the other hand, follistatin correlated positively with MDA (r = 0.235, P = 0.047). Gene expression analysis showed higher follistatin (P = 0.003) and myostatin inhibitor follistatin-like 3 protein (FSTL3) (P = 0.008) and lower expression of activin receptor type 1B (ALK4) (P = 0.034), signal transducer SMAD3 (P = 0.023) and atrophy marker atrogin-1 (P = 0.0009) in IIM muscle tissue compared with controls. CONCLUSION: This study shows lower myostatin and higher follistatin levels in circulation and attenuated expression of myostatin pathway signalling components in skeletal muscle of patients with myositis, a newly emerging pattern of the activin A-myostatin-follistatin system in muscle wasting diseases.

Detection of black market follistatin 344.

Follistatin, a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA 2019 List of Prohibited Substances and Methods". While currently no approved pharmaceutical formulations of follistatin are available, follistatin can be bought on the black market. Most of the products are labeled "follistatin 344" (FS344), a few "follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only nine of the 17 tested products actually contained follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Surprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western blotting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-follistatin antibody can be used. For immunoprecipitation (IP), a polyclonal anti-follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practically all currently available follistatin standards were investigated. The detection limit of the method for black market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 µL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous follistatin is possible.

Role of Activin A in the Pathogenesis of Endothelial Cell Dysfunction in Preeclampsia.

This chapter describes the methodologies which may be used in evaluating in vitro endothelial cell dysfunction in preeclampsia.

Regional localization of activin-ßA, activin-ßC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

Activins and inhibins, members of the TGF-ß superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-ßA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-ß superfamily, the activin-ßC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (ßC-ßC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.

Cardiokines as Modulators of Stress-Induced Cardiac Disorders.

Almost 30 years ago, the protein, atrial natriuretic peptide, was identified as a heart-secreted hormone that provides a peripheral signal from the myocardium that communicates to the rest of the organism to modify blood pressure and volume under conditions of heart failure. Since then, additional peripheral factors secreted by the heart, termed cardiokines, have been identified and shown to coordinate this interorgan cross talk. In addition to this interorgan communication, cardiokines also act in an autocrine/paracrine manner to play a role in intercellular communication within the myocardium. This review focuses on the roles of newly emerging cardiokines that are mainly increased in stress-induced cardiac diseases. The potential of these cardiokines as clinical biomarkers for diagnosis and prognosis of cardiac disorders is also discussed.

Seminal plasma transforming growth factor-ß, activin A and follistatin fluctuate within men over time.

STUDY QUESTION: Do seminal plasma transforming growth factor-ß (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? SUMMARY ANSWER: Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. WHAT IS ALREADY KNOWN: Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. PARTICIPANTS/MATERIALS, SETTING AND METHOD: The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individual's status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.

Expression, Regulation, and Functional Characterization of FST Gene in Porcine Granulosa Cells.

Proliferation, differentiation, and estrogen secretion of granulosa cells are the key factors affecting the estrous after weaning in sows. The objective of this study was to evaluate the expression of Follistatin (FST) in the ovary of Xiushui Hang and Duroc sows at weaning and estrus, the effect of FSH on transcript abundance of FST gene in granulosa cells and the role of FST gene in the weaning to estrus using siRNAs targeted to FST gene. In the present study, expression of the FST mRNA was evaluated by real time PCR. The FST mRNA levels showed a reduction from weaning to the estrus in both Xiushui Hang and Duroc sows, and the mRNA levels in Duroc ovary was higher than in Xiushui Hang sows at the beginning of estrus. Granulosa cells were obtained from the two largest follicles around follicular deviation, FST expression was decreased sharply after treatment with FSH (250 ng/ml). Knockdown of FST by siRNA in porcine granulosa cells significantly increased cell proliferation and estrogen secretion. These results indicate that FST gene is a negative regulator of follicle growth and function during the weaning-estrus interval.

Imbalance of Amniotic Fluid Activin-A and Follistatin in Intraamniotic Infection, Inflammation, and Preterm Birth.

CONTEXT: Microbial invasion of the amniotic fluid (AF) cavity stimulates an inflammatory response that involves activin-A, a pleiotropic mediator member of the TGFß superfamily involved in connective tissue remodeling. The role of AF follistatin, a natural inhibitor of activin-A, in inflammation-induced preterm birth (PTB), has yet to be determined. OBJECTIVE: The objective of the study was to investigate the relationships between AF activin-A and follistatin in physiological gestation and in pregnancies complicated by PTB and to evaluate a possible role played by the activin-A-follistatin balance in processes leading to PTB and preterm premature rupture of membranes (PPROM). STUDY DESIGN: The AF levels of total activin-A and follistatin were immunoassayed in 168 women with a normal pregnancy outcome or PTB with and without intraamniotic inflammation or PPROM. The impact of the activin-A-follistatin imbalance on PTB terminal effector pathways (prostaglandins [prostaglandin E2, prostaglandin F2α] and matrix metalloproteinases [MMP-1, MMP-2, MMP-3, and MMP-9]) was investigated in an amniochorion explant system challenged with lipopolysaccharide (LPS) to mimic inflammation. RESULTS: AF follistatin and the activin-A to follistatin ratio varied with gestational age, both decreasing toward term (P < .001). Activin-A was up-regulated in AF infection (>2-fold elevation in activin-A to follistatin ratio) correlating directly with severity of inflammation (both P < .001). Activin-A increased prostaglandins, MMP-1, and MMP-9 released by amniochorion (P < .05) to LPS-equivalent levels. Follistatin effectively blunted the prostaglandin response to activin-A and LPS and that of MMPs after activin-A but not after LPS challenge. CONCLUSION: Activin-A and follistatin are part of the complex inflammatory response of the gestational sac to infection and modulate effector pathways leading to PTB. The activin-A to follistatin ratio may play a role in determining the clinical phenotype of PTB as preterm labor or PPROM.

Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.

The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

OBJECTIVE: To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. INTERVENTION(S): Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. MAIN OUTCOME MEASURE(S): Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. RESULT(S): Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. CONCLUSION(S): The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility.

Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These results indicate that retrieving COCs 4 hours after slaughter could increase bovine in vitro developmental competence, which is linked to higher levels of oocyte MATER and OCT-4 transcripts and lower follicular progesterone concentration. Moreover, the results of the present study contribute to the identification of factors involved in the developmental competence of immature oocytes.

Expression patterns of bone morphogenetic protein antagonists in colorectal cancer desmoplastic invasion fronts.

Bone morphogenetic proteins (BMPs) are a group of growth factors with dual functions in cancer development and progression. Recently, certain tumor-promoting roles have been identified for selected antagonists/inhibitors (BMPIs) of this developmental pathway. A recent focus on the implication of BMP in colorectal cancer progression has emerged, mainly due to the presence of inactivating mutations in several members of the canonical signaling cascade. However, the detailed expression profiles of BMPIs remain largely unknown. Based on our previous work, whereby three specific BMPIs, gremlin-1 (GREM1), high-temperature requirement A3 (HTRA3) and follistatin (FST) were collectively overexpressed in desmoplastic cocultures of colorectal cancer (CRC), here, we undertook an immunohistochemical approach to describe the patterns of their expression in CRC patients. Two major characteristics described the BMPI expression signature: First, the synchronous and coordinated stromal and epithelial overexpression of individual BMPIs in desmoplastic lesions, which demonstrated that all three of them contribute to increasing levels of BMP antagonism in such areas. Second, the presence of microenvironmental polarity in the BMPI pattern of expression, which was indicated through the preferential expression of HTRA3 in the stromal, and the parallel FST/GREM1 expression in the epithelial component of the investigated sections. In addition, expression of HTRA3 in the epithelial compartment of the tumors demonstrated a significant predictive power to discriminate between tumor-budding-bearing and tumor-budding-free desmoplastic microenvironments. Together, these findings contribute to the understanding of signaling dynamics of BMP antagonism in the colorectal cancer desmoplastic invasion front.

Single-microchannel, multistep assay reports protein size and immunoaffinity.

We introduce a fully integrated multistep protein assay that reports both protein identity and size. To report these two properties, a microfluidic design strategy integrates pore limit electrophoresis (PLE) with a heterogeneous immunoassay in a single microchannel (PLE-IA). PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation. In a single-channel multistage assay approach, an antibody to follistatin was first immobilized in a polyacrylamide PLE gradient gel, near the origin of the separation axis. Immobilization relies on pore-limit exclusion of the antibody and not on chemical functionalization of either the sieving matrix or the antibody, making assay customization by an end-user straightforward. Subsequently, target and ladder protein species were electrophoretically introduced into the antibody-patterned PLE channel. Species having an affinity for the immobilized antibody were detected via heterogeneous immunoassay. Noninteracting and, thus, unbound species electromigrated past the patterned antibodies, along the separation axis, and finally separated according to the pore-size limit of each, yielding a log-linear dependence of molecular weight on migration distance. Separations of 10 min yielded an average peak capacity of 18 ± 1.3 (separation resolution (SR) = 1) in a 10 mm separation distance. Comparison of the separated peaks in two parallel PLE channels in the presence or absence of capture antibody with a protein size ladder revealed good agreement of the target molecular weight with reported values. In addition, a more than 50-fold decrease in the detection limit (0.078 vs 5 nM) was achieved using an electrophoretic "continuous injection" technique in which sample material was continuously loaded for 40 min. On the basis of this proof-of-principle demonstration with follistatin, PLE-IA should find application in study of cell signaling, including questions related to aging and regeneration.

Myostatin levels in skeletal muscle of hibernating ground squirrels.

Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus, at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature (T(b)) falling; early hibernation (EHib), stable T(b) in torpor for 24 h; late hibernation (LHib), stable T(b) in torpor for 3 days; early arousal (EAr), T(b) rising; and arousal (AR), T(b) restored to 34-37°C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin.

Changes in ovarian follistatin levels during the oestrous cycle in sheep may serve as an intraovarian regulator.

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day -1 and 0) to mid-luteal phase (days 11-13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.

Levels of expression for BMP-7 and several BMP antagonists may play an integral role in a fracture nonunion: a pilot study.

Delays in bone healing or even the development of a nonunion could be related to the concentrations and/or functions of the bone morphogenetic proteins (BMPs). The RNA expression profile of the BMPs within fracture nonunion tissue is unknown. This preliminary descriptive study was performed to define the RNA profiles of the BMPs, their receptors, and their inhibitors within human fracture nonunion tissue and correlate them to matched healing bone. All patients had hypertrophic nonunions. Tissue samples taken from the nonunion site of 15 patients undergoing surgical treatment for an established nonunion were analyzed. The RNA expression patterns of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8; BMP receptor Types IA, IB, and II; and the BMP inhibitors chordin, Noggin, Drm (Gremlin), and follistatin were determined in the nonunion (fibrous tissue) and healing bone (callus tissue) using quantitative real-time PCR. Comparison between the nonunion and healing bone samples revealed substantially elevated concentrations of BMP-4, Drm/Gremlin, follistatin, and Noggin in nonunion tissue when compared to healing bone. In contrast, BMP-7 concentration was higher in the healing bone. Our data suggest inhibition of BMP-7, by Drm (Gremlin), follistatin, and Noggin and upregulation of BMP-4 may play an integral role in the development of nonunions.

Effect of activin A on tubulointerstitial fibrosis in diabetic nephropathy.

AIM: The effect of activin A on tubulointerstitial fibrosis in diabetic nephropathy (DN) using streptozotocin (STZ)-induced diabetic rats and high glucose-cultured HK-2 cells was investigated. METHODS: Male Wistar rats were randomized into a normal control group (NC) and diabetes mellitus group (DM). Diabetes was induced by i.p. injection of STZ. Six rats were respectively killed 4, 8, 12 and 16 weeks after model establishment in each group. The changes of kidney weight/bodyweight (KW/BW), urine albumin excretion rate (AER) and creatinine clearance rate (Ccr) were determined. The morphology of tubulointerstitium was observed by light microscopy. Further biochemical analysis was provided using immunohistochemistry and real-time polymerase chain reaction. The different parameters in high glucose-cultured HK-2 cells were monitored by western blotting or enzyme-linked immunosorbent assay (ELISA) and the intervention of rh-follistatin on them was investigated. RESULTS: Compared with the NC group, there was marked enlargement in the levels of KW/BW, AER, Ccr and interstitial fibrosis index, and the production of P-Smad2/3 and fibronectin in the DM group from 8 to 16 weeks. Activin betaA, mainly located in tubular epithelial cells, was significantly higher in the DM group than that in the NC group throughout the study periods. Follistatin was abundant in the NC group, but was diminished gradually in the DM group. High glucose may facilitate the synthesis of activin betaA, transforming growth factor (TGF)-beta, P-Smad2/3 and fibronectin in HK-2 cells while rh-follistatin inhibited them except TGF-beta. CONCLUSION: Activin A is involved in tubulointerstitial fibrosis in DN by inducing the production of fibronectin through Smad signal pathway.

Changes in concentrations of follistatin in maternal plasma and foetal fluids during pregnancy and parturition in sheep.

Studies have shown that follistatin may be involved in the regulation of ovarian development, pregnancy and parturition. The aim of the present study was to measure follistatin levels in maternal plasma and foetal fluids during pregnancy and parturition in sheep. Using a previously described follistatin radioimmunoassay, we found that follistatin in foetal plasma and allantoic fluid was higher in the female than in the male at days 50-75 of gestation. Follistatin concentrations in maternal plasma declined from -9 to -3h before the completion of lamb delivery, and increased from 21 to 39 h after parturition. These results suggest that follistatin may play a role in femaleness development, and that follistatin may be involved in the mechanism of normal parturition and in regulation of the tissue repair after parturition.

Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.