Follistatin mRNA Expression Research Articles

Regulation of Hepatic Follistatin Expression at Rest and during Exercise in Mice.

Follistatin (FST) is a protein with numerous biological roles and was recently identified as an exercise-inducible hepatokine; however, the signals that regulate this are not well understood. The purpose of this study was to delineate potential endocrine factors that may regulate hepatic FST at rest and during exercise. This study used four experiments. First, male and female C57BL/6J mice remained sedentary or were subjected to a single bout of exercise at moderate or exhaustive intensity with liver collected immediately post. Second, mice were injected with glucagon (1 mg·kg, 60 min), epinephrine (2 mg·kg, 30 min), glucagon then epinephrine, or saline. Third, mice were pretreated with propranolol (20-60 mg·kg, 30 min) before epinephrine injection. Fourth, glucagon receptor wild type (Gcgr) or knockout (Gcgr) mice were pretreated with saline or propranolol (20 mg·kg, 30 min) and were subjected to a single bout of exhaustive exercise with liver collected immediately post or after 2 h recovery. In all experiments liver FST mRNA expression was measured, and in experiment four FST protein content was measured. A single bout of treadmill exercise performed at an exhaustive but not moderate-intensity increased FST expression, as did injection of glucagon or epinephrine alone and when combined. Pretreatment of mice with propranolol attenuated the epinephrine-induced increase in FST expression. The exercise-induced increase in FST expression was attenuated in Gcgr mice, with no effect of propranolol. Gcgr mice had higher protein content of FST, but there was no effect of exercise or propranolol. These data suggest that both glucagon and epinephrine regulate hepatic FST expression at rest; however, only glucagon is required for the exercise-induced increase.

PO-306 The effect of four weeks hypoxic resistance training on myostatin and follistatin expression in skeletal muscle of rats

Objective Loss of skeletal muscle weight is a common phenomenon in hypoxic environment. It has been recognized that resistance training can reduce hypoxia-induced skeletal muscle atrophy, but its molecular mechanism is still unclear. Myostatin is a major factor that inhibits muscle growth and differentiation, and Follistatin can inhibit Myostatin. Therefore, this study is to clarify the effect of 4-week hypoxic resistance exercise on Myostatin and Follistatin gene expression in skeletal muscle of rats.
 Methods Twenty four 8-week-old male SD rats were randomly divided into normoxic control group (group C: 6 rats), normoxic exercise group (group R: 6 rats), hypoxic control group (group H: 6 rats) and hypoxic exercise group (group HR: 6 rats). Rats in each hypoxic group were fed in a hypoxic chamber (atmospheric hypoxia) with oxygen concentration of 12.7% (simulated 4000m altitude). Rats in each exercise group were trained according to the rat's resistance training program developed in our laboratory. After all the intervention, DEXA was used to analyze the body composition. The soleus, extensor digitorum longus and biceps brachii muscles of rats were taken and the wet weight of individual muscles was measured. The data were processed by SPSS17.0 statistical software. The expression level of skeletal muscle mRNA was expressed as "median (25-75%)" and the data of body composition and muscle wet weight were expressed as"mean±standard deviation". The differences between the groups were evaluated using a one-way analysis of variance (ANOVA) test. The significance level for the study was less than 0.05.
 Results Body composition analysis after 4 weeks of hypoxic intervention showed that the body weight of rats in group H decreased significantly (p=0.012), and the muscle mass decreased more significantly (p<0.001). But resistance exercises obviously reduced the muscle atrophy (p<0.01) caused by hypoxia. After analyzing the changes of the wet weight of individual muscles, it was found that the wet weight of biceps brachii in HR group was significantly higher than that in H group (p=0.048). After 4 weeks of hypoxic intervention and hypoxic resistance exercise, the expression of Myostatin mRNA in individual muscles of each group changed differently. The expression of Myostatin mRNA in soleus muscle of H group was significantly higher than that of C group (371.2%) after 4 weeks of hypoxia intervention. Myostatin mRNA expression in soleus and biceps brachii of HR group was significantly lower than that of H group (591.1% and 478.4% respectively). However, there was no significant difference in the expression level of Myostatin mRNA in the extensor digitorum longus between each group (p=0.259). The change of Follistatin mRNA expression in different groups also showed a different trend. The expression of Follistatin mRNA in soleus muscle and biceps brachii muscle was significantly different among groups (p=0.003, p=0.004, respectively). However, there was no significant difference in the expression level of Follistatin mRNA in the extensor digitorum longus between each group (p=0.734). Myostatin mRNA/Follistatin mRNA ratio (M/F) showed a more significant difference. The M/F ratio of soleus muscle in group H was significantly lower than that in group C (p<0.001), but the M/F ratio in group HR was significantly higher than that in group H (p<0.001). The M/F ratio of biceps brachii in group H was significantly lower than that in group C (p<0.001), but the M/F ratio in group HR showed a higher trend than that in group H (p=0.051).
 Conclusions Hypoxic exposure results in an increase in Myostatin mRNA expression in skeletal muscle, but hypoxic resistance exercise reduces such an increase. On the contrary, the level of Follistatin mRNA expression in skeletal muscle decreased after hypoxic exposure, and hypoxic resistance exercise could slow down the decline. As a result, rat resistance exercise significantly slowed down hypoxia-induced muscle atrophy. In conclusion, the mutual restriction between Myostatin and Follistatin is one of the main links of resistance exercise to reduce hypoxia-induced skeletal muscle atrophy. However, the process of resistance training to reduce the hypoxia-induced skeletal muscle atrophy is very complex. There are many molecular signaling pathways involved, which need further study.

Hypertrophy Stimulation at the Onset of Type I Diabetes Maintains the Soleus but Not the EDL Muscle Mass in Wistar Rats.

Diabetes mellitus induces a reduction in skeletal muscle mass and strength. Strength training is prescribed as part of treatment since it improves glycemic control and promotes increase of skeletal muscle mass. The mechanisms involved in overload-induced muscle hypertrophy elicited at the establishment of the type I diabetic state was investigated in Wistar rats. The purpose was to examine whether the overload-induced hypertrophy can counteract the hypotrophy associated to the diabetic state. The experiments were performed in oxidative (soleus) or glycolytic (EDL) muscles. PI3K/Akt/mTOR protein synthesis pathway was evaluated 7 days after overload-induced hypertrophy of soleus and of EDL muscles. The mRNA expression of genes associated with different signaling pathways that control muscle hypertrophy was also evaluated: mechanotransduction (FAK), Wnt/β-catenin, myostatin, and follistatin. The soleus and EDL muscles when submitted to overload had similar hypertrophic responses in control and diabetic animals. The increase of absolute and specific twitch and tetanic forces had the same magnitude as muscle hypertrophic response. Hypertrophy of the EDL muscle from diabetic animals mostly involved mechanical loading-stimulated PI3K/Akt/mTOR pathway besides the reduced activation of AMP-activated protein kinase (AMPK) and decrease of myostatin expression. Hypertrophy was more pronounced in the soleus muscle of diabetic animals due to a more potent activation of rpS6 and increased mRNA expression of insulin-like growth factor-1 (IGF-1), mechano-growth factor (MGF) and follistatin, and decrease of myostatin, MuRF-1 and atrogin-1 contents. The signaling changes enabled the soleus muscle mass and force of the diabetic rats to reach the values of the control group.

Grass Carp Follisatin: Molecular Cloning, Functional Characterization, Dopamine D1 Regulation at Pituitary Level, and Implication in Growth Hormone Regulation.

Activin is involved in pituitary hormone regulation and its pituitary actions can be nullified by local production of its binding protein follistatin. In our recent study with grass carp, local release of growth hormone (GH) was shown to induce activin expression at pituitary level, which in turn could exert an intrapituitary feedback to inhibit GH synthesis and secretion. To further examine the activin/follistatin system in the carp pituitary, grass carp follistatin was cloned and confirmed to be single-copy gene widely expressed at tissue level. At the pituitary level, follistatin signals could be located in carp somatotrophs, gonadotrophs, and lactotrophs. Functional expression also revealed that carp follistatin was effective in neutralizing activin’s action in stimulating target promoter with activin-responsive elements. In grass carp pituitary cells, follistatin co-treatment was found to revert activin inhibition on GH mRNA expression. Meanwhile, follistatin mRNA levels could be up-regulated by local production of activin but the opposite was true for dopaminergic activation with dopamine (DA) or its agonist apomorphine. Since GH stimulation by DA via pituitary D1 receptor is well-documented in fish models, the receptor specificity for follistatin regulation by DA was also investigated. Using a pharmacological approach, the inhibitory effect of DA on follistatin gene expression was confirmed to be mediated by pituitary D1 but not D2 receptor. Furthermore, activation of D1 receptor by the D1-specific agonist SKF77434 was also effective in blocking follistatin mRNA expression induced by activin and GH treatment both in carp pituitary cells as well as in carp somatotrophs enriched by density gradient centrifugation. These results, as a whole, suggest that activin can interact with dopaminergic input from the hypothalamus to regulate follistatin expression in carp pituitary, which may contribute to GH regulation by activin/follistatin system via autocrine/paracrine mechanisms.

Protective effect of rimonabant, a canabinoid receptor 1 antagonist, on nonalcoholic fatty liver disease in a rat model through modulation of the hepatic expression of activin A and follistatin.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver morbidity and mortality, and there is still no proven effective therapy. The endocannabinoid system plays an important role in various liver diseases. Activin A is a member of the transforming growth factor beta (TGF-β) superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin A. This study was designed to investigate the effect of rimonabant (a potent cannabinoid receptor1 (CB1) antagonist) on NAFLD induced with a choline-deficient (CD) diet in rats, as well as to detect whether it can alter the hepatic expression of activin A and follistatin. Forty rats were distributed among 4 groups: the control group, the rimonabant treatment group (normal rats that received rimonabant); the CD diet group (NAFLD induced with a CD diet); and the CD diet + rimonabant group (NAFLD treated with rimonabant). It was found that the CD diet caused significant increase in liver index, serum levels of liver enzymes, malondialdehyde (MDA), TGF-β1, activin A, and CB1 expression in liver tissue, with a significant decrease in glutathione peroxidase (GSH-Px) and follistatin mRNA expression in liver tissues. The administration of rimonabant significantly improved all of the studied parameters compared with the group fed the CD diet alone. Histopathological examination supported these results. We concluded that rimonabant significantly counteracted NAFLD induced with the CD diet by decreasing oxidative stress and hepatic expression of TGF-β1, and modulating the hepatic expression of activin A and follistatin.

Habitual physical activity protects against lipopolysaccharide-induced inflammation in mouse adipose tissue

ABSTRACTSepsis is a systemic inflammatory response to infection, with no preventative strategies. In this study, we identify a role for habitual physical activity in the prevention of adipose tissue inflammation induced by a model of sepsis, lipopolysaccharide (LPS). Male C57BL/6J mice (8 weeks old) were housed with access to voluntary wheel running (VWR) or sedentary (SED) for 10 weeks. Mice were then injected with LPS (2 mg/kg) or saline (SAL), and tissues were removed 6 hours post-injection. VWR attenuated body, epididymal adipose tissue (eWAT), and subcutaneous inguinal adipose tissue (iWAT) mass gain, improved glucose tolerance, increased markers of mitochondrial biogenesis in iWAT and eWAT, and increased UCP-1 protein content in iWAT. In iWAT, VWR attenuated the LPS induced increase in mRNA expression of TNF-α, MCP-1, and follistatin, along with phosphorylation of STAT3. In addition, VWR had a main effect for reducing iWAT mRNA expression of IL-1β, IL-6, and SOCS3. In eWAT, VWR had a main effect for reducing mRNA expression of IL-1β, MCP-1, IL-6, and follistatin. Further, VWR increased SOCS3 mRNA expression and phosphorylation of STAT3 in SAL mice, thus the relative change in response to LPS for these markers was attenuated. The protective effect of prior physical activity occurred in conjunction with increases in the protein content of a component of the LPS binding complex, MyD88. Overall, the results from this study demonstrate that habitual physical activity can attenuate the LPS induced inflammatory response in adipose tissue and this occurs to a greater extent in iWAT compare with eWAT.

MRNA expression of Follistatin and follistatin-like 3, bone morphogenetic protein-4 antagonists in lung tissues of hypoxic mice

To observe the expression changes of Follistatin (FSN) and follistatin-like 3 (FSRP) mRNA, both of bone morphogenetic protein-4(BMP4) antagonists, in mice lung tissue under different hypoxic time and its relation with BMP4. Thirty BMP4+/+ C57BL/6J wild type mice were randomly divided into normoxic control group, and 4 groups including 1 day, 3 days, 7 days and 21 days under hypoxic condition. The hypoxic animal model was established by exposing the mice to hypoxic condition for different time. The expressing level of FSN and FSRP mRNA in lung tissues were measured by Quantitative Real-Time PCR. FSN and FSRP mRNA increased in hypoxic 1 day group as (28.0 ± 9.4) and (2.0 ± 0.4), in hypoxic 3 day group, FSN mRNA increased by (6.3 ± 3.2) and FSRP mRNA by (1.67 ± 0.7) (P < 0. 05). Compared with the normoxic group (1.77 ± 0.36) and (1.22 ± 0.15) (P < 0. 01), FSN and FSRP mRNA up-regulated in hypoxic 7 day group. The positive degree of FSN and FSRP mRNA in hypoxic 21 day group were (1.04 ± 0.27) and (0.85 ± 0.10) (P < 0. 05). In normoxic 1 day group, FSN mRNA of lung tissues of BMP4+/- C57BL/6J mice was (0.95 ± 0.05); FSRP mRNA was (1.11 ± 0.03) (P < 0. 05). In BMP4+/- C57BL/6J mice lung tissues, FSN mRNA were (1.10 ± 0.40) (P < 0. 05); FSRP mRNA were (0.85 ± 0.18). The expression of FSN and FSRP mRNA in hypoxic 1;3;7 day mice lung tissues increased obviously, and FSN may play an important role in the hypoxic pulmonary hypertension through BMP4.

Amygdalin isolated from Semen Persicae (Tao Ren) extracts induces the expression of follistatin in HepG2 and C2C12 cell lines.

BackgroundThe Chinese medicine formulation ISF-1 (also known as Bu-Yang-Huan-Wu-Tang) for post-stroke rehabilitation could increase the expression of growth-regulating protein follistatin by approximately 4-fold. This study aims to identify the active compounds of ISF-1 for the induction of follistatin expression.MethodsActive compounds in ISF-1 responsible for induction of follistatin were identified by a bioactivity-guided fractionation procedure involving liquid-liquid extraction, HPLC separation and RT-PCR detection. The aqueous extracts of seven ISF-1 ingredients including Semen Persicae (Tao Ren) and the S. Persicae-derived fractions were assayed for the induction of follistatin mRNA expression in human hepatocarcinoma HepG2 cells by RT-PCR. The concentrations of isolated compounds were proportionally normalized to the reported IC50 concentration (5.8 mg/mL) of the formulation ISF-1 in HepG2. The active fractions were characterized by reverse-phase HPLC on a C18 column and identified by mass spectrometry.ResultsThree ingredients of ISF-1, namely S. Persicae (Tao Ren), Pheretima (Di Long), and Flos Carthami (Hong Hua), induced the expression of follistatin mRNA. Among these, the ingredient S. Persicae were the most active, and amygdalin from S. Persicae extract was identified as a novel follistatin inducer. Amygdalin stimulated the growth of skeletal muscle cell line C2C12 cells in a concentration-dependent manner.ConclusionsAmygdalin isolated from S. Persicae extract in ISF-1 through a bioactivity-guided fractionation procedure induced the expression of follistatin in HepG2 and C2C12 cell lines.

Effect of physical training on liver expression of activin A and follistatin in a nonalcoholic fatty liver disease model in rats

Nonalcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver and is associated with obesity and insulin resistance. Activin A is a member of the transforming growth factor beta (TGF)-β superfamily and inhibits hepatocyte growth. Follistatin antagonizes the biological actions of activin. Exercise is an important therapeutic strategy to reduce the metabolic effects of obesity. We evaluated the pattern of activin A and follistatin liver expression in obese rats subjected to swimming exercise. Control rats (C) and high-fat (HF) diet-fed rats were randomly assigned to a swimming training group (C-Swim and HF-Swim) or a sedentary group (C-Sed and HF-Sed). Activin βA subunit mRNA expression was significantly higher in HF-Swim than in HF-Sed rats. Follistatin mRNA expression was significantly lower in C-Swim and HF-Swim than in either C-Sed or HF-Sed animals. There was no evidence of steatosis or inflammation in C rats. In contrast, in HF animals the severity of steatosis ranged from grade 1 to grade 3. The extent of liver parenchyma damage was less in HF-Swim animals, with the severity of steatosis ranging from grade 0 to grade 1. These data showed that exercise may reduce the deleterious effects of a high-fat diet on the liver, suggesting that the local expression of activin-follistatin may be involved.

Effects of the regulation of follistatin mRNA expression by IGF-1 in duck (Anas platyrhynchos) skeletal muscle

The IGF-1 and TGF-β pathways have been shown to be involved in regulating muscle development. Many mediators that are associated with the regulation of muscle development have been found to participate in the cross-talk between these two pathways. To research the relationships between IGF-1 and the follistatin-mediated TGF-β pathways in duck skeletal muscle development, a series of studies were conducted. The results showed that follistatin had similar expression patterns to IGF-1 during duck embryonic muscle development. The in ovo feeding of IGF-1 to duck eggs was shown to increase follistatin expression in the duck skeletal muscle. Thus, IGF-1 may induce the mRNA expression of follistatin. These results suggest that follistatin may be a key regulator of multiple signaling cascades responding to the cross-talk between the IGF-1 and TGF-β pathways.

Irisin mRNA and circulating levels in relation to other myokines in healthy and morbidly obese humans

Skeletal muscle is considered to be an endocrine organ that secretes a number of myokines including follistatin (FST), myostatin (MSTN), activin A, and the newly identified irisin. Irisin's biology and function exhibit similarities with the functions of the FST-MSTN-activin A axis. It remains unknown whether there is any interplay among these molecules. The aim of this study is to examine potential associations of irisin with the FST, MSTN, and activin A axis. Two observational studies were performed to evaluate the associations of irisin with the other three peptides. Study A included 150 healthy males aged 18.48±0.16 years with BMI 23.18±3.75 kg/m(2). Fasting serum samples were used to measure the levels of the molecules of interest. Study B included 14 morbidly obese individuals, candidates for bariatric surgery, aged 53.14±8.93 years with BMI 50.18±10.63 kg/m(2). Blood samples were obtained after an overnight fast. Eight out of the 14 participants consented to an optional thigh biopsy during their bariatric surgery. Using the above blood and tissue samples, we measured circulating levels and muscle mRNA of irisin, FST, MSTN, and activin A. We report that FNDC5 mRNA in muscle is positively correlated with FST mRNA expression in morbidly obese subjects (ρ=0.93, P<0.001). We also found that circulating irisin is positively correlated with FST circulating levels among lean subjects (ρ=0.17, P=0.05) while this association was suggestive among the obese (ρ=0.56, P=0.07). The newly identified myokine irisin may be positively associated with FST at both the mRNA and circulating protein level.

Activin A and its Regulatory Molecules in Placenta and Fetal Membranes of Women with Preterm Premature Rupture of the Membranes Associated with Acute Chorioamnionitis

LABELED PROBLEM: To investigate regulation of activin A and related molecules in placenta/fetal membranes from preterm premature rupture of membranes (pPROM) associated with acute chorioamnionitis (ACA). Tissues were obtained from women with spontaneous preterm deliveries (PTD), pPROM without ACA, pPROM with ACA. Activin A, follistatin, and nodal and cripto mRNA were measured by RT-PCR. Activin A mRNA was up-regulated in tissues from pPROM, in presence or absence of HCA, respect to PTD and in pPROM with ACA respect to pPROM without ACA. Follistatin mRNA expression did not differ between the groups. In placenta, nodal mRNA showed the same trend of activin A, while cripto was down-regulated in pPROM with ACA than other groups. Nodal and cripto were not expressed by fetal membranes. The study shows the involvement of activin A pathway in pPROM with ACA. Further studies will focus on its role in placental immune functions.

Characterization of Wnt2 Overexpression in a Rat Granulosa Cell Line (DC3): Effects on CTNNB1 Activation1

WNTs comprise a family of secreted glycoproteins that are essential for normal embryonic development of the female reproductive system. The functional role that WNTs play in the postnatal ovary is poorly defined. We have shown previously that Wnt2 and Fzd4 mRNAs are expressed in granulosa cells of the postnatal rat ovary. Here we examine the effects of Wnt2 overexpression in a rat granulosa cell line (DC3) that displays characteristics of granulosa cells at an early stage of follicular development. We show that DC3 cells express a 7.7-kb Fzd4 mRNA transcript similar in size to that detected in the rat and human ovary. Our results demonstrate that Wnt2 overexpression in DC3 promotes cytosolic and nuclear accumulation of beta-catenin (CTNNB1), but does not stimulate CTNNB1/TCF-dependent (pGL3-OT) transcriptional activity. We show that chibby (CBY1), a nuclear CTNNB1-associated antagonist of the WNT pathway, is expressed in DC3 cells and associates with CTNNB1 in the presence and absence of Wnt2 overexpression, suggesting that Cby1 contributes to suppression of CTNNB1/TCF-dependent transcription in these cells. Our results show that Wnt2 overexpression in DC3 cells increases follistatin (Fst) mRNA expression and promotes resistance to activin-induced cell deletion. Taken together, our results suggest that WNT2 opposes activin activity in granulosa cells by up-regulating expression of the activin antagonist Fst in a CTNNB1/TCF-independent manner, and that rat granulosa cells express factors, including Cby1, that suppress CTNNB1/TCF-dependent signal transduction in the presence of a WNT signal.

Expression of myostatin, myostatin receptors and follistatin in diabetic rats submitted to exercise

Myostatin (MSTN) has been implicated in metabolic adaptation to physiological stimuli, such as physical exercise, which is linked to improved glucose homeostasis. The aim of the present study was to evaluate the influence of exercise on the expression of MSTN, MSTN receptors (ActRIIB and ALK4) and follistatin (FS) in the muscle and fat of streptozotocin-induced diabetic rats. Control and diabetic rats were randomly assigned to a swimming training group (EC and ED, respectively) and a sedentary group (SC and SD, respectively). Exercising animals swam for 45min at 0900 and 1700hours, 5day/week, for 4weeks. The mRNA expression of MSTN, ActRIIB, ALK4 and FS mRNA was quantified by real-time reverse transcription-polymerase chain reaction. Expression of MSTN and FS mRNA increased in the muscle and subcutaneous fat of SD compared with SC rats. Expression of ActRIIB mRNA was increased in the muscle, mesenteric fat and brown adipose tissue (BAT) of SD compared with SC rats, whereas ALK4 mRNA expression was only increased in the BAT of SD compared with SC rats. After training, MSTN and ActRIIB expression was lower in the BAT of EC compared with SC rats. Expression of MSTN mRNA increased in the mesenteric fat of ED compared with SD rats, whereas FS mRNA expression decreased in the muscle, mesenteric and subcutaneous fat and BAT. Lower ALK4 mRNA expression was noted in the BAT of ED compared with SD rats. These results indicate that MSTN, its receptors and FS expression change in both the muscle and fat of diabetic rats and that the expression of these factors can be modulated by exercise in diabetes.

Hyal‐1 but not hyal‐3 deficiency has an impact on ovarian folliculogenesis and female fertility by altering the follistatin/activin/Smad3 pathway and the apoptotic process

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.

Gene–environment interactions: The potential role of contaminants in somatic growth and the development of the reproductive system of the American alligator

Developing organisms interpret and integrate environmental signals to produce adaptive phenotypes that are prospectively suited for probable demands in later life. This plasticity can be disrupted when embryos are impacted by exogenous contaminants, such as environmental pollutants, producing potentially deleterious and long-lasting mismatches between phenotype and the future environment. We investigated the ability for in ovo environmental contaminant exposure to alter the growth trajectory and ovarian function of alligators at five months after hatching. Alligators collected as eggs from polluted Lake Apopka, FL, hatched with smaller body masses but grew faster during the first five months after hatching, as compared to reference-site alligators. Further, ovaries from Lake Apopka alligators displayed lower basal expression levels of inhibin beta A mRNA as well as decreased responsiveness of aromatase and follistatin mRNA expression levels to treatment with follicle stimulating hormone. We posit that these differences predispose these animals to increased risks of disease and reproductive dysfunction at adulthood.

IL-1β Stimulates Activin βA mRNA Expression in Human Skin Fibroblasts Through the MAPK Pathways, the Nuclear Factor-κB Pathway, and Prostaglandin E2

During mouse skin wound healing, mRNAs encoding IL-1, activins, and TGF-βs significantly increased. To elucidate involvement of IL-1 in the regulation of activins and related factors in the wounded skin, human foreskin fibroblasts were stimulated with IL-1β, and levels of mRNAs encoding activins, TGF-βs, and follistatin family proteins were examined by quantitative real-time PCR. IL-1β increased activin βA (INHBA) and follistatin (FST) mRNA expression within 6 h. A p38 MAPK inhibitor, SB202190, a MAPK/ERK kinase inhibitor, U0126, and an nuclear factor κB pathway inhibitor, SC-514, significantly suppressed the IL-1β-stimulated INHBA and FST mRNA expression. A prostaglandin-endoperoxide synthase inhibitor indomethacin, a potent inhibitor of prostaglandin E(2) (PGE(2)) synthesis, also significantly suppressed the IL-1β-stimulated INHBA but not FST mRNA expression. Furthermore, stimulation of fibroblasts with PGE(2) significantly increased INHBA mRNA. The PGE(2)-induced INHBA mRNA expression was significantly suppressed by U0126 and a protein kinase C inhibitor, Gö 6983. Although IL-1β stimulated FST mRNA in an acute phase, long-term exposure of fibroblasts to IL-1β revealed time-dependent stimulatory and inhibitory effects of IL-1β on FST mRNA expression. On the other hand, coculture with keratinocytes significantly increased INHBA mRNA expression in dermal equivalents. In summary, the present study indicates that the p38 MAPK, the MAPK/ERK kinase, the nuclear factor κB pathway, and PGE(2) mediate the effects of IL-1β on INHBA mRNA expression. Furthermore, it is indicated that keratinocyte-derived factor of factors stimulate INHBA mRNA expression during wound healing.

Exercise Induces a Marked Increase in Plasma Follistatin: Evidence That Follistatin Is a Contraction-Induced Hepatokine

Follistatin is a member of the TGF-β super family and inhibits the action of myostatin to regulate skeletal muscle growth. The regulation of follistatin during physical exercise is unclear but may be important because physical activity is a major intervention to prevent age-related sarcopenia. First, healthy subjects performed either bicycle or one-legged knee extensor exercise. Arterial-venous differences were assessed during the one-legged knee extensor experiment. Next, mice performed 1 h of swimming, and the expression of follistatin was examined in various tissues using quantitative PCR. Western blotting assessed follistatin protein content in the liver. IL-6 and epinephrine were investigated as drivers of follistatin secretion. After 3 h of bicycle exercise, plasma follistatin increased 3 h into recovery with a peak of 7-fold. No net release of follistatin could be detected from the exercising limb. In mice performing a bout of swimming exercise, increases in plasma follistatin as well as follistatin mRNA and protein expression in the liver were observed. IL-6 infusion to healthy young men did not affect the follistatin concentration in the circulation. When mice were stimulated with epinephrine, no increase in the hepatic mRNA of follistatin was observed. This is the first study to demonstrate that plasma follistatin is increased during exercise and most likely originates from the liver. These data introduce new perspectives regarding muscle-liver cross talk during exercise and during recovery from exercise.

Activin-related proteins in bovine mammary gland: Localization and differential expression during gestational development and differentiation

Bovine mammary gland morphogenesis and differentiation are regulated by actions of growth factors including members of the transforming growth factor β superfamily. Activins A and B, which are members of the transforming growth factor β superfamily, bind selectively to ActRIB and ActRIIA receptors and their biological effects are antagonized by inhibins and follistatins. In the present paper we evaluated gene and protein expression of the activin and inhibin subunits βA, βB, and α-inhibin and follistatin and ActRIB and ActRIIA receptors in the mammary gland of nonpregnant and pregnant heifers. Mammary glands were obtained from nonpregnant Nelore (Bos indicus) heifers (n=9) and from primigravid Nelore heifers during early (n=9), mid (n=6), and late (n=5) pregnancy. Specimens of mammary tissue were analyzed by real-time PCR and immunohistochemistry. The βA and α-inhibin subunits and ActRIB and ActRIIA mRNA expression was higher in the early-pregnancy group compared with the nonpregnant group. In the mid-pregnancy group, the subunits βA, βB, and α-inhibin as much as follistatin mRNA expression was higher compared with the nonpregnant group, whereas ActRIB transcripts were absent in the late-pregnancy group. Immunostaining of these proteins, with the exception of ActRIB, was observed in the mammary tissue sections at all time points analyzed; these findings are in agreement with the observed pattern of mRNA expression. Staining and mRNA expression for ActRIB were undetected in the late-pregnancy group. In summary, the present study demonstrated that the activin-related proteins, βA, βB, and α-inhibin subunits, as much as follistatin and ActRIB and ActRIIA receptors display different patterns of expression regarding time of gestation in the bovine mammary gland. The modulation of the expression pattern during gestation suggests that activin-related proteins may play a key role in regulating bovine mammary branching morphogenesis and epithelial differentiation.

Gonadal mRNA Expression Levels of TGFβ Superfamily Signaling Factors Correspond with Post-Hatching Morphological Development in American Alligators

Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin βA subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin α and βB subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.