Human Activin Research Articles

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

Activin promotes follicular integrity and oogenesis in cultured pre-antral bovine follicles

The aim of this study was to determine the individual and combined effect of activin and follicle stimulating hormone (FSH) on somatic and germ cell development in cultured pre-antral follicles. Pre-antral bovine follicles (mean diameter 157 +/- 3, range 132-199 microm) were cultured for 8 days in serum-free medium in the presence of either 100 ng/ml of recombinant human activin A (rhAct A), 100 ng/ml rhAct A combined with a high (100 ng/ml) or low (50 ng/ml) concentration of recombinant FSH (rFSH) or 50 ng/ml rFSH alone. Intrafollicular connexin 43 expression and actin-based cell adhesion were assessed on Day 2 and 4 of culture. Steroidogenesis was evaluated after Day 4 and 8. Follicles exposed to 100 ng/ml activin maintained expression of connexin 43 at the follicular periphery. In the presence of activin, with or without 100 ng/ml or 50 ng/ml FSH, follicles were steroidogenic undergoing significant growth (P < 0.01), granulosa cell proliferation (P < 0.01) and antral cavity formation (P < 0.05) compared with cultured controls. Maximum oocyte growth occurred in the presence of 100 ng/ml activin alone with a significant percentage of these oocytes maintaining normal morphology over controls (P < 0.05). These results are consistent with a role for activin in maintaining oocyte granulosa cell interactions due to increased peripheral granulosa cell adhesion to the basement membrane and retention of adhesion at the surface of the zona pellucida. Thus, the polarized expression of cell contact interactions promoted by activin supports ongoing folliculogenesis.

Activin A inhibits activation of human primordial follicles in vitro

To determine whether Activin A affects the activation and survival of human primordial follicles in vitro. Ovarian cortical biopsies from eight women undergoing elective caesarean sections or benign gynaecological procedures were taken and cut into small pieces (1-3 mm(3)), cultured in serum-free medium for 7 days with/without human recombinant Activin A at a concentration of either 50 or 100 ng/ml. Ovarian tissue were analysed by histology for follicle viability, development and density. Significant activation of primordial follicles within cultured cortical tissue was observed after 7 days in control medium. However, medium supplemented with Activin A at 50 ng/ml resulted in significant inhibition of follicular activation. Increasing the concentration of Activin A to 100 ng/ml reversed the inhibitory effect. The effect of Activin A appeared to be specific to activation of non-growing (primordial) follicles into the growing population since no significant differences in follicle viability was observed between treatment groups. Activin A at a concentration of 50 ng/ml can inhibit the spontaneous activation of human primordial follicles in vitro indicating that this may be a component of the signalling mechanisms that maintain follicular quiescence.

Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2-10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF-beta proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein.

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1

BackgroundActivin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1.ResultsWe have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1.ConclusionsOur results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Implication of activin E in glucose metabolism: Transcriptional regulation of the inhibin/activin βE subunit gene in the liver

Aims Activin E is a newly identified member of the transforming growth factor-β superfamily. To assess the role of activin E in glucose/energy metabolism, we investigated the transcriptional regulation of activin E in the liver. Main methods Northern blotting, Western blotting, quantitative real-time polymerase chain reaction (PCR), reporter assays and chromatin immunoprecipitation assays were used in this study. Key findings Insulin up-regulated activin E expression at the mRNA and protein level in HepG2 cells. Reporter assays revealed that the putative, functional, promoter sequence of human activin E gene was responsible for the effect of insulin. Mutational analysis of the promoter revealed that CCAAT/enhancer-binding proteins (C/EBPs) play a key role in regulating activin E expression and in the stimulatory effect of insulin on activin E transcription. Chromatin immunoprecipitation assays revealed that the C/EBPs can bind to the activin E promoter in HepG2 cells. The expression of activin E mRNA was up-regulated in the liver of diet-induced obese mice. Significance These observations suggest that activin E plays a pathophysiological role in glucose metabolism.

M.P.1.03 Preliminary results from single subcutaneous administration of ACE-031, a form of the soluble activin type IIB receptor, in healthy postmenopausal volunteers

ACE-031 is a fusion protein derived from the extracellular domain of the human activin receptor type IIB (ActRIIB) linked to the Fc portion of human IgG1. ACE-031 binds with high affinity to GDF-8 (myostatin) and other negative regulators of muscle mass. In numerous nonclinical studies using normal animals and various disease animal models, treatment with a soluble ActRIIB fusion protein results in increased lean mass and decreased adiposity. Pharmacologic responsiveness to ACE-031 has been observed in several species including non-human primates.

The use of serum inhibin A and activin A levels in predicting the outcome of 'pregnancies of unknown location'

The aim was to evaluate the role of serum inhibin A and activin A in the prediction of the outcome of women with 'pregnancies of unknown location' (PULs). Serum human chorionic gonadotrophin (hCG), progesterone, inhibin A and activin A levels were measured at 0 and 48 h. Differences in the mean levels and the change in levels over 48 h expressed as a ratio (48/0 h) were examined between the three outcome groups--failing PUL, intrauterine pregnancy (IUP) or ectopic pregnancy. Variables were incorporated into logistic regression models to predict the pregnancy outcomes, which were evaluated using receiver operator characteristic curves. One hundred and forty-one women were classified as PULs: 67 failing PULs (47.5%), 58 IUPs (41.1%) and 16 ectopic pregnancies (11.4%). Activin A levels were not significantly different between the three outcome groups. Inhibin A levels were significantly lower in failing PULs. The logistic regression model based on serum inhibin levels gave an area under the curve (AUC) of 0.88 for failing PUL, 0.87 for IUP and 0.60 for ectopic pregnancy. The model based on serum activin levels gave an AUC of 0.61 for failing PUL, 0.64 for IUP and 0.51 for ectopic pregnancy, and the model based on serum hCG levels gave an AUC of 0.95 for failing PUL, 0.97 for IUP and 0.67 for ectopic pregnancy. Serum activin A levels at 0 and 48 h are not helpful in predicting the outcome of PULs. Although serum inhibin A levels may be of use in the prediction of failing PULs and IUPs in the PUL populations, they do not perform as well as serum hCG levels.

Identification and characterization of human ARIP2 and its relation to breast cancer

Activin, a member of the TGF-β superfamily, inhibits the proliferation of breast cancer cells. Activin interacts with its type I and type II receptors to induce phosphorylation of intracellular signaling molecules known as Smads. Previous studies showed that mouse ARIP2 can reduce activin signaling by interacting with activin type II receptors (ActRIIs); however, the activity of ARIP2 in breast cancer is still unclear. In this study, we used RT-PCR to obtain a human homologue of mouse ARIP2, human activin receptor-interacting protein 2 (hARIP2). Like murine ARIP2, hARIP2 has a PDZ domain in its NH2-terminal region and can interact specifically with ActRIIs. Overexpression of hARIP2 reduced activin-induced transcriptional activity and enhanced cell proliferation and colony formation in human breast adenocarcinoma MCF-7 cells and MDA-MB-231 cells. However, down-regulation of hARIP2 expression by RNAi enhanced activin-induced transcriptional activity and reduced cell proliferation and colony formation. Immunohistochemistry revealed that hARIP2 was expressed more frequently and much more intensely in malignant breast tissues such as simple carcinoma, invasive ductal carcinoma and mucinous adenocarcinoma than in benign hyperplasia or fibroadenoma cases. These results suggest that hARIP2 is a putative growth-promoting factor involved in breast tumorigenesis and tumor development.

Lineage Restriction and Differentiation of Human Embryonic Stem Cells into Hepatic Progenitors and Zone 1 Hepatocytes

Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

Involvement of ERK, Bcl-2 family and caspase 3 in recombinant human activin A-induced apoptosis in A549

Background Activins are members of the transforming growth factor-β (TGF-β) superfamily. Previous studies have shown that activin A may have a central role in regulating both apoptosis and proliferation. However, direct studies of recombination human activin A on human NSCLC A549 cells have not yet been reported. The purpose of this study was to investigate whether activin A could induce apoptosis in A549 cells and the possible mechanisms via which it worked. Methods Cellular apoptosis induced by activin A was detected by TUNEL assay and the levels of protein expression were detected by western blot. Results Recombination human activin A induced apoptosis in human NSCLC A549 cells in a concentrate-dependent manner. Activin A-induced A549 apoptosis was accompanied by the up-regulation of Bax, Bad and Bcl-Xs and down-regulation of Bcl-2. Moreover, activin A treatment increased the expression of its typeII receptors, activated ERK and caspase 3 in A549. These results clearly demonstrate that the induction of apoptosis by activin-A involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins and caspase 3 participate in activin A-induced apoptotic process in A549 cells. On the other hand, activin A treatment had little effect on primary human small airway epithelial cells (SAECs). Conclusion Recombination human activin A induced apoptosis in A549 cells, at least partially, through ERK and mitochondrial pathway. The result that activin A did not affect the normal SAEC revealed activin A might be considered as a potential anticancer agent and worthy of further studies.

A novel 1,25-dihydroxyvitamin D–activin A pathway in human alveolar macrophages is dysfunctional in patients with pulmonary alveolar proteinosis (PAP)

We have shown that activin A, a cytokine implicated in regulating B-cell proliferation, is severely deficient in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP), an autoimmune disorder characterized by surfactant accumulation and neutralizing autoantibodies to granulocyte–macrophage colony stimulating factor. Mechanisms of activin regulation in alveolar macrophages are not well understood. Based on previous gene array results from PAP bronchoalveolar lavage cells suggesting deficiencies in vitamin D target genes, and on recent evidence of vitamin D receptor elements (VDREs) in the human activin A gene promoter, we investigated the effects of 1,25-dihydroxyvitamin D (vitamin D3) on activin A expression in alveolar macrophages from healthy individuals and PAP patients. Activin A expression was stimulated by LPS in cultures of either healthy control or PAP alveolar macrophages; in contrast, vitamin D3 increased activin A only in healthy controls but not in PAP. Compared to healthy controls, freshly obtained (uncultured) PAP alveolar macrophages displayed healthy intrinsic vitamin D receptor expression but deficient expression of vitamin D target genes, cathelicidin and thioredoxin interacting protein. PAP patients also demonstrated a relative insufficiency of circulating vitamin D. Investigation of activin A in murine alveolar macrophages confirmed a lack of functional response to vitamin D as anticipated since murine activin A does not contain VDREs. Results suggest that mechanisms of activin A deficiency in PAP alveolar macrophages may involve dysregulation of a novel species-specific vitamin D–activin A pathway.

A novel in vitro system for studying cardiomyocyte differentiation with medaka embryonic cells

Our studies revealed that dissociated cells from medaka (the fresh-water fish, Oryzias latipes ) blastula-stage embryos differentiate into many rhythmically contracting cells when incubated with a conditioned medium from a cell line. Analyses of these cells by immunostaining, electron microscopy, expression of marker genes, action potential recordings and pharmacological responses all showed the characteristics of myocardial cells. We succeeded in purifying the cardiac cell-inducing factor from the conditioned medium by 523,100-fold with 8% recovery of the protein. Analysis of its amino-acid sequence by mass spectrometry revealed the factor to be medaka activin A2 (homodimer of inhibin betaA2 subunit). Recombinant human activin A showed the same cardiac cell-inducing activity toward medaka embryonic cells. Our results demonstrate that activin A is a potent factor for medaka myocardial cell differentiation. This culture method should provide a novel and simple experimental system to study cardiomyocyte differentiation in vitro.

Synthesis, purification and bioactivity of recombinant human activin A expressed in the yeast Pichia pastoris

The transforming growth factor-beta (TGF-β) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.

Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes

The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

Effects of non-steroidal gonadal factors on LH secretion in female common carp during the reproductive cycle

The aim of this study was to evaluate the effects of recombinant human inhibin A, recombinant human activin A and desteroidized ovarian extract on LH secretion &lt;I&gt;in vitro&lt;/I&gt; and &lt;I&gt;in vivo&lt;/I&gt; in female common carp during different stages of reproductive cycle. Inhibin stimulated spontaneous as well as GnRH-stimulated LH release &lt;I&gt;in vivo&lt;/I&gt; in fish during gonadal recrudescence. This hormone did not have an influence on spontaneous LH secretion in the periovulatory period, but had a slightly inhibitory effect on GnRH-stimulated LH release in this stage of gonad maturity. Activin decreased spontaneous LH secretion during gonadal recrudescence and increased LH secretion before ovulation, having no effects on GnRH-stimulated LH release during both stages of gonad maturity. The desteroidized ovarian extract failed to modify spontaneous LH secretion, but decreased GnRH-stimulated LH release during recrudescence and especially before ovulation. It is to conclude that these data suggest the differential role of inhibin/activin as substances in the regulation of LH secretion in common carp females.

Development of human preantral follicles in vitro is enhanced by timed exposure to activin-A followed by FSH

OBJECTIVE: The aim of this study was to determine whether exposure to Activin-A followed by FSH would enhance growth and development of isolated human preantral follicles within a recently developed two-step serum free culture system.DESIGN: Ovarian cortical biopsies of approximately 5x4mm were obtained after informed consent during laparoscopic oophorectomy (n=2) and at caesarean section (n=1) from woman aged 40-41 years. Approval for this study was given by the local ethics committee.MATERIALS AND METHODS: Ovarian cortical strips containing primordial and primary follicles were cultured in serum free medium (McCoys + human serum albumen) for 6 days. At the end of this culture period secondary follicles were isolated from the strips. Intact secondary follicles with a mean diameter of 74 +/−5 microns (n = 33) were selected for further culture. Isolated follicles were placed individually within v shaped micro well culture plates in serum free medium in the presence of 100ng/ml of human recombinant activin A. After 2 days 50ng/ml of rhFSH in combination with 100ng/ml of activin A was added to approximately half of the cultured follicles (n=18) whilst the remainder (n=15) were exposed to only activin A for a further 2 days. Follicle diameters were measured on day 2 and day 4, cultured follicles at day 4 were fixed in Bouin's solution and prepared for histology and subsequent morphological analysis to determine oocyte and granulosa cell health.RESULTS: Isolated preantral follicles showed a significant increase in size during the first 2 days of culture in the presence of activin A. No follicular rupture or degeneration was observed during this period. Follicles exposed to FSH after 2 days in vitro showed a significantly higher increase in size (130+/−22 microns) when compared with those cultured in activin alone (91 +/− 13 microns) (p< 0.05). Morphological analysis showed an improvement in follicle health in those follicles exposed to both activin A and FSH.CONCLUSIONS: Primordial follicles can be activated to grow in vitro and develop to preantral follicles within 6 days of culture in cortical strips. Isolated preantral follicles show significant growth during a further 4 day culture period in the presence of activin A. Follicles exposed to a combination of activin A and FSH show a significantly greater rate of growth and improved morphology compared to activin alone. These preliminary results represent further progress towards achieving full in vitro growth of human oocytes. OBJECTIVE: The aim of this study was to determine whether exposure to Activin-A followed by FSH would enhance growth and development of isolated human preantral follicles within a recently developed two-step serum free culture system. DESIGN: Ovarian cortical biopsies of approximately 5x4mm were obtained after informed consent during laparoscopic oophorectomy (n=2) and at caesarean section (n=1) from woman aged 40-41 years. Approval for this study was given by the local ethics committee. MATERIALS AND METHODS: Ovarian cortical strips containing primordial and primary follicles were cultured in serum free medium (McCoys + human serum albumen) for 6 days. At the end of this culture period secondary follicles were isolated from the strips. Intact secondary follicles with a mean diameter of 74 +/−5 microns (n = 33) were selected for further culture. Isolated follicles were placed individually within v shaped micro well culture plates in serum free medium in the presence of 100ng/ml of human recombinant activin A. After 2 days 50ng/ml of rhFSH in combination with 100ng/ml of activin A was added to approximately half of the cultured follicles (n=18) whilst the remainder (n=15) were exposed to only activin A for a further 2 days. Follicle diameters were measured on day 2 and day 4, cultured follicles at day 4 were fixed in Bouin's solution and prepared for histology and subsequent morphological analysis to determine oocyte and granulosa cell health. RESULTS: Isolated preantral follicles showed a significant increase in size during the first 2 days of culture in the presence of activin A. No follicular rupture or degeneration was observed during this period. Follicles exposed to FSH after 2 days in vitro showed a significantly higher increase in size (130+/−22 microns) when compared with those cultured in activin alone (91 +/− 13 microns) (p< 0.05). Morphological analysis showed an improvement in follicle health in those follicles exposed to both activin A and FSH. CONCLUSIONS: Primordial follicles can be activated to grow in vitro and develop to preantral follicles within 6 days of culture in cortical strips. Isolated preantral follicles show significant growth during a further 4 day culture period in the presence of activin A. Follicles exposed to a combination of activin A and FSH show a significantly greater rate of growth and improved morphology compared to activin alone. These preliminary results represent further progress towards achieving full in vitro growth of human oocytes.

GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

Hedgehog (HH) signaling in the epidermis is primarily mediated by the zinc finger transcription factors GLI1 and GLI2. Exquisite regulation of HH/GLI signaling is crucial for proper specification of the epidermal lineage and development of its derivatives, whereas dysregulation of HH/GLI signaling disrupts tissue homeostasis and causes basal cell carcinoma (BCC). Similarly, bone morphogenetic proteins (BMPs) and activins have been described as key signaling factors in the complex regulation of epidermal fate decisions, although their precise interplay with HH/GLI is largely elusive. Here we show that, in human epidermal cells, expression of the activin/BMP antagonist follistatin (FST) is predominantly up-regulated by the HH effector GLI2. Consistently, we found strong FST expression in the outer root sheath of human hair follicles and BCC. Detailed promoter analysis showed that two sequences with homology to the GLI consensus binding site are required for GLI2-mediated activation. Interestingly, activation of the FST promoter is highly GLI2-specific, because neither GLI1 nor GLI3 can significantly increase FST transcription. GLI2 specificity requires the presence of a 518-bp fragment in the proximal FST promoter region. On the protein level, sequences C-terminal to the zinc finger are responsible for GLI2-specific activation of FST transcription, pointing to the existence of GLI-interacting cofactors that modulate GLI target specificity. Our results reveal a key role of GLI2 in activation of the activin/BMP antagonist FST in response to HH signaling and provide new evidence for a regulatory interaction between HH and activin/BMP signaling in hair follicle development and BCC.

A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin

The objective of this study was to determine whether follicles grown within human ovarian cortical strip culture for 6 days in serum-free medium could be isolated at the secondary stage of pre-antral development and grown in vitro to the late pre-antral/early antral stage during a 4 day culture period. Ovarian cortical biopsies were obtained from six women aged 26-40 years, with informed consent, during elective Caesarean section. Small tissue slices of ovarian cortex, with underlying stromal tissue removed, were cultured in serum-free medium for 6 days and at the end of this period pre-antral (secondary) follicles were dissected from the strips. Seventy-four intact pre-antral follicles ranging in size (66-132 microm) (mean size 100 microm +/- 3.4) were selected for further culture. Follicles were placed individually within V-shaped microwell culture plates in serum-free medium in the presence (n = 38) or absence (n = 36) of 100 ng/ml of human recombinant activin A. Pre-antral follicles grown for 4 days in the presence of activin A grew to a larger size (mean diameter 143 microm +/- 7.4) than those grown in control medium (mean diameter 111 microm +/- 8) (P < 0.005). Ninety percent of follicles cultured in the presence of activin A increased in size during the first 2 days of culture compared with only 36% of follicles in control medium (P > 0.005). Of the follicles surviving the entire culture period, 30% of those cultured in the presence of activin A showed normal morphology with intact oocytes and antral formation. None of the follicles grown in control medium developed antral cavities and >90% of those follicles collected at the end of the culture period showed signs of oocyte degeneration. The results reported here demonstrate that under certain conditions, it is possible to achieve accelerated oocyte/follicle development from human primordial/primary follicles. This provides the first encouraging step towards achieving full in vitro growth of human oocytes.