Characterization of inhibin α subunit (inha) in the zebrafish: evidence for a potential feedback loop between the pituitary and ovary

Inhibin and activin are closely related disulphide-linked dimers that belong to the transforming growth factor β superfamily. Although inhibin has been extensively studied in mammals, the information about its existence and function in lower vertebrates is very scarce. Using zebrafish as a model, the present study demonstrated that the inhibin-specific α subunit (inha) was predominantly expressed in the gonads and no transcript could be detected in other tissues including the pituitary and brain. In the ovary, the expression ofinhawas restricted to the somatic follicle cells surrounding the oocyte, together with the β subunits (inhbaaandinhbb). This was further supported by the absence of its expression in the ovulated unfertilized eggs. During folliculogenesis,inhaexpression in the follicles slightly but steadily increased from primary growth to the mid-vitellogenic stage; however, its expression surged dramatically at the full-grown stage. Interestingly, the expression level ofinhadecreased significantly in the follicles whose oocytes were undergoing spontaneous maturation or germinal vesicle breakdown. When tested on cultured ovarian fragments, both goldfish pituitary extract and forskolin significantly stimulatedinhaexpression. Further experiments showed that recombinant zebrafish FSH but not LH significantly increasedinhaexpression in the same assay system. When testedin vitro, human inhibin A exhibited a slight but significant inhibitory effect on 17α, 20β-dihydroxyprogesterone-induced oocyte maturation after 4 h incubation. The stimulation ofinhaexpression by FSH and the potential inhibition of FSH by inhibin suggest a possible existence of a negative feedback loop between the pituitary and ovary in the zebrafish.

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Interleukin-4 and Transforming Growth Factor β Have Opposing Regulatory Effects on Gamma Interferon-Mediated Inhibition of Cryptosporidium parvum Reproduction

Infection and Immunity ◽

10.1128/iai.71.8.4580-4585.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4580-4585 ◽

Cited By ~ 28

Author(s):

I.-Sarah Lean ◽

Stuart A. C. McDonald ◽

Mona Bajaj-Elliott ◽

Richard C. G. Pollok ◽

Michael J. G. Farthing ◽

...

Keyword(s):

Growth Factor ◽

Cryptosporidium Parvum ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Inhibitory Effect ◽

Gamma Interferon ◽

Interleukin 4 ◽

Effector Cells ◽

Ifn Γ

ABSTRACT It was shown previously that enterocytes activated by gamma interferon (IFN-γ) are efficient effector cells in the killing of Cryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor β (TGF-β) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-β might modulate the effect of IFN-γ in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-β can abolish the inhibitory effect on C. parvum development and that neither IL-13 nor IL-10 influenced the action of IFN-γ. In contrast, IL-4 cooperated with low concentrations of IFN-γ (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-γ and IL-4 was intracellular Fe2+ deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-γ signaling, were similar when cells were stimulated with IFN-γ alone and with IFN-γ and IL-4γ, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-β and IL-4 on IFN-γ function did not appear to involve any alteration in the level of expression of IFN-γ receptors.

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Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes

Zygote ◽

10.1017/s0967199407004212 ◽

2007 ◽

Vol 15 (2) ◽

pp. 183-187 ◽

Cited By ~ 9

Author(s):

G. Sánchez Toranzo ◽

O.S. Giordano ◽

L.A. López ◽

M.I. Bühler

Keyword(s):

Germinal Vesicle ◽

Inhibitory Effect ◽

Follicle Cells ◽

Dependent Manner ◽

Bufo Arenarum ◽

M Phase ◽

Amplification Loop ◽

Activation Cascade ◽

P34 Cdc2

SummaryIn amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 μM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

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In Vitro Reconstruction of Xenopus Oocyte Ovulation

International Journal of Molecular Sciences ◽

10.3390/ijms20194766 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4766 ◽

Cited By ~ 2

Author(s):

Alexander A. Tokmakov ◽

Yuta Matsumoto ◽

Takumi Isobe ◽

Ken-Ichi Sato

Keyword(s):

Mapk Pathway ◽

Germinal Vesicle ◽

Ovarian Follicles ◽

Follicle Cells ◽

Germinal Vesicle Breakdown ◽

Follicle Rupture ◽

Ovulation Inhibition ◽

The Matrix ◽

Clawed Frog

Progesterone is widely used to induce maturation of isolated fully grown oocytes of the African clawed frog, Xenopus laevis. However, the hormone fails to release oocytes from the layer of surrounding follicle cells. Here, we report that maturation and follicle rupture can be recapitulated in vitro by treating isolated follicular oocytes with progesterone and low doses of the matrix metalloproteinase (MMP), collagenase, which are ineffective in the absence of the steroid. Using this in vitro ovulation model, we demonstrate that germinal vesicle breakdown (GVBD) and oocyte liberation from ovarian follicles occur synchronously during ovulation. Inhibition of the MAPK pathway in these experimental settings suppresses both GVBD and follicular rupture, whereas inhibition of MMP activity delays follicular rupture without affecting GVBD. These results highlight importance of MAPK and MMP activities in the ovulation process and provide the first evidence for their involvement in the release of oocytes from ovarian follicles in frogs. The in vitro ovulation model developed in our study can be employed for further dissection of ovulation.

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Blocking p38 signalling inhibits chondrogenesis in vitro but not ankylosis in a model of ankylosing spondylitis in vivo

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2011-200377 ◽

2011 ◽

Vol 71 (5) ◽

pp. 722-728 ◽

Cited By ~ 13

Author(s):

Kirsten Braem ◽

Frank P Luyten ◽

Rik J U Lories

Keyword(s):

Proinflammatory Cytokines ◽

Chondrogenic Differentiation ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Inhibitory Effect ◽

Mitogen Activated Protein Kinase ◽

Alternative Activation ◽

Beneficial Effects

ObjectivesTo investigate p38 mitogen activated protein kinase (MAPK) signalling in an in vitro model of bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ)-induced chondrogenesis and in vivo, with specific attention to its potential role in ankylosing enthesitis.MethodsHuman periosteum-derived cells (hPDCs) were cultured in pellets and stimulated with BMP2 or TGFβ1 in the presence or absence of a p38 inhibitor SB203580 or proinflammatory cytokines. Chondrogenic differentiation was evaluated using quantitative PCR. Male DBA/1 mice from different litters were caged together at the age of 8 weeks and treated with SB203580 in both a preventive and therapeutic strategy. The mice were evaluated for prospective signs of arthritis and the toe joints were analysed histologically to assess disease severity.Resultsp38 inhibition by SB203580 and proinflammatory cytokines downregulated chondrogenic markers in pellet cultures stimulated by BMP2 or TGFβ1. In contrast, the in vivo experiments resulted in an increased clinical incidence of arthritis and pathology severity score, reflecting progression towards ankylosis in mice given SB203580.ConclusionInhibition of p38 inhibited chondrogenic differentiation of progenitor cells, showing that not only the SMAD signalling pathways and also alternative activation of MAPKs including p38 contribute to chondrogenesis. Such an inhibitory effect is not found in an in vivo model of joint ankylosis and spondyloarthritis. Increased incidence and severity of disease in preventive experiments and shifts in disease stages in a therapeutic experimental set-up suggest that specific inhibition of p38 may have deleterious rather than beneficial effects.

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Inhibitory action of somatostatin on meiotic maturation of cultured porcine follicular ova

Acta Endocrinologica ◽

10.1530/acta.0.1100408 ◽

1985 ◽

Vol 110 (3) ◽

pp. 408-412 ◽

Cited By ~ 2

Author(s):

Takahide Mori ◽

Minoru Irahara ◽

Haruhiko Saito ◽

Yoshio Ohno ◽

Eiji Hosoi

Keyword(s):

Inhibitory Action ◽

Germinal Vesicle ◽

Inhibitory Effect ◽

Meiotic Maturation ◽

Stimulatory Action ◽

Germinal Vesicle Breakdown ◽

Physiological Importance ◽

Inhibiting Factor ◽

Synthetic Oxytocin

Abstract. To investigate the physiological importance of somatotrophin release-inhibiting factor (SRIF), effects in vitro of synthetic SRIF 14 on germinal vesicle breakdown (GVB) of cultured porcine follicular ova were studied. The proportion of ova with GVB decreased gradually and significantly with increasing concentrations of SRIF 14 in the range from 6 × 10−12 to 6 × 10−7 m during a 22 h period of culture. The inhibitory effect was apparent for the period between 14 and 22 h in the course of culture but was reversed by a concomitant addition of anti-SRIF to the medium. Neither synthetic oxytocin, vasoactive intestinal polypeptide nor substance P exerted any inhibitory or stimulatory action on GVB. These results suggest a limited but definite inhibitory action of SRIF on GVB of porcine ova.

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Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes

Zygote ◽

10.1017/s0967199404002709 ◽

2004 ◽

Vol 12 (3) ◽

pp. 185-195 ◽

Cited By ~ 5

Author(s):

G. Sánchez Toranzo ◽

F. Bonilla ◽

L. Zelarayán ◽

J. Oterino ◽

M. I. Bühler

Keyword(s):

Germinal Vesicle ◽

Peptide Hormone ◽

Inhibitory Effect ◽

Reproductive Period ◽

Follicle Cells ◽

Germinal Vesicle Breakdown ◽

Bufo Arenarum ◽

Hormonal Stimulus ◽

Different Response ◽

Maturation Promoting Factor

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring–summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.

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Role of cyclic AMP in the maturation of Ciona intestinalis oocytes

Zygote ◽

10.1017/s096719941000047x ◽

2010 ◽

Vol 19 (4) ◽

pp. 365-371 ◽

Cited By ~ 6

Author(s):

Francesco Silvestre ◽

Alessandra Gallo ◽

Annunziata Cuomo ◽

Tiziana Covino ◽

Elisabetta Tosti

Keyword(s):

Cyclic Amp ◽

Oocyte Maturation ◽

Sea Water ◽

Germinal Vesicle ◽

Ciona Intestinalis ◽

Follicle Cells ◽

Biological Mechanisms ◽

Germinal Vesicle Breakdown

SummaryImmature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.

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A new vision of the origin and the oocyte development in the Ostariophysi applied to Gymnotus sylvius (Teleostei: Gymnotiformes)

Neotropical Ichthyology ◽

10.1590/s1679-62252010000400008 ◽

2010 ◽

Vol 8 (4) ◽

pp. 787-804 ◽

Cited By ~ 19

Author(s):

Gisleine Fernanda França ◽

Harry J. Grier ◽

Irani Quagio-Grassiotto

Keyword(s):

Basement Membrane ◽

Ovarian Follicle ◽

Secondary Growth ◽

Germinal Vesicle ◽

Oocyte Development ◽

Follicle Cells ◽

Germinal Epithelium ◽

Germinal Vesicle Breakdown ◽

Vitellogenic Stage ◽

New Vision

Based on new knowledge coming from marine perciform species, the origin of oocytes and their development in the Ostariophysi, Gymnotus sylvius is described. In both Gymnotus sylvius and marine perciform fish, oogonia are found in the germinal epithelium that forms the surface of the ovarian lamellae. At the commencement of folliculogenesis, proliferation of oogonia and their entrance into meiosis gives rise to germ cell nests that extend into the stroma from the germinal epithelium. Both cell nests and the germinal epithelium are supported by the same basement membrane that separates them from the stroma. At the time of meiotic arrest, oocytes in a cell nest become separated one from the other as processes of prefollicle cells, these being derived from epithelial cells in the germinal epithelium, gradually encompass and individualize them while also synthesizing a basement membrane around themselves during folliculogenesis. The oocyte enters primary growth while still within the cell nest. At the completion of folliculogenesis, the oocyte and follicle cells, composing the follicle, are encompassed by a basement membrane. The follicle remains connected to the germinal epithelium as the both share a portion of common basement membrane. Cells originating from the stroma encompass the ovarian follicle, except where there is a shared basement membrane, to form the theca. The follicle, basement membrane and theca form the follicular complex. Oocyte development occurs inside the follicular complex. Development is divided into the stages primary and secondary growth, oocyte maturation and ovulation. Cortical alveoli appear in the ooplasm just prior to the beginning of secondary growth, the vitellogenic stage that begins with yolk deposition and proceeds until the oocyte is full-grown and the ooplasm is filled with yolk globules. Maturation is characterized by the germinal vesicle or nuclear migration, germinal vesicle breakdown or nuclear envelop fragmentation and the resumption of meiosis. At the ovulation the egg is released from the follicular complex into the ovarian lumen. When compared to marine Perciformes that lay pelagic eggs, oocyte development in Gymnotus sylvius has fewer steps within the stages of development, the two most remarkable being the absence of oil droplet formation during primary and secondary growth, (and the consequent absence of the oil droplets fusion during maturation), and the hydrolysis of yolf preceding ovulation.

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Trypanosoma cruzi Induces Regulatory Dendritic Cells In Vitro

Infection and Immunity ◽

10.1128/iai.01298-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2633-2641 ◽

Cited By ~ 41

Author(s):

Carolina Verónica Poncini ◽

Catalina Dirney Alba Soto ◽

Estela Batalla ◽

Maria Elisa Solana ◽

Stella Maris González Cappa

Keyword(s):

Dendritic Cells ◽

Trypanosoma Cruzi ◽

Lymphocyte Proliferation ◽

Transforming Growth Factor ◽

Acute Infection ◽

Transforming Growth Factor Β ◽

Interleukin 10 ◽

Inhibitory Effect ◽

Mhc Ii

ABSTRACT A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor β (TGF-β) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-β, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-β and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.

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Nonclinical Development of SRK-181: An Anti-Latent TGFβ1 Monoclonal Antibody for the Treatment of Locally Advanced or Metastatic Solid Tumors

International Journal of Toxicology ◽

10.1177/1091581821998945 ◽

2021 ◽

pp. 109158182199894

Author(s):

Brian T. Welsh ◽

Ryan Faucette ◽

Sanela Bilic ◽

Constance J. Martin ◽

Thomas Schürpf ◽

...

Keyword(s):

Transforming Growth Factor ◽

Checkpoint Inhibitors ◽

Human Peripheral Blood ◽

Phase 1 ◽

Locally Advanced ◽

Acquired Resistance ◽

Transforming Growth Factor Β ◽

Human Antibody ◽

Primary Resistance

Checkpoint inhibitors offer a promising immunotherapy strategy for cancer treatment; however, due to primary or acquired resistance, many patients do not achieve lasting clinical responses. Recently, the transforming growth factor-β (TGFβ) signaling pathway has been identified as a potential target to overcome primary resistance, although the nonselective inhibition of multiple TGFβ isoforms has led to dose-limiting cardiotoxicities. SRK-181 is a high-affinity, fully human antibody that selectively binds to latent TGFβ1 and inhibits its activation. To support SRK-181 clinical development, we present here a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies showed that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood. Four-week toxicology studies with SRK-181 showed that weekly intravenous administration achieved sustained serum exposure and was well tolerated in rats and monkeys, with no treatment-related adverse findings. The no-observed-adverse-effect levels levels were 200 mg/kg in rats and 300 mg/kg in monkeys, the highest doses tested, and provide a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that SRK-181 is a selective inhibitor of latent TGFβ1 that does not produce the nonclinical toxicities associated with nonselective TGFβ inhibition. These data support the initiation and safe conduct of a phase 1 trial with SRK-181 in patients with advanced cancer.

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