Fonction de la protéine Ceroid lipofuscinosis neuronal 5 (CLN5) dans le tri et le recyclage à l’endosome

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi.

Étude du rôle des régions variables 4 et 5 dans les changements de conformation de la gp120 du VIH-1

Le VIH infecte les cellules par fusion de sa membrane avec la membrane de la cellule cible. Cette fusion est effectuée par les glycoprotéines de l'enveloppe (Env) qui sont synthétisées en tant que précurseur, gp160, qui est ensuite clivé en gp120 et gp41. La protéine gp41 est la partie transmembranaire du complexe de l'enveloppe et l’ancre à la particule virale alors que la gp120 assure la liaison au récepteur cellulaire CD4 et corécepteur CCR5 ou CXCR4. Ces interactions successives induisent des changements de conformation d’Env qui alimentent le processus d'entrée du virus conduisant finalement à l'insertion du peptide de fusion de la gp41 dans la membrane de la cellule cible. La sous-unité extérieure gp120 contient cinq régions variables (V1 à V5), dont trois (V1, V2 et V3) étant capables d’empêcher l’adoption spontanée de la conformation liée à CD4. Cependant, le rôle de régions variables V4 et V5 vis-à-vis de ces changements de conformation reste inconnu. Pour étudier leur effet, des mutants de l'isolat primaire de clade B YU2, comprenant une délétion de la V5 ou une mutation au niveau de tous les sites potentiels de N-glycosylation de la V4 (PNGS), ont été générés. L'effet des mutations sur la conformation des glycoprotéines d'enveloppe a été analysé par immunoprécipitation et résonance de plasmon de surface avec des anticorps dont la liaison dépend de la conformation adopté par la gp120. Ni le retrait des PNGS de la V4 ni la délétion de V5 n’a affecté les changements conformationnels d’Env tels que mesurés par ces techniques, ce qui suggère que les régions variables V1, V2 et V3 sont les principaux acteurs dans la prévention de l’adoption de la conformation lié de CD4 d’Env.

The influence of organophosphorus pesticide-methylparathion on protein, lipid metabolism and detoxifying enzymes in rohu (Labeo rohita)

Methylparathion (MP) is an organophosphorus insecticide used world wide in agriculture due to its high activity against a broad spectrum of insect pests. The aim of the study is to understand the effect of methylparathion on the lipid peroxidation, detoxifying and antioxidant enzymes namely catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione Stransferase (GST), total reduced glutathione (GSH), lipid peroxidation (LPO), acetylcholinesterase (AChE) and disease diagnostic marker enzymes in liver, sarcoplasmic (SP) and myofirbirllar (MF) proteins in muscles, lipids and histopathlogical changes in various organs of Labeo rohita of size 75 i 6g at lethal and sublethal level of exposure. The probit analysis showed that the lethal concentration (LC 50%) for 24, 48, 72 and 96h were 15.5mg/L, 12.3mg/L, 11.4mg/L and 10.2mg/L respectively which is much higher compared to the LC50 for juvenile fish. The LPO level and GST activity increased five folds and two folds respectively on exposure to methylparathion at 10.2 mg/L and the level of the enzymes increased, on sub lethal exposure beyond 0.25mg/L. AChE activity was inhibited by 74% at a concentration of 1.8mg/L and 90% at 5.4mg/L. The disease diagnostic marker enzymes AST, ALT, ALP and LDH increased by about 2, 3 ,3 and 2 folds respectively at pesticide concentration of 10.2mg/L when compared to control. On sub lethal exposure, however the enzymes did not show any significant changes up to 0.5mg/L. At a concentration of 10.2 mg/L, there was a three fold increase in myofibrillar proteins while the increase in sarcoplasmic protein was above 1.5 fold. On sub lethal exposure, significant alteration was noticed up to 30 days up to 1mg/L of methylparathion concentration. Further exposure up to 45 days increased sarcoplasmic proteins (upto 0.5mg/L). ln the case of myofibrillar proteins, noticeable changes were observed at 1mg/L concentration right from 15th day. The cholesterol content in brain tissues increased by about 27% at methylparathion concentration of 5.4 mglL. However at 0.25mg/L sub lethal concentration, no significant alteration was observed in enzyme activity, muscle proteins, lipids and histopathology of the tissues. The results suggest that methylparathion has the potential to induce oxidative stress in fish, and that liver, muscle and brains are more sensitive organs of Labeo rohita, with poor antioxidant potentials at higher concentrations of the pesticide. The various parameters studied in this investigation can also be used as biomarkers of methylparathion exposure.

Thermal stability of myofibrillar protein from Indian major carps

The characteristics and stability of natural actomyosin (NAM) from rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were investigated. The total extractable actomyosin (AM) was higher (7.60mgml−1) in the case of rohu compared with that from catla and mrigal (5mgml−1). Although the specific AM ATPase activity was similar (0.43–0.5 μmolPmin−1 mgP−1) among the three species, the total ATPase activity was lower in mrigal (25 μmol g−1 meat) compared with the other species (37 μmol g−1 meat). The inactivation rate constants (kd) of AM Ca ATPase activity showed differences in the stabilities of actomyosin among these fish, the actomyosin from catla being least stable. The NAM from these species was stable up to 20 ◦C at pH 7.0. Catla AM became unstable at 30 ◦C, while rohu and mrigal AM could withstand up to 45 ◦C. The thermal denaturation with respect to solubility, turbidity, ATPase activity, sulphhydryl group and surface hydrophobicity showed noticeable changes at around these temperatures

Analyse von Protein-Ligand Interaktionen zur Charakterisierung zyklonukleotidbindender Proteine

Hyperpolarisations-aktivierte zyklonukleotid-gesteuerte (HCN) Kanäle übernehmen wichtige Funktionen in der Regulation der Herz- und Neuronalaktivität und können über einen dualen Mechanismus aus Membranhyperpolarisation und der Bindung von zyklischen Nukleotiden aktiviert werden. Ein großes Ziel der aktuellen Forschung ist die Entwicklung neuartiger Inhibitoren, die einer Fehlregulation der Kanäle entgegenwirken. In der vorliegenden Arbeit wurde die Regulation von HCN Kanälen durch zyklische Nukleotide im Detail analysiert, indem erstmals ein umfassender Screen mit 48 unterschiedlichen Zyklonukleotid-Analoga am C-terminalen Bereich (bestehend aus C-Linker und Zyklonukleotid-Bindedomäne) der drei Isoformen HCN1, HCN2 und HCN4 durchgeführt wurde. Mit Hilfe eines Fluoreszenzpolarisations-Assays wurde der Einfluss von Modifikationen in der Base, der Ribose und dem zyklischen Phosphat auf die Bindungsaffinitäten innerhalb der Zyklonukleotid-Bindedomäne untersucht. Zyklonukleotid-Analoga mit Modifikationen an der Position 7 und 8 der Base verschoben die apparenten Affinitäten im Vergleich zu den beiden natürlich vorkommenden Zyklonukleotiden cAMP und cGMP vom mikromolaren in den nanomolaren Bereich. Selektiv für die HCN4 Isoform erwiesen sich Zyklonukleotid-Analoga mit Modifikationen an der Position 6 der Base, während Modifikationen an der Position 8 der Base zu einer höheren Affinität für die HCN2 Isoform führten. Im Gegensatz zu HCN2 und HCN4 zeigte die HCN1 Isoform besonders hohe Affinitäten für Zyklonukleotid-Analoga mit Modifikationen an der Position 8 von cGMP. Eine Substitution der 2’-Hydroxylgruppe erlaubte keine Bindung an die HCN Kanäle. Mit 7-CH-cAMP konnte ein hochaffines Bindemolekül für HCN Kanäle identifiziert werden, denn der Austausch eines Stickstoffs gegen eine CH-Einheit an Position 7 der Base führte zu einer 100-fachen Steigerung der Affinität im Vergleich zu cAMP. In Übereinstimmung mit der hochaffinen Bindung konnte in kinetischen Analysen eine langsamere Dissoziationsrate für 7-CH-cAMP gemessen werden. Anhand thermodynamischer Messungen konnte ein entropisch favorisierter Bindungsmodus für 7-CH-cAMP im Vergleich zu cAMP identifiziert werden. Basierend auf einer Kristallstruktur des HCN4 CNBD:7-CH-cAMP Komplexes (2,5 Å) lässt sich erklären, dass 7-CH-cAMP durch seine höhere Lipohilie im Vergleich zu cAMP eine stärkere Präferenz für das hydrophobe Netzwerk zwischen Protein und Base besitzt. In detaillierten, vergleichenden Analysen mit den zyklonukleotidbindenden Proteinen PKA Typ I und II, hPKGIβ und Epac 1 und 2 konnte gezeigt werden, dass 7-CH-cAMP die höchsten Affinitäten für die drei Isoformen der HCN Kanäle aufwies. Somit könnte sich 7-CH-cAMP als vielversprechender Kandidat für die selektive Regulation von HCN Kanälen in vitro und in lebenden Zellen eignen und möglicherweise einen wichtigen Beitrag als krankheitsrelevanter Effektor leisten.

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNALysUUU, tRNAGluUUC and tRNAGlnUUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALysUUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALysUUU hypomodification and malfunction.

Caracterización de la proteína del cuello de las roptrias 5 en plasmodium falciparum (PfRON5)

El conocimiento de las proteínas implicadas en el proceso de invasión de los merozoitos a los eritrocitos por Plasmodium es el punto de partida para el desarrollo de nuevas estrategias para controlar la malaria. Muchas de estas proteínas han sido estudiadas en Toxoplasma gondii, donde se han identificado las proteínas que pertenecen al Tight Junction (TJ), el cual permite una interacción fuerte entre las membranas de la célula huésped y el parásito, necesaria para la invasión parasitaria. En este género, cuatro proteínas del cuello de las roptrias (RON2, RON4, RON5 y RON8) y una proteína de micronemas (TgAMA-1) se han encontrado como parte del TJ. En Plasmodium falciparum, se han caracterizado las proteínas PfRON2 y PfRON4. En el presente estudio se realiza la identificación de la proteína PfRON5, una proteína de ~110 kDa que se expresa en las etapas de merozoitos y esquizontes de la cepa FCB-2 utilizando técnicas de biología molecular, bioinformática e inmuoquímica.

Dynamic behavior of Arabidopsis eif4a-iii, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

A heterologous expression system for bovine lens transmembrane main intrinsic protein (MIP) in Nicotiana tabacum plants

We have developed a heterologous expression system for transmembrane lens main intrinsic protein (MIP) in Nicotiana tabacum plant tissue. A native bovine MIP26 amplicon was subcloned into an expression cassette under the control of a constitutive Cauliflower Mosaic Virus promoter, also containing a neomycin phosphotransferase operon. This cassette was transformed into Agrobacterium tumefaciens by triparental mating and used to infect plant tissue grown in culture. Recombinant plants were selected by their ability to grow and root on kanamycin-containing media. The presence of MIP in the plant tissues was confirmed by PCR, RT-PCR and immunohistochemistry. A number of benefits of this system for the study of MIP will be discussed, and also its application as a tool for the study of heterologously expressed proteins in general.

Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture

Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2x108, 1.1x109 and 3.5x1010 at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2 mg at d18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus shown to be a flexible and powerful tool for manufacturing recombinant proteins.

Hydrolyzable tannin structures influence relative globular and random coil protein binding strengths

Binding parameters for the interactions of pentagalloyl glucose (PGG) and four hydrolyzable tannins (representing gallotannins and ellagitannins) with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction of PGG and isolated mixtures of tara gallotannins and of sumac gallotannins with gelatin and BSA were of the same order of magnitude for each tannin (in the range of 10(4)-10(5) M-1 for stronger binding sites when using a binding model consisting of two sets of multiple binding sites). In contrast, isolated mixtures of chestnut ellagitannins and of myrabolan ellagitannins exhibited 3-4 orders of magnitude greater equilibrium binding constants for the interaction with gelatin (similar to 2 x 10(6) M-1) than for that with BSA (similar to 8 x 10(2) M-1). Binding stoichiometries revealed that the stronger binding sites on gelatin outnumbered those on BSA by a ratio of at least similar to 2:1 for all of the hydrolyzable tannins studied. Overall, the data revealed that relative binding constants for the interactions with gelatin and BSA are dependent on the structural flexibility of the tannin molecule.

Effect of breed, gender, housing system and dietary crude protein content on performance of finishing beef cattle fed maize-silage-based diets

Maize silage-based diets with three dietary crude protein (CP) supplements were offered to 96 finishing cattle of contrasting breed (Holstein Friesian (HF) v. Simmental x HF (SHF)) and gender (bull v. steer) housed in two types of feeding system (group fed v. individually fed). The three protein supplements differed either in CP or protein degradability (degradable (LUDP) v. rumen undegradable (HUDP)) and provided CP concentrations of 142 (Con), 175 (LUDP) and 179 (HUDP) g/kg dry matter (DM) respectively, with ratios of degradable to undegradable of 3.0, 1.4 and 0.9:1 for diets Con, LOP and HUDP respectively. DM intakes were marginally higher (P = 0. 102) for LOP when compared with Con and HOP Rates of daily live-weight gain (DLWG) were higher (P = 0.005) in LUDP and HOP when compared with Con. HF had higher DM intakes than SHF although this did not result in any improvement in HF DLWG. Bulls had significantly better DM intakes, DLWG and feed conversion efficiency than steers. Conformation scores were better in SHF than HF (P < 0.001) and fat scores lower in bulls than steers (p < 0.001). There was a number of first order interactions established between dietary treatment, breed, gender and housing system with respect to rates of gain and carcass fat scores.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Effect of plasma insulin and branched-chain amino acids on skeletal muscle protein synthesis in fasted lambs

The increase in fractional rate of protein synthesis (K-s) in the skeletal muscle of growing rats during the transition from fasted to fed state has been explained by the synergistic action of a rise in plasma insulin and branched-chain amino acids (BCAA). Since growing lambs Also exhibit an increase in K-s with level of feed intake, the objective of the present study was to determine if this synergistic relationship between insulin and BCAA also occurs in ruminant animals. Six 30 kg fasted (72 h) lambs (8 months of age) received each of four treatments, which were based on continuous infusion into the jugular vein for 6 h of: (1) saline (155 mmol NaCl/l); (2) a mixture of BCAA (0.778 mumol leucine, 0.640 mumol isoleucine and 0.693 mumol valine/min.kg); (3) 18.7 mumol glucose/min.kg (to induce endogenous insulin secretion): (4) co-infusion of BCAA and glucose. Within each period all animals received the same isotope of phenylalanine, (Phe) as follows: (1) L-[1-C-13]Phe; (2) L-phenyl-[ring H-2(5)]-alanine; (3) L-[N-15]Phe; (4) L-[ring 2,6-H-3]Phe. Blood was sampled serially during infusions to measure plasma concentrations of insulin, glucose and amino acids, and plasma free Phe isotopic activity; biopsies were taken 6 h after the beginning of infusions to determine K-s in in. longissimus dorsi and vastus muscle. Compared with control (saline-infused) lambs, K-s was increased by an average of 40% at the end of glucose infusion, but this effect was not statistically significant in either of the muscles sampled. BCAA infusion, alone or in combination with glucose, also had no significant effect on K-s compared with control sheep. K-s was approximately 60% greater for vastus muscle than for m. longissimus dorsi (P<0.01), regardless of treatment. It is concluded that there are signals other than insulin and BCAA that are responsible for the feed-induced increase in K-s in muscle of growing ruminant animals.

Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular-luteal transition in cattle

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. in this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-beta A and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to P-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-beta A mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P-4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A (similar to 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.