Cytoskeleton reorganization mediates alpha6beta1 integrin-associated actions of laminin on proliferation and survival, but not on steroidogenesis of ovine granulosa cells

BACKGROUND: Laminin (LN) is one of the most abundant extracellular matrix components of the basal lamina and granulosa cell layers of ovarian follicles. Culture of ovine granulosa cells (GC) on LN substratum induces cell spreading, enhances cell survival and proliferation, and promotes luteinization. Previous investigations have shown that these effects are mostly mediated by the alpha6beta1 integrin, but its signalization pathways have not been investigated. This study aimed to assess the importance of the cytoskeleton in the alpha6beta1 integrin-mediated actions of laminin on survival, proliferation and steroidogenesis of ovine GC. METHODS: The relationships between morphology and functions of ovine GC cultured on substrata containing LN or/and RGD peptides were investigated. The effects of (1) cytochalasin D, an actin cytoskeleton-disrupting drug, (2) a specific function-blocking antibody raised against alpha6 integrin subunit (anti-alpha6 IgG), and (3) an inhibitor of the ERK1/2 signalization pathway (PD98059) were assessed for GC shape, pyknosis and proliferation rates, oestradiol and progesterone secretions. RESULTS: Cytoskeleton disruption by cytochalasin D induced cell rounding, inhibited proliferation, promoted pyknosis, inhibited progesterone secretion and enhanced oestradiol secretion by GC cultured on LN. When GC were cultured on various substrata containing LN and/or RGD peptides in the presence or absence of anti-alpha6 IgG, both the existence of close correlations between the percentage of round cells, and the GC proliferation rate (r = -0.87) and pyknotic rate (r = 0.76) were established, but no relationship was found between cell shape and steroidogenesis. Inhibition of the ERK1/2 signalization pathway by PD98059 had no effect on GC shape, proliferation or pyknotic rates. However, it dramatically reduced progesterone secretion, expression of cytochrome P450 cholesterol side-chain cleavage and 3beta-hydroxysteroid deshydrogenase enzymes, and enhanced oestradiol secretion, thereby reproducing all the effects of the anti-alpha6 IgG on steroidogenesis of GC cultured on LN. CONCLUSION: LN may participate in the paracrine control of follicular development through different mechanisms. It could enhance proliferation and survival of GC through its alpha6beta1 integrin-mediated actions on cytoskeleton. In contrast, its stimulating action on GC luteinization could be partly mediated by the ERK1/2 pathway, irrespective of cell shape

Cell-Kinetics Based Calibration of a Multiscale Model of Structured Cell Populations in Ovarian Follicles

International audienceIn this paper, we present a strategy for tuning the parameters of a multiscale model of structured cell populations in which physiological mechanisms are embedded into the cell scale. This strategy allows one to cope with the technical difficulties raised by such models, that arise from their anchorage in cell biology concepts: localized mitosis, progression within and out of the cell cycle driven by time-and possibly unknown-dependent, and nonsmooth velocity coefficients. We compute different mesoscopic and macroscopic quantities from the microscopic unknowns (cell densities) and relate them to experimental cell kinetic indexes. We study the expression of reaching times corresponding to characteristic cellular transitions in a particle-like reduction of the original model. We make use of this framework to obtain an appropriate initial guess for the parameters and then perform a sequence of optimization steps subject to quantitative specifications. We finally illustrate realistic simulations of the cell populations in cohorts of interacting ovarian follicles. Introduction. In this paper, we deal with the question of the numerical calibration of an existing multiscale model of cell-structured populations in the physiological context of ovulation. This model was formulated as a system of weakly coupled, non conservative transport equations with controlled velocities and sink terms, where the unknowns are the cell densities in each follicle [9, 8]. A number of theoretical studies have established the well-posedness of the model [19], examined optimal control problems related to the ovulatory trajectories in the framework of hybrid optimal control theory [6], and studied the reachability of final states corresponding to either ovulatory or atretic cases in the framework of backwards reachable sets [8]. Implementation of the model in an efficient and reliable computing environment has involved the design of a finite-volume scheme dealing with the discontinuous coefficients [3], embedding this scheme within a dedicated adaptive mesh based on a multi-resolution approach [4], and implementing it on parallel architecture [2]. This has left the question of model calibration to biological specifications to be resolved. We have to face a generic, yet unsolved issue in parameter fitting for physiologically-oriented multiscale mathematical models: although mechanistic knowledge in molecular and cell biology is available on the lower scales, quantitative experimental data are rather available on the higher scales. In our case, the question is how to infer the parameters entering the microscopic functions (on the level of the follicular cells) from mesoscopic (on the level of the individual follicles, i.e. the number of follicular cells) or macroscopic (on the level of the populations of follicles) information. In addition, even on the macroscopic level, data remain rather scarce and are rarely obtained directly a

Coupled somatic cell kinetics and germ cell growth: multiscale model-based insight on ovarian follicular development

International audienceThe development of ovarian follicles is a unique instance of a morphogenesis process still occurring during adult life and resulting from the interactions between somatic and germ cells. In mammals, the initiation of follicular development from the pool of resting follicles is characterized by an increase in the oocyte size concomitant with the surrounding somatic cells proliferating to build an avascular tissue called granulosa. We present a stochastic individual-based model describing the first stages of follicular development, where the cell population is structured with respect to age (progression within the cell cycle) and space (radial distance from the oocyte). The model accounts for the molecular dialogue existing between the oocyte and granulosa cells. Three dynamically interacting scales are considered in the model: (i) a microscopic, local scale corresponding to an individual cell embedded in its immediate environment, (ii) a mesoscopic, semi-local scale corresponding to anatomical or functional areas of follicles and (iii) a macroscopic, global scale corresponding to the morphology of the follicle. Numerical simulations are performed to reproduce the 3D morphogenesis of follicles and follow simultaneously the detailed spatial distribution of individual granulosa cells, their organization as concentric layers or functional cell clones and the increase in the follicle size. Detailed quantitative simulation results are provided in the ovine species, in which well characterized genetic mutations lead to a variety of phenotypic follicle morphogenesis. The model can help to explain pathological situations of imbalance between oocyte growth and follicular cell proliferation 1. Introduction. A highly efficient reproductive capacity is a major advantage for species preservation, faced with the natural selection process, and for individuals within species. In mammalian females, ovarian function is the subject of intensive investigations with the aim to improve the reproductive capacity of domestic and wild animal species and to treat ovarian failures leading to infertility in humans. The issues are crucial for both clinical and zootechnical applications. In humans, the prevalence of the polycystic ovarian syndrome, which is a main cause of infertility, has been estimated at up to 10% among reproductive-age women [19]. Improvement of reproductive biotechnologies, including in vitro fertilization, intra-cytoplasmic sperm injection, frozen embryo replacements and egg donation, is a key issue for a better management of reproduction. Improving the knowledge upon ovarian function and its control will help to improve the success of assisted reproductive technologies, hence to prevent ovarian failure or hyperstimulation syndrome in women and to manag

The use of plasma AMH in sheep as an endocrine marker of the ovarian respnse to FSH in MOET programs: abstract

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Regulation of ovulation rate in mammals: contribution of sheep genetic models

Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate

Determination of Anti-Müllerian hormone plasma concentration can help to select donor sheep for laparoscopic ovum pick-up: abstract

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Regulation of AMH type 2 receptor expression by Bone Morphogenetic Protein 15 in ovine granulosa cells (abstract)

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Caractérisation phénotypique des vaches laitières portant l'haplotype " fertil+/+ " ou " fertil-/- " pour un QTL de fertilité femelle situé sur le chromosome 3

Au cours de ces dernières années, la fertilité des vaches laitières hautes productrices a fortement diminué, notamment en race Prim'Holstein. Cette baisse de fertilité est en partie liée à une intense sélection de la productivité laitière. Des études réalisées par des généticiens ont mis en évidence plusieurs QTLs (Quantitative Trait Locus) de fertilité femelle chez la vache laitière. Nous avons choisi d'étudier des vaches laitières homozygotes à l'haplotype favorable "fertil+/+" ou défavorable "fertil-/-" pour un QTL de fertilité femelle situé sur le chromosome 3 (QTL-Fert-F-BTA3), ce QTL étant impliqué dans les échecs précoces de la gestation. Plusieurs paramètres ont été étudiés sur quarante cinq génisses puis sur quarante et une vaches en première lactation. Nous avons observé que les primipares "fertil-/-" ont perdu plus de poids que les primipares "fertil+/+" dans les sept premières semaines après vêlage. Dans cette même période, la production laitière ainsi que l'ingéré alimentaire des primipares "fertil-/-" ont été inférieurs à ceux des primipares "fertil+/+". Grâce aux profils de progestérone plasmatique, nous avons constaté que les animaux "fertil+/+" ont repris leur activité ovarienne plus vite que les animaux "fertil-/-" après le vêlage. Nous avons enfin constaté un meilleur taux de réussite à la première insémination artificielle pour les animaux "fertil+/+" que pour les animaux "fertil-/-". La perte de poids après vêlage plus importante chez les animaux "fertil-/-" pourrait expliquer un allongement de l'inactivité ovarienne post-partum et une fertilité plus faible chez ces animaux par rapport aux animaux "fertil+/+"